CN113164520A - Methods of treating immunotherapy-related toxicity using GM-CSF antagonists - Google Patents

Abstract

Methods are provided for neutralizing and/or removing human GM-CSF in a subject in need thereof, the methods comprising administering CAR-T cells with a GM-CSF gene knockout (GM-CSF) to the subject^k/oCAR-T cells). Also provided are methods for GM-CSF gene inactivation or GM-CSF knock-out (KO) in a cell, comprising targeted genome editing or GM-CSF gene silencing. Methods are provided for preventing/treating immunotherapy-related toxicity comprising administering to the subject a CAR-T cell with GM-CSF gene inactivation or GM-CSF knock-out (GM-CSF)^k/oCAR-T cells) and/or a recombinant GM-CSF antagonist, wherein the GM-CSF gene is inactivated or knocked out. Methods are provided for reducing the level of a cytokine or chemokine other than GM-CSF in a subject having immunotherapy-related toxicity comprising administering to the subject a recombinant hGM-CSF antagonist. Also provided are methods for treating or preventing immunotherapy-related toxicity in a subject, comprising administering to the subject a chimeric antigen receptor-expressing T cell (CAR-T cell) having a GM-CSF gene knockout (GM-CSF) ^k/oCAR-T cells). Also provided are methods for preventing or reducing blood brain barrier disruption in a subject treated with immunotherapyThe method comprises administering to the subject a CAR-T cell with a GM-CSF gene knockout (GM-CSF)^k/oCAR-T cells).

Description

Methods of treating immunotherapy-related toxicity using GM-CSF antagonists

Cross Reference to Related Applications

The present application is a partial continuation application of U.S. application No. 16/283,694 filed on day 22/2/2019, a partial continuation application of U.S. application No. 16/248,762 filed on day 15/1/2019, a partial continuation application of U.S. application No. 16/204,220 filed on day 29/11/2018, a partial continuation application of U.S. application No. 16/149,346 filed on day 2/10/2018, a priority protection of U.S. provisional application No. 62/567,187 filed on day 2/10/2017 and priority protection of U.S. application No. 62/729,043 filed on day 10/9/10/2018, and a priority protection of PCT application No. PCT/US2018/053933 filed on day 2/10/2018, which are hereby incorporated by reference.

Technical Field

The present invention relates to a method for neutralizing and/or removing human GM-CSF in a subject in need thereof, comprising administering CAR-T cells with GM-CSF gene knockout (GM-CSF) to said subject ^k/oCAR-T cells). The invention also relates to methods for GM-CSF gene inactivation or GM-CSF knock-out (KO) in a cell comprising targeted genome editing or GM-CSF gene silencing. The invention further relates to methods for preventing/reducing immunotherapy-related toxicity comprising administering to a subject a CAR-T cell with GM-CSF gene inactivation or GM-CSF knock-out (GM-CSF)^k/oCAR-T cells), wherein the GM-CSF gene is inactivated or knocked out by the method.

The present invention relates to a method for reducing blood brain barrier disruption in a subject treated with immunotherapy, said method comprising administering to said subject a recombinant GM-CSF antagonist. The invention also relates to a method for preventing blood brain barrier integrity in a subject treated with immunotherapy, said method comprising administering to said subject a recombinant hGM-CSF antagonist. The invention further relates to methods for reducing or preventing CAR-T cell therapy-induced neuroinflammation in a subject in need thereof, the methodsComprising administering to the subject a recombinant GM-CSF antagonist. The present invention relates to methods for reducing the recurrence rate or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy in the absence of the occurrence of immunotherapy-related toxicities. The invention also relates to methods for reducing the recurrence rate or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy in the presence of toxicity associated with the occurrence of immunotherapy. The invention further relates to a method for reducing the level of a cytokine or chemokine other than GM-CSF in a subject who has developed immunotherapy-related toxicity, comprising administering to the subject a recombinant GM-CSF antagonist. The invention also relates to methods for treating or preventing immunotherapy-related toxicity in a subject comprising administering to the subject a chimeric antigen receptor-expressing T cell (CAR-T cell), a CAR-T cell with a GM-CSF gene knockout (GM-CSF) ^k/oCAR-T cells) and recombinant hGM-CSF antagonists. The disclosure herein also provides a method of inhibiting or reducing the incidence and/or severity of immunotherapy-related toxicity in a subject, comprising administering to the subject a recombinant GM-CSF antagonist.

Background

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine secreted by various cell types, including macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts. GM-CSF stimulates the differentiation of granulocytes and monocytes. In turn, monocytes migrate into the tissue and mature into macrophages and dendritic cells. Thus, secretion of GM-CSF results in a rapid increase in macrophage numbers. GM-CSF is also involved in inflammatory reactions of the Central Nervous System (CNS), leading to the influx of blood-borne monocytes and macrophages and the activation of astrocytes and microglia. Immune-related toxicities potentially include life-threatening immune responses due to high levels of immune activation caused by different immunotherapies. Immune-related toxicity is currently a major complication of the use of immunotherapy in cancer patients. Chimeric antigen receptor T (CAR-T) cell therapy has emerged as a novel and potentially revolutionary therapy for the treatment of cancer. Based on the unprecedented response of B-cell malignancies, the FDA approved two CD 19-targeted CAR-T (CART19) cell products in 2017. However, the broader application of CAR-T cell therapy is limited by the emergence of unique and potentially lethal toxicities. These include the development of Cytokine Release Syndrome (CRS) and Neurotoxicity (NT). Up to 50% of patients treated with CART19 cells develop CRS or NT of grade 3 or higher, and several deaths have been reported. These toxicities are associated with prolonged hospitalization, Intensive Care Unit (ICU) stay, and the long-term effects of NTs are unknown. Therefore, control of these CART19 cell-related toxicities is essential to reduce morbidity, mortality, duration of hospitalization, ICU admission, required supportive care, and significant collateral costs associated with CAR-T cell therapy. Clearly, there remains an urgent need for methods of preventing and treating immune-related toxicities. The ideal approach would minimize the risk of these life-threatening complications without affecting the efficacy of immunotherapy, and could even potentially improve efficacy by allowing, for example, a safe increase in the dose of immunotherapeutic compounds and/or expansion of T cells.

Disclosure of Invention

In one aspect, the invention provides a method for neutralizing and/or removing human GM-CSF in a subject in need thereof, the method comprising administering CAR-T cells with a GM-CSF gene knockout (GM-CSF) to the subject^k/oCAR-T cells).

In another aspect, the invention provides methods for GM-CSF gene inactivation or GM-CSF Knock Out (KO) in a cell, comprising targeting genome editing or GM-CSF gene silencing.

In a further aspect, the invention provides a method for preventing/reducing immunotherapy-related toxicity, the method comprising administering to a subject CAR-T cells with GM-CSF gene inactivation or GM-CSF knock-out (GM-CSF)^k/oCAR-T cell), wherein the GM-CSF gene is inactivated or knocked out by the methods described herein.

In one aspect, the invention provides a method for reducing blood brain barrier disruption in a subject treated with immunotherapy, the method comprising administering to the subject a recombinant GM-CSF antagonist.

In another aspect, the invention provides a method for preventing blood brain barrier integrity in a subject treated with immunotherapy, the method comprising administering to the subject a recombinant hGM-CSF antagonist.

In yet another aspect, the invention provides a method for reducing or preventing CAR-T cell therapy-induced neuroinflammation in a subject in need thereof, the method comprising administering to the subject a recombinant GM-CSF antagonist.

In another aspect, the invention provides a method for preventing or reducing blood brain barrier disruption in a subject treated with an immunotherapy, the method comprising administering to the subject a CAR-T cell with a GM-CSF gene knockout (GM-CSF)^k/oCAR-T cells).

In one aspect, the invention provides methods for reducing the recurrence rate or preventing or delaying the occurrence of tumor recurrence in a subject treated with immunotherapy in the absence of the occurrence of immunotherapy-related toxicity, comprising administering to the subject a recombinant hGM-CSF antagonist. In a related aspect, the invention provides methods for reducing the recurrence rate or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy in the presence of toxicity associated with the occurrence of immunotherapy, comprising administering to the subject a recombinant hGM-CSF antagonist.

In another aspect, the invention provides a method of reducing the level of a cytokine or chemokine other than GM-CSF in a subject who has developed immunotherapy-related toxicity, comprising administering to the subject a recombinant hGM-CSF antagonist, wherein the level of the cytokine or the chemokine is reduced compared to the level of the cytokine or the chemokine in the subject during the development of said immunotherapy-related toxicity.

In a further aspect, the invention provides a method for preventing or reducing a subjectA method of immunotherapy-related toxicity, the method comprising administering to the subject a chimeric antigen receptor-expressing T cell (CAR-T cell) having its GM-CSF gene "knock-out" (GM-CSF)^k/oCAR-T cells), and recombinant hGM-CSF antagonists. GM-CSF^k/oCAR-T cells can be administered in combination with a hGM-CSF antagonist (recombinant hGM-CSF antagonist).

In one aspect, disclosed herein is a method of inhibiting or reducing the incidence or severity of immunotherapy-related toxicity in a subject, comprising the step of administering to the subject a recombinant hGM-CSF antagonist.

In related aspects, the immunotherapy comprises adoptive cell transfer, administration of monoclonal antibodies, administration of cytokines or chemokines, administration of cancer vaccines, T cell engagement therapy, or any combination thereof.

In another aspect, adoptive cell transfer includes administering a chimeric antigen receptor expressing T cell (CAR T cell), a T Cell Receptor (TCR) modified T cell, a Tumor Infiltrating Lymphocyte (TIL), a Chimeric Antigen Receptor (CAR) modified natural killer cell, or a dendritic cell, or any combination thereof. In a related aspect, the monoclonal antibody is selected from the group comprising: anti-CD 3, anti-CD 52, anti-PD 1, anti-PD-L1, anti-CTLA 4, anti-CD 20, anti-BCMA antibodies, bispecific antibodies, or bispecific T-cell engager (BiTE) antibodies, or any combination thereof. In a related aspect, the cytokine is selected from the group comprising: IFN alpha, IFN beta, IFN gamma, IFN lambda, IL-1, IL-2, IL-6, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18, GM-CSF, TNF alpha or any combination thereof.

In another aspect, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing the concentration of at least one inflammation-related factor in the serum, tissue fluid, or CSF of the subject. In a related aspect, the inflammation-related factor is selected from the group comprising: c-reactive protein, GM-CSF, IL-1, IL-2, sIL2R α, IL-5, IL-6, IL-8, IL-10, IP10, IL-15, MCP-1(AKA CCL2), MIG, MIP1 β, IFN γ, CX3CR1, or TNF α, or any combination thereof. In another aspect, administration of the recombinant GM-CSF antagonist does not reduce the efficacy of the immunotherapy. In another aspect, administration of a recombinant GM-CSF antagonist increases the efficacy of the immunotherapy. In another aspect, the administration of the recombinant GM-CSF antagonist is performed prior to, simultaneously with, or after immunotherapy. In related aspects, the recombinant GM-CSF antagonist is co-administered with a corticosteroid, an anti-IL-6 antibody, tocilizumab (tocilizumab), an anti-IL-1 antibody, cyclosporin, an antiepileptic, a benzodiazepine, acetazolamide, a hyperventilation therapy, or a hypertonic therapy, or any combination thereof.

In another aspect, the immunotherapy-related toxicity comprises a brain disease, injury, or dysfunction. In a related aspect, the brain disease, injury, or dysfunction includes CAR-T cell associated NT or CAR-T cell associated encephalopathy syndrome (CRES). In related aspects, inhibiting or reducing the incidence of a brain disease, injury, or dysfunction includes reducing headache, delirium, anxiety, tremor, seizure activity, confusion, altered wakefulness, hallucinations, speech impairment, ataxia, apraxia, facial paralysis, motor weakness, seizures, non-tic EEG seizures, altered levels of consciousness, coma, endothelial activation, vascular leakage, intravascular coagulation, or any combination thereof in the subject. In another aspect, the immunotherapy-related toxicity comprises CAR-T induced Cytokine Release Syndrome (CRS). In a related aspect, inhibiting or reducing the incidence of CRS comprises reducing or inhibiting, but is not limited to, high fever, myalgia, nausea, hypotension, hypoxia or shock, or a combination thereof. In a related aspect, immunotherapy-related toxicities are life-threatening.

In another aspect, the subject has a decreased serum concentration of ANG2 or VWF or a decreased serum ANG2: ANG1 ratio. In a related aspect, the body temperature of the subject is greater than 38 ℃, the IL-6 serum concentration is >16pg/ml, or the MCP-1 serum concentration is greater than 1,300pg/ml during the first 36 hours after infusion of the CAR-T cells. In related aspects, the subject is predisposed to having the brain disease, injury, or dysfunction. In a related aspect, the ANG2 to ANG1 ratio in the serum of the subject is greater than 1 prior to infusion of the CAR-T cells.

In another aspect, the immunotherapy-related toxicities include Hemophagocytic Lymphocytosis (HLH) or Macrophage Activation Syndrome (MAS). In related aspects, inhibiting or reducing the incidence of HLH or MAS includes increasing survival time and/or relapse time, decreasing macrophage activation, decreasing T cell activation, decreasing the concentration of IFN γ in peripheral circulation, or decreasing the concentration of GM-CSF in peripheral circulation, or any combination thereof.

In another aspect, the subject exhibits fever, splenomegaly, cytopenia involving two or more lines, hypertriglyceridemia, hypofibrinogenemia, erythrophagia, low or absent NK cell activity, a serum concentration of ferritin above 500U/ml or a serum concentration of soluble CD25 above 2400U/ml, or any combination thereof. In related aspects, the subject is predisposed to acquire HLH or MAS. In related aspects, the subject carries a mutation in a gene selected from the group consisting of: PRF1, UNC13D, STX11, STXBP2, or RAB27A, or having reduced perforin expression, or any combination thereof.

In one embodiment, the GM-CSF antagonist is an anti-hGM-CSF antibody. In another embodiment, the anti-hGM-CSF antibody blocks the binding of hGM-CSF to the alpha subunit of the hGM-CSF receptor. In another embodiment, the anti-hGM-CSF antibody is a polyclonal antibody. In another embodiment, the anti-hGM-CSF antibody is a monoclonal antibody. In another embodiment, the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab ', F (ab')2, scFv, or dAB. In some embodiments, monoclonal anti-hGM-CSF antibodies, single chain Fv and Fab can be produced in chicken; chicken IgY is an avian equivalent of a mammalian IgG antibody. (Park et al, "Biotechnology Letters" (2005)27: 289) 295; Finley et al, "applied and environmental microbiology (appl. environ. Microbiol.), (2006, 5.p., 3343) 3349). The chicken IgY antibody has the following advantages: higher avidity, i.e., overall strength of binding between the antibody and antigen, higher specificity (lower cross-reactivity with mammalian proteins other than the immunogen); high yield of egg yolk, and lower background (structural differences in Fc regions of IgY and IgG result in less false positive staining). In another example, the anti-hGM-CSF antibody may be a camelid, e.g., a heavy chain antibody (also referred to as sdAb, VHH and VHH) lacking a light chain

) (iii) a single variable domain of llama origin; the VHH domain (about 15kDa) is the smallest known antigen recognition site present in mammals with full binding capacity and affinity (equivalent to conventional antibodies). (Garacicoechea et al (2015) public science library: Complex (PLoS ONE) 10(8) e 0133665; Arbabi-ghahroud M (2017) immunologic frontier (front. immunological.) 8: 1589; Wu et al, transformation Oncology (2018)11, 366-. In another embodiment, the antibody fragment is conjugated to polyethylene glycol. In another embodiment, the affinity of the anti-hGM-CSF antibody ranges from about 5pM to about 50 pM. In another embodiment, the anti-hGM-CSF antibody is a neutralizing antibody. In another embodiment, the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. In another embodiment, the anti-hGM-CSF antibody is a human antibody. In another embodiment, the anti-hGM-CSF antibody comprises a human variable region. In another embodiment, an anti-hGM-CSF antibody comprises an engineered human variable region. In another embodiment, the anti-hGM-CSF antibody comprises a humanized variable region. In another embodiment, an anti-hGM-CSF antibody comprises an engineered human variable region. In another embodiment, the anti-hGM-CSF antibody comprises a humanized variable region.

In one embodiment, the anti-hGM-CSF antibody comprises a human light chain constant region. In another embodiment, the anti-hGM-CSF antibody comprises a human heavy chain constant region. In another embodiment, the human heavy chain constant region is a gamma chain. In another embodiment, the anti-hGM-CSF antibody binds the same epitope as the chimeric 19/2 antibody. In another embodiment, an anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of the chimeric 19/2 antibody. In another embodiment, an anti-GM-CSF antibody includes the VH region CDR1, CDR2 and CDR3 and the VL region CDR1, CDR2 and CDR3 of the chimeric 19/2 antibody.

In one embodiment, the anti-hGM-CSF antibody comprises a heavy chain variable region comprising CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V segment, wherein the J segment comprises at least 95% identity to human JH4(YFDYWGQGTLVTVSS) and the V segment comprises at least 90% identity to human germline VH 11-02 or VH 11-03 sequencesThe first property; or a heavy chain variable region comprising a CDR3 binding specificity determinant of RQRFPY. In another embodiment, the J section comprises YFDYWGQGTLVTVSS. In another embodiment, the CDR3 includes RQRFPYYFDY or RDRFPYYFDY. In another embodiment, the heavy chain variable region CDR1 or CDR2 may be a human germline VH1 sequence; or both the CDR1 and CDR2 may be human germline VH 1. In another embodiment, the antibody comprises a heavy chain variable region CDR1 or CDR2 or both CDR1 and CDR2, such as the V shown in fig. 1 _HShown in the section. In another embodiment, the anti-hGM-CSF antibody has a V segment with a V as shown in figure 1_HV segment sequence. In another embodiment, V_HSequences having VH #1, VH #2, VH #3, VH #4, or VH #5 shown in fig. 1.

In another embodiment, for example, an anti-hGM-CSF antibody having a heavy chain variable region as described in the preceding paragraph comprises a light chain variable region comprising a CDR3 binding specificity determinant comprising the amino acid sequence FNK or FNR.

In another embodiment, the anti-hGM-CSF antibody comprises a VL region comprising CDR3 comprising the amino acid sequence FNK or FNR. In one embodiment, the anti-GM-CSF antibody comprises a human germline JK4 region. In another embodiment, antibody V_LRegion CDR3 includes QQFN (K/R) SPLT. In another embodiment, an anti-GM-CSF antibody comprises a VL region comprising CDR3 comprising QQFNKSPLT. In another embodiment, the VL region comprises V as shown in FIG. 1_LThe CDR1, or the CDR2, or both the CDR1 and the CDR2 of the region. In another embodiment, V_LRegions include a V segment that is at least 95% identical to the VKIIIA 27V segment sequence shown in fig. 1. In another embodiment, V _LRegions have the sequence VK #1, VK #2, VK #3 or VK #4 shown in FIG. 1.

In some embodiments, the anti-hGM-CSF antibody has a VH region CDR3 binding specificity determinant RQRFPY or RDRFPY, and a VL region having a CDR3 comprising QQFNKSPLT. In another embodiment, the anti-hGM-CSF antibody has the VH region sequence shown in figure 1 and the VL region sequence shown in figure 1. In another embodiment, the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence include a methionine at the N-terminus. In another embodiment, the GM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibodies or receptor subunits or soluble GM-CSF receptors, cytochrome b562 antibody mimetics, hGM-CSF peptide analogues, mimetibody protein drugs (adnectins), lipocalin scaffold antibody mimetics, calixarene antibody mimetics and antibody-like binding peptide mimetics.

In one embodiment, disclosed herein is a method of increasing the efficacy of CAR-T immunotherapy in a subject, the method comprising the step of administering to the subject a recombinant hGM-CSF antagonist, wherein the administration increases the efficacy of CAR-T immunotherapy in the subject. In another embodiment, the administering of the recombinant hGM-CSF antagonist is prior to, concurrent with, or subsequent to the CAR-T immunotherapy. In another embodiment, the increased efficacy comprises increased CAR-T cell expansion, decreased number of Myeloid Derived Suppressor Cells (MDSCs) that suppress T cell function, synergy with a checkpoint inhibitor, or any combination thereof. In another embodiment, the increased CAR-T cell expansion comprises an increase of at least 50% compared to a control. In another embodiment, the increased CAR-T cell expansion comprises at least one-quarter log expansion compared to a control. In another embodiment, the increased cell expansion comprises at least one-half log expansion as compared to a control. In another embodiment, the increased cell expansion comprises at least one log expansion as compared to a control. In another embodiment, the increased cell expansion comprises more than one log expansion compared to a control.

In one embodiment, the hGM-CSF antagonist comprises a neutralizing antibody. In another embodiment, the neutralizing antibody is a monoclonal antibody.

In one embodiment, disclosed herein is a method of inhibiting or reducing the incidence or severity of CAR-T related toxicity in a subject, comprising the step of administering to the subject a recombinant hGM-CSF antagonist, wherein the administration inhibits or reduces the incidence or severity of CAR-T related toxicity in the subject. In one embodiment, the CAR-T associated toxicity comprises NT, CRS, or a combination thereof. In one embodiment, the methods of treatment provided herein prevent and treat CRS and NT in a subject in need thereof. In some embodiments, the CAR-T cell-associated NT is reduced by about 50% compared to the reduction in NT in a subject treated with the CAR-T cell and the control antibody. In various embodiments, the recombinant hGM-CSF antagonist is an hGM-CSF neutralizing antibody according to the embodiments described herein.

In another embodiment, said inhibiting or reducing the incidence of CRS comprises increasing time to survival and/or time to relapse, reducing macrophage activation, reducing T cell activation, or reducing the concentration of circulating hGM-CSF, or any combination thereof. In another embodiment, the subject exhibits fever (with or without chills, malaise, fatigue, anorexia, myalgia, joint pain, nausea, vomiting, headache, rash, diarrhea, tachypnea, hypoxemia, hypoxia, shock, cardiovascular tachycardia, pulse broadening, hypotension, capillary leakage, increased early cardiac output, decreased late cardiac output, increased D-dimers, hypofibrinogenemia with or without bleeding, azotemia, elevated transaminase, hyperbilirubinemia, altered mental state, confusion, delirium, tandard aphasia, hallucinations, tremors, dysdiscrimination, altered gait, seizures, organ failure, or any combination thereof).

In another embodiment, inhibiting or reducing the incidence or severity of CAR-T associated toxicity comprises preventing the onset of CAR-T associated toxicity.

In another embodiment, disclosed herein is a method of blocking or reducing GM-CSF expression in a cell, comprising knocking out or silencing GM-CSF gene expression in a cell. In one embodiment, the blocking or reduction of GM-CSF expression comprises short interfering rns (sirna), CRISPR, RNAi, DNA-directed RNA interference (ddRNAi), which is a gene silencing technique that uses DNA constructs to activate the endogenous RNA interference (RNAi) pathway of an animal cell, or targeted genome editing with an engineered transcription activator-like effector nuclease (TALEN), an artificial protein consisting of a customizable sequence-specific DNA binding domain fused to a nuclease that cleaves DNA in a non-sequence-specific manner. (Joung and Sander, Nature review of molecular Cell biology (Nat Rev Mol Cell Biol.) 2013, 1 month; 14(1): 49-55, which is incorporated herein by reference in its entirety.) in some embodiments, the cells are CAR-T cells.

In one embodiment, the subject is a human.

In one embodiment, disclosed herein is an hGM-CSF antagonist for use in a method of inhibiting or reducing the incidence or severity of immunotherapy-related toxicity in a subject, comprising the step of administering to the subject a recombinant hGM-CSF antagonist. In one embodiment, disclosed herein is a pharmaceutical composition comprising an anti-hGM-CSF antagonist.

Drawings

FIG. 1 provides exemplary V of anti-GM-CSF antibodies_HAnd V_LAnd (4) sequencing.

FIGS. 2A-2B show binding of GM-CSF to Ab1 (FIG. 2A) or Ab2 (FIG. 2B) as determined by surface plasmon resonance analysis (Biacore 3000) at 37 ℃. Ab1 and Ab2 were captured by anti-Fab polyclonal antibodies immobilized on a Biacore chip. As shown, different concentrations of GM-CSF were injected onto the surface. Assume that global fit analysis was performed with a 1:1 interaction using the Scrubber2 software.

Fig. 3A-3B show binding of Ab1 and Ab2 to glycosylated (fig. 3A) and non-glycosylated GM-CSF (fig. 3B). Binding to glycosylated GM-CSF expressed from human 293 cells or non-glycosylated GM-CSF expressed in E.coli (E.coli) was determined by ELISA. Representative results of a single experiment are shown (experiment 1). A two-fold dilution of Ab1 and Ab2 starting at 1500ng/ml was applied to GM-CSF coated wells. Each point represents the mean ± standard error of triplicate determinations. Sigmoidal curve fitting was performed using Prism 5.0 software (Graphpad).

Fig. 4A-4B show a competition ELISA demonstrating binding of Ab1 and Ab2 to a shared epitope. ELISA plates coated with 50 ng/well of recombinant GM-CSF were incubated with various concentrations of antibody (Ab2, Ab1, or isotype control antibody) and 50nM biotinylated Ab 2. Biotinylated antibody binding was determined using a neutravidin-HRP conjugate. Competition for binding to GM-CSF was 1 hour (FIG. 4A) or 18 hours (FIG. 4B). Each point represents the mean ± standard error of triplicate determinations. Sigmoidal curve fitting was performed using Prism 5.0 software (Graphpad).

FIG. 5 shows GM-CSF-induced inhibition of IL-8 expression. Various amounts of each antibody were incubated with 0.5ng/ml GM-CSF and with U937 cells for 16 hours. Secretion of IL-8 into the culture supernatant was determined by ELISA.

FIG. 6 shows the dose-dependent inhibition of human granulocytes by GM-CSF stimulated CD11b by anti-GM-CSF antibody.

FIG. 7 shows the dose-dependent inhibition of GM-CSF-induced HLA-DR on CD14+ human primary monocytes/macrophages by anti-GM-CSF antibodies.

Figure 8 demonstrates the role of GM-CSF (myeloid inflammatory factor) as a key cytokine in CAR-T related activity and in stimulating leukocyte proliferation, a characteristic feature of certain leukemias, e.g., Acute Myelogenous Leukemia (AML).

FIG. 9 shows the inhibition of GM-CSF dependent human TF-1 cell proliferation (human erythroleukemia) by neutralizing human GM-CSF with an anti-GM-CSF antibody. KB003 is a recombinant monoclonal antibody designed to target and neutralize human GM-CSF. KB002 is a mouse/human chimeric monoclonal antibody that targets and neutralizes hGM-CSF.

Figure 10 is a depiction of a chimeric antigen receptor.

Figure 11 demonstrates that CAR-T19 therapy results in a high response rate in relapsed refractory ALL. The data show historical and in R/R ALL after CAR-T19 therapy. (Maude et al, New England Journal of Medicine (NEJM); 2014).

FIG. 12 presents evidence showing significant association of GM-CSF with NT. GM-CSF levels are associated with severe adverse effects following CAR-T cell therapy. GM-CSF levels precede and regulate other cytokines besides IL-15. The increase in GM-CSF is clearly associated with grade 3 NT. IL-2 is only relevant to other cytokines.

Figure 13 shows the estimated time course of CRS and NT following CD19 CAR-T cell therapy. The time of onset of symptoms and the severity of CRS depends on the inducing agent, the type of cancer, the age of the patient, and the degree of immune cell activation. CAR-T associated CRS symptomatic attack typically occurs days to occasionally weeks after T cell infusion, which coincides with maximal T cell expansion. Similar to the CRS associated with mAb therapy, CRS associated with adoptive T cell therapy has been consistently associated with elevated IFN γ, IL-6, TNF α, IL-1, IL-2, IL-6, GM-CSF, IL-10, IL-8, and IL-5. There is no clear CAR-T cell dose-response relationship for CRS, but very high doses of T cells can lead to earlier onset of symptoms.

FIG. 14 demonstrates that GM-CSF is a key initiator for CAR-T adverse reactions. The figure depicts the central role of GM-CSF in CRS and NT. Perforin allows granzyme to penetrate the tumor cell membrane. GM-CSF produced by CAR-T recruits CCR2+ myeloid cells to the tumor site, which produces CCL2(MCP 1). CCL2 positively enhanced its own production through CCR2+ myeloid cell recruitment. IL-1 and IL-6 from myeloid cells form another positive feedback loop with CAR-T by inducing the production of GM-CSF. The result of the cell membrane disruption by perforin and granzyme is that phosphatidylserine is exposed. Phosphatidylserine stimulates myeloid cell production of CCL2, IL-1, IL-6, and other inflammatory effectors. The net result of this self-enhancing feedback loop is endothelial activation, vascular permeability, and ultimately CRS and NT. Furthermore, animal model evidence suggests that GM-CSF knock-out mice show no signs of CRS, but IL-6 knock-out mice can still develop CRS. GM-CSF receptor k/o from CCR2+ myeloid cells abolished the cascade in the neuroinflammatory model. (sentiman et al, J.Immunol.), "Immunity" (2015 (43) 510-514; Ishii et al, Blood (Blood) 128: 3358; Teaache et al, Cancer discovery (Cancer Discov.) -2016 (6) (664) -679; Lee et al, Blood 124:2: 188; Barrett et al, Blood 2016:128-654, each of which is incorporated herein by reference in its entirety).

FIGS. 15a-15g show that GM-CSF CRISPR knockout T cells exhibit reduced GM-CSF expression, but similar levels of other cytokines and degranulation. a. Generating GM-CSF knock-out CAR-T. (see example 6).

Figures 16a-16i demonstrate that GM-CSF neutralizing antibodies according to the embodiments described herein do not inhibit CAR-T mediated killing, proliferation or cytokine production, but successfully neutralize GM-CSF (see example 7).

FIGS. 17a-17b show the protocol and results from a mouse model of human CRS. (example 5).

Figures 18a-18c demonstrate CAR-T efficacy in xenograft models in combination with GM-CSF neutralizing antibodies according to the examples described herein. GM-CSF neutralizing antibodies were shown not to inhibit CAR-T efficacy in vivo. (see example 8).

Figure 19 demonstrates in vitro and in vivo preclinical data showing that GM-CSF neutralizing antibodies according to the examples described herein do not impair the CAR-T effect on survival. In the absence of PBMCs, GM-CSF neutralizing antibodies do not block CAR-T cell function in vivo. Survival rates of CAR-T + control and CAR-T + GM-CSF neutralizing antibodies were similar. (see example 9).

Figures 20a-20b demonstrate in vitro and in vivo preclinical data showing that GM-CSF neutralizing antibodies according to the examples described herein do increase CAR-T amplification. GM-CSF neutralizing antibodies increase CAR-T cancer cell killing in vitro. Antibody neutralization of GM-CSF increases the proliferation of CAR-T cells in the presence of PBMCs. The GM-CSF neutralizing antibody increases the proliferation of CAR-T in the presence of PBMCs. (unaffected without PBMC). anti-GM-CSF antibodies do not inhibit CAR-T degranulation, intracellular GM-CSF production, or IL-2 production. (see example 10).

Figure 21 demonstrates that CAR-T amplification correlates with improved overall response rate. CAR AUC (area under the curve) is defined as the cumulative level of CAR + cells/microliter of blood within the first 28 days after CAR-T administration. P values were calculated by Wilcoxon rank sum test. (Neelapu et al ICML 2017 Abstract 8). (see example 11).

Figure 22 shows a study protocol for GM-CSF neutralizing antibodies according to the examples described herein. (see example 12). CRS and NT were assessed daily during the hospitalization period and 30 days prior to the first visit. Eligible subjects received GM-CSF neutralizing antibody on days-1, +1, and +3 of CAR-T treatment. Additional doses may be considered to be administered for at least day 7. Tumor assessments were performed at baseline and at 1 st, 3 rd, 6 th, 9 th, 12 th, 18 th and 24 th months. Blood samples (PBMC and serum) at days-5, -1, 0, 1, 3, 5, 7, 9, 11, 13, 21, 28, 90, 180, 270 and 360. (see example 12).

FIGS. 23A-24B demonstrate that GM-CSF depletion increases CAR-T cell expansion. FIG. 23A shows GM-CSF in comparison to control CAR-T cells^k/oIncreased ex vivo expansion of CAR-T cells. Figure 23B demonstrates more robust CAR-T cell proliferation after treatment with GM-CSF neutralizing antibodies according to embodiments described herein. (see example 13).

Figure 24 shows a safety profile of GM-CSF neutralizing antibodies according to the examples described herein. (see example 14).

Figures 25A-25D show that GM-CSF neutralizing antibodies exhibited a 90% reduction in neuroinflammation in a mouse preclinical model when added to CAR-T cell therapy. Figure 25A demonstrates MRI data (T1 high intensity indicates BBB destruction and neuroinflammation) where mouse brains were protected from neuroinflammation following administration of CAR-T cells and GM-CSF neutralizing antibodies according to the examples described herein, compared to mouse brains showing signs of neurotoxicity following administration of CAR-T cells and control antibodies (top row) and compared to untreated (baseline) mouse brains (bottom row). Figure 25B quantitatively demonstrates the percent increase in T1 high intensity relative to baseline: the brain T1 high intensity in mice administered with CAR-T and GM-CSF neutralizing antibodies according to the examples described herein increased by about 10% percent relative to baseline compared to a slightly more than 100% increase in mice already administered with CAR-T cells and control antibodies. As shown in the comparative figures, an approximately 10% increase in brain T1 high intensity relative to baseline in mice administered with CAR-T and GM-CSF neutralizing antibodies compared to the amount of neuroinflammation present in mice receiving CAR-T cells and control antibodies is a 90% decrease in neuroinflammation, as measured by brain T1 high intensity relative to baseline. Figures 25C-25D show that treatment with CAR-T plus GM-CSF neutralizing antibodies according to the embodiments described herein resulted in a significant reduction in the number of leukemic cells (to between 500 and 5,000 cells) and an improvement in overall disease control (see example 15) compared to untreated mice (which had 500,000 to 1.5M leukemic cells) and CAR-T plus control antibody (which had between 15,000 and 100,000 leukemic cells).

Figures 26A-26I show that GM-CSF blockade helps to control CART19 toxicity and indeed improve efficacy. Figure 26A shows that CART19 and ritvolumab-treated CART19 were equally effective in survival outcome in the high tumor burden NALM6 relapse model compared to UTD (untransformed T cells) per group of 7-8 mice, n ═ 2. Figures 26B-26D show that ritzelumab and anti-mouse GM-CSF antibody-controlled CRS-induced weight loss, neutralization of serum human GM-CSF, and reduction of serum mouse MCP-1 (monocyte chemotactic protein-1) expression in the primary ALL xenograft CART19 CRS/NT model (3 mice per group,. p < 0.05). Figure 26E shows that in the primary ALL xenograft CART19 CRS/NT model (3 mice per group p <0.05, p <0.01), rituximab and anti-mouse GM-CSF antibodies reduced encephalitis as shown by MRI. Figures 26F-26G show improved efficacy of CART19+ ritzimab treated mice compared to anti-mouse GM-CSF antibody treated mice (i.e., CART19+ anti-hGM-CSF antibody), which shows a reduction in CD19+ brain leukemia burden and a reduction in the percentage of brain macrophages in the primary ALL xenograft CART19 CRS/NT model (3 mice per group). Figure 26H shows CRISPR Cas 9K/O of GM-CSF reduces its expression by intracellular staining in CART19 and UTD by stimulation with NALM 6. (representative experiment, n ═ 2) figure 26I shows that CART19 and GM-CSF K/O CART19 control tumor burden better than UTD and improved efficacy of GM/CSF K/O CART19 cells controlling tumor burden was slightly better than CART19 in the high tumor burden NALM6 relapse model (6 mice per group, # p <0.05, # p < 0.0001). Error bars SEM.

Figures 27A-27D show that GM-CSF enhances CAR-T cell proliferation and does not impair CAR-T cell effector function in vitro and in the presence of monocytes. Figure 27A graphically depicts litzilucumab (hGM-CSF neutralizing antibody) neutralization of hGM-CSF produced by CAR-T cells in vitro, as determined by multiplex analysis after 3 days of culture with CART19 in culture medium alone or CART19 co-cultured with NALM6, n-2 experiments, 2 replicates per experiment, depicting representative experiments, { p <0.001 between litzilucumab and isotype control treatment, ttest, mean + SEM, compared to isotype control treatment. Figure 27B graphically illustrates the ability of hGM-CSF neutralizing antibody treatment to not inhibit CAR-T cell proliferation as determined by the CSFE flow cytometry proliferation assay of live CD3 cells, n ═ 3 donors, 2 replicates per donor, representative experiments depicted at the 3-day time point, ns p >0.05 between litzizumab and isotype control treatment, T-test, mean + SEM. Separately: CART19, MOLM 13: CART19+ MOLM13, PMA/ION: CART19 plus 5ng/mL PMA and 0.1ug/mL ION, NALM 6: CART19+ NALM 6. Figure 27C graphically depicts that litzizumab enhanced proliferation of CART19 by neutralizing hGM-CSF when co-cultured with human monocytes compared to isotype control treated with CART19, n-3 donors at 3-day time point, 2 replicates per donor,. p <0.0001, mean + SEM. Figure 27D graphically shows that litzizumab treatment did not inhibit cytotoxicity of CART19 or untransformed T cells (UTD) when cultured with NALM6, n ═ 3 donors, 2 replicates per donor, representative experiments depicted at 48 hour time points, ns p >0.05 between litzizumab and isotype control treatment, T-tests, mean + SEM.

FIGS. 28A-28F show that GM-CSF neutralization in vivo enhances CAR-T cell antitumor activity (i.e., tumor cell killing) in xenograft models. Fig. 28A shows the experimental protocol: NSG mice were injected with CD19+ luciferase + cell line NALM6(1 × 106 cells per mouse i.v). 4-6 days later, mice were imaged, randomized, and on the next day received an equal number of total cells of 1-1.5 × 106 CAR-T19 or control UTD cells with Ritzuzumab or control IgG (10mg/Kg, IP given daily for 10 days, starting on the day of CAR-T injection). Starting on day 7 after CAR-T cell injection, mice were subjected to continuous bioluminescent imaging to assess disease burden, and overall survival. Tail vein bleeding was performed 7-8 days after CAR-T cell injection. Figure 28B depicts lizluzumab neutralized CAR-T generated serum hGMCSF in vivo compared to isotype control treatment as determined by hGM-CSF single peak, n ═ 2 experiments, 7-8 mice per group, representative experiments, sera from CAR-T cells/day 8 post UTD injection, p <0.001 between ranibizumab and isotype control treatment, T test, mean + SEM. Figure 28C graphically depicts that ritvolumab treated in vivo CAR-T was equally effective in controlling tumor burden compared to isotype control treated CAR-T in a high tumor burden relapse xenograft model of ALL, with 7 days post CAR-T injection, n ═ 2 experiments, 7-8 mice per group, depicting representative experiments, ×) p <0.001, × p <0.05, ns p >0.05, T-tests, mean + SEM. Fig. 28D depicts the mouse image from fig. 28C. Fig. 28E shows the experimental protocol: NSG mice were injected with blast cells derived from patients with ALL (1 × 106 cells/mouse i.v). Mice were serially bled and when CD19+ cells > 1/microliter, mice were randomly grouped to receive 2.5 x 106 CART19(10mg/Kg, IP given daily for 10 days, starting on the day of CAR-T injection) with either ritzezumab or control IgG. Starting on day 14 post CAR-T cell injection, mice were subjected to serial tail vein bleedings to assess disease burden, and overall survival was performed. Figure 28F graphically depicts that litzimab treatment with CAR-T therapy resulted in more sustained control of tumor burden over time in primary Acute Lymphoblastic Leukemia (ALL) xenograft model compared to isotype control treatment with CAR-T therapy, 6 mice per group, p <0.01, p <0.05, ns p >0.05, T-test, mean + SEM.

Figures 29A-29E demonstrate that GM-CSF CRISPR knock-out CAR-T cells exhibit reduced GM-CSF expression, similar levels of key cytokines and chemokines, and enhanced anti-tumor activity. FIG. 29A shows CRISPR Cas9 GM-CSF compared to wild-type CART19^k/oCART19 exhibited reduced GM-CSF production, but other cytokine production and degranulation were not inhibited by disruption of the GM-CSF gene, CART19 and GM-CSFk/o CART19 stimulated with NALM6, n ═ 3 experiments, 2 replicates per experiment<0.001，*p<0.05，ns p>0.05 mixing of GM-CSF^k/oCART19 and CAR19Comparison, t-test, mean + SEM. FIG. 29B shows GM-CSF in comparison to CAR-T treatment^k/oCAR-T has reduced serum human GM-CSF in vivo, as determined by multiplex analysis, 5-6 mice per group (4-6 at bleeding, 8 days post CAR-T cell injection), between GM-CSFk/o CART19 and wild-type CART19<0.0001，***p<0.001, t test, mean + SEM. FIG. 29C shows GM-CSF in comparison to wild-type CART19^k/oCART19 enhanced overall survival in vivo in a high tumor burden recurrent xenograft model of ALL using the NALM6 cell line, 5-6 mice per group<0.01, log rank. FIGS. 29D-29E show human (FIG. 29D) and mouse (FIG. 29E) cytokines and chemokines from multiple sera, except for hGM-CSF in GM-CSF ^k/oThere were no statistical differences shown between CART19 and wild-type CART19, further suggesting that key T-cell cytokines and chemokines were not disadvantageously depleted by reducing GM-CSF expression, 5-6 mice per group (4-6 at bleeding)<0.0001, t test.

Figures 30A-30D show patient-derived xenograft models directed against neuroinflammation and cytokine release syndrome. Fig. 30A shows the experimental protocol: mice received 1-3 x 106 primary blast cells derived from peripheral blood of patients with primary ALL. The mice were monitored for transplantation by tail vein bleeding for about 10-13 weeks. When serum CD19+ cells ≧ 10 cells/microliter, the mice received CART19 (2-5X 106 cells) and antibody therapy was initiated for a total of 10 days, as indicated. Mice were weighed on a daily basis as a measure of their health status. Mouse brain MRI was performed 5-6 days after CART19 injection and tail vein bleeding was performed 4-11 days after CART19 injection for cytokine/chemokine and T cell analysis, 2 independent experiments. Figure 30B shows that GM-CSF neutralization in combination with CART19 was equally effective as the isotype control antibody in combination with CART19 in controlling CD19+ loading of ALL cells, representative experiments with 3 mice per group, 11 days post-CART 19 injection, p <0.05 between GM-CSF neutralization + CART19 and isotype control + CART19, t-test, mean + SEM. Figure 30C presents brain MRI data showing that CART19 therapy exhibited T1 enhancement, indicating brain blood brain barrier disruption and possible edema. Representative images of 3 mice per group, 5-6 days after CART19 injection. Figure 30D demonstrates that high tumor burden primary ALL xenografts treated with CART19 showed human CD3 cell infiltration of brain compared to untreated PDX control. 3 mice per group, representative image.

Figure 31 shows a typical pathway of alterations in brain from patient-derived xenografts following treatment with CART19 cells. The red boxes indicate gene upregulation in CART19 plus isotype control treated mice compared to untreated patient-derived xenografts.

Figures 32A-32D demonstrate improvement in CRS in GM-CSF and in vivo following CART19 therapy in a xenograft model. Figure 32A shows that ritvolumab and anti-mouse GM-CSF antibodies prevent CRS-induced weight loss compared to mice treated with CART19 and isotype control antibody (3 mice per group, two-way anova, mean + SEM). Figure 32B shows neutralization of human GM-CSF in patient-derived xenografts treated with litzizumab and mouse GM-CSF neutralizing antibody, p <0.001, p <0.05 per group, t-test, mean + SEM. Figure 32C shows that human cytokine/chemokine heatmap (serum collected 11 days after CART19 injection) shows an increase in cytokines and chemokines typical of CRS after CART19 treatment. GMCSF neutralization resulted in a significant reduction of several cytokines and chemokines, including several myeloid related cytokines and chemokines, compared to mice treated with CART19 and isotype control antibodies, as shown in the panel, 3 mice per group, from sera at day 11 after CART19 injection, # p <0.001, # p <0.01, # p <0.05, GM-CSF neutralizing antibody treated and isotype control treated mice receiving CAR-T cell therapy were compared, and T-tested. Figure 32D shows that the mouse cytokine/chemokine heatmap (serum collected on day 11 after CART19 injection) shows an increase in mouse cytokines and chemokines typical of CRS after CART19 treatment. GM-CSF neutralization resulted in a significant reduction of several cytokines and chemokines compared to treatment with CART19 using control antibodies, including several myeloid differentiation cytokines and chemokines, as shown in the panel, 3 mice per group, serum from day 11 post CART19 injection, p <0.05, GM-CSF neutralizing antibody treated mice receiving CAR-T cell therapy were compared to isotype control treated mice, T-test.

Figures 33A-33D demonstrate improvement in neuroinflammation in GM-CSF and in vivo following CART19 therapy in a xenograft model. Fig. 33A-33B depict gadolinium enhanced T1-high intensity (cubic millimeter) MRI showing that GM-CSF neutralization helps to reduce brain inflammation, blood brain barrier disruption, and possible edema compared to isotype control, (a) representative images, (33B) 3 mice per group, × p <0.01, × p <0.05, one-way anova, mean + SD. Fig. 33C shows that human CD 3T cells were present in the brain following treatment with CART19 therapy. Neutralization of GM-CSF resulted in a reduced tendency of CD3 infiltration in the brain, as determined by flow cytometry in the brain hemisphere, 3 mice per group, mean + SEM. Figure 33D depicts the reduction of CD11b + bright macrophages in the brain of mice receiving GM-CSF neutralization during CAR-T therapy compared to isotype control during CAR-T therapy, 3 mice per group, mean + SEM, as determined by flow cytometry in the brain hemisphere.

FIGS. 34A-34B show GM-CSF^k/oProduction of CART19 cells. FIG. 34A shows an experimental protocol; FIG. 34B shows a flowchart for generating GM-CSF^k/ogRNA sequences and primer sequences of CART 19. To generate GM-CSF^k/oCART19 cells, grnas cloned into Cas9 lentiviral vector under control of U6 promoter and used for lentiviral production. T cells from normal donors were stimulated with CD3/CD28 beads and double transduced with CAR19 virus and CRISPR/Cas9 virus after 24 hours. CD3/CD28 magnetic bead removal was performed on day +6, and GM-CSF was cryopreserved on day 8 ^k/oCART19 cells or control CART19 cells.

Fig. 35 shows a flow chart of a procedure used in RNA sequencing. Binary basic call data is converted to fastq using Illumina bcl2fastq software. Adaptor sequences were removed using trimmatic, and the quality was checked using FastQC. The latest human (GRCh38) and mouse (GRCm38) reference genomes were downloaded from NCBI. A genome index file was generated using STAR, and paired end reads were mapped to the genome of each condition. HTSeq was used to generate expression counts for each gene, and DeSeq2 was used to calculate differential expression. The gene ontology was assessed using Enrichr.

Figure 36 shows that litzizumab plus CAR-T cell treated mice have comparable survival rates in a high tumor burden relapsing xenograft model of ALL compared to isotype control antibody plus CAR-T cell treated mice. n-2 experiments, 7-8 mice per group, representative experiments are depicted, p <0.0001, p <0.001, log rank.

FIG. 37 shows representative TIDE sequences to validate genomic alterations in GM-CSF CRISPR Cas9 knockout CAR-T cells. n-2 experiments, representative experiments are depicted.

Figure 38 shows that GM-CSF knockout CAR-T cells show slightly enhanced tumor burden control in vivo compared to wild-type CAR-T cells in a high tumor burden relapsing xenograft model of ALL. Representative experiments are depicted with 5-6 mice per group (2 mice remaining in the UTD group on day 13) on the days post CAR-T cell injection listed on the x-axis, with p <0.0001, p <0.05, two-way anova, mean ± SEM.

FIG. 39 shows a patient-derived xenograft model of neuroinflammation and CRS treated with CART19+ anti-hGM-CSF antibody. High tumor burden primary ALL xenografts treated with CART19+ anti-hGM-CSF antibody showed human CD3 cell infiltration of brain compared to untreated PDX control (fig. 30D) (fig. 39). 3 mice per group, representative image.

Figures 40A-40B show that BBB integrity is preserved and neuroinflammation is significantly reduced after CAR-T and litzizumab therapy. Figure 40A shows that confocal microscopy clearly shows in high resolution images that after CAR-T therapy, BBB was significantly compromised and that integrity of BBB was maintained with CAR-T and litzizumab therapy. FIG. 40B is adapted from Santomasso, BD et al, DOI in Online first publication (OnlineFirst) at 7.6.2018, 10.1158/2159-8290, CD-17-1319, and is incorporated herein by reference in its entirety, showing that high levels of protein in CSF (as shown in the data for Santomasso) are indicative of BBB destruction and protein leakage into the CNS.

Detailed Description

The present subject matter may be understood more readily by reference to the following detailed description that forms a part of this disclosure. It is to be understood that this disclosure is not limited to the specific products, methods, conditions or parameters described and/or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed disclosure.

Immunotherapy-related toxicities

The skilled person will understand that the term "immunotherapy-related toxicity" refers to a series of inflammatory symptoms caused by high levels of immune activation. Different types of toxicity are associated with different immunotherapy approaches. In some embodiments, the immunotherapy-related toxicities include capillary leak syndrome, heart disease, respiratory disease, CAR-T cell-related encephalopathy syndrome (CRES), neurotoxicity, colitis, convulsions, Cytokine Release Syndrome (CRS), cytokine storm, decreased left ventricular ejection fraction, diarrhea, disseminated intravascular coagulation, edema, encephalopathy, rash, gastrointestinal bleeding, gastrointestinal perforation, phagocytic lymphocytosis (HLH), liver disease, hypertension, hypophysitis, immune-related adverse events, immune hepatitis, immunodeficiency, ischemia, hepatotoxicity, Macrophage Activation Syndrome (MAS), pleural effusion, reversible pericardial effusion, pneumonia, polyarthritis, posterior encephalopathy syndrome (PRES), pulmonary hypertension, thromboembolism, and elevated transaminase.

Although the different types of toxicity vary in their pathophysiological and clinical manifestations, they are often associated with an increase in factors associated with inflammation, such as C-reactive protein, GM-CSF, IL-1, IL-2, sIL-2 Ra, IL-5, IL-6, IL-8, IL-10, IP10, IL-15, MCP-1(AKA CCL2), MIG, MIP-1 β, IFN γ, CX3CR1, or TNF α. The skilled person will understand that in some embodiments, the term "inflammation-associated factor" includes molecules, small molecules, peptides, gene transcripts, oligonucleotides, proteins, hormones, and biomarkers that are affected during inflammation. The skilled person will understand that the systems affected during inflammation include up-regulation, down-regulation, activation, inactivation or any kind of molecular modification. Serum concentrations of inflammation-related factors (e.g., cytokines) can be used as an indicator of immunotherapy-related toxicity, and can be expressed as-fold increase, percent increase (%), net increase, or rate of change in cytokine levels or concentrations. The concentration of inflammation-related factors in body fluids other than serum may also be used as an indicator of immunotherapy-related toxicity. In some embodiments, an absolute cytokine level or concentration above a certain level or concentration may be indicative of a subject experiencing or about to experience immunotherapy-related toxicity. In another embodiment, an absolute cytokine level or concentration at a certain level, such as that normally found in a control subject, can be indicative of a method for inhibiting or reducing the incidence of immunotherapy-related toxicity in a subject. The skilled person will appreciate that the term "cytokine level" may encompass a measure of concentration, a measure of fold change, a measure of percent (%) change, or a measure of rate change. Further, methods for measuring cytokines in blood, cerebrospinal fluid (CSF), saliva, serum, urine, and plasma are well known in the art.

Many approaches have been detailed to classify the types of neurotoxicity and manage them accordingly. These classifications are based on clinical and biological conditions such as fever, hypotension, hypoxia, organ toxicity, cardiac dysfunction, respiratory dysfunction, gastrointestinal dysfunction, liver dysfunction, renal dysfunction, coagulopathy, seizures, intracranial pressure, muscle tone, motor performance, ferritin levels, and phagocytosis. Similarly, each type of neurotoxicity can be graded according to its severity. Table 1A (taken from cell Therapy embodiment: the MDACC augmentation: the MDACC Approach, p. kebriaiei, 24/2/2017) discloses a method for ranking neurotoxicity according to its severity into 1, 2, 3 and 4. However, some of the aforementioned symptoms are not typically associated with neurotoxicity. (Lee et al, blood 2014; 124: 188-.

Table 1A: neurotoxicity grading method-criteria for adverse events (CTCAE)

Patients with body temperatures above 38.9 ℃, IL-6 serum concentrations above 16pg/ml, or MCP-1(AKA CCL2) serum concentrations above 1,343.5pg/ml had a higher probability of developing severe neurotoxicity within the first 36 hours after immunotherapy infusion (Gust et al Cancer findings (Cancer Discov.) 2017, 12/10).

CRS is a serious pathology and life threatening, due to dysregulation of cytokines and hence severe inflammation. Symptoms may include, but are not limited to, fever, heart and respiratory disorders, nausea, vomiting, and seizures. CRS may be ranked by assessing symptoms and their severity, for example: level 1 CRS: fever, constitutional symptoms; level 2 CRS: hypotension-response to fluid or a low dose of a pressor agent, hypoxia-response<40％O₂Response, organ toxicity; 2, level; 3-level CRS: hypotension-requires multiple boosters or high doses of boosters, hypoxia-requires ≥ 40% O₂Organ toxicity-grade 3, grade 4 transaminase elevation; 4-level CRS: mechanical ventilation, organ toxicity-grade 4, does not contain elevated transaminases. (Lee et al, blood 2014; 124: 188-.

CRES can be ranked, for example, by combining neurological assessments with other parameters such as papillary edema, CSF opening pressure, imaging assessments, and the presence of seizures and motor weakness. One method for ranking CRES is described in neelpau et al, nature review clinical oncology (Nat Rev Clin Oncol.) 15(1):47-62(2018) (electronic publication 2017, 9, 19), incorporated herein by reference in its entirety. Table 1B (taken from neelpau et al, nature review clinical tumors 15(1):47-62(2018)) discloses a method for classifying CRES according to its severity into grades 1, 2, 3 and 4.

Table 1B: CRES grading method. In CARTOX-10, a score is correctly assigned to each of the following tasks that are performed: the orientation of presidents/presidents of year, month, city, hospital and country of residence (each score of 1 point); three objects were named (each with a score of 1); writing a standard sentence; the reciprocal starts from 100 in units of ten.

NT, CRS, and CRES manifestations may include encephalopathy, headache, delirium, anxiety, tremor, seizure activity, confusion, altered wakefulness, decreased levels of consciousness, hallucinations, speech disturbances, aphasia, ataxia, apraxia, facial paralysis, motor weakness, seizures, non-tic EEG seizures, cerebral edema, and coma. CRES is associated with elevated concentrations of circulating cytokines such as C-reactive protein, GM-CSF, IL-1, IL-2, sIL2R α, IL-5, IL-6, IL-8, IL-10, IP10, IL-15, MCP-1, MIG, MIP1 β, IFN γ, CX3CR1, and TNF α.

The cytokine concentration gradient between serum and CSF observed under normal conditions was reduced or lost during CRES. In addition, CAR T cells and high protein concentrations are observed in the CSF of patients and correlate with the severity of the condition. All these indicate blood brain barrier dysfunction after immunotherapy. Increased vascular permeability may be explained in part by an increase in the concentration of ANG2 and an increase in the ratio of ANG2: ANG1 in neurotoxic patients. ANG1 induced endothelial cell quiescence, while ANG2 induced endothelial cell activation and microvascular permeability. Patients with increased endothelial activation prior to immunotherapy are reported to have a higher probability of developing neurotoxicity (Gust et al, cancer discovery, 2017, 10 months and 12 days).

Phagocytic lymphohistiocytosis (HLH) involves severe excessive inflammation caused by uncontrolled proliferation of benign lymphocytes and macrophages that secrete large amounts of inflammatory cytokines. In some embodiments, HLH may be classified as one of the cytokine storm syndromes. In some embodiments, HLH occurs following a strong immune activation, such as a systemic infection, immunodeficiency, malignancy, or immunotherapy. In some embodiments, the term "HLH" may be used interchangeably with the terms "phagocytic lymphohistiocytosis," "hemophagic cell syndrome," or "hemophagic cell syndrome," all of the same properties and meanings.

Primary HLH includes heterogeneous autosomal recessive genetic disorders. Patients with homozygous mutations in one of several genes show a loss of function of proteins involved in cytolytic granule exocytosis. In some embodiments, HLH may be present during infancy with minimal or no triggering. Secondary or acquired HLH occurs after intense immune activation, such as occurs in systemic infections, immunodeficiency, potential malignancies, or immunotherapy. Both forms of HLH are characterized by overwhelming activation of normal T lymphocytes and macrophages, always resulting in clinical and hematological changes and even death without treatment.

In some embodiments, HLH may be caused by viral infections, EBV, CMV, parvovirus, HSV, VZV, HHV8, HIV, influenza, hepatitis a, hepatitis b, hepatitis c, bacterial infections, gram negative bacilli, mycoplasma species and mycobacterium tuberculosis, parasitic infections, plasmodium species, leishmania species, toxoplasma species, fungal infections, cryptococcus species, candida species, pneumocystis species, and the like.

Macrophage Activation Syndrome (MAS) includes a condition that involves uncontrolled activation and proliferation of macrophages and T lymphocytes with significant increases in circulating cytokine levels (e.g., IFN γ and GM-CSF). MAS is closely related to secondary HLH. MAS manifestations include hyperpyrexia, hepatosplenomegaly, lymphadenopathy, pancytopenia, liver dysfunction, disseminated intravascular coagulation, erythrophagy, hypofibrinogenemia, methemoglobinemia and hypertriglyceridemia.

CRS includes a non-antigen specific immune response similar to that found in severe infections. CRS is characterized by any or all of the following symptoms: fever, malaise, fatigue, anorexia, myalgia, arthralgia, nausea, vomiting, headache, rash, diarrhea, tachypnea, hypoxemia, hypoxia, shock, cardiovascular tachycardia, pulse broadening, hypotension, capillary leakage, increased cardiac output (early stage), reduced potential cardiac output (late stage), increased D-dimer, hypofibrinogenemia with or without bleeding, azotemia, elevated transaminase, hyperbilirubinemia, headache, altered mental state, confusion, delirium, aphasia with difficulty or tandemly found, hallucinations, tremor, dysdiscrimination, altered gait, seizure, organ failure, multiple organ failure. Death has also been reported. It has been reported that up to 60% of patients receiving CAR-T19 develop severe CRS.

Cytokine storms include an immune response consisting of a positive feedback loop between cytokines and leukocytes, with high levels of each cytokine. The term "cytokine storm" can be used interchangeably with the terms "cytokine cascade" and "hypercytoemia" having all the same properties and meanings. In some embodiments, the cytokine storm is characterized by IL-2 release and lymphoproliferation. Cytokine storms result in potential life-threatening complications including cardiac dysfunction, adult respiratory distress syndrome, nervous system toxicity, renal and/or hepatic failure, and disseminated intravascular coagulation.

As noted above, CAR-T cell therapy is currently limited by life-threatening neurotoxicity and risk of CRS. Despite aggressive management, all CAR-T responders experience some degree of CRS. Up to 50% of patients treated with CD19CAR-T have at least grade 3 CRS or neurotoxicity. GM-CSF levels and T cell expansion are the factors most associated with grade 3 or higher CRS and neurotoxicity.

Reducing or eliminating CRS and neurotoxicity is of great value in immunotherapy, such as CAR-T cell therapy, and it is crucial to determine what is driving or exacerbating the characteristic CAR-T inflammatory response. Although many cytokines, signaling molecules and cell types are involved in this pathway, GM-CSF is a cytokine that appears to be centrally located in the pathway. Not normally detectable in human serum, an extreme periodic positive feedback loop driving inflammation to cytokine storm and endothelial cell activation is crucial. Neurotoxicity and cytokine storm are not the result of simultaneous cytokine release, but rather a series of inflammatory responses triggered by GM-CSF, resulting in the trafficking and recruitment of myeloid cells to the tumor site. These myeloid cells produce cytokines that are observed in CRS and neurotoxicity, thereby continuing the inflammatory cascade.

Granulocyte macrophage colony stimulating factor (GM-CSF)

As used herein, "granulocyte macrophage colony stimulating factor" (GM-CSF) refers to a naturally occurring small glycoprotein with internal disulfide bonds having a molecular weight of approximately 23 kDa. In some embodiments, GM-CSF refers to human GM-CSF. In some embodiments, GM-CSF refers to non-human GM-CSF. In humans, it is encoded by genes located within a cytokine cluster on human chromosome 5. The sequences of human genes and proteins are known. Proteins have N-terminal signal sequences and C-terminal receptor binding domains (Rasko and Gough In: The Cytokine Handbook), A.Thomson et al, Academic Press, New York (1994), p.349-369). Although the amino acid sequences are not similar, the three-dimensional structure is similar to that of interleukins. GM-CSF is produced by mesenchymal cells present in the hematopoietic environment and at sites surrounding inflammation in response to a number of inflammatory mediators. GM-CSF is capable of stimulating the production of neutrophils, macrophages and mixed granulocyte-macrophage colonies by bone marrow cells and stimulating the formation of eosinophil colonies by fetal hepatic progenitors. GM-CSF can also stimulate certain functional activities of mature granulocytes and macrophages. GM-CSF, a cytokine present in the bone marrow microenvironment, recruits inflammatory monocyte-derived dendritic cells, stimulates secretion of high levels of IL-6 and CCL2/MCP-1, and causes a feedback loop, thereby recruiting more monocytes, inflammatory dendritic cells, to the site of inflammation.

As mentioned above, CRS is involved in the increase of several cytokines and chemokines, including IFN- γ, IL-6, IL-8, CCL2(MCP-1), CCL3(MIP1 α) and GM-CSF. (teache, d. et al (2016. 6.d.), "cancer discovery," CD-16-0040; Morgan r. et al, (2010. 4.d.), "Molecular Therapy"). IL-6 is one of the key inflammatory cytokines, not produced by CAR-T cells. (Barrett, D. et al (2016), "blood"). Instead, it is produced by myeloid cells that recruit to the tumor site. GM-CSF mediates this recruitment, inducing chemokine production that activates myeloid cells and trafficks them to tumor sites. Elevated levels of GM-CSF can serve as both a predictive biomarker for CRS and an indicator of its severity. More than the key component of the inflammatory cascade, GM-CSF is the key initiator, responsible for both CRS and NT. As described herein, in vivo studies using a murine model indicate that gene silencing of GM-CSF prevents cytokine storm-while still maintaining CAR-T efficacy. GM-CSF knock-out mice have normal levels of INF-. gamma.IL-6, IL-10, CCL2(MCP1), CCL3/4(MIG-1) in vivo and do not develop CRS. (Sentman, M. -L., et al (2016), "J Immunol., 197(12), 4674-. The GM-CSF knock-out CAR-T model recruits fewer NK cells, CD8 cells, myeloid cells, and neutrophils to the tumor site compared to GM-CSF + CAR-T.

The term "soluble granulocyte macrophage colony stimulating factor receptor" (sGM-CSFR) refers to a non-membrane bound receptor that binds to GM-CSF but does not transduce a signal when bound to a ligand.

As used herein, a "peptide GM-CSF antagonist" refers to a peptide that interacts with GM-CSF or its receptor to reduce or block (partial or complete) signal transduction that would otherwise result from binding of GM-CSF to its cognate receptor expressed on a cell. GM-CSF antagonists may act by reducing the amount of GM-CSF ligand available to bind to the receptor (e.g., antibodies increase clearance of GM-CSF once bound to GM-CSF) or by preventing ligand binding to its receptor by binding to GM-CSF or to the receptor (e.g., neutralizing antibodies). The GM-CSF antagonist may also comprise other peptide inhibitors, which may comprise polypeptides that bind to GM-CSF or its receptor to partially or completely inhibit signaling. The peptide GM-CSF antagonist may be, for example, an antibody; natural or synthetic ligands for the GM-CSF receptor that antagonize GM-CSF, or other polypeptides. Example 1 provides an exemplary assay to detect GM-CSF antagonist activity. Typically, peptide GM-CSF antagonists (e.g., neutralizing antibodies) have an EC50 of 10nM or less.

As used herein, a "purified" GM-CSF antagonist refers to a GM-CSF antagonist that is substantially or essentially free of components that normally accompany it in its natural state. For example, a GM-CSF antagonist, e.g., an anti-GM-CSF antibody, purified from blood or plasma is substantially free of other blood or plasma components, such as other immunoglobulin molecules. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein that is the major species present in the preparation is substantially purified. Generally, "purified" means that the protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure, relative to the components of the naturally occurring protein.

Antibodies

As used herein, "antibody" refers to a protein that is defined functionally as a binding protein and structurally as comprising an amino acid sequence recognized by a skilled artisan as a framework region of an immunoglobulin-encoding gene derived from an animal from which the antibody is produced. An antibody may consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

Typical immunoglobulin (antibody) building blocks are known to comprise tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The term variable light chain (V)_L) And a variable heavy chain (V)_H) Refer to these light and heavy chains, respectively.

The term "antibody" encompasses antibody fragments that retain binding specificity. For example, there are many well-characterized antibody fragments. Thus, for example, pepsin digests antibodies C-terminal of disulfide bonds in the hinge region to produce Fab dimer F (ab')2, which is itself a light chain linked to VH-CH1 by disulfide bonds. The F (ab ')2 may be reduced under mild conditions to break the disulfide bond in the hinge region, thereby converting the (Fab ')2 dimer into an Fab ' monomer. The Fab' monomer is essentially an Fab with a portion of the hinge region (see basic Immunology, ed., w.e. paul, reden Press, a more detailed description of other antibody fragments in new york (1993)). Although various antibody fragments are defined in terms of digestion of intact antibodies, one skilled in the art will appreciate that fragments can be synthesized de novo by chemical methods or using recombinant DNA methods. Thus, as used herein, the term antibody also encompasses antibody fragments produced by modifying whole antibodies or synthesized using recombinant DNA methods.

Antibodies comprise dimers, e.g. V_H-V_LDimer, V_HDimer or V_LDimers, comprising single chain antibodies (antibodies in the form of a single polypeptide chain), such as single chain Fv antibodies (sFv or scFv), in which a variable heavy chain region and a variable light chain region are linked together (either directly or through a peptide linker) to form a continuous polypeptide. The single-chain Fv antibody is covalently linked to V_H-V_LHeterodimers, which may be derived from V comprising a direct linkage or a linkage through a peptide-encoding linker_H-and V_LNucleic acid expression of coding sequences (e.g., Huston et al, Proc. Nat. Acad. Sci. USA, 85: 5879-. When V is_HAnd V_LWhen connected as a single polypeptide chain, V_HAnd V_LThe domains associate non-covalently. Alternatively, the antibody may be another fragment, such as a disulfide stabilized fv (dsfv). Other fragments may also be generated, including the use of recombinant techniques. scFv antibodies and many other structures convert naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into molecules that fold into three-dimensional structures that are substantially similar to the structure of the antigen binding site, and are known to those of skill in the art: (See, for example, U.S. patent nos. 5,091,513, 5,132,405, and 4,956,778). In some embodiments, the antibodies comprise those that have been displayed on phage or produced by recombinant techniques using vectors in which the chains are secreted as soluble proteins, such as scFv, Fv, Fab, (Fab')2, or by recombinant techniques using vectors in which the chains are secreted as soluble proteins. The antibodies used in the present invention may also comprise diabodies and minibodies.

The antibodies of the invention also comprise heavy chain dimers, such as camelid antibodies. V due to heavy chain dimer IgG in camelids_HThe regions do not necessarily have to interact hydrophobically with the light chain, so in camelids the regions of the heavy chain that normally contact the light chain become hydrophilic amino acid residues. V of heavy chain dimer IgG_HThe domains are referred to as VHH domains. Antibodies useful in the present invention include single domain antibodies (dAbs) and nanobodies (see, e.g., cortex-Retamozo et al, Cancer research 64:2853-2857, 2004).

As used herein, "V region" refers to an antibody variable region domain comprising segments of framework 1, CDR1, framework 2, CDR2, and framework 3, including CDR3 and framework 4, that are added to V segments as a result of heavy and light chain V region gene rearrangement during B cell differentiation. As used herein, "V segment" refers to a region of the V region (heavy or light chain) encoded by a V gene. The V segment of the heavy chain variable region encodes FR1-CDR1-FR2-CDR2 and FR 3. For the purposes of this invention, the V segment of the light chain variable region is defined as extending through FR3 up to CDR 3.

As used herein, the term "J segment" refers to a subsequence of the encoded variable region that includes the C-terminal portion of CDR3 and FR 4. Endogenous J-segments are encoded by immunoglobulin J genes.

As used herein, "Complementarity Determining Regions (CDRs)" refer to the three hypervariable regions in each chain that interrupt the four "framework" regions created by the light and heavy chain variable regions. The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 numbered sequentially from the N-terminus and are also typically numbered by a particular CDRThe chain in which the gene is identified. Thus, for example, V_HCDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while V_LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.

The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of the antibody (i.e., the combined framework regions that make up the light and heavy chains) are used to locate and align the CDRs in three-dimensional space.

The amino acid sequences of the CDRs and framework regions may be determined using various definitions well known in the art, such as Kabat, Chothia, International ImmunoGeneTiCs database (IMGT) and AbM (see, e.g., Johnson et Al, supra; Chothia and Lesk,1987, Standard Structure of the hypervariable regions of immunoglobulins (Canonical structures for the hypervariable regions of immunoglobulins). J.mol.biol. 196, 901-917; Chothia C. et Al, 1989, conformation of the hypervariable regions of immunoglobulins (constructs of immunoglobulin hypervariable regions). Nature (Nature) 342, 877-883; Chothia C. et Al, 1992, structural group of the human VH segments (Laggen. molecular) 799, 1997, molecular biology 794, 1997, 1989, molecular Strongylon. 794, molecular Strongylon. 35, 797, Strongylon. C. et Al, 1992, structural group of the human VH segments, et Al, 1997, molecular Strongylophilus, 794, molecular Strongylophilus, 1989, molecular Strongylophilus, Strongylon. The definition of antigen binding sites is also described in the following documents: ruiz et al, IMGT, International ImmunoGeneTiCs database (International ImmunoGeneTiCs database) Nucleic Acids research (Nucleic Acids Res.), 28, 219-221 (2000); and Lefranc, M. -P.IMGT, International ImmunoGeneTiCs database, nucleic acids research, 1 month 1; 29(1) 207-9 (2001); MacCallum et al, antibody-antigen interaction: contact analysis and binding site topography (Antibody-interactions: Contact analysis and binding site topographies), journal of molecular biology 262(5),732-745 (1996); and Martin et al, Proc. Natl. Acad. Sci. USA, 86, 9268-9272 (1989); martin et al, Methods in enzymology (Methods Enzymol.), 203, 121-; pedersen et al, "immunization methods (immunology), 1,126, (1992); and Rees et al, Sternberg M.J.E. (eds.), (Protein Structure Prediction) Oxford University Press (Oxford University Press), Oxford, 141-.

An "epitope" or "antigenic determinant" refers to a site on an antigen to which an antibody binds. Epitopes can be formed from both contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. In a unique spatial conformation, an epitope typically comprises at least 3 and more typically at least 5 or 8-10 amino acids. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Methods in Molecular Biology Methods, Epitope Mapping Protocols, Vol.66, Glenn E.Morris, eds (1996).

The term "binding specificity determinant" or "BSD" as used in the context of the present invention refers to the smallest contiguous or non-contiguous amino acid sequence within a CDR region that is necessary to determine the binding specificity of an antibody. In the present invention, the minimal binding specificity determinant is located within a portion or the full length of the CDR3 sequences of the heavy and light chains of the antibody.

As used herein, "anti-GM-CSF antibody" or "GM-CSF antibody" are used interchangeably to refer to an antibody that binds to GM-CSF and inhibits GM-CSF receptor binding and activation. Such antibodies may be identified using any number of art-recognized assays for assessing GM-CSF binding and/or function. For example, a binding assay, such as an ELISA assay, can be used that measures inhibition of binding of GM-CSF to the alpha receptor subunit. Cell-based assays for GM-CSF receptor signaling, such as assays to determine the rate of proliferation of GM-CSF-dependent cell lines in response to a limited amount of GM-CSF, are also conveniently used, as are assays to measure the amount of cytokine production, e.g., IL-8 production, in response to GM-CSF exposure.

As used herein, "neutralizing antibody" refers to an antibody that binds to GM-CSF and inhibits signaling of the GM-CSF receptor or prevents GM-CSF from binding to its receptor.

As used herein, "human granulocyte macrophage colony stimulating factor" (hGM-CSF) refers to a naturally occurring small glycoprotein with internal disulfide bonds having a molecular weight of approximately 23 kDa; the source and target of GM-CSF is human; thus, as described in the examples herein, anti-hGM-CSF antibodies bind only to human and primate GM-CSF, but not to mouse, rat, and other mammalian GM-CSF. As described in the examples herein, hGM-CSF antibodies neutralize human GM-CSF. In some embodiments, hGM-CSF in humans is encoded by a gene located within a cytokine cluster on human chromosome 5. The sequences of human genes and proteins are known. The protein has an N-terminal signal sequence and a C-terminal receptor binding domain (Rasko and Gough in: A.cytokine handbook, A.Thomson et al, academic Press, New York (1994), p.349-369). Although the amino acid sequences are not similar, the three-dimensional structure is similar to that of interleukins. GM-CSF is produced in response to a number of inflammatory mediators present in the hematopoietic environment and at sites surrounding inflammation. GM-CSF is capable of stimulating the production of neutrophils, macrophages and mixed granulocyte-macrophage colonies by bone marrow cells and stimulating the formation of eosinophil colonies by fetal hepatic progenitors. GM-CSF also stimulates some functional activity in mature granulocytes and macrophages and inhibits apoptosis of granulocytes and macrophages.

The term "equilibrium dissociation constant" or "affinity" (K)_D) Refers to the dissociation rate constant (k)_dTime of day^-1) Divided by the association rate constant (K)_aTime of day^-1M^-1). The equilibrium dissociation constant can be measured using any method known in the art. The antibodies of the invention are high affinity antibodies. Such antibodies have a monovalent affinity of better (less) than about 10nM, and often better than about 500pM or better than about 50pM, as determined by surface plasmon resonance analysis performed at 37 ℃. Thus, in some embodiments, the antibodies of the invention have an affinity of less than 50pM, typically less than about 25pM, or even less than 10pM (as measured using surface plasmon resonance).

In some embodiments, the anti-GM-CSF antibodies of the invention have a slow off-rate, wherein the off-rate constant for monovalent interactions with GM-CSF is measured by surface plasmon resonance analysis at 37 ℃(kd) less than about 10^-4s^-1Preferably less than 5 x 10^-5s^-1And most preferably less than 10^-5s^-1。

As used herein, "chimeric antibody" refers to an immunoglobulin molecule in which (a) the constant region or a portion thereof is altered, replaced, or exchanged such that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function, and/or species, or an entirely different molecule that confers novel properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or portion thereof is substituted with a variable region or portion thereof having a different or altered antigenic specificity; or by a corresponding sequence alteration, substitution or exchange from another species or from another antibody class or subclass.

As used herein, "humanized antibody" refers to immunoglobulin molecules in the CDRs from a donor antibody grafted onto human framework sequences. Humanized antibodies may also include residues of donor origin in the framework sequence. The humanized antibody may also comprise at least a portion of a human immunoglobulin constant region. Humanized antibodies may also include residues that are not found in the recipient antibody or in the imported CDR or framework sequences. Humanization can be performed using methods known in the art (e.g., Jones et al, Nature 321: 522-525; 1986; Riechmann et al, Nature 332:323-327, 1988; Verhoeyen et al, Science 239:1534-1536, 1988; Presta, Current Biol., curr. Op. struct. biol., 2:593-596, 1992; U.S. Pat. No. 4,816,567), including techniques such as "super humanization" antibodies (Tan et al, J.Immunol. 169:1119,2002) and "resurfacing" (e.g., Staelens et al, molecular immunology 43:1243,2006; and Rogusa et al, national academy. 969,1994).

In the context of the present invention it is,

an antibody is an engineered human antibody having the binding specificity of a reference antibody. For The engineered human antibodies of the invention have immunoglobulin molecules with minimal sequence derived from the donor immunoglobulin. In some embodiments, the engineered human antibody may retain only the minimal essential binding specificity determinant from the CDR3 region of the reference antibody. Typically, the Binding Specificity Determinant (BSD) from the CDR3 region of the heavy chain of a reference antibody is linked to human V by a DNA sequence encoding the same_HSegment the sequence and attach a DNA sequence encoding the light chain CDR3 BSD from a reference antibody to human V_LThe segment sequences are used to engineer human antibodies. "BSD" refers to the CDR3-FR4 region or a portion of this region that mediates binding specificity. Thus, a binding specificity determinant may be the smallest primary binding specificity determinant of a CDR3-FR4, CDR3, CDR3 (which refers to any region smaller than CDR3 that confers binding specificity when present in the V region of an antibody), the D segment (as opposed to the heavy chain region), or other region of a CDR3-FR4 that confers binding specificity to a reference antibody. Methods for engineering human antibodies are provided in U.S. patent application publication No. 20050255552 and U.S. patent application publication No. 20060134098.

As used herein, the term "human antibody" refers to an antibody that is substantially human, i.e., having FR regions, and often CDR regions, from the human immune system. Thus, the term encompasses humanized and human engineered antibodies, as well as antibodies isolated from mice reconstituted with the human immune system and antibodies isolated from display libraries.

The term "heterologous" when used in reference to portions of a nucleic acid indicates that the nucleic acid includes two or more subsequences that are not normally found in the same relationship to each other in nature. For example, nucleic acids are typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged into a new functional nucleic acid. Similarly, a heterologous protein generally refers to two or more subsequences that are not found in the same relationship to each other in nature.

The term "recombinant" when used in conjunction with a reference, e.g., a cell or nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell originates from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all. The term "recombinant nucleic acid" means herein a nucleic acid that is initially formed in vitro, typically by manipulation of the nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this way, an operable connection of different sequences is achieved. Thus, for the purposes of the present invention, isolated nucleic acids in linear form or expression vectors formed in vitro by ligating DNA molecules that are not normally joined are considered recombinant. It will be appreciated that once a recombinant nucleic acid is prepared and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, are nevertheless considered recombinant for the purposes of the present invention despite subsequent non-recombinant replication. Similarly, a "recombinant protein" is a protein that is prepared using recombinant techniques, i.e., by expressing a recombinant nucleic acid.

The phrase "specifically (or selectively) binds" an antibody or "specifically (or selectively) immunoreactive" refers to a binding reaction of an antibody to an antigen of interest. In the context of the present invention, antibodies typically bind to an antigen (e.g., GM-CSF) with an affinity of 500nM or less, and for other antigens the affinity of the antibody is 5000nM or greater.

The term "identical" or percent "identity," in the context of two or more polypeptide (or nucleic acid) sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues (or nucleotides) that are the same (i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or specified region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm using default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI website). Such sequences are then referred to as "substantially identical". Sequences that are "substantially identical" also include sequences with deletions and/or additions, as well as sequences with substitutions, as well as naturally occurring sequences, such as polymorphisms or allelic and man-made variants. As described below, the preferred algorithm can solve the problem of gaps and the like. Preferably, protein sequence identity exists over a region of at least about 25 amino acids in length, or more preferably over a region of 50-100 amino acids in length or over the length of the protein.

As used herein, a "comparison window" includes reference to a segment of one of the consecutive number of positions selected from the group consisting of: from 20 to 600, typically from about 50 to about 200, more typically from about 100 to about 150, wherein after optimal alignment of two sequences, one sequence can be compared to a reference sequence of the same number of consecutive positions. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by: local homology algorithms of Smith and Waterman, advanced application mathematics (adv.Appl.Math.) 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J.Mol.biol., 48:443 (1970); similarity search by Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988); by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package of the Genetics Computer Group (Genetics Computer Group) No. 575, Madison scientific Dawley, Wisconsin); or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al, 1995, supplement).

An indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with an antibody directed against the second polypeptide. Thus, a polypeptide is typically substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions.

Preferred examples of algorithms suitable for determining sequence identity and percent sequence similarity include the BLAST and BLAST 2.0 algorithms described in: altschul et al, nucleic acids Res.25: 3389-3402(1977) and Altschul et al, J. mol. biol.215: 403-410 (1990). Percent sequence identity for nucleic acids and proteins of the invention was determined using BLAST and BLAST 2.0 with the parameters described herein. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, M-5, N-4, and a comparison of the two strands as defaults. For amino acid sequences, the BLASTP program uses a word length (W) of 3 and an expectation (E) of 10 and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)) as defaults for alignment (B) of 50, an expectation (E) of 10, M-5, N-4 and a comparison of the two strands.

The terms "isolated," "purified," or "biologically pure" refer to a material that is substantially or essentially free of components that normally accompany it in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein that is the major species present in the preparation is substantially purified. In some embodiments, the term "purified" means that the protein produces substantially one band in the electrophoresis gel. Preferably, it means that the protein is at least 85% pure, more preferably at least 95% pure and most preferably at least 99% pure.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues and non-naturally occurring amino acids.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission (the IUPAC-IUB Biochemical Nomenclature Commission). Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

"conservatively modified variants" applies to amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or substantially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to substantially identical or related, e.g., naturally contiguous sequences. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode most proteins. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to another of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid changes are "silent changes," which are conservatively modified changes. Silent changes to nucleic acids are also described herein for each nucleic acid sequence encoding a polypeptide. The skilled artisan will recognize that in certain contexts, each codon in a nucleic acid (except AUG, which is the only codon for methionine, and TGG, which is the only codon for tryptophan) may be modified to yield a functionally identical molecule. Thus, silent changes in a nucleic acid encoding a polypeptide are typically implicit in the sequence relative to the expression product, rather than relative to the actual probe sequence.

With respect to amino acid sequences, those skilled in the art will recognize that a single substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence that alters, adds, or deletes a single amino acid or a small portion of an amino acid in the coding sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables and substitution matrices (e.g., BLOSUM) that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are complements of, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention. Typical conservative substitutions for each other include: 1) alanine (a), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins (Proteins) (1984)).

Methods of preventing or treating immunotherapy-related toxicities

In some embodiments, disclosed herein are methods of inhibiting immunotherapy-related toxicity in a subject. In some embodiments, herein is a method of reducing the incidence of immunotherapy-related toxicity in a subject. In some embodiments, disclosed herein are methods of neutralizing hGM-CSF. In some embodiments, the method comprises the step of administering to the subject a recombinant hGM-CSF antagonist. In some embodiments, the method comprises hGM-CSF gene silencing. In some embodiments, the method comprises hGM-CSF gene knockout. Methods of gene silencing and gene knockout are well known to those of ordinary skill in the art and can include, but are not limited to, RNA interference (RNAi), CRISPR, short interfering rns (sirna), DNA-directed RNA interference (ddRNAi), targeted genome editing to engineer transcription activator-like effector nucleases (TALENs), or other suitable techniques.

For knock-out of GM-CSF: GM-CSF^k/oGene editing technology of

In some embodiments, gene editing techniques are used to knock out the expression of GM-CSF. Genome editing is accomplished by delivering endonucleases (including but not limited to Fok1 or Cas9) that cleave DNA into site-specific segments of the genetic code. Endonucleases cleave DNA, which triggers endogenous DNA repair mechanisms.

Practitioners skilled in the art will recognize that any method to achieve site-specific chromosomal DNA cleavage (which then triggers endogenous DNA repair) will result in the same targeted genomic modification. Whether site-specific by RNA guidance, DNA guidance or by DNA binding proteins or DNA cleavage is achieved with which endonuclease, targeted genomic modifications will not differ. Examples of RNA-guided site-specificity include, but are not limited to, CRISPR/Cas 9. An example of DNA-directed site specificity includes, but is not limited to, flap endonuclease 1 (FEN-1). Examples of DNA binding proteins for achieving site-specificity include, but are not limited to, zinc finger proteins (ZFNs), transcription activator-like effectors (TALENS), homing endonucleases (including ARCUS), meganucleases, and the like.

Other methods that can be used for gene silencing are well known to those of ordinary skill in the art and can include, but are not limited to, RNA interference (RNAi), short interfering rns (sirna), DNA-directed RNA interference (ddRNAi).

In one aspect, the invention provides a method for GM-CSF gene inactivation or GM-CSF knock-out (KO) in a cell, comprising targeted genome editing or GM-CSF gene silencing. In embodiments of the provided methods, targeted genome editing comprises an endonuclease, wherein the endonuclease is a Fok1 restriction enzyme or flap endonuclease 1 (FEN-1). In another embodiment, the endonuclease is Cas9 CRISPR-associated protein 9(Cas 9). In some embodiments, the GM-CSF gene is inactivated by CRISPR/Cas9, which targets and edits GM-CSF at exon 1, exon 2, exon 3, or exon 4. In further embodiments, GM-CSF gene inactivation includes CRISPR/Cas9 target and edits GM-CSF at exon 3. In one embodiment, GM-CSF gene inactivation includes CRISPR/Cas9 target and edits GM-CSF at exon 1. In one embodiment, GM-CSF gene inactivation comprises multiple CRISPR/Cas9 enzymes, wherein each Cas9 enzyme targets and edits a different sequence of GM-CSF at exon 1, exon 2, exon 3, or exon 4. In particular embodiments, GM-CSF gene inactivation includes biallelic CRISPR/Cas9 targeting and knockout/inactivation of the GM-CSF gene. In another aspect of the provided methods for GM-CSF gene inactivation or GM-CSF knock-out (KO), the method further comprises treating the primary T cell with valproic acid to enhance the biallelic knock-out/inactivation. In one aspect of the provided methods for GM-CSF gene inactivation/gene knockout, the targeted genome editing comprises a zinc finger (ZnF) protein. In further aspects, targeted genome editing comprises a transcription activator-like effector (TALENS). In one embodiment, the targeted genome editing comprises a homing endonuclease, wherein the homing endonuclease is an ARC nuclease (ARCUS) or a meganuclease. In particular embodiments of the methods provided herein, the cell is a CAR T cell. In another embodiment, the CAR T cell is a CD19 CAR-T cell. In one embodiment, GM-CSF gene silencing is selected from the group consisting of: RNA interference (RNAi), short interfering RNS (siRNA), and DNA-directed RNA interference (ddRNAi).

Methods of preventing or treating immunotherapy-related toxicities

In some embodiments, the methods comprise administering a CAR-T cell that has been modified to express lower levels of GM-CSF by GM-CSF gene silencing or GM-CSF gene knockout. Methods of gene silencing and gene knockout are well known to those of ordinary skill in the art and can include, but are not limited to, RNAi, CRISPR, siRNA, ddRNAi, TALEN, zinc finger, homing endonucleases and meganucleases or other suitable techniques.

In some embodiments, administration of GM-CSF silenced or gene knocked-out CAR-T cells prevents or significantly reduces the incidence and/or severity of CRS and NT. In some embodiments, administration of GM-CSF silenced or gene knocked-out CAR-T cells prevents or significantly reduces BBB disruption. In some embodiments, administration of GM-CSF silenced or gene knocked-out CAR-T cells prevents or significantly reduces CD14+ myeloid cell activation and trafficking into the CNS. In some embodiments, administration of GM-CSF silenced or knockout CAR-T cells results in lower levels of systemic cytokines IL-3, IL-5, IP10, KC, MCP-1, MIP-1a, MIP-1b, M-CSF, MIP-2, MIG, VEGF, IL-1RA, IL-1b, IL-6, IL-12p40, IL12p70, IL-RA, M-CSF, and G-CSF than are observed when wild-type CAR-T cells are administered.

In some embodiments, administration of GM-CSF silenced or gene knocked-out CAR-T cells is performed with a recombinant GM-CSF antagonist, which further reduces the incidence or severity of CRS, NT, and further prevents or reduces BBB disruption, and further prevents or reduces CD14+ myeloid cell activation and trafficking into the CNS, and further prevents or reduces systemic cytokine levels of IL-3, IL-5, IP10, KC, MCP-1, MIP-1a, MIP-1b, M-CSF, MIP-2, MIG, VEGF, IL-1RA, IL-1b, IL-6, IL-12p40, IL12p70, IL1-RA, M-CSF and G-compared to that observed when wild-type CAR-T cells are administered.

Methods for improving the efficacy of adoptive cell therapy

In some embodiments, the methods comprise administering a CAR-T cell that has been modified to express lower levels of GM-CSF by GM-CSF gene silencing or GM-CSF gene knockout. In some embodiments, GM-CSF gene-silenced or knockout CAR-T cells are less differentiated after expansion and comprise a higher percentage of native, stem cell memory and central memory characteristics after expansion relative to wild-type CAR-T cells. In some embodiments, the GM-CSF gene-silenced or gene-knocked CAR-T cells do not express FAS or express a lower level of FAS than wild-type CAR-T cells. In some embodiments, the GM-CSF gene-silenced or knockout CAR-T cell is more tolerant to activation-induced cell death (AICD), more tolerant to senescence, and more tolerant to anergy compared to a wild-type CAR-T cell. In some embodiments, GM-CSF gene-silenced or gene-knocked CAR-T cells result in lower levels of MDSC formation and better CAR-T cell expansion and persistence compared to wild-type CAR-T cells. In some embodiments, administration of GM-CSF gene-silenced or gene-knocked-out CAR-T cells exhibits greater expansion and persistence than wild-type CAR-T cells. In some embodiments, the GM-CSF gene-silenced or knockout CAR-T cell exhibits a higher level of a targeted response (full response and partial response) as compared to a wild-type CAR-T cell. In some embodiments, the GM-CSF gene-silenced or gene-knocked CAR-T cell exhibits lower levels of relapse at 6 months, 12 months, and 24 months as compared to a wild-type CAR-T cell. In some embodiments, the GM-CSF gene-silenced or gene-knocked CAR-T cell exhibits an improved level of progression-free survival and/or overall survival compared to a wild-type CAR-T cell.

In some embodiments, administration of GM-CSF silenced or knockout CAR-T cells is performed with a recombinant GM-CSF antagonist, which further improves expansion, persistence, tolerance to senescence, and tolerance anergy. In other embodiments, administration of GM-CSF silenced or gene knocked-out CAR-T cells with a recombinant GM-CSF antagonist further reduces MDSC formation. In other embodiments, administration of GM-CSF silenced or gene knocked-out CAR-T cells with a recombinant GM-CSF antagonist further improves objective responses (full and partial responses), reduces the level of relapse at 6 months, 12 months, and 24 months and demonstrates improved levels of progression-free survival and/or overall survival.

In some embodiments, the CAR-T cell is a CD19 CAR-T cell; in other embodiments, the CAR-T cell is a BMCA CAR-T cell; in other embodiments, the CAR-T cell is a duplex CD19/CD22 CAR-T cell. In other embodiments, the CAR-T cell is a duplex CD19/CD20 CAR-T cell.

In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicities comprises reducing immune activation. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises ameliorating capillary leak syndrome. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicities comprises ameliorating cardiac dysfunction. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicities comprises ameliorating encephalopathy. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing colitis. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises inhibiting tics. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises ameliorating CRS. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises ameliorating neurotoxicity. In various embodiments, the CAR-T cell-associated neurotoxicity of the subject is reduced by about 90% as compared to the reduction in neurotoxicity of the subject treated with the CAR-T cells and the control antibody. In certain embodiments, the recombinant GM-CSF antagonist is an antibody, in particular a GM-CSF neutralizing antibody according to the embodiments described herein, including example 15.

In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing cytokine storm symptoms. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises increasing the impaired left ventricular ejection fraction. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises ameliorating diarrhea. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises improving disseminated intravascular coagulation.

In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing edema. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicities comprises reducing skin rash. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing gastrointestinal bleeding. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises treating gastrointestinal perforation. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises treating phagocytic lymphocyte histocytosis (HLH). In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicities comprises treating liver disease. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing hypotension. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing hypophysitis.

In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises inhibiting an immune-related adverse event. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicities comprises reducing immune hepatitis. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicities comprises reducing immunodeficiency. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises treating ischemia. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing hepatotoxicity. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicities comprises treating Macrophage Activation Syndrome (MAS). In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing symptoms of neurotoxicity.

In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing pleural effusion. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing pericardial effusion. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing pneumonia.

In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing polyarthritis. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises treating a Posterior Reversible Encephalopathy Syndrome (PRES). In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing pulmonary hypertension. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises treating thromboembolism. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing transaminase elevation. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing the CRES, Neurotoxicity (NT), and/or Cytokine Release Syndrome (CRS) grade in the patient. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises improving the patient's CARTOX-10 score.

In one aspect, the invention further provides a method for treating or preventing immunotherapy-related toxicity in a subject, the method comprising administering to the subject a chimeric antigen receptor-expressing T cell (CAR-T cell), a CAR-T cell with a GM-CSF gene knockout (GM-CSF) ^k/oCAR-T cells) as well as recombinant hGM-CSF antagonists, as demonstrated in examples 6 and 20-21. In some embodiments, the GM-CSF is compared to the level of GM-CSF expressed by a wild-type CAR-T cell^k/oThe GM-CSF level expressed by the CAR-T cells is reduced. In certain embodiments, the GM-CSF^k/oThe CAR-T cell expresses one or more cytokines and/or chemokines at a level less than or equal to the level of one or more cytokines and/or chemokines expressed by the wild-type CAR-T cell. In particular embodiments, the one or more cytokines are human cytokines selected from the group consisting of: IFN-gamma, GRO, MDC, IL-2, IL-3, IL-5, IL-7, IP-10, CD107a, TNF-a, and VEGF. In some embodiments, the one or more cytokines are selected from the group consisting of: IFN-gamma, IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL7, IL-9, IL-10, IL-12p40, IL-12p70, ILF, IL-13, LIX, IL-15, IP-10, KC, MCP-1, MIP-1a, MIP-1b, M-CSF, MIP-2, MIG, RANTES and TNF-a, eotaxin, G-CSF and combinations thereof. In various embodiments, the recombinant GM-CSF antagonist is an hGM-CSF antagonist. In various embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In particular embodiments, the anti-GM-CSF antibody binds to human GM-CSF. In other embodiments, the anti-GM-CSF antibody binds primate GM-CSF. In various embodiments of the present invention, the, The anti-GM-CSF antibody binds to mammalian GM-CSF. In some embodiments, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. In certain embodiments, the anti-hGM-CSF antibody is a monoclonal antibody. In various embodiments, the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab ', F (ab')2, scFv, or dAB. In some embodiments, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. In certain embodiments, the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. In various embodiments, the anti-hGM-CSF antibody is a human antibody. In some embodiments, the CAR-T cell is a CD19 CAR-T cell. In particular embodiments, the GM-CSF^k/oCAR-T cells enhance the anti-tumor activity of the recombinant hGM-CSF antagonist. In particular embodiments, the GM-CSF is compared to the survival of a subject treated by administration of wild-type CAR-T cells^k/oThe CAR-T cells improve the overall survival of the subject. In particular embodiments, CAR-T cells with GM-CSF gene knock-out (GM-CSF) are administered to the subject^k/oCAR-T cells) and recombinant hGM-CSF antagonists are durable treatments for preventing or treating immunotherapy-related toxicities (such as CRS, neurotoxicity, and neuroinflammation). In some embodiments, the subject has cancer. In various embodiments, the cancer is acute lymphoblastic leukemia.

Methods for removing human GM-CSF from a subject

In one aspect, the invention provides a method for neutralizing and/or removing human GM-CSF in a subject in need thereof, the method comprising administering CAR-T cells with GM-CSF gene knockout (GM-CSF) to the subject^k/oCAR-T cells).

In one embodiment of such a method, the method further comprises administering to the subject a recombinant hGM-CSF antagonist. In particular embodiments, the recombinant GM-CSF antagonist is an hGM-CSF antagonist. In some embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In another embodiment, the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab ', F (ab')2, scFv, or dAB. In one embodiment, the anti-hGM-CSF antibody has the VH region sequence shown in figure 1 and the VL region sequence shown in figure 1. In another embodiment, the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence include a methionine at the N-terminus. In further embodiments, the hGM-CSF antagonist is selected from the group comprising: an anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, a cytochrome b562 antibody mimetic, an hGM-CSF peptide analog, a mimetic antibody protein drug, a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptide mimetic. In another embodiment, the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. In further embodiments, the GM-CSF is a CAR T-derived GM-CSF or a non-CAR T-derived GM-CSF. In some embodiments, the subject has developed immunotherapy-related toxicity.

Methods for reducing blood brain barrier disruption and for preserving/maintaining BBB integrity

In one aspect, the invention provides a method for reducing blood brain barrier disruption in a subject treated with immunotherapy, the method comprising administering to the subject a recombinant GM-CSF antagonist. In some embodiments, the subject has developed immunotherapy-related toxicity.

In certain embodiments, the immunotherapy comprises adoptive cell transfer, administration of monoclonal antibodies, administration of cytokines, administration of cancer vaccines, T cell conjugation therapy, or any combination thereof. In various embodiments, the adoptive cell transfer comprises administering a chimeric antigen receptor expressing T cell (CAR T cell), a T Cell Receptor (TCR) modified T cell, a Tumor Infiltrating Lymphocyte (TIL), a Chimeric Antigen Receptor (CAR) modified natural killer cell, or a dendritic cell, or any combination thereof. In particular embodiments, the CAR T cell is a CD19 CAR-T cell.

In further embodiments, the recombinant GM-CSF antagonist is an hGM-CSF antagonist. In some embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In various embodiments, the anti-GM-CSF antibody binds to mammalian GM-CSF. In certain embodiments, the anti-GM-CSF antibody binds primate GM-CSF. In some embodiments, the primate is a monkey, baboon, macaque, chimpanzee, gorilla, lemur, lazy monkey, spectacle monkey, plexi monkey, tree bear monkey, crown lemur, bizary, lemur ape, simian, or human.

In particular embodiments, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. In various embodiments, the anti-hGM-CSF antibody binds to human GM-CSF. In certain embodiments, the anti-hGM-CSF antibody is a monoclonal antibody. In various embodiments, the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab ', F (ab')2, scFv, or dAB. In some embodiments, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. In further embodiments, the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. In further embodiments, the anti-hGM-CSF antibody is a human antibody. In particular embodiments, the anti-hGM-CSF antibody binds the same epitope as the chimeric 19/2 antibody. In more particular embodiments, the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of the chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody is administered prior to, concurrently with, after, or a combination thereof with an immunotherapy.

In various embodiments, the anti-hGM-CSF antibody comprises the VH region CDR1, antibody CDR2 and CDR3, and the VL region CDR1, CDR2 and CDR3 of the chimeric 19/2 antibody. In certain embodiments, the anti-hGM-CSF antibody comprises: a VH region comprising a CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V segment, wherein the J segment comprises at least 95% identity to human JH4(YFD YWGQGTL VTVSS) and the V segment comprises at least 90% identity to human germline VH 11-02 or VH 11-03 sequences; or a VH region comprising CDR3 binding specificity determinant RQRFPY. In some embodiments, the J segment comprises YFDYWGQGTLVTVSS. In certain embodiments, the CDR3 includes RQRFPYYFDY or RDRFPYYFDY. In further embodiments, the VH region CDR1 is a human germline VH1 CDR 1; the VH region CDR2 is the human germline VH1 CDR 2; or both CDR1 and CDR2 are from human germline VH1 sequences. In still further embodiments, the anti-hGM-CSF antibody comprises VH CDR1 or VH CDR2 or both VH CDR1 and VH CDR2 as shown in the VH region shown in figure 1. In some embodiments, the V segment sequence has the VH V segment sequence shown in fig. 1. In various embodiments, the VH has the sequence of VH #1, VH #2, VH #3, VH #4, or VH #5 shown in fig. 1. In certain embodiments, the anti-hGM-CSF antibody comprises a VL region comprising CDR3 comprising the amino acid sequence FNK or FNR.

In further embodiments, the anti-hGM-CSF antibody comprises the human germline JK4 region. In certain embodiments, the VL region CDR3 comprises QQFN (K/R) SPL. In some embodiments, the anti-hGM-CSF antibody comprises a VL region comprising CDR3 comprising QQFNKSPLT. In particular embodiments, the VL region comprises the CDR1 or CDR2 or both CDR1 and CDR2 of the VL region shown in fig. 1. In certain embodiments, the VL region comprises a V segment that is at least 95% identical to a VKIII a 27V segment sequence as shown in figure 1. In various embodiments, the VL region has the sequence of VK #1, VK #2, VK #3, or VK #4 shown in fig. 1. In some embodiments, the anti-hGM-CSF antibody has a VH region CDR3 binding specificity determinant RQRFPY or RDRFPY, and a VL region having a CDR3 comprising QQFNKSPLT. In further embodiments, the anti-hGM-CSF antibody has the VH region sequence shown in figure 1 and the VL region sequence shown in figure 1. In still further embodiments, the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence include a methionine at the N-terminus.

In various embodiments, the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibodies or soluble hGM-CSF receptors, cytochrome b562 antibody mimetics, hGM-CSF peptide mimetics, mimetic antibody proteinants, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptidomimetics. In particular embodiments, the immunotherapy-related toxicity is CAR-T related toxicity. In a more particular embodiment, the CAR-T associated toxicity is cytokine release syndrome, neurotoxicity, neuroinflammation, or a combination thereof.

In another aspect, the invention provides a method for preserving blood brain barrier integrity in a subject treated with an immunotherapy, the method comprising administering to the subject a recombinant hGM-CSF antagonist.

In a further aspect, the invention provides a method for preventing or reducing blood brain barrier disruption in a subject treated with an immunotherapy, the method comprising administering to the subject a CAR-T cell with a GM-CSF gene knockout (GM-CSF)^k/oCAR-T cells).

In one embodiment of the provided methods, the recombinant hGM-CSF antagonist is an anti-GM-CSF antibody. In another embodiment, the anti-GM-CSF antibody binds to mammalian GM-CSF. In yet another embodiment, the anti-GM-CSF antibody binds primate GM-CSF. In further embodiments, the primate is a monkey, baboon, macaque, chimpanzee, gorilla, lemur, lazy monkey, spectacle monkey, plexi monkey, tree bear monkey, crown lemur, bizary, lemian, simian, or human.

In particular embodiments of the provided methods, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. In further specific embodiments, the anti-hGM-CSF antibody binds to human GM-CSF. In still further embodiments, the anti-hGM-CSF antibody is a monoclonal antibody. In particular embodiments, the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab ', F (ab')2, scFv, or dAB. In one embodiment, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. In another embodiment, the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. In some embodiments, the anti-hGM-CSF antibody is a human antibody. In certain embodiments, the anti-hGM-CSF antibody binds the same epitope as the chimeric 19/2 antibody. In various embodiments, the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of the chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody is administered prior to, concurrently with, after, or a combination thereof with an immunotherapy.

In one embodiment of the provided methods, the anti-hGM-CSF antibody comprises the VH region CDR1, antibody CDR2 and CDR3 and the VL region CDR1, CDR2 and CDR3 of the chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody comprises: a VH region comprising a CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V segment, wherein the J segment comprises at least 95% identity to human JH4(YFD YWGQGTL VTVSS) and the V segment comprises at least 90% identity to human germline VH 11-02 or VH 11-03 sequences; or a VH region comprising CDR3 binding specificity determinant RQRFPY. In certain embodiments, the J segment comprises YFDYWGQGTLVTVSS. In particular embodiments, the CDR3 includes RQRFPYYFDY or RDRFPYYFDY. In some embodiments, the VH region CDR1 is the human germline VH1 CDR 1; the VH region CDR2 is the human germline VH1 CDR 2; or both CDR1 and CDR2 are from human germline VH1 sequences. In particular embodiments, the anti-hGM-CSF antibody comprises VH CDR1 or VH CDR2 or both VH CDR1 and VH CDR2 as shown in the VH region shown in figure 1. In one embodiment, the V segment sequence has the VH V segment sequence shown in fig. 1. In some embodiments, the VH has the sequence of VH #1, VH #2, VH #3, VH #4, or VH #5 shown in fig. 1. In various embodiments, the anti-hGM-CSF antibody comprises a VL region comprising CDR3 comprising the amino acid sequence FNK or FNR. In certain embodiments, the anti-hGM-CSF antibody comprises a human germline JK4 region. In one embodiment, the VL region CDR3 comprises QQFN (K/R) SPL. In certain embodiments, the anti-hGM-CSF antibody comprises a VL region comprising CDR3 comprising QQFNKSPLT. In some embodiments, the VL region comprises the CDR1 or CDR2 or both CDR1 and CDR2 of the VL region shown in fig. 1. In various embodiments, the VL region comprises a V segment that is at least 95% identical to a VKIII a 27V segment sequence as shown in fig. 1. In particular embodiments, the VL region has the sequence VK #1, VK #2, VK #3, or VK #4 as shown. In particular embodiments, the anti-hGM-CSF antibody has a VH region CDR3 binding specificity determinant RQRFPY or RDRFPY, and a VL region having a CDR3 comprising QQFNKSPLT. In further embodiments, the anti-hGM-CSF antibody has the VH region sequence shown in figure 1 and the VL region sequence shown in figure 1. In some embodiments, the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence include a methionine at the N-terminus. In certain embodiments, the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibodies or soluble hGM-CSF receptors, cytochrome b562 antibody mimetics, hGM-CSF peptide mimetics, mimetic antibody proteinants, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptidomimetics. In particular embodiments of the provided methods, the subject has immunotherapy-related toxicity. In a further embodiment, the immunotherapy-related toxicity is CAR-T related toxicity. In further embodiments, the CAR-T associated toxicity is cytokine release syndrome, neurotoxicity, neuroinflammation, or a combination thereof.

In a further aspect, the invention provides a method for reducing or preventing CAR-T cell therapy-induced neuroinflammation in a subject in need thereof, the method comprising administering to the subject a recombinant GM-CSF antagonist.

In some embodiments, administration of the recombinant GM-CSF antagonist reduces disruption of the blood brain barrier, thereby maintaining its integrity. In particular embodiments, reducing said disruption of the blood brain barrier reduces or prevents the influx of proinflammatory cytokines into the central nervous system. In various embodiments, the proinflammatory cytokine is selected from the group consisting of: IP-10, IL-2, IL-3, IL-5, IL-1Ra, VEGF, TNF-a, FGF-2, IFN- γ, IL-12p40, IL-12p70, sCD40L, MDC, MCP-1, MIP-1a, MIP-1b, or combinations thereof. In certain embodiments, the proinflammatory cytokine is selected from the group consisting of: IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-9, IL-10, IP-10, KC, MCP-1, MIP or combinations thereof. In particular embodiments, the neuroinflammation of the subject is reduced by 75% to 95% compared to a subject treated with CAR-T cell therapy and a control antibody. In various embodiments, the 75% to 95% reduction in neuroinflammation is similar to neuroinflammation in untreated control subjects. In some embodiments, the subject is administered a chimeric antigen receptor-expressing T cell (CAR T cell). In certain embodiments, a T Cell Receptor (TCR) -modified T cell, a tumor-infiltrating lymphocyte (TIL), a Chimeric Antigen Receptor (CAR) -modified natural killer cell, or a dendritic cell, or any combination thereof, is administered to the subject. In particular embodiments, the CAR T cell is a CD19 CAR-T cell. In various embodiments, the recombinant GM-CSF antagonist is an hGM-CSF antagonist. In certain embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In some embodiments, the anti-GM-CSF antibody binds to mammalian GM-CSF. In further embodiments, the anti-GM-CSF antibody binds primate GM-CSF. In some embodiments, the primate is a monkey, baboon, macaque, chimpanzee, gorilla, lemur, lazy monkey, spectacle monkey, plexi monkey, tree bear monkey, crown lemur, bizary, lemur ape, simian, or human. In one embodiment, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. In another embodiment, the anti-hGM-CSF antibody binds to human GM-CSF. In further embodiments, the anti-hGM-CSF antibody is a monoclonal antibody. In still further embodiments, the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab ', F (ab')2, scFv, or dAB. In particular embodiments, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody.

In various embodiments, the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. In some embodiments, the anti-hGM-CSF antibody is a human antibody. In particular embodiments, the anti-hGM-CSF antibody binds the same epitope as the chimeric 19/2 antibody. In further embodiments, the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of the chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody is administered prior to, concurrently with, after, or a combination thereof with an immunotherapy.

In still further embodiments, the anti-hGM-CSF antibody comprises the VH region CDR1, antibody CDR2 and CDR3, and the VL region CDR1, CDR2 and CDR3 of the chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody comprises: a VH region comprising a CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V segment, wherein the J segment comprises at least 95% identity to human JH4(YFD YWGQGTL VTVSS) and the V segment comprises at least 90% identity to human germline VH 11-02 or VH 11-03 sequences; or a VH region comprising CDR3 binding specificity determinant RQRFPY. In various embodiments, the J section comprises YFDYWGQGTLVTVSS.

In particular embodiments, the CDR3 includes RQRFPYYFDY or RDRFPYYFDY. In some embodiments, the VH region CDR1 is the human germline VH1 CDR 1; the VH region CDR2 is the human germline VH1 CDR 2; or both CDR1 and CDR2 are from human germline VH1 sequences. In various embodiments, the anti-hGM-CSF antibody comprises VH CDR1 or VH CDR2 or both VH CDR1 and VH CDR2 as shown in the VH region shown in figure 1. In certain embodiments, the V segment sequence has the VH V segment sequence shown in fig. 1. In particular embodiments, the VH has the sequence of VH #1, VH #2, VH #3, VH #4, or VH #5 shown in fig. 1. In further embodiments, the anti-hGM-CSF antibody comprises a VL region comprising CDR3 comprising the amino acid sequence FNK or FNR. In still further embodiments, the anti-hGM-CSF antibody comprises the human germline JK4 region. In some embodiments, the VL region CDR3 comprises QQFN (K/R) SPL. In certain embodiments, the anti-hGM-CSF antibody comprises a VL region comprising CDR3 comprising QQFNKSPLT. In various embodiments, the VL region comprises the CDR1 or CDR2 or both CDR1 and CDR2 of the VL region shown in fig. 1. In further embodiments, the VL region comprises a V segment that is at least 95% identical to a VKIII a 27V segment sequence as shown in fig. 1. In still other embodiments, the VL region has the sequence of VK #1, VK #2, VK #3, or VK #4 shown in fig. 1.

In certain embodiments, the anti-hGM-CSF antibody has a VH region CDR3 binding specificity determinant RQRFPY or RDRFPY, and a VL region having a CDR3 comprising QQFNKSPLT. In some embodiments, the anti-hGM-CSF antibody has the VH region sequence shown in figure 1 and the VL region sequence shown in figure 1. In various embodiments, the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence include a methionine at the N-terminus.

In further embodiments, the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibodies or soluble hGM-CSF receptors, cytochrome b562 antibody mimetics, hGM-CSF peptide mimetics, mimetic antibody proteinants, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptidomimetics. In some embodiments, the subject further has CAR-T related toxicity selected from cytokine release syndrome, neurotoxicity, or a combination thereof.

Methods for reducing relapse rate or preventing occurrence

In one aspect, the invention provides a method for reducing the recurrence rate or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy, the method comprising administering to the subject a recombinant GM-CSF antagonist. In some embodiments, the reducing the recurrence rate or preventing the occurrence of tumor recurrence in the subject occurs in the absence of the occurrence of immunotherapy-related toxicity. In certain embodiments, the reducing the recurrence rate or preventing the occurrence of tumor recurrence in the subject occurs in the presence of immunotherapy-related toxicity. In some embodiments, the recombinant GM-CSF antagonist is an hGM-CSF antagonist. In various embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In some embodiments, the anti-GM-CSF antibody binds to human GM-CSF. In certain embodiments, the anti-GM-CSF antibody binds primate GM-CSF. In various embodiments, the primate is selected from the group consisting of a monkey, baboon, macaque, chimpanzee, gorilla, lemur, lazy monkey, spectacle monkey, plexi monkey, tree bear monkey, crown lemur, bizary monkey, lemur ape, or simian. In some embodiments, the anti-GM-CSF antibody binds to mammalian GM-CSF.

In particular embodiments, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. As described above, the anti-GM-CSF antibody is a monoclonal antibody. In another embodiment, the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab ', F (ab')2, scFv, or dAB. In some embodiments, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. In certain embodiments, the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. In various embodiments, the anti-hGM-CSF antibody is a human antibody. In some embodiments, the anti-hGM-CSF antibody binds the same epitope as the chimeric 19/2 antibody. In certain embodiments, the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of the chimeric 19/2 antibody. In various embodiments, the anti-hGM-CSF antibody comprises the VH region CDR1, antibody CDR2 and CDR3, and the VL region CDR1, CDR2 and CDR3 of the chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody comprises: a VH region comprising a CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V segment, wherein the J segment comprises at least 95% identity to human JH4(YFD YWGQGTL VTVSS) and the V segment comprises at least 90% identity to human germline VH 11-02 or VH 11-03 sequences; or a VH region comprising CDR3 binding specificity determinant RQRFPY. In a particular embodiment, the J section includes YFDYWGQGTLVTVSS. In further embodiments, the CDR3 includes RQRFPYYFDY or RDRFPYYFDY. In some embodiments, the VH region CDR1 is a human germline VH1 CDR 1; the VH region CDR2 is the human germline VH1 CDR 2; or both the CDR1 and the CDR2 are from human germline VH1 sequences.

In certain embodiments, the anti-hGM-CSF antibody comprises VH CDR1 or VH CDR2 or both VH CDR1 and VH CDR2 as shown in the VH region shown in figure 1. In various embodiments, the V segment sequence has the VH V segment sequence shown in fig. 1. In certain embodiments, the VH has the sequence of VH #1, VH #2, VH #3, VH #4, or VH #5 shown in fig. 1. In some embodiments, the anti-hGM-CSF antibody comprises a VL region comprising CDR3 comprising the amino acid sequence FNK or FNR. In some embodiments, the anti-hGM-CSF antibody comprises a human germline JK4 region. In certain embodiments, the VL region CDR3 comprises QQFN (K/R) SPLT. In various embodiments, the anti-hGM-CSF antibody comprises a VL region comprising CDR3 comprising QQFNKSPLT. In some embodiments, the VL region comprises the CDR1 or CDR2 or both CDR1 and CDR2 of the VL region shown in fig. 1. In particular embodiments, the VL region comprises a V segment that is at least 95% identical to the VKIII a 27V segment sequence shown in fig. 1. In some embodiments, the VL region has the sequence of VK #1, VK #2, VK #3, or VK #4 shown in figure 1. In certain embodiments, the anti-hGM-CSF antibody has a VH region CDR3 binding specificity determinant RQRFPY or RDRFPY, and a VL region having a CDR3 comprising QQFNKSPLT. In some embodiments, the anti-hGM-CSF antibody has the VH region sequence shown in figure 1 and the VL region sequence shown in figure 1. In other embodiments, the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence include a methionine at the N-terminus.

In some embodiments, the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibodies or soluble hGM-CSF receptors, cytochrome b562 antibody mimetics, hGM-CSF peptide mimetics, mimetic antibody proteinants, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptidomimetics. In certain embodiments, the CAR-T cell is a CD19 CAR-T cell. In particular embodiments, the immunotherapy-related toxicity is CAR-T related toxicity. In some embodiments, the CAR-T related toxicity is CRS, NT, or neuroinflammation.

In particular embodiments, the tumor recurrence incidence is reduced from 50% to 100% in the first quarter of a year after administration of the recombinant GM-CSF antagonist compared to the tumor recurrence incidence in a subject treated with immunotherapy and not administered a recombinant GM-CSF antagonist. In certain embodiments, the tumor recurrence incidence is reduced from 50% to 95% in the first half year after administration of the recombinant GM-CSF antagonist. In various embodiments, the tumor recurrence incidence is reduced from 50% to 90% in the first year after administration of the recombinant GM-CSF antagonist. In some embodiments, the tumor recurrence is prevented chronically. As used herein, the term "long-term" means an extended period of time of at least one year, i.e., 12 months, from the last day of treatment with the recombinant hGM-CSF antagonist. In some embodiments, the recombinant hGM-CSF antagonist is an hGM-CSF neutralizing antibody. In various embodiments, the recombinant hGM-CSF antagonist is an anti-hGM-CSF antibody, e.g., ritzilumab (Lenzilumab). In certain embodiments, the prophylactic effect of the occurrence of tumor recurrence lasts for 12 to 36 months. In some embodiments, "completely" (100%) prevents tumor recurrence from occurring, as used herein, meaning that the tumor has not recurred for at least 12 months from the last day of treatment with the recombinant hGM-CSF antagonist. In certain embodiments, the subject has acute lymphoblastic leukemia.

In various embodiments of the methods provided herein for reducing the recurrence rate or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy, the subject has a "refractory cancer," which, as used herein, is (a) a malignancy (also referred to herein as "cancer" or "tumor") for which surgery is ineffective, and (b) a malignancy that is initially unresponsive or resistant to treatment, wherein the treatment is chemotherapy, radiation therapy, or a combination thereof, or (b) a malignancy that becomes or has become unresponsive to one or more of the above treatments. In some embodiments, a subject has a "recurrent" cancer, which, as used herein, is a cancer that is responsive but responsive to treatment but has returned. In particular embodiments, the refractory cancer or the recurrent cancer is non-hodgkin's lymphoma (NHL). In various embodiments, the refractory cancer or the recurrent cancer is non-hodgkin's lymphoma (NHL). In certain embodiments, the refractory cancer is refractory aggressive B cell non-hodgkin's lymphoma. In some embodiments, the refractory cancer or relapsed cancer is chemotherapy-refractory B-cell lymphoma. In various embodiments, the refractory cancer or the recurrent cancer is hormone refractory prostate cancer. In certain embodiments, the refractory cancer or the recurrent cancer is a pediatric cancer. In some embodiments, the refractory pediatric cancer or the relapsed pediatric cancer is neuroblastoma. In particular embodiments, the refractory pediatric cancer or the relapsed pediatric cancer is a pediatric leukemia selected from the group consisting of: acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML) or rare pediatric leukemia, which is juvenile myelomonocytic leukemia or chronic myeloid leukemia. In certain embodiments, the refractory cancer or the recurrent cancer is pediatric bone cancer. In some embodiments, the refractory cancer or the recurrent cancer is adrenal cancer. In various embodiments, the refractory cancer or the recurrent cancer is breast cancer. In certain embodiments, the refractory cancer or the recurrent cancer is colon cancer, rectal cancer, or colorectal cancer. In particular embodiments, the refractory cancer or the relapsed cancer is T-cell lymphoma. In some embodiments, the refractory cancer or the recurrent cancer is a head and neck cancer. In some embodiments, the refractory cancer or the recurrent cancer is a brain cancer and/or spinal cord cancer, including but not limited to glioma, glioblastoma. In further embodiments, the refractory cancer or the recurrent cancer is a tumor of bone or soft tissue, including but not limited to chondrosarcoma. In various embodiments, the refractory cancer or the recurrent cancer is bone cancer. In some embodiments, the refractory cancer or the recurrent cancer is esophageal cancer. In certain embodiments, the refractory cancer or the recurrent cancer is gallbladder cancer (gall bladder cancer). In some embodiments, the refractory cancer or the recurrent cancer is renal cancer. In various embodiments, the refractory cancer or the recurrent cancer is melanoma. In some embodiments, the refractory cancer or the recurrent cancer is ovarian cancer. In certain embodiments, the refractory cancer or the recurrent cancer is pancreatic cancer. In some embodiments, the refractory cancer or the recurrent cancer is a skin cancer selected from basal cell carcinoma, squamous cell carcinoma, or melanoma. In various embodiments, the refractory cancer or the recurrent cancer is lung cancer. In some embodiments, the refractory cancer or the recurrent cancer is salivary gland cancer. In further embodiments, the refractory cancer or the recurrent cancer is uterine smooth muscle cancer. In some embodiments, the refractory cancer or the recurrent cancer is testicular cancer. In various embodiments, the refractory cancer or the recurrent cancer is gastric cancer or gastrointestinal cancer. In certain embodiments, the refractory cancer or the recurrent cancer is bladder cancer. In further embodiments, the refractory cancer or the recurrent cancer is an adipose tissue tumor. In some embodiments, the refractory pediatric cancer or the relapsed pediatric cancer is adenocarcinoma. In certain embodiments, the refractory cancer or relapsed cancer is thymoma. In various embodiments, the refractory or recurrent cancer is angiosarcoma, a cancer of the vascular lining, which may occur in any part of the body, including but not limited to skin, breast, liver, spleen, and deep tissues, i.e., deep-seated tumors. In some embodiments, the refractory cancer or the recurrent cancer is a metastasis of any of the refractory or recurrent cancers described above.

In some embodiments, the immunotherapy is an activated immunotherapy. In some embodiments, immunotherapy is provided as a cancer treatment. In some embodiments, the immunotherapy comprises adoptive cell transfer.

In some embodiments, adoptive cell transfer includes administering chimeric antigen receptor expressing T cells (CAR T cells). The skilled person will appreciate that a CAR is an antigen-targeting receptor consisting of an intracellular T cell signalling domain (most commonly a single-chain variable fragment (scFv) from a monoclonal antibody) fused to an extracellular tumour-binding moiety. CARs recognize cell surface antigens directly, independent of MHC-mediated presentation, allowing the use of a single receptor construct specific for any given antigen in all patients. The original CAR fused the antigen recognition domain to the CD3 zeta activation chain of the T Cell Receptor (TCR) complex. These first generation CARs induced T cell effector function in vitro, but were limited by poor antitumor efficacy in vivo. Subsequent CAR iterations included secondary costimulatory signals in tandem with CD3 ζ, including intracellular domains from CD28 or multiple TNF receptor family molecules, such as 4-1BB (CD137) and OX40(CD 134). Further, the third generation receptor contains two costimulatory signals in addition to CD3 ζ, most commonly from CD28 and 4-1 BB. Second and third generation CARs can significantly improve anti-tumor efficacy, in some cases inducing complete remission in patients with advanced cancer. In one embodiment, the CAR T cell is an immunoresponsive cell modified to express a CAR that is activated upon binding of the CAR to its antigen.

In one embodiment, the CAR T cell is an immunoresponsive cell that includes an antigen receptor that is activated when its receptor binds its antigen. In one embodiment, the CAR T cells used in the compositions and methods disclosed herein are first generation CAR T cells. In another embodiment, the CAR T cells used in the compositions and methods disclosed herein are second generation CAR T cells. In another embodiment, the CAR T cells used in the compositions and methods disclosed herein are third generation CAR T cells. In another embodiment, the CAR T cells used in the compositions and methods disclosed herein are fourth generation CAR T cells.

In some embodiments, adoptive cell transfer includes administering T Cell Receptor (TCR) modified T cells. The skilled person will appreciate that TCR-modified T cells are made by isolating T cells from tumour tissue and isolating their TCR α and TCR β chains. These TCR α and TCR β are then cloned and transfected into T cells isolated from peripheral blood, and then TCR α and TCR β are expressed from tumor-recognizing T cells.

In some embodiments, adoptive cell transfer comprises administration of Tumor Infiltrating Lymphocytes (TILs). In some embodiments, adoptive cell transfer includes administering a Chimeric Antigen Receptor (CAR) modified NK cell. The skilled artisan will appreciate that CAR-modified NK cells include NK cells isolated from a patient or commercially available NK cells engineered to express a CAR that recognizes a tumor-specific protein.

In some embodiments, adoptive cell transfer includes administration of dendritic cells.

In some embodiments, immunotherapy comprises administering a monoclonal antibody. In some embodiments, the monoclonal antibody attaches to a specific protein on the cancer cell, thereby labeling the cell for discovery by the immune system and destroying it. In some embodiments, the monoclonal antibody acts by inhibiting the immunodetection site, thereby preventing suppression of the immune system by the cancer cells. In some embodiments, the monoclonal antibody improves the efficacy of the CAR-T in synergy with the checkpoint inhibitor.

In some embodiments, the antibody targets a protein selected from the group comprising: 5AC, 5T4, activin receptor-like kinase 1, AGS-22M6, alpha-fetoprotein, angiogenin 2, angiogenin 3, B7-H3, BAFF, BCMA, C242 antigen, CA-125, carbonic anhydrase 9, CCR4, CD125, CD152, CD184, CD19, CD2, CD20, CD200, CD20, CD221, CD20, CD274, CD276, CD20, EGFL 20, CEA, CSF 4, CG364, CGD, CTDL 20, CTLA-binding protein receptor binding protein, VEGF 72, DPP-binding protein, DPP-binding protein, VEGF-binding protein, binding, GCGR, GD2 ganglioside, GD3 ganglioside, GDF-8, glypican 3, GM-CSF receptor alpha chain, GPNMB, GUCY2C, HER1, HER2/neu, HGF, HHGFR, histone complexes, human scatter factor receptor kinase, human TNF, ICOSL, IFN-alpha, IGF1, IGF2, IGHE, IL-17A, IL-13, IL1A, IL-2, IL-6 receptor, IL-8, IL-9, ILGF2, integrin alpha 4, integrin alpha 5 beta 1, integrin alpha 7 beta 7, integrin alpha v beta 3, IP10, KIR2D, KLRC1, Lewis-Y antigen, MAGE-A, MCP-1, mesothelin, MIPF, MIG, MS 632 beta, MS4A1, MULN 1, MULN 23, NOLN-8, NOOX-6, NRRC 638, NOOX-6, and IFN receptor, PD-1, PDCD1, PDGF-R α, sodium phosphate cotransporter, phosphatidylserine, platelet-derived growth factor receptor β, prostate cancer cells, RHD, RON, RTN4, SDC1, sIL2R α, SLAMF7, SOST, sphingosine-1-phosphate, Staphylococcus aureus, STEAP1, TAG-72, T cell receptor, TEM1, tenascin C, TFPI, TGF β 1, TGF β 2, TGF β, TNFR superfamily member 4, TNF- α, TRAIL-R1, TRAIL-R2, TRP-1, TRP-2, TSLP, tumor antigen CTAA16.88, tumor specific glycosylation of MUC1, tumor associated calcium signaling transducer 2, TWEAK receptor, TYRP1 (75), VEGFA, glycoprotein-1, VEGFR2, vimentin, and VWF.

In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a bispecific T cell engager (BiTE) antibody. In some embodiments, the antibody is selected from the group comprising: ipilimumab (ipilimumab), nivolumab (nivolumab), pembrolizumab (pembrolizumab), atelizumab (atezolizumab), aviluzumab (avelumab), dutvacizumab (durvalumab), rituximab (rituximab), TGN1412, alemtuzumab (alemtuzumab), OKT3, or any combination thereof.

In some embodiments, the immunotherapy comprises administering a cytokine. The skilled person will appreciate that cytokines may be administered in order to enhance the immune system by increasing the recognition and killing of immunotoxic cells to attack the tumour. In some embodiments, the cytokine is selected from the group comprising: IFN alpha, IFN beta, IFN gamma, IFN lambda, IL-1, IL-2, IL-6, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18, GM-CSF, TNF alpha or any combination thereof.

In some embodiments, the immunotherapy comprises administering an immune checkpoint inhibitor. The skilled person will appreciate that an immune checkpoint is a membrane protein that protects T cells from attack by the cells expressing it. Immune checkpoints are typically expressed by cancer cells, thereby preventing T cells from attacking them. In some embodiments, the checkpoint proteins include PD-1/PD-L1 and CTLA-4/B7-1/B7-2. Blocking the checkpoint protein was shown to release the inhibition of T cells, thereby attacking and killing cancer cells. In some embodiments, the checkpoint inhibitor is selected from the group comprising molecules that block CTLA-4, PD-1, or PD-L1. In some embodiments, the checkpoint inhibitor is an antibody or portion thereof.

In some embodiments, the immunotherapy comprises administering a polysaccharide. The skilled person will appreciate that certain polysaccharides found in mushrooms enhance the immune system and its anti-cancer properties. In some embodiments, the polysaccharide is β -glucan or lentinan.

In some embodiments, the immunotherapy comprises administering a cancer vaccine. The skilled person will appreciate that cancer vaccines expose the immune system to cancer specific antigens and adjuvants. In some embodiments, the cancer vaccine is selected from the group comprising: western Pulsai-T, GVAX, ADXS11-001, ADXS31-001, ADXS31-164, ALVAC-CEA vaccine, AC vaccine, Talimogen laherparvec, BiovaxID, Prostvac, CDX110, CDX1307, CDX1401, CimaVax-EGF, CV9104, DNDN, NeuVax, Ae-37, GRNVAC, tarmogens, GI-4000, GI-6207, GI-6301, ImPACT therapy, IMA901, hepcotripelisimu-L, Stimuvavax, VaDCVax-L, DCVax-Direct, DCVax prostate, CBLI, Cvac, RGSH4K, SCIB1, NCT01758328, and PVX-410.

Methods for reducing the levels of cytokines or chemokines other than GM-CSF

In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing the concentration of at least one inflammation-related factor in a bodily fluid. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing the concentration of at least one inflammation-related factor in serum. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing the concentration of at least one inflammation-related factor in cerebrospinal fluid (CSF). In some embodiments, disclosed herein are methods for reducing the concentration of at least one inflammation-related factor in serum. In some embodiments, disclosed herein are methods for reducing the concentration of at least one inflammation-related factor in interstitial fluid. In some embodiments, disclosed herein are methods for reducing the concentration of at least one inflammation-related factor in CSF. In some embodiments, the concentration of at least one inflammation-related factor in the serum is reduced. In some embodiments, the concentration of at least one inflammation-related factor in the interstitial fluid is reduced. In some embodiments, the concentration of at least one inflammation-related factor in the CSF is reduced. The skilled artisan will appreciate that reducing the concentration of an inflammation-related factor includes reducing or inhibiting the production of an inflammation-related factor in a subject, or inhibiting or reducing the incidence or severity of immunotherapy-related toxicity in a subject. In another embodiment, reducing or inhibiting the production of inflammation-related factors comprises treating immunotherapy-related toxicity. In another embodiment, reducing or inhibiting the production of inflammation-related factors comprises preventing immunotherapy-related toxicity. In another embodiment, reducing or inhibiting the production of inflammation-related factor levels comprises reducing immunotherapy-related toxicity. In another embodiment, reducing or inhibiting the production of inflammation-related factors comprises ameliorating immunotherapy-related toxicity.

In some embodiments, the inflammation-associated factor is a cytokine. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing the concentration of at least one cytokine in serum. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing the concentration of at least one cytokine in CSF.

In some embodiments, the cytokine is hGM-CSF. In some embodiments, the cytokine is Interleukin (IL) -1 β. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is sIL2R α. In some embodiments, the cytokine is IL-5. In some embodiments, the cytokine is IL-6. In some embodiments, the cytokine is IL-8.

In some embodiments, the cytokine is IL-10. In some embodiments, the cytokine is IP 10. In some embodiments, the cytokine is IL-13. In some embodiments, the cytokine is IL-15. In some embodiments, the cytokine is tumor necrosis factor alpha (TNF α). In some embodiments, the cytokine is interferon gamma (IFN γ). In some embodiments, the cytokine is a monokine induced by gamma interferon (MIG). In some embodiments, the cytokine is Macrophage Inflammatory Protein (MIP)1 β. In some embodiments, the cytokine is a C-reactive protein. In some embodiments, reducing or inhibiting the production of a cytokine level comprises reducing or inhibiting the production of one cytokine. In some embodiments, reducing or inhibiting production of the cytokine level comprises reducing or inhibiting production of at least one cytokine. In some embodiments, reducing or inhibiting production of cytokine levels comprises reducing or inhibiting production of a plurality of cytokines.

In one aspect, the invention provides a method of reducing the level of a cytokine or chemokine other than GM-CSF in a subject who has developed immunotherapy-related toxicity, comprising administering to the subject a recombinant hGM-CSF antagonist, wherein the level of the cytokine or chemokine is reduced during the development of immunotherapy-related toxicity as compared to the level of the cytokine or chemokine in a subject administered an isotype control antibody. In some embodiments, the immunotherapy comprises adoptive cell transfer, administration of monoclonal antibodies, administration of cancer vaccines, T cell conjugation therapy, or any combination thereof. In certain embodiments, the adoptive cell transfer comprises administering a chimeric antigen receptor expressing T cell (CAR T cell), a T Cell Receptor (TCR) modified T cell, a Tumor Infiltrating Lymphocyte (TIL), a Chimeric Antigen Receptor (CAR) modified natural killer cell, or a dendritic cell, or any combination thereof. In some embodiments, the CAR-T cell is a CD19CAR-T cell. In certain embodiments, the recombinant GM-CSF antagonist is an hGM-CSF antagonist. In various embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In particular embodiments, the anti-GM-CSF antibody binds to human GM-CSF. In other embodiments, the anti-GM-CSF antibody binds primate GM-CSF, as described above. In some embodiments, the anti-GM-CSF antibody binds to mammalian GM-CSF. In certain embodiments, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. In some embodiments, the anti-hGM-CSF antibody is a monoclonal antibody. In various embodiments, the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab ', F (ab')2, scFv, or dAB. In some embodiments, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. In certain embodiments, the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. In some embodiments, the anti-hGM-CSF antibody is a human antibody. In particular embodiments, the cytokine or chemokine is a human cytokine or chemokine selected from the group consisting of: IP-10, IL-2, IL-3, IL-5, IL-1Ra, VEGF, TNF-a, FGF-2, IFN- γ, IL-12p40, IL-12p70, sCD40L, MDC, MCP-1, MIP-1a, MIP-1b, or combinations thereof, as demonstrated in example 22. In some embodiments, the cytokine or chemokine is selected from the group consisting of: IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-9, IL-10, IP-10, KC, MCP-1, MIP or combinations thereof (see example 22). In certain embodiments, the subject has acute lymphoblastic leukemia.

In one embodiment, the methods disclosed herein do not affect the efficacy of immunotherapy. In another embodiment, the methods disclosed herein reduce the efficacy of immunotherapy by less than about 5%. In another embodiment, the methods disclosed herein reduce the efficacy of immunotherapy by less than about 10%. In another embodiment, the methods disclosed herein reduce the efficacy of immunotherapy by less than about 15%. In another embodiment, the methods disclosed herein reduce the efficacy of immunotherapy by less than about 20%. In another embodiment, the methods disclosed herein reduce the efficacy of immunotherapy by less than about 50%.

In one embodiment, the methods described herein increase the efficacy of immunotherapy. In one embodiment, increased efficacy allows for improved clinical management of immunotherapy, patient outcome, and therapeutic index. In another embodiment, the increased efficacy enables administration of higher immunotherapy doses. In another embodiment, increased efficacy reduces hospital stays and additional treatment and monitoring. In one embodiment, the immunotherapy comprises CAR-T.

Any suitable method of quantifying cytotoxicity may be used to determine whether the efficacy of immunotherapy remains substantially unchanged. For example, cell culture-based assays, such as the cytotoxicity assays described in the examples, can be used to quantify cytotoxicity. Cytotoxicity assays may employ dyes that preferentially stain dead cell DNA. In other cases, fluorescence and luminescence measurements that measure the relative number of live and dead cells in a cell population can be used. For such assays, protease activity serves as a marker of cell viability and cytotoxicity, and the labeled cell permeable peptide generates a fluorescent signal proportional to the number of viable cells in the sample. In another embodiment, the measure of cytotoxicity may be qualitative. In another embodiment, the measure of cytotoxicity can be quantitative.

In one embodiment, the increased efficacy comprises increased CAR-T cell expansion, decreased number and/or activity of Myeloid Derived Suppressor Cells (MDSCs) that suppress T cell function, synergy with a checkpoint inhibitor, or any combination thereof. In another embodiment, the increased CAR-T cell expansion comprises an increase of at least 50% compared to a control. In another embodiment, the increased CAR-T cell expansion comprises at least one-quarter log expansion compared to a control. In another embodiment, the increased cell expansion comprises at least one-half log expansion as compared to a control. In another embodiment, the increased cell expansion comprises at least one log expansion as compared to a control. In another embodiment, the increased cell expansion comprises more than one log expansion compared to a control.

In one embodiment, immunotherapy-related toxicities occur between 2 days and 4 weeks after administration of immunotherapy. In one embodiment, immunotherapy-related toxicities occur between 0 and 2 days after administration of immunotherapy. In some embodiments, the hGM-CSF antagonist is administered to the subject concurrently with immunotherapy as a prophylaxis. In another embodiment, the hGM-CSF antagonist is administered to the subject 0-2 days after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-3 days after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 7 days after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 10 days after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 14 days after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-14 days after administration of the immunotherapy.

In another embodiment, the hGM-CSF antagonist is administered to the subject 2-3 hours after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 7 hours after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 10 hours after administration of the immunotherapy. In another embodiment, the GM-CSF antagonist is administered to the subject 14 hours after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-14 hours after administration of the immunotherapy.

In alternative embodiments, the hGM-CSF antagonist is administered to the subject prior to immunotherapy as a prophylaxis. In another embodiment, the hGM-CSF antagonist is administered to the subject 1 day prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-3 days prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 7 days prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 10 days prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 14 days prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-14 days prior to administration of the immunotherapy.

In another embodiment, the hGM-CSF antagonist is administered to the subject 2-3 hours prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 7 hours prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 10 hours prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 14 hours prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-14 hours prior to administration of the immunotherapy.

In another embodiment, an hGM-CSF antagonist can be administered therapeutically once immunotherapy-related toxicity has occurred. In one embodiment, an hGM-CSF antagonist can be administered once a pathophysiological process is detected that causes or evidences the onset of immunotherapy-related toxicity. In one embodiment, hGM-CSF antagonists can terminate pathophysiological processes and avoid sequelae thereof. In some embodiments, the pathophysiological process comprises at least one of: increased cytokine concentration in serum, increased cytokine concentration in CSF, increased C-reactive protein (CRP) in serum, increased ferritin in serum, increased serum IL-6, endothelial activation, Disseminated Intravascular Coagulation (DIC), increased serum ANG2 concentration, increased serum ANG2: ANG1 ratio, presence of CAR T cells in CSF, increased serum concentration of Von Willebrand Factor (VWF), leakage of the Blood Brain Barrier (BBB), or any combination thereof.

In another embodiment, the hGM-CSF antagonist may be administered therapeutically at multiple time points. In another embodiment, the hGM-CSF antagonist is administered at least at two time points. In another embodiment, the hGM-CSF antagonist is administered at least three time points.

In one embodiment, the hGM-CSF antagonist is administered once. In another embodiment, the hGM-CSF antagonist is administered twice. In another embodiment, the hGM-CSF antagonist is administered three times. In another embodiment, the hGM-CSF antagonist is administered four times. In another embodiment, the hGM-CSF antagonist is administered at least four times. In another embodiment, the hGM-CSF antagonist is administered more than four times.

The skilled person will appreciate that immunotherapy-related toxicities are controlled by different treatments. In some embodiments, the hGM-CSF antagonist is co-administered with other therapies. In some embodiments, the additional treatment is selected from the group consisting of: cytokine-directed therapy, anti-IL-6 therapy, corticosteroids, tositumomab, cetuximab (siltuximab), low-dose vasopressors, inotropic agents, supplemental oxygen, diuresis, thoracentesis, antiepileptics, benzodiazepines, levetiracetam, phenobarbital, hyperventilation, hypertonic therapy, and standard therapy for specific organ toxicity.

In some embodiments, the immunotherapy-related toxicity comprises a brain disease, injury, or dysfunction. In some embodiments, the immunotherapy-related toxicity comprises CAR T cell-associated NT. In some embodiments, the immunotherapy-related toxicity comprises CAR T-cell associated encephalopathy syndrome (CRES). In some embodiments, provided herein are methods for inhibiting or reducing the incidence of brain disease, injury, or dysfunction.

In some embodiments, inhibiting or reducing the incidence of CRES comprises ameliorating headache. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing delirium. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing anxiety. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing tremor. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing seizure activity. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing confusion. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing changes in wakefulness.

In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing hallucinations. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing speech impairment. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing ataxia. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing apraxia. In some embodiments, inhibiting or reducing the incidence of CRES comprises improving facial paralysis. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing exercise weakness. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing seizures. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing non-tic EEG seizures. In some embodiments, inhibiting or reducing the incidence or severity of CRES comprises improving coma recovery.

In some embodiments, inhibiting or reducing the incidence or severity of CRES comprises reducing endothelial activation. The skilled person will understand that endothelial activation is an inflammatory and pro-coagulant state of endothelial cells, characterized by increased interaction with leukocytes.

In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing vascular leakage. The term "vascular leak" may be used interchangeably with the terms "vascular leak syndrome" and "capillary leak syndrome" having all the same properties and meanings. The skilled artisan will appreciate that vascular leakage associated with endothelial cells is isolated, allowing leakage of plasma and transendothelial migration of inflammatory cells into body tissues, resulting in tissue and organ damage. In addition, neutrophils can cause microcirculation occlusion, resulting in reduced tissue perfusion. In some embodiments, reducing the incidence of CRES comprises reducing intravascular coagulation.

In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing the concentration of at least one circulating cytokine. In some embodiments, the cytokine is selected from the group comprising: hGM-CSF, IFN gamma, IL-1, IL-15, IL-6, IL-8, IL-10 and IL-2. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing the serum concentration of ANG 2. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing the ANG2 to ANG1 ratio in serum.

In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing the CRES grade. In some embodiments, inhibiting or reducing the incidence of CRES comprises improving the CARTOX-10 score. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing an increase in intracranial pressure. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing seizures. In some embodiments, inhibiting or reducing the incidence of CRES comprises reducing exercise weakness.

In some embodiments, the immunotherapy-related toxicity comprises CAR T cell-associated CRS. In some embodiments, provided herein are methods for inhibiting or reducing the incidence or severity of CRS and/or NT.

In some embodiments, inhibiting or reducing the incidence of CRS or NT includes, but is not limited to, ameliorating fever (with or without chills, malaise, fatigue, anorexia, myalgia, joint pain, nausea, vomiting, headache, rash, diarrhea, tachypnea, hypoxemia, hypoxia, shock, cardiovascular tachycardia, pulse broadening, hypotension, capillary leak, increased early cardiac output, decreased late cardiac output, increased D-dimer, hypofibrinogenemia with or without bleeding, azomia, increased transaminase, hyperbilirubinemia, altered mental state, confusion, delirium, loss of speech at rate, hallucinations, tremor, dysdiscrimination, altered gait, seizure, organ failure, or any combination thereof, or any other symptom or characteristic known in the art to be associated with CRS).

In some embodiments, inhibiting or reducing the incidence of CRS comprises reducing the concentration of at least one circulating cytokine. In some embodiments, the cytokine is selected from the group comprising: GM-CSF, IFN gamma, IL-1, IL-15, IL-6, IL-8, IL-10, and IL-2.

In some embodiments, suppressing or reducing the incidence of CRS comprises reducing CRS rank. In some embodiments, inhibiting or reducing the incidence of NT comprises reducing the level of NT. In some embodiments, inhibiting or reducing the incidence of CRS comprises improving the CARTOX-10 score. In some embodiments, inhibiting or reducing the incidence of NT comprises improving the CARTOX-10 score. In some embodiments, inhibiting or reducing the incidence of CRS comprises reducing an increase in intracranial pressure. In some embodiments, inhibiting or reducing the incidence of CRS comprises reducing seizures. In some embodiments, suppressing or reducing the incidence of CRS comprises reducing motor weakness. In some embodiments, inhibiting or reducing the incidence of NT or CRS comprises inhibiting or reducing its incidence to less than 60%. In some embodiments, inhibiting or reducing the incidence of NT or CRS comprises inhibiting or reducing its incidence to less than 50%. In some embodiments, inhibiting or reducing the incidence of NT or CRS comprises inhibiting or reducing its incidence to less than 40%. In some embodiments, inhibiting or reducing the incidence of NT or CRS comprises inhibiting or reducing its incidence to less than 30%. In some embodiments, inhibiting or reducing the incidence of NT or CRS comprises inhibiting or reducing the incidence of less than 20% in a patient. In some embodiments, suppressing or reducing the incidence of NT or CRS comprises eliminating NT or CRS.

In some embodiments, the subject has grade 1 CRS and/or NT. In some embodiments, the subject has grade 2 CRS and/or NT. In some embodiments, the subject has grade 3 CRS and/or NT. In some embodiments, the subject has grade 4 CRS and/or NT. In some embodiments, the subject has any combination of the above.

In some embodiments, suppressing or reducing the incidence of NT or CRS comprises reducing the CRS rating, NT rating, or both. In some embodiments, the grade is reduced to ≦ 3NT and/or CRS in 95% of patients.

In some embodiments, the body temperature of the subject after administration of the immunotherapy is greater than 37 ℃. In some embodiments, the body temperature of the subject after administration of the immunotherapy is greater than 38 ℃. In some embodiments, the body temperature of the subject after administration of the immunotherapy is greater than 39 ℃. In some embodiments, the body temperature of the subject after administration of the immunotherapy is greater than 40 ℃. In some embodiments, the body temperature of the subject after administration of the immunotherapy is greater than 41 ℃. In some embodiments, the body temperature of the subject after administration of the immunotherapy is greater than 42 ℃.

In some embodiments, the subject has a serum concentration of IL-6 greater than 10pg/mL after administration of the immunotherapy. In some embodiments, the subject has a serum concentration of IL-6 greater than 12pg/mL after administration of the immunotherapy. In some embodiments, the subject has a serum concentration of IL-6 greater than 14pg/mL after administration of the immunotherapy. In some embodiments, the subject has a serum concentration of IL-6 greater than 16pg/mL after administration of the immunotherapy. In some embodiments, the subject has a serum concentration of IL-6 greater than 18pg/mL after administration of the immunotherapy. In some embodiments, the subject has a serum concentration of IL-6 greater than 20pg/mL after administration of the immunotherapy. In some embodiments, the subject has a serum concentration of IL-6 greater than 22pg/mL after administration of the immunotherapy.

In some embodiments, the subject has a serum concentration of MCP-1 above 200pg/ml following administration of the immunotherapy. In some embodiments, the subject has a serum concentration of MCP-1 above 400pg/ml after administration of the immunotherapy. In some embodiments, the subject has a serum concentration of MCP-1 above 600pg/ml after administration of the immunotherapy. In some embodiments, the subject has a serum concentration of MCP-1 above 800pg/ml after administration of the immunotherapy. In some embodiments, the subject has a serum concentration of MCP-1 above 1000pg/ml after administration of the immunotherapy. In some embodiments, the subject has a serum concentration of MCP-1 of greater than 1200pg/ml following administration of the immunotherapy. In some embodiments, the subject has a serum concentration of MCP-1 above 1400pg/ml following administration of the immunotherapy. In some embodiments, the subject has a serum concentration of MCP-1 above 1600pg/ml following administration of the immunotherapy. In some embodiments, the subject has a serum concentration of MCP-1 above 1800pg/ml following administration of the immunotherapy. In some embodiments, the subject has a serum concentration of MCP-1 above 2000pg/ml after administration of the immunotherapy.

In some embodiments, the subject has grade 1 CRES. In some embodiments, the subject has grade 2 CRES. In some embodiments, the subject has grade 3 CRES. In some embodiments, the subject has grade 4 CRES.

In some embodiments, the subject is predisposed to having a brain disease, injury, or dysfunction prior to immunotherapy. In some embodiments, the predisposition is genetic. In some embodiments, the trend is obtained. In some embodiments, the trend takes into account existing medical conditions. In some embodiments, the predisposition is diagnosed prior to immunotherapy. In some embodiments, the predisposition is not diagnosed. In some embodiments, the subject is medically evaluated to determine a predisposition to acquire an immunotherapy-related brain disease, injury, or dysfunction prior to the immunotherapy.

In some embodiments, the medical assessment includes determining ANG1 concentration in the bodily fluid. In some embodiments, the medical assessment includes determining ANG1 concentration in serum. In some embodiments, the medical assessment includes determining ANG2 concentration in the bodily fluid. In some embodiments, the medical assessment includes determining ANG2 concentration in serum. In some embodiments, the medical assessment includes calculating an ANG2 to ANG1 ratio in serum. In some embodiments, subjects with a ratio of serum ANG2: ANG1 greater than 0.5 prior to immunotherapy are predisposed to CRES. In some embodiments, subjects with a ratio of serum ANG2: ANG1 greater than 0.7 prior to immunotherapy are predisposed to CRES. In some embodiments, subjects with a ratio of serum ANG2: ANG1 greater than 0.9 prior to immunotherapy are predisposed to CRES. In some embodiments, subjects with a serum ANG2: ANG1 ratio greater than 1 prior to immunotherapy are predisposed to CRES. In some embodiments, subjects with a ratio of serum ANG2: ANG1 greater than 1.1 prior to immunotherapy are predisposed to CRES. In some embodiments, subjects with a ratio of serum ANG2: ANG1 greater than 1.3 prior to immunotherapy are predisposed to CRES. In some embodiments, subjects with a ratio of serum ANG2: ANG1 greater than 1.5 prior to immunotherapy are predisposed to CRES.

In some embodiments, the immunotherapy-related toxicity comprises phagocytic lymphocytosis (HLH). In some embodiments, the immunotherapy-related toxicity comprises Macrophage Activation Syndrome (MAS). In some embodiments, provided herein are methods for inhibiting or reducing the incidence of HLH. In some embodiments, provided herein are methods for inhibiting or reducing the incidence of MAS.

In some embodiments, inhibiting or reducing the incidence of HLH comprises increasing survival in the subject. In some embodiments, inhibiting or reducing the incidence of HLH comprises increasing the time to relapse. In some embodiments, inhibiting or reducing the incidence of MAS comprises increasing survival in the subject. In some embodiments, inhibiting or reducing the incidence of MAS comprises increasing the time to relapse.

In some embodiments, inhibiting or reducing the incidence of HLH or MAS comprises inhibiting macrophage activation and/or proliferation. In some embodiments, inhibiting or reducing the incidence of HLH or MAS comprises inhibiting T lymphocyte activation and/or proliferation. In some embodiments, inhibiting or reducing the incidence of HLH or MAS comprises reducing the concentration of circulating IFN γ. In some embodiments, inhibiting or reducing the incidence of HLH or MAS comprises reducing the circulating concentration of GM-CSF.

In some embodiments, the subject exhibits fever after immunotherapy. In some embodiments, the subject exhibits splenomegaly following immunotherapy. In some embodiments, the subject exhibits a hemorrhagic cytopenia following immunotherapy. In some embodiments, the subject exhibits a hemorrhagic cytopenia in two or more cell lines following immunotherapy. In some embodiments, the subject exhibits hypertriglyceridemia after immunotherapy. In some embodiments, the subject exhibits hypofibrinogenemia following immunotherapy. In some embodiments, the subject exhibits erythrophagy following immunotherapy. In some embodiments, erythrophagy is observed in the bone marrow. In some embodiments, the subject exhibits low NK cell activity following immunotherapy. In some embodiments, the subject exhibits a lack of NK activity following immunotherapy.

In some embodiments, the subject exhibits a serum concentration of ferritin above 100U/ml following immunotherapy. In some embodiments, the subject exhibits a serum concentration of ferritin above 300U/ml following immunotherapy. In some embodiments, the subject exhibits a serum concentration of ferritin above 500U/ml following immunotherapy. In some embodiments, the subject exhibits a serum concentration of ferritin above 700U/ml following immunotherapy. In some embodiments, the subject exhibits a serum concentration of ferritin above 900U/ml following immunotherapy.

In some embodiments, the subject exhibits a serum concentration of soluble CD25 above 1200U/ml following immunotherapy. In some embodiments, the subject exhibits a serum concentration of soluble CD25 above 1500U/ml after immunotherapy. In some embodiments, the subject exhibits a serum concentration of soluble CD25 above 1800U/ml after immunotherapy. In some embodiments, the subject exhibits a serum concentration of soluble CD25 above 2000U/ml after immunotherapy. In some embodiments, the subject exhibits a serum concentration of soluble CD25 above 2200U/ml after immunotherapy. In some embodiments, the subject exhibits a serum concentration of soluble CD25 above 2400U/ml after immunotherapy. In some embodiments, the subject exhibits a serum concentration of soluble CD25 above 2700U/ml after immunotherapy. In some embodiments, the subject exhibits a serum concentration of soluble CD25 above 3000U/ml following immunotherapy.

In some embodiments, the subject is predisposed to have HLH. In some embodiments, the predisposition is genetic. In some embodiments, the trend takes into account existing medical conditions. The skilled person will appreciate that sporadic HLH has been associated with many genetic mutations. In some embodiments, the subject carries a mutation in a gene selected from the group consisting of: PRF1, UNC13D, STX11, STXBP2, or RAB27A, or any combination thereof. In some embodiments, the subject has reduced or absent perforin expression.

hGM-CSF antagonists

Antagonists of hGM-CSF suitable for use selectively interfere with the induction of signaling by the hGM-CSF receptor by causing a decrease in the binding of hGM-CSF to the receptor. Such antagonists may comprise antibodies that bind to the hGM-CSF receptor, antibodies that bind to hGM-CSF, GM-CSF analogs (e.g., E21R), and other proteins or small molecules that compete with hGM-CSF for binding to its receptor or inhibit signaling normally caused by ligand binding to the receptor.

In many embodiments, the hGM-CSF antagonist used in the invention is a polypeptide, such as an anti-hGM-CSF antibody, an anti-hGM-CSF receptor antibody, a soluble hGM-CSF receptor, or a modified GM-CSF polypeptide, that competes with hGM-CSF for binding to the receptor, but is inactive. Such proteins are typically produced using recombinant expression techniques. Such methods are well known in the art. General molecular biological methods, including expression methods, can be found, for example, in instruction manuals, such as Sambrook and Russell (2001), "molecular cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 3 rd edition; current protocols in molecular biology (2006), John Wiley parent-child publishing company (John Wiley and Sons), ISBN: 0-471-.

Various prokaryotic and/or eukaryotic based protein expression systems can be used to produce hGM-CSF antagonist proteins. Many such systems are widely available from commercial suppliers.

hGM-CSF antibodies

The hGM-CSF antibodies of the invention are antibodies that bind with high affinity to hGM-CSF and are antagonists of hGM-CSF. Antibodies include those related to human germline V_HAnd V_LVariable regions of high sequence identity. In a preferred embodiment, the BSD sequence in CDRH3 of the antibody of the invention comprises the amino acid sequence RQRFPY or RDRFPY. The BSD in CDRL3 includes FNK or FNR.

A complete V region was generated in which the BSD formed part of CDR3, and additional sequences were used to complete CDR3 and add the FR4 sequence. Typically, the portion of CDR3 that does not contain a BSD and the complete FR4 consists of human germline sequences. In some embodiments, the CDR3-FR4 sequence that does not include a BSD differs from human germline sequences by no more than 2 amino acids per chain. In some embodiments, the J segment comprises a human germline J segment. Human germline sequences can be determined, for example, by the publicly available international immunegenetics database (IMGT) and V-base (on the world wide web vbase. mrc-cpe. cam. ac. uk).

The human germline V-segment repertoire consists of 51 heavy chain V-regions, 40K light chain V-segments and 31 λ light chain V-segments, forming a total of 3,621 germline V-region pairs, and in addition, there are a majority of stable allelic variants of these V-segments, but these variants have limited contribution to the structural diversity of the germline repertoire. The sequences of all human germline V segment genes are known and can be accessed in the V-base database provided by the MRC Protein Engineering center of Cambridge, UK (MRC Centre for Protein Engineering, Cambridge, United Kingdom) (see also Chothia et al, 1992, J. mol. biol. 227: 776-798; Tomlinson et al, 1995; EMBO J. 14: 4628-4638; and Williams et al, 1996, J. mol. biol. 264: 220-232).

The antibodies or antibody fragments as described herein may be expressed in prokaryotic or eukaryotic microbial systems or in cells of higher eukaryotes (e.g., mammalian cells).

The antibodies used in the present invention may be in any form. For example, in some embodiments, the antibody can be an intact antibody comprising constant regions, e.g., human constant regions, or can be an intact antibody, e.g., Fd, Fab ', F (ab')₂scFv, Fv fragments or single domain antibodies such as nanobodies or fragments or derivatives of camelid antibodies. Such antibodies may additionally be recombinantly engineered by methods well known to those skilled in the art. As described above, such antibodies can be produced using known techniques.

In some embodiments, the hGM-CSF antagonist is an antibody that binds to hGM-CSF or to an α or β subunit of hGM-CSF receptor. Antibodies can be raised against hGM-CSF (or hGM-CSF receptor) proteins or fragments, or recombinantly produced. Antibodies directed to GM-CSF for use in the present invention may be neutralizing antibodies or may be non-neutralizing antibodies that bind to GM-CSF and increase the rate of clearance of hGM-CSF in vivo, thereby reducing circulating hGM-CSF levels. The hGM-CSF antibody is typically a neutralizing antibody.

Methods for preparing polyclonal Antibodies are known to the skilled worker (e.g., Harlow and Lane, Antibodies, A Laboratory Manual (1988), Methods in Immunology (Methods in Immunology)). Polyclonal antibodies can be raised in a mammal by one or more injections of an immunizing agent and, if desired, an adjuvant. The immunizing agent comprises GM-CSF or a GM-CSF receptor protein (e.g., human GM-CSF or GM-CSF receptor protein), or a fragment thereof.

In some embodiments, the GM-CSF antibodies used in the invention are purified from human plasma. In such embodiments, the GM-CSF antibody is typically a polyclonal antibody that is isolated from other antibodies present in human plasma. Such a separation procedure may be performed, for example, using known techniques such as affinity chromatography.

In some embodiments, the GM-CSF antagonist is a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal, is typically immunized with an immunizing agent, such as human GM-CSF, to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro. The immunizing agent preferably comprises human GM-CSF protein, a fragment thereof, or a fusion protein thereof.

Human monoclonal antibodies can be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. mol. biol. 227:381 (1991); Marks et al, J. mol. biol. 222:581 (1991)). The techniques of Cole et al and Boerner et al can also be used to prepare human Monoclonal Antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy (Monoclonal Antibodies and Cancer Therapy), 77(1985), and Boerner et al, J. Immunol. 147(1):86-95 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated. After challenge, human antibody production was observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and repertoire of antibodies. For example, in U.S. patent nos. 5,545,807; nos. 5,545,806; U.S. Pat. No. 5,569,825; 5,625,126 No; 5,633,425 No; 5,661,016, and in the following scientific publications: marks et al, Biotechnology (Bio/Technology) 10:779-783 (1992); lonberg et al, Nature 368:856-859 (1994); morrison, Nature 368:812-13 (1994); fishwild et al, Nature Biotechnology (Nature Biotechnology) 14:845-51 (1996); neuberger, Nature Biotechnology 14:826 (1996); lonberg and Huszar, International immunologic review (Intern.Rev.Immunol.) 13:65-93 (1995).

In some embodiments, the anti-GM-CSF antibody is a chimeric antibody or a humanized monoclonal antibody. As described above, humanized forms of antibodies are chimeric immunoglobulins in which residues from a Complementarity Determining Region (CDR) of a human antibody are substituted with CDR residues from a non-human species (e.g., mouse, rat, or rabbit) having the desired specificity, affinity, and capacity.

In some embodiments of the invention, the antibody is additionally engineered to reduce immunogenicity, e.g., to render the antibody suitable for repeated administration. Methods of generating antibodies with reduced immunogenicity include humanization/human engineering procedures and modification techniques, such as deimmunization, in which antibodies are further engineered in, for example, one or more framework regions to remove T cell epitopes.

In some embodiments, the antibody is a human engineered antibody. The human engineered antibody is an engineered human antibody having the binding specificity of the reference antibody obtained by linking a DNA sequence encoding a Binding Specificity Determinant (BSD) from a CDR3 region of a heavy chain of the reference antibody to a human VH segment sequence and a DNA sequence encoding a light chain CDR3 BSD from the reference antibody to a human VL segment sequence. Methods for human engineering are provided in U.S. patent application publication No. 20050255552 and U.S. patent application publication No. 20060134098. In U.S. patent application 20070020685, a method for the signal-free secretion of antibody fragments from E.coli is described.

The antibody may be further de-immunized to remove one or more predicted T cell epitopes from the V region of the antibody. Such a procedure is described, for example, in WO 00/34317.

The heavy chain constant region is typically a gamma chain constant region, such as a gamma-1, gamma-2, gamma-3 or gamma-4 constant region. In some embodiments, for example where the antibody is a fragment, the antibody may be conjugated to another molecule, for example to provide an extended in vivo half-life, such as polyethylene glycol (pegylation) or serum albumin. In Knight et al (2004) Platelets (Platelets) 15:409 (for abciximab); pedley et al (1994) J.Cancer in British cancer 70:1126 (for anti-CEA antibodies); an example of pegylation of an antibody fragment is provided in Chapman et al (1999) Nature Biotechnology 17: 780.

The antibodies used in the present invention bind to hGM-CSF or the hGM-CSF receptor. Antibody binding specificity can be determined using a variety of techniques. See, e.g., Harlow and Lane, antibodies, A laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine the specific immunoreactivity of an antibody.

An exemplary antibody suitable for use in the present invention is c19/2 (mouse/human chimeric anti-hGM-CSF antibody). In some embodiments, monoclonal antibodies that compete with c19/2 for binding to the same epitope or with c19/2 for binding to the same epitope are used. The ability of a particular antibody to recognize the same epitope as another antibody is typically determined by the ability of the first antibody to competitively inhibit the binding of the second antibody to the antigen. Any of a variety of competitive binding assays can be used to measure competition between two antibodies directed against the same antigen. For example, a sandwich ELISA assay can be used for this purpose. This is done by coating the surface of the wells with a capture antibody. A sub-saturating concentration of labeled antigen is then added to the capture surface. Such proteins will bind to the antibody through specific antibody-epitope interactions. After washing, a second antibody that has been covalently linked to a detectable moiety (e.g., HRP, labeled antibody is defined as the detection antibody) is added to the ELISA. If this antibody recognizes the same epitope as the capture antibody, it will not bind to the target protein because the particular epitope will no longer be available for binding. However, if this second antibody recognizes a different epitope on the target protein, it will be able to bind, and this binding can be detected by quantifying the level of activity (and thus the bound antibody) using a relevant substrate. The background is defined by using a single antibody as both the capture and detection antibodies, while the maximum signal can be established by capture with an antigen-specific antibody and detection with an antibody directed against a label on the antigen. By using the background and maximum signal as references, antibodies can be evaluated in pairs to determine epitope specificity.

A first antibody is considered to competitively inhibit binding of a second antibody in the presence of the first antibody using any of the assays described above if the binding of the second antibody to the antigen is reduced by at least 30%, typically at least about 40%, 50%, 60% or 75%, and typically by at least about 90%.

In some embodiments of the invention, antibodies are used that compete for binding to the same epitope as a known antibody, e.g., c19/2, or to the same epitope as a known antibody. Methods of mapping epitopes are well known in the art. For example, one method of locating a functionally active region of human granulocyte-macrophage colony stimulating factor (hGM-CSF) is to map an epitope recognized by a neutralizing anti-hGM-CSF monoclonal antibody. For example, the epitope that binds to c19/2 (having the same variable region as the neutralizing antibody LMM 102) has been defined using a proteolytic fragment obtained by enzymatic digestion of bacterial-synthesized hGM-CSF (Dempsey et al, Hybridoma (Hybridoma) 9:545-558, 1990). RP-HPLC fractionation of the trypsin digest resulted in the identification of an immunoreactive "trypsin core" peptide containing 66 amino acids (52% of the protein). Further digestion of this "trypsin core" with S.aureus (S.aureus) V8 protease produced a unique immunoreactive hGM-CSF product comprising two peptides, residues 86-93 and 112-127, linked by a disulfide bond between residues 88 and 121. Antibodies do not recognize a single peptide.

In some embodiments, antibodies suitable for use in the present invention have high affinity binding to human GM-CSF or hGM-CSF receptor. If the dissociation constant (KD) of the antibody<About 10nM, typically<1nM and preferably<100pM, there is high affinity binding between the antibody and the antigen. In some embodiments, the off-rate of the antibody is about 10^-4Every second or higher.

As is well known to those skilled in the art, the binding affinity of an antibody to its target antigen can be determined using a variety of methods, such as surface plasmon resonance assays, saturation assays, or immunoassays, such as ELISA or RIA. An exemplary method of determining binding affinity is by in BIAcore^TMSurface plasmon resonance analysis using a CM5 sensor chip on a 2000 instrument (Biacore AB, fleabag, germany), such as Krinner et al, (2007) analytical february immunology (moi. immunol. feb); 44(5) 916-25. (5.H.2006 electronic publication)).

In some embodiments, the hGM-CSF antagonist neutralizes the antibody hGM-CSF, its receptor, or its receptor subunit in a manner that interferes with the binding of hGM-CSF to its receptor or receptor subunit. In some embodiments, an anti-hGM-CSF antibody for use in the invention inhibits binding to the alpha subunit of the hGM-CSF receptor. Such antibodies can bind to hGM-CSF, for example, at the region where hGM-CSF binds to the receptor, thereby inhibiting binding. In another embodiment, the anti-hGM-CSF antibody inhibits hGM-CSF function without blocking its binding to the α subunit of hGM-CSF receptor.

Heavy chain II

The heavy chain of the anti-hGM-CSF antibody of the present invention comprises a heavy chain V region comprising the following elements:

1) a human heavy chain V segment sequence comprising FR1-CDR1-FR2-CDR2-FR 3;

2) the CDRH3 region comprising the amino acid sequence R (Q/D) RFPY;

3) FR4 contributed by a human germline J gene segment.

In FIG. 1 is shown the support of the CDR3-FR4 segment and complementary V_LRegion combinations with hGM-CSF binding V segment sequences. The V segment may be, for example, from the human VH1 subclass. In some embodiments, V segment is human V_HA subclass 1 segment having a high degree of amino acid sequence identity, e.g., at least 80%, 85% or 90% or more identity, to the germline segment VH 11-02 or VH 11-03. In some embodiments, the V segment differs from VH 11-02 or VH 11-03 by no more than 15 residues and preferably no more than 7 residues.

The FR4 sequence of the antibody of the invention is provided by a human JH1, JH3, JH4, JH5 or JH6 gene germline segment, or a sequence with high amino acid sequence identity to a human germline JH segment. In some embodiments, the J segment is a human germline JH4 sequence.

CDRH3 also includes sequences derived from human J segments. Typically, the CDRH3-FR4 sequence that does not contain a BSD differs from the human germline J segment by no more than 2 amino acids. In typical embodiments, the J segment sequences in CDRH3 are from the same J segment used for FR4 sequences. Thus, in some embodiments, the CDRH3-FR4 region includes the BSD and the intact human JH4 germline gene segment. An exemplary combination of CDRH3 and FR4 sequences is shown below, where BSD is shown in bold and human germline J segment JH4 residues are underlined:

CDR3.

In some embodiments, the antibodies of the invention comprise a heavy chain variable region that binds to germline segment VH 1-02 or VH 1-03; or V as shown in FIG. 1_HOne of the V segments of a region, e.g., a V segment portion of VH #1, VH #2, VH #3, VH #4, or VH #5, is a V segment that is at least 90% identical or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, V_HThe V segments of the regions have CDR1 and/or CDR2 as shown in fig. 1. For example, an antibody of the invention may have CDR1 with the sequence GYYMH or NYYIH; or a CDR2 having the sequence WINPNSGGTNYAQKFQG or WINAGNGNTKYSQKFQG.

In particular embodiments, the antibody has a V from that shown in figure 1_HBoth CDR1 and CDR2 of one of the region V segments and CDR3 including R (Q/D) RFPY, e.g., RDRFPYYFDY or RQRFPYYFDY. Thus, in some embodiments, an anti-GM-CSF antibody of the invention may, for example, have CDR3-FR4 and CDR1 and/or CDR2 comprising the sequence R (Q/D) RFPYYFDYWGQGTLVTVSS, as shown in fig. 1.

In some embodiments, the V of an antibody of the invention_HThe regions have a CDR3 containing the binding specificity determinant R (Q/D) RFPY, a CDR2 from a human germline VH1 segment, or a CDR1 from a human germline VH 1. In some embodiments, both CDR1 and CDR2 are from a human germline VH1 segment.

Light chain

The light chain of the anti-hGM-CSF antibody of the present invention is comprised in the light chain V region, which comprises the following elements:

1) a human light chain V segment sequence comprising FR1-CDR1-FR2-CDR2-FR 3;

2) including the CDRL3 region of sequence FNK or FNR, e.g. QQFNRSPLT or QQFNKSPLT.

3) FR4 contributed by a human germline J gene segment.

V_LRegions include V.lambda.or V.kappa.V segments. In the figure1, examples of V.kappa.sequences supporting a complementary V_HThe zones are combined.

V_LThe region CDR3 sequence includes J segment derived sequences. In typical embodiments, the J segment sequence in CDRL3 is from the same J segment used for FR 4. Thus, in some embodiments, the sequences may differ from the human kappa germline V segment and J segment sequences by no more than 2 amino acids. In some embodiments, the CDRL3-FR4 region includes BSD and the entire human JK4 germline gene segment. An exemplary CDRL3-FR4 combination of kappa chains is shown below, with the minimum essential binding specificity determinant shown in bold and the JK4 sequence underlined:

CDR3

the vk segment usually belongs to the VKIII subclass. In some embodiments, the segment has at least 80% sequence identity to the human germline VKIII subclass, e.g., at least 80% identity to the human germline VKIIIA27 sequence. In some embodiments, the vk segment may differ from VKIIIA27 by no more than 18 residues. In other embodiments, the V of the antibodies of the invention _LZone V segment and V shown in FIG. 1_LThe human kappa V segment sequences of a region, e.g., VK #1, VK #2, VK #3, or VK #4, have at least 85% identity or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

In some embodiments, the variable region consists of a human V gene sequence. For example, the variable region sequence may be at least 80% identical or at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical or greater to the human germline V gene sequence.

In some embodiments, V_LThe V segments of the regions have CDR1 and/or CDR2 as shown in fig. 1. For example, an antibody of the invention may have a CDR1 sequence of RASQSVGTNVA or RASQSIGSNLA; or CDR2 sequenceSTSSRAT。

In particular embodiments, an anti-GM-CSF antibody of the invention may have a V as shown in FIG. 1_LThe combined CDR1 and CDR2 and CDR3 sequence including FNK or FNR shown in one of the V segments of the region, e.g., CDR3 can be QQFNKSPLT or QQFNRSPLT. In some embodiments, such GM-CSF antibodies may include the FR4 region that is FGGGTKVEIK. Thus, an anti-GM-CSF antibody of the invention may include, for example, a V from that shown in FIG. 1 _LBoth CDR1 and CDR2 of one of the regions and the CDR3-FR4 region that is FGGGTKVEIK.

Preparation of hGM-CSF antibody

The antibodies of the invention may comprise a V as shown in FIG. 1_HAny one of the regions VH #1, VH #2, VH #3, VH #4 or VH # 5. In some embodiments, an antibody of the invention may comprise a V as shown in figure 1_LAny one of the regions VK #1, VK #2, VK #3 or VK # 4. In some embodiments, the antibody has a V as shown in figure 1_HThe region VH #1, VH #2, VH #3, VH #4 or VH # 5; and V as shown in FIG. 1_LThe regions VK #1, VK #2, VK #3, or VK #4, as described, for example, in U.S. patent nos. 8,168,183 and 9,017,674, each of which is incorporated herein by reference in its entirety.

The antibody can be tested to confirm that the antibody retains activity against hGM-CSF activity. Any number of endpoints can be used to determine antagonist activity, including proliferation assays. Neutralizing antibodies and other hGM-CSF antagonists can be identified or evaluated using a number of assays for assessing hGM-CSF function. For example, cell-based assays for hGM-CSF receptor signaling are conveniently used, such as assays to determine the rate of proliferation of hGM-CSF-dependent cell lines in response to limited amounts of hGM-CSF. The human TF-1 cell line is suitable for this assay. See, Krinner et al, (2007) molecular immunology (mol. In some embodiments, the neutralizing antibodies of the invention inhibit hGM-CSF-stimulated TF-1 cell proliferation by at least 50% when using a concentration of hGM-CSF that stimulates 90% of maximal TF-1 cell proliferation. Thus, in general, the neutralizing antibodies or other hGM-CSF antagonists of the invention have an EC50 of less than 10nM (e.g., table 2). Additional assays suitable for identifying neutralizing antibodies suitable for use in the present invention are well known to those skilled in the art. In other embodiments, the neutralizing antibody inhibits hGM-CSF-stimulated proliferation by at least about 75%, 80%, 90%, 95%, or 100% of the antagonist activity of antibody chimera c19/2 (e.g., WO03/068920) having the variable regions of the mouse monoclonal antibody LMM102 and CDRs.

An exemplary chimeric antibody suitable for use as an antagonist of hGM-CSF is c 19/2. The c19/2 antibody bound hGM-CSF with a monovalent binding affinity of about 10pM as determined by surface plasmon resonance analysis. The variable region sequences of the heavy and light chains of the c19/2 antibody are known (e.g., WO 03/068920). The CDRs defined according to Kabat are:

CDRs can also be determined using other definitions well known in the art, such as Chothia, international imminogenetics database (IMGT), and AbM.

In some embodiments, the antibodies used in the present invention compete with c19/2 for binding to the same epitope or with c19/2 for binding to the same epitope. The GM-CSF epitope recognized by c19/2 has been identified as a product having two peptides linked by a disulfide bond between residues 88 and 121, residues 86-93 and residue 112-127. The c19/2 antibody inhibited GM-CSF-dependent proliferation of human TF-1 leukemia cell line with an EC50 of 30pM when cells were stimulated with 0.5ng/ml GM-CSF. In some embodiments, the antibodies used in the present invention bind the same epitope as c 19/2.

Antibodies for administration, such as c19/2, may additionally be engineered. For example, the c19/2 antibody can be further engineered to contain human V gene segments.

High affinity antibodies can be identified using well known assays to determine binding activity and affinity. Such techniques include ELISA assays as well as binding assays using surface plasmon resonance or interferometry. For example, affinity can be determined by biolayer interferometry using ForteBio (mountain view, ca) Octet biosensors. The antibodies of the invention typically bind to both glycosylated and non-glycosylated forms of hGM-CSF with similar affinity.

The antibodies of the invention compete with the c19/2 antibody for binding to hGM-CSF. The ability of the antibodies described herein to block the c19/2 antibody or compete with the c19/2 antibody for binding to hGM-CSF indicates that the antibodies bind to the same epitope as the c19/2 antibody or to an epitope that is close to, e.g., overlapping, the epitope bound by the c19/2 antibody. In other embodiments, the antibodies described herein, for example, comprise V as shown in the table provided in figure 1_HAnd V_LThe antibody of the domain combination can be used as a reference antibody for assessing whether another antibody competes for binding to hGM-CSF. In the presence of the test antibody, the test antibody is considered to competitively inhibit binding of the reference antibody if binding of the reference antibody to the antigen is reduced by at least 30%, typically at least about 40%, 50%, 60% or 75%, and often by at least about 90%. A number of assays can be used to assess binding, including ELISA, as well as other assays, such as immunoblots. In some embodiments, the antibodies of the invention have an off-rate that is at least 2 to 3 fold slower than a reference chimeric c19/2 monoclonal antibody determined under the same conditions, but are at least 6-10 fold more potent than a reference antibody in neutralizing hGM-CSF activity in a cell-based assay that measures hGM-CSF activity.

Methods for isolating antibodies having V region sequences close to human germline sequences have been previously described (U.S. patent application publication nos. 20050255552 and 20060134098). The antibody library may be expressed in a suitable host cell, including mammalian cells, yeast cells, or prokaryotic cells. For expression in some cell systems, a signal peptide may be introduced at the N-terminus to direct secretion into the extracellular medium. The antibody may be secreted from bacterial cells (e.g.E.coli) with or without a signal peptide. In U.S. patent application 20070020685, a method for the signal-free secretion of antibody fragments from E.coli is described.

In some embodiments of the present invention, the,the hGM-CSF binding antibodies of the invention are generated wherein an antibody having CDRs from one of the VH regions of the invention shown in figure 1 is combined with an antibody having CDRs from one of the VL regions shown in figure 1 and expressed in any of a variety of forms in a suitable expression system. Thus, antibodies may be expressed as scFv, Fab '(containing immunoglobulin hinge sequences), F (ab')₂(formed by disulfide bonds between the hinge sequences of the two Fab' molecules), intact or truncated immunoglobulins, or expressed as fusion proteins (either inside the host cell or by secretion) in prokaryotic or eukaryotic host cells. Methionine residues may optionally be present at the N-terminus, for example in polypeptides produced in a signal-free expression system. Each V described herein _HThe region may be associated with each V_LThe regions are paired to generate anti-hGM-CSF antibodies. In one embodiment, the fusion protein comprises an anti-hGM-CSF binding antibody or fragment thereof of the invention (in a non-limiting example, the anti-hGM-CSF antibody fragment is a Fab, Fab ', F (ab')2, scFv, or dAB) and human transferrin, wherein the human transferrin is in heavy chain constant region 1 (C)_H1) End, hinged back or C _H3 followed by fusion with an antibody, as described in Shin, S-U.S. et al, Proc. Natl. Acad. Sci. USA, Vol. 92, p. 2820-2824, 1995, which is incorporated herein by reference in its entirety.

Exemplary combinations of heavy and light chains are shown in the table provided in fig. 1. In some embodiments, an antibody VL region (e.g., VK #1, VK #2, VK #3, or VK #4 of fig. 1) is combined with a human kappa constant region to form a complete light chain. Further, in some embodiments, the VH region is combined with a human γ -1 constant region. Any suitable gamma-1 allotype, such as the f-allotype, may be selected. Thus, in some embodiments, the antibody is, for example, an IgG having an f-allotype with a VH selected from VH #1, VH #2, VH #3, VH #4, or VH #5 (fig. 1), and a VL selected from VK #1, VK #2, VK #3, or VK #4 (fig. 1).

The antibodies of the invention inhibit the activation of the hGM-CSF receptor, e.g., by inhibiting the binding of hGM-CSF to the receptor, and exhibit high affinity binding to hGM-CSF, e.g., 500 pM. In some embodiments, the antibody has a dissociation constant of about 10^-4Per second or less. Without being bound by theory, antibodies with slower dissociation constants may provide improved therapeutic benefits. For example, an antibody of the invention with an off-rate three times slower than the c19/2 antibody produced hGM-CSF neutralizing activity 10-fold more effective, e.g., in cell-based assays, such as IL-8 production (see, e.g., example 2).

Antibodies can be produced using a number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell expression, such as a CHO cell expression system. Many such systems are widely available from commercial suppliers. In that the antibody comprises V_HAnd V_LIn the example of both zones, V_HAnd V_LThe regions may be expressed using a single vector, for example in a bicistronic expression unit or under the control of different promoters. In other embodiments, V may be expressed using a separate vector_HAnd V_LAnd (4) a zone. V as described herein_HOr V_LThe region may optionally include a methionine at the N-terminus.

The antibodies of the invention can be produced in a variety of forms, including Fab, Fab ', F (ab')₂scFv or dAB. The antibodies of the invention may also comprise human constant regions. The constant region of the light chain may be a human kappa or lambda constant region. The heavy chain constant region is typically a gamma chain constant region, such as a gamma-1, gamma-2, gamma-3 or gamma-4 constant region. In other embodiments, the antibody may be IgA.

In some embodiments of the invention, antibody V_LRegions (e.g., VK #1, VK #2, VK #3, or VK #4 of FIG. 1) are combined with human kappa constant regions (e.g., SEQ ID NO:10) to form complete light chains.

In some embodiments of the invention, V_HThe regions are combined with human gamma-1 constant regions. Any suitable gamma-1 f allotype, such as the f-allotype, may be selected. Thus, in some embodiments, the antibody is an IgG having an f-allotype constant region, e.g., SEQ ID NO 11, having a V selected from VH #1, VH #2, VH #3, VH #4, or VH #5 (FIG. 1)_H. In some embodiments, the antibody has a V selected from VK #1, VK #2, VK #3, or VK #4_L(FIG. 1). In particular embodiments, the antibody has the amino acid sequence set forth in SEQ ID NO 10A kappa constant region as shown, and a heavy chain constant region as shown in SEQ ID NO:11, wherein the heavy and light chain variable regions comprise one of the following combinations from the sequences shown in FIG. 1: a) VH #2, VK # 3; b) VH #1, VK # 3; c) VH #3, VK # 1; d) VH #3, VL # 3; e) VH #4, VK # 4; f) VH #4, VK # 2; g) VH #5, VK # 1; h) VH #5, VK # 2; i) VH #3, VK # 4; or j) VH #3, VL # 3).

In some embodiments, for example where the antibody is a fragment, the antibody may be conjugated to another molecule, such as polyethylene glycol (pegylation) or serum albumin, to provide an extended half-life in vivo. Examples of pegylation of antibody fragments are provided in the following references: knight et al, platelets 15:409,2004 (for abciximab); pedley et al, J.K. cancer 70:1126,1994 (for anti-CEA antibodies); chapman et al, Nature Biotechnology 17:780,1999; and Humphreys et al, Protein engineering, Des 20:227,2007.

In some embodiments, the antibodies of the invention are in the form of Fab' fragments. Full-length light chain is prepared by mixing V_LThe regions are generated by fusion to human kappa or lambda constant regions. Any constant region can be used for any light chain; however, in typical embodiments, a kappa constant region is used in combination with a vk variable region, and a lambda constant region is used in combination with a vk variable region.

The heavy chain of Fab' is prepared by mixing the V of the invention_HThe Fd' fragment resulting from the fusion of the region with the human heavy chain constant region sequence, the first constant (CH1) domain and the hinge region. The heavy chain constant region sequence may be from any immunoglobulin class, but is typically from IgG, and may be from IgG1, IgG2, IgG3, or IgG 4. The Fab' antibodies of the invention may also be hybrid sequences, e.g., the hinge sequence may be from one immunoglobulin subclass and the CH1 domain may be from a different subclass.

Administering an anti-hGM-CSF antibody to treat a disease in which GM-CSF is a target.

The invention also provides methods of treating a patient having a disease involving hGM-CSF, wherein inhibition of hGM-CSF activity is desired, i.e., wherein hGM-CSF is the therapeutic target. In some embodiments, such patients have chronic inflammatory diseases, such as arthritis, e.g., rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, systemic-onset Still's disease, and other inflammatory diseases of the joints; inflammatory bowel diseases, such as ulcerative colitis, Crohn's disease, Barrett's syndrome, ileitis, enteritis, eosinophilic esophagitis, and gluten-sensitive bowel disease; inflammatory diseases of the respiratory system such as asthma, eosinophilic asthma, adult respiratory distress syndrome, allergic rhinitis, silicosis, chronic obstructive pulmonary disease, allergic pulmonary disease, interstitial lung disease, diffuse parenchymal lung disease, bronchiectasis; inflammatory diseases of the skin including psoriasis, scleroderma and inflammatory dermatoses such as eczema, atopic dermatitis, urticaria and pruritus; diseases involving inflammation of the central and peripheral nervous system include multiple sclerosis, idiopathic demyelinating polyneuropathy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, neurofibromatosis, and neurodegenerative diseases such as Alzheimer's disease. Various other inflammatory diseases may be treated using the methods of the present invention. These diseases include systemic lupus erythematosus, immune-mediated kidney diseases such as glomerulonephritis and spondyloarthropathies; and diseases with undesirable chronic inflammatory components, such as systemic sclerosis, idiopathic inflammatory myopathy, Sjogren's syndrome, vasculitis, sarcoidosis, thyroiditis, gout, otitis, conjunctivitis, sinusitis, sarcoidosis, Behcet's syndrome, autoimmune lymphoproliferative syndrome (or ALPS, also known as canarle-Smith syndrome), Ras-related autoimmune leukoproliferative disorder (or RALD), Noonan syndrome, hepatobiliary diseases, such as hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis. In some embodiments, the patient has inflammation following injury to the cardiovascular system. Other inflammatory diseases include Kawasaki's Disease, multicenter Castleman's Disease, tuberculosis, and chronic cholecystitis. Additional chronic inflammatory diseases are described, for example, in Harrison's Principles of Internal Medicine, 12 th edition, edited by Wilson et al, McGraw-Hill, Inc. In some embodiments, the patient treated with the antibody has a cancer in which GM-CSF contributes to tumor or cancer cell growth, including, but not limited to, for example, acute myelogenous leukemia, plexiform neurofibroma, autoimmune lymphoproliferative syndrome (or ALPS, also known as kaneller-smith syndrome), Ras-associated autoimmune leukoproliferative disorder (or RALD), noonan's syndrome, chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, and acute myelogenous leukemia. In some embodiments, a patient treated with an antibody of the invention has or is at risk of heart failure, for example due to ischemic injury to the cardiovascular system, such as ischemic heart disease, stroke, and atherosclerosis. In some embodiments, a patient treated with an antibody of the invention has asthma. In some embodiments, a patient treated with an antibody of the invention has alzheimer's disease. In some embodiments, a patient treated with an antibody of the invention has a reduction in bone mass, e.g., osteoporosis. In some embodiments, a patient treated with an antibody of the invention has thrombocytopenic purpura. In some embodiments, the patient has type I or type II diabetes. In some embodiments, the patient may have more than one disease in which GM-CSF is a therapeutic target, e.g., the patient may have rheumatoid arthritis and heart failure or osteoporosis and rheumatoid arthritis, etc.

Two other examples of neutralizing anti-GM-CSF antibodies are the human E10 antibody and the human G9 antibody described in Li et al, (2006) PNAS 103(10): 3557-3562. E10 and G9 are IgG class antibodies. E10 has a binding affinity of 870pM for GM-CSF, while G9 has an affinity of 14pM for GM-CSF. Both antibodies were specific for binding to human GM-CSF and showed strong neutralizing activity as assessed by the TFl cell proliferation assay.

An additional exemplary neutralizing anti-GM-CSF antibody is the MT203 antibody described by Krinner et al (molecular immunology 44:916-25,2007; electronic publication 2006, 5-month 112006). MT203 is a class IgG1 antibody that binds to GM-CSF with picomolar affinity. The antibodies showed potent inhibitory activity and their ability to block IL-8 production in U937 cells as assessed by TF-1 cell proliferation assay.

Additional antibodies suitable for use in the present invention are known to those skilled in the art.

An antagonist of hGM-CSF that is an antibody to hGM-CSF receptor may also be employed with the methods of the present disclosure. Such hGM-CSF antagonists comprise antibodies directed against the alpha or beta chain of the hGM-CSF receptor. The anti-hGM-CSF receptor antibody for use in the present invention may be in any antibody form as explained above, e.g. intact, chimeric, monoclonal, polyclonal, antibody fragments, humanized, human engineered etc. Examples of anti-hGM-CSF receptor antibodies suitable for use in the invention, such as neutralizing high affinity antibodies, are known (see, e.g., U.S. Pat. No. 5,747,032 and Nicola et al, blood 82:1724,1993).

Non-antibody GM-CSF antagonists

Other proteins that can interfere with the productive interaction of hGM-CSF with its receptor include mutant hGM-CSF proteins and secreted proteins that include at least a portion of the extracellular portion of one or both hGM-CSF receptor chains that bind to hGM-CSF and compete with cell surface receptor binding. For example, soluble hGM-CSF receptor antagonists may be prepared by fusing the coding region of sGM-CSFR α to the CH2-CH3 region of murine IgG2 a. Exemplary soluble hGM-CSF receptors are described by Raines et al (1991) Proc. Natl. Acad. Sci. USA 88: 8203. Examples of GM-CSFR α -Fc fusion proteins are provided, for example, in Brown et al (1995) blood 85: 1488. In some embodiments, the Fc component of such fusions can be engineered to modulate binding to an Fc receptor, e.g., to increase binding to an Fc receptor.

Other hGM-CSF antagonists include hGM-CSF mutants. For example, hGM-CSF, described by Hercus et al, Proc. Natl. Acad. Sci. USA 91:5838,1994, having mutation of amino acid residue 21 of hGM-CSF to arginine or lysine (E21R or E21K), has been shown to have in vivo activity in preventing the spread of hGM-CSF-dependent leukemia cells in a mouse xenograft model (Iversen et al, blood 90:4910,1997). As will be appreciated by those skilled in the art, such antagonists may comprise conservatively modified variants of hGM-CSF with substitutions, such as those indicated at amino acid residue 21, or hGM-CSF variants with, for example, amino acid analogs, to increase half-life.

In some embodiments, the hGM-CSF antagonist can be a peptide. For example, an hGM-CSF peptide antagonist can be a peptide designed to structurally mimic specific residue positions on the B and C helices of human GM-CSF involved in receptor binding and biological activity (e.g., Monfardii et al, J. biol. chem 271:2966-2971, 1996).

In other embodiments, the hGM-CSF antagonist is an "antibody mimetic" that targets and binds an antigen in a manner similar to an antibody. Some of these "antibody mimetics" use non-immunoglobulin protein frameworks as alternative protein frameworks to antibody variable regions. For example, Ku et al (Proc. Natl. Acad. Sci. USA 92(14):6552-6556(1995)) disclosed an alternative to antibodies based on cytochrome b562 in which the two loops of cytochrome b562 were randomized and selected for binding to bovine serum albumin. Each mutant was found to selectively bind BSA similarly to anti-BSA antibodies. U.S. patent nos. 6,818,418 and 7,115,396 disclose antibody mimetics characterized by a fibronectin or fibronectin-like protein scaffold and at least one variable loop. These fibronectin-based antibody mimetics are known as mimobody protein drugs and share many of the properties of natural or engineered antibodies, including high affinity and specificity for any targeting ligand. The structure of these fibronectin based antibody mimetics is similar to that of the IgG heavy chain variable region. Thus, these mimetics exhibit antigen binding properties with properties and affinities similar to those of natural antibodies. Further, these fibronectin-based antibody mimetics exhibit certain benefits over antibodies and antibody fragments. For example, these antibody mimetics do not rely on disulfide bonds to achieve natural folding stability, and are therefore stable under conditions that would normally break down the antibody. In addition, since the structure of these fibronectin based antibody mimetics is similar to that of the IgG heavy chain, a method of loop randomization and shuffling can be employed in vitro, which is similar to that of antibody affinity maturation in vivo.

Beste et al (Proc. Natl. Acad. Sci. 96(5):1898-1903(1999)) disclose an antibody mimetic based on a lipocalin scaffold

Lipocalins consist of a beta barrel with four hypervariable loops at the protein end. The loops are subjected to random mutagenesis and selected for binding to, for example, fluorescein. Three variants showed specific binding to fluorescein, with one variant showing similar binding to the anti-fluorescein antibody. Further analysis showed that all random positions were variable, indicating that Anticalin would be suitable as a surrogate for antibodies. Therefore, Anticalin is a small single chain peptide, typically between 160 and 180 residues, with several advantages over antibodies, including reduced production costs, improved storage stability and reduced immune response.

U.S. patent No. 5,770,380 discloses a synthetic antibody mimetic using a rigid non-peptide organic scaffold of calixarene linked to multiple variable peptide loops that serve as binding sites. The peptide loops project geometrically from the same side relative to each other from the calixarene. Due to this geometric confirmation, all loops are available for binding, thereby increasing the binding affinity to the ligand. However, in contrast to other antibody mimetics, calixarene-based antibody mimetics do not consist solely of peptides and are therefore not susceptible to attack by proteases. The scaffold is also not composed of only peptides, DNA or RNA, which means that such antibody mimetics are relatively stable and long-lived under extreme environmental conditions. Further, since the calixarene-based antibody mimics are relatively small, it is unlikely that an immunogenic response will be generated.

Murali et al (Cell MoI Biol) 49(2):209-216(2003)) describe a method for reducing antibodies to smaller peptidomimetics, known as "antibody-like binding peptidomimetics" (ABiP), which can also be used as a substitute for antibodies.

In addition to the non-immunoglobulin protein framework, antibody properties have also been mimicked in compounds including RNA molecules and non-natural oligomers (e.g., protease inhibitors, benzodiazepines, purine derivatives, and β -turn mimetics). Thus, non-antibody GM-CSF antagonists may also comprise such compounds.

Therapeutic administration

In some embodiments, the methods of the present disclosure comprise administering to a subject with CRS or a cytokine storm an hGM-CSF antagonist (e.g., an anti-hGM-CSF antibody) as a pharmaceutical composition. In some embodiments, the hGM-CSF antagonist is administered in a therapeutically effective amount using a dosing regimen appropriate for the treatment of the disease.

In some embodiments, a therapeutically effective amount is an amount that at least partially arrests the condition or a symptom thereof. For example, a therapeutically effective amount may prevent immune activation, may reduce the level of circulating cytokines, may reduce T cell activation, or may reduce fever, malaise, fatigue, anorexia, myalgia, joint pain, nausea, vomiting, headache, rash, nausea, vomiting, diarrhea, tachypnea, hypoxemia, cardiovascular tachycardia, pulse broadening, hypotension, increased cardiac output (early), decreased potential cardiac output (late), increased D-dimer, hypofibrinogenemia with or without bleeding, azomia, elevated transaminases, hyperbilirubinemia, headache, altered mental state, confusion, delirium, aphasia with difficulty or rate of word finding, hallucinations, tremors, dysdiscrimination, altered gait, or seizure.

The methods of the invention comprise administering to a patient an anti-hGM-CSF antibody as a pharmaceutical composition in a therapeutically effective amount using a dosing regimen suitable for the treatment of the disease. The compositions can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition for proper formulation. Suitable formulations for use in the present invention are found in the following documents: remington: pharmaceutical sciences and practices (Remington: The Science and Practice of Pharmacy), 21 st edition, Philadelphia, Lippincott Williams and Wilkins, Pa., 2005. For a brief review of drug delivery methods, see Langer, science 249: 1527-.

The anti-hGM-CSF antibodies used in the methods of the invention are provided in a solution suitable for injection into a patient, such as a sterile isotonic aqueous injection solution. The antibody is dissolved or suspended in an acceptable carrier at a suitable concentration. In some embodiments, the carrier is aqueous, such as water, saline, phosphate buffered saline, and the like. The composition may contain auxiliary drugs required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and the like.

Administering to a patient a pharmaceutical composition of the invention, for example a patient suffering from: osteopenia, rheumatoid arthritis, juvenile idiopathic arthritis, systemic-onset still's disease, asthma, eosinophilic esophagitis, multiple sclerosis, psoriasis, atopic dermatitis, plexiform neurofibromas, autoimmune lymphoproliferative syndrome (or ALPS, also known as Carnelle-Smith syndrome), Ras-associated autoimmune leukoproliferative disorder (or RALD), Knoop syndrome, chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, acute myelogenous leukemia, multicenter castleman disease, chronic obstructive pulmonary disease, interstitial lung disease, diffuse parenchymal lung disease, idiopathic thrombocytopenic purpura, alzheimer's disease, heart failure, kawasaki disease, heart injury or diabetes caused by ischemic events, in an amount sufficient to cure or at least partially arrest the disease or symptoms of the disease and its complications. An amount sufficient to accomplish this is defined as a "therapeutically effective dose". A therapeutically effective dose is determined by monitoring the patient's response to treatment. Typical benchmarks indicating therapeutically effective doses include improving the patient's symptoms of the disease. An effective amount for such use will depend on the severity of the disease and the general health of the patient, including other factors such as age, weight, sex, route of administration and the like. Single or multiple administrations of the antibody may be administered depending on the dose and frequency required and tolerated by the patient. Regardless, the methods provide a sufficient amount of anti-hGM-CSF antibody to effectively treat the patient.

The antibodies can be administered alone, or in combination with other therapies to treat a disease of interest.

The antibody may be administered by injection or infusion by any suitable route, including but not limited to intravenous, subcutaneous, intramuscular, or intraperitoneal routes. In some embodiments, the antibody may be administered by insufflation. In an exemplary embodiment, the antibody may be stored at a concentration of 10mg/ml in sterile isotonic saline solution for injection at 4 ℃ and diluted in 100ml or 200ml of 0.9% sodium chloride for injection prior to administration to a patient. The antibody is administered by intravenous infusion at a dose of between 0.2 and 10mg/kg over the course of 1 hour. In other embodiments, the antibody is administered, for example, by intravenous infusion over a period of between 15 minutes and 2 hours. In still other embodiments, the administration procedure is by subcutaneous or intramuscular injection.

In some embodiments, the hGM-CSF antagonist, e.g., an anti-hGM-CSF antibody, is administered by the perispinal route. Perispinal administration involves anatomically localized delivery, which is performed to place the therapeutic molecule directly near the spine at the time of initial administration. Perispinal administration is described, for example, in U.S. patent No. 7,214,658 and in Tobinick and Gross, journal of neuroinflammation (j.neuroinflammation) 5:2,2008.

The dose of hGM-CSF antagonist is selected to provide an effective therapy for a subject who has been diagnosed with CRS or a cytokine storm. The dose is typically in the range of about 0.1mg/kg body weight to about 50mg/kg body weight or in the range of about 1mg to about 2g per patient. The dose is typically in the range of about 1 to about 20mg/kg or about 50mg to about 2000 mg/patient. The dosage may be repeated at an appropriate frequency, which may range from once a day to once every three months, depending on the pharmacokinetics of the antagonist (e.g., half-life of the antibody in circulation) and pharmacodynamics response (e.g., duration of therapeutic effect of the antibody). In some embodiments where the antagonist is an antibody or modified antibody fragment, the in vivo half-life and antibody dose of about 7 to about 25 days is repeated between once a week and once every 3 months. In other embodiments, the antibody is administered about once a month.

V of the invention_HZone and/or V_LThe regions may also be used for diagnostic purposes. For example, V_HAnd/or V_LThe regions may be used in clinical assays, such as for detecting GM-CSF levels in a patient. V of the invention_HOr V_LThe regions may also be used, for example, to generate anti-Id antibodies.

Unless defined otherwise herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of skill in the art. Further, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

In one embodiment, "treating" includes therapeutic treatment, "preventing" includes prophylactic or preventative measures, wherein the object is to prevent or alleviate the targeted pathological condition or disorder as described above. Thus, in one embodiment, treatment may comprise directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with a disease, disorder, or condition, or a combination thereof. Thus, in one embodiment, "treating", "improving" and "alleviating" refer in particular to delaying progression, accelerating remission, inducing remission, increasing remission, accelerating recovery, increasing efficacy or decreasing resistance to alternative therapy or a combination thereof. In one embodiment, "preventing" refers to, inter alia, delaying onset of symptoms, preventing recurrence of disease, reducing the number or frequency of recurrent episodes, increasing latency between symptom onset, or a combination thereof. In one embodiment, "suppressing" or "inhibiting" refers to, inter alia, reducing the severity of symptoms, reducing the severity of acute episodes, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, alleviating secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

In this disclosure, the singular forms "a", "an" and "the" include plural references and reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. The term "plurality," as used herein, refers to more than one. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value.

Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable. In some embodiments, the term "about" refers to a deviation of between 0.0001-5% from the indicated number or range of numbers. In some embodiments, the term "about" refers to a deviation of 1-10% from the indicated number or range of numbers. In some embodiments, the term "about" means that there is a deviation of up to 25% from the indicated number or range of numbers. The term "comprising" is intended to cover all of the listed elements but may also include additional unnamed elements and may be used interchangeably with the terms "comprises", "comprising" or "includes" having all of the same properties and meanings. The term "consisting of … …" means consisting of the listed elements or steps, and is used interchangeably with the term "consisting of … …" having all the same properties and meanings.

Examples of the invention

Example 1-exemplary human engineered antibodies to GM-CSF

A panel of engineered Fab' molecules with specificity c19/2 was generated from a library of epitope-focused human V segments as described in U.S. patent application publication nos. 20060134098 and 20050255552. The library in the epitope pool was constructed from a library sequence of human V segments linked to a CDR3-FR4 region containing the BSD sequences in CDRH3 and CDRL3 and the human germline J segment sequences. For the heavy chain, a human germline JH4 sequence is used, and for the light chain, a human germline JK4 sequence is used.

The full-length human engineered V-regions supporting binding to recombinant human GM-CSF were selected from a Vh1 restriction library. As described in U.S. patent application publication No. 20060134098, a "full-length" V-kappa library was used as the basis for constructing a "cassette" library in which only a portion of the murine c19/2V segment was initially replaced with a library of human sequences. Two types of cassettes were constructed. The V-kappa chain cassette was prepared by bridge PCR with overlapping consensus sequences in the framework 2 region. In this way, a "front-end" and "middle" human cassette library was constructed for the human V-kappa III isoforms. Human V-kappa III cassettes that support binding to GM-CSF were identified by colony-elevating binding assays and ranked according to affinity in ELISA. The V-kappa human "front end" and "middle" cassettes were fused together by bridge PCR to reconstitute the fully human V-kappa region that supports GM-CSF binding activity. Thus, the human engineered Fab consists of artificially engineered V-heavy and V-kappa regions that support binding to human GM-CSF.

Binding activity was determined by surface plasmon resonance (spr) analysis. Biotinylated GM-CSF was captured by a streptavidin-coated CM5 biosensor chip. The engineered Fab fragments expressed from E.coli were diluted to an initial concentration of 30nM at pH 7.4 in 10mM HEPES, 150mM NaCl, 0.1mg/ml BSA and 0.005% P20. Each Fab was serially diluted 4-fold using a 3-fold dilution and each concentration was tested twice at 37 degrees celsius to determine binding kinetics to antigen surfaces of different densities. Data from all three surfaces were globally fitted to extract the dissociation constants.

Binding kinetics were analyzed by Biacore 3000 Surface Plasmon Resonance (SPR). Recombinant human GM-CSF antigen was biotinylated and immobilized on a streptavidin CM5 sensor chip. Fab samples were diluted to an initial concentration of 3nM and performed in a 3-fold dilution series. The assay was performed in 10mM HEPES, 150mM NaCl, 0.1mg/mL BSA and 0.005% p20 at pH 7.4 and 37 ℃. Each concentration was tested twice. Fab' binding assays were performed on two antigen density surfaces, providing duplicate data sets. The mean affinities (KD) of each of 6 different human engineered anti-GM-CSF Fab clones calculated using the 1:1Langmuir binding model are shown in table 2.

TF-1 cell proliferation assay was used to test GM-CSF neutralization of Fab. GM-CSF-dependent proliferation of human TF-1 cells was determined after 4 days incubation with 0.5ng/ml GM-CSF using the MTS assay (cell titer 96, Promega) to identify viable cells. In this assay, all fabs inhibited cell proliferation, indicating that they are neutralizing antibodies. There was a good correlation between the relative affinity of anti-GM-CSF Fab and EC50 in cell-based assays. anti-GM-CSF antibodies with monovalent affinities in the range of 18pM to 104pM demonstrated effective neutralization of GM-CSF in cell-based assays.

Exemplary engineered anti-GM-CSF V region sequences are shown in FIG. 1.

Table 2: affinity of anti-GM-CSF Fab as compared to Activity (EC50) in a GM-CSF dependent TF-1 cell proliferation assay as determined by surface plasmon resonance analysis

Fab	Monovalent binding affinity (pM) determined by SPR	EC in TF-1 cell proliferation assay50(pM)
			94	18	165
104	19	239
			77	29	404
92	58	539
			42	104	3200
44	81	7000

Example 2 evaluation of human engineered GM-CSF antibodies

This example evaluated the binding activity and biological potency of artificially engineered anti-GM-CSF antibodies in cell-based assays, in comparison to chimeric IgG1k antibody (Ab2) with variable regions from the mouse antibody LMM102 (Nice et al, Growth Factors 3:159,1990). Ab1 is a human engineered IgG1k antibody against GM-CSF, with the same constant regions as Ab 2.

Surface plasmon resonance analysis of binding of human GM-CSF to Ab1 and Ab2

Surface plasmon resonance analysis was used to compare the binding kinetics and monovalent affinity of Ab1 and Ab2 for interaction with glycosylated human GM-CSF using a Biacore 3000 instrument. Ab1 or Ab2 were captured onto the Biacore chip surface using polyclonal anti-human F (Ab') 2. Glycosylated recombinant human GM-CSF expressed from human 293 cells was used as an analyte. Kinetic constants were determined in 2 independent experiments (see fig. 2A-2B and table 3). The results show that GM-CSF binds Ab2 and Ab1 with comparable monovalent affinity in this experiment. However, Ab1 was "association rate" two times slower than Ab2, but "dissociation rate" was approximately three times slower.

Table 3: showing the kinetic constants at 37 ℃ determined by surface plasmon resonance analysis in FIGS. 2A-2B; association constant (k)_a) Dissociation constant (k)_d) And calculated affinity (KD).

GM-CSF is occupied at both N-and O-linked glycosylation sitesBut glycosylation is not required for biological activity. To determine whether GM-CSF glycosylation affected Ab1 or Ab2 binding, antibodies were compared in an ELISA using recombinant GM-CSF from two different sources; GM-CSF expressed in E.coli (unglycosylated) and GM-CSF expressed in human 293 cells (glycosylated). The results in FIGS. 3A-3B and Table 4 show that both antibodies bind glycosylated and non-glycosylated GM-CSF with equivalent activity. In this assay, both antibodies also showed comparable EC ₅₀The value is obtained.

TABLE 4 EC for Ab2 and Ab1 binding to human GM-CSF from two different sources as determined by ELISA₅₀Summary of (1). Binding to recombinant GM-CSF from human 293 cells (glycosylated) or from E.coli (non-glycosylated) was determined from two independent experiments. Experiment 1 is shown in FIGS. 3A-3B.

	Non-glycosylation (experiment 1)	Non-glycosylation (experiment 2)	Glycosylation (experiment 1)
				Ab2	400pM	433pM	387pM
Ab1	373pM	440pM	413pM

Ab1 is a human engineered antibody derived from the mouse variable region present in Ab 2. Overlapping epitope specificity of Ab1 was tested by competition ELISA (Ab 2).

Biotinylated Ab2 was prepared using known techniques. Biotinylation did not affect the binding of Ab2 to GM-CSF as determined by ELISA. In the assay, different concentrations of Ab2 or Ab1 were added at fixed amounts of biotinylated Ab 2. Detection of biotinylated Ab2 was determined in the presence of unlabeled Ab or Ab1 competitor (fig. 4A-4B). Ab1 and Ab2 both competed with biotinylated Ab2 for binding to GM-CSF, thus indicating binding to the same epitope. Ab1 competes more effectively for binding to GM-CSF than Ab2, consistent with the slower dissociation kinetics of Ab1 when compared to Ab2 by surface plasmon resonance analysis.

Ab1 and Ab2 neutralize GM-CSF activity

Cell-based assays for neutralizing GM-CSF activity were used to assess biological efficacy. The assay measures IL-8 secretion from U937 cells induced with GM-CSF. After 16 hours of induction with 0.5ng/ml E.coli-derived GM-CSF, secretion of IL-8 into the culture supernatant was determined by ELISA.

A comparison of the neutralizing activity of Ab1 and Ab2 in this assay is shown in the representative assay in fig. 5. In three independent experiments, Ab1 inhibited GM-CSF activity more effectively than Ab2 when IC50 was compared (table 5).

TABLE 5 IC50 comparison for inhibition of GM-CSF-induced IL-8 expression. Data and mean IC of three independent experiments shown in fig. 5₅₀Expressed in ng/ml and nM.

Experiment of	Ab2(ng/ml)	Ab2(nM)	Ab1(ng/ml)	Ab1(nM)
					A	363	2.4	31.3	0.21
B	514	3.4	92.5	0.62
					C	343	2.2	20.7	0.14
Mean value of	407	2.7	48.2	0.32

SUMMARY

The engineered Ab1 bound to GM-CSF and the equilibrium binding constant (KD) calculated was 25 pM. Ab2 bound to GM-CSF with a KD of 30.5 pM. Association constant (k) of Ab2 for GM-CSF_a) Two times higher than Ab1, and dissociation kinetics of Ab1 (k)_d) Showing three times slower than Ab 2. In the antigen binding ELISA, Ab2 and Ab1 showed similar binding activity to glycosylated and non-glycosylated GM-CSF. Competitive ELISA demonstrated twoAntibodies all compete for the same epitope; ab1 showed higher competitive binding activity than Ab 2. In addition, Ab1 showed higher GM-CSF neutralizing activity than Ab2 in the GM-CSF induced IL-8 induction assay.

Example 3-administration of neutralizing anti-GM-CSF antibodies in mouse models of immunotherapy-related toxicity

A mouse model of immunotherapy-related toxicity can be used to show the efficacy of anti-GM-CSF antibodies in preventing and treating immunotherapy-related toxicity. In one model of immunotherapy-related toxicity, CAR T cells are injected into mice at doses that induce toxicity. For example, van der Stegen et al (J Immunol 191:4589-4598(2013)) which is incorporated herein by reference describe a method of 30X 10 ⁶Is called T4⁺CRS model induced by a single dose of intraperitoneal injection of cells of T cells. T4⁺The T cells are engineered T cells expressing a Chimeric Ag Receptor (CAR) T1E28 z. T cells engineered to express T1E28z were activated by cells expressing dimers based on ErbB1 and ErbB4, as well as ErbB2/3 heterodimers.

To assess the efficacy of anti-GM-CSF antibodies in preventing and treating CRS, mice were divided into several groups (n-10), each group receiving either of the following: a) single intra-peritoneal saline injection; b) intraperitoneal injection of 30X 10⁶A T4⁺A T cell; c) intraperitoneal injection of 30X 10⁶A T4⁺T cells and 0.25mg intravenous (i.v.) and T4⁺T cell co-administered anti-GM-CSF monoclonal antibody 22E9 (recombinant rat anti-mouse GM-CSF antibody); d) intraperitoneal injection of 30X 10⁶A T4⁺T cells and 0.25mg intranasally (i.n.) with T4⁺T cell co-administered anti-GM-CSF antibody 22E 9; e) intraperitoneal injection of 30X 10⁶A T4⁺T cells and methods of use in administering T4⁺6 hours prior to T cells, 0.25mg of intravenous anti-GM-CSF antibody 22E 9; f) intraperitoneal injection of 30X 10⁶A T4⁺T cells and 0.25mg of intranasal anti-GM-CSF antibody 22E9 6 hours prior to administration of T4+ T cells; g) intraperitoneal injection of 30X 10⁶A T4⁺T cells and methods of use in administering T4⁺2 hours after T cells, 0.25mg of intravenous anti-GM-CSF antibody 22E 9; or h) intraperitoneal injection of 30X 10 ⁶A T4⁺T cells and inAdministration of T4⁺2 hours after T cells, 0.25mg of intranasal anti-GM-CSF antibody 22E 9. Additional doses, administration times and routes of administration will be evaluated.

To evaluate the anti-GM-CSF antibody 22E9 effect, organs will be collected from mice, fixed with formalin and then histopathological analysis. Blood will be collected and the concentrations of human IFN γ, human IL-2 and mouse IL-6, IL-2, IL-4, IL-6, IL-10, IL-17, IFN γ and TNF α will be assessed by good methods described in the literature, such as ELISA assays. The mice will be observed for body weight, behavior and clinical appearance.

Example 4 Effect of anti-GM-CSF antibodies on immunotherapy

Mouse models can be used to show that GM-CSF antagonists do not negatively impact the efficacy of cancer immunotherapy. SCID beige mice can be inoculated with cancer cell lines and used with immunotherapeutics known to induce CRS (e.g., T4)⁺T cells), with or without anti-GM-CSF antibodies.

To assess whether anti-GM-CSF antibodies affect the efficacy of immunotherapy, mice were divided into several groups (n ═ 10), each group receiving either of the following: a) subcutaneous (s.c.) injection of 30X 10⁶SKOV3 cells; b) subcutaneous injection 30X 10⁶SKOV3 cells and intraperitoneal injection of 30X 10 ⁶A T4⁺A T cell; or c) subcutaneous implantation of 30X 10⁶ 30X 10 intraperitoneal injection of SKOV3 cells⁶A T4⁺T cells and intravenous injection of 0.25mg of anti-GM-CSF antibody 22E 9.

To evaluate anti-GM-CSF antibody 22E9 versus T4⁺Effect of T cell efficacy, tumor size will be measured by calipers every four days, and tumor volume calculated by the following formula: 0.5 (larger diameter) x (smaller diameter)². The mice will be observed for body weight, behavior and clinical appearance. At the end of the experiment, animals were sacrificed and tumor tissue was harvested and weighed.

Example 5-mouse model of human CRS

A mouse model for CRS has been developed to study the role of humanized anti-GM-CSF monoclonal antibodies in the treatment or prevention of CRS. (FIG. 17a. -17 b.).

The method comprises the following steps: the model used was the primary AML model. Immunocompromised NSG-S mice additionally transgenic for human SCF, IL-3 and GM-CSF are transplanted with AML blasts from CD123 positive AML patients. After 2-4 weeks, they are bled to confirm transplantation and achieve high disease burden. Then 1X 10 with high dose of CAR-T123⁶Mice were treated with one cell, which was 10-fold the dose previously studied.

As a result: it was observed that within 1-2 weeks after CAR-T cell injection, these mice developed a disease characterized by weakness, wasting, a hunched body, flinching and poor motor response. The mice eventually succumb to disease within 7-10 days. Symptoms are associated with massive T cell expansion in mice and elevation of various human cytokines (e.g., IL-6, MIP1 α, IFN- γ, TNF α, GM-CSF, MIP1 β, and IL-2) and are in a pattern similar to that seen in human CRS following CAR-T cell therapy. The fold change in GM-CSF was significantly greater than other cytokines. (FIGS. 17 a-b).

Example 6 Generation of GM-CSF knock-out CAR-T

GM-CSF CRISPR knock-out T cells were generated and showed reduced expression of GM-CSF, but similar levels of other cytokines and degranulation, indicating immune cell function. (see FIGS. 15a-15 g).

Example 7-anti-GM-CSF neutralizing antibodies do not inhibit CAR-T mediated killing, proliferation, or cytokine production but neutralize GM-CSF

anti-GM-CSF neutralizing antibodies do not inhibit CAR-T mediated killing, proliferation, or cytokine production, but successfully neutralize GM-CSF. (see FIGS. 16a-16 i).

Example 8-anti-GM-CSF neutralizing antibodies do not inhibit CAR-T efficacy in vivo

Humanized anti-GM-CSF monoclonal antibody, neutralizing hGM-CSF antibody did not inhibit the efficacy of CAR-T in vivo (FIGS. 18a-18 c). CAR-T efficacy in combination with an anti-GM-CSF neutralizing antibody according to the embodiments described herein in a xenograft model. As shown in fig. 18a, NSG mice were injected with NALM-6-GFP/luciferase cells (human peripheral blood pre-leukemic B cells) and bioluminescent imaging (BLI0) was performed to confirm tumor growth. Mice were treated with (1) anti-GM-CSF antibody (10 mg/Kg per day for ten days) and (a) CART19 or (b) untransduced human T cellsCell (UTD) 1X 10⁶Individual cells or (2) IgG control antibody (10 mg/Kg per day for ten days) and (a) CART19 or (b) untransduced human T cells (UTD) 1X 10 ⁶And (4) treating the cells. FIGS. 18b and 18c demonstrate that anti-GM-CSF neutralizing antibodies do not inhibit CAR-T efficacy in vivo.

Example 9-anti-GM-CSF neutralizing antibodies do not impair the CAR-T effect on survival

Preclinical data in vitro and in vivo show that anti-GM-CSF neutralizing antibodies (humanized anti-GM-CSF monoclonal antibodies) do not impair the CAR-T's effect on survival in a mouse model. (FIG. 19).

In the absence of PBMCs, anti-GM-CSF neutralizing antibodies do not block CAR-T cell function in vivo. The survival rates of the CAR-T + control and CAR-T + anti-GM-CSF neutralizing antibodies have been shown to be similar.

Example 10 anti-GM-CSF neutralizing antibodies can increase CAR-T amplification

Preclinical data in vitro and in vivo showed that anti-GM-CSF neutralizing antibodies (humanized anti-GM-CSF monoclonal antibodies) can increase CAR-T amplification (figure 20). anti-GM-CSF neutralizing antibodies can increase CAR-T's cancer cell killing in vitro. The antibodies increase proliferation of CAR-T cells and may improve efficacy. The GM-CSF neutralizing antibody increases the proliferation of CAR-T in the presence of PBMCs. (unaffected without PBMC). The antibody does not inhibit degranulation, intracellular GM-CSF production, or IL-2 production.

Example 11 CAR-T amplification associated with Overall response Rate improvement

CAR-T amplification is associated with an increase in overall response rate. (FIG. 21). CAR AUC (area under the curve) is defined as the cumulative level of CAR + cells/microliter of blood within the first 28 days after CAR-T administration. P values were calculated by Wilcoxon rank sum test. (Neelapu et al ICML 2017 Abstract 8).

Example 12-study protocol for anti-GM-CSF neutralizing antibodies according to the examples described herein

Study protocol for anti-GM-CSF neutralizing antibodies (engineered anti-GM-CSF monoclonal antibodies) according to the examples described herein. (see FIG. 22). CRS and NT were assessed daily during the hospitalization period and 30 days prior to the first visit. Eligible subjects received GM-CSF neutralizing antibody on days-1, +1, and +3 of CAR-T treatment. Tumor assessments were performed at baseline and at 1 st, 3 rd, 6 th, 9 th, 12 th, 18 th and 24 th months. Blood samples (PBMC and serum) at days-5, -1, 0, 1, 3, 5, 7, 9, 11, 13, 21, 28, 90, 180, 270 and 360.

Example 13 GM-CSF consumption increases CAR-T cell expansion

GM-CSF consumption increases CAR-T cell expansion. (FIGS. 23A-23B) FIG. 23A shows GM-CSF in comparison to control CAR-T cells^k/oIncreased ex vivo expansion of CAR-T cells. Figure 23b demonstrates more robust proliferation after in vivo treatment with an anti-GM-CSF neutralizing antibody (a humaneered anti-GM-CSF monoclonal antibody) according to the examples described herein.

Example 14-safety profile of anti-GM-CSF neutralizing Ab in >100 human patients

Stage I: single dose, escalation of dose in healthy adult volunteers. The aim was to analyze safety/tolerability, PK and immunogenicity.

Number of participants/dose:

(n＝12)

3/1mg/kg

3/3mg/kg

3/10mg/kg

3/placebo

Safety results:

net safety profile:

without drug-related Serious Adverse Effects (SAE)

Non-immunogenic

Stage II: 1) dose for rheumatoid arthritis patients at weeks 0, 2, 4, 8, 12. The aim was to analyze efficacy, safety/tolerability, PK and immunogenicity.

Number of participants/dose:

(n＝9)

7/600mg

2/placebo

Safety results:

net safety profile:

without drug-related Serious Adverse Effects (SAE)

Non-immunogenic

2) Dose in severe asthma patients at weeks 0, 2, 4, 8, 12, 16, 20. The aim was to analyze efficacy, safety/tolerability, PK and immunogenicity.

Number of participants/dose:

(n＝160)

78/400mg

82/placebo

Safety results:

net safety profile:

without drug-related Serious Adverse Effects (SAE)

Non-immunogenic

94 patients in the study above, plus 12 patients in an ongoing CMML phase I trial, wherein drug tolerance is good; an additional 76 patients received GM-CSF neutralized Ab (KB002) chimeric form and showed similar safety profile.

All studies were randomized for double-blind placebo-controlled IV administration. (see FIG. 24.)

Example 15 Effect of anti-GM-CSF antibodies on CART Activity and toxicity

The effect of blocking of GMCSF by anti-GM-CSF antibodies on chimeric antigen receptor T Cell (CART) activity and toxicity will be investigated. This can be achieved by two objectives:

purpose # 1: investigation of the Effect of blocking GMCSF by anti-GM-CSF antibodies on CART cell effector function

Purpose # 2: the effect of blocking GMCSF by anti-GM-CSF antibodies on reducing cytokine release syndrome after CART cell therapy study strategy was investigated. The following experiments are suggested:

in vitro studies of four different doses of anti-GM-CSF antibodies blocking GMCSF binding to CART cells (cytokine production (30plex Lumiex, comprising GM-CSF, IL-2, INFg, IL-6, IL-8, MCP-1), antigen-specific killing, degranulation, proliferation and depletion) in the presence or absence of myeloid cells using the following model: CART19 antagonizes ALL.

The binding of GMCSF blockade (with or without murine GMCSF blockade) to CART cells was studied in vivo using the following two models at different doses of anti-GM-CSF antibodies:

xenografts transplanted with a CD19 positive cell line (NALM6), treated with CART19 with or without anti-GM-CSF antibodies; and

patient derived primary ALL xenografts were then treated with CART19 with or without anti-GM-CSF antibodies.

Mice were injected intraperitoneally with 10mg/kg of anti-GM-CSF antibody immediately prior to CART cell implantation and administered 10 mg/kg/day for 10 days. The tumor response and survival of the mice will be followed. One week after CART cell therapy, starting once a week thereafter, retroorbital bleeding will be obtained. Disease burden, T cell expansion kinetics, expression of depletion markers and cytokine levels (30Plex) will be analyzed. After completion of the experiment, spleen and bone marrow will be collected and analyzed for tumor characteristics and CAR-T cell number.

In vivo studies of GMCSF blockade in combination with anti-GM-CSF antibodies (with or without murine GMCSF blockade) and CART cells in the CRS model (in this model, high doses of CART cells will be used to prime CRS) in the presence of PBMCs using the following model:

primary ALL patients were xenografted and then treated with CART19 with or without anti-GM-CSF antibodies.

Mice were injected intraperitoneally with 10mg/kg of anti-GM-CSF antibody immediately prior to CART cell implantation and administered 10 mg/kg/day for 10 days. Mice will be followed for tumor response, CRS toxicity symptoms and survival. Retroorbital bleeding will be obtained at baseline, 2 days, one week after CART cell therapy, and weekly thereafter. Disease burden, T cell expansion kinetics, expression of depletion markers and cytokine levels (30Plex) will be analyzed. After completion of the experiment, spleen and bone marrow will be collected and analyzed for tumor characteristics and CAR-T cell number.

In vivo neurotoxicity assay

Using the model discussed in #3 above, mice will be imaged by MRI at the time of disease to assess the development of neurotoxicity following CART cell therapy. Images will be compared between mice receiving CART cells and anti-GM-CSF antibodies and control antibodies. The experiment was repeated. In these repeated experiments, mice were euthanized 14 days after CART cells. Brain tissue will be analyzed for cytokines by multiplex assays, and examined for the presence of monocytes, human T cells, and the integrity of the blood brain barrier by IHC, flow and microscopy.

Example 16

anti-hGM-CSF neutralizing antibodies reduce neuroinflammation in CAR-T cell-associated Neurotoxicity (NT)

There is extensive scientific evidence suggesting that GM-CSF is essential for the Cytokine Release Syndrome (CRS), Neurotoxicity (NT) and inflammatory cascades seen following initiation of CAR-T cell therapy. The hypothesis of the study was that blocking soluble GM-CSF with neutralizing antibody (ritzimab) would abrogate or prevent the onset and severity of CRS and NT observed with CAR-T cell therapy. Importantly, CAR-T cell activity should be preserved or improved if possible. Experimental design the effect of anti-GM-CSF antibody (ritzimab) on GM-CSF blockade on effector function of CAR-T cells, CAR-T efficacy in tumor xenograft models, development of CRS and development of NTs in CRS xenograft models was tested using MRI imaging and volume analysis to quantify neuroinflammation observed with CAR-T cell therapy. In vitro and in vivo experiments with CAR-T +/-Ritzuzumab in the presence and absence of human PBMC were studied. (see examples 9 and 10, FIGS. 19 and 20a-20 b).

Method

In vitro studies were performed to evaluate the GM-CSF neutralizing antibody litzizumab in combination with human CD19+ CAR-T cells for antigen-specific killing, degranulation, proliferation and depletion in the presence or absence of human PBMCs.

To evaluate the effect of anti-GM-CSF antibodies (ritzimab) on CAR-T cell proliferation and efficacy, in vivo studies (with or without murine GM-CSF blockade) were subsequently performed using the following model:

effector/target control experiment: xenografts transplanted from a CD19 positive cell line (NALM6) with or without anti-GM-CSF antibody (litteruzumab) were treated with CART19 in the absence of human PBMCs.

NSG mice were injected intraperitoneally with 10mg/kg of anti-GM-CSF antibody (ritelumab) immediately prior to CAR-T cell implantation and thereafter administered at the same dose daily for 10 days, and tumor response and survival were then assessed. CAR-T cell therapy was initiated one week after, once a week after, and retroorbital bleeding was obtained. Disease burden, T cell expansion kinetics, expression of depletion markers and cytokine levels (30Plex) were also analyzed. After completion of the experiment, spleen and bone marrow were collected and analyzed for tumor characteristics and CAR-T cell number.

CRS/NT experiments: patient-derived primary ALL xenografts, followed by treatment with CART19 in combination or not with rituximab in the presence of human PBMCs:

To assess the effect of ritzimab on the elimination or prevention of the onset and severity of CAR-T induced CRS and NT, in a CRS model (in which high doses of CAR-T cells were used to elicit CRS), treatment with CART19 in combination or not in combination with ritzimab was performed using primary ALL patient-derived xenografts in the presence of PBMCs, and in vivo studies with human CAR-T cells (with and without murine GM-CSF blockade). The NSG mice were given ritzizumab 10mg/kg intraperitoneally immediately prior to CAR-T cell implantation and daily for 10 days thereafter. Mice were followed for tumor response, survival, CRS and NT symptoms. Brain MRI scans were performed at baseline, during and at the end of CAR-T cell therapy, and volumetric analysis was performed to assess and quantify neuroinflammation and MRI T1 high levels across treatment groups (treatment arm). Body weight and retro-orbital bleeding were obtained at baseline, 2 days, one week, and weekly after CAR-T cell therapy. Disease burden, T cell expansion kinetics, expression of depletion markers and cytokine levels (30Plex) were analyzed. After completion of the experiment, spleen and bone marrow were collected and analyzed for tumor characteristics and CAR-T cell number.

Results

In vitro model

In this experiment, the effect of neutralizing GM-CSF with Rizelizumab on CAR-T cell effector function was studied. It was demonstrated that CAR-T cells secrete GM-CSF at very high levels (over 1,500pg/ml), and that GM-CSF was completely neutralized using Rizerumab, but did not inhibit CAR-T degranulation, intracellular GM-CSF production, or IL2 production. Furthermore, Ritzuzumab does not inhibit CAR-T antigen specific proliferation or CAR-T lethality. The effector-to-target ratio (E: T) of CAR-T + litteruzumab to CAR-T + control antibody was similar, p ═ ns (fig. 16a-16d and 16 j).

In vivo model:

effector/target control experiment:

to investigate the effect of ritvolumab on CART19 cell function in vivo, immunocompromised NOD-SCID-g-/-and CD19+ ALL cell line NALM6 were transplanted in the absence of human PBMC. Although GM-CSF levels were completely neutralized in these mice, treatment with CART19 in combination with ritvolumab resulted in potent anti-tumor activity and improved overall survival, similar to CART19 using the control antibody, suggesting that GM-CSF does not impair CAR-T cell activity in mice in vivo in the absence of PBMCs (fig. 16f and 16 g).

CRS and NT experiments:

using human ALL blast cells, human CD19 CAR-T and human PBMC, litziuzumab was found to reduce neuroinflammation by about 90% in combination with CAR-T cell therapy compared to CAR-T alone, as assessed by quantifying MRI T1 high intensity. This is a milestone finding and the first in vivo demonstration that neuroinflammation caused by CAR-T cell therapy can be effectively eliminated. MRI images after litzizumab plus CAR-T cell therapy are similar to baseline pre-treatment scans, in sharp contrast to MRI images after control antibody plus CAR-T cell therapy, which show a significant increase in inflammation. In addition, a reduction in myeloid cells was seen in the brain of mice treated with Ritzuzumab plus CAR-T compared to mice treated with CAR-T and control antibodies. This finding is consistent with data reported in clinical trials of CD19 CAR-T cell therapy in which an increase in myeloid cells was observed in CSF in neurotoxic patients with severity grade > 3. Additionally, the use of litzizumab in combination with CAR-T cell therapy reduces the onset and severity of CRS compared to CAR-T plus control antibody. This finding is supported by a statistically significant reduction in body weight seen in mice treated with CAR-T plus controls, the reduction in body weight being the most objective marker and the hallmark symptom of CRS observed in vivo. Body weight was maintained at baseline levels (p <0.05) in mice treated with Ritzuzumab plus CAR-T compared to CAR-T plus control. In addition, mice treated with CAR-T plus control antibody showed physical symptoms consistent with CRS, including hunched posture, flinching and weakness, while mice treated with CAR-T plus rituximab showed health. Importantly, in these CRS/NT experiments with PBMCs, rituzumab plus CAR-T also showed a significant 5-fold increase in CAR-T cell proliferation compared to CAR-T plus control. It has been previously shown in various clinical trials of CD19 CAR-T cell therapy that improved CAR-T proliferation or expansion correlates with improved efficacy (including ORR, CR), suggesting that rituximab may improve the anti-tumor response. This finding can be explained in part by the known reduction in amplification and transport of MDSCs transmitted by GM-CSF. Finally, the combination of litzizumab plus CAR-T significantly improved leukemia control as compared to CAR-T and control antibodies, as quantified by flow cytometry. Treatment with CAR-T plus litzimab resulted in a significant reduction in the number of leukemic cells (down to between 500 and 5,000 cells) and improved overall disease control compared to untreated mice (which had 500,000 to 1.5M leukemic cells) and CAR-T plus control antibody (which had between 15,000 and 100,000 leukemic cells) (see figures 25A-25D).

Figure 25A MRI images show a significant improvement in Neurotoxicity (NT) (neuroinflammation) in the brain of mice administered CAR-T cells and anti-GM-CSF neutralizing antibodies according to the examples described herein. In contrast, the brains of mice administered CAR-T cells and control antibodies showed signs of neurotoxicity in MRI images. Figure 25B graphically illustrates a 90% reduction in NT in group 1 mice compared to an increase in NT in group 2 mice. The quantitative improvement (90% reduction in NT) following administration of CAR-T cells and anti-GM-CSF neutralizing antibodies according to the examples described herein was an unexpected finding.

Conclusion

anti-GM-CSF antibody (ritzimab) when combined with CAR-T cell therapy suggests that using human ALL blast cells, human CD19 CAR-T and human PBMC, it is possible to prevent the onset and severity of CRS and NT, while improving CAR-T expansion/proliferation and overall in vivo leukemia control. This demonstrates for the first time that CAR-T induced neurotoxicity can be eliminated in vivo. Key clinical trials were planned using ritzizumab in combination with CAR-T cell therapy to validate these findings of improved safety and efficacy.

Example 17

GM-CSF blockade reduces cytokine release syndrome and neurotoxicity during chimeric antigen receptor T cell therapy, and may enhance effector function thereof

Despite its therapeutic efficacy, chimeric antigen receptor T cell therapy (CART) is limited by the development of Cytokine Release Syndrome (CRS) and Neurotoxicity (NT). Although CRS is associated with an extreme increase in cytokines and extensive T cell expansion, the exact mechanism of NT has not been elucidated. Preliminary studies have shown that NT may be mediated by myeloid cells that cross the blood brain barrier. This was supported by a correlation analysis of the CART19 key test, in which patients with severe NT had an increased number of CD14+ cells in the cerebrospinal fluid (Locke et al, ASH 2017). Therefore, the objective of this study was to investigate the effect of GM-CSF neutralization on prevention of CRS and NT following CART cell therapy by monocyte control.

First, the effect of GM-CSF blockade on CART cell effector function was investigated. Here, human GM-CSF neutralizing antibody (Ritzuzumab, human antigen Co., Humanigen, Berlingham, Calif.) t, which has been shown to be safe in phase II clinical trials, was used. Upon stimulation of CART19 cells with CD19+ luciferase + Acute Lymphoblastic Leukemia (ALL) cell line NALM6, litzillumab (10 μ g/kg) neutralized GM-CSF, but did not impair CART cell function in vitro. Malignant tumor-associated macrophages were found to reduce CART proliferation. Neutralization of GM-CSF with ritzeuzumab in the presence of monocytes resulted in enhanced CART cell antigen-specific proliferation. To confirm this in vivo, NOD-SCID-g-/-mice were transplanted with NALM6 with a high disease burden and treated with low doses of CART19 or control T cells (to induce tumor recurrence) in combination with litzizumab or isotype control antibodies. The combination of CART19 and ritzimab resulted in significant anti-tumor activity and overall survival benefit compared to control T cells (fig. 26A), similar to mice treated with CART19 in combination with isotype control antibody, indicating that GM-CSF neutralization did not compromise the activity of CART cells in vivo. This antitumor activity has been demonstrated in a xenograft model derived from ALL patients.

Next, the impact of GM-CSF neutralization on CART cell-associated toxicity was explored in a new patient-derived xenograft model. Here, NOD-SCID-g-/-mice were transplanted with leukemic blast cells (1-3X 106 cells) derived from patients with high risk of relapsing ALL. Mice were then treated with high doses of CART19 cells (intravenous 2-5 x 106). Five days after CART19 treatment, mice began to develop progressive motor weakness, a loss of body and weight from hunchback, which is associated with a large-scale increase in circulating human cytokine levels. Magnetic Resonance Imaging (MRI) of the brain during this syndrome shows diffuse enhancement and edema, associated with Central Nervous System (CNS) infiltration of CART cells and murine activated myeloid cells. This is similar to what has been reported in the CART19 clinical trial of patients with severe NT. The combination of CART19, litzizumab (for neutralizing human GM-CS) and murine GM-CSF blocking antibody (for neutralizing mouse GM-CSF) prevented weight loss (fig. 26B), reduced key myeloid cytokines (fig. 26C-26D), reduced brain edema (fig. 26E), enhanced control of leukemia disease in the brain (fig. 26F), and reduced brain macrophages (fig. 26G).

Finally, it was hypothesized that disruption of GM-CSF by CRISPR/Cas9 gene editing during the CART cell manufacturing process would result in reduced GM-CSF secretion by functional CART cells. Design the guide RNA of GM-CSF gene to target exon 3 and generate GM-CSF ^k/oCART19 cells. Preliminary data indicate that these CART produced significantly less GM-CSF upon activation, but continued to exhibit similar production of other cytokines and normal effector function in vitro (fig. 26H). GM-CSF compared to CART19 cells using NALM6 high tumor burden recurrent xenograft model as described above^k/oCART19 cells resulted in a slight enhancement of disease control (fig. 26I).

Thus, modulation of bone marrow cell behavior by GM-CSF blockade can help control CART-mediated toxicity and can reduce its immunosuppressive characteristics, thereby improving leukemia control. These studies demonstrate a novel approach to eliminate NT and CRS by GM-CSF neutralization, which also may enhance CART cell function. Based on these results, a phase II clinical trial was designed using ritzimab as a modality to prevent CART-related toxicity in patients with diffuse large B-cell lymphoma.

Example 18

GM-CSF neutralization in vitro enhances CAR-T cell proliferation in the presence of monocytes and does not impair CAR-T cell effector function

Cell lines and primary cells

The NALM6 cell line was purchased from ATCC of Masnasas, Virginia, USA, and the MOLM13 cell line was a gift from Jelinek Laboratory (Jelinek Laboratory) located in the Meiji Clinic (Mayo Clinic) (from DSMZ of Blancer, Germany). These cell lines were transduced with luciferase-ZsGreen lentivirus (Addgene, cambriqi, ma, usa) and sorted to 100% purity. Cell lines were cultured in R10 (prepared with RPMI 1640 (Gibco corporation, gaithersburg, maryland), 10% fetal bovine serum (FBS, Millipore Sigma, Ontaria, Canada) and 1% penicillin-streptomycin-glutamine (Gibco corporation, gaithersburg, maryland). Primary cells were obtained from the meiosis Clinic biological specimen bank (Mayo clinical Biobank) for patients with acute leukemia according to protocols approved by the institutional review board of meiosis Clinic (IRB). The use of recombinant DNA in the laboratory was approved by the Biosafety Committee (IBC) of the Mei You clinical institute.

Primary cells and CAR-T cells

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from de-identified cones of normal donor blood apheresis blood components obtained according to protocols approved by the miniclinic IRB using SepMate tubes (STEMCELL Technologies, wengowa, canada). Using EasySep^TMHuman T cell isolation kit (Wingonian Stem cell technology, Inc., Canada) used negative selection magnetic beads to isolate T cells. Using a catalyst from Belgischrad, GermanyIsolation of monocytes human monocyte isolation kit from american whirlpool biotechnology limited of bach (Miltenyi Biotec, Bergisch Gladbach, Germany) which isolates CD14+ monocytes. Primary cells were cultured in T cell culture medium made with X-Vivo 15 supplemented with 10% human serum albumin (Corning, NY, USA) and 1% penicillin-streptomycin-glutamine (Gibco, gaithersburg, maryland, USA). CART19 cells were generated by lentiviral transduction of normal donor T cells as described below. The second generation CART19 construct (IDT) was synthesized de novo under the control of the EF-1 α promoter and cloned into a third generation lentivirus. The CD19 directed single chain variable fragment was derived from clone FMC 63. Second generation 4-1BB co-stimulatory (FMC63-41BBz) CAR constructs were synthesized and used in these experiments. Lentivirus particles were generated by transient transfection of plasmids into 293T virus producing cells (gift from Ikeda laboratories in the pact clinic) in the presence of Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), VSV-G, and a packaging plasmid (addge from cambrix, ma). T cells isolated from normal donors were stimulated at a 1:3 ratio using the cell therapy system Dynabeads CD3/CD28 (Life Technologies, Oslo, Norway) and then transduced with lentiviral particles at a multiplicity of infection (MOI) of 3.0 24 hours after stimulation. To determine titre and subsequent MOI, after concentration of the lentiviral particles, 1X 10 in 100. mu.l of T cell medium was transduced by 50. mu.l of lentivirus ⁵Titer was determined for each primary T cell. First, T cells were stimulated with CD3/CD28 beads and then transduced with lentiviral particles 24 hours later. Transduction was performed in triplicate and at serial dilutions. Fresh T cell medium was added one day later. After two days, cells were harvested, washed twice with PBS, and CAR expression on T cells was determined by flow cytometry. Titers were determined based on the percentage of CAR positive cells (percentage of CAR + cells x T cell count at transduction x specific dilution/volume) and expressed as transduction units/mL (TU/mL). Bead removal at day 6, and CAR-T cells harvested and cryopreserved at day 8For future experiments. CAR-T cells were thawed and left for 12 hours in T cell culture media before being used in the experiment.

GM-CSF neutralizing antibodies and isotype controls

Ritzuzumab (human antigen Co., Burlingham, Calif.), according to the examples described herein and as described in U.S. Pat. Nos. 8,168,183 and 9,017,674, the hGM-CSF neutralizing antibody (each of which is incorporated herein by reference in its entirety) is a novel, first-class antibody

A monoclonal antibody that neutralizes human GM-CSF. For in vitro experiments, 10. mu.g/mL of Ritzuzumab or InVivoMAb human IgG1 isotype control (BioXCell, West Lebanon, NH, USA), West, N.H.) was used. For in vivo experiments, from the day of CART19 injection, 10mg/kg of litzuzumab or isotype control was injected intraperitoneally daily for 10 days. In some experiments, anti-mouse GM-CSF neutralizing antibodies (InVivoMAb anti-mouse GM-CSF, BioXCell of cetraria, new hampshire, usa) or corresponding isotype controls (InVivoMAb rat IgG2a isotype control, BioXCell of cetraria, new hampshire, usa) were also used as indicated in the experimental protocol.

Functional assay of T cells

Cytokine assays were performed 24 or 72 hours after co-culturing CAR-T cells with their targets at a 1:1 ratio as indicated. As indicated, a panel of high sensitivity T-cell MAGNETIC BEADs (nimobabfe, ontario), a 38Plex kit of nimobo human cytokine/chemokine MAGNETIC BEAD premix (nimobabfe, ontario) or a 32Plex kit of nimobo mouse cytokine/chemokine MAGNETIC BEAD premix (nimobibio, ontario) was performed on the supernatants or sera collected from these experiments. This was analyzed using Luminex (michigan sigma, ontario, canada). In monensin (monensin) (BioLegend (Bi) of san Diego, Calif., USA)oelengd, San Diego, CA, USA)), hCD49d (BD Biosciences, San Diego, CA, USA) and hCD28 (BD Biosciences, San Diego, CA, USA) in the presence of CAR-T cells incubated for 4 hours at a 1:5 ratio with targets and a T cell degranulation assay was performed. After 4 hours, the cells were harvested and after surface staining intracellular staining was performed followed by fixation and permeabilization with fixation media a and B (life technologies of oslo, norway). For proliferation assays, CFSE (life technologies, oslo, norway) labeled effector cells (CART19) and irradiated target cells were co-cultured at a 1:1 ratio. In some experiments, CD14+ monocytes were added to the co-culture at a 1:1:1 ratio as indicated. The cells were co-cultured for 3-5 days, as indicated in the specific experiment, and then the cells were harvested and treated with anti-hCD 3 (eBioscience, San Diego, CA, USA) and LIVE/DEAD (r) anti-hCD 3 (eBioscience, San Diego, CA, USA) ^TMFixable light green DEAD cell staining kit (LIVE/DEAD)^TMFixable Aqua Cell Stain Kit (Invitrogen, Calsbad, Calif.) was surface stained. PMA/ionomycin (michigan sigma, ontario, canada) was used as a positive non-specific stimulator of T cells at different concentrations as indicated in the specific experiments. For the kill assay, CD19 was determined⁺Luciferase enzyme⁺ALL cell line NALM6 or CD19^-Luciferase enzyme⁺Control MOLM13 cells were incubated with effector T cells at the indicated rates for 24, 48, or 72 hours, as listed in the specific experiment. Lethality was calculated as a measure of residual viable cells by bioluminescence imaging on a Xenogen IVIS-200 spectral camera (PerkinElmer, Hopkinton, MA, USA) in perkin elmer, Hopkinton, massachusetts. Prior to imaging, the samples were treated with 1 μ l D-fluorescein (30 μ g/mL) per 100 μ l sample volume (Gold Biotechnology, st. louis, MO, USA) for 10 minutes.

Multiparameter flow cytometry

Anti-human antibody and anti-mouseAntibodies were purchased from Biolegend, eBioscience, or BD biosciences (san diego, ca, usa). Cells are isolated from in vitro cultures or from the peripheral blood of animals. After BD FACS lysis (BD biosciences, san diego, california, USA), it was washed twice in phosphate buffered saline supplemented with 2% FBS (milan bosch sigma, ontarit, canada) and 1% sodium azide (lica chemicals, Arlington, TX, USA) and stained at 4 ℃. For cell number quantification, Countbright beads (invitrogen, calsbard, ca, usa) were used according to the manufacturer's instructions (invitrogen, calsbard, usa). In all analyses, the population of interest was gated based on forward and side scatter properties, followed by single line gating, and LIVE/DEAD was used ^TMStaining with a fixable, light green dead cell staining kit (invitrogen, carlsbad, ca, usa) was followed by gating of live cells. The surface expression of the CAR was detected by staining with goat anti-mouse F (ab')2 antibody (invitrogen, calsbad, ca). Flow cytometry was performed on a triple laser cytometer, Canto II (BD biosciences, san diego, california), and CytoFLEX (Beckman Coulter, Chaska, MN, USA). Analysis was performed using FlowJo x10.0.7r2 software (Ashland, OR, USA, a group of asia-charles, oregon) and Kaluza 2.0 software (beckmann coulter, charcol, mn, USA).

Results

If GM-CSF neutralization following CAR-T cell therapy is used as a strategy to prevent CRS and neurotoxicity, then it must not inhibit CAR-T cell efficacy. Therefore, initial experiments aimed at studying the effect of GM-CSF neutralization on CAR-T cell effector function. CART19 cells with or without CD19 in the presence of Rizlumab (hGM-CSF neutralizing antibody) or isotype control (IgG)⁺ALL cell line NALM6 was co-cultured. It was established that Rizizumab but not IgG control antibody was indeed able to completely neutralize hGM-CSF (FIG. 27A) but not inhibit CAR-T cell antigen specific proliferation was made (figure 27B). CART19 cells and CD19 when combined in the presence of monocytes⁺Upon co-culture of the cell line NALM6, ritzizumab in combination with CART19 demonstrated an exponential increase in antigen-specific CART19 proliferation compared to CART19 plus isotype control IgG (P)<0.0001, fig. 27C). To study CAR-T specific cytotoxicity, CART19 or control UTD T cells were treated with luciferase + CD19⁺NALM6 cell lines were cultured and treated with isotype control antibody or GM-CSF neutralizing antibody (FIG. 27D). GM-CSF neutralizing antibody treatment did not inhibit the ability of CAR-T cells to kill NALM6 target cells (figure 27D). Collectively, these results indicate that ritzilutumab does not inhibit CAR-T cell function in vitro and enhances CART19 cell proliferation in the presence of monocytes, suggesting that GM-CSF neutralization may improve CAR-T cell-mediated efficacy.

Example 19

In vivo GM-CSF neutralization enhances CAR-T cell xenograft models in xenograft models

Xenograft mouse model

NOD-SCID-IL2r gamma for male and female 8-12 week old were generated according to a breeding protocol approved by the Animal Care and Use Committee (IACUC) of the Meiji Clinic organization ^-/-(NSG) mice were bred and cared for within the comparative medical department of the Meiji clinic. Mice were maintained in the animal barrier space approved by IBC for BSL2+ level experiments.

NALM6 cell line xenografts

CD19 was used according to IACUC approved protocol⁺Luciferase⁺ALL NALM6 cell line was used to establish ALL xenografts. Here, Intravenous (IV) injection was performed by tail vein injection at 1X 10⁶And (4) cells. 4-6 days after injection, the mice were bioluminescent imaged using a Xenogen IVIS-200 spectral camera (perkin elmer, hopkinton, ma, usa) to confirm the transplantation. Imaging was performed 10 minutes after Intraperitoneal (IP) injection of 10. mu.l/g D-fluorescein (15mg/mL, St. Louis, Mo.). Mice were then randomized into groups based on bioluminescent imaging of the mice to acceptDifferent treatments as outlined in the specific experiments. Typically, each mouse is injected IV 1-1.5X 10⁶Individual CAR-T cells (and equivalent of total T cell number of Untransformed (UTD) T cells). The transduction efficiency of CAR-T cells is typically about 50%. For example, with 50% transduction efficiency of CAR-T cells, receive 1.5X 10⁶One CAR-T cell mouse received 3 million total T cells and the corresponding UTD mouse received 3 × 10 ⁶And (4) UTD. Imaging was performed weekly to assess and follow disease burden. Bioluminescent images were acquired using a Xenogen IVIS-200 spectral camera (perkin elmer, hopkinton, massachusetts, usa) and analyzed using live Image version 4.4 (Living Image version 4.4) (Caliper life sciences, perkin elmer). Tail vein bleeding was performed 7-8 days after CAR-T cell injection to assess T cell expansion and cytokines and chemokines, and then as needed. Peripheral blood of mice was subjected to red blood cell lysis using BD FACS lysate (BD biosciences, san diego, california, usa) and then used for flow cytometry studies. Antibody-treated mice began daily antibody therapy (10mg/kg litzizumab or isotype control) IP on the same day of CART cell therapy for a total of 10 days.

RNA-Seq on mouse brain tissue

RNA was isolated using miRNeasy mini kit (Qiagen, Gaithersburg, MD, USA) and treated with RNase-free DNase groups. RNA-seq was performed on Illumina HTseq 4000 (Illumina, San Diego, Calif., USA) by the genome analysis core of the Meiji clinic. Binary basic call data is converted to fastq using Illumina bcl2fastq software. Removal of adaptor sequences using trimmatic, as by Bolger, AM et al, Bioinformatics 2014; 30(15) 2114-2120.10.1093/bioinformatics/btu170, which is incorporated herein by reference in its entirety, and FastQC, such as by Leggett Rm et al, "Front genetics" (Front gene.) 2013; 288 as DOI 10.3389/fgene.2013.00288 in month 01 and 02 of 2014 Incorporated herein by reference in its entirety for quality checking purposes. The latest human (GRCh38) and mouse (GRCm38) reference genomes were downloaded from NCBI. Use of STAR to generate a genomic index file, as described by Dobin a et al, bioinformatics 2013; 29(1) 15-21.10.1093/bioinformatics/bts635, which is incorporated herein by reference in its entirety and for each case mapped paired end reads to the genome. HTSeq, as determined by Anders S et al, bioinformatics 2015; 31(2) 166-169 Pre-release at 28 days 09/2014 as DOI 10.1093/bioinformatics/btu638, which is incorporated herein by reference in its entirety, and Deseq2³⁸2014, as by Love Mi et al, "Genome Biol. (Genome Biol.); 15(12) 550 as described in 12/18 th month of 2014 as DOI 10.1186/s13059-014-0550-8, which is herein incorporated by reference in its entirety for the calculation of differential expression. The gene ontology was assessed using Enrichr, as described by kuleshoov Mv et al, Nucleic Acids Research 2016; 44(W1) W90-W97.10.1093/Nar/gkw377, which is incorporated herein by reference in its entirety. FIG. 35 summarizes the steps detailed above. RNA sequencing data are available in the Gene Expression concentrate (Gene Expression Omnibus) under accession number GSE 121591.

Statistics of

Data were analyzed using Prism Graph Pad (ralasia, ca, USA) and Microsoft Excel (Microsoft, Redmond, WA, USA) from Redmond, washington. High cytokine concentrations in the heatmap were normalized to "1" and low concentrations to "0" by Prism. The statistical tests are described in the legend.

Results

To confirm that GM-CSF depletion does not inhibit CART19 effector function, the effect of GM-CSF with ritbruumab on neutralization of CART19 anti-tumor activity was studied in a xenograft model. First, a relapse model aimed at vigorously investigating whether the antitumor activity of CART19 cells was affected by GM-CSF neutralization was used. NOD/SCID/interleukin-2 receptor gamma Null (NSG) mice were treated with 1X 10⁶Luciferase enzyme⁺NALM6 cells were injected, and thenImaging was performed 6 days later, allowing sufficient time for the mice to reach a very high tumor burden. Mice were randomized to receive a single injection of CART19 or UTD cells and 10 days of isotype control antibody or litzlumab (fig. 28A). GM-CSF assays on sera collected 8 days after CART19 injection showed that ritzimab successfully neutralized GM-CSF in the context of CART19 therapy (fig. 28B). Bioluminescence imaging one week after CART19 injection showed that CART19 in combination with ritzimab effectively controlled leukemia and was significantly better than control UTD cells in this high tumor burden recurrence model (fig. 28C-28D). Despite neutralization of GM-CSF levels, treatment with CART19 in combination with rituzumab resulted in potent anti-tumor activity and improved overall survival, similar to CART19 with control antibody, suggesting that GM-CSF does not impair CAR-T cell activity in vivo (figure 36). Second, these experiments were performed in a primary ALL patient-derived xenograft model in the presence of human PBMCs, as this represents a more relevant heterogeneous model. Following treatment with busulfan conditioning chemotherapy, mice were injected with blast cells derived from patients with relapsed ALL. Mice were monitored for transplantation by continuous tail vein bleeding for several weeks and when CD19 in the blood ⁺At approximately 1/microliter blast, starting on the day of CART19 injection, mice were randomly grouped to receive CART19 treatment with PBMCs and in combination with ritvolumab plus anti-mouse GM-CSF neutralizing antibody or isotype control IgG antibody for 10 days (fig. 28E). In this primary ALL xenograft model, GM-CSF neutralization in combination with CART19 therapy resulted in significant improvement of leukemic controls that lasted at least 35 days with time following CART19 administration compared to CART19 plus isotype control (fig. 28F). This suggests that GM-CSF neutralization may play a role in reducing relapse and increasing sustained complete responses following CART19 cell therapy.

Example 20

GM-CSF^k/oGeneration of CART19

Selection of guide RNAs (gRNAs) targeting exon 3 of human GM-CSF by screening previously reported gRNAs with high efficiency for human GM-CSF, e.g., in Sanjana Ne et al, improved vectors and whole genome libraries for CRISPR screening(Improved vectors and genes-with ligands for CRISPR screening) 2014; 11(8): 783-. The gRNA was sequenced in a CAS9 third generation lentivirus construct (lentiCRISPRV2), under the control of the U6 promoter (GenScript, Township, NJ, USA, N.J.). Lentiviral particles encoding this construct were produced as described above. T cells were double transduced with CAR19 and GM-CSFgRNA-lentiCRISPRv2 lentivirus 24 hours after stimulation with CD3/CD28 beads. CAR-T cell expansion then continues as described above. To analyze the efficiency of targeting GM-CSF, PureLink Genomic DNA Mini Kit (Invitrogen, Calsbad, Calif.) was used to determine the efficiency of targeting GM-CSF from GM-CSF ^k/oGenomic DNA was extracted from CART19 cells. The DNA of interest was PCR amplified using Choice Taq Blue Mastermix (Thomas Scientific, Minneapolis, MN, USA) and Gel extracted using the QIAquick Gel Extraction Kit (Kjegen, Hillman, Md.) to determine editing. PCR amplicons were sent for european house sequencing (lewis verval, kentucky, usa) and allele modification frequency was calculated (by resolving follow-up indels) using TIDE, a method that requires only two parallel PCR reactions followed by a pair of standard capillary sequencing analyses; the two sequencing tracks generated were then analyzed using specially designed software provided as a simple website tool and as R-code available at tide. nki. nl, as by Brinkman Ek et al, Easy quantitative assessment of genome editing by sequence trace decomposition (Easy quantitative assessment of genome editing by sequence trace composition) nucleic acid research 2014; 42(22) e168 as published in 11 days 10/2014 as DOI 10.1093/nar/gku936, which is incorporated herein by reference in its entirety. FIG. 34B depicts gRNA sequences and primer sequences, and FIGS. 34A (i) -34A (iii) depict methods for generating GM-CSF ^k/oMode of CART19 mode.

Example 21

GM-CSF CRISPR knock-out CAR-T cells exhibit reduced GM-CSF expression, similar levels of key cytokines and chemokines, and enhanced antitumor activity

To be confident not to include any role of GM-CSF critical in CAR-T cell function, GM-CSF gene was disrupted during CAR-T cell manufacture using grnas that have been reported to generate high efficiency knockouts and cloned into CRISPR lentiviral backbone, as by Sanjana Ne et al, nature 2014; 11(8): 783-. Using this gRNA, a knockout efficiency of about 60% was achieved in CART19 cells (fig. 37). When using CD19⁺When the cell line NALM6 stimulated CAR-T cells, GM-CSF compared to CART19 with the wild-type GM-CSF locus ("wild-type CART19 cells")^k/oCAR-T cells produced statistically significantly less GM-CSF. GM-CSF knock-out in CAR-T cells did not impair the production of other key T cell cytokines including IFN- γ, IL-2 or CAR-T cell antigen specific degranulation (CD107a) (fig. 29A), but did exhibit reduced expression of GM-CSF (fig. 29B). To confirm GM-CSF ^k/oCAR-T cells continued to exhibit normal function, testing their in vivo efficacy in a high tumor burden relapsing xenograft model of ALL (as described in figure 28A). In this xenograft model, M-CSF was utilized 7 days after CART19 treatment^k/oCART19 replaced wild-type CART19 significantly reduced serum levels of human GM-CSF (fig. 29B). Bioluminescent imaging data suggests GM-CSF as compared to CART19^k/oCART19 cells showed an enhanced leukemia control in this model (fig. 38). Importantly, GM-CSF compared to wild-type CART19 cells^k/oCART19 cells showed significant improvement in overall survival (fig. 29C). Human GM-CSF is in GM-CSF as compared to wild-type CART19^k/oStatistically significant reduction by t-test in CART19 cells (fig. 29D). Mouse GM-CSF appeared visually increased, but this was not statistically significant by t-test (P-0.472367) (fig. 29E). This lack of reduction of mouse GM-CSF is not necessarily surprising, since GM-CSF^k/oCART19 cell (which)Is human) is the only cell in the mouse that possesses the knockout, and therefore mouse GMCSF would not likely be directly affected. By visual inspection, mouse IP-10, a chemokine that attracts multiple cell types, including T cells and monocytes, was found to be present in GM-CS as compared to CART19 ^Fk/oCART19 appeared to be paradoxically increasing, but this was also not statistically significant by the t-test, P-0.4877 (fig. 29E). By visual inspection, mouse MIP1 a (an inflammatory cytokine important in neutrophil induction) and mouse M-CSF (a cytokine critical in macrophage differentiation) appeared to be reduced, but it was not statistically significant at P0.2437 and P0.3619 (fig. 29E). In GMCSF, in contrast to CART19 (FIG. 29E)^k/oMouse IL-1b (a key inflammatory cytokine produced by macrophages) and mouse IL-15 (a cytokine produced by macrophages that contributes to NK cell proliferation) in CART19 appeared to be reduced, with P values of 0.0741 and 0.0900 (fig. 29E), respectively. Key human T cell cytokines are not subject to GM-CSF^k/oInhibition (fig. 29D). It should be emphasized that these xenografts were generated with a high load of NALM6 cell line and that the CRS/NI model (FIGS. 30A-30D, 31, 32A-32D and 33A-33D) required the use of primary ALL cells to be generated. Thus, the cytokine profile did not differ surprisingly between the two models, as the NALM6 xenografts (fig. 29A-29E) did not develop CRS or NI. In summary, in the context of the NALM6 high tumor burden model without CRS, the results of FIGS. 29A-29E demonstrate FIGS. 27A-27D and 28A-28F, indicating that GM-CSF depletion does not impair normal cytokines or chemokines critical for CAR-T therapeutic function. In addition, the results in FIGS. 29A to 29E show that GM-CSF ^k/oCART can represent a therapeutic option for the "built in" GM-CSF control as a modification during CAR-T cell manufacture.

Example 22

Patient-derived xenograft models for Neuroinflammation (NI) and cytokine release syndrome/GM-CSF neutralization in vivo improved cytokine release syndrome and neuroinflammation following CART19 therapy in xenograft models

Primary patient derived ALL xenografts

To establish primary ALL xenografts, NSG mice first received 30mg/kg Busulfan IP (Selleckchem, Houston, TX, USA) from Selleckchem, Houston, USA). The following day, mice were injected with 2.5 × 10 of peripheral blood derived from patients with relapsed or refractory ALL⁶And (3) primary mother cells. The mice were monitored for transplantation for about 10-13 weeks. When CD19 was consistently observed in blood⁺Cells (approximately 1 cell/microliter), they were randomly grouped to receive CART19(2.5 x 10) derived from the same donor with or without antibody therapy⁶Individual cell IV) and PBMC (1X 10)⁵Individual cells IV) (10mg/kg of litzizumab or isotype control IP for a total of 10 days, starting from the day it received CAR-T cell therapy). Mice were monitored regularly for leukemic burden by tail vein bleeding.

Primary patient derived ALL xenografts of CRS/NI

Similar to the above experiment, mice were IP injected with 30mg/kg busulfan (Seik chemical Co., Houston, Tex., USA). The following day, mice received 1-3X 10 of peripheral blood from patients with relapsed ALL⁶And (3) primary mother cells. The mice were monitored for transplantation by tail vein bleeding for about 10-13 weeks. When serum CD19⁺When the number of cells is 10 cells/microliter, the mice receive CART19 (2-5X 10)⁶Individual cell IV) and antibody therapy was initiated for a total of 10 days as indicated. Mice were weighed on a daily basis as a measure of their health status. Mouse brain MRI was performed 5-6 days after CART19 injection and tail vein bleedings were performed 4-11 days after CART19 injection for cytokine/chemokine and T cell analysis.

MRI acquisition

A Bruker Avance II 7Tesla vertical borehole small animal MRI system (Bruker Avance II 7Tesla vertical bore MRI system) (Bruker Biospin) was used for image acquisition to assess Central Nervous System (CNS) vascular permeability. Inhalation anesthesia was induced and maintained by 3% to 4% isoflurane. Use of MRI compatible Vital sign monitoring System (model 1030; SA Instruments, Stoni Brooks) during an acquisition session School, new york) monitors the respiration rate. IP injections of gadolinium were given to mice using a weight-based dose of 100mg/kg and after a standard delay of 15 minutes T1-weighted images were obtained using a volume acquisition T1-weighted spin echo sequence (repetition time 150ms, echo time 8ms, field of view: 32mm × 19.2mm × 19.2mm, matrix: 160 × 96 × 96; average 1). Gadolinium enhanced MRI changes indicate blood brain barrier disruption.²⁴The volumetric analysis was performed using an analysis software package developed by biomedical imaging resources of the meiosis clinic.

Results

In this model, regulated NSG mice were transplanted with primary ALL blast cells and the transplantation was monitored for several weeks until they developed a high disease burden (fig. 30A). CD19 in peripheral blood⁺Level of blast cells>At 10/microliter, mice were randomized to receive different treatments as indicated (fig. 30A). Treatment with CART19 (either with control IgG antibody or with GM-CSF neutralizing antibody) successfully eradicated the disease (figure 30B). Within 4-6 days after treatment with CART19, the mice began to develop a motor weakness, a hunched body and a progressive weight loss; symptoms consistent with CRS and NI. This correlates with an increase in key serum cytokines and chemokines 4-11 days after CART19 injection, similar to that seen in human CRS after CAR-T cell therapy (including human GM-CSF, TNF- α, IFN- γ, IL-10, IL-12, IL-13, IL-2, IL-3, IP-10, MDC, MCP-1, MIP-1 α, MIP-1 β, and mouse IL-6, GM-CSF, IL-4, IL-9, IP-10, MCP-1, and MIG). These mice treated with CART19 also developed NI as indicated by brain MRI analysis, indicating abnormal T1 enhancement, suggesting blood brain barrier disruption and possible brain edema (fig. 30C), along with flow cytometric analysis of the harvested brain, indicating infiltration of human CART19 cells (fig. 30D). In addition, RNA-seq analysis of brain sections harvested from mice that developed these signs of NI showed significant up-regulation of genes that regulate T cell receptors, cytokine receptors, T cell immune activation, T cell trafficking, and T cell and myeloid cell differentiation (fig. 31, table 6).

Table 6: table of typical pathways altered in brain from patient-derived xenografts following treatment with CART19 cells in tabular form.

The effect of GM-CSF neutralization on CART19 toxicity was studied using the xenograft patient derived model of NI and CRS shown in fig. 30A. To rule out the co-invasive effect of mouse GM-CSF, mice received CART19 cells in combination with 10 days of GM-CSF antibody therapy (10mg/kg Ritzuzumab and 10mg/kg anti-mouse GM-CSF neutralizing antibody) or isotype control antibody. GM-CSF neutralizing antibody therapy statistically significantly reduced CRS-induced weight loss after CART19 therapy (figure 32A). Cytokine and chemokine analysis 11 days after CART19 cell therapy showed that human GM-CSF was neutralized by antibodies (fig. 32B). In addition, GM-CSF neutralization resulted in significant reductions in several human (IP-10, IL-3, IL-2, IL-1Ra, IL-12p40, VEGF, GM-CSF) (FIG. 32C) and mouse (MIG, MCP-1, KC, IP-10) (FIG. 32D) cytokines and chemokines. Interferon gamma-induced proteins (IP-10, CXCL10) are produced by monocytes and other cell types and serve as chemoattractants for many cell types, including monocytes, macrophages and T cells. IL-3 plays a role in myeloid progenitor cell differentiation. IL-2 is a key T cell cytokine. Interleukin-1 receptor antagonists (IL-1Ra) inhibit IL-1. (IL-1 is produced by macrophages and is a key inflammatory cytokine family.) IL-12p40 is a subunit of IL-12 that is produced by macrophages in other cell types and can promote Th1 differentiation. Vascular Endothelial Growth Factor (VEGF) promotes angiogenesis. Monokines induced by gamma interferon (MIG, CXCL9) are T cell chemoattractants. Monocyte chemoattractant protein 1(MCP-1, CCL2) attracts monocytes, T cells, and dendritic cells. KC (CXCL1) is produced by macrophages and other cell types and attracts myeloid cells, such as neutrophils. There were also several non-statistically significant reductions in other human and mouse cytokines and chemokines following neutralization by GM-CSF. This suggests that GMCSF plays a role in downstream activity of several cytokines and chemokines that are helpful in the cascade of CRS and NI generation.

Brain MRI 5 days after CAR19 treatment showed that GM-CSF neutralization reduced T1 enhancement as a measure of brain inflammation, blood brain barrier disruption, and possible edema compared to CART19 plus control antibody. MRI images after GM-CSF neutralization (with ritzeuzumab and anti-mouse GM-CSF antibody) were similar to baseline pretreatment scans, indicating that GM-CSF neutralization effectively helped abrogate NI associated with CART19 therapy (fig. 33A and 33B). Using human ALL blast cells and human CART19 in this patient-derived xenograft model, GM-CSF neutralization after CART19 reduced neuroinflammation by 75% compared to CART19 plus isotype control (fig. 33B). This is a significant finding and for the first time in vivo it was demonstrated that NI caused by CART19 could be effectively eliminated. Human CD 3T cells were present in the brain following CART19 therapy, and the raw mean differed from the reduction of brain CD 3T cells in the case of GM-CSF neutralization, but did not meet statistical significance, as determined by flow cytometry (fig. 33C, 30D and 39). Finally, differences in the original mean (but this did not reach statistical significance) and the reduction in CD11b + bright macrophages were observed in the brains of mice receiving GM-CSF neutralization during CAR-T cell therapy compared to isotype controls during CAR-T therapy (fig. 33D), which may suggest that GM-CSF neutralization contributes to the reduction of macrophages within the brain.

The results of examples 18-19 and 22 demonstrate that neutralization of GM-CSF eliminates toxicity following CAR-T cell therapy and can enhance its therapeutic activity. In particular, GM-CSF neutralization in combination with CART19 therapy was shown to prevent the development of CRS and significantly reduce the severity of NI in a xenograft model using human ALL blast cells and human CART 19. GM-CSF neutralization results in a reduction in chemokines (such as IP-10, MCP-1, KC and other inflammatory cytokines and chemokines) associated with myeloid trafficking, and is associated with the original mean (but not statistically significant) of the reduction in T cell infiltration and myeloid cell activation in the brain. Interestingly, the experiments herein also show that GM-CSF inhibition enhances CART19 proliferation, antitumor activity and overall survival in vivo. Based on these results, GM-CSF neutralization can be considered as a potential next generation strategy to enable conventional CAR-T cell immunotherapy.

In the studies described herein, neutralization of GM-CSF with Ritzuzumab did not impair any CART19 effector function in vitro. In two different xenograft models (NALM6 xenograft and patient-derived xenograft), despite GM-CSF neutralization, CART19 in combination with ritbruumab effectively eradicated tumors and significantly improved leukemic disease control 35 days post-treatment, while CART19 plus isotype control failed to maintain disease control 35 days post-treatment. Finally, in examples 21-22, GM-CSF ^k/oCART19 cells displayed potent effector functions in vitro and significantly improved overall survival compared to CART19 in vivo.

The CRS and NI models described herein are unique and relevant ALL patient-derived xenograft models to develop toxic therapies after human CAR-T cell therapy. In the model described herein, the time intervals between CAR-T cell infusion to symptom onset, brain MRI changes, cytokine and chemokine elevation, and effector cell infiltration into the CNS were all similar to those reported for patients who developed toxicity following CART19 therapy. Mice develop symptoms of CRS and NI (weight loss, motor function decline and hunched body). Changes in brain MRI were detected 4-6 days after the infusion of CART19 cells. Brain MRI T1 uptake suggests blood brain barrier disruption and possible brain edema, and is comparable to the changes noted on human brain MRI in cases of severe neurotoxicity, as measured by Gust et al, 2017 cancer discovery 2017; 7(12) 1404-.

Interestingly, Gust et al 2017 further described that blood-brain barrier permeability prevents CSF from being protected by systemic cytokines that induce perivascular cell stress and secretion of endothelial activating cytokines, and patients showed evidence of endothelial activation. In the CRS/NI model described herein, GM-CSF was found to be neutralized in the serum of mice receiving CART19 therapy with GM-CSF neutralizing antibodies, compared to CART19 and isotype control antibodies. Thus, T cells in the mouse brain may themselves provide GM-CSF production, and serum GM-CSF, as well as other cytokines and chemokines, may be able to reach CSF. In addition, endothelial cells are capable of producing GM-CSF, which may lead to worsening the circulation. NI is associated with T cell infiltration and activation of myeloid cells in the CNS, similar to CSF changes in patients with CAR-T induced neurotoxicity as well as in non-human primate models. The model described herein is similar to the previously reported patient-derived xenograft model, in which CRS was developed after CAR-T cell therapy. Recent reports have shown that IL-1 blockade prevents NI by depletion of myeloid cells. However, the development of NI in the model was delayed and correlated with meningeal thickening, unlike what was observed in the model described herein and in patients receiving CART19 therapy. Thus, the model described herein is provided as a reliable way to study novel interventions for prevention and treatment of CRS and neurotoxicity following CART19 cell therapy. The results described herein show that GM-CSF neutralization results in a reduction of key myeloid and several inflammatory cytokines and chemokines, suggesting that GM-CSF is a key cytokine in the downstream activation of several cytokines and chemokines; blockade contributes to a reduction in the original mean of myeloid and T cell infiltration in the brain/CNS (but not to statistical significance); and block neuroinflammation which helps reduce apparent neurotoxicity.

Interestingly, an exponential increase in CART19 cell proliferation, enhanced anti-tumor activity and improved overall survival by GM-CSF blockade were observed. For example, following neutralization by GM-CSF, CART19 antigen-specific proliferation was increased in vitro in the presence of monocytes. Furthermore, in ALL patient-derived xenografts, CART19 cells resulted in more durable disease control when combined with litzizumab. In addition, GM-CSF was found^k/oCAR-T cells were more effective in controlling leukemia in NALM6 xenografts and demonstrated improved overall survival. Although the mechanism for enhanced CART effector function following GM-CSF depletion is currently unclear, the results provided herein are comparable to previous onesConsistent with the report, the previous report indicated that monocytes ex vivo damaged T cell expansion and that M2 polarized macrophages inhibited CART19 antigen-specific proliferation. This is an important finding, as improved CAR-T cell proliferation is consistently associated with improved efficacy and response (i.e., overall and complete response rates) in CAR-T clinical trials.

Activated T cells are known to produce GM-CSF. T cells do not have all subunits of the GM-CSF receptor, so GM-CSF is not normally fed back directly on T cells in normal cases, but can be at very high levels in some cases. In contrast, this GM-CSF affects the behavior of many other cell types, including macrophages and dendritic cells. Subsequent activation of these cells results in actions that act to stimulate T cells, such as cytokine production and antigen presentation. T cell stimulation may further drive the production of GM-CSF and other cytokines to act on other cell types such as macrophages and dendritic cells, thereby driving circulation. In CAR-T cell therapy, the large number of activated T cells generated in a very short time line is likely to push this cycle to an extreme situation. The results described herein indicate that blocking GM-CSF helps to prevent this immune hyperstimulation without compromising T cell function, in fact enhancing said cell function. The exact mechanism of enhanced CAR-T cell effector function following GM-CSF blockade is unclear.

Finally, the results provided herein additionally demonstrate GM-CSF^k/oThe development of CART19 cells could represent a new approach for partially controlling GM-CSF production, which could be incorporated into current CAR-T cell manufacturing. These results indicate that these cells function normally and may represent independent therapeutic approaches to enhance the therapeutic window following CAR-T cell therapy. anti-GM-CSF antibodies (such as ritzimab) are clinical stage therapeutic solutions for neutralizing GM-CSF, eliminating both CRS and neuroinflammation of apparent neurotoxicity and potentially improving CAR-T cell function.

The studies described herein represent a significant advance in understanding and preventing toxicity following CAR-T cell therapy. These results strongly suggest that modulation of myeloid cell behavior by GM-CSF blockade contributes to control CAR-T cell-mediated toxicity and reduces its immunosuppressive characteristics, thereby improving leukemia control. These studies illustrate a new approach to eliminate apparent neurotoxicity and neuroinflammation of CRS by GM-CSF neutralization, which also potentially enhances CAR-T cell function.

Example 23

Administration of an anti-GM-CSF monoclonal antibody (Ritzuzumab) significantly reduced neuroinflammation caused by CAR-T therapy and maintained the integrity of the blood brain barrier in a xenograft model

This preclinical study was designed to closely replicate the findings observed in CAR-T clinical trials, and was performed in mice using human Acute Lymphoblastic Leukemia (ALL), human CD19 to target CAR-T (CART19), and human Peripheral Blood Mononuclear Cells (PBMCs).

Primary patient derived ALL xenografts

ALL xenografts were established in mice essentially as described in example 22.

MRI acquisition

The integrity of the BBB can be monitored non-invasively by Magnetic Resonance Imaging (MRI). Conventional MR Contrast Agents (CA) containing gadolinium are used in conjunction with MRI to detect and quantify BBB leakage. Under normal conditions, CA does not cross the entire BBB. However, due to its small size, CA spills from the blood into the brain tissue even when the BBB is partially damaged.

MRI was obtained essentially as described in example 22. Based on T taken before and after CA injection₁The gadolinium enhanced MRI method of weighted images is consistent with the method used in the present preclinical study of ritvolumab and CART19, as described by Ku, Mc et al, methods of molecular biology 2018; 1718:395-408.doi:10.1007/978-1-4939-7531-0_23, which are incorporated herein by reference in their entirety. This gadolinium enhanced MRI method is useful for studying BBB permeability in an in vivo mouse model, and can be readily applied in a variety of experimental disease conditions, including neuroinflammatory disorders, or to assess desirable (undesirable) drug effects.

Confocal microscope

Confocal microscopy was used to assess the damage/disruption of the blood brain barrier (also referred to herein as BBB). This microscopy technique uses spatial filtering to eliminate out-of-focus or flash light in a sample that is thicker than the focal plane; thus, such confocal microscopes offer several advantages over conventional optical microscopes, including controllable depth of field, elimination of image degradation defocus information, and the ability to collect successive optical slices from thick specimens.

Results

BBB integrity is preserved and neuroinflammation is significantly reduced following CAR-T and Ritzuzumab therapy

In this study, MRI images taken on day 5 qualitatively revealed diffuse neuroinflammation using CAR-T therapy, while MRI images taken after the combination of litzizumab + CAR-T showed significantly less neuroinflammation, similar to the untreated control group (see fig. 33A). Quantification of T1 high intensity MRI enhanced by gadolinium showed a significant 75% reduction in neuroinflammation and BBB injury using litzuzumab + CAR-T versus CAR-T + control antibody (fig. 33A). Furthermore, confocal microscopy clearly showed in high resolution images that after CAR-T therapy, BBB was significantly impaired (fig. 40A), consistent with MRI images showing qualitatively diffuse neuroinflammation using CAR-T therapy (see fig. 33A). In contrast, confocal microscopy showed maintenance of the integrity of BBB using litzizumab in combination with CAR-T (fig. 40A), consistent with qualitative and quantitative MRI images taken after the litzizumab + CAR-T combination, which showed significant reduction in neuroinflammation compared to CAR-T plus isotype control. Fig. 40A shows confocal microscope BBB data. Fig. 33A is a confirmatory quantitative MRI of T1-high intensity using gadolinium enhancement, showing three treatment groups: untreated control CART19+ litzizumab versus CART19+ isotype control. Confocal microscopy results are critical as they help explain the pathology of CAR-T induced neuroinflammation. This data suggests that following CAR-T administration, the BBB becomes compromised, allowing massive influx of pro-inflammatory cytokines into the CNS, which is thought to spread neuroinflammation. This data is consistent with data reported in the CAR-T clinical trial. In that At 2X 10⁶In a ZUMA-1 study of intravenously administered Chimeric Antigen Receptor (CAR) -transduced autologous T cells at a target dose of anti-CD 19 CAR T cells per kilogram (yescata), a significant increase in pro-inflammatory cytokines was observed in the CNS of patients developing grade 3+ neurotoxicity. The confocal microscopy data of the present invention help explain why and how addition of litzizumab significantly reduces CAR-T induced neuroinflammation.

MRI images qualitatively showed diffuse neuroinflammation and damage of the BBB on day 5 after CAR-T therapy (CART19) administration. When rituzumab was co-administered with CAR-T, the integrity of the BBB was preserved/maintained, and MRI images taken on day 5 after the combination of rituzumab + CAR-T showed significantly less neuroinflammation, similar to the untreated control group (see figure 33A). Furthermore, confocal microscopy showed that this result is in complete agreement with MRI imaging data showing a 75% reduction in neuroinflammation and BBB injury following rituzumab and CAR-T compared to CAR-T and control antibodies (Y-axis in this analysis is gadolinium enhanced T1 high intensity).

Following CART and litzizumab therapy, the CART19 cell proliferation index increased and leukemia control significantly improved

As described in example 22, administration of litzizumab following CART19 therapy also resulted in an increase in the CART19 cell proliferation index and a significant improvement in leukemia controls that lasted at least 35 days over time following CART19 infusion compared to CART19 plus controls. These results indicate that neutralization of GM-CSF with an anti-GM-CSF monoclonal antibody (ritbrumab) can reduce relapse and increase play a role in a sustained complete response following CART19 therapy. This is a significant finding given that more than 50% of adult lymphoma patients who initially responded to CART19 therapy subsequently relapsed within the first year of follow-up.

FIG. 40B (adapted from Santomasso, BD et al, "on-line priority publication", published 6/7.2018, DOI:10.1158/2159-8290.CD-17-1319, which is incorporated herein by reference in its entirety) shows that high levels of protein in CSF (as shown in the data for Santomasso) indicate BBB disruption and protein leakage into the CNS (due to increased permeability of the blood cerebrospinal fluid (CSF) barrier during neurotoxicity). This suggests that BBB destruction is central to the pathophysiology of NI and links the xenograft model findings provided herein to the clinical findings of Santomasso. In embodiments of the methods provided herein, the methods further comprise performing lumbar puncture (by a clinician) and measuring CSF levels of protein/albumin that can predict NT grade and subsequent clinical expectations of preemptive measures.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Example 24

Gene editing technique for knocking out GM-CSF gene in T cell

Several strategies are being pursued by each group to incorporate gene editing into the development of next generation Chimeric Antigen Receptor (CAR) T cells for use in the treatment of various cancers. Severe toxicity (cytokine release syndrome and neurotoxicity) is associated with CAR T cell therapy and may lead to poor patient outcomes. The key initiator in the toxicity process appears to be the CART cell-derived GM-CSF.

Gene editing (with, for example, engineered nucleases) can be used for the KO GM-CSF gene in T cells and/or genes encoding proteins necessary for GM-CSF gene expression. Nucleases that can be used for such genome editing include, but are not limited to, CRISPR-associated (Cas) nucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector (TALE) nucleases, and Homing Endonucleases (HE) (also known as meganucleases).

Zinc finger nucleases for GM-CSF

The GM-CSF gene in CART cells can be inactivated using Zinc Finger Nuclease (ZFN) technology. DNA sequence specific nucleases cleave the GM-CSF gene and DNA double strand break repair results in inactivation of the gene. Sequence-specific nucleases were generated by combining a sequence-specific DNA binding domain (zinc finger) with the Fok1 endonuclease domain. The targeted nuclease acts as a dimer and employs two different DNA recognition domains to provide site-specific cleavage. Engineering of the Fok1 endonuclease ensures that heterodimers are formed rather than homodimers. Thus, the obligate heterodimer Fok1-EL variant provides a higher level of specificity.

To date, the clinical experience of the gene KO approach using ZFN technology has been limited. However, in small safety studies using ZFN technology to knock out CCR5 receptors and reintroduction of T cells into HIV patients, there is a significant survival advantage for modified versus unmodified cells when antiretroviral drug therapy is discontinued.

The best effect was observed when biallelic gene disruption was achieved. This indicates that the KO technique that achieves the greatest% gene disruption rate is probably the most effective (Singh 2017, Tebas 2014). In some human cell types, biallelic targeting efficiency was increased by RAD51 overexpression and valproic acid treatment (Takayama 2017).

Exons 1-4 of the human GM-CSF gene can be targeted with ZFNs that form pairs within selected target regions. A potential advantage of targeting the access to the translation initiation codon in a DNA sequence is that it ensures that gene knockouts do not result in large protein fragments that are still synthesized. Such protein fragments may have undesirable biological activity.

A variety of tools are available to identify potential Zinc Finger Nuclease (ZFN) sites in a specific target sequence. Examples of such tools can be found at the following websites:http://bindr.gdcb.iastate.edu/ZiFiT/. The vectors used to express the ZFN pairs identified in this way (for the GM-CSF gene KO) were tested in human cells expressing GM-CSF, and the effectiveness of the gene disruption of each pair was measured by the changes in GM-CSF production within the cell bank. The ZFN pair that demonstrated the highest reduction in GM-CSF levels was selected for testing in human CART cells.

For example, autologous T cells can be transduced ex vivo with a replication-defective recombinant Ad5 viral vector encoding a GM-CSF-specific ZFN pair, resulting in modification of the GM-CSF gene. The vector supports only transient expression of the gene encoded by the vector. These two ZFNs bind to a complex bp sequence that is specifically found in the region (within exons 1, 2, 3 or 4) selected for mutagenesis of the GM-CSF gene. Expression of GM-CSF specific ZFNs induces double strand breaks in cellular DNA that are repaired by the cellular machinery, resulting in random sequence insertions or deletions in the transduced cells. These insertions and deletions disrupt the GM-CSF coding sequence, resulting in frame shift mutations and termination of protein expression.

T cell manufacturing/patient specific samples

Study subjects underwent 10 liter leukopheresis for collection>10⁹And (4) white blood cells. Leukapheresis products were depleted of monocytes by countercurrent centrifugation elutriation and enriched for CD4+ cells by magnetic depletion of CD8+ T cells, both using a disposable closed system disposable device. The resulting enriched CD4+ T cells were activated with anti-CD 3/anti-CD 28 mAb coated paramagnetic beads and transduced with vectors encoding CAR T and vectors encoding ZFN. The cells were then expanded and cultured in a closed system. T cell expansion continues after transfer to the WAVE bioreactor for additional expansion under perfusion conditions. At the end of the incubation period, the cells were depleted of magnetic beads, washed, concentrated and cryopreserved.

Primary T cells can also be treated with other agents, such as valproic acid, in order to increase the biallelic targeting efficiency of ZFNs.

Putative targeting sequences

Exon 1

ATG TGG CTG CAG AGC CTG CTG CTC TCG GGC

TAC ACC GAC GTC TCG GAC GAC GAG AGC CCG

CTC GCC CAG CCC CAG CAC GCA GCC

GAG CGG GTC GGG GTC GTG CGT CGG

Exon 2

AAT GAA ACA GTA GAA GTC ATC TCA GAA ATG

TTA CTT TGT CAT CTT CAG TAG AGT CTT TAC

GAA GTC ATC TCA GAA ATG TTT GAC

CTT CAG TAG AGT CTT TAC AAA CTG

Design exon 3

GAG CCG A CC TGC CTA CAG ACC CGC CTG GAG

CTC GGC TGG ACG GAT GTC TGG GCG GAC CTC

GCC TAC AGA CCCGCCT GGA GCT GTA

CGG ATG TCT GGGCGGA CCT CGA CAT

Exon 4

GAA ACT TCC TGT GCA ACC CAG ATT ATC ACC

CTT TGA AGG ACA CGT TGG GTC TAA TAG TGG

TGC AAC CCA GAT TATC ACC TTT GAA

ACG TTG GGT CTA ATAG TGG AAA CTT

TALENS

The GM-CSF gene in T cells can also be inactivated using a transcription activator-like effector nuclease (TALENS). TALENS is similar to ZFNs in that it includes a Fok1 nuclease domain fused to a sequence-specific DNA binding domain. The targeted nuclease then creates a double-strand break in the DNA and is error prone to repair the mutated target gene. TALENS can be easily designed using a simple protein-DNA code that uses DNA to bind TALE (transcription activator-like effector) repeat domains to a single base in the binding site. The robustness of TALENs means that genome editing is a reliable and easy process (Reyon d. et al, 2012 "natural biotechnology" 5 month 2012; 30(5):460-5.doi:10.1038/nbt.2170, which is incorporated herein by reference in its entirety).

For example, some TALE target sequences within exon 1 of the human GM-CSF gene are:

1.TGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCA

2.TTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCA

examples of TALE target sequences in exon 4 of the human GM-CSF gene:

1.TGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGA

2.TCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAA

3.TTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCA

CRISPR Cas-9 mediates GM-CSF gene KO in primary T cells.

CRISPR (clustered regularly interspaced short palindromic repeats), Cas-9 system consists of Cas9, an RNA-guided nuclease and a short guide RNA (grna), which promotes the generation of site-specific DNA breaks that are repaired by cellular endogenous mechanisms. Delivery of Cas9/gRNA RNP to primary human T cells resulted in highly efficient target gene modification. CRISPR/Cas 9-mediated methods for knocking out GM-CSF genes are described by detailed Protocols, see Oh, s.a., Seki, a., & Rutz, S. (2018) & Immunology laboratory guidelines in Immunology, 124, e69.doi:10.1002/cpim.69, and Seki and Rutz, journal of experimental medicine, 2018, volume 215, phase 3985-.

Inactivation of GM-CSF by the gene KO reportedly reduces the cytokine release syndrome and neurotoxicity of CAR T-treated mice bearing tumor xenografts and improves the antitumor activity of the mice (as by Sterner Rm et al, 2018, blood 2018: blood-2018-10-881722; doi: https:// doi.org/10.1182/blood-2018-10-881722), which are incorporated herein by reference in their entirety.

Inactivation of the GM-CSF gene by CRISPR methods targeting exon 1 or 2 or 3 or 4.

Multiple (e.g., 3) Cas9 constructs targeting 3 different sequences within the GM-CSF gene may be used in order to ensure effective gene inactivation in all samples. This is easily accomplished with CRISPR compared to other gene editing methods.

Cas9 was used to report high frequencies of biallelic KO (as by Zhang, y. et al,. Methods) in 2014 for 9 months; 69(2) 171-178. doi:10.1016/j.ymeth.2014.05.003, which are incorporated herein by reference in their entirety). This high frequency of biallelic KO provides a potential advantage.

Additional Gene silencing techniques for GM-CSF KO in CAR T cells

Other methods that can be used for gene silencing are well known to those of ordinary skill in the art and can include, but are not limited to, Homing Endonucleases (HEs) (also known as meganucleases), RNA interference (RNAi), short interfering RNS (siRNA), DNA directed RNA interference (ddRNAi).

A combination of GM-CSF gene KO in CAR T cells and neutralizing antibodies for non-CAR T-derived GM-CSF.

Removal/neutralization of all GM-CSF in patients requires anti-GM-CSF antibodies or anti-receptor antibodies or soluble receptor-Fc fusions to be used in combination with the GM-CSF gene KO in CART cells (these combinations need to be claimed).

All publications, accession numbers, patents, and patent applications cited in this specification are herein incorporated by reference as if each were specifically and individually indicated to be incorporated by reference.

Exemplary V of anti-GM-CSF antibodies of the invention_HThe sequence of the region:

SEQ ID NO:1(VH #1, FIG. 1)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRRDRFPYYFDYWGQGTLVTVSS

SEQ ID NO:2(VH #2, FIG. 1)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRDRFPYYFDYWGQGTLVTVSS

SEQ ID NO 3(VH #3, FIG. 1)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRQRFPYYFDYWGQGTLVTVSS

SEQ ID NO 4(VH #4, FIG. 1)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCVRRQRFPYYFDYWGQGTLVTVSS

SEQ ID NO:5(VH #5, FIG. 1)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCVRRQRFPYYFDYWGQGTLVTVSS

Exemplary V of anti-GM-CSF antibodies of the invention_LThe sequence of the region:

SEQ ID NO 6(VK #1, FIG. 1)

EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNRSPLTFGGGTKVEIK

SEQ ID NO 7(VK #2, FIG. 1)

EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNKSPLTFGGGTKVEIK

SEQ ID NO 8(VK #3, FIG. 1)

EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNRSPLTFGGGTKVEIK

SEQ ID NO 9(VK #4, FIG. 1)

EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNKSPLTFGGGTKVEIK

Exemplary kappa constant region of SEQ ID NO 10

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Exemplary heavy chain constant region of SEQ ID NO 11, f-allotype:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

Claims (342)

1. A method for neutralizing and/or removing the human GM-CSF (hGM-CSF) of a subject treated with a CAR-T cell, the method comprising administering to the subject a method with GM-CSF Gene-inactivated or gene-knocked CAR-T cells (GM-CSF ^k/o CAR-T cells). 2. The method of claim 1, further comprising administering to the subject a recombinant hGM-CSF antagonist. 3. The method of claim 2, wherein the recombinant hGM-CSF antagonist is an anti-hGM-CSF antibody. 4. The method of claim 3, wherein the anti-hGM-CSF antibody is a recombinant antibody fragment that is a Fab, Fab', F(ab')2, scFv, Fv, or dAB. 5. The method of claim 4, wherein the anti-hGM-CSF antibody has the VH region sequence shown in Figure 1 and the VL region sequence shown in Figure 1 . 6. The method of claim 4, wherein the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence comprise a methionine at the N-terminus. 7. The method of claim 2, wherein the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, adnectins, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 8. The method of claim 7, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 9. The method of claim 1, wherein the GM-CSF is CAR T-derived GM-CSF and/or non-CAR T-derived GM-CSF. 10. The method of claim 1, wherein the subject develops immunotherapy-related toxicity. 11. The method of claim 1, further comprising prophylactically administering to the subject (a) GM-CSF ^k/o CAR-T cells and /or (b) an anti-hGM-CSF antagonist, wherein following prophylactic administration of (a) and/or (b), the subject is prevented from developing immunotherapy-related toxicity. 12. The method of claim 1, wherein the subject is treated with immunotherapy comprising administration of GM-CSF ^k/o CAR-T cells. 13. A method for GM-CSF gene inactivation or GM-CSF knockout (KO) in a cell, the method comprising targeted genome editing or GM-CSF gene silencing. 14. The method of claim 13, further comprising an endonuclease as a nucleic acid cleaving enzyme. 15. The method of claim 14, wherein the endonuclease is Fokl restriction enzyme or Flap Endonuclease 1 (FEN-1). 16. The method of claim 14, wherein the endonuclease is Cas9 CRISPR-associated protein 9 (Cas9). 17. The method of claim 13, wherein the GM-CSF gene inactivation by CRISPR/Cas9 targets and edits exon 1, exon 2, exon 3, or exon 4 GM-CSF gene. 18. The method of claim 13, wherein the GM-CSF gene inactivation comprising CRISPR/Cas9 targets and edits the GM-CSF gene at exon 3. 19. The method of claim 13, wherein the GM-CSF gene inactivation comprising CRISPR/Cas9 targets and edits the GM-CSF gene at exon 1. 20. The method of claim 14, wherein the GM-CSF gene inactivation comprises a plurality of CRISPR/Cas9 enzymes, wherein each Cas9 enzyme targets and edits exon 1, exon 2, exon 3 or different sequences of the GM-CSF gene at exon 4. 21. The method of claim 14, wherein the GM-CSF gene inactivation comprises biallelic CRISPR/Cas9 targeting and knockout/inactivation of the GM-CSF gene. 22. The method of claim 21, further comprising treating primary T cells with valproic acid to enhance biallelic knockout/inactivation. 23. The method of claim 13, wherein the targeted genome editing comprises zinc finger (ZnF) proteins. 24. The method of claim 13, wherein the targeted genome editing comprises transcription activator-like effector nucleases (TALENS). 25. The method of claim 13, wherein the targeted genome editing comprises a homing endonuclease, wherein the homing endonuclease is an ARC nuclease (ARCUS) or a meganuclease. 26. The method of claim 13, wherein the targeted genome editing comprises a flap endonuclease (FEN-1). 27. The method of claim 13, wherein the cells are CAR T cells. 28. The method of claim 27, wherein the CAR T cells are CD19 CAR-T cells. 29. The method of claim 27, wherein the CAR T cells are BCMA CAR-T cells. 30. The method of claim 13, wherein the GM-CSF gene silencing is selected from the group consisting of RNA interference (RNAi), short interfering RNS (siRNA), and DNA-directed RNA interference (ddRNAi). 31. A method for preventing or reducing immunotherapy-related toxicity, the method comprising administering to the subject a CAR-T cell with GM-CSF gene inactivation or GM-CSF knockout (GM-CSF ^{k /o} CAR-T cells), wherein the GM-CSF gene is inactivated or knocked out by the method according to any one of claims 13 to 26. 32. The method of claim 31, wherein the immunotherapy-related toxicity is a CAR-T-related toxicity selected from cytokine release syndrome, neurotoxicity, or a combination thereof. 33. The method of claim 31, further comprising administering the GM-CSF K/O CAR T cells in combination with an hGM-CSF antagonist. 34. The method of claim 33, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 35. The method of claim 31, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, Fv, scFv, or dAB. 36. The method of claim 35, wherein the anti-hGM-CSF antibody has the VH region sequence shown in Figure 1 and the VL region sequence shown in Figure 1 . 37. The method of claim 36, wherein the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence comprise a methionine at the N-terminus. 38. The method of claim 37, wherein the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 39. The method of claim 38, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 40. A method for reducing the rate of recurrence or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy, the method comprising administering to the subject a recombinant GM-CSF antagonist. 41. The method of claim 40, wherein the immunotherapy comprises adoptive cell transfer, administration of monoclonal antibodies, administration of cytokines, administration of cancer vaccines, T cell conjugation therapy, or any combination thereof. 42. The method of claim 40, wherein the subject has diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and follicular lymphoma DLBCL due to lymphoma. 43. The method of claim 41, wherein the adoptive cell transfer comprises administering chimeric antigen receptor expressing T cells (CAR T cells), T cell receptor (TCR) modified T cells, tumor infiltrative Lymphocytes (TILs), chimeric antigen receptor (CAR) modified natural killer cells or dendritic cells or any combination thereof. 44. The method of claim 40, wherein the recombinant GM-CSF antagonist is an hGM-CSF antagonist. 45. The method of claim 40, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 46. The method of claim 45, wherein the anti-GM-CSF antibody binds to human GM-CSF. 47. The method of claim 45, wherein the anti-GM-CSF antibody binds to primate GM-CSF. 48. The method of claim 47, wherein the primate is selected from the group consisting of monkeys, baboons, macaques, chimpanzees, gorillas, lemurs, lorises, tarsiers, bush monkeys, koalas, Crested lemur, great lemur, lemur or great ape. 49. The method of claim 45, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF. 50. The method of claim 45, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 51. The method of claim 50, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 52. The method of claim 50, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, Fv, scFv, or dAB. 53. The method of claim 50, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 54. The method of claim 50, wherein the anti-hGM-CSF antibody is a recombinant antibody, a humanized antibody, a CDR-grafted antibody, or a chimeric antibody. 55. The method of claim 50, wherein the anti-hGM-CSF antibody is a human antibody. 56. The method of any one of claims 50-55, wherein the anti-hGM-CSF antibody binds the same epitope as chimeric 19/2. 57. The method of any one of claims 50-55, wherein the anti-hGM-CSF antibody comprises VH region CDR3 and VL region CDR3 of chimeric 19/2. 58. The method of any one of claims 50 to 55, wherein the anti-hGM-CSF antibody comprises the VH regions CDR1, CDR2 and CDR3 and the VL regions CDR1, CDR2 and CDR3 of chimeric 19/2. 59. The method of any one of claims 50 to 55, wherein the anti-hGM-CSF antibody comprises: a VH region comprising a CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V segment, wherein The J segment comprises at least 95% identity to human JH4 (YFD YWGQGTL VTVSS) and the V segment comprises at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or comprises CDR3 Binds to the VH region of the specificity determinant RQRFPY. 60. The method of claim 59, wherein the J segment comprises YFDYWGQGTLVTVSS. 61. The method of any one of claims 59 or 60, wherein the CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. 62. The method of any one of claims 59 or 60, wherein the VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2 is a human germline VH1 CDR2; or both the CDR1 and the CDR2. All were derived from human germline VH1 sequences. 63. The method according to any one of claims 59 or 60, wherein the anti-hGM-CSF antibody comprises VH CDR1 or VH CDR2 or VH CDR1 and VH CDR2 as shown in the VH region as shown in Figure 1 both. 64. The method of any one of claims 59 or 60, wherein the V segment sequence has the VH V segment sequence shown in Figure 1 . 65. The method of any one of claims 59 or 60, wherein the VH has the sequence of VH#1, VH#2, VH#3, VH#4 or VH#5 shown in Figure 1 . 66. The method of any one of claims 50-55, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising the amino acid sequence FNK or FNR. 67. The method of claim 66, wherein the anti-hGM-CSF antibody comprises a human germline JK4 region. 68. The method of claim 66 or claim 67, wherein the VL region CDR3 comprises QQFN(K/R)SPLT. 69. The method of claim 68, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. 70. The method of claim 66, wherein the VL region comprises CDRl or CDR2 or both CDRl and CDR2 of the VL region shown in Figure 1 . 71. The method of claim 66, wherein the VL region comprises a V segment that is at least 95% identical to the VKIIIA27 V segment sequence shown in Figure 1 . 72. The method of claim 66, wherein the VL region has the sequence of VK#1, VK#2, VK#3, or VK#4 shown in Figure 1 . 73. The method of any one of claims 50-55, wherein the anti-hGM-CSF antibody has a VH region CDR3 binding specificity determinant RQRFPY or RDRFPY and a VL region with a CDR3 comprising QQFNKSPLT. 74. The method of any one of claims 50 to 55, wherein the anti-hGM-CSF antibody has the VH region sequence shown in Figure 1 and the VL region sequence shown in Figure 1 . 75. The method of any one of claims 50 to 55, wherein the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence comprise methyl sulfide at the N-terminus amino acid. 76. The method of claim 44, wherein the hGM-CSF antagonist is selected from the group consisting of: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM- CSF peptide analogs, antibody-mimicking protein drugs, lipocalin scaffolding antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 77. The method of claim 76, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 78. The method of claim 43, wherein the CAR-T cells are CD19 CAR-T cells. 79. The method of claim 40, wherein the reducing the rate of relapse in the subject or preventing the occurrence of tumor relapse in the subject occurs in the absence of immunotherapy-related toxicity. 80. The method of claim 40, wherein said reducing the rate of recurrence in said subject or said preventing the occurrence of tumor recurrence in said subject occurs in the presence of the occurrence of immunotherapy-related toxicity. 81. The method of claim 80, wherein the immunotherapy-related toxicity is a CAR-T-related toxicity. 82. The method of claim 81, wherein the CAR-T-related toxicity is cytokine release syndrome, neurotoxicity, or neuroinflammation. 83. The method of claim 40, wherein one year after administration of the recombinant GM-CSF antagonist occurs compared to the occurrence of tumor recurrence in a subject treated with immunotherapy and not administered a recombinant GM-CSF antagonist In the top quartile, the incidence of tumor recurrence was reduced from 50% to 100%. 84. The method of claim 40, wherein the incidence of tumor recurrence is reduced from 50% to 95% in the first half year following administration of the recombinant GM-CSF antagonist. 85. The method of claim 40, wherein the preventive effect of the occurrence of tumor recurrence persists for 12 to 36 months. 86. The method of claim 40, wherein the complete preventive effect (100%) of the occurrence of tumor recurrence persists for about 10 years. 87. The method of claim 40, wherein the subject has acute lymphoblastic leukemia. 88. The method of claim 40, further comprising administering the GM-CSF K/O CAR T cells in combination with an hGM-CSF antagonist. 89. A method for reducing the levels of cytokines or chemokines other than GM-CSF in a subject treated with immunotherapy (and developing immunotherapy-related toxicity), the method comprising in the administering a recombinant hGM-CSF antagonist to the subject before or during immunotherapy, wherein compared to the level of the cytokine or the chemokine in the subject during the occurrence of the immunotherapy-related toxicity , the level of the cytokine or the chemokine is reduced. 90. The method of claim 89, wherein the immunotherapy comprises adoptive cell transfer, administration of monoclonal antibodies, administration of a cancer vaccine, T cell conjugation therapy, or any combination thereof. 91. The method of claim 90, wherein the adoptive cell transfer comprises administering chimeric antigen receptor expressing T cells (CAR T cells), T cell receptor (TCR) modified T cells, tumor infiltrative Lymphocytes (TILs), chimeric antigen receptor (CAR) modified natural killer cells or dendritic cells or any combination thereof. 92. The method of claim 91, wherein the CAR-T cells are CD19 CAR-T cells or BCMA CAR-T cells. 93. The method of claim 89, wherein the recombinant GM-CSF antagonist is a hGM-CSF antagonist. 94. The method of claim 90, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 95. The method of claim 90, wherein the anti-GM-CSF antibody binds to human GM-CSF. 96. The method of claim 90, wherein the anti-GM-CSF antibody binds to primate GM-CSF. 97. The method of claim 90, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF. 98. The method of claim 90, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 99. The method of claim 94, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 100. The method of claim 94, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, Fv, scFv, or dAB. 101. The method of claim 94, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 102. The method of claim 94, wherein the anti-hGM-CSF antibody is a recombinant antibody, a humanized antibody, a CDR-grafted antibody, or a chimeric antibody. 103. The method of claim 94, wherein the anti-hGM-CSF antibody is a human antibody. 104. The method of claim 89, wherein the cytokine or the chemokine is a human cytokine or chemokine selected from the group consisting of: IFN-γ, GRO, MDC, IL-2, IL-3, IL-5, IL-7, IP-10, CD107a, TNF-a, IL-1Ra, FGF-2, IL-12p40, IL-12p70, sCD40L, VEGF, MCP-1, MIP-1a, MIP-1b and combinations thereof. 105. The method of claim 89, wherein the cytokine or the chemokine is selected from the group consisting of: IFN-γ, IL-1a, IL-1b, IL-2, IL-3, IL -4, IL-5, IL-6, IL7, IL-9, IL-10, IL-12p40, IL-12p70, ILF, IL-13, LIX, IL-15, IP-10, KC, MCP-1 , MIP-1a, MIP-1b, M-CSF, MIP-2, MIG, RANTES, TNF-a, eotaxin, G-CSF, IL-1Ra, FGF-2, sCD40L and its combination. 106. The method of claim 89, wherein the subject has acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma cell lymphoma or DLBCL caused by follicular lymphoma. 107. The method of claim 89, wherein the hGM-CSF antagonist is selected from the group consisting of: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 108. The method of claim 107, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 109. A method for treating or preventing immunotherapy-related toxicity in a subject, the method comprising administering to the subject a chimeric antigen receptor expressing T cell (CAR-T cell), the CAR -T cells with GM-CSF gene inactivation or gene knockout (GM-CSF ^k/o CAR-T cells). 110. The method of claim 109, wherein the level of GM-CSF expressed by the GM-CSF ^k/o CAR-T cell is reduced compared to the level of GM-CSF expressed by the wild-type CAR-T cell . 111. The method of claim 109, wherein the level of one or more cytokines and/or chemokines expressed by the GM-CSF ^k/o CAR-T cells is lower than or equal to that produced by wild-type CAR-T cells. The level of one or more cytokines and/or chemokines expressed by T cells. 112. The method of claim 109, wherein the one or more cytokines are human cytokines and/or chemokines selected from the group consisting of: IFN-γ, GRO, MDC, IL-2 , IL-3, IL-5, IL-7, IP-10, CD107a, TNF-a, IL-1Ra, FGF-2, IL-12p40, IL-12p70, sCD40L, VEGF, MCP-1, MIP-1a , MIP-1b and combinations thereof. 113. The method of claim 109, wherein the one or more cytokines are selected from the group consisting of: IFN-γ, IL-1a, IL-1b, IL-2, IL-3, IL- 4. IL-5, IL-6, IL7, IL-9, IL-10, IL-12p40, IL-12p70, ILF, IL-13, LIX, IL-15, IP-10, KC, MCP-1, MIP-1a, MIP-1b, M-CSF, MIP-2, MIG, RANTES, TNF-a, eotaxin, G-CSF, IL-IRa, FGF-2, sCD40L, and combinations thereof. 114. The method of claim 109, wherein the CAR-T cells are CD19 CAR-T cells or BCMACAR-T cells. 115. The method of claim 109, wherein the GM-CSF ^k/o CAR-T cells improve relapse rates compared to relapse rates in subjects treated with wild-type CAR-T cells. 116. The method of claim 109, wherein the GM-CSF ^k/o CAR-T cells improve the objective response rate (complete response and partial answer). 117. The method of claim 109, wherein the GM-CSF ^k/o CAR-T cells improve the GM-CSF k/o CAR-T cells compared to progression-free survival in subjects treated with wild-type CAR-T cells. Progression-free survival of subjects. 118. The method of claim 109, wherein the subject has acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma cell lymphoma or DLBCL caused by follicular lymphoma. 119. The method of claim 109, wherein the GM-CSF ^k/o CAR-T cells enhance the antitumor activity of the recombinant hGM-CSF antagonist. 120. The method of claim 109, wherein the GM-CSF ^k/o CAR-T cells improve the subject compared to survival in a subject treated by administering wild-type CAR-T cells. overall survival rate. 121. The method of claim 109, wherein the subject has acute lymphoblastic leukemia. 122. The method of claim 109, further comprising administering a recombinant hGM-CSF antagonist. 123. The method of claim 122, wherein the recombinant GM-CSF antagonist is an hGM-CSF antagonist. 124. The method of claim 122, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 125. The method of claim 124, wherein the anti-GM-CSF antibody binds to human GM-CSF. 126. The method of claim 124, wherein the anti-GM-CSF antibody binds to primate GM-CSF. 127. The method of claim 124, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF. 128. The method of claim 124, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 129. The method of claim 128, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 130. The method of claim 128, wherein the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab', F(ab')2, scFv, or dAB. 131. The method of claim 128, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 132. The method of claim 128, wherein the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. 133. The method of claim 128, wherein the anti-hGM-CSF antibody is a human antibody. 134. The method of claim 123, wherein the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 135. The method of claim 134, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 136. A method for reducing the rate of recurrence or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy, the method comprising administering to the subject a recombinant hGM-CSF antagonist. 137. The method of claim 136, wherein the subject has acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma cell lymphoma or DLBCL caused by follicular lymphoma. 138. The method of claim 136, wherein the subject has refractory/relapsed cancer that is non-Hodgkin lymphoma (non-Hodgkin lymphoma). NHL) or chemotherapy-refractory B-cell lymphoma. 139. The method of claim 136, wherein the recombinant GM-CSF antagonist is an hGM-CSF antagonist. 140. The method of claim 136, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 141. The method of claim 136, wherein the anti-GM-CSF antibody binds to human GM-CSF. 142. The method of claim 136, wherein the anti-GM-CSF antibody binds to primate GM-CSF. 143. The method of claim 136, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF. 144. The method of claim 136, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 145. The method of claim 144, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 146. The method of claim 144, wherein the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab', F(ab')2, scFv, or dAB. 147. The method of claim 144, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 148. The method of claim 144, wherein the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. 149. The method of claim 144, wherein the anti-hGM-CSF antibody is a human antibody. 150. The method of claim 139, wherein the hGM-CSF antagonist is selected from the group consisting of: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 151. The method of claim 150, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 152. The method of claim 136, further comprising administering the GM-CSF K/O CAR T cells in combination with an hGM-CSF antagonist. 153. The method of claim 139, wherein the GM-CSF K/O CAR T cells have GM-CSF gene inactivation or GM-CSF knockout (GM-CSF ^k/o CAR-T cells), wherein The GM-CSF gene is inactivated or knocked out by the method according to any one of claims 12 to 25. 154. A method for reducing blood-brain barrier disruption in a subject treated with immunotherapy, the method comprising administering to the subject a recombinant GM-CSF antagonist. 155. The method of claim 154, wherein the subject develops immunotherapy-related toxicity. 156. The method of claim 154, wherein the immunotherapy comprises adoptive cell transfer, administration of monoclonal antibodies, administration of cytokines, administration of cancer vaccines, T cell conjugation therapy, or any combination thereof. 157. The method of claim 156, wherein the adoptive cell transfer comprises administering chimeric antigen receptor expressing T cells (CAR T cells), T cell receptor (TCR) modified T cells, tumor infiltrative Lymphocytes (TILs), chimeric antigen receptor (CAR) modified natural killer cells or dendritic cells or any combination thereof. 158. The method of claim 157, wherein the CAR T cells are CD19 CAR-T cells or BCMACAR-T cells. 159. The method of claim 154, wherein the recombinant GM-CSF antagonist is an hGM-CSF antagonist. 160. The method of claim 154, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 161. The method of claim 160, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF or binds to primate GM-CSF. 162. The method of claim 161, wherein the primate is a monkey, baboon, macaque, chimpanzee, gorilla, lemur, loris, tarsier, bush monkey, koala monkey, crested lemur, Great Lemur, Lemur, Great Ape or Human. 163. The method of claim 159, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 164. The method of claim 163, wherein the anti-hGM-CSF antibody is administered before, concurrently with, after, or a combination of immunotherapy. 165. The method of claim 163, wherein the anti-hGM-CSF antibody binds to human GM-CSF. 166. The method of claim 163, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 167. The method of claim 163, wherein the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab', F(ab')2, scFv, or dAB. 168. The method of claim 163, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 169. The method of claim 163, wherein the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. 170. The method of claim 163, wherein the anti-hGM-CSF antibody is a human antibody. 171. The method of any one of claims 163-170, wherein the anti-hGM-CSF antibody binds the same epitope as the chimeric 19/2 antibody. 172. The method of any one of claims 163 to 170, wherein the anti-hGM-CSF antibody comprises VH region CDR3 and VL region CDR3 of a chimeric 19/2 antibody. 173. The method of any one of claims 163 to 170, wherein the anti-hGM-CSF antibody comprises the VH region CDRl, antibody CDR2 and CDR3 and VL regions CDRl, CDR2 and CDR3 of the chimeric 19/2 antibody. 174. The method of any one of claims 163 to 170, wherein the anti-hGM-CSF antibody comprises: a VH region comprising a CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V segment, wherein The J segment comprises at least 95% identity to human JH4 (YFD YWGQGTL VTVSS) and the V segment comprises at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or comprises CDR3 Binds to the VH region of the specificity determinant RQRFPY. 175. The method of claim 174, wherein the J segment comprises YFDYWGQGTLVTVSS. 176. The method of any one of claims 174 or 175, wherein the CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. 177. The method of any one of claims 174 or 175, wherein the VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2 is a human germline VH1 CDR2; or both the CDR1 and the CDR2. All were derived from human germline VH1 sequences. 178. The method of any one of claims 174 or 175, wherein the anti-hGM-CSF antibody comprises VH CDR1 or VH CDR2 or VH CDR1 and VH CDR2 as shown in the VH region as shown in Figure 1 both. 179. The method of any one of claims 174 or 175, wherein the V segment sequence has the VH V segment sequence shown in Figure 1. 180. The method of any one of claims 174 or 175, wherein the VH has the sequence of VH#1, VH#2, VH#3, VH#4 or VH#5 shown in Figure 1 . 181. The method of claim 163, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising the amino acid sequence FNK or FNR. 182. The method of claim 181, wherein the anti-hGM-CSF antibody comprises a human germline JK4 region. 183. The method of claim 181 or claim 182, wherein the VL region CDR3 comprises QQFN(K/R)SPL. 184. The method of claim 183, wherein the anti-hGM-CSF antibody comprises a VL region comprising CDR3 comprising QQFNKSPLT. 185. The method of claim 181, wherein the VL region comprises CDRl or CDR2 or both CDRl and CDR2 of the VL region shown in Figure 1 . 186. The method of claim 181, wherein the VL region comprises a V segment that is at least 95% identical to the VKIIIA27 V segment sequence shown in Figure 1 . 187. The method of claim 181, wherein the VL region has the sequence of VK#1, VK#2, VK#3, or VK#4 shown in Figure 1 . 188. The method of any one of claims 163 to 170, wherein the anti-hGM-CSF antibody has a VH region CDR3 binding specificity determinant RQRFPY or RDRFPY and a VL region with a CDR3 comprising QQFNKSPLT. 189. The method of claim 188, wherein the anti-hGM-CSF antibody has the VH region sequence shown in FIG. 1 and the VL region sequence shown in FIG. 1 . 190. The method of claim 188, wherein the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence comprise a methionine at the N-terminus. 191. The method of claim 159, wherein the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 192. The method of claim 155, wherein the immunotherapy-related toxicity is a CAR-T-related toxicity. 193. The method of claim 155, wherein the CAR-T-related toxicity is cytokine release syndrome, neurotoxicity, neuroinflammation, or a combination thereof. 194. The method of claim 159, wherein the hGM-CSF antagonist is selected from the group consisting of: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 195. The method of claim 194, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 196. A method for preventing blood brain barrier integrity in a subject treated with immunotherapy, the method comprising administering to the subject a recombinant hGM-CSF antagonist. 197. The method of claim 196, wherein the recombinant hGM-CSF antagonist is an anti-GM-CSF antibody. 198. The method of claim 197, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF or binds to primate GM-CSF. 199. The method of claim 198, wherein the primate is a monkey, baboon, macaque, chimpanzee, gorilla, lemur, loris, tarsier, bush monkey, koala monkey, crested lemur, Great Lemur, Lemur, Great Ape or Human. 200. The method of claim 197, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 201. The method of claim 200, wherein the anti-hGM-CSF antibody is administered before, concurrently with, after, or a combination of immunotherapy. 202. The method of claim 200, wherein the anti-hGM-CSF antibody binds to human GM-CSF. 203. The method of claim 200, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 204. The method of claim 200, wherein the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab', F(ab')2, scFv, or dAB. 205. The method of claim 200, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 206. The method of claim 200, wherein the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. 207. The method of claim 200, wherein the anti-hGM-CSF antibody is a human antibody. 208. The method of any one of claims 200-207, wherein the anti-hGM-CSF antibody binds the same epitope as the chimeric 19/2 antibody. 209. The method of any one of claims 200 to 207, wherein the anti-hGM-CSF antibody comprises VH region CDR3 and VL region CDR3 of a chimeric 19/2 antibody. 210. The method of any one of claims 200 to 207, wherein the anti-hGM-CSF antibody comprises the VH region CDRl, antibody CDR2 and CDR3 and VL regions CDRl, CDR2 and CDR3 of the chimeric 19/2 antibody. 211. The method of any one of claims 200 to 207, wherein the anti-hGM-CSF antibody comprises: a VH region comprising a CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V segment, wherein The J segment comprises at least 95% identity to human JH4 (YFD YWGQGTL VTVSS) and the V segment comprises at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or comprises CDR3 Binds to the VH region of the specificity determinant RQRFPY. 212. The method of claim 211, wherein the J segment comprises YFDYWGQGTLVTVSS. 213. The method of any one of claims 211 or 212, wherein the CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. 214. The method of any one of claims 211 or 212, wherein the VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2 is a human germline VH1 CDR2; or both the CDR1 and the CDR2. All were derived from human germline VH1 sequences. 215. The method of any one of claims 211 or 212, wherein the anti-hGM-CSF antibody comprises VH CDR1 or VH CDR2 or VH CDR1 and VH CDR2 as shown in the VH region as shown in Figure 1 both. 216. The method of any one of claims 211 or 212, wherein the V segment sequence has the VH V segment sequence shown in Figure 1 . 217. The method of any one of claims 211 or 212, wherein the VH has the sequence of VH#1, VH#2, VH#3, VH#4 or VH#5 shown in Figure 1 . 218. The method of claim 200, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising the amino acid sequence FNK or FNR. 219. The method of claim 218, wherein the anti-hGM-CSF antibody comprises a human germline JK4 region. 220. The method of claim 218 or claim 219, wherein the VL region CDR3 comprises QQFN(K/R)SPL. 221. The method of claim 220, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. 222. The method of claim 220, wherein the VL region comprises CDRl or CDR2 or both CDRl and CDR2 of the VL region shown in Figure 1 . 223. The method of claim 218, wherein the VL region comprises a V segment that is at least 95% identical to the VKIIIA27 V segment sequence shown in Figure 1 . 224. The method of claim 218, wherein the VL region has the sequence of VK#1, VK#2, VK#3, or VK#4 shown in Figure 1 . 225. The method of any one of claims 200 to 207, wherein the anti-hGM-CSF antibody has a VH region CDR3 binding specificity determinant RQRFPY or RDRFPY and a VL region with a CDR3 comprising QQFNKSPLT. 226. The method of claim 225, wherein the anti-hGM-CSF antibody has the VH region sequence shown in Figure 1 and the VL region sequence shown in Figure 1 . 227. The method of claim 225, wherein the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence comprise a methionine at the N-terminus. 228. The method of claim 196, wherein the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 229. The method of claim 196, wherein the subject has immunotherapy-related toxicity. 230. The method of claim 229, wherein the immunotherapy-related toxicity is a CAR-T-related toxicity. 231. The method of claim 230, wherein the CAR-T-related toxicity is cytokine release syndrome, neurotoxicity, neuroinflammation, or a combination thereof. 232. The method of claim 196, wherein the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 233. The method of claim 232, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 234. A method for reducing or preventing CAR-T cell therapy-induced neuroinflammation in a subject in need thereof, the method comprising administering to the subject a recombinant hGM-CSF antagonist. 235. The method of claim 234, wherein administering the recombinant hGM-CSF antagonist reduces disruption of the blood-brain barrier, thereby maintaining its integrity. 236. The method of claim 235, wherein reducing the disruption of the blood-brain barrier reduces or prevents influx of proinflammatory cytokines into the central nervous system. 237. The method of claim 236, wherein the pro-inflammatory cytokine is a human cytokine selected from the group consisting of: IFN-γ, GRO, MDC, IL-2, IL-3, IL-5 , IL-7, IP-10, CD107a, TNF-a, IL-IRa, FGF-2, IL-12p40, IL-12p70, sCD40L, VEGF, MCP-1, MIP-1a, MIP-1b, and combinations thereof. 238. The method of claim 236, wherein the pro-inflammatory cytokine is selected from the group consisting of: IFN-γ, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL7, IL-9, IL-10, IL-12p40, IL-12p70, ILF, IL-13, LIX, IL-15, IP-10, KC, MCP-1, MIP- 1a, MIP-1b, M-CSF, MIP-2, MIG, RANTES, TNF-a, eotaxin, G-CSF, IL-IRa, FGF-2, sCD40L, and combinations thereof. 239. The method of claim 234, wherein neuroinflammation in the subject is reduced by 75% to 95% compared to a subject treated with CAR-T cell therapy and a control antibody. 240. The method of claim 239, wherein the 75% to 95% reduction in neuroinflammation is similar to neuroinflammation in untreated control subjects. 241. The method of claim 234, wherein chimeric antigen receptor expressing T cells (CAR T cells) are administered to the subject. 242. The method of claim 234, wherein the subject is administered T cell receptor (TCR) modified T cells, tumor infiltrating lymphocytes (TIL), chimeric antigen receptor (CAR) modified T cells Natural killer cells or dendritic cells or any combination thereof. 243. The method of claim 241, wherein the CAR T cells are CD19 CAR-T cells or BCMACAR-T cells. 244. The method of claim 234, wherein the recombinant hGM-CSF antagonist is an hGM-CSF antagonist. 245. The method of claim 234, wherein the recombinant hGM-CSF antagonist is an anti-GM-CSF antibody. 246. The method of claim 245, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF or binds to primate GM-CSF. 247. The method of claim 245, wherein the primate is a monkey, baboon, macaque, chimpanzee, gorilla, lemur, loris, tarsier, bush monkey, koala monkey, crested lemur, Great Lemur, Lemur, Great Ape or Human. 248. The method of claim 246, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 249. The method of claim 248, wherein the anti-hGM-CSF antibody is administered before, concurrently, after, or a combination of immunotherapy. 250. The method of claim 248, wherein the anti-hGM-CSF antibody binds to human GM-CSF. 251. The method of claim 248, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 252. The method of claim 248, wherein the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab', F(ab')2, scFv, or dAB. 253. The method of claim 248, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 254. The method of claim 248, wherein the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. 255. The method of claim 248, wherein the anti-hGM-CSF antibody is a human antibody. 256. The method of any one of claims 248-255, wherein the anti-hGM-CSF antibody binds the same epitope as the chimeric 19/2 antibody. 257. The method of any one of claims 248 to 255, wherein the anti-hGM-CSF antibody comprises VH region CDR3 and VL region CDR3 of a chimeric 19/2 antibody. 258. The method of any one of claims 248 to 255, wherein the anti-hGM-CSF antibody comprises the VH region CDRl, antibody CDR2 and CDR3 and VL regions CDRl, CDR2 and CDR3 of the chimeric 19/2 antibody. 259. The method of any one of claims 248 to 255, wherein the anti-hGM-CSF antibody comprises: a VH region comprising a CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V segment, wherein The J segment comprises at least 95% identity to human JH4 (YFD YWGQGTL VTVSS) and the V segment comprises at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or comprises CDR3 Binds to the VH region of the specificity determinant RQRFPY. 260. The method of claim 259, wherein the J segment comprises YFDYWGQGTLVTVSS. 261. The method of any one of claims 259 or 260, wherein the CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. 262. The method of any one of claims 259 or 260, wherein the VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2 is a human germline VH1 CDR2; or both the CDR1 and the CDR2. All were derived from human germline VH1 sequences. 263. The method of any one of claims 259 or 260, wherein the anti-hGM-CSF antibody comprises VH CDR1 or VH CDR2 or VH CDR1 and VH CDR2 as shown in the VH region as shown in Figure 1 both. 264. The method of any one of claims 259 or 260, wherein the V segment sequence has the VH V segment sequence shown in Figure 1. 265. The method of any one of claims 259 or 260, wherein the VH has the sequence of VH#1, VH#2, VH#3, VH#4 or VH#5 shown in Figure 1 . 266. The method of claim 248, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising the amino acid sequence FNK or FNR. 267. The method of claim 248, wherein the anti-hGM-CSF antibody comprises a human germline JK4 region. 268. The method of claim 266 or claim 267, wherein the VL region CDR3 comprises QQFN(K/R)SPL. 269. The method of claim 268, wherein the anti-hGM-CSF antibody comprises a VL region comprising CDR3 comprising QQFNKSPLT. 270. The method of claim 268, wherein the VL region comprises CDRl or CDR2 or both CDRl and CDR2 of the VL region shown in Figure 1 . 271. The method of claim 268, wherein the VL region comprises a V segment that is at least 95% identical to the VKIIIA27 V segment sequence shown in Figure 1 . 272. The method of claim 268, wherein the VL region has the sequence of VK#1, VK#2, VK#3, or VK#4 shown in Figure 1 . 273. The method of any one of claims 248 to 255, wherein the anti-hGM-CSF antibody has a VH region CDR3 binding specificity determinant RQRFPY or RDRFPY and a VL region with a CDR3 comprising QQFNKSPLT. 274. The method of claim 273, wherein the anti-hGM-CSF antibody has the VH region sequence shown in FIG. 1 and the VL region sequence shown in FIG. 1 . 275. The method of claim 268, wherein the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence comprise a methionine at the N-terminus. 276. The method of claim 234, wherein the hGM-CSF antagonist is selected from the group consisting of: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 277. The method of claim 234, wherein the subject further has a CAR-T-related toxicity selected from cytokine release syndrome, neurotoxicity, or a combination thereof. 278. The method of claim 234, wherein the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. hGM-CSF antagonists 279. The method of claim 278, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 280. A method for preventing or reducing blood-brain barrier disruption in a subject treated with immunotherapy, the method comprising administering to the subject a CAR-T cell (GM-CSF knockout) having a GM-CSF gene knockout. -CSF ^k/o CAR-T cells). 281. The method of claim 280, further comprising administering to the subject a recombinant hGM-CSF antagonist. 282. The method of claim 281, wherein the recombinant GM-CSF antagonist is an hGM-CSF antagonist. 283. The method of claim 282, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 284. The method of claim 283, wherein the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab', F(ab')2, scFv, or dAB. 285. The method of claim 283, wherein the anti-hGM-CSF antibody has the VH region sequence shown in FIG. 1 and the VL region sequence shown in FIG. 1 . 286. The method of claim 283, wherein the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence comprise a methionine at the N-terminus. 287. The method of claim 282, wherein the hGM-CSF antagonist is selected from the group comprising: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 288. The method of claim 280, wherein the subject further has a CAR-T-related toxicity selected from cytokine release syndrome, neurotoxicity, or a combination thereof. 289. The method of claim 282, wherein the hGM-CSF antagonist is selected from the group consisting of: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody Mimics, hGM-CSF peptide analogs, mimetic antibody protein drugs, lipocalin scaffold antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 290. The method of claim 289, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 291. The method of claim 280, further comprising administering the GM-CSF K/O CAR T cell in combination with a hGM-CSF antagonist, wherein by the method of any one of claims 12 to 25 The GM-CSF gene is inactivated or knocked out. 292. A method for blocking or reducing hGM-CFS expression in a subject treated with immunotherapy to prevent or treat immunotherapy-related toxicity, the method comprising administering to the subject a recombinant GM-CSF antagonist agent. 293. The method of claim 292, wherein the immunotherapy comprises adoptive cell transfer, administration of monoclonal antibodies, administration of cytokines, administration of cancer vaccines, T cell conjugation therapy, or any combination thereof. 294. The method of claim 293, wherein the adoptive cell transfer comprises administering chimeric antigen receptor expressing T cells (CAR T cells), T cell receptor (TCR) modified T cells, tumor infiltrative Lymphocytes (TILs), chimeric antigen receptor (CAR) modified natural killer cells or dendritic cells or any combination thereof. 295. The method of claim 292, wherein the recombinant GM-CSF antagonist is an hGM-CSF antagonist. 296. The method of claim 292, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 297. The method of claims 292-294, wherein the anti-GM-CSF antibody binds to human GM-CSF. 298. The method of claim 296, wherein the anti-GM-CSF antibody binds to primate GM-CSF. 299. The method of claim 298, wherein the primate is selected from the group consisting of monkey, baboon, macaque, chimpanzee, gorilla, lemur, slow loris, tarsier, bush monkey, koala monkey, crested lemur , great lemur, lemur or great ape. 300. The method of claim 296, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF. 301. The method of claim 296, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 302. The method of claim 301, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 303. The method of claim 301, wherein the anti-hGM-CSF antibody is an antibody fragment that is a Fab, Fab', F(ab')2, scFv, or dAB. 304. The method of claim 301, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 305. The method of claim 301, wherein the anti-hGM-CSF antibody is a recombinant antibody or a chimeric antibody. 306. The method of claim 301, wherein the anti-hGM-CSF antibody is a human antibody. 307. The method of any one of claims 301-302, wherein the anti-hGM-CSF antibody binds the same epitope as chimeric 19/2. 308. The method of any one of claims 301-302, wherein the anti-hGM-CSF antibody comprises a VH region CDR3 and a VL region CDR3 of chimeric 19/2. 309. The method of any one of claims 301 to 302, wherein the anti-hGM-CSF antibody comprises the VH regions CDRl, CDR2 and CDR3 and the VL regions CDRl, CDR2 and CDR3 of chimeric 19/2. 310. The method of any one of claims 301 to 302, wherein the anti-hGM-CSF antibody comprises: a VH region comprising a CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V segment, wherein The J segment comprises at least 95% identity to human JH4 (YFD YWGQGTL VTVSS) and the V segment comprises at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or comprises CDR3 Binds to the VH region of the specificity determinant RQRFPY. 311. The method of claim 310, wherein the J segment comprises YFDYWGQGTLVTVSS. 312. The method of any one of claims 310 or 311, wherein the CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. 313. The method of any one of claims 310 or 311, wherein the VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2 is a human germline VH1 CDR2; or both the CDR1 and the CDR2. All were derived from human germline VH1 sequences. 314. The method of any one of claims 310 or 311, wherein the anti-hGM-CSF antibody comprises VH CDR1 or VH CDR2 or VH CDR1 and VH CDR2 as shown in the VH region as shown in Figure 1 both. 315. The method of any one of claims 310 or 311, wherein the V segment sequence has the VH V segment sequence shown in Figure 1 . 316. The method of any one of claims 310 or 311, wherein the VH has the sequence of VH#1, VH#2, VH#3, VH#4, or VH#5 shown in Figure 1 . 317. The method of any one of claims 301-306, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising the amino acid sequence FNK or FNR. 318. The method of claims 313-315, wherein the anti-hGM-CSF antibody comprises a human germline JK4 region. 319. The method of claim 317 or claim 318, wherein the VL region CDR3 comprises QQFN(K/R)SPLT. 320. The method of claim 319, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. 321. The method of claim 317, wherein the VL region comprises CDRl or CDR2 or both CDRl and CDR2 of the VL region shown in Figure 1 . 322. The method of claim 317, wherein the VL region comprises a V segment that is at least 95% identical to the VKIIIA27 V segment sequence shown in Figure 1 . 323. The method of claim 317, wherein the VL region has the sequence of VK#1, VK#2, VK#3, or VK#4 shown in Figure 1 . 324. The method of any one of claims 301 to 306, wherein the anti-hGM-CSF antibody has a VH region CDR3 binding specificity determinant RQRFPY or RDRFPY and a VL region with a CDR3 comprising QQFNKSPLT. 325. The method of any one of claims 301 to 306, wherein the anti-hGM-CSF antibody has the VH region sequence shown in FIG. 1 and the VL region sequence shown in FIG. 1 . 326. The method of any one of claims 301 to 306, wherein the VH region amino acid sequence or the VL region amino acid sequence or both the VH region amino acid sequence and the VL region amino acid sequence comprise methylsulfide at the N-terminus amino acid. 327. The method of claim 295, wherein the hGM-CSF antagonist is selected from the group consisting of: anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM- CSF peptide analogs, antibody-mimicking protein drugs, lipocalin scaffolding antibody mimetics, calixarene antibody mimetics, and antibody-like binding peptide mimetics. 328. The method of claim 327, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 329. The method of claim 292, wherein the CAR-T cells are CD19 CAR-T cells. 330. The method of claim 292, wherein blocking or reducing hGM-CFS expression further reduces the recurrence rate of the experimenter or prevents the occurrence of tumor recurrence in the experimenter, wherein the reduction in the experimenter The recurrence rate in the subject or the prevention of tumor recurrence in the subject occurs in the absence of immunotherapy-related toxicity. 331. The method of claim 329, wherein said reducing the rate of relapse in said subject or said preventing the occurrence of tumor relapse in said subject occurs in the presence of immunotherapy-related toxicity. 332. The method of claim 330, wherein the immunotherapy-related toxicity is a CAR-T-related toxicity. 333. The method of claim 331, wherein the CAR-T-related toxicity is cytokine release syndrome, neurotoxicity, or neuroinflammation. 334. The method of claim 330, one year after administration of the recombinant GM-CSF antagonist, compared to the occurrence of tumor recurrence in a subject treated with immunotherapy and not administered a recombinant GM-CSF antagonist. In the top quartile, the incidence of tumor recurrence was reduced from 50% to 100%. 335. The method of claim 330, wherein the incidence of tumor recurrence is reduced from 50% to 95% in the first half year following administration of the recombinant GM-CSF antagonist. 336. The method of claim 330, wherein the incidence of tumor recurrence is reduced from 50% to 90% in the first year following administration of the recombinant GM-CSF antagonist. 337. The method of claim 330, wherein the occurrence of recurrence of the tumor is prevented long-term. 338. The method of claim 330, wherein the preventive effect of the occurrence of tumor recurrence persists for 12 to 36 months. 339. The method of claim 292, wherein recurrence of the tumor is completely prevented (100%). 340. The method of claim 292, wherein the subject has acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma cell lymphoma or DLBCL caused by follicular lymphoma. 341. The method of claim 292, wherein the subject has acute lymphoblastic leukemia. 342. The method of claim 292, further comprising administering GM-CSFK/O CAR T cells in combination with an hGM-CSF antagonist, wherein by performing GM-CSF gene inactivation or GM-CSF knockout in the cell ( KO) methods for inactivating or knocking out the GM-CSF gene comprising targeted genome editing or GM-CSF gene silencing.

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