HK1201852B - Anti-cd134 (ox40) antibodies and uses thereof - Google Patents

Description

anti-CD 134(OX40) antibodies and uses thereof

Technical Field

The present invention relates to antibodies, uses of such antibodies, and in particular to antibodies that bind CD134, which are useful for treating cancer.

Background

Enhancing antitumor T cell function represents a unique approach to the treatment of cancer. There is a great deal of evidence that tumor cells "evade" the immune system by inducing active immune tolerance mediated primarily by regulatory T lymphocytes (Treg; Quezda et al. Immunol Rev 2011; 241: 104-118). Therefore, the balance between effector (i.e. direct or indirect eradication of tumor cells) T lymphocytes (Teff) and tolerogenic (i.e. inhibition of Teff effector function and survival) tregs appears to be crucial for effective anti-tumor immunotherapy. In other words, an effective anti-tumor immune response may be obtained by enhancing the effector function of tumor-specific Teff and/or by attenuating the suppressive function of tumor-specific tregs. The CD134(OX40) receptor has been shown to be a key receptor mediating these responses (Sugamura, K, Ishii, N, Weinberg, A. therapeutic targeting of the effector T-cell co-stimulation receptor OX40.Nature RevImm 2004; 4: 420-431).

CD134 (also known as OX40, TNFRSF4 and ACT35) is a member of the tumor necrosis factor receptor superfamily. This CD134 surface costimulatory receptor is expressed on activated T lymphocytes and plays an important role in their survival and function. CD134 expressing T lymphocytes have been demonstrated in the draining lymph nodes of various human malignancies and cancer patients (Ramstadet al. am J Surg 2000; 179: 400-406; Vetto et al. am J Surg 1997; 174: 258-265).

In tumor-bearing mice, in vivo ligation of the mouse CD134 receptor (via soluble mouse OX40 ligand (OX40L) -immunoglobulin fusion protein or mouse OX40L mimetic, such as anti-mouse CD 134-specific antibody) enhances anti-tumor immunity, resulting in tumor-free survival in mouse models of various mouse malignant cell lines, such as lymphoma, melanoma, sarcoma, colon cancer, breast cancer, and glioma (Sugamura et al. Nature Rev Imm 2004; 4: 420-431).

It has been suggested to enhance the immune response to an antigen in mammals by binding OX40R with an OX40R binding agent (WO 99/42585; Weinberg, 2000). Although this document mentions OX40 binding agents in general, the focus is on using OX40L or parts thereof; anti-OX 40 antibodies are disclosed as equivalents to OX 40L. Indeed, when the Weinberg group used this study in a study with non-human primates, they again intentionally selected antibodies that bind to the OX40L binding site and mimic OX40L in general.

Al-Shamkhani et Al (Eur J Chem 1996; 26:1695-1699) used an anti-OX 40 antibody, called OX86, which did not block OX40L binding to explore the differential expression of OX40 on activated mouse T cells; Hirschhorn-Cymerman et al (J Exp Med 2009; 206: 1103-. However, OX86 is not expected to bind to human OX40, and when selecting an antibody that is effective in humans, one would select an antibody that binds to the OX40L binding site according to the Weinberg study.

In Severe Combined Immunodeficiency (SCID) mice, in vivo ligation of the human CD134 receptor (by anti-human CD134 specific antibodies which interact with the OX40L binding domain on human CD 134; US2009/0214560a1) enhances anti-tumor immunity, which results in tumor growth inhibition of various human malignant tumor cell lines such as lymphoma, prostate, colon and breast cancers.

The precise mechanism of the anti-tumor immune response mediated by human CD134 ligation has not been demonstrated in humans, but is thought to be mediated via the CD134 transmembrane signaling pathway, which is stimulated by interaction with OX 40L. This interaction is mediated by binding of trimeric OX40L to CD 134. Trimerized OX40 ligand is recommended as a more potent drug than anti-OX 40 antibody in current anti-cancer treatments (Morris et al. mol Immunol 2007; 44: 3112-3121).

Summary of The Invention

The applicant has now surprisingly found that, in order to induce T cell mediated anti-tumour activity, the use of an isolated binding molecule which binds human CD134, wherein the binding molecule does not prevent the binding of human CD134(CD134) receptor to OX40 ligand (OX40L), results in an enhanced immune response characterised by enhancing the immunostimulatory/effector function of T effector cells and/or proliferating these cells and/or down regulating the immunosuppressive function of T regulatory cells.

The invention thus provides isolated binding molecules that bind to human CD134, wherein the binding molecules do not prevent binding of human CD134(OX40) receptor to OX40 ligand (OX 40L).

Such binding molecules include suitable anti-CD 134 antibodies, antigen-binding fragments of anti-CD 134 antibodies, and derivatives of anti-CD 134 antibodies. In some embodiments, the binding molecule is at 1x10^-7K of M or less_dBinds to human CD 134. The binding molecules have agonist activity for human CD134 on T effector cells and/or antagonist activity for human CD134 on T regulatory cells. In some further embodiments, the binding molecule is at a K of 100nM or less, preferably less than 50nM, more preferably less than 20nM_dA human monoclonal antibody that specifically binds to human CD 134.

The invention also provides compositions comprising one or more binding molecules and a pharmaceutically acceptable carrier. In some embodiments, the binding molecule is a human monoclonal anti-CD 134 antibody or antigen-binding fragment thereof. The composition may further comprise additional agents such as immunotherapeutic agents, chemotherapeutic agents, and hormonal therapy agents.

The invention further provides diagnostic and therapeutic methods using the binding molecules. In some embodiments, there is provided a method of treating or preventing cancer in a mammal comprising administering to the mammal a therapeutically effective amount of a binding molecule or composition comprising a binding molecule disclosed herein. In some other embodiments, provided herein are methods of enhancing an immune response in a mammal comprising administering to the mammal a therapeutically effective amount of a binding molecule or a composition comprising a binding molecule. In certain embodiments, the binding molecule used in the methods is a human monoclonal anti-CD 134 antibody or antigen-binding fragment thereof that binds to human CD134, wherein the antibody does not prevent human CD134(OX40) receptor binding to OX40 ligand (OX 40L).

The invention further provides nucleic acid molecules encoding the amino acid sequences of the binding molecules, vectors comprising such nucleic acids, host cells comprising said vectors and methods of making said binding molecules.

Other aspects are also provided herein, which are apparent from the entire disclosure, including the claims.

Drawings

The invention is described with reference to the accompanying drawings:

FIG. 1: time course and dose effects of exposure to PHA-M on surface human CD134 expression of human T lymphocytes.

FIG. 2: human CD134 expression on resting (resting) and PHA-M activated human CD4T lymphocytes.

FIG. 3: binding characteristics of mouse anti-human CD134 antibodies clone ACT35, clone 12H3, and clone 20E5 on PHA-M stimulated human CD134 expressing T lymphocytes.

FIG. 4: the binding of mouse anti-human CD134 antibodies clone 12H3 and clone 20E5 on PHA-M stimulated CD4T and CD8T lymphocytes expressing human CD 134.

FIG. 5: cross-competition of unlabeled mouse anti-human CD134 antibody clone 12H3 or clone 20E5 with PE-conjugated commercial mouse anti-CD 134 antibody clone ACT35 or clone L106 on PHA-M stimulated human CD134 expressing T lymphocytes.

FIG. 6: simultaneous binding of the mouse anti-human CD134 antibody clone 12H3 or clone 20E5 to human OX40L on PHA-M stimulated human CD134 expressing T lymphocytes.

FIG. 7: exposure to anti-human CD 3/anti-human CD28 clones stimulated the time course effect of beads on surface human CD134 expression of human effector T lymphocytes (Teff) and regulatory T lymphocytes (tregs).

FIG. 8: dose effect of exposure to mouse anti-human CD134 antibody clone 12H3 or clone 20E5 or exposure to human OX40L on PHA-M stimulated human CD134 expressing T lymphocytes.

FIG. 9: the effect of combining mouse anti-human CD134 antibody clone 12H3 with human OX40L or combining mouse anti-human CD134 antibody clone 20E5 with human OX40L on PHA-M stimulated proliferation of human CD134 expressing T lymphocytes.

FIG. 10: effect of exposure to mouse anti-human CD134 antibody clone 12H3 or clone 20E5 or to human OX40L on anti-human CD 3/anti-human CD28 antibody stimulated proliferation of human CD134 expressing human effector T lymphocytes.

FIG. 11: effect of exposure to mouse anti-human CD134 antibody clone 12H3 or clone 20E5 or to human OX40L on human CD134 expressing human CD134 to stimulate bead stimulation with anti-human CD 3/anti-human CD28 antibody to modulate proliferation of T lymphocytes.

FIG. 12: effect of mouse anti-human CD134 antibody clone 12H3 on human OX 40L-mediated anti-human CD 3/anti-human CD28 antibody stimulated bead-stimulated human effects expressing human CD134 (a) and modulating (B) proliferation of T lymphocytes.

FIG. 13: effect of exposure to mouse anti-human CD134 antibody clone 12H3 or clone 20E5 or to human OX40L on human CD134 expressing human regulatory T lymphocyte mediated inhibition of human CD134 expressing human effector T lymphocyte proliferation.

FIG. 14: binding of chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 on human CD134 expressing CD4T and CD8T lymphocytes stimulated by CD3/CD28 beads (without and with IL-2).

FIG. 15: effect of chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 or human OX40L on PHA-M stimulated proliferation of human CD134 expressing T lymphocytes.

FIG. 16: dose effect of exposure to chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 or to human OX40L on PHA-M stimulated proliferation of human CD134 expressing T lymphocytes.

FIG. 17: the effect of combining chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 with human OX40L on PHA-M stimulated proliferation of human CD134 expressing T lymphocytes.

FIG. 18: effect of chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 or human OX40L on proliferation of human CD134 expressing T lymphocytes stimulated by (without and with IL-2) CD3/CD28 beads.

FIG. 19: binding of mouse anti-human CD134 antibody clones 12H3 and 20E5 to non-reduced and reduced recombinant human CD134 human Fc γ fusion proteins. (A) Non-reducing (a, b) and reducing (c, d) conditions. (B) Recombinant human CD134 stained with Coomassie Brilliant blue human Fc gamma fusion protein (rhuCD134) migration patterns by electrophoresis under non-reducing (a, b) and reducing (c, d) conditions. (C) Western blot of non-reduced (a, b) and reduced (c, d) recombinant human CD134: human Fc γ fusion proteins exposed to either mouse IgG1 kappa isotype control antibody (mIgG1) or to mouse anti-human CD134 antibody clones 12H3 and 20E5 (m 12H3 and m20E5, respectively).

FIG. 20: schematic representation of cysteine-rich domains (CRDs) in full-length human CD134 (designated "CRD 1") and various truncated human CD134 forms (designated "CRD 2", "CRD 3", "CRD 4", and "truncated (tc) CRD 4").

FIG. 21: mouse anti-human CD134 antibody clones 12H3 and 20E5 bound on the 239-F cell line transiently transfected with either a full-length human CD134 construct (designated "CRD 1") or with various truncated human CD134 constructs (designated "CRD 2", "CRD 3", "CRD 4" and "truncated (tc) CRD 4").

FIG. 22: binding of chimeric human IgG4 κ and/or IgG1 κ anti-human CD134 antibody clones 12H3 and 20E5 on 239-F cell lines transiently transfected with either full-length human CD134 constructs (referred to as "CRD 1") or with various truncated human CD134 constructs (referred to as "CRD 2", "CRD 3", "CRD 4" and "truncated (tc) CRD 4").

FIG. 23: mouse anti-human CD134 antibody clone 12H3(A) and chimeric human IgG4 kappa anti-human CD134 antibody clone 12H3(B) bind to human CD134 derived peptides corresponding to the amino acid sequence of the truncated CRD3A 1-module-CRD 4 subdomain A1-module (according to the definition of Latza et al Eur J Immunol 1994; 24: 677-683).

Description of the invention

T cell activation is mediated not only by antigenic stimulation through the T cell receptor, but also by costimulatory signals through costimulatory molecules. Among some costimulatory molecules, the Tumor Necrosis Factor (TNF) receptor family member OX40(CD134) plays a key role in the survival and homeostasis of effector and memory T cells. According to the traditional understanding of OX40 co-stimulation, an interaction between OX40 and OX40 ligand (OX40L) occurs when activated T cells bind to professional Antigen Presenting Cells (APC). T cell functions include cytokine production, expansion and survival followed by enhancement of co-stimulatory signals by OX40. During T cell-Dendritic Cell (DC) interactions, i.e., 2-3 days post antigen recognition, an interaction occurs between OX40 and OX 40L. OX40 expressing T cells may also interact with OX40L expressing cells other than DCs and receive OX40 signals from the cells that can provide key signals for generating memory T cells, enhancing Th2 responses and prolonging inflammatory responses. Thus, the optimal interaction between OX40 and OX40L can be formed in two steps: OX40L expressed on activated CD4T cells interacts with OX40 expressed on other responsive (responder) CD4T cells, leading to optimal production of memory CD4T cells (Soroosh et al, 2006), or OX40L expressed on CD4+ accessory cells (accession cells) can promote Th2 cell survival through interaction with OX40 on Th2 cells (Kim et al, 2003). In addition, OX40L expression on B cells was required for development of Th2 in vivo, but not Th1 (Linton et al 2003), and OX 40L-expressing mast cells directly enhanced effector T cell function through interaction between OX40 on T cells and OX40L on mast cells (Kashiwakura et al JImmunal 2004; 173: 5247-. In addition, because endothelial cells also express OX40L (Imura et al 1996), OX40 bound to endothelial cells may be involved in vascular inflammation. Excess OX40 signaling for responding T cells and T regulatory cells inhibits Treg-mediated immunosuppression. OX40 signals transmitted into responder T cells render them resistant to Treg-mediated suppression. On the other hand, OX40 signaling into Treg cells directly inhibits Treg suppressive function, although there is controversy as to whether OX40 signaling can control Foxp3 expression levels in Treg cells. In addition, intentional OX40 stimulated TFG- β dependent differentiation of suppressor iTreg cells (inducible Treg cells). The inhibition may be mediated in part by effector cytokines such as IL4 and IFN- γ produced by effector T cells stimulated with OX40. Importantly, blocking OX40L significantly promoted iTreg differentiation and induced graft tolerance, which is likely mediated by Treg cells. Thus, OX40 is a potential molecular target for controlling T cell-mediated autoimmunity. In addition, recent studies have reported that the interaction between OX40L expressed by mast cells and OX40 expressed by Treg cells can mutually inhibit mast cell function and Treg cell suppressive function (Gri et al.2008; Picosese et al.2009).

Mice are the experimental tool of choice for immunologists and the study of their immune response has provided a great insight into the working of the human immune system. The general structure of the mouse and human systems appears very similar; however, there are also significant differences. For example, in mice, CD134 is expressed on activated Teff, while Tregs constitutively express CD134 (Picosene et. J Exp Med 2008; 205: 825-. In humans, CD134 is expressed on both Teff and tregs, but only upon activation (see, e.g., example 2(g) below, "CD 134 expression on human effector and regulatory T lymphocytes after stimulation with anti-human CD 3/anti-human CD28 antibody-stimulated beads"). In addition, mouse Tregs induce apoptosis of mouse Teff to achieve suppression (Pandiyan et al. nat Immunol 2007; 8: 1353; Scheffold et al. nat Immunol 2007; 8: 1285-. Taken together, these data suggest a different role for CD134 in Treg suppression function between the human and mouse immune systems.

The term "binding molecule" includes (1) an antibody, (2) an antigen-binding fragment of an antibody, and (3) an antibody derivative, each as defined herein. The term "binds to CD 134" means that the binding molecule as defined herein binds to the CD134 receptor in an in vitro assay such as the BIAcore assay or by Octet (surface plasmon resonance). The binding molecule preferably has a binding affinity (K)_d) Is 1x10^-6M or less, more preferably less than 50x10^-7M, still more preferably less than 1x10^-7M。

The term "isolated antibody" or "isolated binding molecule" refers to an antibody or binding molecule that: (1) does not bind to a naturally associated component with which it coexists in its natural state; (2) does not contain other proteins from the same species; (3) expressed by cells from different species; or (4) does not occur in nature. Examples of isolated antibodies include anti-CD 134 antibodies affinity purified with CD134, anti-CD 134 antibodies produced in vitro by hybridomas or other cell lines, and human anti-CD 134 antibodies derived from transgenic animals.

The term "agonist" refers to a binding molecule as defined herein that, upon binding to CD134, (1) stimulates or activates CD134, (2) enhances, promotes, induces, increases or prolongs the activity, presence or function of CD134, or (3) enhances, promotes, increases or induces the expression of CD 134. The term "antagonist" refers to a binding molecule as defined herein that, when bound to CD134, (1) inhibits (inhibit or suppress) CD134, (2) inhibits the activity, presence or function of CD134, or (3) inhibits the expression of CD 134.

The term "antibody" refers to an immunoglobulin molecule, typically composed of 2 pairs of identical polypeptide chains, each pair having one "heavy" (H) chain and one "light" (L) chain. Human light chains are classified as kappa (κ) and lambda (λ), heavy chains as mu, delta, gamma, alpha, or epsilon, and define the antibody isotypes IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The constant region of the heavy chains of IgD, IgG and IgA consists of 3 domains CH1, CH2 and CH3, and the constant region of the heavy chains of IgM and IgE consists of 4 domains CH1, CH2, CH3 and CH 4. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain CL. The constant region of the antibody may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells). The VH and VL regions can be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of each heavy/light chain pair (VH and VL) form antibody binding sites, respectively. The amino acid profile of each region or domain conforms to the definition of Kabat Sequences of proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and1991)) or to the definition of Chothia et al.formulations of Immunological reagents (Nature 1989; 342(6252): 877-83). The term "antibody" includes antibodies in the form of multimers of antibodies, such as dimers, trimers or higher order multimers of monomeric antibodies. It also includes antibodies linked or attached to non-antibody moieties. In addition, the term "antibody" is not limited by any particular method of producing the antibody. For example, it includes monoclonal antibodies, recombinant antibodies and polyclonal antibodies.

The term "antibody derivative" or "derivative" of an antibody refers to a molecule that binds to the same antigen (i.e., human CD134) to which the antibody binds and comprises the amino acid sequence of the antibody linked to another molecular entity. The amino acid sequence of the antibody contained in the antibody derivative may be the full-length antibody, or may be any portion of the full-length antibody. Examples of additional molecular entities include chemical groups, peptides, proteins (e.g., enzymes, antibodies), amino acids, and compounds. The additional molecular entity may be used as a detection agent, label, therapeutic agent or pharmaceutical agent. The amino acid sequence of the antibody may be attached or linked to the further entity by non-covalent binding, chemical coupling, genetic fusion or other means. The term "antibody derivative" also includes chimeric antibodies, humanized antibodies, and molecules derived by modification of the amino acid sequence of a CD134 antibody, such as conservative amino acid substitutions, insertions, and additions.

The term "antigen-binding fragment" of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen to which the antibody binds (i.e., human CD 134). The term "antigen-binding fragment" also includes a portion of an antibody that is part of a larger molecule formed by the noncovalent or covalent association of an antibody moiety with one or more additional molecular entities. Examples of additional molecular entities include amino acids, peptides or proteins, such as the streptavidin core region, which can be used to prepare tetrameric scFv molecules (Kipriyanov et al. hum Antibodies hybrids 1995; 6(3): 93-101).

The term "chimeric antibody" refers to an antibody comprising amino acid sequences derived from two or more different antibodies. The two or more different antibodies may be from the same species, or from two or more different species.

The term "epitope" refers to an antigenic moiety that is capable of specifically binding to an antibody or T cell receptor or otherwise interacting with a molecule. An "epitope" is also referred to in the art as an "antigenic determinant". Epitopes are usually composed of a chemically active surface assembly of molecules such as amino acids or carbohydrates or sugar side chains. Epitopes can be "linear" or "non-linear/conformational". Once the desired epitope is determined (e.g., by epitope mapping), antibodies can be raised against the epitope. The production and characterization of antibodies may also provide information about the desired epitope. From this information, antibodies that bind the same epitope can then be screened, for example, by performing a cross-competition study, to find antibodies that compete for binding to each other, i.e., antibodies that compete for binding to the antigen.

The term "host cell" refers to a cell into which an expression vector is introduced. The term includes not only the particular subject cell, but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either environmental influences or mutation, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".

The term "human antibody" refers to an antibody consisting only of the amino acid sequence of a human immunoglobulin sequence. The human antibody may contain mouse carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Human antibodies can be made by various methods known in the art.

The term "humanized antibody" refers to a chimeric antibody that contains amino acid residues derived from human antibody sequences. Humanized antibodies may contain some or all of the CDRs from a non-human animal antibody, while the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

The term "mammal" refers to any animal species of the class mammalia. Examples of mammals include: a human; experimental animals such as rats, mice, apes and guinea pigs; livestock such as rabbits, cattle, sheep, goats, cats, dogs, horses, pigs, and the like.

The term "isolated nucleic acid" refers to a nucleic acid molecule of genomic, cDNA, or synthetic origin, or a combination thereof, which is isolated from other nucleic acid molecules present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences located at the 5 'and 3' ends of the nucleic acid of interest in the genomic DNA of the organism from which the nucleic acid is derived.

The term "off-rate" or "K_d"refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, and is used to describe the binding affinity between a ligand (e.g., an antibody) and a protein (e.g., CD 134). The smaller the equilibrium dissociation constant, the more tightly bound the ligand, or the higher the affinity between the ligand and the protein. K_dThe measurement can be by surface plasmon resonance, for example using BIACORE1 or Octet system. The term "anti-CD 134 antibody" refers to an antibody as defined herein that binds human CD 134.

The terms "OX 40 receptor" and "CD 134 receptor" are used interchangeably in this application and include human CD134 and variants, isoforms and species homologs thereof. Thus, the human binding molecules disclosed herein may also bind CD134 from non-human species in some cases. In other cases, the binding molecule may be completely specific for human CD134 and may not exhibit species or other types of cross-reactivity. In particular, they do not bind mouse or rat CD 134.

The term "specifically binds to human CD 134" refers to the K binding molecule that binds to human CD134_dPreferably K which binds to, for example, human CD40_d10-fold, 50-fold, or most preferably 100-fold higher as determined using assays described herein or known to those skilled in the art (e.g., BIAcore assays). Alternatively, the determination that a particular substance specifically binds to the OX40 receptor can be readily made by using or adapting conventional procedures. One suitable in vitro assay utilizes the Western blot procedure (described in a number of standard textbooks, including "Antibodies, A Laboratory Manual" by Harlow and Lane). To determine that a given OX40 receptor binding substance specifically binds human OX40 protein, total cellular protein is extracted from mammalian cells that do not express OX40 antigen, such as non-lymphoid cells (e.g., COS cells or CHO cells), and transformed with a nucleic acid molecule encoding OX40. As a negative control, total cellular protein was also extracted from the corresponding untransformed cells. These protein preparations were then electrophoresed on native or denaturing polyacrylamide gels (PAGE). Thereafter, the protein is transferred to a membrane (e.g., nitrocellulose) by Western blotting, and the test substance is incubated with the membrane. Washing the membrane to removeFollowing non-specific binding of the substance, the presence of the bound substance is detected with an antibody against the test substance conjugated to a detection agent such as alkaline phosphatase, the use of the substrate 5-bromo-4-chloro-3-indolylphosphate/nitrobluetetrazolium resulting in the production of a dense blue compound by the immuno-immobilized alkaline phosphatase. By this technique, substances that specifically bind to human OX40 will show a band of human OX40 in extracts of OX40 transformed cells (whose molecular mass determines that they will be located at a given position on the gel), while little or no binding is observed in extracts of non-transformed cells. This material can bind non-specifically to other proteins and a weaker signal can be detected on the Western blot. The non-specific nature of this binding can be appreciated by those skilled in the art by the weak signal obtained on Western blotting as compared to the strong primary signal resulting from specific substance/human OX40 protein binding. Ideally, the OX40 receptor binding substance does not bind to proteins extracted from non-transformed cells. In addition to binding assays using extracted proteins, putative OX40 receptor binding substances may be tested to confirm their ability to bind essentially only OX40 receptor in vivo by conjugating the binding substance to a fluorescent label, such as FITC, and analyzing its binding to antigen activated CD4+ T cells and the non-activated T cell population by flow cytometry (FACS). Substances that bind substantially only to the OX40 receptor stain only activated CD4+ T cells.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule into a host cell. Examples of vectors include plasmid, viral, cosmid or phage vectors, and naked DNA or RNA expression vectors. Some vectors are capable of autonomous replication in a host cell into which they are introduced. Some vectors, when introduced into a host cell, can integrate into the host cell genome and thereby replicate together with the host genome. Certain vectors are capable of directing the expression of genes to which they are operatively linked and may therefore be referred to as "expression vectors".

As used herein, the 20 conventional amino acids and their abbreviations follow a customary usage.

The present invention provides isolated knots that bind human CD134Synthetic molecules, including anti-CD 134 antibodies, antigen-binding fragments of anti-CD 134 antibodies, and derivatives of anti-CD 134 antibodies. The binding molecule is characterized by at least one of the following functional properties: (a) at 1x10^-6K of M or less_dBinds to human CD 134; (b) does not prevent human CD134(OX40) receptor from binding OX40 ligand (OX 40L); (c) agonist activity on human CD134 on T effector cells and/or antagonist activity on human CD134 on T regulatory cells; (d) does not bind CD40 receptor at concentrations up to 500 nM; (e) does not bind the CD137 receptor at concentrations up to 500 nM; (f) does not bind CD271 receptor at concentrations up to 500 nM; (g) (ii) enhances IL-2 production by isolated human T cells; (h) can enhance immune response; (i) can inhibit the growth of tumor cells; and (j) has a therapeutic effect on cancer. In some embodiments, the binding molecule is at 1x10^-7M or less, or 1x10^-8M or less, or 5x1x10^-9K of M or less_dBinds to human CD 134.

Antibodies and other binding molecules of the invention can be prepared by conventional techniques and then screened to identify and obtain binding molecules that do not prevent OX40L from binding CD 134. For example, binding molecules can be selected that bind to CD134 even when CD134 has been exposed to saturating concentrations of OX 40L.

In one embodiment of the invention, human antibodies that bind to human CD134 are provided. In some embodiments, the human antibody is a monoclonal antibody that has a K of 100nM or less, preferably 10nM or less_dSpecifically binds to human CD134, and/or has agonist activity against human CD134 on T effector cells and/or antagonist activity against human CD134 on T regulatory cells. An example of such a human antibody is human monoclonal antibody clone 12H 3. The amino acid sequence of the complete heavy chain variable region of antibody clone 12H3 and the amino acid sequences of the 3 CDRs of the heavy chain variable region (VH) are shown in SEQ ID NOs 12 and 14-16, respectively. The amino acid sequence of the entire light chain variable region of antibody clone 12H3 and the amino acid sequences of the 3 CDRs of the light chain variable region (VL) are shown in SEQ ID NOs 13 and 17-19, respectively. Another exemplary antibody disclosed is human monoclonal antibody clone 20E 5. Complete heavy chain variable region of antibody clone 20E5The amino acid sequences of the amino acid sequences and the 3 CDRs of the heavy chain variable region (VH) are shown in SEQ ID NOS: 4 and 6-8, respectively. The amino acid sequence of the entire light chain variable region of antibody clone 20E5 and the amino acid sequences of the 3 CDRs of the light chain variable region (VL) are shown in SEQ ID NOs 5 and 9-11, respectively.

The antibodies of the invention may comprise one or more of these CDRs, or one or more of these CDRs with 1, 2 or 3 amino acid substitutions per CDR. The substitution is preferably a "conservative" substitution. Conservative substitutions that provide functionally similar amino acids are well known in the art, for example, as described in table 1 of WO2010/019702, which is incorporated herein by reference.

Whereas clone 12H3 and clone 20E5 bound human CD134, their respective VH and VL sequences could be "mixed and matched" with other anti-CD 134 antibodies to produce additional antibodies. Binding of such "mixed and matched" antibodies to human CD134 can be detected using binding assays known in the art, including those described in the examples. In one case, the VH sequences from a particular VH/VL pairing are replaced with structurally similar VH sequences when the VH and VL regions are mixed and matched. Similarly, in another instance, VL sequences from a particular VH/VL pairing are replaced with structurally similar VL sequences.

Molecules containing only one or two CDR regions (in some cases, even only a single CDR or portion thereof, particularly CDR3) are capable of retaining the antigen binding activity of the antibody from which the CDR is derived. See, e.g., Laune et al.jbc1997; 272: 30937-44; monnet et al.jbc1999; 274: 3789-96; qiu et al naturebiotechnology 2007; 25: 921-9; ladner et al, nature Biotechnology 2007; 25: 875-7; heap et al.J Gen Virol 2005; 86: 1791-1800; nicaise et al protein Science 2004; 1882-91; vaughan and Sollazzo composite Chemistry & High through screening 2001; 4: 417-; quiocho Nature 1993; 362: 293-4; pessi et al, nature 1993; 362: 367-9; bianchi et al j Mol Biol 1994; 236: 649-59; and Gao et al J BiolChem 1994; 269:32389-93.

Accordingly, one embodiment of the invention is an isolated anti-human CD134 antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 12; (b) 13, comprising the amino acid sequence of SEQ ID NO.

In a further embodiment of the invention, there is provided an isolated CD134 binding molecule comprising: (a) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 14; and/or (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 15; and/or a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 16.

In a further embodiment of the invention, there is provided an isolated CD134 binding molecule comprising: (a) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 17; and/or (b) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 18; and/or a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 19.

Accordingly, one embodiment of the invention is an isolated anti-human CD134 antibody comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 4; (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO 5.

In a further embodiment of the invention, there is provided an isolated CD134 binding molecule comprising: (a) heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 6; and/or (b) a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 7; and/or a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 8.

In a further embodiment of the invention, there is provided an isolated CD134 binding molecule comprising: (a) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 9; and/or (b) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 10; and/or a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 11.

Whereas clone 12H3 and clone 20E5 bound human CD134, and antigen binding specificity was provided primarily by the CDR1, CDR2, and CDR3 regions, VH CDR1, CDR2, and CDR3 sequences and VL CDR1, CDR2, and CDR3 sequences can be "mixed and matched" to produce additional anti-CD 134 antibodies. For example, the CDRs from different anti-CD 134 antibodies may be mixed and matched, although each antibody will typically contain VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR 3. Binding of such "mixed and matched" antibodies to CD134 can be tested using binding assays (e.g., ELISA, Biacore analysis) as described above and in the examples. In one case, when VH CDR sequences are mixed and matched, the CDR1, CDR2, and/or CDR3 sequences from a particular VH sequence are replaced with structurally similar CDR sequences. Similarly, when VL CDR sequences are mixed and matched, CDR1, CDR2, and/or CDR3 sequences from a particular VL sequence are replaced with structurally similar CDR sequences. It will be apparent to those skilled in the art that one or more VH and/or VL CDR region sequences may be replaced with structurally similar sequences from the CDR sequences disclosed herein to produce novel VH and VL sequences.

The class (e.g., IgG, IgM, IgE, IgA, or IgD) and subclass (e.g., IgG1, IgG2, IgG3, or IgG4) of anti-CD 134 antibodies can be determined by any suitable method, such as ELISA or Western blotting, among other techniques. Alternatively, the class and subclass of antibodies can be determined by sequencing all or part of the constant domains of the heavy and/or light chains of the antibody, comparing their amino acid sequences to known amino acid sequences of immunoglobulins of various classes and subclasses. The anti-CD 134 antibody may be an IgG, IgM, IgE, IgA, or IgD molecule. For example, the anti-CD 134 antibody may be an IgG that is an IgG1, IgG2, IgG3, or IgG4 subclass. Accordingly, another aspect of the invention provides methods for converting a class or subclass of anti-CD 134 antibodies to another class or subclass.

Binding molecules of embodiments of the invention include monoclonal antibodies, fragments thereof, peptides, and other chemical entities. Monoclonal antibodies can be prepared by conventional methods of immunizing a mammal, followed by isolation of the B plasma cells producing the monoclonal antibody of interest and fusion with myeloma cells.

In various embodiments, the binding moiety may be an antibody mimetic (e.g., based on a non-antibody backbone), an RNA aptamer, a small molecule, or a CovX-body, rather than an actual antibody.

It will be appreciated that antibody mimetics (e.g. non-antibody framework structures which have high stability but still allow variation to be introduced at a position) can be used to generate libraries of molecules from which binding moieties can be derived. Many such molecules are familiar to those skilled in the art of biochemistry. Such molecules may be used as binding moieties in the agents of the invention.

Typical antibody mimetics are discussed in Skerra et al (2007, Curr, Opin, Biotech, 18:295- & 304) and include: affibodies (also known as Trinectins; Nygren,2008, FEBS J,275, 2668-; CTLDs (also known as Tetranectins; Innovations Pharmac. technol. (2006), 27-30); adnectins (also known as monobodies; meth.mol.biol.,352(2007), 95-109); anticalins (Drug discovery today (2005),10, 23-33); DARPins (ankyrns; nat. Biotechnol. (2004),22, 575-; avimers (nat. Biotechnol. (2005),23, 1556-; microbodies (FEBS J, (2007),274, 86-95); peptide aptamers (expert. opin. biol. ther. (2005),5,783-797); kunitz domains (j. pharmacol. exp. ther. (2006)318, 803-; affilins (trends Biotechnol. (2005),23, 514-.

Thus, it is preferred that the antibody mimetic is selected from the group comprising or consisting of: affibodies, tetranectins (CTLDs), adnectins (monobodies), anticalins, DARPins (ankyrns), avimers, iMabs, microbodies, peptide aptamers, Kunitz domains, aptamers, and affilins.

By "small molecule" is meant a low molecular weight organic compound of 900 daltons or less. Although large biopolymers such as nucleic acids, proteins and polysaccharides (e.g. starch or cellulose) are not included in the "small molecules", their constituent monomers (ribonucleotides or deoxyribonucleotides, amino acids and monosaccharides respectively) and oligomers (i.e. short polymers such as dinucleotides, peptides such as antioxidant glutathione and disaccharides such as sucrose) are included. Small molecules are generated as described by Mayes & Whitcomb, 2005, adv. drug Deliv. Rev.57:1742-78 and Root-Bernstein & Dillon,2008, curr. pharm. Des.14: 55-62.

CovX-Bodies are produced by covalently linking a pharmacophore to the binding site of a specifically designed antibody via a linker, which effectively reprograms the antibody (Tryder et al, 2007, bioorg.med.chem.lett.,17: 501-6). The result is the formation of a new class of chemical entities, each component of which contributes to the desirable properties of the complete CovX-Body-in particular, the entity has the biological effect of a peptide and the extended half-life of an antibody.

Human antibodies can be made by several different methods, including the generation of human Antibody fragments (VH, VL, Fv, Fd, Fab or (Fab')₂) And the use of these fragments to construct fully human antibodies by fusing appropriate portions using techniques similar to those used to generate chimeric antibodies. Human antibodies can also be produced in transgenic mice having a human immunoglobulin genome. Such mice are available, for example, from Abgenix, inc., Fremont, California, and Medarex, inc., anandale, New Jersey. In addition to linking the Fv regions of the heavy and light chains to form a single chain peptide, Fab can also be constructed and expressed in a similar manner (M.J.Evans et al.J.Immunol Meth 1995; 184: 123-138).

Delmmunized^TMAntibodies are antibodies in which potentially immunogenic T cell epitopes have been eliminated, as described in International patent application PCT/GB 98/01473. Thus, their immunogenicity in humans is expected to be eliminated or substantially reduced when they are used in vivo. The immunoglobulin-based binding molecules of the invention may have their immunogenic T cell epitopes (if present) removed by this method.

Such fully or partially human antibodies are less immunogenic than fully mouse antibodies or non-human derived antibodies, as are fragments and single chain antibodies. All these molecules (or derivatives thereof) are thus less likely to elicit an immune or allergic response. Thus, they are more suitable for administration in humans than fully non-human antibodies, particularly when repeated or prolonged administration is required.

Bispecific antibodies can be used as cross-linkers between human CD134 on the same human target cell, or human CD134 on two different human target cells. This dual specificityThe antibody has a specificity for each of two different epitopes on human CD 134. These antibodies and methods of making them are described in U.S. Pat. No.5,534,254 (Creative biomoles, Inc.). Various embodiments of bispecific antibodies described in this patent include the use of peptide conjugates (including Ser-Cys, (Gly)₄-Cys、(His)₆-(Gly)₄-Cys), chelating agents and chemical or disulfide bonding (including bismaleimidohexane and bismaleimidocaproyl) linked single chain Fv.

Non-antibody molecules can be isolated or screened from compound libraries by conventional means. Automated systems for generating and screening compound libraries are described in U.S. patents 5,901,069 and 5,463,564. More significant approaches involve three-dimensional modeling of the binding sites, followed by the preparation of a family of molecules that fit the model. These families of molecules are then screened for those with the best binding characteristics.

Another approach is to generate a library of recombinant peptides and then screen for those that bind to an epitope of human CD134 of interest. See, for example, U.S. patent 5,723,322. This epitope is the same as that to which the monoclonal antibody described in the examples below binds. In fact, once an epitope is known, molecules can be generated or isolated relatively easily according to techniques well known in the art.

Further embodiments provide derivatives of any of the above anti-CD 134 antibodies. In a particular aspect, the antibody derivative is derived from a modification of the amino acid sequence of clone 12H3 and/or clone 20E 5. The amino acid sequence of any region of an antibody chain may be modified, such as a framework region, a CDR region, or a constant region. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can comprise natural and unnatural amino acids. The type of modification includes insertions, deletions, substitutions of one or more amino acids of the anti-CD 134 antibody, or combinations thereof. In some embodiments, the antibody derivative comprises a substitution of 1, 2, 3, or 4 amino acids of a heavy chain CDR and/or a substitution of 1 amino acid of a light chain CDR. In some embodiments, the derivative of the anti-CD 134 antibody comprises one or more amino acid substitutions relative to the germline amino acid sequence of the human gene. In particular embodiments, one or more such substitutions from the germline are located in the CDR2 region of the heavy chain. In another specific embodiment, the amino acid substitutions relative to the germline are at one or more of the same positions as the substitutions relative to the germline in antibody clone 12H3 and clone 20E 5. In another embodiment, the amino acid substitution is a change of one or more cysteines in the antibody to another residue, such as, but not limited to, alanine or serine. The cysteine may be a canonical or non-canonical cysteine. Substitutions may be made in the CDRs or framework regions of the variable domain or in the constant domain of the antibody. Another type of amino acid substitution is the elimination of the asparagine-glycine pair by changing one or both residues, which forms a potential deamidation site. In other embodiments, the amino acid substitution is a conservative amino acid substitution. In one embodiment, the antibody derivative has 1, 2, 3, or 4 conservative amino acid substitutions in the heavy chain CDR regions relative to the amino acid sequence of clone 12H3 and/or clone 20E 5. Another type of modification of anti-CD 134 antibodies is to alter the original glycosylation pattern of the antibody. The term "altering" refers to the deletion of one or more carbohydrate moieties in an antibody, and/or the addition of one or more glycosylation sites that are not present in an antibody.

Glycosylation of antibodies is typically N-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. Examples of other modifications include acylation, amidation, acetylation, cross-linking, cyclization, formylation, hydroxylation, iodination, methylation, myristoylation, disulfide bond formation, demethylation, covalent cross-link formation, cysteine formation, oxidation, phosphorylation, prenylation, PEGylation, proteolytic processing, and sulfation.

A further embodiment provides an antibody derivative comprising an anti-CD 134 antibody or antigen-binding fragment thereof as described herein linked to an additional molecular entity. Examples of additional molecular entities include agents, peptides or proteins and detection agents or labels. Specific examples of agents that can be linked to the anti-CD 134 antibody include cytotoxic or other cancer therapeutic agents, and radioisotopes. Specific examples of peptides or proteins that can be linked to the anti-CD 134 antibody include antibodies, which can be the same anti-CD 134 antibody or different antibodies. Specific examples of the detection agent or label that can be linked to the anti-CD 134 antibody include (1) fluorescent compounds such as fluorescein, fluorescein isothiocyanate, phycoerythrin, rhodamine, 5-dimethylamine-l-naphthalenesulfonyl chloride and lanthanide phosphors; (2) enzymes such as horseradish peroxidase, alkaline phosphatase, luciferase and glucose oxidase; (3) biotin; (4) a predetermined polypeptide epitope recognized by a second reporter molecule, such as a leucine zipper pair sequence, a metal binding domain, an epitope tag, and a binding site for a second antibody. Further embodiments provide antibody derivatives that are multimeric forms of the anti-CD 134 antibodies, such as antibody dimers, trimers, or higher order multimers of monomeric antibodies. The individual monomers within an antibody multimer can be the same or different, i.e., they can be heteromeric or homomeric antibody multimers. Multimerization of antibodies can be accomplished by natural aggregation. For example, a certain percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers. Alternatively, antibody homodimers can be formed by chemical ligation techniques known in the art. Suitable crosslinking agents include heterobifunctional crosslinking agents such as m-maleimidobenzoyl-N-hydroxysuccinimide ester, N-succinimidoS-acetylthioacetate and succinimido 4- (maleimidomethyl) cyclohexane-1-carboxylate or homobifunctional crosslinking agents such as disuccinimidosuberate. Such cross-linking agents are commercially available. Antibodies can also be made by multimerization by recombinant DNA techniques known in the art.

A further embodiment provides an antibody derivative which is a chimeric antibody comprising the amino acid sequence of an anti-human CD134 antibody as described herein above. In another example, all CDRs of the chimeric antibody are derived from an anti-human CD134 antibody. In another example, CDRs from more than one anti-human CD134 antibody are combined in a chimeric antibody. Further, a chimeric antibody may comprise framework regions derived from one anti-human CD134 antibody and one or more CDRs from one or more different human antibodies. Chimeric antibodies can be produced by conventional methods known in the art. In some particular embodiments, the chimeric antibody comprises 1, 2, or 3 CDRs from the heavy chain variable region or from the light chain variable region of an antibody selected from antibody clone 12H3 and/or clone 20E 5.

Examples of other antibody derivatives provided by the present invention include single chain antibodies, bivalent antibodies, domain antibodies, nanobodies, and unibody. In a preferred embodiment, the monoclonal antibody may be a chimeric antibody, a humanized antibody, a human antibody, Delmmmunized^TMAntibodies, single chain antibodies, fragments, including Fab, F (ab')₂Fv or other fragments that retain the antigen binding function of the parent antibody. Single chain antibodies ("ScFv") and methods for their construction are described in U.S. Pat. No.4,946,778.

A "single chain antibody" (scFv) consists of a single polypeptide chain comprising a VL domain linked to a VH domain, wherein the VL domain and VH domain pair to form a monovalent molecule. Single chain antibodies can be prepared according to methods known in the art (see, e.g., Bird et al, (1988) Science242:423-426 and Huston et al, (1988) Proc. Natl. Acad. Sci. USA85:5879-5883), "diabodies" consist of two chains, each comprising a heavy chain variable region and a light chain variable region joined by a short peptide linker on the same polypeptide chain, wherein the two regions on the same chain do not pair with each other, but with complementary domains on the other chain to form a bispecific molecule. Methods for the preparation of bivalent antibodies are known in the art (see, e.g., Holliger P.et al, (1993) Proc. Natl. Acad. Sci. USA90: 6444-. Domain antibodies (dabs) are small functional binding units of antibodies, corresponding to the variable region of the heavy or light chain of an antibody. Domain antibodies are well expressed in bacterial, yeast and mammalian cell systems. Further details of domain antibodies and methods for their production are known in the art (see, e.g., U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; WO04/003019 and WO 03/002609). Nanobodies are derived from the heavy chain of an antibody. Nanobodies typically comprise a single variable domain and two constant domains (CH2 and CH3) and retain the antigen binding ability of the original antibody. Nanobodies can be prepared by methods known in the art (see, e.g., U.S. Pat. No.6,765,087, U.S. Pat. No.6,838,254, WO 06/079372). The Unibody consists of one light chain and one heavy chain of the IgG4 antibody. The Unibody can be prepared by removing the hinge region of the IgG4 antibody. Further details of the Unibody and its preparation can be found in WO 2007/059782.

In addition to the binding moiety, the molecules of the invention may further comprise a moiety for increasing the in vivo half-life of the molecule, such as, but not limited to, polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids, and dextran. Such additional moieties may be conjugated or otherwise combined with the binding moiety using methods well known in the art.

In a further aspect of the invention there is provided a nucleic acid molecule encoding the amino acid sequence of the CD134 binding molecule of the first aspect of the invention. The amino acid sequence encoded by the nucleic acid molecule can be any portion of a complete antibody, such as a CDR, a sequence comprising 1, 2, or 3 CDRs, or a variable region of a heavy or light chain, or can be a full-length heavy or light chain. In some embodiments, the nucleic acid molecule encodes an amino acid sequence comprising (1) a CDR3 region, particularly a heavy chain CDR3 region, of antibody clone 12H3 and/or 20E 5; (2) the heavy chain variable region or the light chain variable region of antibody clones 12H3 and/or 20E 5; or (3) the heavy or light chain of antibody clone 12H3 and/or 20E 5. In other embodiments, the nucleic acid molecule encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NO 12, 13, 14, 15, 16, 17, 18 or 19 or selected from SEQ ID NO 4, 5, 6,7, 8, 9, 10 or 11.

The nucleic acid molecules provided herein can be obtained from any source that produces the CD134 antibodies of the invention. mRNA from cells producing anti-CD 134 antibodies can be isolated by standard techniques, cloned and/or amplified using PCR and library construction techniques, and screened using standard protocols to obtain nucleic acid molecules encoding the amino acid sequences of anti-CD 134 antibodies. The mRNA can be used to generate cDNA for use in Polymerase Chain Reaction (PCR) or cDNA cloning of antibody genes. In one embodiment, the nucleic acid molecule is derived from a hybridoma expressing an anti-CD 134 antibody as described above, preferably a hybridoma having non-human transgenic animal cells expressing human immunoglobulin genes as one of its fusion partners. In another embodiment, the hybridoma is derived from a non-human, non-transgenic animal.

A nucleic acid molecule encoding the heavy chain of an anti-CD 134 antibody can be constructed by fusing a nucleic acid molecule encoding the variable region of the heavy chain to a nucleic acid molecule encoding the constant region of the heavy chain. Similarly, a nucleic acid molecule encoding the light chain of an anti-CD 134 antibody can be constructed by fusing a nucleic acid molecule encoding the variable region of the light chain to a nucleic acid molecule encoding the constant region of the light chain. Nucleic acid molecules encoding the VH and VL chains can be converted to full-length antibody genes by insertion into expression vectors that already encode the heavy and light chain constant regions, respectively, such that the VH segments are operably linked to heavy chain constant region (CH) segments within the vector and the VL segments are operably linked to light chain constant region (CL) segments within the vector. Alternatively, nucleic acid molecules encoding a VH or VL chain are converted to full-length antibody genes by linking, for example, using standard molecular biology techniques, a nucleic acid molecule encoding a VH chain to a nucleic acid molecule encoding a CH chain. The same can be achieved using nucleic acid molecules encoding VL and CL chains. The nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed from the cells into which they are introduced, and the anti-CD 134 antibody isolated.

The nucleic acid molecules can be used to recombinantly express large amounts of anti-CD 134 antibodies, as described below. The nucleic acid molecules may also be used to generate other binding molecules provided herein, such as chimeric antibodies, single chain antibodies, immunoadhesins, bivalent antibodies, mutated antibodies and antibody derivatives, as described elsewhere herein. In one embodiment, the nucleic acid molecule is used as a probe or PCR primer for a particular antibody sequence. For example, nucleic acid molecule probes may be used in diagnostic methods, or nucleic acid molecule PCR primers may be used to amplify DNA regions that may be particularly useful for isolating nucleic acid sequences for the production of the variable regions of anti-CD 134 antibodies.

Once the DNA molecules encoding the VH and VL segments of the anti-CD 134 antibody are obtained, these DNA molecules can be further manipulated by recombinant DNA techniques, such as conversion of the variable region genes to full-length antibody chain genes, Fab fragment genes, or scFv genes.

In another aspect of the invention there is provided a vector comprising a nucleic acid molecule as described herein above. The nucleic acid molecule may encode an amino acid sequence of a light chain or a portion of a heavy chain (e.g., a CDR or variable region), a full-length light chain or heavy chain, a polypeptide comprising a portion or the full length of a heavy chain or light chain, or an antibody derivative or antigen-binding fragment.

Examples of suitable expression vectors are expression vectors encoding functionally intact human CH or CL immunoglobulin sequences with suitable restriction sites engineered so that any VH or VL sequence can be inserted and expressed. The expression vector may also encode a signal peptide that facilitates secretion of the amino acid sequence of the antibody chain from the host cell. The DNA encoding the amino acid sequence of the antibody chain may be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the amino acid sequence of the antibody chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein). In addition to the nucleic acid sequence encoding the amino acid sequence of the anti-CD 134 antibody (antibody chain gene), the expression vector carries regulatory sequences that control the expression of the antibody chain gene in the host cell. The design of the expression vector, including the choice of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of protein expression desired, and the like. Regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, Cytomegalovirus (CMV) (e.g., CMV promoter/enhancer), simian virus 40(SV40) (e.g., SV40 promoter/enhancer), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), polyoma viruses, and strong mammalian promoters such as native immunoglobulin and actin promoters.

The host cell may be a mammalian, insect, plant, bacterial or yeast cell. Examples of mammalian cell lines suitable as host cells include Chinese Hamster Ovary (CHO) cells, NSO cells, PER-C6 cells, SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, African green monkey kidney Cells (COS), human hepatoma cells (e.g., Hep G2), human lung cells, A549 cells, and many other cell lines. Examples of insect cell lines include Sf9 or Sf21 cells. Examples of plant host cells include tobacco (Nicotiana), Arabidopsis (Arabidopsis), duckweed (duckweed), maize, wheat, potato, and the like. Bacterial host cells include the species escherichia coli (e.coli) and Streptomyces (Streptomyces). Examples of yeast host cells include Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris).

The amino acid sequences of the binding molecules expressed by different cell lines or in transgenic animals may have different glycosylation. However, all binding molecules encoded by or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the binding molecule.

In another aspect the invention provides a method of producing a CD134 binding molecule as defined above using phage display. The method comprises (a) synthesizing a human antibody library on a bacteriophage, (b) screening the library with CD134 or a portion thereof, (c) isolating the bacteriophage that binds to CD134 or a portion thereof, and (d) obtaining the antibody from the bacteriophage. One exemplary method for preparing an antibody library comprises the steps of: (a) immunizing a non-human animal comprising a human immunoglobulin locus with CD134 or an antigenic portion thereof to generate an immune response; (b) extracting antibody-producing cells from the immunized animal; (c) isolating RNA encoding the heavy and light chains of the anti-CD 134 antibody from the extracted cells; (d) reverse transcription of RNA to produce cDNA; (e) amplifying the cDNA; and (f) inserting the cDNA into a phage display vector, thereby expressing the antibody on the phage. Recombinant anti-human CD134 antibodies or antigen-binding fragments thereof can be isolated by screening recombinant combinatorial antibody libraries. The library may be a scFv phage display library, which is generated with human VL and VH cdnas prepared from mRNA isolated from B cells. Methods of making and screening such libraries are known in the art. Kits for generating phage display libraries are commercially available.

In a preferred embodiment of the invention, there is provided a composition, e.g. a pharmaceutical composition, comprising one or a combination of binding molecules as described herein, and optionally a pharmaceutically acceptable carrier. The compositions may be prepared by conventional methods known in the art. In some embodiments, the composition comprises an anti-CD 134 antibody or antigen-binding fragment thereof. In particular embodiments, the composition comprises the antibody clone 12H3 and/or clone 20E5, or an antigen-binding fragment of either antibody. In other embodiments, the composition comprises a derivative of antibody clone 12H3 and/or clone 20E 5. The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for delivery of the binding molecule in a formulation. The carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (e.g., antioxidant, antibacterial or antifungal agent), sweetener, absorption delaying agent, wetting agent, emulsifier, buffer, etc.

The non-peptide molecules of the invention may be administered orally, including via suspension, tablets, and the like. Liquid formulations may be administered by inhalation of lyophilized or aerosol microcapsules. Suppositories may also be used. Additional pharmaceutical carriers may be used to control the duration of action of the molecules of the invention. The dosage and timing of the selected formulation can be determined by standard procedures well known in the art. This procedure involves extrapolating an estimated dose regime from an animal model and then determining the optimal dose in a human clinical dose range study.

The composition may be in any suitable form, such as liquid, semi-solid and solid dosage forms. The various dosage forms of the compositions can be prepared using conventional techniques known in the art.

The relative amounts of binding molecules included in the compositions will vary depending on a number of factors, such as the desired release and pharmacokinetic characteristics, the particular binding molecule and carrier used, and the dosage form. The amount of binding molecule in a single dosage form is generally that amount which produces a therapeutic effect, but may be smaller. Typically, this amount ranges from about 0.001% to about 99%, from about 0.1% to about 70%, or from about 1% to about 30%, relative to the total weight of the dosage form.

In addition to the binding molecule, one or more additional therapeutic agents may be included in the composition or separately as part of the same therapeutic regimen. Examples of such additional therapeutic agents are described below. The appropriate amount of additional therapeutic agent to include in the composition can be readily selected by one of skill in the art and will vary depending on factors such as the particular agent and carrier used, the dosage form, and the desired release and pharmacokinetic profile. The amount of additional therapeutic agent included in a single dosage form is typically that amount of the agent which produces a therapeutic effect, but may be a lesser amount.

The binding molecules and pharmaceutical compositions comprising the binding molecules provided herein are useful for therapeutic, diagnostic or other purposes, such as enhancing immune responses, treating cancer, enhancing the efficacy of other cancer treatments, or enhancing vaccine efficacy, and have a number of uses, such as use as a pharmaceutical or diagnostic agent. Thus, in a preferred aspect of the invention, methods of using the binding molecules or pharmaceutical compositions are provided.

A further aspect of the invention provides a method of modulating a human CD 134-mediated anti-tumor immune response comprising enhancing the human Teff effector function of expressing human CD134 and/or attenuating the human Treg suppression function of expressing human CD134 with a binding molecule that binds human CD134, said binding molecule comprising an anti-human CD134 antibody that (1) prevents the interaction of naturally occurring human OX40L with a human CD134 receptor and/or (2) does not block human CD 134-mediated cell signaling after human CD134 is occupied by naturally occurring human OX 40L.

In another aspect, the invention provides methods of modulating a human CD 134-mediated anti-tumor immune response, wherein the methods do not include binding molecules that bind human CD134, including anti-human CD134 antibodies, such as human OX40L mimetics, that interact with an OX40L binding domain on the human CD134 receptor and/or block human OX 40L-human CD134 cell signaling.

The present invention discloses binding molecules, including anti-human CD134 antibodies, that bind to human CD134 for anti-tumor therapeutic purposes. The anti-human CD134 antibody binds to the extracellular domain of human CD 134. More specifically, the anti-human CD134 antibodies bind to non-OX 40L binding regions on the extracellular domain of human CD134 on activated human Teff and human tregs (i.e., the anti-human CD134 antibodies do not completely block the binding of human OX40L to human CD 134).

In a particular aspect, there is provided a method of enhancing an immune response in a mammal comprising administering to the mammal a therapeutically effective amount of a binding molecule described herein. In some embodiments, the binding molecule is an anti-CD 134 antibody or antigen-binding fragment thereof, and the mammal is a human. In additional embodiments, the binding molecule is antibody clone 12H3 and/or clone 20E5, or an antigen-binding fragment of either antibody. The term "enhancing an immune response" refers to any response that stimulates, provokes, increases, ameliorates, or enhances the immune system of a mammal. The immune response may be a cellular response (i.e. cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (i.e. antibody-mediated), and may be a primary or secondary immune response. Examples of enhancing the immune response include increased CD4+ helper T cell activity and the generation of cytotoxic T cells. Enhancement of immune response can be assessed using several in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assays, cytokine release (e.g., production of IL-2), tumor regression, survival of tumor bearing animals, antibody production, immune cell proliferation, cell surface marker expression, and cytotoxicity. In one embodiment, the method enhances a cellular immune response, in particular a cytotoxic T cell response.

One aspect of the invention provides a binding molecule that binds to human CD134, wherein at or above the saturation concentration of the binding molecule, as determined by fluorescence-based flow cytometry as described in example 2(f), the binding of OX40L to CD134 is reduced by no more than 70% on human CD134 expressing T cells. More preferably, the binding of OX40L to CD134 is reduced by no more than about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% or less, or preferably not reduced at all.

Another aspect of the invention provides a binding molecule wherein the binding of OX40L to CD134 is reduced by no more than 70% on human CD134 expressing T cells as determined by fluorescence-based flow cytometry at a binding molecule concentration of 70nM as described in example 2 (f). More preferably, the binding of OX40L to CD134 is reduced by no more than about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% or less, or preferably not reduced at all.

In another aspect, the invention provides a binding molecule that competes for binding to human CD134 with an antibody comprising (1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:12 and (2) a light chain variable region comprising the amino acid sequence of SEQ ID NO:13, as shown by the cross-competition between the binding molecule unlabeled on PHA-stimulated human CD 134-expressing T-lymphocytes and the fluorescently labeled antibody as measured by flow cytometry (further described in example 2 (e)). Preferably, the binding of the antibody is reduced, preferably abolished, by at least about 50%, or about 60%, or about 70%, or about 80%, or about 90% or more at or above its saturating concentration, as measured by cross-competition for the binding molecule.

In another aspect, the invention provides a binding molecule that competes for binding to human CD134 with an antibody comprising (1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and (2) a light chain variable region comprising the amino acid sequence of SEQ ID NO:5, as shown by the cross-competition between the binding molecule unlabeled on PHA-stimulated human CD 134-expressing T-lymphocytes and the fluorescently labeled antibody as measured by flow cytometry (further described in example 2 (e)). Preferably, the binding of the antibody is reduced, preferably abolished, by at least about 50%, or about 60%, or about 70%, or about 80%, or about 90% or more at or above its saturating concentration, as measured by cross-competition for the binding molecule.

In another aspect, the invention provides binding molecules that bind to human CD134, wherein the binding of OX40L to CD134 is reduced by no more than about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% or less on human CD134 expressing T cells, and wherein the binding molecules further do not prevent an immunostimulatory and/or proliferative response of human OX40L on human CD134 expressing T effector cells.

In another aspect, the invention provides a binding molecule that binds to human CD134, wherein the binding molecule does not prevent binding of human CD134(OX40) receptor to OX40 ligand (OX40L), and wherein the binding molecule further does not prevent an immunostimulatory and/or proliferative response of human OX40L on human CD134 expressing T effector cells.

In another aspect, the invention provides binding molecules that bind to human CD134, wherein the binding of OX40L to CD134 is reduced by no more than about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% or less on human CD134 expressing T cells, and wherein the binding molecules enhance the immune stimulatory and/or proliferative response of human OX40L on human CD134 expressing T effector cells.

In another aspect, the invention provides a binding molecule that binds to human CD134, wherein the binding molecule does not prevent binding of human CD134(OX40) receptor to OX40 ligand (OX40L), and wherein the binding molecule enhances an immunostimulatory and/or proliferative response of human OX40L on human CD134 expressing T effector cells.

In another aspect, the invention provides binding molecules that bind to human CD134, wherein the binding of OX40L to CD134 is reduced by no more than about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% or less on human CD134 expressing T cells, and wherein the binding molecules further do not prevent an inhibitory functional response of human OX40L on human CD134 expressing T regulatory cells.

In another aspect, the invention provides a binding molecule that binds to human CD134, wherein the binding molecule does not prevent binding of human CD134(OX40) receptor to OX40 ligand (OX40L), and wherein the binding molecule further does not prevent an inhibitory functional response of human OX40L on human CD134 expressing T regulatory cells.

In another aspect, the invention provides binding molecules that bind to human CD134, wherein the binding of OX40L to CD134 is reduced by no more than about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% or less on human CD134 expressing T cells, and wherein the binding molecules further enhance the inhibitory functional response of human OX40L on human CD134 expressing T regulatory cells.

In another aspect, the invention provides a binding molecule that binds to human CD134, wherein the binding molecule does not prevent the binding of human CD134(OX40) receptor to OX40 ligand (OX40L), and wherein the binding molecule enhances the inhibitory functional response of human OX40L on human CD134 expressing T regulatory cells.

In another aspect, the invention provides binding molecules that bind to human CD134, wherein the binding of OX40L to CD134 is reduced by no more than about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% or less on human CD134 expressing T cells, and wherein the binding molecules further do not prevent a proliferative response of human OX40L on human CD134 expressing T regulatory cells.

Another aspect of the invention provides a binding molecule that binds to human CD134, wherein the binding molecule does not inhibit or prevent the binding of human CD134(OX40) receptor to OX40 ligand (OX40L), and wherein the binding molecule further does not prevent a proliferative response of human OX40L on human CD134 expressing T regulatory cells.

In another aspect, the invention provides binding molecules that bind to human CD134, wherein the binding of OX40L to CD134 is reduced by no more than about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% or less on human CD134 expressing T cells, and wherein the binding molecules inhibit the proliferative response of human OX40L on human CD134 expressing T regulatory cells.

In another aspect, the invention provides a binding molecule that binds to human CD134, wherein the binding molecule does not inhibit or prevent the binding of human CD134(OX40) receptor to OX40 ligand (OX40L), and wherein the binding molecule inhibits the proliferative response of human OX40L on human CD134 expressing T regulatory cells.

Suitable methods for measuring simultaneous binding of OX40L to anti-CD 134 antibodies are described below. In the absence of anti-human CD134 antibody, the FITC fluorescence signal (geometric mean or mean fluorescence density (MFI)) of human OX40L bound on PHA-stimulated human CD134 expressing PBMC was set at 100%. In the absence of human OX40L, the PE fluorescence signal (MFI) of anti-human CD134 antibody bound to PHA-stimulated human CD134 expressing PBMC was set at 100%. When human OX40L and anti-human CD134 antibodies are simultaneously added to PHA-stimulated human CD 134-expressing PBMCs, the decrease in FITC fluorescence signal and PE fluorescence signal is preferably no more than about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10% or less.

A suitable method for measuring the loss of inhibition of the proliferative response to OX 40L-mediated Teff is as follows. Tritiated thymidine or BrdU incorporation in Teff expressing human CD134 was set at 100% following human OX40L treatment. When human OX40L and anti-human CD134 antibodies are added simultaneously to activated (e.g., PHA-stimulated or anti-CD 3/anti-CD 28 bead-stimulated) human CD 134-expressing Teff, the change (i.e., depletion or increase) in such tritiated thymidine or BrdU incorporation is preferably no more than about 30%, or about 20%, or about 10% or less.

Suitable methods for measuring the enhancement of the proliferative response to OX 40L-mediated Teff are as follows. Tritiated thymidine or BrdU incorporation in Teff expressing human CD134 was set at 100% following human OX40L treatment. When human OX40L and anti-human CD134 antibodies are added simultaneously to activated (e.g., PHA-stimulated or anti-CD 3/anti-CD 28 bead-stimulated) human CD 134-expressing Teff, the enhancement of such tritiated thymidine or BrdU incorporation is preferably greater than about 30%, or about 40%, or about 50%, or about 60%, or about 70% or greater.

A suitable method of measuring the loss of inhibition of OX 40L-mediated Treg suppressive function is as follows. Tritiated thymidine or BrdU incorporation in human CD134 expressing teffs co-cultured with human CD134 expressing teffs (e.g., Teff/Treg ratio 1:1) was set to 100% following human OX40L treatment. When human OX40L and anti-human CD134 antibodies are added simultaneously to activated (e.g., PHA-stimulated or anti-CD 3/anti-CD 28 bead-stimulated) human CD134 expressing Teff (e.g., Teff/Treg ratio of 1:1) co-cultured with human CD134 expressing Teff, the change (i.e., depletion or increase) in such tritiated thymidine or BrdU incorporation is preferably no more than about 30%, or about 20%, or about 10% or less.

Suitable methods for measuring enhancement of OX 40L-mediated Treg suppressive function are as follows. Tritiated thymidine or BrdU incorporation in human CD134 expressing teffs co-cultured with human CD134 expressing teffs (e.g., Teff/Treg ratio 1:1) was set to 100% following human OX40L treatment. When human OX40L and anti-human CD134 antibodies are added simultaneously to activated (e.g., PHA-stimulated or anti-CD 3/anti-CD 28 bead-stimulated) human CD134 expressing Teff (e.g., Teff/Treg ratio of 1:1) co-cultured with human CD134 expressing Teff, the enhancement of such tritiated thymidine or BrdU incorporation is preferably greater than about 30%, or about 40%, or about 50%, or about 60%, or about 70% or greater.

A suitable method of measuring the loss of obstruction to the OX 40L-mediated proliferative response of tregs is as follows. Tritiated thymidine or BrdU incorporation in tregs expressing human CD134 was set at 100% following human OX40L treatment. When human OX40L and anti-human CD134 antibodies are added simultaneously to activated (e.g., PHA-stimulated or anti-CD 3/anti-CD 28 bead-stimulated) tregs expressing human CD134, the change (i.e., depletion or increase) in such tritiated thymidine or BrdU incorporation is preferably no more than about 30%, or about 20%, or about 10% or less.

A suitable method of measuring inhibition of the OX 40L-mediated proliferative response of tregs is as follows. Tritiated thymidine or BrdU incorporation in tregs expressing human CD134 was set at 100% following human OX40L treatment. When human OX40L and anti-human CD134 antibodies are added simultaneously to activated (e.g., PHA-stimulated or anti-CD 3/anti-CD 28 bead-stimulated) tregs expressing human CD134, the reduction in tritiated thymidine or BrdU incorporation is preferably greater than about 30%, or about 40%, or about 50%, or about 60%, or about 70% or greater.

In another aspect, the invention provides a method of treating cancer in a mammal comprising administering to the mammal a therapeutically effective amount of a binding molecule described herein.

In a further preferred embodiment of the invention, the binding molecule is antibody clone 12H3 and/or clone 20E5, or an antigen-binding fragment of either antibody. In a further embodiment, the mammal is a human.

In another preferred embodiment of the present invention, there is provided a method of preventing cancer in a mammal comprising administering to the mammal a therapeutically effective amount of a binding molecule as described herein.

The term "preventing cancer" refers to delaying, inhibiting or preventing the onset of cancer in a mammal in which the onset of carcinogenesis or tumorigenesis has not been confirmed, but a susceptibility to cancer has been identified, as determined by, for example, genetic screening or other methods. The term also includes treating a mammal having a pre-cancerous condition to stop the progression or cause regression of said pre-cancerous condition to a malignant tumor. Examples of pre-cancerous conditions include hyperplasia, dysplasia and metaplasia. In some embodiments, the binding molecule is an anti-CD 134 antibody or fragment thereof described herein. In a further embodiment of the invention, there is provided a binding molecule selected from the group consisting of antibody clone 12H3 and/or clone 20E5 or an antigen-binding fragment of either antibody. In a further embodiment, the mammal is a human.

Various cancers, including malignant or benign and/or primary or secondary, may be treated or prevented by the methods of the present invention. Examples of such cancers are known to those skilled in the art and are described in standard textbooks such as Merck Manual of Diagnosis and Therapy (published by Merck).

In another embodiment of the invention, the binding molecule may be administered alone as a monotherapy (monotherapy) or in combination with one or more additional therapeutic agents or treatments. Accordingly, in another embodiment of the invention, there is provided a method of treatment or prevention of cancer by combination therapy, the method comprising administering a binding molecule disclosed herein in combination with one or more additional therapeutic or therapeutic agents. The term "additional treatment" refers to a treatment that does not employ a binding molecule provided herein as a therapeutic agent. The term "additional therapeutic agent" refers to any therapeutic agent that is not a binding molecule provided herein. In some embodiments, the binding molecule is anti-human CD134 antibody clone 12H3 and/or clone 20E5 or an antigen-binding fragment of either antibody. In a particular aspect, the invention provides combination therapy for treating cancer in a mammal comprising administering to the mammal a therapeutically effective amount of a binding molecule provided herein in combination with one or more additional therapeutic agents. In a further embodiment, the mammal is a human.

A wide variety of cancer therapeutics can be used in combination with the binding molecules. One skilled in the art is aware of the existence and development of other cancer treatments that can be used in conjunction with the methods and binding molecules herein and is not limited to the treatment modalities described herein. Examples of classes of additional therapeutic agents that may be used in combination therapy for cancer include (1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormonal therapeutic agents.

The term "chemotherapeutic agent" refers to a chemical or biological substance that can cause death of cancer cells or interfere with the division, repair, growth and/or function of cancer cells. Examples of chemotherapeutic agents include those disclosed in WO2006/088639, WO2006/129163, and US20060153808, the disclosures of which are incorporated herein by reference.

The term "immunotherapeutic" refers to a chemical or biological substance that enhances the immune response in a mammal. Examples of immunotherapeutic agents include: bacille calmette-guerin (BCG); cytokines such as interferon; vaccines such as MyVax personalized immunotherapy, Onyvax-P, Oncophage, GRNVACl, Favld, Provenge, GVAX, Lovaxin C, BiovaxID, GMXX and NeuVax; and antibodies such as alemtuzumab (CAMPATH), bevacizumab (AVASTIN), cetupuzumab (ERBITUX), gemtuzumab ozogamicin (MYLOTARG), ibritumomab (ibritumomab tiuxetan) (ZEVALIN), panitumumab (VECTIBIX), rituximab (RITUXAN, mabthhera), trastuzumab (HERCEPTIN), tositumomab (BEXXAR), tremelimumab, CAT-3888, and agonist antibodies against the CD40 receptor disclosed in WO 2003/040170.

The term "hormonal therapeutic agent" refers to a chemical or biological substance that inhibits or eliminates the production of hormones, or inhibits or counteracts the effects of hormones on the growth and/or survival of cancer cells. Examples of such therapeutic agents suitable for the methods herein include those disclosed in US 20070117809. Examples of specific hormonal therapy agents include tamoxifen (NOLVADEX), toremifene (Fareston), Fulvestrant (FASLODEX), Anastrozole (ARIMIDEX), exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate (MEGACE), goserelin (ZOLADEX) and ropham (LUPRON). The binding molecules herein can also be used in combination with non-pharmaceutical hormone therapies, such as (1) surgical procedures to remove all or a portion of the organs or glands involved in hormone production, such as the ovary, testis, adrenal gland, and pituitary, and (2) radiation therapy, wherein the patient's organ or gland is subjected to an amount of radiation sufficient to inhibit or eliminate the production of the target hormone.

In another embodiment of the invention, there is provided a method of treating or preventing cancer by combination therapy, the method comprising administering a binding molecule disclosed herein and surgically removing the tumor. The binding molecule may be administered to the mammal before, during or after the surgery.

Combination therapy for the treatment of cancer also includes combinations of the binding molecules provided herein and radiation therapy, such as ionizing (electromagnetic) radiation therapy (e.g., X-rays or gamma rays) and particle beam radiation therapy (e.g., high linear energy radiation). The radiation source may be external or internal to the mammal. The binding molecule may be administered to the mammal before, during or after radiation therapy.

The binding molecules and compositions provided herein can be administered via any suitable enteral or parenteral route. The term "enteral route" administration refers to administration via any portion of the gastrointestinal tract. Examples of enteral routes include oral, mucosal, buccal (buccal) and rectal routes, or the intragastric route. By "parenteral route" administration is meant a route of administration that is not an enteral route. Suitable routes of administration and methods may vary depending on factors such as the particular antibody used, the desired rate of absorption, the particular formulation or dosage form used, the type or severity of the disease being treated, the particular site of action, and the condition of the patient, and can be readily selected by one skilled in the art.

The term "therapeutically effective amount" of a binding molecule refers to an amount effective for the intended therapeutic purpose. For example, in the context of enhancing an immune response, a "therapeutically effective amount" is any amount effective to stimulate, provoke, augment, improve or enhance any response of the mammalian immune system. In the context of treating cancer, a "therapeutically effective amount" is any amount sufficient to produce any desired or beneficial effect in the treated mammal, such as inhibiting further growth or spread of cancer cells, cancer cell death, inhibiting cancer recurrence, reducing pain associated with cancer, or improving survival of the mammal. In a method of preventing cancer, a "therapeutically effective amount" is any amount effective to delay, inhibit, or prevent the onset of cancer in a mammal to which the binding molecule is administered.

A therapeutically effective amount of the binding molecule typically ranges from about 0.001 to about 500mg/kg, more typically from about 0.05 to about 100mg/kg of mammalian body weight. For example, the amount may be about 0.3mg/kg, 1mg/kg, 3mg/kg, 5mg/kg, 10mg/kg, 50mg/kg or 100mg/kg of the mammalian body weight. In some embodiments, a therapeutically effective amount of an anti-human CD134 antibody is in the range of about 0.1-30mg/kg of mammal body weight. The precise dosage level to be administered can be readily determined by one of skill in the art and will depend upon a variety of factors, such as the type and severity of the disease being treated, the particular binding molecule employed, the route of administration, the time of administration, the duration of the treatment, the particular additional treatment employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the art.

The binding molecules or compositions are typically administered in multiple instances. The interval between individual doses may be, for example, weekly, monthly, every 3 months, or yearly. Exemplary treatment regimens are once weekly, once every two weeks, once every three weeks, once every four weeks, once every month, once every 3 months, or once every 3-6 months. A typical dosing regimen for anti-human CD134 antibodies includes intravenous administration of 1mg/kg body weight or 3mg/kg body weight using one of the following dosing regimens: (i) once every 4 weeks, 6 doses were administered, then once every 3 months; (ii) once every 3 weeks; (iii) once at 3mg/kg body weight, followed by 1mg/kg body weight once every 3 weeks.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way. On the contrary, it is to be understood that the description of the invention is provided to enable one of ordinary skill in the art to make various other embodiments, modifications, and equivalents without departing from the spirit of the invention and/or scope of the appended claims.

Examples

Example 1: production of mouse anti-human CD134(═ OX40) monoclonal antibodies

(a) Production of Sf9 insect cells expressing surface CD134

cDNA encoding human CD134 protein (GenBank ref CAB 96543.1; ref.)See SEQ ID No.1) was optimized for Sf9 insect cell (Spodotera frugiperda) expression and was synthesized by GENEART, Regensburg, Germany (see SEQ ID No. 2). This cDNA was subcloned into the baculovirus transfer plasmid pVL1393(BD transfer kitcat No. 560129; BD Biosciences). Sf9 cells (ATCC) were then co-transfected with the transfer plasmid pVL1393 containing cDNA encoding human CD134 together with Baculogold baculovirus DNA (BD transfer kit) and incubated for 4-5 days at 27 ℃. Following this co-transfection step, the supernatant was collected and stored at 4 ℃ and used to infect more Sf9 insect cells for virus expansion. For this purpose, Sf9 insect cells were transfected with the amplified recombinant baculovirus and then incubated at 27 ℃ for 3-5 days. These Sf9 insect cells were harvested, washed with sterile PBS, and washed at 5x10 in PBS⁶Cells/250 μ l aliquot, stored at-80 ℃ to obtain cell lysate. Prior to storage, surface expression of human CD134 on transfected Sf9 insect cells was confirmed with 1:10 Phycoerythrin (PE) conjugated mouse anti-human CD134(cloneACT 35; BD Biosciences) and flow cytometry.

(b) Immunization and production of mouse anti-human CD134 monoclonal antibodies

BALB/c mice (female, 6 weeks old; Charles River Laboratories) were injected subcutaneously with approximately 400. mu.L of human CD134 transfected Sf9 insect cell lysate (250. mu.L aliquots of cell lysate + 250. mu.L complete Freund's adjuvant; Sigma) on day 0. Similar subcutaneous injections were performed on days 21 and 42 with Sf9 insect cell lysate transfected with human CD134 and incomplete Freund's adjuvant (Sigma). Intraperitoneal booster injections were performed on day 61 and day 62 with Sf9 insect cell lysates (250 μ L/mouse) transfected with human CD134 without adjuvant. On day 65, the application was initiated withand Milstein (Nature 1975; 256: p495-497) describe standard hybridoma technology that spleen cells from immunized mice are fused with SP2/0 myeloma cells (ATCC). Hybridomas producing antibodies against human CD134 (mouse IgG class) were amplified, cryopreserved and cloned by limiting dilution (recombinant human CD134: human Fc. gamma. fusion protein was used respectively)(R&D Systems) and pha (roche) expressing human CD134 stimulated CD4T blast cells (see example 2 below) as targets, screened using conventional ELISA and flow cytometry techniques. Purification of anti-human CD134 specific monoclonal antibody by protein G column (GE Healthcare) yielded the mouse anti-human CD134 monoclonal antibody clone 12H3 (mouse IgG1 kappa isotype; using IsoTrip from Roche)^TMMouse Monoclonal antibody Isotype Kit) and clone 20E5 (Mouse IgG1 κ Isotype; as above).

Example 2: flow cytometry characterization of mouse anti-human CD134 monoclonal antibodies clone 12H3 and 20E5

(a) Expression of CD134 on PHA-stimulated human T lymphocytes

Human Peripheral Blood Mononuclear Cells (PBMC) from healthy donors (informed consent) were density centrifuged on Lymphoprep (1.077 g/mL; Nycomed). Then, at 1-2x10⁶PBMC/mL RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (Bodinco) and 50. mu.g/mL gentamicin (Gibco) supplemented with 0, 0.1, 1.0 or 10.0. mu.g/mL phytohemagglutinin-M (PHA-M; Roche) at 37 ℃ with 5% CO₂And culturing for 1-3 days. After culture, PBMCs were harvested and cultured at 1-2X10⁶cells/mL were placed in ice-cold phosphate buffered saline (PBS/BSA/NaN)₃) (containing 0.1% bovine serum albumin (Sigma)/0.05% NaN₃And supplemented with 10% human pooled serum (HPS; blocking Fc. gamma. receptors; BioWhittaker)). Cells were incubated with 10. mu.g/mL of The commercially available mouse anti-human CD134 antibody clone ACT35 (mouse IgG1 isotype; BD Biosciences, Alphen aande Rijn, The Netherlands) for 30 minutes at 4 ℃. In PBS/BSA/NaN₃After extensive washing, the cells were then incubated with PE conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch) diluted 1:200 at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After extensive washing, the cells were incubated with a 1:20 dilution of a mouse anti-human CD3 antibody conjugated with Fluorescein Isothiocyanate (FITC) to detect T lymphocytes (BD Biosciences) for 30 minutes at 4 ℃. In PBS/BSA/NaN₃After washing well, cells were washed in 2% formaldehyde in PBS/BSA/NaN₃Medium 4 deg.CFixation was performed for 30 minutes. Binding of the antibody was measured by flow cytometry (FACSCalibur; BD Biosciences).

As shown in figure 1 (n ═ 1 from each donor), peripheral blood-derived unstimulated/resting human T lymphocytes did not express any CD134, however, PHA dose-dependent stimulation of human CD3⁺T lymphocytes express surface CD 134. Activated human CD3 when exposed to 10 μ g/mL PHA⁺The expression level of CD134 on T lymphocytes appeared to be stable between "day 1" and "day 2", however, human CD134 was present during the experiment⁺/CD3⁺The percentage of T lymphocytes increases time-dependently.

(b) Expression of CD134 on PHA-stimulated human CD4T lymphocyte subpopulation

PHA stimulation (1 day at 0 and 10. mu.g/mL, see above) produced T lymphocytes expressing human CD 134. Cells were harvested and cultured at 1-2X10⁶cells/mL were placed in ice-cold PBS/BSA/NaN supplemented with 10% HPS (blocking Fc. gamma. receptors; BioWhittaker)₃. Cells were incubated with either FITC-conjugated mouse anti-human CD4 antibody (BD Biosciences) at 1:10 dilution, or a combination of FITC-conjugated mouse anti-human CD8 antibody (BD Biosciences) at 1:10 dilution and commercially available PE-conjugated mouse anti-human CD134 antibody clone ACT35(BD Biosciences) at 1:10 dilution for 30 minutes at 4 ℃. In PBS/BSA/NaN₃After washing well, cells were washed in 2% formaldehyde in PBS/BSA/NaN₃Fixed at medium 4 ℃ for 30 minutes. Binding of the antibody was measured by flow cytometry (FACSCalibur; BD Biosciences).

As shown in FIG. 2, in PHA stimulated human CD4⁺CD134 expression was observed on T lymphocytes, but at resting human CD4⁺Not observed on T lymphocytes. In PHA-activated human CD8⁺Low expression of CD134 was found on T lymphocytes, but at rest human CD8⁺Not found on T lymphocytes (data not shown).

(c) Binding of mouse anti-human CD134 monoclonal antibodies clone 12H3 and 20E5 on PHA-stimulated human CD134 expressing T lymphocytes

PHA stimulation (2 days at 10. mu.g/mL, see above) produced T lymphocytes expressing human CD 134. Cells were harvested and cultured at 1-2X10⁶cells/mL were placed in ice-cold PBS/BSA/NaN supplemented with 10% HPS (blocking Fc. gamma. receptors; BioWhittaker)₃In (1). Cells were incubated for 30 minutes at 4 ℃ with 0, 0.007, 0.02, 0.07, 0.2, 0.6, 1.9, 5.6, 16.7, 50.0 μ g/mL of the commercially available mouse anti-human CD134 antibody clone ACT35 (mouse IgG1 isotype; BD Biosciences), and the home-made mouse anti-human CD134 antibody clone 12H3 or clone 20E 5. In PBS/BSA/NaN₃After extensive washing, the cells were then incubated with PE conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch) diluted 1:200 at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After extensive washing, the cells were incubated with a 1:20 dilution of FITC conjugated mouse anti-human CD3 antibody (BD Biosciences) detecting T lymphocytes for 30 minutes at 4 ℃. In PBS/BSA/NaN₃After washing well, cells were washed in 2% formaldehyde in PBS/BSA/NaN₃Fixed at medium 4 ℃ for 30 minutes. Binding of the antibody was measured by flow cytometry (FACSCalibur; BDbiosciences).

As shown in FIG. 3 (mean. + -. SD; results observed in two donors), the mouse anti-human CD134 antibodies clone ACT35, clone 12H3 and clone 20H5 gave PHA-stimulated CD3 at approximately 5.0-10.0. mu.g/mL⁺Human CD134 surface molecules on T lymphocytes are saturated. With these two donors, half maximal binding was observed for mouse anti-human CD134 antibody clone 12H3 at about 0.5 μ g/mL, and half maximal binding was observed for mouse anti-human CD134 antibody clones ACT35 and 20E5 at about 2.5 μ g/mL.

(d) Binding of mouse anti-human CD134 monoclonal antibodies, clone 12H3 and 20E5, on PHA-stimulated CD 4-positive and CD 8-positive T-lymphocytes expressing human CD134

PHA stimulation (1 day at 20. mu.g/mL, see above) produced T lymphocytes expressing human CD 134. Cells were harvested and cultured at 1-2X10⁶cells/mL were placed in ice-cold PBS/BSA/NaN supplemented with 10% HPS (blocking Fc. gamma. receptors; BioWhittaker)₃In (1). Cells were incubated at 4 ℃ with 20.0. mu.g/mL mouse IgG1 kappa isotype control (BD Biosciences), or with 20.0. mu.g/mL mouse anti-human CD134Monoclonal antibody clone 12H3 or clone 20E5 was incubated for 30 minutes. In PBS/BSA/NaN₃After extensive washing, the cells were then incubated with PE conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch) diluted 1:100 at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After extensive washing, cells were incubated with FITC conjugated mouse anti-human CD4 antibody (BD Biosciences) diluted 1:20, or FITC conjugated mouse anti-human CD8 antibody (BD Biosciences) diluted 1:20 to detect T lymphocyte subpopulations at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After washing well, cells were washed in 2% formaldehyde in PBS/BSA/NaN₃Fixed at medium 4 ℃ for 30 minutes. Binding of the antibody was measured by flow cytometry (FACSCalibur; BDbiosciences).

As shown in FIG. 4, it was confirmed that the mouse anti-human CD134 monoclonal antibody clone 12H3 and clone 20E5 were present in activated human CD4⁺Positive staining on a subset of T lymphocytes, on activated human CD8⁺Low positive staining on T lymphocyte subpopulations.

(e) Cross-competition of unlabeled mouse anti-human CD134 antibody clones 12H3 and 20E5 on PHA-stimulated human CD134 expressing T lymphocytes with PE-conjugated commercial mouse anti-human CD134 antibody

Stimulation with PHA (at 10. mu.g/mL or 20. mu.g/mL for 4 days or 1 day, respectively, see above) produced T lymphocytes expressing human CD 134. Cells were harvested and cultured at 1-2X10⁶cells/mL were placed in ice-cold PBS/BSA/NaN supplemented with 10% HPS (blocking Fc. gamma. receptors; BioWhittaker)₃In (1). Cells were incubated with 20. mu.g/mL of unlabeled mouse anti-human CD134 monoclonal antibody clone 12H3 or with 10. mu.g/mL of unlabeled clone 20E5 for 30 minutes at 4 ℃. The cells were then incubated with PE conjugated, commercially available mouse anti-human CD134 antibody clone ACT35(BD Biosciences) or clone L106 (BDbiosciences; see also Godfrey) diluted 1:20 at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After washing well, cells were washed in 2% formaldehyde in PBS/BSA/NaN₃Fixed at medium 4 ℃ for 30 minutes. Binding of PE conjugated commercially available anti-CD 134 antibodies was measured by flow cytometry (FACSCalibur; BD Biosciences).

As shown in fig. 5, pre-incubation with unlabeled mouse anti-human CD134 antibody clone 12H3 partially blocked the binding of commercial PE-conjugated mouse anti-human CD134 antibody clone L106 to human CD134 on PHA-stimulated T lymphocytes. Preincubation with unlabeled mouse anti-human CD134 antibody clone 20E5 slightly blocked the binding of commercial PE-conjugated mouse anti-human CD134 antibody clone L106 to human CD134 on PHA-stimulated T lymphocytes. Preincubation with unlabeled mouse anti-human CD134 antibody clone 12H3 and clone 20E5 showed no effect on the binding of commercial PE-conjugated mouse anti-human CD134 antibody clone ACT35 to human CD134 on PHA-stimulated T lymphocytes.

These results demonstrate that the mouse anti-human CD134 antibody clone 12H3 specifically recognizes human CD134 on PHA-stimulated T lymphocytes (partially blocks clone L106 binding), and binds to (ii) an epitope on human CD134 that is different from the epitope recognized by the commercial mouse anti-human CD134 antibody clone L106. These results also demonstrate that mouse anti-human CD134 antibody clone 20E5(i) specifically recognizes human CD134 on PHA-stimulated T lymphocytes (slightly blocks L106 binding), and (ii) binds to a different epitope than the epitope recognized by commercial mouse anti-human CD134 antibody clone L106. In addition, these results demonstrate that the mouse anti-human CD134 antibody clone 12H3 and clone 20E5 appear to recognize human CD134 epitopes on PHA-stimulated T lymphocytes, which are different from the epitope recognized by the commercial mouse anti-human CD134 antibody clone ACT 35. In addition, these results demonstrate that the mouse anti-human CD134 antibodies clone 12H3 and clone 20E5 appear to recognize dissimilar human CD134 epitopes on PHA-stimulated T lymphocytes (as evidenced by the partial blockade of vs slightly blocking L106 binding, respectively).

(f) Simultaneous binding of recombinant human OX40 ligand on PHA-stimulated human CD134 expressing T lymphocytes with mouse anti-human CD134 antibody clones 12H3 and 20E5

PHA stimulation (1 day at 10. mu.g/mL; see above) produced T lymphocytes expressing human CD 134. Cells were harvested and cultured at 1-2X10⁶cells/mL were placed in ice-cold PBS/BSA/NaN supplemented with 10% HPS (blocking Fc. gamma. receptors; BioWhittaker)₃In (1). Cells were incubated with 10.0. mu.g/mL polyhistidineAcid-labeled recombinant human OX40 ligand (OX 40L; R)&D Systems) and 50.0. mu.g/mL anti-polyhistidine antibody (mouse IgG)₁,clone AD1.1.10；R&D Systems) was incubated at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After extensive washing, the cells were then incubated with a 1:100 dilution of FITC-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch) at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After extensive washing, the cells were incubated with 10.0. mu.g/mL of biotinylated (using N-hydroxysuccinimidyl biotin from Pierce) mouse anti-human CD134 monoclonal antibody clone 12H3 or clone 20E5 for 30 minutes at 4 ℃. In PBS/BSA/NaN₃After extensive washing, cells were incubated with PE conjugated streptavidin (Jackson ImmunoResearch) diluted 1:100 at 4 ℃ for 30 min. In PBS/BSA/NaN₃After washing well, cells were washed in 2% formaldehyde in PBS/BSA/NaN₃Fixed at medium 4 ℃ for 30 minutes. Binding of human OX40L and anti-human CD134 antibodies was measured by flow cytometry (FACSCalibur; BD Biosciences).

As shown in FIG. 6, both mouse anti-human CD134 monoclonal antibody clone 12H3 and mouse anti-human CD134 monoclonal antibody clone 20E5 bound simultaneously to human OX40L on PHA-stimulated human CD134 expressing T lymphocytes. This indicates that the mouse anti-human CD134 monoclonal antibodies clone 12H3 and clone 20E5 do not interact with an epitope within the OX40L binding region on the human CD134 receptor. This finding is in contrast to the commercially available mouse anti-human CD134 monoclonal antibody clone L106(Stanford University/Godfrey patent EP 0726952B 1), clone L106 recognizes an epitope within the human OX40L binding region of the human CD134 receptor (Taylor and Schwarz. J Immunol Methods 2001; 255: 67-72; Kirin & La Jolla institute/Croft patent WO2007/062235A 2).

(g) CD134 expression on human effector and regulatory T lymphocytes following stimulation of the beads with anti-human CD 3/anti-human CD28 antibodies

Human CD4T lymphocytes were generated by using microbead-conjugated mouse anti-human CD4 antibody (Miltenyi Biotec) and VarioMACS^TMMagnet/LS columns (Miltenyi Biotec) were purified from PBMC by positive selection. Subsequently, these CD4T lymphocytes were conjugated with FITC in miceAnti-human CD4 antibody (Dako) and PE-conjugated mouse anti-human CD25 antibody (BDBiosciences). CD4⁺/CD25^-Conventional effector T lymphocytes (Teff) and CD4⁺/CD25^highRegulatory T lymphocytes (tregs) were sorted using an Altra flow cytometry cell sorter (Beckman-Coulter). This resulted in an enrichment of greater than 95% Teff and greater than 95% tregs. Teff and Treg at 2.5x10⁵cells/mL were plated with 0.02mM pyruvate (Gibco), 100U/mL penicillin (Gibco), 100. mu.g/mL streptomycin (Gibco), and 10% heat inactivated HPS (HPS)_i(ii) a RPMI-1640/glutamax medium (Gibco) from LMI). Then, the cells were incubated at 2.5 × 10⁴Cells/200. mu.L/well seeded in 96-well round bottom plates (Greiner) and at 25U/mL recombinant human interleukin-2: (G-IL)From novartis pharmaceuticals UK Ltd) was stimulated with mouse anti-human CD 3/mouse anti-human CD28 antibody beads (CD3/CD28 beads; invitrogen) at 1 bead/2 cells at 37 ℃, 5% CO₂Stimulating for 2-8 days. After culture, cells were harvested and cultured at 1-2X10⁶cells/mL were plated in ice-cold PBS/0.2% BSA and were concomitantly diluted 1:50 with FITC-conjugated mouse anti-human CD4 antibody (Dako), 1:10 with PE-conjugated mouse anti-human CD25 antibody (BD Biosciences), 1:50 with ECD^TMConjugated mouse anti-human CD3 antibody (Beckman-Coulter), 1:10 diluted PE-Cy^TM5 conjugated mouse anti-human CD134 antibody (clone ATC 35; BD Biosciences) and 1:10 diluted PE-Cy^TM7 conjugated mouse anti-human CD127 antibody (eBiosciences). Binding of the antibody was measured by flow cytometry (FACSCalibur; BD Biosciences).

As shown in figure 7 (n ═ 1 from each donor), peripheral blood purified unstimulated/restimulated (day 0) human Teff and human tregs did not express any CD134, however, CD3/CD28 bead stimulated human Teff and human tregs expressed surface CD 134. CD134 expression on activated human Teff and human tregs peaked after 2 days of culture and was attenuated after 5 and 8 days of culture.

Example 3: biological characterization of mouse anti-human CD134 monoclonal antibodies clone 12H3 and 20E5

(a) PHA-stimulated proliferation of human CD134 expressing T lymphocytes after treatment with mouse anti-human CD134 antibody clones 12H3 and 20E5

PHA stimulation (1 day at 0 and 10. mu.g/mL; see above) produced T lymphocytes expressing human CD 134. Cells were harvested and cultured at 2X10⁶cells/mL were suspended in RPMI medium (Gibco) containing 10% fetal bovine serum (Bodinco) and 50. mu.g/mL gentamicin (Gibco). Cells were cultured at 0.1X10⁶Cells/100. mu.L/well (i.e., 1X 10)⁶cells/mL) were seeded in 96-well round bottom plates (Corning) and incubated at 37 deg.C, 5% CO₂Exposure to 0, 0.025, 0.25, 2.5 or 25.0. mu.g/mL mouse anti-human CD134 monoclonal antibody clone 12H3 or mouse anti-human CD134 monoclonal antibody clone 20E5, and/or to 0, 0.01, 0.1 or 1.0. mu.g/mL polyhistidine-tagged recombinant human OX40L (in the presence of a 1:5 molar ratio mouse anti-polyhistidine antibody; R < u > in M >)&DSystems) for 6 days. After 6 days, colorimetric (BrdU incorporation) Cell Proliferation ELISA was used^TM(Roche) and ELISA Analyzer (BioRad) cell proliferation was measured at A450 nm.

As shown in fig. 8 (mean ± SD, n-4 with 1 donor), the mouse anti-human CD134 monoclonal antibody clone 12H3 and the mouse anti-human CD134 monoclonal antibody clone 20E5 dose-dependently induced PHA-stimulated proliferation of human CD 134-expressing T lymphocytes. Mouse anti-human CD134 monoclonal antibody clone 12H3 induced proliferation at 0.25, 2.5 and 25. mu.g/mL. Mouse anti-human CD134 monoclonal antibody clone 12H3 induced proliferation at 2.5 and 25. mu.g/mL. In addition, human OX40L also dose-dependently induced PHA-stimulated proliferation of human CD134 expressing T lymphocytes. Human OX40L induced proliferation at 0.1 and 1.0. mu.g/mL. Resting (no PHA stimulation) human CD134^-T lymphocytes did not show any proliferative response after treatment with mouse anti-human CD134 monoclonal antibody clone 12H3, mouse anti-human CD134 monoclonal antibody clone 20E5, or human OX40L (data not shown).

As shown in figure 9 (mean ± SD, n ═ 2 using 1 donor), mouse anti-human CD134 monoclonal antibody clone 12H3 (at 2.5 and 25 μ g/mL), mouse anti-human CD134 monoclonal antibody clone 20E5(2.5 and 25 μ g/mL), and human OX40L (1.0 μ g/mL) induced PHA-stimulated proliferation of human CD134 expressing T lymphocytes. Untreated (medium only) or treatment with the mouse IgG1 kappa isotype control (2.5 and 25. mu.g/mL; BD Biosciences) did not show any effect on PHA-stimulated proliferation of human CD 134-expressing T-lymphocytes. 2.5 and 25. mu.g/mL (or lower concentrations; data not shown) of mouse anti-human CD134 monoclonal antibody clone 12H3 or 2.5 and 25. mu.g/mL (or lower concentrations; data not shown) of mouse anti-human CD134 monoclonal antibody clone 20E5 in combination with 1.0. mu.g/mL (or lower concentrations; data not shown) of human OX40L did not show any reciprocal (i.e., synergistic or additive or even inhibitory) effect on PHA-stimulated proliferation of human CD134 expressing T lymphocytes.

(b) Proliferation of human CD134 expressing T effector and T regulatory lymphocytes stimulated by anti-human CD 3/anti-CD 28 beads after treatment with mouse anti-human CD134 antibody clones 12H3 and 20E5

Human CD4T lymphocytes were purified from PBMCs by negative selection using a mouse antibody cocktail (BD BioSciences) against human CD8 (clone RPA-T8), CD14 (clone M5E2), CD16 (clone 3G8), CD19 (clone 4G7), CD33 (clone P67.6), CD56 (clone B159) and CD235a (HIR 2). In and withConjugated sheep anti-mouse IgG (Invitrogen) incubated, and collected from Dynal Magnetic Particle Concentrator, MPC^TMUnbound CD4T lymphocytes were collected in-6 (Invitrogen). From these enriched CD4T lymphocytes, mouse anti-human CD25 antibody (Miltenyi Biotec)/10 conjugated with 10. mu.L microbeads⁷Cells and MiniMACS^TMMagnet/MS column (Miltenyi BiotecVarioMACS)^TMMagnet/LS column (Miltenyi Biotec) separation of CD25 by MACS sorting^highTreg and CD25^-Teff. This resulted in an enrichment of greater than 90% Teff and greater than 90% Treg. Teff and Treg at 0.25x10⁶cells/mL were plated with 0.02mM pyruvate (Gibco), 100U/mL penicillin (Gibco), 100. mu.g/mL streptomycin (Gibco), and 10% HPS_iRPMI-1640/glutamax medium (Gibco). Then Teff and Treg at 2.5x10⁴Cells/200. mu.L/well (i.e., 0.125X 10)⁶cells/mL) were seeded in 96-well round bottom plates (Greiner) and1 bead/5 cells with CD3/CD28 beads (Invitrogen) at 37 ℃, 5% CO₂Stimulation was performed with or without 5.0. mu.g/mL mouse anti-human CD134 monoclonal antibody clone 12H3, 5.0. mu.g/mL mouse anti-human CD134 monoclonal antibody clone 20E5, 1.0. mu.g/mL polyhistidine-tagged recombinant human OX40L (in the presence of mouse anti-polyhistidine antibody at a 1:5 molar ratio; R.sub.&D Systems), 5.0. mu.g/mL mouse anti-human CD134 monoclonal antibody clone 12H3 in combination with 1.0. mu.g/mL polyhistidine-tagged recombinant human OX40L (in the presence of a 1:5 molar ratio of mouse anti-polyhistidine antibody), or 5.0. mu.g/mL mouse anti-human CD134 monoclonal antibody clone 20E5 in combination with 1.0. mu.g/mL polyhistidine-tagged recombinant human OX40L (in the presence of a 1:5 molar ratio of mouse anti-polyhistidine antibody). After 4 or 5 days, cell proliferation was followed by 0.5. mu. Ci tritiated thymidine (Perkin)&Elmer) incorporation and β -counter (Canberra-Packard).

As shown in figure 10 (mean ± SD), although CD3/CD28 stimulated beads alone induced considerable proliferation (i.e., culture medium) in human CD134 expressing teffs, mouse anti-human CD134 monoclonal antibody clone 12H3 or human OX40L induced additional proliferation in human CD134 expressing teffs stimulated by CD3/CD28 beads. The mouse anti-human CD134 monoclonal antibody clone 20E5 did not induce additional proliferation in CD3/CD28 bead-stimulated Teff expressing human CD 134.

As shown in figure 11 (mean ± SEM, from 5 donors), mouse anti-human CD134 monoclonal antibody clone 12H3 and mouse anti-human CD134 monoclonal antibody clone 20E5 induced no or low proliferation in human CD134 expressing tregs stimulated with CD3/CD28 beads, whereas human OX40L induced very strong proliferation in human CD134 expressing tregs stimulated with CD3/CD28 beads.

As shown in figure 12A (mean ± SD), the combination of mouse anti-human CD134 monoclonal antibody clone 12H3 with human OX40L did not show any interactive (i.e., inhibitory, synergistic, or additive) effect in CD3/CD28 bead stimulated human CD134 expressing Teff. In addition, the combination of mouse anti-human CD134 monoclonal antibody clone 20E5 with human OX40L did not show any interactive (i.e., inhibitory, synergistic or additive) effects in CD3/CD28 bead stimulated human CD134 expressing teffs (data not shown).

As shown in figure 12B (mean ± SD), mouse anti-human CD134 monoclonal antibody clone 12H3 strongly inhibited human OX 40L-mediated proliferative responses in CD3/CD28 bead-stimulated human CD 134-expressing tregs, compared to the effect (lack of any) observed in human OX 40L-mediated proliferative responses in CD3/CD28 bead-stimulated human CD 134-expressing teffs. .

(c) Inhibitory function of human CD 134-expressing T regulatory lymphocytes stimulated with anti-human CD 3/anti-CD 28 beads after treatment with mouse anti-human CD134 antibody clones 12H3 and 20E5

Human CD4T lymphocytes were purified from PBMCs and enriched for Teff and tregs as described in example 3(b) above. Teff and Treg at 0.25x10⁶cells/mL were plated with 0.02mM pyruvate (Gibco), 100U/mL penicillin (Gibco), 100. mu.g/mL streptomycin (Gibco), and 10% HPS_iRPMI-1640/glutamax medium (Gibco). Then Teff at 2.5x10⁴Cells/200. mu.L/well (i.e., 0.125X 10)⁶Teff/mL) were seeded in 96-well round bottom plates (Greiner) and incubated with 2.5 × 10⁴Suppressive tregs/200 μ L/well (i.e., 0.125x 10)⁶Treg/mL; Teff/Treg ratio 1: 1). These Teff/Treg cocultures were cultured using CD3/CD28 beads (Invitrogen) at 1 bead/10 cells at 37 ℃, 5% CO₂5 days of stimulation with or without 5.0. mu.g/mL of mouse anti-human CD134 monoclonal antibody clone 12H3, 5.0. mu.g/mL of mouse anti-human CD134 monoclonal antibody clone 20E5, and 1.0. mu.g/mL of polyhistidine-tagged recombinant human OX40L (in the presence of mouse anti-polyhistidine antibody at a 1:5 molar ratio; R&D Systems). After 5 days, cell proliferation was followed by 0.5. mu. Ci tritiated thymidine (Perkin)&Elmer) incorporation and β -counter (Canberra-Packard).

As shown in figure 13 (mean ± SD), human tregs inhibited the human Teff proliferative response (i.e. culture medium) induced by CD3/CD28 beads. This suppressive function of human tregs was reduced in the presence of the mouse anti-human CD134 monoclonal antibody clone 12H3 or in the presence of human OX40L (dampened). Mouse anti-human CD134 monoclonal antibody clone 20E5 showed no effect on human Treg suppression function.

Example 4: molecular genetic characterization of mouse anti-human CD134 monoclonal antibodies clone 20E5 and 12H3

(a) Isotyping (Isotyping) and Edman degradation

Using IsoTrip^TMMouse Monoclonal Antibody Isotype Kit (Roche), protein G purified Mouse anti-human CD134 Monoclonal Antibody clone 20E5 and 12H3 immunoglobulin class, Isotype and light chain type were determined, showing that Mouse anti-human CD134 Monoclonal Antibody clone 20E5 and 12H3 are both Mouse IgG1 with kappa light chain.

In the use of preformed gelsSystem (Invitrogen) following standard LDS-PAGE electrophoresis under reducing (DTT and 70 ℃ heating) conditions, the mouse anti-human CD134 monoclonal antibody clone 20E5 was electroblotted onto polyvinylidene fluoride (PDVF/Immobilon-P) transfer membrane (Millipore) and stained with Coomassie Brilliant blue (BioRad). Then, light and heavy chain bands (50 kDa and 25kDa, respectively) were excised from The PVDF membrane and used in Edman degradation analysis (performed by EuroSequence, Groningen, The Netherlands) to determine The N-terminal amino acid sequence. The results of the mouse anti-human CD134 monoclonal antibody clone 20E5 are shown in SEQ ID NO.3 and SEQ ID NO. 61. The 11 amino acids from the N-terminus of the heavy chain and the 11 amino acids from the N-terminus of the light chain were determined.

(b).RT PCR

Hybridoma cells of clones 20E5 and 12H3 were harvested from cell culture. Cells were washed with PBS and aliquoted to 5X10 each⁶Individual cells were in vials and stored as pellets at-80 ℃. RNA from the cell pellet was isolated using RNeasy Mini Isolation Kit (QIAGEN). Determining the RNA concentration (A260nm) andRNA was stored at-80 ℃. Total yield of isolated RNA: clone 20E5 was 27.3. mu.g, and clone 12H3 was 58.4. mu.g (both A260/A280 ratios were 1.9). By reverse transcriptase, RevertAId was used^TMH Minus First Strand cDNA Synthesis Kit (Fermentas) cDNA was synthesized from 1. mu.g of RNA and stored at-20 ℃.

Based on the isotype (mouse kappa/IgG 1) and Edman degradation analysis of the mouse anti-human CD134 monoclonal antibody clone 20E5, the following primers were designed to amplify the V region of the mouse anti-human CD134 monoclonal antibody clone 20E 5:

numbering according to the Bioceros internal coding system;

and (3) N ═ A, C, G or T, Y ═ C or T, R ═ A or G, W ═ A or T, S ═ G or C.

Based on the isotype of the mouse anti-human CD134 monoclonal Antibody clone 12H3 (mouse κ/IgG1) and a sense primer that complementarily binds to the cDNA encoding the mouse signal peptide (based in part on Antibody Engineering Volume1Kontermann, Roland e.; bubel, Stefan (Eds.), Springer Lab Manuals,2nd ed.,2010), the following primers were designed to amplify the V region of the mouse anti-human CD134 monoclonal Antibody clone 12H 3:

numbering according to the Bioceros internal coding system;

and (C) N ═ a, C, G or T, Y ═ C or T, R ═ a or G, W ═ a or T, S ═ G or C, M ═ C or a, K ═ G or T.

Primers 201 and 266 are antisense primers designed to complementarily bind to positions 214-232 and 236-255 (based on accession number V00807[ version V00807.1]) within the constant region of the mouse kappa gene, respectively.

Primers 203 and 204 are antisense primers designed to bind complementarily to the constant region of mouse IgG1 at positions 115-134 and 221-240, respectively (based on accession number J00453[ version J00453.1 ]).

Primers 259 and 260 are sense degenerate primers (degeneracy 512 and 256, respectively) that complementarily bind to the N-terminus (amino acids 1-8 and 2-9, respectively) of the heavy chain of Edman degradation-based mouse anti-human CD134 antibody clone 20E 5.

Primer 265 is a sense degenerate primer (16 degeneracy) that complementarily binds to the N-terminus (amino acids 1-7) of the light chain of the Edman-based degraded mouse anti-human CD134 antibody clone 20E 5.

Primer 416 is antisense and is designed to bind complementarily to mouse IgG1 constant region position 111-131 (based on accession number J00453[ version J00453.1 ]).

Primer 394 is antisense and is designed to complementarily bind to constant region position 235-254 (based on accession number V00807[ version V00807.1]) of the mouse kappa gene.

Primers 389, 405 and 410 are degenerate primers (degeneracy 2, 8 and 8, respectively) that bind complementary to the signal peptide sequence of the mouse antibody. Primer 389 was designed for the light chain and primers 405 and 410 for the heavy chain.

Primers 201, 266, 203, 204, 259, 260, and 265 were used in various combinations to amplify the variable region of mouse anti-human CD134 antibody clone 20E5, and primers 416, 394, 405, 410, and 389 were used in various combinations to amplify the variable region of mouse anti-human CD134 antibody clone 12H 3. Various PCR were performed using the cDNA of the two clones produced as a template.

Using Accuprime^TMPfx DNA polymerase (Invitrogen) amplified the variable regions of the heavy and light chains of mouse anti-human CD134 antibody clone 20E5 and clone 12H 3. The PCR products were analyzed on a 1% agarose gel. The PCR reaction product was gel purified and cloned into pCR-Blunt II-Vectors were used for sequence analysis. From the plasmid containing the PCR insert sequence,the cloned insert was analyzed by DNA sequencing using T7 (performed by servicexs b.v., Leiden, The Netherlands orMacrogen, Amsterdam, The Netherlands) to obtain a consensus sequence of The V regions of mouse anti-human CD134 antibody clones 20E5 and 12H 3. 11 message heavy chain responses and 3 message light chain sequence responses were obtained for mouse anti-CD 134 antibody clone 20E 5. 5 informative-sequence heavy-chain responses and 3 informative light-chain-sequence responses were obtained for mouse anti-CD 134 antibody clone 12H 3. Based on this information, the consensus sequences of the V regions of the two antibodies were determined (see SEQ ID NO.4, 5, 12 and 13).

Where documents are listed or discussed in this specification as being explicitly published a priori, this should not necessarily be taken as an acknowledgement that the documents are part of the state of the art or are common general knowledge.

Example 5: generation of chimeric human IgG 4/kappa and/or human IgG 1/kappa (i.e., mouse constant domain exchanged for human IgG/kappa constant domain) anti-human CD134 monoclonal antibody clones 20E5 and 12H3

Chimeric human antibody versions were designed based on the determined mouse V regions of mouse anti-CD 134 antibody clones 20E5 and 12H3 (see example 4(b) above). For this purpose, the CHO cell optimized cDNA sequence (see SEQ ID NO.20 (encoding chimeric human IgG4 heavy chain clone 20E5), SEQ ID NO.21 (encoding chimeric human kappa light chain clone 20E5), SEQ ID NO.22 (encoding chimeric human IgG1 heavy chain clone 20E5), SEQ ID NO.23 (encoding chimeric human IgG4 heavy chain clone 12H3), and SEQ ID NO.24 (encoding chimeric human kappa light chain clone 12H3)) was purchased from GENEART (Regensburg, Germany) and encodes a mouse signal peptide followed by a variable light chain linked to a human kappa constant region, or a variable heavy chain linked to a human IgG constant region. This design was performed for two antibodies, for clone 20E5, the variable heavy chain was linked to either human IgG4 or to the human IgG1 constant region; for clone 12H3, the variable heavy chain was linked to the human IgG4 constant region. The resulting cDNA was subcloned into pcDNA3.1-derived expression plasmids using appropriate restriction enzymes. FreeStyle for chimeric antibody^TMMAX CHO (CHO-S cells) Expression System (Invitrogen). Expressed antibodiesPurifying with affinity chromatography protein A column (GE Healthcare). The chimeric amino acid sequences are shown in SEQ ID NO.25, 26, 27, 28 and 29.

Example 6: binding characterization of chimeric human IgG 4/kappa and/or IgG 1/kappa anti-human CD134 monoclonal antibody clone 20E5

(a) Characterization of binding of human IgG4 kappa anti-human CD134 monoclonal antibody clone 20E5 on PHA-stimulated CD4 positive T-lymphocytes expressing human CD134

PHA stimulation (1 day at 10. mu.g/mL; see above) produced T lymphocytes expressing human CD 134. Cells were harvested and cultured at 1-2X10⁶cells/mL were plated in ice-cold PBS/BSA/NaN₃In (1). Cells were incubated with 0, 0.007, 0.02, 0.07, 0.2, 0.6, 1.9, 5.6, 16.7, 50.0. mu.g/mL chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 for 30 min at 4 ℃. In PBS/BSA/NaN₃After extensive washing, the cells were then incubated with FITC-conjugated mouse anti-human IgG4 antibody (Sigma) diluted 1:50 at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After extensive washing, cells were incubated with PE conjugated mouse anti-human CD4 antibody (BD Biosciences) diluted 1:10 at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After washing well, cells were washed in 2% formaldehyde in PBS/BSA/NaN₃Fixed at medium 4 ℃ for 30 minutes. Binding of the antibody was measured by flow cytometry (FACSCalibur; BDbiosciences).

The chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 stimulated PHA CD4 at approximately 5.0-10.0. mu.g/mL⁺Human CD134 surface molecules on T lymphocytes were saturated (data not shown). Half-maximal binding of chimeric human IgG4 κ anti-human CD134 antibody clone 20E5 was observed at about 1.0 μ g/mL (data not shown).

(b) Binding of chimeric human IgG4 kappa anti-human CD134 monoclonal antibody clone 20E5 on PHA-stimulated CD 4-positive and CD 8-positive T-lymphocytes expressing human CD134

PHA stimulation (1 day at 10. mu.g/mL; see above) produced T lymphocytes expressing human CD 134. Cells were harvested and cultured at 1-2X10⁶cells/mL were plated in ice-cold PBS/BSA/NaN₃In (1). Cells were incubated at 4 ℃ for 30 minutes with or without 20.0. mu.g/mL chimeric human IgG4 kappa anti-human CD134 antibody clone 20E 5. In PBS/BSA/NaN₃After extensive washing, the cells were then incubated with a 1:200 dilution of PE conjugated goat anti-human IgG (Fc gamma specific) antibody (Jackson ImmunoResearch) for 30 minutes at 4 ℃. In PBS/BSA/NaN₃After extensive washing, cells were incubated with FITC conjugated mouse anti-human CD4 antibody (BDbiosciences) diluted 1:10 or FITC conjugated mouse anti-human CD8 antibody (BDbiosciences) diluted 1:10 to detect T lymphocyte subpopulations at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After washing well, cells were washed in 2% formaldehyde in PBS/BSA/NaN₃Fixed at medium 4 ℃ for 30 minutes. Binding of the antibody was measured by flow cytometry (FACSCalibur; BDbiosciences).

Chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 was demonstrated in PHA-activated human CD4⁺Positive staining on T lymphocyte subpopulation, and PHA-activated human CD8⁺Low positive staining on T lymphocyte subpopulations (data not shown).

(c) Binding of chimeric human IgG4 kappa anti-human CD134 monoclonal antibody clone 20E5 on human CD 3/anti-human CD28 antibody-stimulated bead-stimulated human CD134 expressing CD 4-positive and CD 8-positive T-lymphocytes

Human Peripheral Blood Mononuclear Cells (PBMC) from healthy donors (informed consent) were density centrifuged on Lymphoprep (1.077 g/mL; Nycomed). Then, at 1x10⁶PBMC/mL RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (Bodinco) and 50. mu.g/mL gentamicin (Gibco), stimulated beads with mouse anti-human CD 3/mouse anti-human CD28 antibody (CD3/CD28 beads; Invitrogen) at 1 bead/4 cells in the absence or presence of 25U/mL recombinant human interleukin-2 (PeproTech) at 37 ℃, 5% CO₂Stimulating for 1 day. After culture, PBMCs were harvested and cultured at 1-2X10⁶cells/mL were plated in ice-cold PBS/BSA/NaN₃In (1). Cells were incubated at 4 ℃ for 30 minutes with or without 20.0. mu.g/mL chimeric human IgG4 kappa anti-human CD134 antibody clone 20E 5. In PBS/BSA/NaN₃After extensive washing, cells were then washed with 1:200 dilution of PE conjugated mountainSheep anti-human IgG (Fc γ -specific) antibody (Jackson ImmunoResearch) was incubated at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After extensive washing, cells were incubated with FITC-conjugated mouse anti-human CD4 antibody (BD Biosciences) diluted 1:10 or FITC-conjugated mouse anti-human CD8 antibody (BD Biosciences) diluted 1:10 to detect T lymphocyte subpopulations at 4 ℃ for 30 minutes. In PBS/BSA/NaN₃After washing well, cells were washed in 2% formaldehyde in PBS/BSA/NaN₃Fixed at medium 4 ℃ for 30 minutes. Binding of the antibody was measured by flow cytometry (FACSCalibur; BD Biosciences).

As shown in FIG. 14, chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 demonstrated activation of human CD4 on CD3/CD28 beads⁺Positive staining on T lymphocyte subpopulation, and human CD8 activated on CD3/CD28 beads⁺Low positive staining on T lymphocyte subpopulations. No significant effect was observed with the recombinant human IL-2 supplement.

Example 7: biological characterization of chimeric human IgG 4/kappa anti-human CD134 monoclonal antibody clone 20E5

(a) PHA-stimulated proliferation of human CD134 expressing T lymphocytes after treatment with chimeric human IgG4 kappa anti-human CD134 monoclonal antibody clone 20E5

PHA stimulation (1 day at 10. mu.g/mL; see above) produced T lymphocytes expressing human CD 134. Cells were harvested and cultured at 2X10⁶cells/mL were suspended in RPMI medium (Gibco) containing 10% fetal bovine serum (Bodinco) and 50. mu.g/mL gentamicin (Gibco). Cells were cultured at 0.1X10⁶Cells/100. mu.L/well (i.e., 1X 10)⁶cells/mL) were seeded in 96-well flat-bottom plates (Corning) and incubated at 37 ℃, 5% CO₂Clone 20E5 exposed to 25.0. mu.g/mL chimeric human IgG4 kappa anti-human CD134 antibody or to 25.0. mu.g/mL control human IgG4 kappa anti-human CD40 antibody (PG 102; Pannetetics), or to 1.0. mu.g/mL polyhistidine-tagged recombinant human OX40L (in the presence of 1:5 molar ratio mouse anti-polyhistidine antibody; R&DSystems)6 days. After 6 days, colorimetric (BrdU incorporation) Cell Proliferation ELISA was used^TM(Roche) and ELISA Analyzer (B)ioRad) cell proliferation was measured at a450 nm.

As shown in fig. 15 (mean ± SD), chimeric human IgG4 κ anti-human CD134 antibody clone 20E5(hu20E5) and human OX40L induced PHA-stimulated proliferation of human CD134 expressing T lymphocytes. Untreated (medium only) or treatment with the control human IgG4 kappa anti-human CD40 antibody (huIgG4) did not show any effect on PHA-stimulated proliferation of human CD134 expressing T lymphocytes.

(b) PHA-stimulated proliferation of human CD134 expressing T lymphocytes after treatment with the combination of chimeric human IgG4 kappa anti-human CD134 monoclonal antibody clone 20E5 and recombinant human OX40L

PHA stimulation (1 day at 10. mu.g/mL; see above) produced T lymphocytes expressing human CD 134. Cells were harvested and cultured at 2X10⁶cells/mL were suspended in RPMI medium (Gibco) containing 10% fetal bovine serum (Bodinco) and 50. mu.g/mL gentamicin (Gibco). Cells were cultured at 0.1X10⁶Cells/100. mu.L/well (i.e., 1X 10)⁶cells/mL) were seeded in 96-well flat-bottom plates (Corning) and incubated at 37 ℃, 5% CO₂Exposure to either 0, 0.025, 0.25, 2.5 or 25.0. mu.g/mL chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 or/and recombinant human OX40L labeled with 0, 0.01, 0.1 or 1.0. mu.g/mL polyhistidine (in the presence of a 1:5 molar ratio mouse anti-polyhistidine antibody; R&D Systems) for 6 days. After 6 days, colorimetric (BrdU incorporation) Cell Proliferation ELISA was used^TM(Roche) and ELISA Analyzer (BioRad) cell proliferation was measured at A450 nm.

As shown in figure 16 (mean ± SD), chimeric human IgG4 κ anti-human CD134 antibody clone 20E5(hu20E5) and human OX40L dose-dependently induced PHA-stimulated proliferation of human CD134 expressing T lymphocytes. Chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 induced proliferation at 2.5 and 25. mu.g/mL (donor 1) or at 0.25, 2.5 and 25. mu.g/mL (donor 2) donor-dependent. In addition human OX40L induced proliferation donor-dependently at 0.1 and 1.0. mu.g/mL (donor 1) or at 0.01, 0.1 and 1.0. mu.g/mL (donor 2).

As shown in FIG. 17 (mean. + -. SD), the combination of chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5(hu20E5) at 2.5 and 25. mu.g/mL (or lower concentrations; data not shown) with human OX40L at 0.1 and 1.0. mu.g/mL (or lower concentrations; data not shown) did not show any interactive (i.e., synergistic or additive or even inhibitory) effect on PHA stimulated proliferation of human CD134 expressing T lymphocytes.

(c) Human CD134 expressing T lymphocyte proliferation stimulated by anti-human CD 3/anti-human CD28 antibody stimulation beads after treatment with chimeric human IgG4 kappa anti-human CD134 monoclonal antibody clone 20E5

Human Peripheral Blood Mononuclear Cells (PBMC) from healthy donors (informed consent) were density centrifuged on Lymphoprep (1.077 g/mL; Nycomed). Subsequently, PBMC were dosed at 0.1x10⁶Cells/100. mu.L/well (i.e., 1X 10)⁶cells/mL) were inoculated in RPMI-1640 medium (Gibco) (containing 10% fetal bovine serum (Bodinco) and 50 μ g/mL gentamicin (Gibco)) in 96-well flat-bottom plates (Corning) and stimulated with mouse anti-human CD 3/mouse anti-human CD28 antibody beads (CD3/CD28 beads; invitrogen) 1 bead/2 cells in the absence or presence of 25U/mL recombinant human interleukin-2 (PeproTech) at 37 ℃, 5% CO₂And (5) performing stimulation. After 1 or 2 days, these (without or with IL-2) CD3/CD28 bead-stimulated human CD134 expressing T lymphocytes were incubated at 37 ℃ with 5% CO₂Exposure to 25.0. mu.g/mL chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 or to 1.0. mu.g/mL polyhistidine-tagged recombinant human OX40L (in the presence of a 1:5 molar ratio of mouse anti-polyhistidine antibody; R)&D Systems) for 6 or 5 days. Prior to cell proliferation measurements, cells initially stimulated with the recombinant human interleukin-2 combination, aggravated with CD3/CD28 beads, were restimulated with 25U/mL recombinant human interleukin-2 for 1 day. Colorimetric (BrdU incorporation) CellProlification ELISA after exposure to chimeric human IgG4 kappa anti-human CD134 antibody clone 20E5 or to human OX40L6 or 5 days^TM(Roche) and ELISA Analyzer (BioRad) cell proliferation was measured at A450 nm.

As shown in figure 18 (mean ± SD, n ═ 3, using 1 donor), while CD3/CD28 stimulated beads alone induced considerable proliferation of human CD134 expressing T lymphocytes (i.e. culture medium), chimeric human IgG4 κ anti-human CD134 antibody clone 20E5(hu20E5) and human OX40L induced additional proliferation in CD3/CD28 bead stimulated human CD134 expressing T lymphocytes. The addition of interleukin-2 appeared to increase basal (i.e., culture medium) proliferation only in human CD134 expressing T lymphocytes stimulated by CD3/CD28 beads.

(d) Immunostimulatory responses in rhesus monkeys (rhesumacaque monkeys) after treatment with human (chimeric) anti-human CD134 antibody clones 12H3 and 20E5

Non-human primate rhesus monkeys can be immunized with the simian immunodeficiency virus protein gp130 as described by Weinberg et al (J Immunother 2006; 29: 575-.

Draining lymph nodes from immunized monkeys treated with anti-human CD134 antibody clones 12H3 and 20E5 with human (e.g. chimeric or humanized or de-immunized (deimmunized); e.g. sub-human IgG1 or IgG4) are expected to show swollen lymph nodes compared to control immunized monkeys. Human (e.g., chimeric or humanized or deimmunized) anti-human CD134 antibody clones 12H 3-treated and 20E 5-treated monkeys were expected to show increased gp 130-specific antibody titers, and increased long-term T cell responses, compared to controls. There should be no significant signs of toxicity in monkeys treated with anti-human CD134 antibody clone 12H3 or clone 20E5 (e.g., chimeric or humanized or deimmunized).

Example 8: characterization of human CD134 domains and epitopes recognized by mouse anti-human CD134 monoclonal antibodies clone 12H3 and 20E5

(a) Binding of mouse anti-human CD134 monoclonal antibodies clone 12H3 and 20E5 to non-reduced and reduced recombinant human CD134: human Fc γ fusion proteins (western blot)

Denaturing electrophoresis on preformed LDS-PAGE under various non-reducing and reducing conditions (see FIG. 19-A)In the system, 1300 or 650 ng/lane (for Coomassie blue staining) or250 ng/lane (for western blot) recombinant human CD134: human Fc γ (IgG1) fusion protein (R)&D Systems) were subjected to electrophoresis. Recombinant human CD134: human Fc γ fusion protein was then stained with coomassie brilliant blue (BioRad) or electroblotted onto polyvinylidene fluoride (PDVF) transfer membrane (Millipore). After blocking with PBS/0.05% Tween 20/1% BSA fraction V (Roche) for 20 minutes at room temperature, PDVF membranes were incubated with 100ng/mL mouse anti-human CD134 monoclonal antibody clone 12H3 or 20E5 for 1 hour at room temperature. In parallel, 100ng/mL mouse IgG1 kappa isotype control antibody (BD Biosciences) was used as a negative control. After extensive washing in PBS/0.05% Tween20, the binding of the mouse anti-human CD134 monoclonal antibody clone 12H3 or 20E5 was assayed with a 1:5000 dilution of horseradish peroxidase conjugated goat anti-mouse Fc γ specific antibody (Jackson ImmunoResearch) at room temperature for 1 hour, followed by colorimetric detection with a ready-to-use solution of TMB substrate (Sigma).

As shown in FIG. 19-B, the recombinant human CD134: human Fc γ fusion protein was confirmed to have a molecular mass of about 130-140kDa under non-reducing (and LDS denaturation without and with thermal denaturation, conditions a and B, respectively) conditions. Non-reduced without heating (condition a) showed 2 bands in close proximity, suggesting that the fraction of recombinant human CD134: human Fc γ fusion protein was not completely denatured/unfolded. Heated non-reduction (condition b) showed 1 band, suggesting complete denaturation/unfolding of recombinant human CD134: human Fc γ fusion protein. Recombinant human CD134 human Fc gamma fusion protein in the reduction (and without and with thermal denaturation LDS denaturation, conditions c and d respectively) under conditions of about 110kDa band (condition c) and about 60-65kDa band (condition d). The former observation suggested incomplete reduction of the recombinant human CD134: human Fc γ fusion protein, the latter observation suggested complete reduction/cleavage of the disulfide bond linking the 2 human IgG 1-derived Fc γ fragment within each recombinant human CD134: human Fc γ fusion protein molecule.

As shown in fig. 19-C, both the mouse anti-human CD134 antibody clone 12H3 and clone 20E5 recognized recombinant human CD134: human Fc γ fusion protein, predominantly at about 130kDa, under non-reducing (and no and heat-denatured LDS denaturation, conditions a and b, respectively) conditions. In contrast, mouse anti-human CD134 antibody clone 12H3 showed only slight binding to recombinant human CD134: human Fc γ fusion protein under reducing (and no and thermal denaturation of LDS, conditions c and d, respectively) conditions, while mouse anti-human CD134 antibody clone 20E5 showed strong binding to recombinant human CD134: human Fc γ fusion protein under reducing (and no and thermal denaturation of LDS, conditions c and d, respectively) conditions.

These results confirmed that mouse anti-human CD134 clone 12H3 and clone 20E5 specifically recognized human CD 134. In addition, these results demonstrate that the mouse anti-human CD134 antibodies clone 12H3 and clone 20E5 appear to recognize different human CD134 epitopes as evidenced by the strong binding of the slightly (clone 12H3) vs (clone 20E5) to recombinant human CD134: human Fc γ fusion protein under reducing (and LDS denaturation without and with heat denaturation) conditions. These results suggest that mouse anti-human CD134 antibody clone 12H3 recognizes an epitope on human CD134 that is insensitive to denaturation (LDS and heat treatment) and sensitive to reduction (i.e., DTT is associated with a disulfide bond that is likely to be cysteine-rich domain (CRD)). These results suggest that mouse anti-human CD134 antibody clone 20E5 recognizes an epitope on human CD134 that is insensitive to denaturation (LDS and heat treatment) and to reduction (i.e. DTT is associated with a disulfide bond cleavage, most likely a cysteine-rich domain (CRD)).

(b) Binding of mouse anti-human CD134 monoclonal antibodies clone 12H3 and 20E5 to full-length human CD134 constructs and various truncated human CD134 constructs on 293-F cell line (domain localization)

To analyze the fine specificity of mouse anti-human CD134 monoclonal antibodies clone 12H3 and 20E5, the location of the epitope recognized by mouse anti-human CD134 monoclonal antibodies clone 12H3 and 20E5 was determined by domain localization. The ability of mouse anti-human CD134 monoclonal antibodies clone 12H3 and 20E5 to bind to truncated human CD134 constructs expressed on the surface of (HEK-derived) 297-F cells was determined by FACS analysis.

Cysteine-rich in the extracellular region of human CD134 was identified based on literature (Swiss-Prot: P43489.1; Latza et al Eur J Immunol 1994; 24: 677-683; Bodmer et al trends Biochem Sci 2002; 27: 19-26; Comepan et al Structure 2006; 14: 1321-1330; US patent 2011/0028688A1)The amino acid domain (CRD) and the hinge-like structure. The CRDs are numbered CRD1, CRD2, (truncated) CRD3, (truncated) CRD4 (see fig. 20). The CRD contains topologically independent module types, called a-module and B-module (see also fig. 20). The A module is in a C-shaped structure, and the B module is in an S-shaped structure. A typical CRD is usually composed of an a1-B2 module or a2-B1 module (or a less common pair of different modules, such as a1-B1), with 6 conserved cysteine residues, where the numbers represent the number of disulfide bonds in each module (see also fig. 20). As shown in fig. 20, 5 different human CD134 constructs were prepared and expressed: (1) the full-length human CD134 construct, which originates from the N-terminal CRD1 (i.e.the CRD1A1-B2 module covers amino acids 29-65), is therefore referred to as "CRD 1" and comprises the amino acids 1-277 (see SEQ ID NO.1), (2) "CRD 2" construct, which originates from the N-terminal CRD2 (i.e.the CRD2A1-B2 module covers amino acids 66-107) and comprises the amino acids 66-277 (see SEQ ID NO.30) linked to the signal peptide amino acids 1-28, (3) "CRD 3" construct, which originates from the N-terminal CRD3 (i.e.the CRD3A1-B1 module covers amino acids 108-146 (according to composite et al.Structure2006; 14:1321-1330)) or a truncated CRD3A1 module covers amino acids 108-126 (according to Latz et al J J. Eur J24: 677; 1994: 677-D1-31) and comprises the signal peptide linked to amino acids 277 (see SEQ ID NO.31), (4) a "CRD 4" construct consisting of an N-terminal CRD4 or CRD3 sub-domain B1 module/truncated CRD4A1 module (i.e. CRD4A 1-B1-module covering amino acids 127 & 167(Latza et al. Eur JImmunol 1994; 24:677 & 683) or a combination of CRD3 sub-domain B1 module and truncated CRD4A1 module (not shown in FIG. 20) covering amino acids 127 & 146 and 147 & 167 (Comepan et al. structural 2006; 14:1321 & 1330)), respectively, and comprising amino acids 2006 & 277 & 2006 & 277 (see SEQ ID NO.32) linked to signal peptide amino acids 1-28, (5) truncated (CRD 4 "construct consisting of an N-terminal CRD4 or CRD4 sub-domain B1 module (i.e. CRD 167A. 32; 5)" truncated CRD4 "module covering amino acids # 20 & 6714. J.11 & gt # 11 & gt; SEQ ID NO. 11 & gt # 11 & gt # 20 & gt # 11 & gt # 20 & gt # 19 & gt Amino acid 147-. By using Accuprime^TMOf Pfx DNA polymerase (Invitrogen)PCR was assembled, and the 5 human CD134 constructs were generated using the primers in the following table:

primer numbering according to the Bioceros internal coding system

Briefly, the cDNA encoding signal peptide amino acids 1-28 and the cDNA encoding amino acids 66-277 of human CD134 were amplified in a PCR reaction using full length human CD134 as a template with primer pairs 362/365 and 364/363, respectively. Subsequently, these two PCR products were used in an assembly PCR using primer pair 362/363 to generate the "CRD 2" construct. The cDNA encoding the "CRD 2" construct was subcloned into pcDNA3.1-derived expression plasmids using appropriate restriction sites. Similarly, the corresponding primers shown in the above table were used to generate the "CRD 3" construct (signal peptide amino acids 1-28 linked to amino acids 108-277 of human CD134), "CRD 4" construct (signal peptide amino acids 1-28 linked to amino acids 127-277) and "truncated CRD 4" construct (signal peptide amino acids 1-28 linked to amino acids 147-277) and subcloned into the pcDNA3.1-derived expression plasmid. In addition, full-length human CD134(SEQ ID NO.1) was also recloned into pcDNA3.1-derived expression plasmid.

Using FreeStyle^TM293 expression System (Invitrogen), transiently transfected FreeStyle with 5 generated human CD134 variants^TM293-cells (Invitrogen). After 48-72 hours, FACS was used to analyze the expression of surface human CD134 on the transfected cells. To do this, the transfected cells were harvested and cultured at 1-2X10⁶cells/mL were plated in ice-cold PBS/BSA/NaN₃In (1). Cells were incubated with 20.0. mu.g/mL mouse anti-human CD134 monoclonal antibodies, clone 12H3 and 20E5, for 30 minutes at 4 ℃. In parallel, 20.0. mu.g/mL mouse IgG1 kappa isotype control antibody (BD Biosciences) was used as a negative control. In PBS/BSA/NaN₃After extensive washing, the cells were then incubated with a 1:200 dilution of PE conjugated goat anti-mouse IgG (Fc gamma specific) antibody (Jackson ImmunoResearch) for 30 minutes at 4 ℃. In PBS/BSA/NaN₃After thorough washing, the cells are washed2% Formaldehyde in PBS/BSA/NaN₃Fixed at medium 4 ℃ for 30 minutes. Binding of the antibody was measured by flow cytometry (FACSCalibur; BDbiosciences).

As shown in FIG. 21, both mouse anti-human CD134 antibody clones 12H3 and 20E5 recognized full-length (termed the "CRD 1" construct) human CD134 on transfected 293-F cells, while both mouse anti-human CD134 antibody clones 12H3 and 20E5 showed no binding on 293-F cells transfected with transfection reagent control. In addition, the mouse anti-human CD134 antibody clones 12H3 and 20E5 recognized a truncated human CD134 variant on transfected 293-F cells, which lacks CRD1 and CRD1-CRD2 (referred to as the "CRD 2" construct and the "CRD 3" construct, respectively). In contrast, mouse anti-human CD134 antibody clone 12H3 binds very weakly to the truncated human CD134 variant lacking the CRD1-CRD 2-truncated CRD3A1 module (referred to as the "CRD 4" construct), mouse anti-human CD134 antibody clone 12H3 binds very weakly to the truncated human CD134 variant lacking the CRD1-CRD 2-truncated CRD3A 1-module-CRD 4 subdomain A1-module (defined by Latza et al EurJ Immunol 1994; 24:677-683) or the truncated human CD134 variant lacking the CRD1-CRD2-CRD3A 1-B1-module (defined by Compiaetaan. Structurel 2006; 14: 1321-1330; referred to as the "tcCRD 4" construct), whereas mouse CD134 antibody clone 20E 53 binds completely to the truncated human CD134 variant lacking the CRD2-CRD subdomain A1 (referred to as the "CRD 1-CRD 1-module 1-CRD 1-TRD 1-III-CRD 3A-module) and -683) or CRD1-CRD2-CRD3a 1-B1-module (according to Compaan et al structure 2006; 14: 1321-; construct designated "tcCRD 4") showed strong binding.

These results confirm that mouse anti-human CD134 antibody clones 12H3 and 20E5 specifically recognize human CD134 (compare full-length human CD134 transfection vs transfection reagent control transfection). In addition, these results demonstrate that mouse anti-human CD134 antibody clones 12H3 and 20E5 appear to recognize different human CD134 epitopes as evidenced by the lack of strong binding (using clone 12H3) of vs to the truncated human CD134 variant lacking the CRD1-CRD 2-truncated CRD3A 1-module (referred to as the 'CRD 4' construct) and strong binding to the human CD134 variant lacking the CRD1-CRD 2-truncated CRD3A 1-module-CRD 4 sub-domain A1-module (defined according to Lajitsa et al Eur. Eummunol 1994; 24: 677. sub-683) or CRD1-CRD2-CRD3A 1-B1-module (defined according to Compuetaan. structural 2006; 14: 1321. sub. 1330; referred to "CRD 4" construction 39134 ") using clone 5, respectively. These results demonstrate that mouse anti-human CD134 antibody clone 12H3 does not appear to recognize the human CD134 epitope in CRD1 and CRD2, mouse anti-human CD134 antibody clone 20E5 does not appear to recognize the human CD134 epitope in CRD1, CRD2 and truncated CRD3A 1-module CRD4 sub-domain A1-module (according to the definition of Latza et al Eur J Immunol 1994; 24:677-683) or CRD1-CRD2-CRD3A 1-B1-module (according to the definition of Compaan et al 1330 Structure 2006; 14: 1321-one). These results confirm that mouse anti-human CD134 antibody clone 12H3 appears to recognize a linear or nonlinear/conformational epitope in the truncated CRD3A 1-module (according to the definition of Latza et al. Eur J Immunol 1994; 24:677-683) with amino acid sequence 108-126 (i.e. 19-mer peptide RCRAGTQPLDSYKPGVDCA; see SEQ ID No.34) on extracellular human CD134, or that amino acid sequence 108-126 (i.e. 19-mer peptide RCRAGTQPLDSYKPGVDCA; see SEQ ID No.34) forms a critical part to bind to the truncated CRD3A 1-module/CRD 4A 1-B1-module (according to the definition of Latza et al. Eur J munol 1994; 24:677-683) with amino acid sequence 108-214 (see SEQ ID No.35) on extracellular human CD134 and possibly a nonlinear/conformational epitope in the hinge-like structure. These results confirm that the mouse anti-human CD134 antibody clone 20E5 appears to recognize linear or nonlinear/conformational epitopes in the truncated CRD4A 1-module (according to the definitions of Compaan et al, Structure 2006; 14:1321-1330) with the amino acid sequence 147-214 (see SEQ ID NO.36) and possibly the hinge-like structure on extracellular human CD 134.

Using crystallography, CRD1, CRD2 (particularly the A1 loop and residues immediately following it) and CRD3 (mainly the A1 loop) on CD134 were recently found to be critical in OX40 ligand (CD252)/CD134(═ OX40) interactions. This finding is in good agreement with our finding that (1, see above) mouse anti-human CD134 antibody clone 20E5 does not appear to recognize the human CD134 epitope in either CRD1, CRD2 and truncated CRD3A 1-module-CRD 4 subdomain A1-module (according to the definition of Latza et al. Eur J Immunol 1994; 24:677-683) or CRD1-CRD2-CRD3A 1-B1-module (according to the definition of Compaan et al. Structure 2006; 14:1321-1330), on extracellular human CD134, and (2, see above) mouse anti-human CD134 antibody clone 20E5 binds simultaneously to human OX40L on PHA-stimulated human CD134 expressing T lymphocytes. This suggests that mouse anti-human CD134 antibody clone 20E5 recognizes an epitope on human CD134 that is not important for the interaction of human CD134 with human OX 40L. In addition, we found that (1, see above) mouse anti-human CD134 antibody clone 12H3 appeared to recognize a linear or nonlinear/conformational epitope in the truncated CRD3A 1-module (according to the definition of Latza et al. Eur J Immunol 1994; 24:677-683) with amino acid sequence 108-126 (i.e. 19-mer peptide RCRAGTQPLDSYKPGVDCA; see SEQ ID NO.34) on extracellular human CD134, or that amino acid sequence 108-126 (i.e. 19-mer peptide RCRAGTQPLDSYKPGVDCA; see SEQ ID NO.34) formed a key part for binding to the truncated CRD3A 1-module/CRD 4A 1-B1-module (according to the definition of Latza et al. 1994J Immunol; 24: 677-mer 683) with amino acid sequence 108-214 (see SEQ ID NO.35) on extracellular human CD134 and possibly the nonlinear/linear/PHA-like structure in the hinge-like structure, and that PHA (1, see also PHA 2, see CD134) bound to the aforementioned human lymphocyte T134-expressing epitope on human CD134 Human OX40L, supporting the notion that the epitope on human CD134 (as described above) recognized by mouse anti-human CD134 antibody clone 12H3 the interaction of human CD134 with human OX40L is not important.

(c) Epitope mapping of mouse anti-human CD134 monoclonal antibody clone 12H3 using human CD 134-derived peptide ELISA (1)

To further analyze the fine specificity of mouse anti-human CD134 monoclonal antibody clone 12H3, the position of the epitope recognized by mouse anti-human CD134 monoclonal antibody clone 12H3 was determined by epitope mapping. The ability of the mouse anti-human CD134 monoclonal antibody clone 12H3 to bind to a human CD 134-derived peptide corresponding to the amino acid sequence of the truncated CRD3A 1-module-CRD 4 subdomain A1-module (according to the definition of Latza et al. Eur J Immunol 1994; 24:677-683) was determined by ELISA.

96-well flat-bottom ELISA plates (Corning) were coated overnight at 4 ℃ in PBS o/n with 10 ng/well of a peptide derived from human CD134 (synthesized by Pepscan Presto, Lelystad, The Netherlands) corresponding to The amino acid sequence of The truncated CRD3A 1-module-CRD 4 sub-domain A1-module (see SEQ ID NO.38), or with 10 ng/well of a control peptide derived from human fibronectin (synthesized by Pepscan Presto, Lelystad, The Netherlands) corresponding to The amino acid sequence of The outer type III domain (see SEQ ID NO. 37). After extensive washing in PBS/0.05% Tween20, the plates were blocked in PBS/0.05% Tween 20/1% BSA fraction V (Roche) for 1 hour at room temperature. The plates were then incubated with 0, 0.00005-50.0 (10 fold gradient dilution in blocking buffer) μ g/mL mouse anti-human CD134 monoclonal antibody clone 12H3 or mouse IgG₁Kappa isotype control antibody (BD Biosciences) was incubated at room temperature for 1 hour. After extensive washing in PBS/0.05% Tween20, binding of the antibodies was determined with a 1:5000 dilution of horseradish peroxidase conjugated goat anti-mouse IgG Fc γ -specific antibody (Jackson ImmunoResearch) at room temperature for 1 hour, followed by colorimetric detection by a ready-to-use solution of TMB substrate (Invitrogen). Add 1M H₂SO₄Thereafter, the optical density was measured at a wavelength of 450nm (reference wavelength 655nm) with a microplate analyzer (BioRad)

As shown in figure 23-a (n ═ 1), the mouse anti-human CD134 monoclonal antibody clone 12H3 dose-dependently and specifically bound to human CD 134-derived peptides, while mouse IgG bound to human CD 134-derived peptides₁The kappa isotype control antibody demonstrated no binding to human CD 134-derived peptides. Mouse anti-human CD134 monoclonal antibodies clone 12H3 and IgG₁The kappa isotype control antibodies all demonstrated no binding to the human fibronectin derived control peptide.

These results demonstrate that the mouse anti-human CD134 antibody clone 12H3 specifically recognizes an epitope on human CD134 (vs human CD134 derived peptide vs human fibronectin derived control peptide). In addition, these results demonstrate that the mouse anti-human CD134 antibody clone 12H3 appears to recognize a linear or nonlinear/conformational epitope within the truncated CRD3A 1-module-CRD 4 subdomain A1-module (according to the definition of Latza et al Eur J Immunol 1994; 24:677-683) having amino acid sequence 108-146 (i.e., 39-mer peptide RCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTN; see SEQ ID NO.38) on extracellular human CD 134.

(d) Epitope mapping of mouse anti-human CD134 monoclonal antibodies clone 12H3 and 20E5 using the CLIPS epitope mapping technique of Pepscan (2)

The epitopes recognized by mouse anti-human CD134 antibody clones 12H3 and 20E5 can be determined using The CLIPS epitope mapping technique of Pepscan (Lelystad, The Netherlands). The CLIPS technology enables the determination of linear, conformational, discontinuous and complex epitopes involved in dimeric or multimeric protein complexes. For this purpose, the linear amino acid sequence of human CD134 ═ OX40 (SEQ id No.1) was used as target protein.

Example 9: characterization of human CD134 domains and epitopes recognized by chimeric human IgG 4/kappa and/or IgG 1/kappa anti-human CD134 monoclonal antibody clones 12H3 and 20E5

(a) Binding (domain localization) of chimeric human IgG4 κ and/or IgG1 κ anti-human CD134 monoclonal antibody clones 12H3 and 20E5 to full-length human CD134 constructs and various truncated human CD134 constructs expressed on 293-F cell line

To analyze the fine specificity of chimeric human IgG4 κ and/or IgG1 κ anti-human CD134 monoclonal antibody clones 12H3 and 20E5, the positions of the epitopes recognized by chimeric human IgG4 κ and/or IgG1 κ anti-human CD134 monoclonal antibody clones 12H3 and 20E5 were determined by domain localization. The ability of chimeric human IgG4 κ and/or IgG1 κ anti-human CD134 monoclonal antibody clones 12H3 and 20E5 to bind to truncated human CD134 constructs (see example 8(b) above) expressed on the surface of (HEK-derived) 297-F cells was determined by FACS analysis.

Using FreeStyle^TM293 expression System (Invitrogen), transient transfection of FreeStyle with 5 generated human CD134 variants (see above)^TM293-cells (Invitrogen). After 48-72 hours, FACS was used to analyze the expression of surface human CD134 on the transfected cells. To do this, the transfected cells were harvested and cultured at 1-2X10⁶cells/mL were plated in ice-cold PBS/BSA/NaN₃In (1). Cells were incubated at 4 ℃ for 30 minutes with or without 20.0. mu.g/mLChimeric human IgG4 κ anti-human CD134 antibody clone 20E 5. In PBS/BSA/NaN₃After extensive washing, the cells were then incubated with a 1:200 dilution of PE conjugated goat anti-human IgG (Fc gamma specific) antibody (Jackson ImmunoResearch) for 30 minutes at 4 ℃. In PBS/BSA/NaN₃After washing well, cells were washed in 2% formaldehyde in PBS/BSA/NaN₃Fixed at medium 4 ℃ for 30 minutes. Binding of the antibody was measured by flow cytometry (FACSCalibur; BDbiosciences).

As shown in FIG. 22, both chimeric human IgG4 κ and IgG1 κ anti-human CD134 monoclonal antibody clone 12H3 and chimeric human IgG4 κ anti-human CD134 monoclonal antibody clone 20E5 demonstrated the same binding characteristics to various truncated human CD134 constructs on transfected cells as their corresponding parent mouse anti-human CD134 antibody clone 12H3 and 20E5 counterparts (see example 8 (b); compare FIG. 22 vs. FIG. 21).

(b) Epitope mapping of the chimeric human IgG4 kappa anti-human CD134 monoclonal antibody clone 12H3 using human CD 134-derived peptide ELISA

To further analyze the fine specificity of chimeric human IgG4 κ anti-human CD134 monoclonal antibody clone 12H3, the location of the epitope recognized by chimeric human IgG4 κ anti-human CD134 monoclonal antibody clone 12H3 was determined by epitope mapping. The ability of the chimeric human IgG4 kappa anti-human CD134 monoclonal antibody clone 12H3 to bind to human CD 134-derived peptides was determined by ELISA, which corresponds to the amino acid sequence of the truncated CRD3A 1-module-CRD 4 subdomain A1-module (according to the definition of Latza et. Eur J Immunol 1994; 24: 677-683).

96-well flat-bottom ELISA plates (Corning) were coated overnight at 4 ℃ in PBS o/n with 10 ng/well of a peptide derived from human CD134 (synthesized by Pepscan Presto, Lelystad, The Netherlands) corresponding to The amino acid sequence of The truncated CRD3A 1-module-CRD 4 sub-domain A1-module (see SEQ ID NO.38), or with 10 ng/well of a control peptide derived from human fibronectin (synthesized by Pepscan Presto, Lelystad, The Netherlands) corresponding to The amino acid sequence of The outer type III domain (see SEQ ID NO. 37). After extensive washing in PBS/0.05% Tween20, the plates were washed in PBS/0.05% Tween 20/1% BSA fraction V (Roche)Blocking at room temperature for 1 hour. The plates were then incubated with either 0, 0.00005-50.0 (10 fold gradient step in blocking buffer) μ g/mL chimeric human IgG4 κ anti-human CD134 monoclonal antibody clone 12H3 or control human IgG4 κ anti-human CD40 antibody (Biocult) for 1 hour at room temperature. After extensive washing in PBS/0.05% Tween20, binding of the antibodies was determined with a 1:5000 dilution of horseradish peroxidase conjugated goat anti-human IgG Fc γ -specific antibody (Jackson ImmunoResearch) at room temperature for 1 hour followed by colorimetric detection by a ready-to-use solution of TMB substrate (Invitrogen). Add 1M H₂SO₄Thereafter, the optical density (reference wavelength 655nm) was measured at a wavelength of 450nm with a microplate analyzer (BioRad).

As shown in fig. 23-B (n ═ 1), the chimeric human IgG4 κ anti-human CD134 monoclonal antibody clone 12H3 dose-dependently and specifically bound to human CD 134-derived peptides, while the control human IgG4 κ anti-human CD40 antibody demonstrated no binding to human CD 134-derived peptides. Chimeric human IgG4 kappa anti-human CD134 monoclonal antibody clone 12H3 and control human IgG4 kappa anti-human CD40 antibody both demonstrated no binding to human fibronectin-derived control peptide.

These results demonstrate that the chimeric human IgG4 κ anti-human CD134 monoclonal antibody clone 12H3 specifically recognizes an epitope on human CD134 (vs human CD134 derived peptide vs human fibronectin derived control peptide). In addition, these results demonstrate that the chimeric human IgG4 kappa anti-human CD134 monoclonal antibody clone 12H3 appears to recognize a linear or nonlinear/conformational epitope within the truncated CRD3A 1-module-CRD 4 subdomain A1-module (according to the definition of Latza et al. Eur J Immunol 1994; 24:677-683) having the amino acid sequence 108-146 (i.e. 39-mer peptide RCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTN; see SEQ ID NO.38) on extracellular human CD 134.

The accompanying sequence listing forms a part of the specification.

In SEQ ID NO:1, which is the amino acid sequence of human CD134 (GenBank ref CAB 96543.1; aa1-277), the signal peptide is at amino acids (aa)1-28 and the transmembrane region is at aa 215-235.

Also of interest is SEQ ID NO 61, which forms the 11N-terminal amino acids of SEQ ID NO 5. It is the 20E5 light chain equivalent of SEQ ID NO.3, which is the 11N-terminal amino acids of the 20E5 heavy chain.

SEQ ID NO.37(TYSSPEDGIHELFPAPDGEEDTAELQGGC), the amino acid sequence from a human fibronectin-derived peptide, the amino acid sequence corresponding to the outer type III (extra type III) domain (ED 1; Peters et al am Rev Resp Dis 1988; 138: 167-71).

Claims (25)

1. A binding molecule that binds to human CD134, wherein the binding molecule is selected from an antibody or an antigen-binding fragment of an antibody, said binding molecule comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises:

(a) heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO 6;

(b) heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO. 7; and

(c) heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO. 8,

and the light chain variable region comprises:

(a) a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO. 9;

(b) a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO. 10; and

(c) light chain CDR3 consisting of the amino acid sequence of SEQ ID NO. 11.

2. The binding molecule of claim 1, comprising:

(a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID No.4 or a variant of said sequence having amino acid substitutions in 1, 2 or 3 framework regions; and/or

(b) A light chain variable region consisting of the amino acid sequence of SEQ ID No.5 or a variant of said sequence having amino acid substitutions in 1, 2 or 3 framework regions.

3. A binding molecule according to claim 1 or 2, wherein at or above the saturation concentration of the molecule, the binding of OX40L to CD134 is reduced by no more than 50% on human CD134 expressing T cells.

4. The binding molecule of claim 1 or 2, wherein the binding molecule does not prevent human CD134(OX40) receptor binding to OX40 ligand (OX 40L).

5. The binding molecule of claim 1 or 2, which is a human antibody.

6. The binding molecule of claim 1 or 2, which is chimeric, humanized or DeImmunized^TMAn antibody, or a fragment thereof.

7. The binding molecule of claim 1 or 2, which is an IgA, IgD, IgE, IgG or IgM antibody.

8. The binding molecule of claim 7 which is an IgG1, IgG2, IgG3 or IgG4 antibody.

9. The binding molecule of claim 1 or 2, wherein the antibody is an antigen-binding fragment of an antibody.

10. The binding molecule of claim 9, wherein the antigen-binding fragment is an scFv.

11. The binding molecule of claim 1 or 2, wherein the binding molecule is a recombinant antibody.

12. The binding molecule of claim 1 or 2, wherein the binding molecule is a monoclonal antibody.

13. A nucleic acid molecule encoding the binding molecule of any one of claims 1-12.

14. A vector comprising at least one nucleic acid molecule of claim 13.

15. A host cell comprising the vector of claim 14.

16. The host cell of claim 15, wherein said host cell is derived from a mammal or an insect.

17. Use of a binding molecule according to any one of claims 1 to 12 and optionally a pharmaceutically acceptable carrier for the manufacture of a medicament for enhancing an immune response in a human subject.

18. Use according to claim 17, wherein the enhanced immune response comprises an increase in the immunostimulatory/effector function of T effector cells and/or a down-regulation of the immunosuppressive function of T regulatory cells.

19. Use according to claim 18, wherein the enhanced immune response comprises an increase in the immunostimulatory/effector function of T effector cells as a result of proliferation of these cells and/or a down-regulation of the immunosuppressive function of T regulatory cells, the number of these cells not being expanded.

20. Use of a binding molecule according to any one of claims 1 to 12 in the manufacture of a medicament for reducing the size of a tumor or inhibiting the growth of cancer cells in a subject, or reducing or inhibiting the development of metastatic cancer in a subject suffering from cancer.

21. Use of a binding molecule according to any one of claims 1 to 12 in the manufacture of a medicament for the treatment or prevention of cancer.

22. The use of claim 21, wherein the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, hematological cancer, or any other disease or disorder characterized by uncontrolled cell growth.

23. A pharmaceutical composition comprising a binding molecule according to any one of claims 1 to 12 and one or more pharmaceutically acceptable diluents or excipients.

24. The composition of claim 23, wherein the composition is suitable for parenteral administration to a human.

25. The composition of claim 24, wherein the composition is administered intravenously, intramuscularly, intradermally, intraperitoneally, intratumorally, intravesically, intraarterially, intrathecally, intracapsularly, intraorbitally, intracardially, transtracheally, intraarticularly, subcapsulally, subarachnoid, intraspinal, epidural, intrasternally, or subcutaneously.