CN120437287A

CN120437287A - Pharmaceutical composition comprising bispecific antibody specifically binding to GUCY2C and CD3

Info

Publication number: CN120437287A
Application number: CN202510134834.XA
Authority: CN
Inventors: 杜敏; 李润智; 吴婷婷
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2024-02-07
Filing date: 2025-02-07
Publication date: 2025-08-08

Abstract

The present disclosure relates to a pharmaceutical composition comprising a bispecific antibody that specifically binds to GUCY2C and CD 3. In particular, the disclosure relates to a pharmaceutical composition comprising a bispecific antibody that specifically binds to GUCY2C and CD3 and a buffer that is a histidine salt buffer, an acetate buffer, a citrate buffer, a succinate buffer, or a Tris salt buffer.

Description

Pharmaceutical composition comprising bispecific antibody specifically binding to GUCY2C and CD3

Technical Field

The present disclosure is in the field of biotechnology, and more specifically, the disclosure relates to a pharmaceutical composition comprising bispecific antibodies that specifically bind to GUCY2C and CD 3.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

GUCY2C (Guanylyl Cyclase C) belongs to the family of transmembrane guanylate cyclases, consisting of an extracellular domain, a single transmembrane domain and an intracellular catalytic domain, anchored to the cell membrane in homodimeric or trimeric fashion. Its main function is to catalyze the formation of cGMP from GTP to maintain normal physiological functions such as intestinal homeostasis (Michaela Kuhn, physiological Reviews (2016), 96 (2): 751-804). In normal tissues, GUCY2C is expressed only in intestinal epithelial cells, but not or very low in other tissues. At the same time, GUCY2C is expressed in gastrointestinal malignancies, including more than 90% of colorectal cancers of each stage and more than 50% of gastric or gastroesophageal junction cancers (Ruth Birbe MD., human pathway, (2005), 36 (2): 170-179;Hadi Danaee,PLoS One, (2017), 12 (12): e 0189953). In addition, since the expression polarity at the cellular level and the enrichment degree of blood vessels are obviously different in normal intestinal tissues and intestinal cancers, the probability of contact of GUCY2C in the intestinal cancers with the blood vessels is higher than that of the normal tissues (Divya Mathur, CLINICAL CANCER RESEARCH, (2020), 26 (9): 2188-2202), so that the GUCY2C is a specific and potential target point for gastrointestinal cancers, particularly intestinal cancers.

Bispecific antibodies targeting CD3 and TAA are a novel immunotherapy that can bind T cells and tumor cells simultaneously, mimic the interaction of MHC and TCR, and release perforin and granzyme-specific killing of tumor cells after T cells form lytic synapse. The activated T cells can release cytokines, initiate other immune cells and amplify immune responses against the tumor, ultimately leading to proliferation of T cells and a cascade of killing tumor cells.

Disclosure of Invention

The present disclosure provides a pharmaceutical composition of bispecific antibodies that specifically bind to GUCY2C and CD3 and uses thereof.

In one aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody that specifically binds to GUCY2C and CD3 and a buffer, wherein:

the bispecific antibody specifically binding GUCY2C and CD3 comprises a first chain with a structure shown in a formula I and a second chain with a structure shown in a formula II,

Formula I [ GUCY2C-VL ] - [ linker 1] - [ CD3-VH ] - [ linker 2] - [ Fc1],

Formula II [ CD3-VL ] - [ linker 3] - [ GUCY2C-VH ] - [ linker 2] - [ Fc2],

The GUCY2C-VH has a GUCY2C-HCDR1 comprising the amino acid sequence of SEQ ID NO. 4, a GUCY2C-HCDR2 comprising the amino acid sequence of SEQ ID NO. 5 and a GUCY2C-HCDR3 comprising the amino acid sequence of SEQ ID NO. 6, and the GUCY2C-VL has a GUCY2C-LCDR1 comprising the amino acid sequence of SEQ ID NO. 18, a GUCY2C-LCDR2 comprising the amino acid sequence of SEQ ID NO. 8, and a GUCY2C-LCDR3 comprising the amino acid sequence of SEQ ID NO. 9, and

The CD3-VH has a CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:35, a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and a CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO:37, and the CD3-VL has a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:38, a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:39, and a CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 40;

The structure shown in the formula I and the formula II is arranged from the N end to the C end, the linker 1, the linker 2 and the linker 3 are identical or different peptide linkers, and the Fc1 and the Fc2 are Fc region structural sequences which can be mutually associated by a pestle-mortar technology;

the buffer is histidine salt buffer, acetate buffer, citrate buffer, succinate buffer or Tris salt buffer.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein in the bispecific antibody that specifically binds to GUCY2C and CD 3:

The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO 27 or an amino acid sequence having at least 80% (e.g. at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO 27, and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO 25 or an amino acid sequence having at least 80% (e.g. at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO 25, and

The CD3-VH comprises the amino acid sequence of SEQ ID No. 41 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID No. 41, and the CD3-VL comprises the amino acid sequence of SEQ ID No. 42 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID No. 42.

The GUCY2C-VH comprises the amino acid sequence of SEQ ID NO. 27 and the GUCY2C-VL comprises the amino acid sequence of SEQ ID NO. 25, and

The CD3-VH comprises the amino acid sequence of SEQ ID NO. 41, and the CD3-VL comprises the amino acid sequence of SEQ ID NO. 42.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region in a bispecific antibody that specifically binds to GUCY2C and CD 3.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the Fc1 has a raised structure according to the knob and hole technique in a bispecific antibody that specifically binds to GUCY2C and CD3, and the Fc2 has a hole structure according to the knob and hole technique. In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein said Fc1 comprises the amino acid sequence of SEQ ID NO. 43 and said Fc2 comprises the amino acid sequence of SEQ ID NO. 44 in a bispecific antibody that specifically binds GUCY2C and CD 3.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein in the bispecific antibody that specifically binds to GUCY2C and CD3, the linker 1, linker 2 and linker 3 are all peptide linkers known in the art, so long as the bispecific antibody is capable of exhibiting the desired antigen binding activity. For example, the peptide linker may be a flexible peptide comprising 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linker is 1-15 amino acid residues in length. In some embodiments, the linker 1 has a structure as shown in the general sequence formula (GGGS) nGm, where n is 1-5, preferably 1, 2 or 3, and m is 1-10, preferably 4, 5,6,7 or 8. In some embodiments, the sequence of linker 1 is GGGSGGGG (SEQ ID NO: 45). In some embodiments, the linker 2 has a structure as shown in the general sequence formula Gm, wherein m is 1-10, preferably 1-5, more preferably 1, 2 or 3. In some embodiments, the sequence of linker 2 is G (SEQ ID NO: 46). In some embodiments, the linker 3 has a structure as shown in the general sequence formula (GGGGS) nGm, where n is 1-5, preferably 1, 2 or 3, and m is 1-10, preferably 4, 5,6,7 or 8. In some embodiments, the sequence of linker 3 is GGGGSGGGG (SEQ ID NO: 47).

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the linker 1, linker 2, and linker 3 described in the bispecific antibody that specifically binds to GUCY2C and CD3 are the same or different, each independently selected from SEQ ID No. 45, SEQ ID No. 46, and SEQ ID No. 47.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the amino acid sequences of linker 1, linker 2 and linker 3 in the bispecific antibody that specifically binds to GUCY2C and CD3 are SEQ ID No. 45, SEQ ID No. 46 and SEQ ID No. 47, respectively.

the first strand comprises the amino acid sequence of SEQ ID NO. 48, and the second strand comprises the amino acid sequence of SEQ ID NO. 49.

the first strand is shown in the amino acid sequence of SEQ ID NO. 48 and the second strand is shown in the amino acid sequence of SEQ ID NO. 49.

In some embodiments, the buffer of any one of the preceding claims is a histidine-histidine hydrochloride buffer, a histidine-histidine acetate buffer, an acetic acid-sodium acetate buffer, a succinic acid-sodium succinate buffer, a citric acid-sodium citrate buffer, or a Tris-HCl salt buffer.

In some embodiments, the buffer as described above is a histidine-histidine hydrochloride buffer, a histidine-histidine acetate buffer, or a Tris-HCl salt buffer.

In some embodiments, the buffer of any one of the preceding claims is a histidine-histidine hydrochloride buffer.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the concentration of the buffer is 5mM to 100mM. In some embodiments, the buffer is at a concentration of 10mM to 50mM. In some embodiments, the buffer is at a concentration of 10mM to 30mM. In some embodiments, the buffer is at a concentration of 10mM to 25mM. In some embodiments, the buffer is at a concentration of 10mM to 23mM. In some embodiments, the buffer is at a concentration of 10mM to 20mM. In some embodiments, the buffer is at a concentration of 11mM to 13mM. In some embodiments, the buffer is at a concentration of 15mM to 17mM. In some embodiments, the buffer is at a concentration of 11mM to 20mM. In some embodiments, the buffer is at a concentration of 11mM to 17mM. In some embodiments, the buffer is at a concentration of 13mM to 20mM. In some embodiments, the buffer is at a concentration of 13mM to 17mM. In some embodiments, the buffer is at a concentration of 13mM to 15mM. In some embodiments, the buffer is at a concentration of about 20mM. In some embodiments, the buffer is at a concentration of about 14mM. In some embodiments, the buffer is at a concentration of about 13mM. In some embodiments, the buffer is at a concentration of about 10mM. In some embodiments, the buffer is at a concentration of about 30mM. In some embodiments, the buffer is at a concentration of about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, about 25mM, about 27mM, about 30mM, about 33mM, about 36mM, about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM, or about 100mM. In some embodiments, the buffer is present at a concentration of 5mM、6mM、7mM、8mM、9mM、10mM、11mM、12mM、13mM、14mM、15mM、16mM、17mM、18mM、19mM、20mM、21mM、22mM、23mM、24mM、25mM、27mM、30mM、33mM、36mM、40mM、50mM、60mM、70mM、80mM、90mM or 100mM, and any range between these spot values. In some embodiments, the buffer is at a concentration of 20mM. In some embodiments, the buffer is at a concentration of 14mM. In some embodiments, the buffer is at a concentration of 13mM. In some embodiments, the buffer is at a concentration of 10mM. In some embodiments, the buffer is at a concentration of 30mM.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the concentration of histidine-hcl histidine buffer is 5mM to 100mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 10mM to 50mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 10mM to 30mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 10mM to 25mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 10mM to 23mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 10mM to 20mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 11mM to 13mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 15mM to 17mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 11mM to 20mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 11mM to 17mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 13mM to 20mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 13mM to 17mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 13mM to 15mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of about 20mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of about 14mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of about 13mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of about 10mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of about 30mM. In some embodiments, the histidine-histidine hydrochloride buffer is at a concentration of about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, about 25mM, about 27mM, about 30mM, about 33mM, about 36mM, about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about, about 90mM or about 100mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 5mM、6mM、7mM、8mM、9mM、10mM、11mM、12mM、13mM、14mM、15mM、16mM、17mM、18mM、19mM、20mM、21mM、22mM、23mM、24mM、25mM、27mM、30mM、33mM、36mM、40mM、50mM、60mM、70mM、80mM、90mM or 100mM, and any range between these spot values. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 20mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 14mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 13mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 10mM. In some embodiments, the histidine-hcl histidine buffer is at a concentration of 30mM. In some embodiments, the pharmaceutical composition of any one of the preceding claims, having a pH of 5.0 to 7.5. In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the pH of the pharmaceutical composition is from 5.0 to 6.5. In some embodiments, the pharmaceutical composition of any one of the preceding claims, having a pH of 5.0 to 6.2. In some embodiments, the pharmaceutical composition of any one of the preceding claims, having a pH of 5.1 to 6.2. In some embodiments, the pharmaceutical composition of any one of the preceding claims, having a pH of 5.5 to 6.2. In some embodiments, the pharmaceutical composition of any one of the preceding claims, having a pH of 5.5 to 6.0. In some embodiments, the pharmaceutical composition of any one of the preceding claims, having a pH of 5.0 to 6.0. In some embodiments, the pharmaceutical composition of any one of the preceding claims, having a pH of 5.5 to 6.5.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, having a pH of 5.2 to 6.2. In some embodiments, the pharmaceutical composition has a pH of about 5.0. In some embodiments, the pharmaceutical composition has a pH of about 5.1. In some embodiments, the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition has a pH of about 5.5. In some embodiments, the pH of the pharmaceutical composition is about 5.7. In some embodiments, the pharmaceutical composition has a pH of about 6.0. In some embodiments, the pharmaceutical composition has a pH of about 6.2. In some embodiments, the pharmaceutical composition has a pH of about 6.5. In some embodiments, the pharmaceutical composition has a pH of about 7.0. In some embodiments, the pharmaceutical composition has a pH of about 7.5. In some embodiments, the pharmaceutical composition has a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 7.0, or about 7.5. In some embodiments, the pH of the pharmaceutical composition is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 7.0, or 7.5, or any range between these point values. In some embodiments, the pharmaceutical composition has a pH of 5.0. In some embodiments, the pharmaceutical composition has a pH of 5.1. In some embodiments, the pharmaceutical composition has a pH of 5.2. In some embodiments, the pharmaceutical composition has a pH of 5.5. In some embodiments, the pharmaceutical composition has a pH of 5.7. In some embodiments, the pharmaceutical composition has a pH of 6.0. In some embodiments, the pharmaceutical composition has a pH of 6.2. In some embodiments, the pharmaceutical composition has a pH of 6.5. The pH of the pharmaceutical composition was 7.0. In some embodiments, the pharmaceutical composition has a pH of 7.5. When referring to a point value in this disclosure, it should be understood that the point value encompasses an error range. Such error ranges are due to factors such as laboratory environment, personnel handling, instrumentation, methodology, measurement errors, and the like. Taking pH as an example, when measured at about 5.7, it is understood that this includes the error range. As an example, "about 5.7" means 5.7.+ -. 0.2 (i.e., pH 5.5 to 5.9) when the formulation is measured using an industrial pH meter.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 1mg/mL to 250mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 1mg/mL to 200mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 1mg/mL to 150mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 1mg/mL to 120mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 1mg/mL to 100mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 2mg/mL to 200mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 2mg/mL to 150mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 2mg/mL to 100mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 70mg/mL to 150mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 80mg/mL to 120mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 90mg/mL to 110mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of bispecific antibody that specifically binds to GUCY2C and CD3 is about 2mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of bispecific antibody that specifically binds to GUCY2C and CD3 is about 100mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of bispecific antibody that specifically binds to GUCY2C and CD3 is about 200mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of bispecific antibody that specifically binds GUCY2C and CD3 is about 1mg/mL, about 2mg/mL, about 4mg/mL, about 5mg/mL, about 6mg/mL, about 10mg/mL, about 20mg/mL, about 30mg/mL, about 40mg/mL, about 45mg/mL, about 50mg/mL, about 55mg/mL, about 60mg/mL, about 65mg/mL, about 70mg/mL, about 75mg/mL, about 80mg/mL, about 85mg/mL, About 90mg/mL, about 95mg/mL, about 100mg/mL, about 105mg/mL, about 110mg/mL, about 120mg/mL, about 130mg/mL, about 140mg/mL, about 150mg/mL, about 160mg/mL, about 170mg/mL, about 180mg/mL, about 190mg/mL, about 200mg/mL, about 210mg/mL, about 220mg/mL, about 230mg/mL, about 240mg/mL, or about 250mg/mL. in some embodiments, the pharmaceutical composition, wherein the concentration of bispecific antibody that specifically binds to GUCY2C and CD3 is 1mg/mL、2mg/mL、4mg/mL、5mg/mL、6mg/mL、10mg/mL、20mg/mL、30mg/mL、40mg/mL、45mg/mL、50mg/mL、55mg/mL、60mg/mL、65mg/mL、70mg/mL、75mg/mL、80mg/mL、85mg/mL、90mg/mL、95mg/mL、100mg/mL、105mg/mL、110mg/mL、120mg/mL、130mg/mL、140mg/mL、150mg/mL、160mg/mL、170mg/mL、180mg/mL、190mg/mL、200mg/mL、210mg/mL、220mg/mL、230mg/mL、240mg/mL or 250mg/mL, or any range between these spot values. In some embodiments, the pharmaceutical composition, wherein the concentration of bispecific antibody that specifically binds to GUCY2C and CD3 is 2mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of bispecific antibody that specifically binds to GUCY2C and CD3 is 100mg/mL. in some embodiments, the pharmaceutical composition, wherein the concentration of bispecific antibody that specifically binds to GUCY2C and CD3 is 200mg/mL.

In some embodiments, the pharmaceutical composition of any one of the above, wherein the pharmaceutical composition comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from the group consisting of polysorbate (e.g., polysorbate 80, polysorbate 20), poloxamer (e.g., P188), polyhydroxyene, triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, oleamidopropyl-betaine, myristamidopropyl-dimethylamine, palmamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoa acyl, sodium methyl taurate, polyethylene glycol, polypropylene glycol, and the like. In some embodiments, the surfactant is polysorbate or poloxamer. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is a poloxamer. In some embodiments, the surfactant is polysorbate 80 or poloxamer 188 (P188). In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is poloxamer 188.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the concentration of the surfactant is 0.01mg/mL to 2.0mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the surfactant is from 0.1mg/mL to 1.0mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the surfactant is from 0.2mg/mL to 0.6mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the surfactant is from 0.3mg/mL to 0.5mg/mL. In some embodiments, the concentration of the surfactant is about 0.1mg/mL. In some embodiments, the concentration of the surfactant is about 0.4mg/mL. In some embodiments, the concentration of the surfactant is about 1.0mg/mL. In some embodiments, the surfactant concentration is about 0.01mg/mL, about 0.05mg/mL, about 0.1mg/mL, about 0.15mg/mL, about 0.2mg/mL, about 0.3mg/mL, about 0.4mg/mL, about 0.5mg/mL, about 0.6mg/mL, about 0.7mg/mL, about 0.8mg/mL, about 0.9mg/mL, about 1.0mg/mL, about 1.5mg/mL, or about 2.0mg/mL. In some embodiments, the surfactant concentration is 0.01mg/mL、0.05mg/mL、0.1mg/mL、0.15mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1.0mg/mL、1.5mg/mL or 2.0mg/mL, or any range between these point values. In some embodiments, the concentration of the surfactant is 0.1mg/mL. In some embodiments, the concentration of the surfactant is 0.4mg/mL. In some embodiments, the concentration of the surfactant is 1.0mg/mL.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the concentration of polysorbate 80 is 0.01mg/mL to 2.0mg/mL. In some embodiments, the concentration of polysorbate 80 is 0.1mg/mL to 1.0mg/mL of the pharmaceutical composition of any one of the preceding claims. In some embodiments, the concentration of polysorbate 80 is 0.2mg/mL to 0.6mg/mL of the pharmaceutical composition of any one of the preceding claims. In some embodiments, the concentration of polysorbate 80 is 0.3mg/mL to 0.5mg/mL of the pharmaceutical composition of any one of the preceding claims. In some embodiments, the concentration of polysorbate 80 is about 0.1mg/mL. In some embodiments, the concentration of polysorbate 80 is about 0.4mg/mL. In some embodiments, the concentration of polysorbate 80 is about 1.0mg/mL. In some embodiments, the polysorbate 80 concentration is about 0.01mg/mL, about 0.05mg/mL, about 0.1mg/mL, about 0.15mg/mL, about 0.2mg/mL, about 0.3mg/mL, about 0.4mg/mL, about 0.5mg/mL, about 0.6mg/mL, about 0.7mg/mL, about 0.8mg/mL, about 0.9mg/mL, about 1.0mg/mL, about 1.5mg/mL, or about 2.0mg/mL. In some embodiments, the polysorbate 80 concentration is 0.01mg/mL、0.05mg/mL、0.1mg/mL、0.15mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1.0mg/mL、1.5mg/mL or 2.0mg/mL, or any range between these point values. In some embodiments, the polysorbate 80 concentration is 0.1mg/mL. In some embodiments, the polysorbate 80 concentration is 0.4mg/mL. In some embodiments, the concentration of polysorbate 80 is 1.0mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the concentration of poloxamer 188 is between 0.01mg/mL and 2.0mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the poloxamer 188 is at a concentration of 0.1mg/mL to 1.0mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the poloxamer 188 is at a concentration of 0.2mg/mL to 0.6mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the poloxamer 188 is at a concentration of 0.3mg/mL to 0.5mg/mL. In some embodiments, the poloxamer 188 is at a concentration of about 0.1mg/mL. In some embodiments, the poloxamer 188 is at a concentration of about 0.4mg/mL. In some embodiments, the poloxamer 188 is at a concentration of about 1.0mg/mL. In some embodiments, the poloxamer 188 concentration is about 0.01mg/mL, about 0.05mg/mL, about 0.1mg/mL, about 0.15mg/mL, about 0.2mg/mL, about 0.3mg/mL, about 0.4mg/mL, about 0.5mg/mL, about 0.6mg/mL, about 0.7mg/mL, about 0.8mg/mL, about 0.9mg/mL, about 1.0mg/mL, about 1.5mg/mL, or about 2.0mg/mL. In some embodiments, the poloxamer 188 concentration is 0.01mg/mL、0.05mg/mL、0.1mg/mL、0.15mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1.0mg/mL、1.5mg/mL or 2.0mg/mL, or any range between these spot values. In some embodiments, the poloxamer 188 is at a concentration of 0.1mg/mL. In some embodiments, the poloxamer 188 is at a concentration of 0.4mg/mL. In some embodiments, the poloxamer 188 is at a concentration of 1.0mg/mL.

In some embodiments, the pharmaceutical composition of any one of the above comprises a sugar. In some embodiments, the sugar is selected from the group consisting of conventional compositions (CH ₂O)_n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like; in some embodiments, the sugar may be selected from the group consisting of sucrose, trehalose, glucose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol sorbitol, mannitol, melibiose, melezitose, raffinose, mannitinose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, isomaltulose, and the like, in some embodiments, in some embodiments, the sugar is sucrose.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the concentration of the sugar is 10mg/mL to 120mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the sugar is 30mg/mL to 100mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the sugar is 65mg/mL to 95mg/mL. In some embodiments, the concentration of the sugar is 72mg/mL to 88mg/mL. In some embodiments, the concentration of the sugar is about 30mg/mL. In some embodiments, the concentration of the sugar is about 50mg/mL. In some embodiments, the concentration of the sugar is about 80mg/mL. In some embodiments, the concentration of the sugar is about 100mg/mL. In some embodiments, the sugar is at a concentration of about 10mg/mL, about 20mg/mL, about 30mg/mL, about 35mg/mL, about 37.5mg/mL, about 40mg/mL, about 45mg/mL, about 50mg/mL, about 55mg/mL, about 60mg/mL, about 64mg/mL, about 65mg/mL, about 68mg/mL, about 70mg/mL, about 72mg/mL, about 75mg/mL, about 80mg/mL, about 85mg/mL, about 88mg/mL, about 90mg/mL, about 92mg/mL, about 95mg/mL, about 96mg/mL, about 100mg/mL, or about 120mg/mL. In some embodiments, the concentration of the sugar is 10mg/mL、20mg/mL、30mg/mL、35mg/mL、37.5mg/mL、40mg/mL、45mg/mL、50mg/mL、55mg/mL、60mg/mL、64mg/mL、65mg/mL、68mg/mL、70mg/mL、72mg/mL、75mg/mL、80mg/mL、85mg/mL、88mg/mL、90mg/mL、92mg/mL、95mg/mL、96mg/mL、100mg/mL or 120mg/mL, and any range between these point values. In some embodiments, the concentration of the sugar is 30mg/mL. In some embodiments, the concentration of the sugar is 50mg/mL. In some embodiments, the concentration of the sugar is 80mg/mL. In some embodiments, the concentration of the sugar is 100mg/mL.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the concentration of sucrose is 10mg/mL to 120mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of sucrose is 30mg/mL to 100mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of sucrose is 65mg/mL to 95mg/mL. In some embodiments, the sucrose is at a concentration of 72mg/mL to 88mg/mL. In some embodiments, the sucrose concentration is about 30mg/mL. In some embodiments, the sucrose concentration is about 50mg/mL. In some embodiments, the sucrose concentration is about 80mg/mL. In some embodiments, the sucrose is at a concentration of about 100mg/mL. In some embodiments, the sucrose is at a concentration of about 10mg/mL, about 20mg/mL, about 30mg/mL, about 35mg/mL, about 37.5mg/mL, about 40mg/mL, about 45mg/mL, about 50mg/mL, about 55mg/mL, about 60mg/mL, about 64mg/mL, about 65mg/mL, about 68mg/mL, about 70mg/mL, about 72mg/mL, about 75mg/mL, about 80mg/mL, about 85mg/mL, about 88mg/mL, about 90mg/mL, about 92mg/mL, about 95mg/mL, about 96mg/mL, about 100mg/mL, or about 120mg/mL. In some embodiments, the sucrose concentration is 10mg/mL、20mg/mL、30mg/mL、35mg/mL、37.5mg/mL、40mg/mL、45mg/mL、50mg/mL、55mg/mL、60mg/mL、64mg/mL、65mg/mL、68mg/mL、70mg/mL、72mg/mL、75mg/mL、80mg/mL、85mg/mL、88mg/mL、90mg/mL、92mg/mL、95mg/mL、96mg/mL、100mg/mL or 120mg/mL, and any range between these point values. In some embodiments, the sucrose concentration is 30mg/mL. In some embodiments, the sucrose is at a concentration of 50mg/mL. In some embodiments, the sucrose concentration is 80mg/mL. In some embodiments, the sucrose is at a concentration of 100mg/mL.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition further comprises an adjuvant, preferably the adjuvant is ethylenediamine tetraacetic acid or a salt thereof, ethylenediamine tetraacetic acid hydrate or a salt thereof, DTPA (diethylenetriamine pentaacetic acid), arginine hydrochloride, glycine, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride or calcium chloride, more preferably the adjuvant is the sodium salt of ethylenediamine tetraacetic acid hydrate, most preferably the adjuvant is disodium ethylenediamine tetraacetate dihydrate.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, wherein the concentration of the adjuvant is 0.01mg/mL to 3mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the adjuvant is 0.01mg/mL to 1mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the adjuvant is 0.01mg/mL to 0.5mg/mL. In some embodiments, the adjuvant is at a concentration of 0.01mg/mL to 0.2mg/mL. In some embodiments, the adjuvant is at a concentration of 0.01mg/mL to 0.12mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the adjuvant is 0.01mg/mL to 0.1mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the adjuvant is 0.02mg/mL to 0.08mg/mL. In some embodiments, the pharmaceutical composition of any one of the preceding claims, the concentration of the adjuvant is 0.04mg/mL to 0.06mg/mL. In some embodiments, the concentration of the adjuvant is about 0.01mg/mL. In some embodiments, the concentration of the adjuvant is about 0.05mg/mL. in some embodiments, the concentration of the adjuvant is about 0.1mg/mL. In some embodiments, the concentration of the adjunct is about 1mg/mL. In some embodiments, the adjuvant is present at a concentration of about 0.01mg/mL, about 0.02mg/mL, about 0.03mg/mL, about 0.04mg/mL, about 0.05mg/mL, about 0.06mg/mL, about 0.08mg/mL, about 0.09mg/mL, about 0.1mg/mL, about 0.11mg/mL, about 0.12mg/mL, about 0.13mg/mL, about 0.14mg/mL, about 0.15mg/mL, about 0.2mg/mL, about 0.3mg/mL, about 0.4mg/mL, about 0.5mg/mL, About 0.6mg/mL, about 0.7mg/mL, about 0.8mg/mL, about 0.9mg/mL, about 1mg/mL, about 1.1mg/mL, about 1.2mg/mL, about 1.3mg/mL, about 1.4mg/mL, about 1.5mg/mL, about 1.6mg/mL, about 1.7mg/mL, about 1.8mg/mL, about 1.9mg/mL, about 2mg/mL, about 2.1mg/mL, about 2.2mg/mL, about 2.3mg/mL, about 2.4mg/mL, about 2.5mg/mL, about 2.6mg/mL, about 2.7mg/mL, about 2.8mg/mL, about 2.9mg/mL, or about 3mg/mL. In some embodiments, the concentration of the adjuvant is 0.01mg/mL、0.02mg/mL、0.03mg/mL、0.04mg/mL、0.05mg/mL、0.06mg/mL、0.08mg/mL、0.09mg/mL、0.1mg/mL、0.11mg/mL、0.12mg/mL、0.13mg/mL、0.14mg/mL、0.15mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1mg/mL、1.1mg/mL、1.2mg/mL、1.3mg/mL、1.4mg/mL、1.5mg/mL、1.6mg/mL、1.7mg/mL、1.8mg/mL、1.9mg/mL、2mg/mL、2.1mg/mL、2.2mg/mL、2.3mg/mL、2.4mg/mL、2.5mg/mL、2.6mg/mL、2.7mg/mL、2.8mg/mL、2.9mg/mL or 3mg/mL, and any range between these point values. In some embodiments, the adjuvant is at a concentration of 0.01mg/mL. In some embodiments, the adjuvant is at a concentration of 0.05mg/mL. In some embodiments, the adjuvant is at a concentration of 0.1mg/mL. In some embodiments, the adjuvant is at a concentration of 1mg/mL.

In some embodiments, the pharmaceutical composition of any one of the above, wherein the concentration of disodium edetate dihydrate is 0.01mg/mL to 3mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of 0.01mg/mL to 1mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of 0.01mg/mL to 0.5mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of 0.01mg/mL to 0.2mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of 0.01mg/mL to 0.12mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of 0.01mg/mL to 0.1mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of 0.02mg/mL to 0.08mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of 0.04mg/mL to 0.06mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of about 0.01mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of about 0.1mg/mL. in some embodiments, the disodium edetate dihydrate is at a concentration of about 1mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of about 0.05mg/mL. In some embodiments, the disodium edetate dihydrate is present at a concentration of about 0.01mg/mL, about 0.02mg/mL, about 0.03mg/mL, about 0.04mg/mL, about 0.05mg/mL, about 0.06mg/mL, about 0.08mg/mL, about 0.09mg/mL, about 0.1mg/mL, about 0.11mg/mL, about 0.12mg/mL, about 0.13mg/mL, about 0.14mg/mL, about 0.15mg/mL, about 0.2mg/mL, about 0.3mg/mL, about 0.4mg/mL, About 0.5mg/mL, about 0.6mg/mL, about 0.7mg/mL, about 0.8mg/mL, about 0.9mg/mL, about 1mg/mL, about 1.1mg/mL, about 1.2mg/mL, about 1.3mg/mL, about 1.4mg/mL, about 1.5mg/mL, about 1.6mg/mL, about 1.7mg/mL, about 1.8mg/mL, about 1.9mg/mL, about 2mg/mL, about 2.1mg/mL, about 2.2mg/mL, about 2.3mg/mL, about 2.4mg/mL, about 2.5mg/mL, about 2.6mg/mL, about 2.7mg/mL, about 2.8mg/mL, about 2.9mg/mL, or about 3mg/mL. In some embodiments, the disodium edetate dihydrate is present at a concentration of 0.01mg/mL、0.02mg/mL、0.03mg/mL、0.04mg/mL、0.05mg/mL、0.06mg/mL、0.08mg/mL、0.09mg/mL、0.1mg/mL、0.11mg/mL、0.12mg/mL、0.13mg/mL、0.14mg/mL、0.15mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1mg/mL、1.1mg/mL、1.2mg/mL、1.3mg/mL、1.4mg/mL、1.5mg/mL、1.6mg/mL、1.7mg/mL、1.8mg/mL、1.9mg/mL、2mg/mL、2.1mg/mL、2.2mg/mL、2.3mg/mL、2.4mg/mL、2.5mg/mL、2.6mg/mL、2.7mg/mL、2.8mg/mL、2.9mg/mL or 3mg/mL, and any range between these point values. In some embodiments, the adjuvant is 0.01mg/mL disodium ethylenediamine tetraacetate dihydrate. In some embodiments, the disodium edetate dihydrate is at a concentration of 0.1mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of 0.05mg/mL. In some embodiments, the disodium edetate dihydrate is at a concentration of 1mg/mL.

In some embodiments, the pharmaceutical composition of any one of the preceding claims, comprising the following components:

(a) 1mg/mL to 250mg/mL of the bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.01mg/mL to 2.0mg/mL of surfactant,

(C) 10mg/mL to 120mg/mL sugar, and

(D) 5mM to 100mM buffer, the pH of the pharmaceutical composition being from 5.0 to 7.5.

(B) 0.01mg/mL to 2.0mg/mL of surfactant,

(C) Sugar of 10mg/mL to 120mg/mL,

(D) 0.01mg/mL to 3mg/mL of ethylenediamine tetraacetic acid hydrate or salt thereof, and

(E) 5mM to 100mM buffer, the pH of the pharmaceutical composition being from 5.0 to 7.5.

In some embodiments, the pharmaceutical composition of any one of the preceding claims comprises the following components:

(a) 1mg/mL to 200mg/mL of the bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.1mg/mL to 1.0mg/mL of polysorbate 80 or poloxamer 188,

(C) Sucrose 30mg/mL to 100mg/mL, and

(D) 10mM to 50mM histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer, acetic acid-acetic acid sodium salt buffer, succinic acid-succinic acid sodium salt buffer, citric acid-sodium citrate buffer or Tris-HCl salt buffer, the pH of the pharmaceutical composition being 5.0 to 7.5.

(B) 0.1mg/mL to 1.0mg/mL of polysorbate 80 or poloxamer 188,

(C) Sucrose of 30mg/mL to 100mg/mL,

(D) 0.01mg/mL to 1mg/mL of sodium salt of ethylenediamine tetraacetic acid hydrate, and

(E) 10mM to 50mM histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer, acetic acid-acetic acid sodium salt buffer, succinic acid-succinic acid sodium salt buffer, citric acid-sodium citrate buffer or Tris-HCl salt buffer, the pH of the pharmaceutical composition being 5.0 to 7.5.

(B) 0.1mg/mL to 1.0mg/mL of polysorbate 80 or poloxamer 188,

(C) Sucrose 30mg/mL to 100mg/mL, and

(D) 10mM to 50mM histidine-HCl buffer, the pH of the pharmaceutical composition being 5.0 to 6.5.

(B) 0.1mg/mL to 1.0mg/mL of polysorbate 80 or poloxamer 188,

(C) Sucrose of 30mg/mL to 100mg/mL,

(E) 10mM to 50mM histidine-HCl buffer, the pH of the pharmaceutical composition being 5.0 to 6.5.

(a) 70mg/mL to 150mg/mL of the bispecific antibody that specifically binds GUCY2C and CD3,

(B) Polysorbate 80 or poloxamer 188 at 0.2mg/mL to 0.6mg/mL,

(C) 65mg/mL to 95mg/mL sucrose, and

(D) 10mM to 30mM histidine-HCl buffer, the pH of the pharmaceutical composition being 5.0 to 6.5.

(B) Polysorbate 80 or poloxamer 188 at 0.2mg/mL to 0.6mg/mL,

(C) 65mg/mL to 95mg/mL sucrose,

(D) 0.01mg/mL to 0.5mg/mL disodium edetate dihydrate, and

(E) 10mM to 30mM histidine-HCl buffer, the pH of the pharmaceutical composition being 5.0 to 6.5.

(a) 80mg/mL to 120mg/mL of the bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.3mg/mL to 0.5mg/mL of polysorbate 80 or poloxamer 188,

(C) 65mg/mL to 95mg/mL sucrose, and

(D) 10mM to 20mM histidine-hcl histidine buffer, the pH of the pharmaceutical composition is 5.2 to 6.0.

(B) 0.3mg/mL to 0.5mg/mL of polysorbate 80 or poloxamer 188,

(C) 65mg/mL to 95mg/mL sucrose,

(D) 0.01mg/mL to 0.1mg/mL of disodium edetate dihydrate, and

(E) 10mM to 20mM histidine-hcl histidine buffer, the pH of the pharmaceutical composition is 5.2 to 6.0.

(B) From 0.01mg/mL to 2.0mg/mL of polysorbate 80,

(C) Sugar of 10mg/mL to 120mg/mL,

(B) From 0.1mg/mL to 1.0mg/mL of polysorbate 80,

(C) Sucrose of 30mg/mL to 100mg/mL,

(B) From 0.1mg/mL to 1.0mg/mL of polysorbate 80,

(C) Sucrose of 30mg/mL to 100mg/mL,

(B) From 0.2mg/mL to 0.6mg/mL of polysorbate 80,

(C) 65mg/mL to 95mg/mL sucrose,

(D) 0.01mg/mL to 0.5mg/mL disodium edetate dihydrate, and

(B) From 0.3mg/mL to 0.5mg/mL of polysorbate 80,

(C) 65mg/mL to 95mg/mL sucrose,

(D) 0.01mg/mL to 0.1mg/mL of disodium edetate dihydrate, and

(B) Poloxamer 188 at 0.01mg/mL to 2.0mg/mL,

(C) Sugar of 10mg/mL to 120mg/mL,

(B) Poloxamer 188 at 0.1mg/mL to 1.0mg/mL,

(C) Sucrose of 30mg/mL to 100mg/mL,

(B) Poloxamer 188 at 0.1mg/mL to 1.0mg/mL,

(C) Sucrose of 30mg/mL to 100mg/mL,

(B) Poloxamer 188 at 0.2mg/mL to 0.6mg/mL,

(C) 65mg/mL to 95mg/mL sucrose,

(D) 0.01mg/mL to 0.5mg/mL disodium edetate dihydrate, and

(B) Poloxamer 188 at 0.3mg/mL to 0.5mg/mL,

(C) 65mg/mL to 95mg/mL sucrose,

(D) 0.01mg/mL to 0.1mg/mL of disodium edetate dihydrate, and

(a) About 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL of sucrose per mL of sucrose,

(D) About 0.05mg/mL disodium edetate dihydrate, and

(E) About 20mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is 5.2 to 6.0.

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.4mg/mL polysorbate 80,

(C) 80mg/mL of sucrose, and the concentration of sucrose,

(D) 0.05mg/mL disodium edetate dihydrate, and

(E) 20mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is from 5.2 to 6.0.

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL of sucrose per mL of sucrose,

(D) About 0.05mg/mL disodium edetate dihydrate, and

(E) About 14mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is 5.2 to 6.0.

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.4mg/mL polysorbate 80,

(C) 80mg/mL of sucrose, and the concentration of sucrose,

(D) 0.05mg/mL disodium edetate dihydrate, and

(E) 14mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is from 5.2 to 6.0.

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL of sucrose per mL of sucrose,

(D) About 0.05mg/mL disodium edetate dihydrate, and

(E) About 14mM histidine-histidine buffer, the pH of the pharmaceutical composition is about 5.7.

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.4mg/mL polysorbate 80,

(C) 80mg/mL of sucrose, and the concentration of sucrose,

(D) 0.05mg/mL disodium edetate dihydrate, and

(E) 14mM histidine-histidine buffer, the pH of the pharmaceutical composition is 5.7.

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL of sucrose per mL of sucrose,

(D) About 0.05mg/mL disodium edetate dihydrate, and

(E) About 10mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is about 5.7. In some embodiments, the pharmaceutical composition of any one of the preceding claims comprises the following components:

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.4mg/mL polysorbate 80,

(C) 80mg/mL of sucrose, and the concentration of sucrose,

(D) 0.05mg/mL disodium edetate dihydrate, and

(E) 10mM histidine-histidine buffer, the pH of the pharmaceutical composition is 5.7. In some embodiments, the pharmaceutical composition of any one of the preceding claims comprises the following components:

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL of sucrose per mL of sucrose,

(D) About 0.05mg/mL disodium edetate dihydrate, and

(E) About 30mM histidine-histidine buffer, the pH of the pharmaceutical composition is about 5.7. In some embodiments, the pharmaceutical composition of any one of the preceding claims comprises the following components:

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.4mg/mL polysorbate 80,

(C) 80mg/mL of sucrose, and the concentration of sucrose,

(D) 0.05mg/mL disodium edetate dihydrate, and

(E) 30mM histidine-histidine buffer, the pH of the pharmaceutical composition is 5.7. In some embodiments, the pharmaceutical composition of any one of the preceding claims comprises the following components:

(a) About 200mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL of sucrose per mL of sucrose,

(D) About 0.05mg/mL disodium edetate dihydrate, and

(E) About 14mM histidine-histidine buffer, the pH of the pharmaceutical composition is about 5.7. In some embodiments, the pharmaceutical composition of any one of the preceding claims comprises the following components:

(a) 200mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.4mg/mL polysorbate 80,

(C) 80mg/mL of sucrose, and the concentration of sucrose,

(D) 0.05mg/mL disodium edetate dihydrate, and

(E) 14mM histidine-histidine buffer, the pH of the pharmaceutical composition is 5.7. In some embodiments, the pharmaceutical composition of any one of the preceding claims comprises the following components:

(a) About 2mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL of sucrose per mL of sucrose,

(D) About 0.05mg/mL disodium edetate dihydrate, and

(a) 2mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.4mg/mL polysorbate 80,

(C) 80mg/mL of sucrose, and the concentration of sucrose,

(D) 0.05mg/mL disodium edetate dihydrate, and

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL sucrose, and

(D) About 20mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is 5.2 to 6.0.

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.4mg/mL polysorbate 80,

(C) 80mg/mL sucrose, and

(D) 20mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is from 5.2 to 6.0.

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL sucrose, and

(D) About 14mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is about 5.2 to 6.0.

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.4mg/mL polysorbate 80,

(C) 80mg/mL sucrose, and

(D) 14mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is from 5.2 to 6.0.

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL sucrose, and

(D) About 14mM histidine-histidine buffer, the pH of the pharmaceutical composition is about 5.7.

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) 0.4mg/mL polysorbate 80,

(C) 80mg/mL sucrose, and

(D) 14mM histidine-histidine buffer, the pH of the pharmaceutical composition is 5.7.

(B) About 0.4mg/mL poloxamer 188,

(C) About 80mg/mL of sucrose per mL of sucrose,

(D) About 0.05mg/mL disodium edetate dihydrate, and

(E) About 20mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is 5.0 to 6.2.

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) Poloxamer 188 at 0.4mg/mL,

(C) 80mg/mL of sucrose, and the concentration of sucrose,

(D) 0.05mg/mL disodium edetate dihydrate, and

(E) 20mM histidine-histidine buffer, the pH of the pharmaceutical composition is from 5.0 to 6.2.

(B) About 0.4mg/mL poloxamer 188,

(C) About 80mg/mL sucrose, and

(D) About 20mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is 5.0 to 6.2.

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) Poloxamer 188 at 0.4mg/mL,

(C) 80mg/mL sucrose, and

(D) 20mM histidine-histidine buffer, the pH of the pharmaceutical composition is from 5.0 to 6.2.

(B) About 0.4mg/mL poloxamer 188,

(C) About 80mg/mL of sucrose per mL of sucrose,

(D) About 0.05mg/mL disodium edetate dihydrate, and

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) Poloxamer 188 at 0.4mg/mL,

(C) 80mg/mL of sucrose, and the concentration of sucrose,

(D) 0.05mg/mL disodium edetate dihydrate, and

(B) About 0.4mg/mL poloxamer 188,

(C) About 80mg/mL sucrose, and

(a) 100mg/mL of bispecific antibody that specifically binds GUCY2C and CD3,

(B) Poloxamer 188 at 0.4mg/mL,

(C) 80mg/mL sucrose, and

(D) 14mM histidine-histidine buffer, the pH of the pharmaceutical composition is about 5.7.

In one aspect, the present disclosure provides a pharmaceutical composition according to any one of the preceding claims, which is a subcutaneous injection formulation, an intravenous injection formulation, an intraperitoneal injection formulation or an intramuscular injection formulation, preferably a subcutaneous injection formulation or an intravenous injection formulation.

In one aspect, the present disclosure provides a method of preparing a lyophilized formulation comprising the step of lyophilizing the pharmaceutical composition of any one of the preceding claims.

In one aspect, the present disclosure provides a lyophilized formulation obtained by the method as described previously.

In one aspect, the present disclosure also provides a lyophilized formulation comprising the pharmaceutical composition of any one of the above.

In one aspect, the present disclosure also provides a lyophilized formulation that upon reconstitution can form a pharmaceutical composition as described in any one of the above.

In one aspect, the present disclosure also provides a reconstitution solution, characterized in that the reconstitution solution is obtained by reconstitution of a lyophilized formulation as described in any one of the above.

In one aspect, the present disclosure also provides a reconstituted solution that is a reconstituted form formulation of the lyophilized formulation described above.

In some embodiments, the reconstituted solution of any one of the above, has the same components and amounts as the pharmaceutical composition of any one of the above.

In one aspect, the present disclosure also provides an article of manufacture comprising a container containing a pharmaceutical composition as defined in any one of the above, or a lyophilized formulation as defined in any one of the above, or a reconstituted solution as defined in any one of the above.

In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution of any one of the above is an intravenous formulation, a subcutaneous formulation, an intraperitoneal formulation, or an intramuscular formulation. In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution of any one of the above is an intravenous formulation.

In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution of any one of the above is suitable for intravenous injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection. In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution of any one of the above is suitable for intravenous injection.

In some embodiments, the pharmaceutical composition, lyophilized formulation or reconstituted solution of any one of the above is used to prepare a medicament for intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection. In some embodiments, the pharmaceutical composition, lyophilized formulation or reconstituted solution of any one of the above is used to prepare an intravenous drug.

In another aspect, the present disclosure also provides a method of treating a tumor or cancer, the method comprising administering to a subject a therapeutically effective amount of a composition of any one of the preceding claims, a lyophilized formulation of any one of the preceding claims, or a reconstituted solution of any one of the preceding claims.

In another aspect, the present disclosure also provides a composition of any one of the preceding claims, a lyophilized formulation of any one of the preceding claims, or a reconstituted solution of any one of the preceding claims for use as a medicament. In some embodiments, the medicament is for treating a tumor or cancer.

In one aspect, the present disclosure provides the use of a composition according to any one of the preceding claims, a lyophilized formulation according to any one of the preceding claims, or a reconstituted solution according to any one of the preceding claims, in the manufacture of a medicament for the treatment of a tumor or cancer.

In one aspect, the tumor or cancer described in the present disclosure is selected from:

Gastrointestinal cancer (gastric adenocarcinoma) and intestinal cancer (small intestine cancer, colorectal cancer (colon cancer and rectal cancer))), esophageal cancer (i.e., esophageal cancer), anal cancer, liver cancer (hepatocellular carcinoma), gallbladder cancer, bile duct cancer, pancreatic cancer, prostate cancer, renal cancer (renal cell carcinoma), urothelial cancer, breast cancer, bladder cancer, thymus cancer, melanoma, glioma (glioblastoma), sarcoma (osteosarcoma), ovarian cancer, thyroid cancer, cervical squamous cell carcinoma, endometrial cancer, lung cancer (non-small cell lung cancer (lung squamous cell carcinoma, lung adenocarcinoma) and small cell lung cancer), skin cancer, head and neck cancer (head and neck squamous cell carcinoma), brain cancer, glioblastoma multiforme, gastroesophageal cancer (gastroesophageal adenocarcinoma), metastatic adenocarcinoma of the liver, multiple myeloma, lymphoma (hodgkin's lymphoma, non-hodgkin's lymphoma or B-cell lymphoma), or leukemia (such as Acute Myelogenous Leukemia (AML), acute Lymphoblastic Leukemia (ALL)), chronic Lymphoblastic Leukemia (CLL)).

In some embodiments, cells of a tumor or vascular endothelial cells adjacent to the tumor as described above express GUCY2C.

In some embodiments, the tumor as described previously is a solid tumor.

In some embodiments, the tumor as previously described is a solid tumor that is gastrointestinal cancer (gastric adenocarcinoma) and intestinal cancer (small intestine and colorectal (colon and rectum))), esophageal cancer (i.e., esophageal cancer), anal cancer, liver cancer (hepatocellular carcinoma), gallbladder cancer, bile duct cancer, pancreatic cancer, prostate cancer, renal cancer (renal cell carcinoma), urothelial cancer, breast cancer, bladder cancer, thymus cancer, melanoma, glioma (glioblastoma), sarcoma (osteosarcoma), ovarian cancer, thyroid cancer, cervical squamous cell carcinoma, endometrial adenocarcinoma, lung cancer (non-small cell lung cancer (lung squamous cell carcinoma, lung adenocarcinoma) and small cell lung cancer), skin cancer, head and neck cancer (head and neck squamous cell carcinoma), brain cancer, glioblastoma multiforme, gastroesophageal cancer (gastroesophageal adenocarcinoma), or liver metastatic adenocarcinoma.

In some embodiments, the tumor as described above is a gastrointestinal cancer, preferably a gastric or intestinal cancer, more preferably a gastric adenocarcinoma, a small intestinal cancer or a colorectal cancer.

In some embodiments, the tumor as described above is a non-solid tumor.

In some embodiments, the tumor as described previously is a non-solid tumor that is multiple myeloma, lymphoma (hodgkin's lymphoma, non-hodgkin's lymphoma, B-cell lymphoma), or leukemia (acute myeloid leukemia (AML), acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL)).

In some embodiments, the tumor or cancer as described above is a disease associated with GUCY2C, and in some embodiments, the tumor or cancer as described above is a disease that expresses GUCY 2C.

In some embodiments, the use of a second therapeutic agent to treat a tumor or cancer as previously described further comprises administering a second therapeutic agent. In some embodiments, the second therapeutic agent comprises an anti-tumor agent, radiation therapy, an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, an immune checkpoint inhibitor, or a combination thereof. In some embodiments, the second therapeutic agent is a CD28-EGFR bispecific antibody that specifically binds CD28 and EGFR. In some embodiments, the CD28-EGFR bispecific antibody is 97H2L3OT-ZAL. In some embodiments, the CD28-VH of CD28-EGFR bispecific antibody 97H2L3OT-ZAL comprises the amino acid sequence of SEQ ID NO:78 and the CD28-VL comprises the amino acid sequence of SEQ ID NO:79, the EGFR-VH comprises the amino acid sequence of SEQ ID NO:76 and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 77. In some embodiments, the amino acid sequence of CD28-EGFR bispecific antibody 97H2L3OT-ZAL comprises chain 1 shown as SEQ ID NO. 80, chain 2 shown as SEQ ID NO. 81, chain 3 shown as SEQ ID NO. 82 and chain 4 shown as SEQ ID NO. 83.

In some embodiments, the use of a second therapeutic agent to treat a tumor or cancer as described previously is administered simultaneously, sequentially or separately with the pharmaceutical composition of any of the present disclosure.

The bispecific antibody preparation provided by the disclosure has the characteristics of good therapeutic activity, safety, pharmacokinetic properties and patent drug properties (such as stability).

Drawings

FIG. 1 is a schematic diagram of the structure of Format 1 of GUCY2C-CD3 bispecific antibody.

FIG. 2A binding capacity of GUCY2C mab (P3-C7L 7H 1) to HCT116/hGUCY C cells and FIG. 2B binding capacity of GUCY2C mab (P3-C7L 7H 1) to HT-55 cells.

FIG. 3A binding capacity of GUCY2C-CD3 bispecific antibody (P3-C7L 7H 1-1) to HCT116/hGUCY C cells, FIG. 3B binding capacity of GUCY2C-CD3 bispecific antibody (P3-C7L 7H 1-1) to HT55 cells, and FIG. 3C binding capacity of GUCY2C-CD3 bispecific antibody (P3-C7L 7H 1-1) to Jurkat cells.

FIG. 4A HCT116/hGUCY C as target cells test the ability of the antibody (P3-C7L 7H 1-1) to activate Jurkat/NFAT cells and FIG. 4B LS1034 as target cells test the ability of the antibody (P3-C7L 7H 1-1) to activate Jurkat/NFAT cells.

FIG. 5A shows IFN-. Gamma.release results for the GUCY2C-CD3 bispecific antibody (P3-C7L 7H 1-1) co-acting with LS1034 cells and FIG. 5B shows IFN-. Gamma.release results for the GUCY2C-CD3 bispecific antibody (P3-C7L 7H 1-1) co-acting with LS174T cells.

FIG. 6A shows the results of IL-6 release by the GUCY2C-CD3 bispecific antibody (P3-C7L 7H 1-1) in combination with LS1034 cells, and FIG. 6B shows the results of IL-6 release by the GUCY2C-CD3 bispecific antibody (P3-C7L 7H 1-1) in combination with LS174T cells.

Detailed Description

Terminology

The terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, in the description and the claims, the words "comprise", "have", "include" and the like are to be construed in an inclusive, but not exclusive, sense as opposed to an exclusive or exhaustive sense.

The term "and/or" is intended to include both "and" or "meanings. For example, the phrase "A, B and/or C" is intended to encompass each of A, B and C, A, B or C, A or B, B or C, A and B, B and C, A (alone), B (alone), and C (alone).

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not.

It will be appreciated by those of skill in the art that when used with reference to a range of values, a cut-off value, or a particular value, an "about" may mean within 1 or more than 1 standard deviation. Or "about" may represent a range differing by up to 20% (i.e., ±20%). Since many of the numerical values used herein are determined experimentally, those of skill in the art will appreciate that such determinations may vary from experiment to experiment and generally from experiment to experiment. Because of this inherent discrepancy, it is believed that the values used herein should not be unduly limited. Thus, the term "about" is used to encompass a variation of ±20% or less, a variation of ±10% or less, a variation of ±5% or less, a variation of ±1% or less, a variation of ±0.5% or less, or a variation of ±0.1% or less from the specified value.

Although the present disclosure provides content ranges or content values, one of ordinary skill in the art will appreciate that the content ranges or content values encompass acceptable error ranges for the particular values determined.

The amino acid three-letter codes and one-letter codes used in the present disclosure are as described in J.biol. Chem,243, p3558 (1968).

The term "and/or", e.g. "X and/or Y", should be understood to mean "X and Y" or "X or Y" and should be used to provide explicit support for both meanings or either meaning.

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

The term "amino acid mutation" includes amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions, and modifications may be made to achieve the final construct, provided the final construct possesses the desired properties, such as reduced or binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino-and/or carboxy-terminus of the polypeptide chain. The specific amino acid mutation may be an amino acid substitution. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., the substitution of one amino acid with another amino acid having a different structure and/or chemical property. Amino acid substitutions include substitutions by non-naturally occurring amino acids or by derivatives of 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Genetic or chemical methods known in the art may be used to generate amino acid mutations. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that other methods than genetic engineering, such as chemical modification, may be used to alter the amino acid side chain groups. Various names may be used herein to indicate the same amino acid mutation. Herein, the amino acid residue at a particular position may be represented by position +amino acid residue, e.g., 366W, and then the amino acid residue at position 366 is represented as W. T366W indicates that the amino acid residue at position 366 is mutated from the original T to W.

The term "antigen binding molecule" is used in its broadest sense to encompass a variety of molecules that specifically bind to an antigen, including but not limited to antibodies, other polypeptides having antigen binding activity, and antibody fusion proteins fused to them, so long as they exhibit the desired antigen binding activity. The antigen binding molecules herein comprise a variable region (VH) and a variable region (VL) that together comprise an antigen binding domain. Illustratively, the antigen binding molecules herein are bispecific antigen binding molecules (e.g., bispecific antibodies).

The term "antibody" is used in its broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), so long as they exhibit the desired antigen-binding activity. For example, a natural IgG antibody is an iso-tetrasaccharide protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide bonded. From N to C-terminal, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by three constant domains (CH 1, CH2 and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

The term "bispecific antibody" refers to an antibody (including an antibody or antigen binding fragment thereof, such as a single chain antibody) capable of specifically binding to two different antigens or at least two different epitopes of the same antigen. Bispecific antibodies of various structures have been disclosed in the art, which can be classified into IgG-like bispecific antibodies and antibody fragment type bispecific antibodies according to the integrity of IgG molecules, bivalent, trivalent, tetravalent or more multivalent bispecific antibodies according to the number of antigen binding regions, and symmetrical structure bispecific antibodies and asymmetrical structure bispecific antibodies according to whether the structures are symmetrical or not. Of these, bispecific antibodies based on antibody fragments, such as Fab fragments lacking an Fc fragment, are formed by binding 2 or more Fab fragments in one molecule, which have lower immunogenicity and small molecular weight, have higher tumor tissue permeability, typical antibody structures of this type such as F (ab) ₂、scFv-Fab、(scFv)₂ -Fab, igG-like bispecific antibodies (e.g. with an Fc fragment), which have a relatively large molecular weight, fc fragments facilitate purification of the antibody and increase its solubility, stability, fc moieties may also bind to the receptor FcRn, increasing antibody serum half-life, typical bispecific antibody structure models such as KiH、CrossMAb、Triomab quadroma、FcΔAdp、ART-Ig、BiMAb、Biclonics、BEAT、DuoBody、Azymetric、XmAb、2:1 TCBs、1Fab-IgG TDB、FynomAb、two-in-one/DAF、scFv-Fab-IgG、DART-Fc、LP-DART、CODV-Fab-TL、HLE-BiTE、F(ab)2-CrossMAb、IgG-(scFv)2、Bs4Ab、DVD-Ig、Tetravalent-DART-Fc、(scFv)4-Fc、CODV-Ig、mAb2、F(ab)4-CrossMAb et al (see Aran F. Labrijn et al Nature Reviews Drug Discovery volume, pages585-608 (2019); chen S1 et al, J Immunol res.2019 Feb 11; 2019:4516041).

The term "variable region" or "variable domain" refers to the antigen-binding domain of an antigen-binding molecule. Herein, the heavy chain variable region in the antigen binding moiety that specifically binds to GUCY2C is designated GUCY2C-VH, the light chain variable region is designated GUCY2C-VL, the heavy chain variable region in the antigen binding moiety that specifically binds to CD3 is designated CD3-VH, and the light chain variable region is designated CD3-VL. VH and VL each comprise four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). Wherein the term "complementarity determining region" or "CDR" refers to the region within the variable domain that primarily contributes to antigen binding, and "framework" or "FR" refers to variable domain residues other than CDR residues. VH contains 3 CDR regions HCDR1, HCDR2 and HCDR3, and VL contains 3 CDR regions LCDR1, LCDR2 and LCDR3. Herein, 3 CDR regions in GUCY2C-VH are labeled GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3, 3 CDR regions in GUCY2C-VL are labeled GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3, 3 CDR regions in CD3-VH are labeled CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, and 3 CDR regions in CD3-VL are labeled CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3, respectively. Each of VH and VL is FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 in the order from N-terminal to C-terminal. A single VH or VL may be sufficient to confer antigen binding specificity.

The amino acid sequence boundaries of the CDRs can be determined by various well-known schemes, such as "Kabat" numbering convention (see Kabat et al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR.protein Sequence and Structure Analysis of Antibody Variable Domains [ J ]. 2001) and ImMunoGenTics (IMGT) numbering convention (Lefranc, M.P. et al, dev.Comp. Immunol.,27,55-77 (2003); front immunol.2018 Oct 16; 9:2278), etc., the correspondence between the various numbering systems being well known to those skilled in the art. The numbering convention of the present disclosure is shown in table 1 below.

TABLE 1 relationship between CDR numbering systems

CDR	IMGT	Kabat	AbM	Chothia	Contact
						HCDR1	27-38	31-35	26-35	26-32	30-35
HCDR2	56-65	50-65	50-58	52-56	47-58
						HCDR3	105-117	95-102	95-102	95-102	93-101
LCDR1	27-38	24-34	24-34	24-34	30-36
						LCDR2	56-65	50-56	50-56	50-56	46-55
LCDR3	105-117	89-97	89-97	89-97	89-96

Unless otherwise indicated, the variable region and CDR sequences in the examples of the present disclosure apply the "Kabat" numbering convention.

The term "antibody fragment" or "antigen binding fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that retains the antigen binding ability of the intact antibody. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂, single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), and multispecific antibodies (e.g., bispecific antibodies) formed from antibody fragments.

The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including the native Fc region and engineered Fc region. In some embodiments, the Fc region comprises two subunits, which may be the same or different. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. Suitable native sequence Fc regions for antibodies described herein include human IgG1, igG2 (IgG 2A, igG B), igG3, and IgG4. The numbering convention for the Fc region is EU index, unless otherwise indicated.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chains in the antibody are derived from a particular source or species, while the remainder of the heavy and/or light chains are derived from a different source or species.

The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be accomplished by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterparts (i.e., the framework regions portions of the constant and variable regions).

The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an internal binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be expressed by the equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. The term "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, while the term "kdis" or "kd" as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of KD to ka (i.e., KD/ka) and is expressed as molar concentration (M). The KD value of an antibody can be determined using methods known in the art, such as surface plasmon resonance, ELISA, or Solution Equilibrium Titration (SET).

The term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules comprised in the population are identical, except for natural mutations that may be present in minor amounts. In contrast, polyclonal antibody preparations typically include a plurality of different antibodies having different amino acid sequences in their variable domains, which are typically specific for different epitopes. "monoclonal" refers to the characteristics of the antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

The term "antigen" refers to a molecule or portion of a molecule that is capable of being bound by a selective binding agent of an antigen binding protein (e.g., an antibody). An antigen may have one or more epitopes that are capable of interacting with different antigen binding proteins (e.g., antibodies).

The term "epitope" refers to a region (area or region) on an antigen that is capable of specifically binding to an antibody or antigen binding fragment thereof. Epitopes can be formed from or comprise non-contiguous amino acids (conformational epitopes), for example, which are spatially accessed by folding of the antigen (i.e., tertiary folding of the antigen by the nature of the protein). Conformational epitopes differ from linear epitopes in that the binding of the antibody to the conformational epitope is lost in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for Antibodies that bind a particular epitope (i.e., those that bind the same epitope) can be performed using routine methods in the art, such as, but not limited to, alanine scanning, peptide blotting (see meth. Mol. Biol.248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see prot. Sci.9 (2000) 487-496), and cross-blocking (see "Antibodies", harlow and Lane (Cold Spring Harbor Press, cold Spring harbor., NY)).

The term "capable of specifically binding", "specifically binding" or "binding" refers to an antibody that is capable of binding to a certain antigen or epitope with a higher affinity than other antigens or epitopes. Typically, an antibody binds an antigen or epitope with an equilibrium dissociation constant (KD) of about 1 x 10 ^-7 M or less (e.g., about 1 x 10 ^-8 M or less). In some embodiments, the antibody binds to an antigen with a KD of 10% or less (e.g., 1%) of the KD of the antibody to a non-specific antigen (e.g., BSA, casein). KD can be measured using known methods, for example by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, e.g., cynomolgus macaque (Macaca fascicularis) (cyno), chimpanzee (Pan troglodes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset).

The term "non-binding" refers to an antibody that is not capable of binding to an antigen or epitope within the antigen in the manner described above for specific binding. For example, when an antibody binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of about 1 x 10 ^-6 M or greater.

The term "linker" refers to a linking unit that links two polypeptide fragments. In this context, the linkers present in the same structural formula may be the same or different. The linker may be a peptide linker comprising one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. The linkers used herein may be the same or different. When "-" appears in the formula, it means that the units on both sides are directly linked by covalent bonds.

The term "sequence identity" refers to the degree (percent) to which the amino acids/nucleic acids of two sequences are identical at equivalent positions when optimally aligned. During the alignment, gaps may be allowed to be introduced as necessary to obtain the maximum percent sequence identity, but any conservative substitutions are not considered to form part of the sequence identity. To determine percent sequence identity, alignment may be accomplished by techniques known in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences compared.

The term "fusion" or "linkage" refers to covalent attachment of components (e.g., antigen binding moiety and Fc domain) directly or via a linker.

The term "subject" or "individual" includes both human and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein unless specifically indicated. As used herein, the term "cynomolgus monkey (cyno)" or "cynomolgus monkey (cynomolgus)" refers to cynomolgus monkey (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

"Surfactant" means a surfactant (surface-ACTIVE AGENT), preferably a nonionic surfactant. The use of surfactants may reduce aggregation of the protein in the formulation and/or reduce particle formation. The amount of surfactant added is such that it can reduce aggregation of the protein in the formulation and minimize particle formation.

"Poloxamer" is a block copolymer of ethylene oxide and propylene oxide that is water soluble and is used as a surfactant in pharmaceutical formulations. Examples of poloxamers include poloxamer 188.

The "viscosity modifier" is a conventional pharmaceutical excipient added to adjust the viscosity of the formulation. The viscosity modifier may be an inorganic salt, preferably selected from sodium chloride, calcium chloride, magnesium chloride, and an amino acid salt, preferably selected from arginine hydrochloride, histidine hydrochloride, lysine hydrochloride, histidine acetate, etc.

"Buffering agent" refers to a buffering agent that is resistant to pH changes by the action of its acid-base conjugate components. Examples of buffers that control the pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate (also known as citrate), tartrate, fumarate, glycylglycine and other organic acid buffers.

A "histidine buffer" is a buffer that comprises histidine. Examples of histidine buffers include histidine-acetate histidine, histidine-hydrochloride histidine, histidine-phosphate histidine, histidine-sulfate histidine and the like buffers, preferably histidine-hydrochloride histidine buffers. The histidine-histidine hydrochloride buffer may be formulated from histidine and hydrochloric acid, or from histidine and histidine hydrochloride.

"Displacement" refers to the displacement of a solvent system that solubilizes an antibody protein, e.g., a physically manipulated high salt or hypertonic solvent system containing an antibody protein using a buffer system of a stable formulation, such that the antibody protein is present in the stable formulation. The physical modes of operation include, but are not limited to, ultrafiltration, dialysis, or centrifugation.

"Pharmaceutical composition" means a mixture comprising one or more antibodies described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and not react with the immune system of a subject. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is Phosphate Buffered Saline (PBS) or physiological (0.9%) saline. Compositions comprising such carriers are formulated by well known conventional methods (see, e.g., remington's Pharmaceutical Sciences, 18 th edition, A. Gennaro, eds., mack Publishing Co., easton, pa., 1990; and R Remington, THE SCIENCE AND PRACTICE of Pharmacy, 20 th edition Mack Publishing, 2000).

By "lyophilized formulation" is meant a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a vacuum freeze-drying step of a liquid or solution formulation. Typically, freeze-drying includes pre-freezing, primary drying, and secondary drying. The pre-freezing is to freeze the product to obtain a crystalline solid, and in some embodiments, the pre-freezing temperature is set at-45 ℃ and the pre-freezing rate is set at 1 ℃ per minute. Primary drying, also known as primary drying, is the primary stage of sample freeze drying, and aims to remove ice from the product, maintain the shape of the product, minimize damage to the product, cause collapse of the product if the primary drying temperature and vacuum are not properly selected, and increase freeze drying efficiency at higher temperatures and vacuum, but also increase the risk of product collapse. In some embodiments, the temperature of the primary drying may be a temperature conventional in the art, such as-30 ℃ to 0 ℃. Secondary drying, also known as resolution drying, is the primary step of removing bound water from the product by pulling a final vacuum (0.01 mbar) and elevated temperature (20 ℃ to 40 ℃). Since most biologicals are relatively sensitive to temperature, the secondary drying temperature is often chosen at a low point in the temperature range, e.g. 25 ℃. The time of lyophilization is related to the freezer, the dose of lyophilized formulation, and the container of lyophilized medicament. Such time adjustments are well known to those skilled in the art.

The pharmaceutical compositions described in this disclosure are in the form of solutions, wherein the solvent is water unless otherwise specified.

In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

The pharmaceutical compositions of the present disclosure are capable of achieving a stabilizing effect in which the antibodies substantially retain their physical and/or chemical stability and/or biological activity after storage, preferably the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are a number of analytical techniques for measuring protein stability that measure stability after storage at a selected temperature for a selected period of time.

Stable formulations include those in which no significant change is observed when stored at refrigeration temperatures (2 ℃ to 8 ℃) for at least 1 month, at least 3 months, at least 5 months, at least 6 months, preferably 1 year, and even more preferably up to 2 years. In addition, stable liquid formulations also include liquid formulations that exhibit desirable characteristics after storage at temperatures including 25 ℃ for periods including 1 month, 3 months, or 6 months. In addition, stable liquid formulations also exhibit desirable characteristics after a period of time including 4 weeks, 1 month, 3 months, or 6 months of temperature storage at 40 ℃. Typical examples of stability are aggregation or degradation of antibodies, as measured by SEC-HPLC, of generally no more than about 10%, preferably no more than about 5%. The formulations were pale yellow near colorless clear liquid or colorless clear liquid, or clear to slightly milky color by visual analysis. The concentration, pH, weight, molecular osmolality of the formulation has no more than about 10%, preferably no more than about 5% aggregation.

An antibody "retains its physical stability" in a pharmaceutical formulation if it does not exhibit a significant increase in aggregation, precipitation and/or denaturation after visual inspection of color and/or clarity, or as measured by UV light scattering, size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS). The change in protein conformation can be assessed by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not exhibit a significant chemical change. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often alter the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide spectroscopy in combination with mass spectrometry or MALDI/TOF/MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide spectroscopy, isoaspartic acid measurement, etc.), and isomerization (assessed by measuring isoaspartic acid content, peptide spectroscopy, etc.).

An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of biological activities exhibited when the pharmaceutical formulation is prepared.

"Administration," "administering," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administration," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration," "administration," and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering an internally or externally used therapeutic agent, such as a pharmaceutical composition comprising any of the present disclosure, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to a subject patient or population in an amount effective to alleviate one or more symptoms of the disease to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. While embodiments of the present disclosure (e.g., therapeutic methods or articles of manufacture) may be ineffective in alleviating each target disease symptom, it should be determined according to any statistical test method known in the art, such as Student t-test, chi-square test, U-test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test, and Wilcoxon test, that the target disease symptom should be alleviated in a statistically significant number of patients.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on such factors as the condition to be treated, the general health of the subject, the route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

The pharmaceutical compositions of the present disclosure may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if topical treatment is desired, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration and pulse infusion. In some embodiments, the pharmaceutical compositions of the present disclosure are administered by subcutaneous injection.

The pharmaceutical compositions of the present disclosure will be formulated, administered and administered in a manner consistent with good medical practice. Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the medical practitioner. Optionally, the pharmaceutical composition may also be formulated with one or more other agents for preventing or treating the condition. The effective amount of such other agents depends on the amount of antigen binding molecule present in the pharmaceutical composition, the type of disorder or treatment, and other factors. May be used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage, and by any route of experience/clinical determination as appropriate.

The disease associated with GUCY2C in the disclosure is not limited so long as it is a disease associated with GUCY2C, for example, therapeutic response induced by antibodies of the disclosure can kill cells that overexpress GUCY2C by binding to human GUCY2C, or inhibit the growth of cells that overexpress GUCY 2C.

Examples and test examples

The present disclosure is further described below in conjunction with examples and test examples, which are not intended to limit the scope of the present disclosure. The experimental methods of the examples and test examples of the present disclosure, in which specific conditions are not specified, are generally conducted according to conventional conditions such as a laboratory manual of antibody technology in cold spring harbor, a molecular cloning manual, or according to the conditions recommended by the manufacturer of raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

1. Preparation of bispecific antibodies that specifically bind to GUCY2C and CD3

Example 1 screening and identification of murine antibodies against human GUCY2C

1.1 Construction of cell lines

Construction of CHO-K1/hGUCY C and HCT116/hGUCY C cell lines, and the related protein sequences were as follows:

human GUCY2C protein (UniProtKB: P25092):

MKTLLLDLALWSLLFQPGWLSFSSQVSQNCHNGSYEISVLMMGNSAFAEPLKNLEDAVNEGLEIVRGRLQNAGLNVTVNATFMYSDGLIHNSGDCRSSTCEGLDLLRKISNAQRMGCVLIGPSCTYSTFQMYLDTELSYPMISAGSFGLSCDYKETLTRLMSPARKLMYFLVNFWKTNDLPFKTYSWSTSYVYKNGTETEDCFWYLNALEASVSYFSHELGFKVVLRQDKEFQDILMDHNRKSNVIIMCGGPEFLYKLKGDRAVAEDIVIILVDLFNDQYFEDNVTAPDYMKNVLVLTLSPGNSLLNSSFSRNLSPTKRDFALAYLNGILLFGHMLKIFLENGENITTPKFAHAFRNLTFEGYDGPVTLDDWGDVDSTMVLLYTSVDTKKYKVLLTYDTHVNKTYPVDMSPTFTWKNSKLPNDITGRGPQILMIAVFTLTGAVVLLLLVALLMLRKYRKDYELRQKKWSHIPPENIFPLETNETNHVSLKIDDDKRRDTIQRLRQCKYDKKRVILKDLKHNDGNFTEKQKIELNKLLQIDYYNLTKFYGTVKLDTMIFGVIEYCERGSLREVLNDTISYPDGTFMDWEFKISVLYDIAKGMSYLHSSKTEVHGRLKSTNCVVDSRMVVKITDFGCNSILPPKKDLWTAPEHLRQANISQKGDVYSYGIIAQEIILRKETFYTLSCRDRNEKIFRVENSNGMKPFRPDLFLETAEEKELEVYLLVKNCWEEDPEKRPDFKKIETTLAKIFGLFHDQKNESYMDTLIRRLQLYSRNLEHLVEERTQLYKAERDRADRLNFMLLPRLVVKSLKEKGFVEPELYEEVTIYFSDIVGFTTICKYSTPMEVVDMLNDIYKSFDHIVDHHDVYKVETIGDAYMVASGLPKRNGNRHAIDIAKMALEILSFMGTFELEHLPGLPIWIRIGVHSGPCAAGVVGIKMPRYCLFGDTVNTASRMESTGLPLRIHVSGSTIAILKRTECQFLYEVRGETYLKGRGNETTYWLTGMKDQKFNLPTPPTVENQQRLQAEFSDMIANSLQKRQAAGIRSQKPRRVASYKKGTLEYLQLNTTDKESTYF

SEQ ID NO:1

The full-length gene encoding human GUCY2C was cloned into the mammalian cell expression vector pCDH, and HEK293T cells (ATCC, CRL-11268) were co-transfected with three plasmids pVSV-G, pCMV-dR8.91 and pCDH-human GUCY2C to package the virus. After 48 hours of transfection, virus-infected CHO-K1 (ATCC, CCL-61) or HCT116 cells (ATCC, CCL-247) were collected, and after two weeks of screening under pressure, subcloning of the cells was performed, and cell lines CHO-K1/hGUCY C and HCT116/hGUCY C, which highly expressed GUCY2C, were obtained by FACS detection.

1.2 Construction of fusion proteins for immunization or screening

Human GUCY2C extracellular region is selected, and His or Fc label is connected at C end, so that fusion protein is constructed for immunization and screening detection. The related protein sequences are as follows:

hGUCY2C ECD-hFc:

SQVSQNCHNGSYEISVLMMGNSAFAEPLKNLEDAVNEGLEIVRGRLQNAGLNVTVNATFMYSDGLIHNSGDCRSSTCEGLDLLRKISNAQRMGCVLIGPSCTYSTFQMYLDTELSYPMISAGSFGLSCDYKETLTRLMSPARKLMYFLVNFWKTNDLPFKTYSWSTSYVYKNGTETEDCFWYLNALEASVSYFSHELGFKVVLRQDKEFQDILMDHNRKSNVIIMCGGPEFLYKLKGDRAVAEDIVIILVDLFNDQYFEDNVTAPDYMKNVLVLTLSPGNSLLNSSFSRNLSPTKRDFALAYLNGILLFGHMLKIFLENGENITTPKFAHAFRNLTFEGYDGPVTLDDWGDVDSTMVLLYTSVDTKKYKVLLTYDTHVNKTYPVDMSPTFTWKNSKLPNDITGRGPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:2

hGUCY2C ECD-His:

SQVSQNCHNGSYEISVLMMGNSAFAEPLKNLEDAVNEGLEIVRGRLQNAGLNVTVNATFMYSDGLIHNSGDCRSSTCEGLDLLRKISNAQRMGCVLIGPSCTYSTFQMYLDTELSYPMISAGSFGLSCDYKETLTRLMSPARKLMYFLVNFWKTNDLPFKTYSWSTSYVYKNGTETEDCFWYLNALEASVSYFSHELGFKVVLRQDKEFQDILMDHNRKSNVIIMCGGPEFLYKLKGDRAVAEDIVIILVDLFNDQYFEDNVTAPDYMKNVLVLTLSPGNSLLNSSFSRNLSPTKRDFALAYLNGILLFGHMLKIFLENGENITTPKFAHAFRNLTFEGYDGPVTLDDWGDVDSTMVLLYTSVDTKKYKVLLTYDTHVNKTYPVDMSPTFTWKNSKLPNDITGRGPQHHHHHH

SEQ ID NO:3

1.3 screening and identification of murine antibodies against GUCY2C

The monoclonal antibody aiming at the human GUCY2C is prepared by a Single B technology, and the obtained antibody specifically binds with the human GUCY2C with higher affinity, can be in cross-binding with the GUCY2C of a cynomolgus monkey, and has better binding activity with the human GUCY2C on the cell surface.

HGUCY2C ECD-hFc, hGUCY C ECD-His, CHO-K1/hGUCY C cells or HCT116/hGUCY C cells were used as immunogens. 50 μg of protein was first immunized and 25 μg of protein was boosted each time toGold Adjuvant (Sigma Cat No. T2684) and ThermoAlum (Thermo Cat No. 77161) was used as an adjuvant for cross immunization. Cellular immunization was performed at 1 x 10 ⁷/time per immunization. Mice with high antibody titers in serum were selected for singleB cell selection after primary and 4 to 7 booster immunizations.

Screening of Single B cells a series of antibody variable region sequences were obtained mainly by positive and negative screening, 10 x genomic and bioinformatics techniques. The obtained sequence is subjected to CDR classification, a murine variable region sequence is selected to be connected with a human antibody constant region sequence and express chimeric antibodies, and the chimeric antibodies are screened by combining with hGUCY C ECD-His and cynoGUCY C ECD-His (ACRO # GUC-C52H 6) proteins, surface plasmon resonance, cell binding and other methods, so that antibody sequences specifically combined with hGUCY C ECD and cynoGUCY C ECD are selected. The heavy and light chain variable region sequences of the antibodies obtained were screened as follows:

TABLE 2 CDR of anti-GUCY 2C antibody obtained by single B screening

>P3-C7 VH:

EVQLQQSGPVLLKPGASVKMSCKASGYTFTDHYIHWVKLSHGKSLEWIGVINPYNDGATYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARPPRAYGNYAGYWGQGTTLTVSS

SEQ ID NO:10

>P3-C7 VL:

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFIGSGSRTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTRLEIK

SEQ ID NO:11

Note that the single underlined part is the CDR according to Kabat rules, the rest is FR.

The variable region sequence of the murine anti-GUCY 2C antibody is combined with the constant regions shown by SEQ ID NO. 29 and SEQ ID NO. 30 to obtain a chimeric antibody. Illustratively, P3-C7 CHI represents a chimeric antibody comprising a P3-C7 murine heavy chain variable region, a light chain variable region, and said constant region, the specific sequences being shown below.

P3-C7 CHI heavy chain:

P3-C7 CHI light chain:

EXAMPLE 2 humanization of anti-GUCY 2C monoclonal antibodies

Humanization of murine antibodies was performed according to methods well known in the art from a number of literature. Briefly, on the basis of the obtained typical structure of the VH/VL CDR of a murine antibody, the homologous sequences of the variable region (VL) and the variable region (VH) of the light chain are searched from a human germline database, the germline with the highest FR homology is selected as a template according to the sequence from high to low of the homology of the FRs, the CDR region of the murine antibody is transplanted to the human template, certain amino acids of the variable region are mutated, and the constant region of the murine antibody is replaced by a human constant region, so that the final humanized antibody is obtained.

Humanization of P3-C7 antibodies

Humanized antibodies of P3-C7 antibodies selected IGKV2-40 x 01 or IGKV1-39 x 01 and IGKJ4 x 01 as light chain framework region templates, and IGHV1-46 x 01 and IGHJ6 x 01 as heavy chain framework region templates. The CDR region of the murine antibody is transplanted to a selected humanized template, and certain amino acids of the variable region are substituted, so that the light chain and heavy chain variable regions of the humanized antibody are obtained, and the specific framework substitution and amino acid substitution are as follows:

TABLE 3 framework substitutions and amino acid substitutions of humanized antibodies P3-C7

Note that amino acid numbering follows the Kabat numbering system.

TABLE 4 CDR of P3-C7 humanized antibodies

The variable region sequence of the P3-C7 humanized antibody is as follows:

>hP3-C7 L1:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIK

SEQ ID NO:19

>hP3-C7 L2:

>hP3-C7 L3:

>hP3-C7 L4:

>hP3-C7 L5:

>hP3-C7 L6:

>hP3-C7 L7:

>hP3-C7 L8:

>hP3-C7 H1:

>hP3-C7 H2:

note that the single underlined part is the CDR, the dot underlined part is the amino acid substitution site, and the rest is the FR.

The heavy chain variable region and the light chain variable region of the anti-GUCY 2C humanized antibody are recombined with the heavy chain constant region hIgG1: CH1-Fc and the light chain constant region CL respectively to obtain the full-length humanized antibody.

>hIgG1:CH1-Fc:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:29

>CL:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:30

Humanized antibodies against GUCY2C obtained by the present disclosure are as follows:

TABLE 5 humanized antibodies of P3-C7 series

Illustratively, the full length sequence of the anti-GUCY 2C humanized antibody is as follows:

P3-C7L 7H1 heavy chain:

P3-C7L 7H1 light chain:

note that the single underlined part is the CDR, the bold dotted underline is the amino acid substitution site, and the italic part is the constant region.

Control antibodies for anti-GUCY 2C antibodies used in the present disclosure are as follows:

1608 (variable region sequence is constructed by referring to GUCY2C-1608 bispecific antibody in WO2019224716A2, the constant region sequence is shown as SEQ ID NO:29 and SEQ ID NO: 30), and the full-length sequence thereof is as follows:

heavy chain > 1608:

>1608 light chain:

example 3 preparation of anti-GUCY 2C-CD3 bispecific antibodies

The GUCY2C arm of the GUCY2C-CD3 bispecific antibody of the disclosure is an anti-GUCY 2C antibody in the disclosure, and the CD3 arm can be derived from any suitable antibody. Specifically, the CD3 arm employed in the present disclosure is S107E.

TABLE 6 CDR of S107E

The S107E variable region sequence is as follows:

>S107E-VH:

EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSS

SEQ ID NO:41

>S107E-VL:

QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL

SEQ ID NO:42

the GUCY2C-CD3 bispecific antibodies in the disclosure have the following molecular structure:

format 1 is an asymmetric structural molecule, and comprises two chains of an intact molecule, wherein the two chains are different, and the method specifically comprises the following steps:

Chain 1 [ GUCY2C-VL ] - [ linker 1] - [ CD3-VH ] - [ linker 2] - [ IgG1Fc (Knob-DI) ]

Chain 2 [ CD3-VL ] - [ linker 3] - [ GUCY2C-VH ] - [ linker 2] - [ IgG1Fc (Hole-DI) ];

A schematic diagram thereof is shown in fig. 1.

The Fc regions of the two chains of a bispecific antibody molecule are Fc regions that are capable of associating with each other by a knob technique. Illustratively, the Fc region of chain 1 is of a convex structure according to the pestle and mortar technique (e.g., SEQ ID NO: 43) and the Fc region of chain 2 is of a pore structure according to the pestle and mortar technique (e.g., SEQ ID NO: 44).

The related sequences are as follows:

>IgG1Fc(Knob-DI,L234A/L235A/G237A/Y349C/T366W)

>IgG1Fc(Hole-DI,L234A/L235A/G237A/S354C/T366S/L368A/Y407V)

connector 1:

GGGSGGGG

SEQ ID NO:45

Connector 2:

G

SEQ ID NO:46

Connector 3:

GGGGSGGGG

SEQ ID NO:47

P3-C7L 7H1-1 indicates that the variable region of the antibody P3-C7L 7H1 is adopted as GUCY2C binding domain, format 1 is adopted as molecular structure, and the CD3 arm is S107E. The specific amino acid sequence is as follows:

Full-length sequence of P3-C7L 7H1-1

Chain 1 (P3-C7L 7H 1-1-1):

DVVMTQTPLSLPVTPGEPASISCRSSQSLVHSNQNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSRTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIKGGGSGGGGEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO:48

Chain 2 (P3-C7L 7H 1-1-2):

QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGGGSGGGGEVQLVQSGAEVKKPGASVKVSCKASGYTFTDHYIHWVRQAPGQGLEWMGVINPYNDGATYNQKFKGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARPPRAYGNYAGYWGQGTTVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO:49

In addition, the present disclosure also uses the GUCY2C-CD3 bispecific antibody GUCY2C-1608 (designated 1608-BsAb in the present disclosure), prepared with reference to patent WO2019224716A2, which has a DIABODY structure, and the complete molecule comprises two chains, as follows:

chain 1 (GUCY 2C-1608-Knob sequence):

chain 2 (GUCY 2C-1608-Hole sequence):

TABLE 7.1608 CDR sequences of BsAb

Furthermore, the present disclosure also uses EGFR-CD28 bispecific antibody 97H2L3OT-ZAL, prepared with reference to patent CN202210371538.8, now published under WO2023198042A1, the sequence of which is specifically as follows:

TABLE 8 CDR of EGFR-CD28 bispecific antibody 97H2L3OT-ZAL

EGFR-VH:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS

SEQ ID NO:76

EGFR-VL:

DIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK

SEQ ID NO:77

CD28-VH:

EVQLVQSGAEVKKPGASVKVSCKASGYTLTSYWITWVRQAPGQGLEWMGDIYPGSGRTNYNEMFKNRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRHYGTGYESFDVWGQGTTVTVSS

SEQ ID NO:78

CD28-VL:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPDGTIKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIK

SEQ ID NO:79

Full-length sequence of EGFR-CD28 bispecific antibody 97H2L3 OT-ZAL:

chain 1:

EVQLVQSGAEVKKPGASVKVSCKASGYTLTSYWITWVRQAPGQGLEWMGDIYPGSGRTNYNEMFKNRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRHYGTGYESFDVWGQGTTVTVSSGGGGSGIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSIDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:80

chain 2:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPDGTIKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKVEIKGGGGSSGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA

SEQ ID NO:81

Chain 3:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:82

Chain 4:

DIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:83

Test case

Test example 1 affinity of the GUCY2C monoclonal antibodies of the disclosure for antigens

To test the affinity of the GUCY2C mab of the disclosure for GUCY2C, the present test example tested the binding capacity of antibodies to hGUCY C-ECD-His (SEQ ID NO: 3) using the instrument Biacore 8K and Biacore T200. The method comprises the steps of using a Protein A biosensing chip coupled with an anti-human antibody to affinity and capture the antibody, then flowing a solution containing GUCY2C Protein on the surface of the chip, and detecting a reaction signal in real time by an instrument to obtain a binding and dissociation curve. After dissociation was completed in each experimental cycle, the biochip was washed and regenerated with 10mM glycine-hydrochloric acid (pH 1.5). The data fitting model uses a 1:1 model. Affinity data for the binding of the GUCY2C mab of the present disclosure to the corresponding antigen are shown in the table below.

TABLE 9 binding of monoclonal antibodies to human GUCY2C antigen

Antibodies to	ka(1/Ms)	kd(1/s)	KD(nM)
				P3-C7 CHI	2.26E+04	1.49E-04	6.61
P3-C7 L1H1	2.37E+04	1.56E-04	6.55
				P3-C7 L2H1	3.10E+04	1.66E-04	5.35
P3-C7 L3H1	2.84E+04	1.35E-03	47.50
				P3-C7 L4H1	2.85E+04	2.09E-03	73.40
P3-C7 L5H1	2.69E+04	7.19E-04	26.70
				P3-C7 L7H1	2.39E+04	1.86E-04	7.77

The results show that both the GUCY2C monoclonal antibodies disclosed by the disclosure have better binding affinity with human GUCY 2C.

Test example 2 binding Activity of anti-GUCY 2C monoclonal antibodies against cell surface antigens

The test example uses flow cytometry to detect the binding activity of GUCY2C monoclonal antibody on human GUCY2C on the surface of stable transgenic cell lines HCT116/hGUCY C and intestinal cancer cells HT-55 which over express human GUCY 2C. The method comprises the following steps:

Cells of 1E5 resuspended in 1% BSA were added to the reaction wells and the supernatant was removed after centrifugation. The diluted antibody of the corresponding concentration was added, incubated at room temperature for 30 minutes, and washed once with 1% BSA. Then, F (ab') 2-goat anti-human IgG Fc secondary antibody, PE (Cat.#H2104, invitrogen) secondary antibody was added, and the mixture was incubated at room temperature for 30 minutes in the absence of light after the resuspension. After washing twice with 1% bsa, fluorescence signal values were read using a flow cytometer. Exemplary FACS results are shown in fig. 2A-2B.

The results show that the GUCY2C monoclonal antibody disclosed by the disclosure has better specific binding with GUCY2C on the cell surface, and the binding activity is better than that of 1608.

Test example 3 nonspecific binding of the humanized antibodies to GUCY2C of the disclosure

This test example detects non-specific binding of a GUCY2C humanized antibody to a non-target cell (e.g., T cell). The method comprises the following steps:

Experiment one the supernatant was removed after centrifugation using flow cytometry, 1% BSA resuspended in 1E5 Jurkat cells (ATCC # TIB-152) per well. The diluted antibody of the corresponding concentration was added, incubated at room temperature for 30 minutes, and washed once with 1% BSA. F (ab') ₂ -goat anti-human IgG Fc secondary antibody, PE (Cat. # H10104, invitrogen) was then added and incubated at room temperature in the absence of light for 30 minutes after resuspension. After washing twice with 1% bsa, fluorescence signal values were read using a flow cytometer. Subtracting the background fluorescence intensity of cells without the incubated antibody from the average fluorescence intensity of the antibody binding to obtain a relative average fluorescence intensity value. The results are shown in Table 10 below.

TABLE 10 binding of GUCY2C humanized monoclonal antibodies to Jurkat

The results show that the GUCY2C humanized monoclonal antibody disclosed herein has no nonspecific binding with Jurkat cells without GUCY2C expression, and 1608 has obvious binding with Jurkat cells.

Experiment two flow cytometry was used to examine whether the GUCY2C humanized monoclonal antibodies of the disclosure bind non-specifically to human PBMC and CD3+ T cells in the PBMC, 3E5 human peripheral blood cells resuspended in 1% BSA were added to each well, and the supernatant was removed after centrifugation. The diluted antibody of the corresponding concentration was added, incubated at room temperature for 30 minutes, and washed once with 1% BSA. Then, the secondary antibody working solution of PE-Cy7 anti-human CD3 (BD# 557851) and APC anti-human IgG Fc (biolegend# 409306) was added, and the mixture was incubated at room temperature for 30 minutes in a dark place after the resuspension. After washing twice with 1% bsa, fluorescence signal values were read using a flow cytometer. Subtracting the background fluorescence intensity of cells without the incubated antibody from the average fluorescence intensity of the antibody binding to obtain a relative average fluorescence intensity value. The results are shown in Table 11 below.

TABLE 11 binding of GUCY2C humanized monoclonal antibodies to human PBMC

The results showed that the present disclosure showed no non-specific binding of the GUCY2C humanized mab to PBMC and T cells without GUCY2C expression, and that the control antibody 1608 had significant non-specific binding to PBMC and T cells. The target specificity of the disclosed GUCY2C humanized monoclonal antibody is better than 1608.

Test example 4 epitope analysis of GUCY2C monoclonal antibodies of the disclosure

To test the epitopes bound by the GUCY2C monoclonal antibodies of the present disclosure, the difference in binding epitopes of the antibodies to the antigen was detected using ELISA methods. The method comprises the following steps:

The tested mab was coated at 100ng overnight at 4 ℃ and blocked with 1% bsa after plate washing. 50ng of hGUCC Y2C ECD-His antigen was then added and incubated at 37℃for 1 hour, the plates were washed, 100ng of biotinylated antibody was added and incubated at 37℃for 1 hour, and finally signal detection was performed with HRP-streptavidin. Relative competition coefficient= (signal value of antibody self-competition/signal value of his anti-competition) ×100. Two antibodies with identical epitopes have a competition coefficient of 100 in competition binding, and antibodies with larger epitope differences have smaller competition coefficients therebetween. The details are shown in table 12 below:

Competitive binding of Gucy2C mab to antigen

The results showed that the epitope to which the disclosed GUCY2C mab bound was significantly different from 1608 and was unable to competitively bind to the GUCY2C antigen.

Test example 5 protein binding Activity of GUCY2C-CD3 bispecific antibody against antigen

The binding activity of the GUCY2C-CD3 double antibody to human GUCY2C was detected by ELISA method in the test example. The method comprises the following steps:

100ng of streptavidin (ACRO # STN-N5116) was added to the wells and plated at 4℃overnight. 1% BSA (PBS) was added and incubated at 100ng hGUCY2C ECD-His 37℃for 1 hour. After washing the plates, diluted gradient antibodies were added and incubated at 37 ℃ for 1 hour. Then Peroxidase AffiniPure goat anti-human IgG (h+l) (cat.# 109-035-003,Jackson ImmunoResearch) was added and incubated at 37 ℃ for 1 hour. After washing the plate, adding a color developing solution, and after color development is complete, adding a stop solution to stop the reaction. The signal values were read by a microplate reader 450 nM. The binding activity of the antibodies is shown in table 13 below.

Table 13 binding Activity (absorbance) of GUCY2C-CD3 bispecific antibodies to human GUCY2C protein

The results show that the GUCY2C-CD3 bispecific antibodies of the disclosure have good binding to human GUCY2C protein.

Test example 6 binding Activity of GUCY2C-CD3 bispecific antibody against cell surface antigen

The present test example uses flow cytometry to examine the binding activity of GUCY2C-CD3 diabodies to human GUCY2C overexpressing stably transformed cell lines HCT116/hGUCY C and intestinal cancer cells HT55 (Nanjac Bai#CBP 60012) and CD3 expressing Jurkat cells (ATCC#TIB-152). GUCY2C expression in HCT116/hGUCY C cells and intestinal cancer cells HT55 the amounts are respectively high expression (++ +) plus) and moderate expression (++).

The method comprises the following steps:

Experiment one the supernatant was removed after centrifugation by adding 1% BSA resuspended 1E5 cells to the reaction wells. Diluted antibodies of the corresponding concentration were added, incubated at room temperature for 30 minutes, and washed twice with 1% BSA. Then adding goat anti-human IgG (gamma) R-PE conjugate F (ab') secondary antibody (life technologies # H10104) diluted by 1:400, and applying for 30 minutes at room temperature in dark place after re-suspending. After washing twice with 1% bsa, fluorescence signal values were read using a flow cytometer. Exemplary FACS results are shown in fig. 3A-3B.

The results show that the disclosed GUCY2C-CD3 double antibody has better specific binding activity on cells expressing different levels of GUCY2C, and the binding activity is superior to 1608-BsAb.

And in the second experiment, the same method as in the first experiment is adopted. The binding activity of the antibodies to Jurkat cells is shown in figure 3C.

The results show that the GUCY2C-CD3 double antibody has better specific binding activity on the cells expressing Jurkat, and the binding activity is better than that of 1608-BsAb.

Test example 7 activation of T cells by GUCY2C-CD3 bispecific antibody

To test the activation of T cells by the GUCY2C-CD3 bispecific antibodies of the disclosure under the combined action of target cells HCT116/GUCY2C and LS1034 cells. This test example uses Jurkat-Lucia ^TM NFAT (InvivoGen, jktl-NFAT) for cell detection. The method comprises the following steps:

The Jurkat Lucia ^TM NFAT cells and target cells are respectively subcultured in complete medium and corresponding medium, the Jurkat-NFAT lum cell set is collected by centrifugation, the culture medium resuspension count of 1640+10% FBS is used for adjusting the cell number to 1E6 cells/mL, the target cells HCT116/GUCY2C and LS1034 cells are resuspended and counted, and then the cell numbers are respectively adjusted to 2E5 cells/mL and 4E5 cells/mL. The JurkatLucia ^TM NFAT and target cells were mixed in equal volumes and 100. Mu.L was added to each well to ensure that the HCT116/GUCY2C and LS1034 cells E:T ratios were 5:1 and 2.5:1. The antibody was diluted with 1640+10% FBS medium, 20. Mu.L was added to the cell well, and left at 37℃for 5 hours. According to the requirements of the specification, the QUANTI-Luc ^TM Gold reagent is dissolved with 25 mL ddH ₂ O. After adding 50 mu L QUANTI-Luc ^TM Gold and incubating for 5 minutes at room temperature, luminescence was detected with the Vendor to calculate the activation of JurkatLucia ^TM NFAT cells by the antibody at different concentrations. The relative fold of the antibody-post-exposure Lumin value divided by the background Lumin value was used for the graph. The relative fold values and EC ₅₀ results are shown in FIGS. 4A-4B.

The results show that the GUCY2C-CD3 diabodies disclosed by the invention act together with target cells, have better activation effect on T cells, and the activation effect is superior to 1608-BsAb.

Test example 8 cytotoxic Activity of the GUCY2C-CD3 bispecific antibodies of the disclosure

This test example investigated the killing activity of the GUCY2C-CD3 bispecific antibodies of the disclosure as T cell adapter molecules against tumor cells. The method comprises the following steps:

The target-specific cytotoxic activity of bispecific antibodies of the present disclosure was tested using LucG stable transgenic cell lines constructed using the GUCY2C expressing intestinal cancer cell lines LS1034, HT-55, and LS174T (ATCC #CL-188) as high, medium, and low expressing target cells, respectively. Cryopreserved PBMC (purchased from Miaoshun Biotechnology) were resuspension with 1640+10% FBS complete medium after resuscitation and incubated in T75 flasks for 4 hours (density 2E6 cells/mL). PBMC were collected and centrifuged, resuspended in 1640+10% fbs, counted and the cell number was adjusted to 1.5e6 cells/mL. Target cells were collected, centrifuged at 1000 rpm for 3 min, resuspended, counted, and cell concentration adjusted to 1.5E5 cells/mL. The PBMC suspension and the target cell suspension were mixed in equal volumes, and 100. Mu.L of each well was added to ensure that the E:T Ratio was 10:1. PBMC ONLY groups were additionally provided and the PBMC suspensions described above were mixed with 1640+10% fbs complete medium in equal volumes. Antibodies were diluted in 1640+10% fbs medium at an initial concentration of 600 nM (6 x final concentration), 5-fold dilution, 9 dose points, and 20 μl added per well. The treated cells were incubated in an incubator at 37℃with 5% CO ₂ for 48 hours and then subjected to luminescence detection. The maximum inhibition of antibody-mediated killing (Imax) and IC ₅₀ were fitted by using Graphpad prism8.0 software to make a dose-response plot based on the log concentration of antibody and signal values. The maximum inhibition of the antibody was calculated by setting the wells to 0% inhibition with only target cells and PBMCs without antibody. The results are shown in Table 14 below.

Table 14 cytotoxic Activity of GUCY2C-CD3 bispecific antibodies against target cells

Note that +indicates the amount of GUCY2C expressed, wherein the higher +indicates the higher amount of GUCY2C expressed.

The results show that the disclosed GUCY2C-CD3 bispecific antibody has better cytotoxic activity on target cells with different expression levels.

Test example 9 release of GUCY2C-CD3 bispecific antibody cytokines of the disclosure

This test example examined the changes in secretion of cytokines IFN-. Gamma.and IL-6 in PBMC under the combined action of GUCY2C-CD3 diabody and target cells (LS 1034 cells and LS174T cells in test example 8).

The supernatant collected in the cell killing experiment (test example 8) and frozen in a-20 refrigerator was thawed at normal temperature, mixed by shaking, and the sample was diluted 20 times with 1640+4% FBS medium for use. IL-6 and IFN-gamma standard powders were dissolved using sterile water and the standard was diluted to the desired concentration using 1640+4% FBS medium. The kit for taking out the cytokine to be detected includes a human IL6 ELISA kit (euphausia biotechnology limited, EHC 007.96) and a human IFN- γ kit (CISBIO, 62, HIFNGPEG), and the kit is equilibrated to normal temperature and detected according to the procedure of the specification. The results are shown in FIGS. 5A-5B and FIGS. 6A-6B.

The results show that the GUCY2C-CD3 bispecific antibodies of the disclosure are effective in inducing release of IFN-gamma associated with cytotoxic activity, and that the release level of the inflammatory-related cytokine IL-6 is relatively low.

Biological evaluation of in vivo Activity

Test example 10 efficacy of the GUCY2C-CD3 bispecific antibodies of the disclosure in LS1034 subcutaneous transplantation tumor model

The present disclosure uses the human colon cancer cell LS1034 mouse subcutaneous engraftment model to evaluate the in vivo efficacy of GUCY2C-CD3 bispecific antibodies.

Female NOG mice were purchased from beijing velutinib Shanghai company. 6-8 weeks of age at purchase. Human PBMCs used in this test case were from Miao-living commercial purchases.

LS1034 cells 1X 10 ⁶ cells/200. Mu.L (containing 50% basement membrane matrix) were inoculated subcutaneously in the right rib of NOG mice, the next day after inoculation, the resuscitated well-conditioned human PBMCs were injected into the abdominal cavity of NOG mice at 5X 10 ⁶ cells/100. Mu.L, and when tumor volume of tumor-bearing mice reached about 150mm ³, oversized and undersized mice were removed and randomly grouped, 8 mice per group. The day of the grouping was defined as day 0 of the experiment, and intraperitoneal injection of each equimolar amount of antibody was started on day 0, 2 times a week for two to three weeks. Tumor volume, animal weight were monitored 2 times per week and data recorded. Tumor-bearing animals were euthanized as the end point of the experiment when tumor volumes of the experimental animals exceeded 1000mm ³ or most tumors had crumbed or lost 20% of weight.

The calculation formula of the tumor volume (V) is V=1/2×a×b ², wherein a and b respectively represent length and width.

Relative tumor proliferation rate T/C (%) = (T-T ₀)/(C-C₀) ×100%, where T, C is the tumor volume of the treatment group and control group at the end of the experiment, and T ₀、C₀ is the tumor volume at the beginning of the experiment.

Tumor inhibition rate TGI (%) =1-T/C (%).

Anti-tumor Activity of GUCY2C-CD3 bispecific antibodies

Note that p <0.05, p <0.01, p <0.001vs vehicle control, T test.

The results showed that the dual GUCY2C-CD3 antibodies of the present disclosure have significant tumor suppression effect on this model.

Test example 11 efficacy of bispecific antibodies of the present disclosure in LS174T subcutaneous transplantation tumor model

The present disclosure uses the human colon cancer cell LS174T mouse subcutaneous engraftment model to evaluate the in vivo efficacy of GUCY2C-CD3 bispecific antibodies.

LS174T cells 1X 10 ⁶ cells/100. Mu.L were mixed with cryopreserved resuscitated PBMC (E: T=1:1) and inoculated subcutaneously in the right rib of NOG mice. When tumor volume of tumor-bearing mice reached about 150 mm ³, mice with oversized tumor volume and undersized body weight were removed, randomly grouped, 8 mice per group. The day of the grouping was defined as day 0 of the experiment and each antibody was intraperitoneally injected on day 0, day 3, day 7, and day 10, administered 4-5 times, tumor volume, animal weight monitored 2 times a week and data recorded. Tumor-bearing animals were euthanized as the end point of the experiment when tumor volumes of the experimental animals exceeded 1000 mm ³ or most tumors had collapsed or body weight was reduced by 20%.

Anti-tumor Activity of GUCY2C-CD3 bispecific antibodies

Note that p <0.05, p <0.01, p <0.001vs vehicle control, T test.

The results showed that the dual GUCY2C-CD3 antibodies of the present disclosure have significant tumor suppression effect on this model. Tumor weight data was substantially consistent with tumor volume trend during the experiment.

Test example 12 anti-tumor Activity of GUCY2C-CD3 diabodies in combination with EGFR-CD28 diabodies

The present disclosure uses the LS174T subcutaneous tumor model to study the effect of the anti-tumor activity of the combination of GUCY2C-CD3 diabody with EGFR-CD28 diabody (97H 2L3OT-ZAL, prepared with reference to patent CN202210371538.8, now published as WO2023198042A 1).

LS174T cells 1X 10 ⁶ cells/100. Mu.L were mixed with cryopreserved resuscitated PBMC (E: T=1:1) and inoculated subcutaneously in the right rib of NOG mice. When tumor volume of tumor-bearing mice reaches about 150mm ³, mice with oversized tumor volume and undersized body weight are removed, randomly grouped, and 8 mice are in each group. The day of the grouping was defined as day 0 of the experiment and each antibody was intraperitoneally administered 4 times on day 0, day 3, day 7, and day 11, with the combination group drug being administered simultaneously after separate formulation, tumor volume, animal weight were monitored 2 times per week and data recorded. Tumor-bearing animals were euthanized as the end point of the experiment when tumor volumes of the experimental animals exceeded 1000mm ³ or most tumors had crumbed or lost 20% of weight. The specific results are shown in Table 17 below.

Table 17 anti-tumor Activity of GUCY2C-CD3 diabodies in combination with EGFR-CD28 diabodies

Note that p <0.05, p <0.01, p <0.001vs vehicle control, T test.

The results show that the EGFR-CD28 diabodies and GUCY2C-CD3 diabodies can effectively improve the drug effect of the single drugs.

2. Formulation example-bispecific antibody formulations that specifically bind to GUCY2C and CD3

SEC size exclusion chromatography:

A method for analyzing the separation of solutes according to the relative relationship between the pore size of the gel pores and the coil size of the polymer sample molecules.

SEC% (SEC monomer content percentage) =a monomer/atotal×100% (a monomer is the peak area of the main peak monomer in the sample, atotal is the sum of all peak areas). Δsec% = SEC% of pre-stability experiment formulation-SEC% of post-stability experiment formulation.

SEC measuring instrument Agilent HPLC 1260;

Column Waters BioResolve ^TM SEC mAb 2.5μm 7.8×300mm Column。

NR-CE capillary gel electrophoresis:

A method of electrophoresis by moving the gel into a capillary as a supporting medium and separating according to the molecular weight of the sample under a certain voltage.

NR-CE% (NR-CE main peak content percentage) =A main peak/A total×100% (A main peak is peak area of main peak in sample, A total is sum of all peak areas). Δnr-CE% = NR-CE% of pre-stability experiment formulation NR-CE% of post-stability experiment formulation.

The CE measuring instrument is a Beckman capillary electrophoresis apparatus, and the model is PA800 plus.

IEC ion exchange chromatography:

The chromatographic method uses ion exchange resin or chemically bonded ion exchanger as stationary phase and uses the difference of ion exchange capacity or selectivity coefficient of separated components to implement separation.

IEC% (IEC neutral peak content percentage) =a neutral peak area/a total area×100% (a total area is the sum of the acid peak, neutral peak and alkaline peak areas). Δiec% = IEC of pre-stability experiment formulation%iec of post-stability experiment formulation.

IEC measurement instrument Agilent HPLC 1260.

Osmotic pressure measurement:

the freezing point method is used for measuring the osmotic pressure, the freezing point of the solution is measured by adopting a high-sensitivity temperature sensing element based on the direct proportional relation between the freezing point depression value and the molar concentration of the solution, and the solution is converted into the osmotic pressure through electric quantity.

The osmotic pressure measuring instrument is Luo Ze Loser, model number OM815.

Proteins

The proteins used in the following examples were bispecific antibody proteins (P3-C7L 7H1-1, hereinafter referred to as "proteins") that specifically bind GUCY2C and CD 3.

The instrument for measuring the concentration of the protein is an ultraviolet-visible spectrophotometer, the model is Nano Drop oneC, and the optical path is 1mm.

Formulation example 1pH and buffer System screening

A formulation of 100mg/mL protein, 50mg/mL sucrose, 50mM NaCl, 0.4mg/mL poloxamer 188 (P188) was prepared using the buffer system shown in Table 18. The samples were subjected to forced degradation studies (25 ℃ C., 200rpm shaking for 7 days) using SEC as an evaluation index to investigate the effect of different pH and buffer systems on protein stability.

The results are shown in Table 18, and SEC data show that the protein has better stability in each buffer system after shaking for 7 days at 25 ℃ and 200rpm, wherein the monomer purity in the preparation of the His-HCl buffer system is better than that of other buffer systems, the preparation prepared by His-HCl pH5.5-6.5 has better stability, and the preparation prepared by His-HCl pH5.5 has optimal stability.

TABLE 18 pH and buffer System screening results

Note that AA represents acetic acid-acetic acid sodium salt, his-AA represents histidine-acetic acid histidine salt system, his-HCl represents histidine-histidine hydrochloride, SA represents succinic acid-succinic acid sodium salt, CA represents citric acid-sodium citrate salt, tris-HCl represents Tris-hydroxymethyl aminomethane hydrochloride, and the same applies.

Formulation example 2pH Range confirmation

A formulation of 100mg/mL protein, 80mg/mL sucrose, 0.4mg/mL poloxamer 188 (P188) was prepared using His-HCl buffer systems of different pH values shown in Table 19. Because of the southwest effect, the pH of the preparation has 0.1-0.2 drift compared with a buffer system, the forced degradation research (placing for 4 weeks at 40 ℃) is carried out on the sample, SEC, CE, IEC is taken as an evaluation index, and the influence of different pH values on the stability of the protein is examined.

The results are shown in Table 19. The SEC, NR-CE and IEC results show that no significant difference exists between groups, the pH value of the preparation prepared from the His-HCl buffer with the pH value of 5.0-6.0 is 5.1-6.2, and the stability is good.

TABLE 19 pH Range confirmation results

Group of	Buffer system	Formulation pH	Conditions of placement	ΔSEC%	ΔNR-CE%	ΔIEC%
							1	20mM His-HCl 5.0	5.1	40°CW4	1.3	4.3	23.7
2	20mM His-HCl 5.5	5.7	40°CW4	1.4	3.7	22.6
							3	20mM His-HCl 6.0	6.2	40°CW4	2.0	4.1	24.7

Note that 40 ° CW4:40 ℃ is left for 4 weeks, as follows.

Formulation example 3 His-HCl buffer System ion concentration screening

Preparations of 100mg/mL protein, 80mg/mL sucrose, 0.4mg/mL PS80, 0.05mg/mLEDTA-2Na, his-HCl buffers of different ionic strengths as shown in Table 20, pH5.7 were prepared. The samples were subjected to forced degradation studies (4 weeks at 40 ℃) and the effect of buffers of different His-HCl ion concentrations on protein stability was investigated using SEC, NR-CE and IEC as evaluation indicators.

The results are shown in Table 20.SEC, NR-CE and IEC data show that after the preparation is placed for 4 weeks at 40 ℃, the preparation with different His-HCl ion intensities has no obvious difference and has good stability.

TABLE 20 His-HCl buffer concentration screening results

Group of	Buffer solution	Conditions of placement	ΔSEC%	ΔNR-CE%	ΔIEC%
						1	10mM His-HCl pH5.7	40°CW4	1.8	2.0	22.6
2	14mM His-HCl pH 5.7	40°CW4	2.2	2.0	24.3
						3	30mM His-HCl pH 5.7	40°CW4	2.5	2.4	25.1

Note that EDTA-2Na represents disodium ethylenediamine tetraacetate dihydrate (C ₁₀H₁₄N₂O₈Na₂·2H₂ O), the same applies below.

Formulation example 4 ph range was again confirmed

100Mg/mL protein, 80mg/mL sucrose, 0.4mg/mL PS80, 0.05mg/mLEDTA-2Na, 14mM His-HCl, formulations of different pH as shown in Table 21 were prepared. The samples were subjected to forced degradation studies (4 weeks at 40 ℃) and the effect of different pH on protein stability was investigated using SEC, NR-CE and IEC as evaluation indexes.

The results are shown in Table 21.SEC, NR-CE and IEC data showed that the formulations at pH 5.2-6.2 were stable with no significant differences between groups after 4 weeks of 40 ℃.

TABLE 21 screening results at different pH

Group of	pH	Conditions of placement	ΔSEC%	ΔNR-CE%	ΔIEC%
						1	5.2	40°CW4	2.3	2.9	23.9
2	5.7	40°CW4	2.2	2.0	24.3
						3	6.2	40°CW4	2.3	3.2	28.3

Formulation example 5 surfactant species screening

A preparation of 100mg/mL protein, 80mg/mL sucrose, different types of surfactants shown in Table 22, 14mM His-HCl, pH5.7 was prepared. The samples were subjected to forced degradation studies (4 weeks at 40 ℃) and the effect of different surfactants on protein stability was investigated using SEC, NR-CE and IEC as evaluation indicators.

The results are shown in Table 22.SEC, NR-CE and IEC data showed no significant differences between the PS80 and poloxamer 188 containing formulations after 4 weeks of 40 ℃.

TABLE 22 surfactant species screening results

Group of	Surface active agent	Conditions of placement	ΔSEC%	ΔNR-CE%	ΔIEC%
						1	0.4mg/mL P188	40°CW4	1.4	3.7	22.6
2	0.4mg/mL PS80	40°CW4	1.7	3.7	24.2

Formulation example 6 surfactant concentration screening

Preparations of 100mg/mL protein, 80mg/mL sucrose, 0.05mg/mL EDTA-2Na, different surfactant concentrations shown in Table 23, 14mM His-HCl, pH5.7 were prepared. The samples were subjected to forced degradation studies (4 weeks at 40 ℃) and the effect of different surfactant concentrations on protein stability was investigated using SEC, NR-CE and IEC as evaluation indices.

The results are shown in Table 23.SEC, NR-CE and IEC data show that after the preparation is placed for 4 weeks at 40 ℃, the preparations with different surfactant concentrations have no obvious difference and the stability is good.

TABLE 23 screening results for different surfactant concentrations

Group of	Surface active agent	Conditions of placement	ΔSEC%	ΔNR-CE%	ΔIEC%
						1	0.1mg/mL PS80	40°CW4	2.7	2.1	24.2
2	0.4mg/mL PS80	40°CW4	2.2	2.0	24.3
						3	1.0mg/mL PS80	40°CW4	2.5	2.1	23.6

Formulation example 7 adjuvant screening

A preparation of 100mg/mL protein, with or without EDTA-2Na, 80mg/mL sucrose and 0.4mg/mL PS80, 14mM His-HCl, pH 5.7 as shown in Table 24 was prepared.

The sample is subjected to forced degradation research (placed at 40 ℃ for 4 weeks), and the degradation rate of PS80 is taken as an evaluation index to examine the influence of different auxiliary materials on the stability of the preparation. The results are shown in Table 24, and the data shows that PS80 in the EDTA-2 Na-containing formulation degraded less after 4 weeks at 40℃over the EDTA-2 Na-free formulation.

TABLE 24 auxiliary material screening results

Group of	Auxiliary materials	Conditions of placement	PS80 degradation rate%
				1	N/A	40°CW4	61
2	0.1mg/mL EDTA-2Na	40°CW4	22

Formulation example 8 adjuvant concentration screening

Preparations of 100mg/mL protein, different concentrations of EDTA-2Na, 80mg/mL sucrose, and 0.4mg/mL PS80, 14mM His-HCl, pH 5.7 as shown in Table 25 were prepared.

The forced degradation research (placing for 4 weeks at 40 ℃) is carried out on the sample, SEC, NR-CE and IEC are taken as evaluation indexes, and the influence of different auxiliary material concentrations on the stability of the preparation is examined. The results are shown in Table 25, and after the preparation is placed at 40 ℃ for 4 weeks, the stability of the preparation of EDTA-2Na with different concentrations is not obviously different, and the stability is good.

TABLE 25 screening results for adjuvant concentration

Group of	Auxiliary materials	Conditions of placement	ΔSEC%	ΔNR-CE%	ΔIEC%
						1	0.01mg/mL EDTA-2Na	40°CW4	2.5	2.2	24.7
2	0.05mg/mL EDTA-2Na	40°CW4	2.2	2.0	24.3
						3	1.0mg/mL EDTA-2Na	40°CW4	2.5	2.3	19.8

Formulation example 9 sugar concentration screening

Preparations of 100mg/mL protein, 0.05mg/mL EDTA-2Na, 0.4mg/mL PS80 and different sucrose concentrations, 14mM His-HCl, pH 5.7 as shown in Table 226 were prepared.

The samples were subjected to forced degradation studies (4 weeks at 40 ℃) and the effect of different sucrose concentrations on protein stability was investigated using SEC, NR-CE and IEC as evaluation indices.

The results are shown in Table 26.SEC, NR-CE and IEC data showed no significant differences in the stability of the formulations at different sucrose concentrations after 4 weeks of 40 ℃.

TABLE 26 screening results for different sucrose concentrations

Group of	Sucrose	Conditions of placement	ΔSEC%	ΔNR-CE%	ΔIEC%
						1	30Mg/mL sucrose	40°CW4	3.0	2.2	23.8
2	80Mg/mL sucrose	40°CW4	2.2	2.0	24.3
						3	Sucrose 100mg/mL	40°CW4	2.4	2.1	23.0

When sucrose concentration was 80mg/mL, the osmotic pressure of the formulation was actually measured to be 300mOsm, which is nearly isotonic. Therefore, the sucrose concentration is preferably 80mg/mL.

Formulation example 10 protein concentration screening

Preparation of 80mg/mL sucrose, 0.05mg/mL EDTA-2Na, 0.4mg/mL PS80 and different protein concentrations shown in Table 27, 14mM His-HCl, pH 5.7.

The samples were subjected to forced degradation studies (4 weeks at 40 ℃) and the effect of different protein concentrations on protein stability was investigated using SEC, NR-CE and IEC as evaluation indices.

The results are shown in Table 27. After 4 weeks of 40 ℃ placement, the greater the SEC decrease with increasing protein concentration, but within acceptable ranges, there was no difference between the NR-CE and IEC data sets.

TABLE 27 screening results for different protein concentrations

Group of	Proteins	Conditions of placement	ΔSEC%	ΔNR-CE%	ΔIEC%
						1	2Mg/mL protein	40°CW4	0.1	1.8	23.7
2	100Mg/mL protein	40°CW4	2.2	2.0	24.3
						3	200Mg/mL protein	40°CW4	4.0	2.3	22.5

Formulation example 11 prescription validation

A preparation of 100mg/mL protein, 80mg/mL sucrose, 0.4mg/mL PS80, 0.05mg/mLEDTA-2Na, 14mM His-HCl, pH5.7 was prepared. And (3) carrying out long-term stability study on the sample, and taking SEC, NR-CE and IEC as evaluation indexes to investigate the long-term stability.

The results are shown in Table 28. Appearance, SEC, NR-CE and IEC data showed that the preparation purity items were not significantly changed when left for 5 months at 2-8℃and the protein had good stability.

TABLE 28 Long term stability results

Note that 2-8 ℃ CM5:2-8 ℃ is left for 5 months.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. A pharmaceutical composition comprising a bispecific antibody that specifically binds to GUCY2C and CD3 and a buffer, wherein:

Formula I [ GUCY2C-VL ] - [ linker 1] - [ CD3-VH ] - [ linker 2] - [ Fc1],

Formula II [ CD3-VL ] - [ linker 3] - [ GUCY2C-VH ] - [ linker 2] - [ Fc2],

The buffer is histidine salt buffer, acetate buffer, citrate buffer, succinate buffer or Tris salt buffer;

Preferably, the buffer is histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer, acetic acid-sodium acetate buffer, succinic acid-sodium succinate buffer, citric acid-sodium citrate buffer or Tris-HCl salt buffer;

more preferably, the buffer is histidine-histidine hydrochloride buffer.

2. The pharmaceutical composition of claim 1, wherein in the bispecific antibody that specifically binds to GUCY2C and CD 3:

The CD3-VH comprises the amino acid sequence of SEQ ID NO. 41, and the CD3-VL comprises the amino acid sequence of SEQ ID NO. 42;

preferably, the Fc1 has a convex structure according to a pestle-and-socket technique, and the Fc2 has a hole structure according to a pestle-and-socket technique;

more preferably, said Fc1 comprises the amino acid sequence of SEQ ID NO. 43 and said Fc2 comprises the amino acid sequence of SEQ ID NO. 44;

The linker 1, the linker 2 and the linker 3 are the same or different and are respectively selected from SEQ ID NO. 45, SEQ ID NO. 46 and SEQ ID NO. 47;

most preferably, the first strand comprises the amino acid sequence of SEQ ID NO. 48 and the second strand comprises the amino acid sequence of SEQ ID NO. 49.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pH of the pharmaceutical composition is from 5.0 to 7.5;

Preferably, the pH of the pharmaceutical composition is from 5.0 to 6.5;

more preferably, the pH of the pharmaceutical composition is from 5.1 to 6.5;

further preferably, the pH of the pharmaceutical composition is from 5.1 to 6.2;

most preferably, the pH of the pharmaceutical composition is from 5.2 to 6.0.

4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the buffer is at a concentration of 5mM to 100mM, preferably at a concentration of 10mM to 50mM, more preferably at a concentration of 10mM to 30mM, most preferably at a concentration of 10mM to 20mM.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 1mg/mL to 250mg/mL;

Preferably, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is from 1mg/mL to 200mg/mL;

More preferably, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is from 70mg/mL to 150mg/mL;

Most preferably, the concentration of the bispecific antibody that specifically binds to GUCY2C and CD3 is 80mg/mL to 120mg/mL.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the pharmaceutical composition comprises a surfactant;

preferably, the surfactant is polysorbate or poloxamer;

more preferably, the surfactant is polysorbate 80 or poloxamer 188;

most preferably, the surfactant is polysorbate 80.

7. The pharmaceutical composition of claim 6, wherein the concentration of the surfactant is 0.01mg/mL to 2.0mg/mL;

Preferably, the concentration of the surfactant is from 0.1mg/mL to 1.0mg/mL;

More preferably, the concentration of the surfactant is from 0.2mg/mL to 0.6mg/mL;

most preferably, the concentration of the surfactant is from 0.3mg/mL to 0.5mg/mL.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the pharmaceutical composition comprises a sugar;

Preferably, the sugar is sucrose, trehalose, mannitol or sorbitol;

More preferably, the sugar is sucrose.

9. The pharmaceutical composition of claim 8, wherein the concentration of the sugar is 10mg/mL to 120mg/mL;

preferably, the concentration of the sugar is 30mg/mL to 100mg/mL;

more preferably, the sugar concentration is 65mg/mL to 95mg/mL.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition further comprises an adjuvant;

Preferably, the auxiliary material is selected from one or more of ethylenediamine tetraacetic acid or salt thereof, ethylenediamine tetraacetic acid hydrate or salt thereof, DTPA, arginine hydrochloride, glycine, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride and calcium chloride;

More preferably, the adjuvant is the sodium salt of ethylenediamine tetraacetic acid hydrate;

most preferably, the adjuvant is disodium edetate dihydrate.

11. The pharmaceutical composition of claim 10, wherein the concentration of the adjuvant is 0.01mg/mL to 3mg/mL;

preferably, the concentration of the auxiliary material is 0.01mg/mL to 1mg/mL;

More preferably, the concentration of the auxiliary material is 0.01mg/mL to 0.5mg/mL;

most preferably, the concentration of the auxiliary material is 0.01mg/mL to 0.1mg/mL.

12. The pharmaceutical composition according to any one of claims 1 to 11, comprising the following components:

(B) 0.01mg/mL to 2.0mg/mL of surfactant,

(C) Sugar of 10mg/mL to 120mg/mL,

(E) 5mM to 100mM buffer, the pH of the pharmaceutical composition being from 5.0 to 7.5;

preferably, the pharmaceutical composition comprises the following components:

(B) 0.1mg/mL to 1.0mg/mL of polysorbate 80 or poloxamer 188,

(C) Sucrose of 30mg/mL to 100mg/mL,

(E) 10mM to 50mM histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer, acetic acid-acetic acid sodium salt buffer, succinic acid-succinic acid sodium salt buffer, citric acid-sodium citrate buffer or Tris-HCl salt buffer, the pH of the pharmaceutical composition being 5.0 to 7.5;

further preferably, the pharmaceutical composition comprises the following components:

(B) 0.1mg/mL to 1.0mg/mL of polysorbate 80 or poloxamer 188,

(C) Sucrose of 30mg/mL to 100mg/mL,

(D) 0.01mg/mL to 1mg/mL disodium edetate dihydrate, and

(E) 10mM to 50mM histidine-hcl histidine buffer, the pH of the pharmaceutical composition is 5.0 to 6.5;

More preferably, the pharmaceutical composition comprises the following components:

(B) Polysorbate 80 or poloxamer 188 at 0.2mg/mL to 0.6mg/mL,

(C) 65mg/mL to 95mg/mL sucrose,

(D) 0.01mg/mL to 0.5mg/mL disodium edetate dihydrate, and

(E) 10mM to 30mM histidine-hcl histidine buffer, the pH of the pharmaceutical composition is 5.0 to 6.5;

still further preferably, the pharmaceutical composition comprises the following components:

(B) 0.3mg/mL to 0.5mg/mL of polysorbate 80 or poloxamer 188,

(C) 65mg/mL to 95mg/mL sucrose,

(D) 0.01mg/mL to 0.1mg/mL of disodium edetate dihydrate, and

(E) 10mM to 20mM histidine-hcl histidine buffer, the pH of the pharmaceutical composition is 5.2 to 6.0;

most preferably, the first and second regions are,

The pharmaceutical composition comprises the following components:

(a) About 100mg/mL of the bispecific antibody that specifically binds GUCY2C and CD3,

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL of sucrose per mL of sucrose,

(D) About 0.05mg/mL disodium edetate dihydrate, and

13. The pharmaceutical composition according to any one of claims 1 to 11, comprising the following components:

(B) 0.01mg/mL to 2.0mg/mL of surfactant,

(C) 10mg/mL to 120mg/mL sugar, and

(D) 5mM to 100mM buffer, the pH of the pharmaceutical composition being from 5.0 to 7.5;

preferably, the pharmaceutical composition comprises the following components:

(B) 0.1mg/mL to 1.0mg/mL of polysorbate 80 or poloxamer 188,

(C) Sucrose 30mg/mL to 100mg/mL, and

(D) 10mM to 50mM histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer, acetic acid-acetic acid sodium salt buffer, succinic acid-succinic acid sodium salt buffer, citric acid-sodium citrate buffer or Tris-HCl salt buffer, the pH of the pharmaceutical composition being 5.0 to 7.5;

(B) 0.1mg/mL to 1.0mg/mL of polysorbate 80 or poloxamer 188,

(C) Sucrose 30mg/mL to 100mg/mL, and

(D) 10mM to 50mM histidine-hcl histidine buffer, the pH of the pharmaceutical composition is 5.0 to 6.5;

(B) Polysorbate 80 or poloxamer 188 at 0.2mg/mL to 0.6mg/mL,

(C) 65mg/mL to 95mg/mL sucrose, and

(D) 10mM to 30mM histidine-hcl histidine buffer, the pH of the pharmaceutical composition is 5.0 to 6.5;

(B) 0.3mg/mL to 0.5mg/mL of polysorbate 80 or poloxamer 188,

(C) 65mg/mL to 95mg/mL sucrose, and

(D) 10mM to 20mM histidine-hcl histidine buffer, the pH of the pharmaceutical composition is 5.2 to 6.0;

most preferably, the first and second regions are,

The pharmaceutical composition comprises the following components:

(B) About 0.4mg/mL polysorbate 80,

(C) About 80mg/mL sucrose, and

14. The pharmaceutical composition according to any one of claims 1 to 13, which is a subcutaneous injection formulation, an intravenous injection formulation, an intraperitoneal injection formulation or an intramuscular injection formulation, preferably a subcutaneous injection formulation or an intravenous injection formulation.

15. A method of preparing a lyophilized formulation comprising the step of lyophilizing the pharmaceutical composition of any one of claims 1 to 14.

16. A lyophilized formulation comprising the pharmaceutical composition of any one of claims 1 to 14.

17. A reconstituted solution, characterized in that it is obtained by reconstitution of the lyophilized formulation of claim 16.

18. An article of manufacture comprising a container containing the pharmaceutical composition of any one of claims 1 to 14, or the lyophilized formulation of claim 16, or the reconstituted solution of claim 17.

19. The pharmaceutical composition according to any one of claims 1 to 14, or the lyophilized formulation according to claim 16, or the use of the reconstituted solution according to claim 17, for the preparation of a medicament for the treatment of a tumor or cancer, preferably wherein the tumor or cancer is selected from the group consisting of gastric cancer, gastrointestinal cancer, intestinal cancer, gastric adenocarcinoma, small intestine cancer, colorectal cancer, esophageal cancer, anal cancer, liver cancer, gall bladder cancer, bile duct cancer, pancreatic cancer, prostate cancer, kidney cancer, urothelial cancer, breast cancer, bladder cancer, thymus cancer, melanoma, glioma, sarcoma, osteosarcoma, ovarian cancer, thyroid cancer, lung squamous cell carcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, endometrial cancer, lung cancer, skin cancer, head and neck cancer, brain cancer, glioblastoma multiforme, gastroesophageal cancer, gastroesophageal adenocarcinoma, liver metastatic adenocarcinoma, multiple myeloma, lymphoma, leukemia or B-cell lymphoma, more preferably wherein

The cells of the tumor or the vascular endothelial cells near the tumor express GUCY2C.