Anti-GUCY2c antibody and use of multi-specific antibody

Abstract

Provided are a multi-specific antibody specifically binding to GUCY2C or an antigen-binding fragment thereof, an isolated polynucleotide encoding the antibody or the antigen-binding fragment thereof, a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, and a use thereof.

Description

Use of an anti-GUCY2C antibody and its multispecific antibody Technical Field

[0001] The present disclosure generally relates to multispecific antibodies and antigen-binding fragments directed against GUCY2C. Background Art

[0002] Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a leading cause of cancer death worldwide. Guanylate cyclase 2C (GUCY2C) belongs to the guanylate cyclase receptor family and is the brush border membrane receptor for the hormones guanylin and uroguanylin, as well as the heat-stable enterotoxin (Sta) derived from the enteric bacterium Escherichia coli. GUCY2C is reported to be expressed in a variety of gastrointestinal cancers, including over 90% of CRC at all stages, and therefore may serve as an effective target for immunotherapy of gastrointestinal cancers. GUCY2C×CD3 bispecific T cell-engaging antibodies have been developed to promote the formation of immune synapses on tumor cells overexpressing GUCY2C, thereby redirecting T cells to mediate killing specifically against tumor cells, reducing the risk of off-target effects.

[0003] However, CD3 antibodies may mediate overactivation of T cells, leading to adverse reactions such as cytokine storms. Furthermore, high-level expression of GUCY2C×CD3 bispecific antibodies is often a technical challenge. Therefore, there is a need to develop new GUCY2C×CD3 multispecific antibodies with improved therapeutic efficacy and expression levels. Summary of the Invention

[0004] Throughout this disclosure, as used herein, the articles "a", "an" and "the" refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an antibody" means one antibody or more than one antibody.

[0005] The present disclosure provides a multispecific antibody or an antigen-binding fragment thereof that specifically binds to GUCY2C and CD3, an isolated polynucleotide encoding the antibody or the antigen-binding fragment thereof, a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, and uses thereof.

[0006] In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof, the antibody comprising

[0007] (a) a GUCY2C binding domain, wherein the GUCY2C binding domain comprises GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 contained in the VL of the GUCY2C antibody shown in Table 1 or Table 3, and GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 contained in the VH of the GUCY2C antibody shown in Table 1 or Table 3;

[0008] and

[0009] (b) one or more T cell antigen binding domains.

[0010] In certain embodiments, the GUCY2C-LCDR1 comprises the GUCY2C-LCDR1 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, the GUCY2C-LCDR2 comprises the GUCY2C-LCDR2 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, and the GUCY2C-LCDR3 comprises the GUCY2C-LCDR1 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4. R3 sequence, the GUCY2C-HCDR1 includes the GUCY2C-HCDR1 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, the GUCY2C-HCDR2 includes the GUCY2C-HCDR2 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, and the GUCY2C-HCDR3 includes the GUCY2C-HCDR2 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4.

[0011] In certain embodiments, the GUCY2C-LCDR1 comprises SASQGISNYLN (SEQ ID NO: 116) and / or RASQDINNYLN (SEQ ID NO: 292).

[0012] In certain embodiments, the GUCY2C-LCDR2 comprises YTSTLHS (SEQ ID NO: 117) and / or YTSRLHS (SEQ ID NO: 293).

[0013] In certain embodiments, the GUCY2C-LCDR3 comprises LQYRKFPYT (SEQ ID NO: 118) and / or QQTKMMYT (SEQ ID NO: 294).

[0014] In certain embodiments, the GUCY2C-HCDR1 comprises NFGMH (SEQ ID NO: 128) and / or SYAMS (SEQ ID NO: 304).

[0015] In certain embodiments, the GUCY2C-HCDR2 comprises YISSGSGTIYYADTVKG (SEQ ID NO: 129) and / or TISSGGSYIYYSDSVKG (SEQ ID NO: 305).

[0016] In certain embodiments, the GUCY2C-HCDR3 comprises QRVLTGTLFDY (SEQ ID NO: 130) and / or HDSGDYAMDY (SEQ ID NO: 306).

[0017] In certain embodiments, the GUCY2C binding domain comprises a combination of 6 CDRs shown in Table 2 or Table 4.

[0018] In certain embodiments, the VH and VL of the GUCY2C binding domain comprise the VH and VL pairs of the M0047, M0055, GCAb0002, and GCAb0005 antibodies in Table 1 or Table 3, respectively.

[0019] In certain embodiments, the GUCY2C binding domain is a fragment antigen binding domain (Fab).

[0020] In certain embodiments, the GUCY2C binding domain is a variable region domain (Fv).

[0021] In certain embodiments, the GUCY2C binding domain is a single-chain variable region domain (scFv).

[0022] In certain embodiments, the amino acid sequence of the GUCY2C binding domain comprises the scFv sequence shown in Table 1.

[0023] In certain embodiments, the one or more T cell antigens are selected from CD3 or CD28 or both.

[0024] In certain embodiments, the CD3 binding domain comprises the CDR sequence of the CD3 antibody shown in Table 5.

[0025] In certain embodiments, the CD3 binding domain comprises the VH and VL sequences of the CD3 antibodies shown in Table 5.

[0026] In certain embodiments, the CD3 binding domain is a scFv.

[0027] In certain embodiments, the CD3 binding domain comprises the scFv sequence of the CD3 antibody shown in Table 5.

[0028] In certain embodiments, the CD3 binding domain is in Fab or IgG form. In certain embodiments, the CD3 binding domain comprises the VH and VL sequences of the CD3 antibodies shown in Table 5.

[0029] In certain embodiments, the antibody further comprises a CD28 binding domain.

[0030] In certain embodiments, the CD28 binding domain comprises the CDR sequence of the CD28 antibody shown in Table 6.

[0031] In certain embodiments, the CD28 binding domain comprises the VH and VL sequences of the CD28 antibodies shown in Table 6.

[0032] In certain embodiments, the CD28 binding domain is a scFv.

[0033] In certain embodiments, the CD28 binding domain comprises the scFv sequence of the CD28 antibody shown in Table 6.

[0034] In certain embodiments, the CD28 binding domain is in the form of Fab or IgG. In certain embodiments, the CD28 binding domain comprises the VH and VL sequences of the CD28 antibodies shown in Table 6.

[0035] In one aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, comprising:

[0036] (a) a GUCY2C binding domain, wherein the GUCY2C binding domain comprises GUCY2C-LCDR1, GUCY2C-LCDR2, GUCY2C-LCDR3, GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 of a GUCY2C antibody shown in Table 2 or Table 4; and

[0037] (b) a CD3 binding domain, wherein the CD3 binding domain comprises the CDR sequence of the CD3 antibody shown in Table 5.

[0038] In one aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, comprising:

[0039] (a) a GUCY2C binding domain, wherein the GUCY2C binding domain comprises GUCY2C-LCDR1, GUCY2C-LCDR2, GUCY2C-LCDR3, GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 of a GUCY2C antibody shown in Table 2 or Table 4; and

[0040] (b) a CD28 binding domain, wherein the CD28 binding domain comprises the CDR sequence of the CD28 antibody shown in Table 6.

[0041] In one aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, comprising:

[0042] (a) a GUCY2C binding domain, wherein the GUCY2C binding domain comprises GUCY2C-LCDR1, GUCY2C-LCDR2, GUCY2C-LCDR3, GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 of a GUCY2C antibody shown in Table 2 or Table 4;

[0043] (b) a CD3 binding domain, wherein the CD3 binding domain comprises the CDR sequence of a CD3 antibody shown in Table 5; and

[0044] (c) a CD28 binding domain, wherein the CD28 binding domain comprises the CDR sequence of the CD28 antibody shown in Table 6.

[0045] In some embodiments, it has the structure shown in Figures 9A to 9F or Figures 9G to 9M.

[0046] In certain embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof comprising the sequences shown in Table 7.

[0047] In certain embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof, which further comprise an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG.

[0048] In certain embodiments, the antibody is a murine, rodent, rabbit, chimeric, humanized or human antibody.

[0049] In certain embodiments, the disclosure provides antibodies, or antigen-binding fragments thereof, linked to one or more conjugate moieties.

[0050] In certain embodiments, the conjugate moiety comprises an immunomodulatory agent, an anti-tumor drug, a radioisotope, a clearance modulator, a toxin, a detectable label, RNA, DNA, a cytokine, or a purification moiety.

[0051] In one aspect, the present disclosure provides a GUCY2C antibody or an antigen-binding fragment thereof, wherein the GUCY2C antibody comprises GUCY2C-LCDR1, GUCY2C-LCDR2, and GUCY2C-LCDR3 contained in the VL of the GUCY2C antibody shown in Table 1 or Table 3, and GUCY2C-HCDR1, GUCY2C-HCDR2, and GUCY2C-HCDR3 contained in the VH of the GUCY2C antibody shown in Table 1 or Table 3.

[0052] In certain embodiments, the GUCY2C-LCDR1 comprises the GUCY2C-LCDR1 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, the GUCY2C-LCDR2 comprises the GUCY2C-LCDR2 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, and the GUCY2C-LCDR3 comprises the GUCY2C-LCDR1 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4. R3 sequence, the GUCY2C-HCDR1 includes the GUCY2C-HCDR1 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, the GUCY2C-HCDR2 includes the GUCY2C-HCDR2 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, and the GUCY2C-HCDR3 includes the GUCY2C-HCDR2 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4.

[0053] In certain embodiments, the GUCY2C antibody comprises the sequence of the VH and VL pairings of the GUCY2C antibody in Table 1 or Table 3.

[0054] In certain embodiments, the GUCY2C antibody comprises the sequences of the VH and VL pairs of the GUCY2C antibodies in Table 29.

[0055] In one aspect, the present disclosure provides a GUCY2C antibody or an antigen-binding fragment thereof, wherein the GUCY2C antibody binds to one or more epitopes selected from the group consisting of: S15N, S62F, I66L, L80V, E55A, D56A, E101A, L105E, R107A.

[0056] In certain embodiments, the GUCY2C antibody binds to one or more epitopes selected from the group consisting of S15N, S62F, I66L, and L80V.

[0057] In certain embodiments, the GUCY2C antibody binds to epitopes E55A and / or D56A.

[0058] In certain embodiments, the GUCY2C antibody binds to one or more epitopes selected from the group consisting of: E101A, L105E, and R107A.

[0059] In one aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof provided by the present disclosure, and a pharmaceutically acceptable carrier.

[0060] In one aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof provided by the present disclosure.

[0061] In one aspect, the present disclosure provides a vector comprising the isolated polynucleotide provided by the present disclosure.

[0062] In one aspect, the present disclosure provides a host cell comprising a vector provided by the present disclosure.

[0063] In one aspect, the present disclosure provides a method for producing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell provided by the present disclosure under conditions that express the antibody or the antigen-binding fragment thereof, and recovering the antibody or the antigen-binding fragment thereof.

[0064] In one aspect, the present disclosure provides a method for treating or ameliorating a disease that benefits from T lymphocyte killing and clearance or a GUCY2C-related disease in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided by the present disclosure, or a pharmaceutical composition provided by the present disclosure.

[0065] In certain embodiments, the methods provided herein comprise co-administration of the antibodies or antigen-binding fragments thereof provided herein.

[0066] In certain embodiments, the disease is cancer.

[0067] In certain embodiments, the cancer is selected from the group consisting of adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioalveolar lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.

[0068] In certain embodiments, the subject is a human.

[0069] In certain embodiments, the antibody or antigen-binding fragment thereof or the pharmaceutical composition is administered intravenously, intraarterially, intratumorally, intramuscularly or subcutaneously.

[0070] In certain embodiments, the methods provided herein further comprise administering to the subject one or more additional therapeutic agents.

[0071] In certain embodiments, the additional therapeutic agent is selected from chemotherapeutic agents, anticancer drugs, radiotherapeutic agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapy agents, gene therapy agents, hormone therapy agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, cytokines, anti-infective agents, and anti-inflammatory agents.

[0072] In certain embodiments, the additional therapeutic agent is selected from a monospecific antibody, a bispecific antibody, a multispecific antibody, a fusion protein, an ADC, an LDC, an RDC, a cell therapy, a small molecule drug, an antisense nucleic acid, an siRNA, an mRNA, and a PROTAC.

[0073] In certain embodiments, the additional therapeutic agent acts directly on GUCY2C or its variants, such as a monospecific antibody targeting GUCY2C or its variants, a bispecific antibody targeting GUCY2C or its variants, a multispecific antibody targeting GUCY2C or its variants, a fusion protein targeting GUCY2C or its variants, an ADC targeting GUCY2C or its variants, an LDC targeting GUCY2C or its variants, an RDC targeting GUCY2C or its variants, a cell therapy targeting GUCY2C or its variants, a small molecule drug targeting GUCY2C or its variants, an antisense nucleic acid targeting GUCY2C or its variants, an siRNA targeting GUCY2C or its variants, an mRNA expressing GUCY2C or its variants, or a PROTAC targeting GUCY2C or its variants.

[0074] In certain embodiments, the one or more additional therapeutic agents are administered concurrently or sequentially with the antibody or antigen-binding fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] FIG1 shows the binding of T84, GUCY2C-HEK293, and HEK293 to anti-huGUCY2C chimeric antibodies determined by flow cytometry.

[0076] FIG2 shows the binding activity of the bi-epitope antibody on T84 cells determined by flow cytometry.

[0077] FIG3 shows the huGUCY2C immunogenicity prediction.

[0078] FIG4 shows the predicted binding epitopes of M0055 and GCAb0002.

[0079] FIG5 shows flow cytometric analysis of antibody binding of huGUCY2C ECD mutants (S15N, S62F, I66L, and L80V).

[0080] FIG6A shows the flow cytometry analysis of the binding of the M0055 predicted epitope mutant E55A to the antibody.

[0081] FIG6B shows flow cytometry analysis of the binding of the M0055 predicted epitope mutant D56A to the antibody.

[0082] FIG6C shows the flow cytometry analysis of the binding of the M0055 predicted epitope mutant N59A to the antibody.

[0083] FIG6D shows the flow cytometry analysis of the binding of the M0055 predicted epitope mutant E63A to the antibody.

[0084] FIG6E shows flow cytometry analysis of the binding of the M0055 predicted epitope mutant Q70A to antibodies.

[0085] FIG6F shows flow cytometry analysis of the binding of the M0055 predicted epitope mutant K403D to the antibody.

[0086] FIG6G shows flow cytometry analysis of the binding of the M0055 predicted epitope mutant Y405T to the antibody.

[0087] FIG6H shows flow cytometry analysis of the binding of the M0055 predicted epitope mutant M409T to the antibody.

[0088] FIG7A shows flow cytometry analysis of the binding of the predicted epitope mutant E101A of GCAb0002 to antibodies.

[0089] FIG7B shows flow cytometry analysis of the binding of GCAb0002 predicted epitope mutant D104A to antibodies.

[0090] FIG7C shows the flow cytometry analysis of the binding of the GCAb0002 predicted epitope mutant L105E to the antibody.

[0091] FIG7D shows flow cytometry analysis of the binding of the predicted epitope mutant R107A of GCAb0002 to antibodies.

[0092] FIG7E shows flow cytometry analysis of the binding of the predicted epitope mutant Q130A of GCAb0002 to antibodies.

[0093] FIG7F shows flow cytometry analysis of the binding of GCAb0002 predicted epitope mutant D134A to antibodies.

[0094] FIG7G shows flow cytometry analysis of the binding of GCAb0002 predicted epitope mutant K154A to antibodies.

[0095] FIG8 shows that M0055 and GCAb0002 bind to the precise epitope of huGUCY2C ECD.

[0096] FIG9A shows the GUCY2C×CD3 multispecific antibody configuration GCbi0007.

[0097] FIG9B shows the GUCY2C×CD3 multispecific antibody configuration GCbi0019.

[0098] FIG9C shows the GUCY2C×CD3 multispecific antibody configuration GCbi0021.

[0099] FIG9D shows the GUCY2C×CD3 multispecific antibody configuration GCbi0023.

[0100] FIG9E shows the GUCY2C×CD3 multispecific antibody configuration GCbi0025.

[0101] FIG9F shows the GUCY2C×CD3 multispecific antibody configuration GCbi0026.

[0102] FIG9G shows the GUCY2C×CD3×CD28 multispecific antibody configuration GCTr0001.

[0103] FIG9H shows the GUCY2C×CD3×CD28 multispecific antibody configuration GCTr0002.

[0104] FIG9I shows the GUCY2C×CD3×CD28 multispecific antibody configuration GCTr0006.

[0105] FIG9J shows the GUCY2C×CD3×CD28 multispecific antibody configuration GCTr0009.

[0106] FIG9K shows the GUCY2C×CD3×CD28 multispecific antibody configuration GCTr0010.

[0107] FIG9L shows the GUCY2C×CD3×CD28 multispecific antibody configuration GCTr0011.

[0108] FIG9M shows the GUCY2C×CD3×CD28 multispecific antibody configuration GCTr0012.

[0109] FIG. 9N shows the GUCY2C-negative anti-HEL×anti-huCD3ed bispecific antibody IsoB0001.

[0110] FIG10A shows the affinity of the GUCY2C×CD3 chimeric bispecific antibody GCbi0007 binding to huGUCY2C.

[0111] FIG10B shows the affinity of the GUCY2C×CD3 chimeric bispecific antibody GCbi0019 binding to huGUCY2C.

[0112] FIG10C shows the affinity of the GUCY2C×CD3 chimeric bispecific antibody GCbi0021 binding to huGUCY2C.

[0113] FIG10D shows the affinity of the GUCY2C×CD3 chimeric bispecific antibody GCbi0023 binding to huGUCY2C.

[0114] FIG10E shows the affinity of GUCY2C×CD3 chimeric bispecific antibody GCbi0026 binding to huGUCY2C.

[0115] FIG10F shows the affinity of positive control antibody PF-07062119 binding to huGUCY2C.

[0116] FIG10G shows the affinity of the GUCY2C×CD3 chimeric bispecific antibody GCbi0007 binding to huGUCY2C.

[0117] FIG10H shows the affinity of GUCY2C×CD3 chimeric bispecific antibody GCbi0019 binding to huGUCY2C.

[0118] FIG10I shows the affinity of the GUCY2C×CD3 chimeric bispecific antibody GCbi0021 binding to huGUCY2C.

[0119] FIG11A shows the affinity of GUCY2C×CD3 chimeric bispecific antibody GCbi0007 binding to huCD3ed.

[0120] FIG11B shows the affinity of GUCY2C×CD3 chimeric bispecific antibody GCbi0019 binding to huCD3ed.

[0121] FIG11C shows the affinity of GUCY2C×CD3 chimeric bispecific antibody GCbi0021 binding to huCD3ed.

[0122] FIG11D shows the affinity of GUCY2C×CD3 chimeric bispecific antibody GCbi0023 binding to huCD3ed.

[0123] FIG. 11E shows the affinity of GUCY2C×CD3 chimeric bispecific antibody GCbi0025 binding to huCD3ed.

[0124] FIG11F shows the affinity of GUCY2C×CD3 chimeric bispecific antibody GCbi0026 binding to huCD3ed.

[0125] FIG. 11G shows the affinity of the negative bispecific antibody IsoB0001 binding to huCD3ed.

[0126] FIG. 11H shows the affinity of positive control antibody PF-07062119 binding to huCD3ed.

[0127] FIG12 shows the binding activity of the GUCY2C×CD3 chimeric bispecific antibody to T84 cells as determined by flow cytometry.

[0128] FIG13 shows the binding activity of the GUCY2C×CD3 chimeric bispecific antibody to panT cells as determined by flow cytometry.

[0129] FIG. 14A shows that the GUCY2C×CD3 chimeric bispecific antibody recruits pan T cells to induce killing of T84 tumor cells.

[0130] FIG14B shows that the GUCY2C×CD3 chimeric bispecific antibody recruits pan T cells to induce killing of HT55 tumor cells.

[0131] FIG14C shows that the GUCY2C×CD3 chimeric bispecific antibody recruits pan T cells to induce killing of HCT15 tumor cells.

[0132] FIG15A and FIG15B show the T cell activation effect of the GUCY2C×CD3 chimeric bispecific antibody in the T84 TDCC assay.

[0133] FIG15C and FIG15D show the T cell activation effect of the GUCY2C×CD3 chimeric bispecific antibody in the HT55 TDCC assay.

[0134] FIG15E and FIG15F show the T cell activation effect of the GUCY2C×CD3 chimeric bispecific antibody in the HCT15 TDCC experiment.

[0135] FIG16A shows the level of cytokine IFN-γ release by the GUCY2C×CD3 chimeric bispecific antibody in TDCC of T84 cells.

[0136] FIG16B shows the cytokine IL-2 release level of the GUCY2C×CD3 chimeric bispecific antibody in TDCC of T84 cells.

[0137] FIG16C shows the cytokine IL-6 release level of the GUCY2C×CD3 chimeric bispecific antibody in TDCC of T84 cells.

[0138] FIG16D shows the level of cytokine TNF-α release by the GUCY2C×CD3 chimeric bispecific antibody in TDCC of T84 cells.

[0139] FIG17A shows the cytokine IFN-γ release level of the GUCY2C×CD3 chimeric bispecific antibody in TDCC of HT55 cells.

[0140] FIG17B shows the cytokine IL-2 release level of the GUCY2C×CD3 chimeric bispecific antibody in TDCC of HT55 cells.

[0141] FIG17C shows the cytokine IL-6 release level of the GUCY2C×CD3 chimeric bispecific antibody in TDCC of HT55 cells.

[0142] FIG17D shows the level of cytokine TNF-α release by the GUCY2C×CD3 chimeric bispecific antibody in TDCC of HT55 cells.

[0143] FIG18A shows the cytokine IFN-γ release level of the GUCY2C×CD3 chimeric bispecific antibody in the GUCY2C-negative cell line HCT15.

[0144] FIG18B shows the cytokine IL-2 release level of the GUCY2C×CD3 chimeric bispecific antibody in the GUCY2C-negative cell line HCT15.

[0145] FIG18C shows the cytokine IL-6 release level of the GUCY2C×CD3 chimeric bispecific antibody in the GUCY2C-negative cell line HCT15.

[0146] FIG18D shows the level of cytokine TNF-α release by the GUCY2C×CD3 chimeric bispecific antibody in the GUCY2C-negative cell line HCT15.

[0147] FIG19A shows the level of non-specific cytokine IFN-γ release by the GUCY2C×CD3 chimeric bispecific antibody at 24 h.

[0148] FIG19B shows the non-specific cytokine IL-2 release level of the GUCY2C×CD3 chimeric bispecific antibody at 24 h.

[0149] FIG19C shows the level of non-specific cytokine IL-6 release by the GUCY2C×CD3 chimeric bispecific antibody at 24 h.

[0150] FIG19D shows the level of non-specific cytokine TNF-α release by the GUCY2C×CD3 chimeric bispecific antibody at 24 h.

[0151] FIG20A shows the level of nonspecific cytokine IFN-γ release by the GUCY2C×CD3 chimeric bispecific antibody at 72 h.

[0152] FIG20B shows the level of nonspecific cytokine IL-2 release by the GUCY2C×CD3 chimeric bispecific antibody at 72 h.

[0153] FIG20C shows the level of nonspecific cytokine IL-6 release by the GUCY2C×CD3 chimeric bispecific antibody at 72 h.

[0154] FIG20D shows the level of non-specific cytokine TNF-α release by the GUCY2C×CD3 chimeric bispecific antibody at 72 h.

[0155] FIG21A shows that the GUCY2C×CD3 chimeric bispecific antibody recruits pan T cells to induce killing of T84 cells.

[0156] FIG21B shows that the GUCY2C×CD3 chimeric bispecific antibody recruits pan T cells to induce killing of HT55 cells.

[0157] FIG21C shows that the GUCY2C×CD3 chimeric bispecific antibody recruits pan T cells to induce killing of LS1034 cells.

[0158] FIG21D shows that the GUCY2C×CD3 chimeric bispecific antibody recruits pan T cells to induce killing of HEK293 cells.

[0159] FIG. 22 shows that the GUCY2C×CD3 chimeric bispecific antibody recruits pan T cells to induce killing of SW403 tumor cells.

[0160] FIG. 23 shows that the GUCY2C×CD3 chimeric bispecific antibody recruits pan T cells to induce killing of T84 tumor cells.

[0161] FIG. 24A shows that the GUCY2C×CD3×CD28 chimeric trispecific antibody recruits pan T cells to induce killing of T84 tumor cells.

[0162] FIG. 24B shows that the GUCY2C×CD3×CD28 chimeric trispecific antibody recruits pan T cells to induce killing of HT55 tumor cells.

[0163] FIG. 24C shows that the GUCY2C×CD3×CD28 chimeric trispecific antibody recruits pan T cells to induce killing of LS1034 tumor cells.

[0164] FIG. 25A shows tumor volume in the HT55 mouse xenograft tumor model.

[0165] FIG. 25B shows the body weight of mice in the HT55 mouse transplant tumor model.

[0166] FIG26A shows the configuration of the humanized GUCY2C×CD3 antibody GCbi0031.

[0167] FIG26B shows the configuration of the humanized GUCY2C×CD3 antibody GCbi0032.

[0168] FIG27A shows the affinity of humanized GUCY2C×CD3 antibody GCbi0031 binding to huGUCY2C.

[0169] FIG27B shows the affinity of humanized GUCY2C×CD3 antibody GCbi0032 binding to huGUCY2C.

[0170] FIG. 27C shows the affinity of humanized GUCY2C×CD3 antibody GCbi0031 binding to huCD3ed.

[0171] FIG27D shows the affinity of humanized GUCY2C×CD3 antibody GCbi0032 binding to huCD3ed.

[0172] FIG28 shows the binding activity of GUCY2C×CD3 antibody to T84 cells as determined by flow cytometry.

[0173] FIG29 shows the binding activity of GUCY2C×CD3 antibody to pan T cells as determined by flow cytometry.

[0174] FIG. 30A shows that GUCY2C×CD3 antibody recruits pan T cells to induce killing of T84 tumor cells.

[0175] FIG. 30B shows that GUCY2C×CD3 antibody recruits pan T cells to induce killing of HT55 tumor cells.

[0176] FIG31 shows the T cell activation effect of GUCY2C×CD3 antibody in T84 ( FIG31A and FIG31B ) and HT55 ( FIG31C and FIG31D ) TDCC experiments.

[0177] FIG31A and FIG31B show the T cell activation effect of GUCY2C×CD3 antibody in T84 TDCC assay.

[0178] FIG31C and FIG31D show the T cell activation effect of GUCY2C×CD3 antibody in HT55 TDCC assay.

[0179] FIG32A shows the level of cytokine TNF-α release in TDCC of T84 cells induced by GUCY2C×CD3 antibody.

[0180] FIG32B shows the level of cytokine IL-6 release in TDCC of T84 cells by GUCY2C×CD3 antibody.

[0181] FIG32C shows the level of cytokine IL-2 release in TDCC of T84 cells by GUCY2C×CD3 antibody.

[0182] FIG32D shows the level of cytokine IFN-γ release by GUCY2C×CD3 antibody in TDCC of T84 cells.

[0183] FIG33A shows the level of cytokine TNF-α release by GUCY2C×CD3 antibody in TDCC of HT55 cells.

[0184] FIG33B shows the cytokine IL-6 release level of GUCY2C×CD3 antibody in TDCC of HT55 cells.

[0185] FIG33C shows the level of cytokine IL-2 release by GUCY2C×CD3 antibody in TDCC of HT55 cells.

[0186] FIG33D shows the level of cytokine IFN-γ release by GUCY2C×CD3 antibody in TDCC of HT55 cells.

[0187] FIG34A shows the changes in tumor volume of the GUCY2C×CD3 antibody in the HT55 mouse transplant tumor model.

[0188] FIG34B shows the changes in mouse body weight in the HT55 mouse transplanted tumor model induced by GUCY2C×CD3 antibody.

[0189] FIG35 shows the configuration of the humanized GUCY2C×CD3 antibody GCbi0043.

[0190] FIG36A shows the binding affinity of the GUCY2C×CD3 antibody GCbi0031 to huGUCY2C ECD.

[0191] FIG36B shows the binding affinity of the GUCY2C×CD3 antibody GCbi0043 to huGUCY2C ECD.

[0192] FIG37A shows the binding affinity of the GUCY2C×CD3 antibody GCbi0031 to huGUCY2C ECD.

[0193] FIG37B shows the binding affinity of the GUCY2C×CD3 antibody GCbi0043 to huGUCY2C ECD.

[0194] FIG37C shows the binding affinity of the GUCY2C×CD3 antibody GCbi0031 to cyGUCY2C ECD.

[0195] FIG37C shows the binding affinity of the GUCY2C×CD3 antibody GCbi0031 to cyGUCY2C ECD.

[0196] FIG37D shows the binding affinity of the GUCY2C×CD3 antibody GCbi0043 to cyGUCY2C ECD.

[0197] FIG38A shows the binding affinity of GUCY2C×CD3 antibody GCbi0043 to huGUCY2C_D56A.

[0198] FIG38B shows the binding affinity of GUCY2C×CD3 antibody GCbi0043 to huGUCY2C_E101A.

[0199] FIG. 39A shows that GUCY2C×CD3 antibody recruits T cells to induce killing of HT55 tumor cells.

[0200] FIG. 39B shows that GUCY2C×CD3 antibody recruits T cells to induce killing of SW403 tumor cells.

[0201] FIG. 39C shows that GUCY2C×CD3 antibody recruits T cells to induce killing of LS174T tumor cells.

[0202] FIG. 39D shows that GUCY2C×CD3 antibody recruits pan T cells to induce killing of AGS tumor cells.

[0203] Figures 39E and 39F show the T cell activation effect of GUCY2C×CD3 antibody in HT55 TDCC experiments.

[0204] Figures 39G and 39H show the T cell activation effect of GUCY2C×CD3 antibody in SW403 TDCC experiments.

[0205] Figures 39I and 39J show the T cell activation effect of GUCY2C×CD3 antibody in LS174T TDCC assay.

[0206] Figures 39K and 39L show the T cell activation effect of GUCY2C×CD3 antibody in AGS TDCC experiments.

[0207] FIG. 40A shows that GUCY2C×CD3 antibody recruits T cells (Donor Y1584) to induce killing of HT55 tumor cells.

[0208] FIG40B shows the cytokine IL-2 release level of GUCY2C×CD3 antibody in the TDCC experiment with Donor Y1584.

[0209] FIG40C shows the cytokine IL-4 release level of GUCY2C×CD3 antibody in the TDCC experiment accompanied with Donor Y1584.

[0210] FIG40D shows the cytokine IL-6 release level of GUCY2C×CD3 antibody in the TDCC experiment accompanied with Donor Y1584.

[0211] FIG40E shows the cytokine IL-10 release level of GUCY2C×CD3 antibody in the TDCC experiment with Donor Y1584.

[0212] FIG40F shows the level of cytokine TNF-α release in the GUCY2C×CD3 antibody accompanied with Donor Y1584 TDCC experiment.

[0213] FIG40G shows the cytokine IFN-γ release level of GUCY2C×CD3 antibody in the TDCC experiment accompanied with Donor Y1584.

[0214] FIG. 41A shows that GUCY2C×CD3 antibody recruits T cells (NF0035) to induce killing of HT55 tumor cells.

[0215] FIG41B shows the cytokine IL-2 release level of GUCY2C×CD3 antibody in the TDCC experiment accompanied with Donor NF0035.

[0216] FIG41C shows the cytokine IL-4 release level of GUCY2C×CD3 antibody in the TDCC experiment accompanied with Donor NF0035.

[0217] FIG41D shows the cytokine IL-6 release level of GUCY2C×CD3 antibody in the TDCC experiment accompanied with Donor NF0035.

[0218] FIG41E shows the cytokine IL-10 release level in the TDCC experiment with GUCY2C×CD3 antibody accompanied by Donor NF0035.

[0219] FIG41F shows the level of cytokine TNF-α released by GUCY2C×CD3 antibody in the TDCC experiment accompanied with Donor NF0035.

[0220] FIG41G shows the level of cytokine IFN-γ released by GUCY2C×CD3 antibody in the TDCC experiment accompanied by Donor NF0035.

[0221] FIG. 42A shows that GUCY2C×CD3 antibody recruits T cells (Donor XW0801211W) to induce killing of HT55 tumor cells.

[0222] FIG. 42B shows the cytokine IL2 release level of GUCY2C×CD3 antibody in a TDCC experiment accompanied by Donor XW0801211W.

[0223] FIG. 42C shows the cytokine IL-4 release level of GUCY2C×CD3 antibody in a TDCC experiment accompanied by Donor XW0801211W.

[0224] FIG42D shows the cytokine IL-6 release level of GUCY2C×CD3 antibody in a TDCC experiment accompanied by Donor XW0801211W.

[0225] FIG42E shows the cytokine IL-10 release level of GUCY2C×CD3 antibody in a TDCC experiment accompanied by Donor XW0801211W.

[0226] FIG42F shows the level of cytokine TNF-α release in the TDCC experiment with GUCY2C×CD3 antibody accompanied by Donor XW0801211W.

[0227] FIG42G shows the cytokine IFN-γ release level of GUCY2C×CD3 antibody in the TDCC experiment accompanied by Donor XW0801211W.

[0228] Figure 43 shows that GUCY2C×CD3 antibody recruited T cells to induce killing of A431 (Figure 43A), ARPE-19 (Figure 43B), BEAS-2β (Figure 43C), HACAT (Figure 43D), HFL-1 (Figure 43E), HK-2 (Figure 43F), IMR-90 (Figure 43G), MRC-5 (Figure 43H), Nthy-ori3-1 (Figure 43I), PNT1A (Figure 43J), RWPE-1 (Figure 43K) and WI38 / VA13 (Figure 43L) cells.

[0229] FIG44A shows the changes in tumor volume of the GUCY2C×CD3 antibody in the HT55 mouse transplant tumor model.

[0230] FIG44B shows the changes in mouse body weight in the HT55 mouse transplanted tumor model induced by GUCY2C×CD3 antibody.

[0231] FIG45 shows exemplary configurations of GUCY2C antibodies. DETAILED DESCRIPTION

[0232] The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Thus, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that such equivalent embodiments will be included herein. All documents cited in this article, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0233] definition

[0234] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, single domain antibody, multispecific antibody or bispecific antibody that binds to a specific antigen. Natural intact IgG antibodies include two heavy (H) chains and two light (L) chains. Mammalian heavy chains are divided into α, δ, ε, γ and μ, each of which consists of a variable region (V H ) and the first constant region, the second constant region and the third constant region (respectively C H1 、C H2 、C H3 ); mammalian light chains are classified as λ or κ, and each light chain consists of a variable region (V L) and constant regions. Antibodies are "Y" shaped, where the stem of the Y consists of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y includes the variable region and first constant region of a single heavy chain that binds to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region of each chain typically contains three highly variable loops called complementarity determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, LCDR3, and heavy chain CDRs include HCDR1, HCDR2, HCDR3). The CDR boundaries of the antibodies and antigen binding domains disclosed herein may be defined or identified by the Kabat, IMGT, AbM, Chothia, or Al-Lazikani conventions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); NR Whitelegg et al., Protein Eng. Engineering, Vol. 13(12), 819-824 (2000); Chothia, C. et al., Nature. Dec. 21-28; 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Ch. 26, 481-514, (2015)). The three CDRs are interposed between flanking extensions called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are divided into multiple classes based on the amino acid sequence of their heavy chain constant regions.The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0235] As used herein, the term "antibody" may also encompass single-domain antibodies, such as heavy-chain antibodies. "Heavy-chain antibodies" or "HCAbs" refer to antibodies that contain two VH domains but no light chains (Riechmann L. and Muyldermans S., J Immunol Methods Dec 10; 231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74(4): 277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Pat. No. 6,005,079). Heavy-chain antibodies were originally derived from the Camelidae family (camels, dromedaries, and llamas). Despite the absence of light chains, camelized antibodies have a well-established antigen-binding repertoire (Hamers-Casterman C. et al., Nature Jun 3;363(6428):446-8 (1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK et al., Immunology. May;109(1):93-101 (2003)). The variable domain of a heavy-chain antibody (VHH domain) represents the smallest known antigen-binding unit produced by the adaptive immune response (Koch-Nolte F. et al. FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0236] As used herein, the term "antigen binding domain" or "antigen binding fragment" refers to an antibody fragment formed by a portion of an antibody comprising one or more CDRs or any other antibody fragment that binds to an antigen but does not comprise a complete native antibody structure. Examples of antigen binding domains include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (divalent bifunctional antibodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and divalent domain antibodies. An antigen binding domain is capable of binding to the same antigen as the antigen bound by a parent antibody. In certain embodiments, an antigen binding domain may comprise one or more CDRs from a specific human antibody that is transplanted to a framework region from one or more different human antibodies. Further and detailed forms of antigen binding domains are described in Spiess et al., 2015 (supra) and Brinkman et al., Monoclonal Antibodies (mAbs), 9(2), pp. 182-212 (2017), which are incorporated herein by reference in their entirety.

[0237] As used herein, the term "antigen" refers to a compound, composition, peptide, polypeptide, protein, or substance that can stimulate the production of antibodies or immune cells (e.g., T cells or myeloid cells) in a cell culture or animal, including compositions that are added to a cell culture (e.g., a hybridoma), injected or absorbed into an animal, or expressed on the surface of a cell (e.g., a composition comprising a cancer-specific protein). Antigens react with products of specific humoral or cellular immunity (e.g., antibodies).

[0238] "Fab" with respect to an antibody refers to the portion of an antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by disulfide bonds. "F(ab)2" refers to a dimer of Fab.

[0239] "Fab'" refers to the Fab fragment including a portion of the hinge region.

[0240] "F(ab')2" refers to a dimer of Fab'.

[0241] "Fragment difficult (Fd)" with respect to antibodies refers to the amino-terminal half of a heavy chain fragment that can combine with a light chain to form a Fab. For example, an Fd fragment can consist of the VH and CH1 domains.

[0242] "Fv" with respect to antibodies refers to the smallest fragment of an antibody that carries a complete antigen binding site. An Fv fragment consists of the variable region of a single light chain combined with the variable region of a single heavy chain. Many Fv designs have been provided, including dsFv, in which the association between the two domains is enhanced by an introduced disulfide bond; and a peptide linker can be used to bind the two domains together as a single polypeptide to form an scFv. Fv constructs containing variable domains of immunoglobulin heavy or light chains associated with the variable domains and constant domains of the corresponding immunoglobulin heavy or light chains have also been produced. Fv has also been multimerized to form bifunctional and trifunctional antibodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0243] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region, which are directly linked to each other or linked to each other through a peptide linker sequence (Huston JS et al., Proceedings of the National Academy of Sciences of the United States of America, 85:5879 (1988)). ScFv can also be used as a basic module for developing multimeric structures (dimer: "diabody"; trimer: "tribody"; tetramer: "tetrabody").

[0244] "Diabodies" or "dAbs" include small antibody fragments with two antigen-binding sites, wherein the fragments comprise a V-binding site joined to a V-binding site in the same polypeptide chain. L Domain V H Domain (V H -V L or V L -V H (See, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA Jul 15;90(14):6444-8 (1993); EP 404097; WO 93 / 11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. The antigen-binding sites can target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds bifunctional antibody" is a bifunctional antibody that targets two different antigens (or epitopes).

[0245] "dsFv" refers to a disulfide-stabilized Fv fragment in which the variable region of a single light chain is connected to the variable region of a single heavy chain by a disulfide bond. In some embodiments, "(dsFv)2" or "(dsFv-dsFv')" comprises three peptide chains: two V HThe moieties are connected by a peptide linker (e.g., a long flexible linker) and are connected to the two V L In some embodiments, the dsFv-dsFv' has bispecificity, wherein each pair of heavy and light chains paired by disulfide bonds has a different antigenic specificity.

[0246] As used herein, the term "valence" refers to the presence of a specified number of antigen binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen binding fragment having only one single antigen binding site; and the term "multivalent" refers to an antibody or antigen binding fragment having multiple (i.e., more than one) antigen binding sites. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites in an antigen binding molecule, respectively. In some embodiments, the antibody or its antigen binding fragment is bivalent.

[0247] "Domain antibodies" or "single domain antibodies" or "sdAbs" refer to antibody fragments that contain only the variable region of a heavy chain or a variable region of a light chain. In some cases, two or more VH domains are covalently joined by a peptide linker to create a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0248] "Fc" with respect to an antibody refers to the portion of the antibody consisting of the second and third constant regions of the first heavy chain bound to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and phagocytosis.

[0249] As used herein, the term "chimeric" means an antibody or antigen-binding domain having a portion of a heavy chain and / or light chain derived from a species and the remainder of the heavy chain and / or light chain derived from another different species. In an illustrative example, a chimeric antibody may include a constant region derived from people and a variable region derived from a non-human animal (e.g., derived from a mouse). In another illustrative example, a chimeric antibody may include a FR region derived from people and a CDR region derived from a non-human animal (e.g., derived from a mouse). In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0250] As used herein, the term "humanized" means an antibody or antigen-binding domain that includes CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (when applicable).

[0251] The term "operably linked" or "operably linked" refers to the juxtaposition of two or more biological sequences of interest, with or without a spacer or linker or intervening sequence, in such a manner that the biological sequences of interest are in a relationship that allows them to function in the intended manner. When applied to polypeptides, the term means that the polypeptide sequences are linked in a manner that allows the linked product to have the intended biological function. For example, an antibody variable region can be operably linked to a constant region to provide a stable product with antigen binding activity. For another example, an antigen binding domain can be operably linked to another antigen binding domain with an intervening sequence therebetween, and such intervening sequence can be a spacer or can include a much longer sequence, such as the constant region of an antibody. The term can also be applied to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it means that the polynucleotide sequences are linked in a manner that allows regulated expression of the polypeptide from the polynucleotide.

[0252] The term "fusion" or "fused" when applied to amino acid sequences (e.g., peptides, polypeptides, or proteins) refers to the combination of two or more amino acid sequences into a single amino acid sequence, for example, by chemical bonding or recombinant means. A fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

[0253] As used herein, "CD3" refers to cluster of differentiation 3, a protein complex and T cell co-receptor involved in activating cytotoxic T cells and T helper cells. In mammals, the CD3 complex comprises a CD3γ chain, a CD3δ chain, two CD3ε chains, and a CD3-ζ (zeta) chain. Human, mouse, and cynomolgus monkey CD3 amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. As used herein, the term CD3 includes full-length wild-type CD3 and proteins thereof comprising mutations (e.g., point mutations), fragments, insertions, deletions, and splice variants.

[0254] As used herein, " CD28 " refers to. Cluster of differentiation 28 (cluster of differentiation 28), it is expressed on T cells, for providing costimulatory signals in T cell activation and survival pathways. Through CD28 and T cell receptor co-action, T cells can be stimulated and effectively activated, thereby producing a large amount of cytokines (such as IL-6). The CD28 amino acid and nucleotide sequences of people, mice and cynomolgus monkeys can be found in public databases, such as GenBank, UniProt and Swiss-Prot. As used herein, the term CD28 includes full-length wild-type CD28 and its proteins comprising mutations (e.g., point mutations), fragments, insertions, deletions and splice variants.

[0255] As used herein, "GUCY2C" refers to guanylyl cyclase C, which is a type I transmembrane protein expressed by intestinal epithelial cells from the duodenum to the rectum. Importantly, the expression of GUCY2C remains unchanged at all stages of tumor transformation, from precancerous polyps to distal colorectal cancer metastasis. Many physiological processes, including intestinal cell proliferation, differentiation and metabolism, are regulated by GUCY2C signals, so it is a potential ideal target antigen for colorectal cancer immunotherapy. The amino acid and nucleic acid sequences of GUCY2C in humans, mice and cynomolgus monkeys can be found in public databases such as GenBank, UniProt and Swiss-Prot. As used herein, the term GUCY2C includes full-length wild-type GUCY2C and proteins thereof containing mutations (e.g., point mutations), fragments, insertions, deletions and splice variants.

[0256] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody or its antigen binding domain and an antigen. In certain embodiments, the antibody molecules or antigen binding domains provided herein specifically bind to human CD3, human CD28 and / or human GUCY2C, wherein the binding affinity (K D )≤10 -6 M (e.g., ≤5×10 -7 M, ≤2×10 -7 M, ≤10 -7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≤10 -8 M, ≤5×10 -9 M, ≤4×10 -9 M). K used in this paper D The ratio of the dissociation rate to the association rate (k off / kon ), which ratio can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance, microthermophoresis, HPLC-MS, and flow cytometry (e.g., FACS). In certain embodiments, K D The value can be suitably determined by using flow cytometry.

[0257] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on the antigen to which an antibody is bound. An epitope can be formed by continuous amino acids (also referred to as linear or sequential epitopes) or by non-continuous amino acids juxtaposed by the tertiary folding of a protein (also referred to as configurational or conformational epitopes). Epitopes formed by continuous amino acids are typically arranged linearly along the primary amino acid residues on a protein, and small segments of continuous amino acids can be digested from antigen binding to major histocompatibility complex (MHC) molecules or retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when treated with denaturing solvents. In a unique spatial conformation, an epitope typically includes at least 3 and more commonly at least 5, about 7, or about 8-10 amino acids. If two antibodies exhibit competitive binding for an antigen, they can bind to the same or closely related epitopes within the antigen. For example, if an antibody or antigen-binding domain blocks at least 85% or at least 90% or at least 95% binding of a reference antibody to an antigen, the antibody or antigen-binding domain can be considered to bind to the same / closely related epitope as the reference antibody.

[0258] As used herein, the term "amino acid" refers to an organic compound containing amino (-NH2) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. Amino acid names are also represented in this disclosure as standard single-letter or three-letter codes, which are summarized below.

[0259] "Conservative substitutions" with respect to amino acid sequences refer to substitutions of amino acid residues with different side chains having similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not cause significant changes in the conformational structure of the protein, and therefore the biological activity of the protein can be retained.

[0260] As used herein, the term "subject" or "individual" or "animal" or "patient" refers to a human or non-human animal, including a mammal or primate, for whom diagnosis, prognosis, alleviation, prevention and / or treatment of a disease or condition is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, and the like.

[0261] As used herein, the term "vector" refers to a polynucleotide encoding a protein that can be operably inserted therein to cause the expression of the protein. A vector can be used for transforming, transducing, or transfecting a host cell so that the genetic elements it carries are expressed in the host cell. Examples of vectors include plasmids, phagemids, cosmids, and artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), etc.), bacteriophages (such as lambda phage or M13 phage, etc.), and animal viruses. The classification of animal viruses used as vectors includes retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). Vectors can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, vectors can contain an origin of replication. Vectors can also include materials that assist in entering cells, including but not limited to viral particles, liposomes, or protein coatings. The vector can be an expression vector or a cloning vector.

[0262] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0263] As used herein, "cancer" interchangeably with "tumor" refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis, and includes solid tumors and non-solid cancers (malignant blood tumors) such as leukemia. As used herein, "solid tumor" refers to a solid mass of neoplastic and / or malignant cells. The example of a cancer or tumor includes hematological malignancies, oral cancer (e.g., lip cancer, tongue cancer or pharyngeal cancer), digestive organ cancer (e.g., esophageal cancer, gastric cancer, small intestine cancer, colon cancer, large intestine cancer or rectal cancer), peritoneal cancer, liver cancer and bile duct cancer, pancreatic cancer, respiratory system cancer such as laryngeal cancer or lung cancer (small cell and non-small cell), bone cancer, connective tissue cancer, skin cancer (e.g., melanoma), breast cancer, reproductive organ cancer (fallopian tube cancer, uterine cancer, cervical cancer, testicular cancer, ovarian cancer or prostate cancer), urinary tract cancer (e.g., bladder cancer or kidney cancer), brain cancer and endocrine gland cancer such as thyroid cancer. In certain embodiments, the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, kidney cancer, bladder cancer, hepatocellular carcinoma and colorectal cancer. In certain embodiments, the cancer is selected from lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma and B cell lymphoma.

[0264] The term "pharmaceutically acceptable" means that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0265] A. Antibodies or Antigen-Binding Fragments thereof

[0266] In one aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof that binds to GUCY2C. In certain embodiments, the multispecific antibody or antigen-binding fragment thereof provided by the present disclosure comprises GUCY2C and one or more T cell antigen binding domains. In certain embodiments, the multispecific antibody or antigen-binding fragment thereof provided by the present disclosure, wherein the one or more T cell antigens are selected from CD3 or CD28 or both. As used herein, the term "binding domain" refers to an antigen-binding domain derived from an antibody and includes at least one antibody fragment (such as a CDR and / or variable region sequence).

[0267] GUCY2C binding domains screened using i.Beacon single B cell technology

[0268] In certain embodiments, provided herein is a series of anti-huGUCY2C murine monoclonal antibodies screened based on Beacon single B cell technology, the variable region sequences of which are shown in Table 1.

[0269] Table 1: Anti-huGUCY2C mouse antibody sequences screened using Beacon single B cell technology

[0270] Exemplarily, M0047 and M0055 were selected for analysis of CDR regions and expression of chimeric human IgG1 antibodies.

[0271] Table 2: CDRs of anti-huGUCY2C mouse monoclonal antibodies screened using Beacon single B cell technology

[0272] The full length of the M0047 chimeric human IgG1 antibody heavy chain is:

[0273] The full length of the light chain of the M0047 chimeric human IgG1 antibody is:

[0274] The full length of the M0055 chimeric human IgG1 antibody heavy chain is:

[0275] The full length of the light chain of the M0055 chimeric human IgG1 antibody is:

[0276] ii. GUCY2C binding domain screened by phage display technology

[0277] In certain embodiments, provided herein is a series of anti-huGUCY2C murine monoclonal antibodies screened based on phage display technology, and their variable region sequences are shown in Table 3.

[0278] Table 3: Screening of anti-huGUCY2C mouse antibody sequences using phage display technology

[0279] For example, GCAb0002 and GCAb0005 were selected for analysis of CDR regions and expression of chimeric human IgG1 antibodies.

[0280] Table 4: CDRs of anti-huGUCY2C mouse monoclonal antibodies screened using phage display technology

[0281] The full length of the heavy chain of GCAb0002 chimeric human IgG1 antibody is:

[0282] The full length of the light chain of the GCAb0002 chimeric human IgG1 antibody is:

[0283] The full length of the heavy chain of GCAb0005 chimeric human IgG1 antibody is:

[0284] The full length of the light chain of GCAb0005 chimeric human IgG1 antibody is:

[0285] iii. CD3 and CD28 binding domains

[0286] In certain embodiments, provided herein are a series of optimized CD3 binding domains, whose variable region and scFv amino acid sequences are shown in Table 5.

[0287] Table 5: CD3 binding domain variable region and scFv amino acid sequence (CDR is underlined)

[0288] In certain embodiments, the present disclosure provides a series of optimized CD28 binding domains, whose variable region and scFv amino acid sequences are shown in Table 6.

[0289] Table 6: CD28 binding domain variable region and scFv amino acid sequence (CDRs divided according to IMGT rules are underlined)

[0290] iv. Anti-GUCY2C multispecific antibodies

[0291] In one aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof targeting GUCY2C. In certain embodiments, the multispecific antibody or antigen-binding fragment thereof provided herein comprises GUCY2C and one or more T cell antigen-binding domains. In certain embodiments, the multispecific antibody or antigen-binding fragment thereof provided herein comprises one or more T cell antigens selected from CD3, CD28, or both.

[0292] In one aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, comprising:

[0293] (a) a GUCY2C binding domain, wherein the GUCY2C binding domain comprises GUCY2C-LCDR1, GUCY2C-LCDR2, GUCY2C-LCDR3, GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 of a GUCY2C antibody shown in Table 2 or Table 4; and

[0294] (b) a CD3 binding domain, wherein the CD3 binding domain comprises the CDR sequence of the CD3 antibody shown in Table 5.

[0295] In one aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, comprising:

[0296] (a) a GUCY2C binding domain, wherein the GUCY2C binding domain comprises GUCY2C-LCDR1, GUCY2C-LCDR2, GUCY2C-LCDR3, GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 of a GUCY2C antibody shown in Table 2 or Table 4; and

[0297] (b) a CD28 binding domain, wherein the CD28 binding domain comprises the CDR sequence of the CD28 antibody shown in Table 6.

[0298] In one aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, comprising:

[0299] (a) a GUCY2C binding domain, wherein the GUCY2C binding domain comprises GUCY2C-LCDR1, GUCY2C-LCDR2, GUCY2C-LCDR3, GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 of a GUCY2C antibody shown in Table 2 or Table 4;

[0300] (b) a CD3 binding domain, wherein the CD3 binding domain comprises the CDR sequence of a CD3 antibody shown in Table 5; and

[0301] (c) a CD28 binding domain, wherein the CD28 binding domain comprises the CDR sequence of the CD28 antibody shown in Table 6.

[0302] In certain embodiments, based on the results of antibody screening using Beacon single B cell technology and phage display technology, the VH, VL, and scFv sequences of the anti-GUCY2C murine antibodies M0055 and GCAb0002, the VH, VL, and scFv sequences of the anti-huCD3ed antibody CD3-002 / 007, and the VH, VL, and scFv sequences of the anti-CD28-065 antibody were used to design antibodies GCbi0007, GCbi0019, GCbi0021, GCbi0023, GCbi0025, and GCbi0026 with bispecific binding activity.

[0303] Antibodies GCTr0001, GCTr0002, GCTr0006, GCTr0009, GCTr0010, GCTr0011 and GCTr0012 with trispecific binding activity were designed using the VH and VL sequences of the anti-GUCY2C murine antibodies M0055 and GCAb0002, the VH and VL and scFv sequences of the anti-huCD3ed antibody CD3-002 / 007, and the VH, VL and scFv sequences of the anti-CD28 antibody.

[0304] At the same time, the GUCY2C negative bispecific antibody IsoB0001 was designed using the VH and VL sequences of anti-HEL and the scFv sequence of the anti-huCD3ed antibody CD3-002.

[0305] The sequences of the above multispecific antibodies are shown in Table 7, and the configurations are shown in Table 8.

[0306] Table 7: Sequences of multispecific antibodies and their mutants

[0307] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have the structure shown in Figure 9. Table 8 below summarizes the configurations of the multispecific antibodies provided herein.

[0308] Table 8: Configurations of the multispecific antibodies provided herein

[0309] In one aspect, the present disclosure provides a GUCY2C antibody or an antigen-binding fragment thereof, wherein the GUCY2C antibody binds to one or more epitopes selected from the group consisting of: S15N, S62F, I66L, L80V, E55A, D56A, E101A, L105E, R107A.

[0310] In certain embodiments, the GUCY2C antibody binds to one or more epitopes selected from the group consisting of S15N, S62F, I66L, and L80V.

[0311] In certain embodiments, the GUCY2C antibody binds to epitopes E55A and / or D56A.

[0312] In certain embodiments, the GUCY2C antibody binds to one or more epitopes selected from the group consisting of: E101A, L105E, and R107A. v. Conjugates

[0313] In some embodiments, the antibody or its Fab provided herein is connected to one or more conjugate moieties. A conjugate moiety is a non-protein portion that can be connected to an antibody or its Fab. It is envisioned that a variety of conjugate moieties can be connected to an antibody or its Fab provided herein (see, for example, "Conjugate Vaccines," in Contributions to Microbiology and Immunology, JM Cruse and R E Lewis, Jr. (eds.), Carger Press, New York (1989)). These conjugate moieties can be connected to an antibody or its Fab by methods such as covalent bonding, affinity bonding, embedding, coordination bonding, complexing, association, blending or addition.

[0314] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein can be engineered to contain specific sites other than the epitope binding moiety that can be used to bind to one or more conjugates. For example, such sites can include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent attachment to the conjugate.

[0315] In certain embodiments, the antibody or antigen-binding fragment thereof can be linked to a conjugate moiety indirectly or through another conjugate moiety. For example, an antibody or antigen-binding fragment thereof can be conjugated to biotin and then indirectly conjugated to a second conjugate moiety conjugated to avidin. The conjugate moiety can be a scavenging modifier, a toxin (e.g., a chemotherapeutic agent), a detectable label (e.g., a radioisotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme substrate label), or a purification moiety.

[0316] A "toxin" can be any agent that is harmful to cells or can damage or kill cells. Examples of toxins include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, and d-actinomycin. D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C ( C) and dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotics (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin-binding agents.

[0317] Examples of detectable labels can include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase, or β-D-galactosidase), radioactive isotopes (e.g., 123 I. 124 I. 125 I. 131 I. 35 S. 3 H. 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y. 88 Y. 90 Y. 177 Lu, 211 At 186 Re、 188 Re、 153 Sm, 212 Bihe 32 P, other lanthanides), luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0318] In certain embodiments, the conjugate moiety can be a clearance modifier that helps increase the half-life of the antibody or its antigen-binding fragment. Illustrative examples include water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol / propylene glycol, and the like. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody or its antigen-binding fragment can vary, and if more than one polymer is attached, the polymers can be the same or different molecules.

[0319] In certain embodiments, the conjugate moiety can be a purification moiety, such as a magnetic bead.

[0320] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein serve as the base of the conjugate.

[0321] B. Pharmaceutical Compositions

[0322] The present disclosure further provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0323] Pharmaceutically acceptable carriers for the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / partitioning agents, sequestering or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0324] Suitable components can include, for example, antioxidants, fillers, binding agents, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers, such as sugar and cyclodextrin. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole (butylated hydroxanisol), butylated benzyl alcohol and / or propyl gallate. As disclosed herein, including one or more antioxidants such as methionine in a composition comprising an antibody or its Fab and a conjugate as provided herein reduces the oxidation of the antibody or its Fab. This oxidative reduction prevents or reduces the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Therefore, in some embodiments, there is provided a composition comprising one or more antibodies or its Fab as disclosed herein and one or more antioxidants such as methionine. Further provided are methods for preventing oxidation of, extending the shelf life of, and / or improving the efficacy of, an antibody or antigen-binding fragment thereof as provided herein by mixing the antibody or antigen-binding fragment thereof with one or more antioxidants, such as methionine.

[0325] To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextran and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextran; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. The antimicrobial agent used as a carrier can be added to the pharmaceutical composition in the multidose container, and the antimicrobial agent includes phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextran, glycerol or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or medicaments such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0326] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained-release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0327] In some embodiments, pharmaceutical composition is formulated into injectable composition.Injectable pharmaceutical composition can be prepared in any conventional form, and described conventional form is liquid solution, suspension, emulsion or is applicable to the solid form producing liquid solution, suspension or emulsion for example.Injection preparation can comprise the sterile and / or pyrogen-free solution of preparation injection, the sterile dry soluble product of preparation and solvent combination before use, such as lyophilized powder, comprise subcutaneous injection tablet, the sterile suspension of preparation injection, the sterile dry insoluble product of preparation and vehicle combination before use and sterile and / or pyrogen-free emulsion.Solution can be aqueous or non-aqueous.

[0328] In certain embodiments, the unit dose parenteral formulation is packaged in an ampoule, a vial, or a syringe with a needle.As is known and practiced in the art, all preparations for parenteral administration should be sterile and pyrogen-free.

[0329] In certain embodiments, a sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment thereof as disclosed herein in a suitable solvent. The solvent may contain an excipient that improves the stability of the powder or a reconstituted solution prepared from the powder or other pharmacological components. Excipients that can be used include, but are not limited to, water, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may contain a buffer such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, and in one embodiment, the pH is about neutral. The solution is then sterile filtered and then lyophilized under standard conditions known to those skilled in the art to provide the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial can contain a single dose or multiple doses of an antibody or antigen-binding fragment thereof, or a combination thereof. It is acceptable to overfill the vial with a slightly higher amount (e.g., about 10%) than required for each dose or a set of doses to facilitate accurate sampling and dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0330] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. In one embodiment, for reconstitution, sterile and / or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder. The exact amount depends on the selected therapy to be administered and can be determined empirically.

[0331] C. Host cells

[0332] i. Polynucleotides

[0333] The present disclosure provides isolated polynucleotides encoding the antibodies or antigen-binding fragments thereof provided herein.

[0334] As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses polynucleotides containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, specific polynucleotide sequences also implicitly encompass conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly specified. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acids Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0335] ii. Carrier

[0336] Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0337] The present disclosure provides a vector (e.g., an expression vector) comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), λ phage and M13 phage, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0338] iii. Host cells

[0339] The carrier comprising the isolated polynucleotide of the antibody or its Fab provided by this paper can be introduced into host cell to carry out cloning or genetic expression.The suitable host cell for cloning or expressing the DNA in this paper carrier is above-mentioned prokaryotic cell, yeast cell or higher eukaryotic cell.The suitable prokaryotic organism that is used for this purpose comprises true bacteria, such as Gram-negative or Gram-positive organism, for example Enterobacteriaceae (Enterobacteriaceae), such as Escherichia (Escherichia) (for example, Escherichia coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) (for example, Salmonella typhimurium (Salmonella typhimurium)), Serratia (Serratia) (for example, Serratia marcescens (Serratia marcescans) and Shigella as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0340] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for the provided vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly used and suitable for use herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0341] Suitable host cells for expressing the glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains and variants and corresponding permissive insect host cells have been identified, and the permissive insect host cells are derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses herein according to the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0342] However, of greatest interest are vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). In some preferred embodiments, the host cell is a 293F cell.

[0343] D. Methods for Producing Antibodies

[0344] The present disclosure further provides a method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell provided herein under conditions where the antibody or antigen-binding fragment thereof is expressed, and recovering the antibody or antigen-binding fragment thereof.

[0345] With above-mentioned expression or cloning vector transformation host cell that is used to produce antibody or its Fab that this paper provides, and described host cell is cultivated in conventional nutrient medium, described conventional nutrient medium is modified into the gene that is suitable for inducing promoter, selecting transformant or amplifying coding desired sequence.In another embodiment, antibody or its Fab that this paper provides can produce by homologous recombination known in the art.

[0346] Host cells for producing antibodies or Fabs thereof provided herein can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Rep. 30,985 can be used as culture medium for the host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN TM Drugs), trace elements (defined as inorganic compounds with final concentrations generally in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions (such as temperature, pH, etc.) are those previously used with the host cell selected for expression and will be apparent to those of ordinary skill in the art.

[0347] When using recombinant technology, antibody or its Fab can be produced in intracellular, periplasmic space, or directly secreted into culture medium.If antibody is produced in intracellular, then as first step, can for example remove the particulate debris of host cell or dissolved fragment by centrifugation or ultrafiltration.Carter et al., " biology / technology (Bio / Technology) " 10:163-167 (1992) describes the program for separating the antibody that is secreted into the periplasmic space of Escherichia coli.In short, cell paste is thawed about 30 minutes when there is sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF).Cell debris can be removed by centrifugation.When antibody or its Fab is secreted into culture medium, usually first use commercially available protein concentration filter, for example Amicon or Millipore Pellicon ultrafiltration unit concentrates the supernatant from this type of expression system.Protease inhibitors such as PMSF can be included in any of the aforementioned steps to inhibit proteolysis, and can include antibiotic to prevent the growth of foreign contaminants.

[0348] Antibodies or antigen-binding fragments thereof produced by the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.

[0349] In certain embodiments, protein A fixed on a solid phase is used for immunoaffinity purification of antibodies or their antigen-binding fragments. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in an antibody or its antigen-binding fragment. Protein A can be used for purifying antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., Journal of Immunological Methods 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., Journal of the European Molecular Biology Association (EMBO J.) 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Compared with the flow rate and processing time that can be achieved with agarose, mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene can achieve faster flow rates and shorter processing times. In the case where an antibody or its antigen-binding fragment includes a CH3 domain, Bakerbond ABX TMResins (JT Baker, Phillipsburg, NJ) can be used for purification. Other techniques for protein purification are available, depending on the antibody to be recovered, such as separation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE TM Chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0350] Following any preliminary purification steps, the mixture comprising the antibody molecule of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 and 4.5, preferably at low salt concentration (e.g., about 0-0.25 M salt).

[0351] E. Multispecific Molecules and Their Characterization

[0352] In certain embodiments, the multispecific antibodies or antigen-binding fragments thereof provided herein are designed in the configuration shown in FIG9 .

[0353] In certain embodiments, the binding of the multispecific antibodies or antigen-binding fragments thereof provided herein to an antigen can be determined by a "half maximal effective concentration" (EC 50 ) value indicates that the value refers to the concentration of the antibody at which 50% of the maximum effect (eg, binding or inhibition, etc.) of the antibody is observed. EC 50 Values ​​can be measured by methods known in the art, for example, sandwich assays such as ELISA, Western blot, flow cytometry assays, and other binding assays.

[0354] The binding affinity of the antigen binding domains provided herein can also be measured by K D The value represents the ratio of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium (k off / k on Antigen binding affinity (e.g., K) can be suitably determined using suitable methods known in the art, including, for example, flow cytometry assays. D In some embodiments, binding of the antigen binding domain to different concentrations of antigen can be determined by flow cytometry, and the determined mean fluorescence intensity (MFI) can first be plotted against the concentration of the antigen binding domain, and then K can be calculated by fitting the dependence of the specific binding fluorescence intensity (Y) and the antibody concentration (X) to a site saturation equation. D Value: Y = B max *X / (K D+X), using Prism version 5 (GraphPad Software, San Diego, CA), where B max It refers to the maximum specific binding of the antigen-binding domain being tested to the antigen.

[0355] In certain embodiments, the binding affinity (K) of the multispecific antibodies provided herein that specifically bind to human CD3, GUCY2C, or CD28 is D ) measured by AI determination.

[0356] In certain embodiments, the binding affinity (K) of the multispecific molecules provided herein that specifically bind to human CD3, GUCY2C, or CD28 is D ) measured by Octet assay.

[0357] In certain embodiments, the binding affinity (K) of the multispecific molecules provided herein that specifically bind to human CD3, GUCY2C, or CD28 is D ) as measured by ELISA assay.

[0358] In certain embodiments, the binding affinity (K) of the multispecific molecules provided herein that specifically bind to human CD3, GUCY2C, or CD28 is D ) as measured by FACS assay.

[0359] F. Variant

[0360] The multispecific molecules provided herein also encompass various variants thereof. In certain embodiments, one or more CDR sequences, one or more variable region sequences (but not in any CDR sequence) and / or one or more modifications or substitutions in the constant region (e.g., Fc region). Such variants retain the specific binding affinity of their parent antibody to CD3, GUCY2C or CD28, but have one or more desirable properties conferred by the modification or substitution. For example, the variant may have improved antigen binding affinity, improved productivity, improved stability, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, reduced or depleted effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity and / or compatibility with conjugation (e.g., one or more introduced cysteine ​​residues).

[0361] Methods known in the art, such as "alanine scanning mutagenesis", can be used to screen the parent antibody sequence to identify suitable or preferred residues to be modified or substituted (see, for example, Cunningham and Wells (1989) Science, 244: 1081-1085). In short, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are generated and screened for properties of interest. If the substitution at a particular amino acid position shows a functional change of interest, the position can be identified as a potential residue for modification or substitution. Potential residues can be further evaluated by replacing them with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0362] In certain embodiments, the CD3 binding domains, CD28 binding domains, and / or GUCY2C binding domains provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences and / or one or more variable region sequences. In certain embodiments, the variant comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in a total of CDR sequences and / or FR sequences and / or one or more variable region sequences.

[0363] In certain embodiments, the CD3 binding domain comprises 1, 2, 3, 4, 5, or 6 CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to 1, 2, 3, 4, 5, or 6 sequences in Table 5, while retaining binding affinity for CD3 at a similar or even higher level relative to its parent antibody.

[0364] In certain embodiments, the CD28 binding domain comprises 1, 2, 3, 4, 5, or 6 CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to 1, 2, 3, 4, 5, or 6 sequences in Table 6, while retaining binding affinity for CD28 at a similar or even higher level relative to its parent antibody.

[0365] In certain embodiments, the GUCY2C binding domain comprises 1, 2, 3, 4, 5 or 6 CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to 1, 2, 3, 4, 5 or 6 sequences in Table 2 or Table 4, and at the same time retains binding affinity for GUCY2C at a level similar to or even higher than that of its parent antibody.

[0366] i. Glycosylation variants

[0367] The multispecific molecules provided herein also encompass glycosylation variants, which can be obtained to increase or decrease the extent of glycosylation of the antigen binding domain or activating receptor domain of the multispecific molecule.

[0368] The multispecific molecules provided herein can include one or more amino acid residues having side chains to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of the antibody antigen-binding domain is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue (e.g., an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine), where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Natural glycosylation sites can be conveniently removed, for example, by altering the amino acid sequence such that one of the tripeptide sequences (for N-linked glycosylation sites) or the serine or threonine residues (for O-linked glycosylation sites) present in the sequence is substituted. New glycosylation sites can be generated in a similar manner by introducing such a tripeptide sequence or a serine or threonine residue.

[0369] ii. Cysteine ​​engineered variants

[0370] The multispecific molecules provided herein also encompass cysteine ​​engineered variants comprising one or more introduced free cysteine ​​amino acid residues.

[0371] Free cysteine ​​residues are not part of a disulfide bond. Cysteine ​​engineered variants can be used to conjugate, for example, cytotoxic compounds and / or imaging compounds, labels, or radioisotopes at the site of the engineered cysteine ​​via, for example, maleimide or haloacetyl groups. Methods for engineering antibody polypeptides to introduce free cysteine ​​residues are known in the art, see, for example, WO2006 / 034488.

[0372] iii. Fc variants

[0373] The multispecific molecules provided herein also encompass Fc variants comprising one or more amino acid residue modifications or substitutions at the Fc region and / or hinge region thereof, e.g., to provide altered effector functions such as ADCC, ADCP, and CDC. Methods for altering ADCC activity by antibody engineering have been described in the art, see, for example, Shields RL. et al., J Biol Chem. 2001. 276(9):6591-604; Idusogie EE. et al., J Immunol. 2000. 164(8):4178-84; Steurer W. et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE. et al., J Immunol. 2001, 166(4):2571-5; Lazar GA. et al., PNAS, 2006, 103(11):4005-4010; Ryan MC. et al., Mol. Cancer Ther., 2007, 6:3009-3018; Richards et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE. et al., J Immunol. 2001, 166(4):2571-5; Lazar GA. et al., PNAS, 2006, 103(11):4005-4010; Ryan MC. et al., Mol. Cancer Ther., 2007, 6:3009-3018; Richards et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE. et al., J Immunol. 2001, 166(4):2571-5. JO, et al., Mol Cancer Therapeutics 2008, 7(8):2517-27; Shields RL et al., J Biol Chem, 2002, 277:26733-26740; Shinkawa T. et al., J Biol Chem, 2003, 278:3466-3473.

[0374] The CDC activity of the antibodies provided herein can also be altered, for example, by improving or reducing C1q binding and / or CDC (see, e.g., WO 99 / 51642; Duncan and Winter, Nature, 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821); and WO 94 / 29351 for other examples of Fc region variants. One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region can be replaced with a different amino acid residue to alter C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC) (see U.S. Patent No. 6,194,551 to Idusogie et al.). One or more amino acid substitutions can also be introduced to alter the ability of an antibody to fix complement (see PCT Publication WO 94 / 29351 to Bodmer et al.).

[0375] The terms "antibody-dependent cellular phagocytosis" and "ADCP" refer to a process by which antibody-coated cells or particles are internalized in whole or in part by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) bound to the Fc region of an immunoglobulin. Methods for changing the ADCP activity of an antibody by antibody engineering are known in the art, see, for example, Kellner C et al., Transfusion Medicine and Blood Therapy (Transfus Med Hemother), (2017) 44: 327-336 and Chung AW et al., AIDS (AIDS), (2014) 28: 2523-2530. Examples of Fc variants are known in the art, see, for example, Wang et al., Protein Cell 2018, 9 (1): 63-73 and Kang et al., Experimental and Molecular Medicine (Exp & Mol., Med.) (2019) 51: 138, which are incorporated herein by reference in their entirety.

[0376] i) Fc variants with enhanced effector function

[0377] In certain embodiments, the Fc variants provided herein have increased ADCC and / or increased affinity for Fcγ receptors (e.g., FcγRI (CD64), FcγRII (CD32) and / or FcγRIII (CD16)) relative to wild-type Fc (e.g., the Fc of IgG1). In certain embodiments, the Fc variant comprises one or more amino acid substitutions at one or more of the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 246, 247, 248, 249, 252, 254, 255, 256, 258, 260, 262, 263, 264, 265, 267, 268, 269, 270, 272, 274, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 , 298, 299, 300, 301, 303, 304, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 345, 360, 373, 376, 378, 382, ​​388, 389, 396, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, 439 and 440 (see Presta's WO 00 / 42072 to Lazar, WO2006 / 019447 and WO2016 / 196228 to Lazar, which are incorporated herein in their entireties), wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat (see, Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th ed. National Institutes of Health, Bethesda, MD, (1991)).Exemplary substitutions for increased effector function include, but are not limited to, 234Y, 235Q, 236A, 236W, 239D, 239E, 239M, 243L, 247I, 268D, 267E, 268D, 268E, 268F, 270E, 280H, 290S, 292P, 298A, 298D, 298V, 300L, 305I, 324T, 326A, 326D, 326W, 330L, 330M, 333S, 332D, 332E, 298A, 333A, 334A, 334E, 326A, 247I, 339D, 339Q, 345R, 280H, 290S, 298D, 298V, 3L, 292P, 300L, 396L, 305I, 396L, 430G, 440Y, or any combination thereof (e.g., 239D / 332E, 239D / 332E / 330L, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F / 324T) (see, WO 2016 / 196228; Richards et al. (2008) Mol Cancer Therapeutics 7:2517; Moore et al. (2010) Monoclonal Antibodies 2:181; and Strohl (2009) Current Opinion in Biotechnology 20:685-691).

[0378] Specific mutations at positions 256, 290, 298, 333, 334, and 339 are shown to improve binding to FcγRIII. In addition, the following combination mutants are shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, F243L / R292P / Y300L / V305I / P396L, S298A / E333A / K334A, and L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the opposite heavy chain (with enhanced FcγRIII binding and ADCC activity). Other Fc variants with strongly enhanced binding to FcγRIIIa include variants with S239D / I332E and S239D / I332E / A330L mutations (which showed the greatest increase in affinity for FcγRIIIa, decreased binding to FcγRIIb, and potent cytotoxic activity) and variants with L235V, F243L, R292P, Y300L, V305I, and P396L mutations (which exhibited enhanced FcγRIIIa and concomitant enhanced ADCC activity). (See Lazar et al. (2006) Proc. Natl. Acad. Sci. USA 103:4005; Awan et al. (2010) Blood 115:1204; Desjarlais and Lazar (2011) Exp. Cell Res; Stavenhagen et al. (2007) Cancer Res 67:8882). Modifications that increase binding to C1q can be introduced to enhance CDC activity. Exemplary modifications include K326 (e.g., K326W) and / or E333 modifications in IgG2, or S267E / H268F / S324T modifications, alone or in combination, in IgG1 (see Idusogie et al. (2001) J. Immunol. 166:2571; Moore et al. (2010) Mabs 2:181). Other exemplary modifications include K326W / E333S, S267E / H268F / S324T, and E345R / E430G / S440Y.

[0379] ii) Fc with reduced effector function

[0380] In certain embodiments, the Fc variants provided herein have reduced effector function relative to wild-type Fc (e.g., Fc of IgG1) and comprise one or more amino acid substitutions at positions selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237, 238, 267, 268, 269, 270, 297, 309, 318, 320, 322, 325, 328, 329, 330, and 331 of the Fc region (see WO 2016 / 196228; Richards et al. (2008) Mol Cancer Therapeutics 7:2517; Moore et al. (2010) Mabs 2:181; and Strohl (2009) Biotechnol Current Rev 20:685-691), wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat. Exemplary substitutions that reduce effector function include, but are not limited to, 220S, 226S, 228P, 229S, 233P, 234V, 234G, 234A, 234F, 234A, 235A, 235G, 235E, 236E, 236R, 237A, 237K, 238S, 267R, 268A, 268Q, 269R, 297A, 297Q, 297G, 309L, 318A, 322A, 325L, 328R, 330S, 331S, or any combination thereof (see WO 2016 / 196228; and Strohl (2009), Biotechnol. Current Rev. 20:685-691).

[0381] In certain embodiments, the Fc variants provided herein are of the IgG1 isotype and include one or more amino acid substitutions selected from the group consisting of: L234A, L234F, L234V, F234A, V234A, L235A, L235E, G237A, P238S, H268Q, H268A, N297A, N297Q, N297G, V309L, A330S, and P331S, or any combination thereof (e.g., L234A / L235A). In certain embodiments, the Fc variants provided herein are of the IgG2 isotype and include one or more amino acid substitutions selected from the group consisting of: H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, and any combination thereof. In certain embodiments, the Fc variants provided herein are of the IgG4 isotype and comprise one or more amino acid substitutions selected from the group consisting of S228P, F234A, L235E, L235A, G237A, E318A, N297A, N297Q, N297G, and any combination thereof.

[0382] iii) Fc with altered binding to FcRn

[0383] In certain embodiments, the Fc variant comprises one or more amino acid substitutions that improve binding affinity to the neonatal Fc receptor (FcRn) at pH 6.0 while retaining minimal binding at pH 7.4. Such variants may have a prolonged pharmacokinetic half-life because the variant binds to FcRn at acidic pH, thereby protecting it from degradation in lysosomes and allowing it to be transported and released outside the cell. Methods for engineering antibodies and antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, for example, Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Vol. 1, Chapter 27: Engineering of the Fc region for improved PK, Springer, 2010; Yeung, Y. et al., Cancer Res, 70:3269-3277 (2010); Hinton, P. et al., J. Immunol., 176:346-356 (2006); Petkova et al. (2006) Int. Immunol. 18:1759; Ball ... Acqua et al., J. Immunol. 2002, 169:5171-5180; Dall'Acqua WF. et al., J. Biol. Chem. 281:23514-23524 (2006); Zalevsky J et al., Nat. Biotechnol. 28:157-159 (2010); WO 2009 / 086320; US 6,277,375; US 6,821,505; WO 97 / 34631; and WO 2002 / 060919.

[0384] Non-limiting examples of Fc modifications that may result in an increase in the serum half-life of the antibody when administered include, for example, substitutions at one or more positions selected from the group consisting of: 234 (e.g., with F), 235 (e.g., with Q), 238 (e.g., with D), 250 (e.g., with E or Q), 252 (e.g., with L / Y / F / W or T), 254 (e.g., with S or T), 256 (e.g., with S / R / Q / E / D or T); 259 (e.g., with I); 272 (e.g., with A), 305 (e.g., with A), 307 (e.g., with A or P), 308 (e.g., with F, C or P); ), 311 (e.g., having A or R), 312 (e.g., having A), 322 (e.g., Q), 328 (e.g., E), 331 (e.g., having A), 378 (e.g., having A), 380 (e.g., having A), 382 (e.g., having A), 428 (e.g., having L or F), 432 (e.g., having C), 433 (e.g., having H / L / R / S / P / Q or K), 434 (e.g., having H / F or Y or S or A or W), 435 (e.g., having H), 436 (e.g., having L) and 437 (e.g., having C) (all positions are numbered by EU) (see WO 2016049000A2; WO 2020052692; WO 2016196228). In some embodiments, the Fc variant comprises one or more amino acid substitutions selected from the group consisting of 234F, 235Q, 238D, 250Q, 252T, 252Y, 254T, 256E, 259I, 272A, 305A, 307A, 308F, 311A, 322Q, 328E, 331S, 380A, 428L, 432C, 433K, 433S, 434S, 434Y, 434F, 434W, 434A, 435H, 436L, 437C, and any combination thereof.In some embodiments, the Fc modification comprises one or a pair or group of modifications selected from the group consisting of: a) 428L (e.g., M428L) and 434S (e.g., N434S) substitutions; b) 433K (e.g., H433K) and 434 (e.g., N434Y or N434F) substitutions; c) 252Y, 254T, and 256E (e.g., M252Y, S254T, and d) 250Q and 428L substitutions (e.g., T250Q and M428L); e) 307A, 380A and 434A substitutions (e.g., T307A, E380A and N434A); f) P238D and L328E substitutions; g) L234F, L235Q, K322Q, M252T, S254T and T256E substitutions; and h) and L432C, H433S, N434W, Y436L and T437C substitutions.

[0385] In some embodiments, hybrid IgG isotypes can be used to increase the FcRn binding and half-life of antibodies. Hybrid Ig can be produced from two or more isotypes. For example, an IgG1 / IgG3 hybrid variant can be constructed by replacing the IgG1 position in the CH2 and / or CH3 region with an amino acid from IgG3 at positions where the two isotypes differ. In some embodiments, a hybrid Ig can include one or more modifications (e.g., substitutions) disclosed herein.

[0386] G. Treatment methods

[0387] The present disclosure further provides a method for treating or ameliorating a disease that benefits from T lymphocyte killing and clearance or a GUCY2C-related disease in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof provided herein, or the pharmaceutical composition provided herein.

[0388] In certain embodiments, the methods provided herein comprise the combined use of one or more of the antibodies or antigen-binding fragments thereof provided herein.

[0389] In certain embodiments, the subject is a human.

[0390] In certain embodiments, the antibody or antigen-binding fragment thereof or the pharmaceutical composition is administered intravenously, intraarterially, intratumorally, intramuscularly or subcutaneously.

[0391] In certain embodiments, the methods provided herein further comprise administering to the subject one or more additional therapeutic agents, wherein the additional therapeutic agent is selected from a chemotherapeutic agent, an anticancer drug, a radiotherapeutic agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic agent, an antioxidant, a metal chelator, a cytokine, an anti-infective agent, or an anti-inflammatory agent.

[0392] In certain embodiments, the additional therapeutic agent is selected from a monospecific antibody, a bispecific antibody, a multispecific antibody, a fusion protein, an ADC, an LDC, an RDC, a cell therapy, a small molecule drug, an antisense nucleic acid, an siRNA, an mRNA, and a PROTAC.

[0393] In certain embodiments, the additional therapeutic agent acts directly on GUCY2C or its variants, such as a monospecific antibody targeting GUCY2C or its variants, a bispecific antibody targeting GUCY2C or its variants, a multispecific antibody targeting GUCY2C or its variants, a fusion protein targeting GUCY2C or its variants, an ADC targeting GUCY2C or its variants, an LDC targeting GUCY2C or its variants, an RDC targeting GUCY2C or its variants, a cell therapy targeting GUCY2C or its variants, a small molecule drug targeting GUCY2C or its variants, an antisense nucleic acid targeting GUCY2C or its variants, an siRNA targeting GUCY2C or its variants, an mRNA expressing GUCY2C or its variants, or a PROTAC targeting GUCY2C or its variants.

[0394] In certain embodiments, the one or more additional therapeutic agents are administered concurrently or sequentially with the antibody or antigen-binding fragment thereof.

[0395] In some embodiments, the subject has been diagnosed with or is at risk for a disease, disorder, or condition selected from the group consisting of cancer (e.g., solid tumors, hematological malignancies), inflammatory diseases, infectious diseases (e.g., chronic infections), autoimmune diseases (e.g., multiple sclerosis), neurological diseases, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant dysfunction, and arthritis. In a preferred embodiment, the subject has been diagnosed with or is at risk for one or more solid tumors.

[0396] In some embodiments, the condition or illness that can be treated by the method provided herein include tumors and cancers. In some embodiments, the condition or illness that can be treated by the method provided herein include solid tumors and hematologic malignancies. Examples of cancers and tumors include non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin's lymphoma (CHL), primary longitudinal myeloma, myeloma, mycosis fungoides, Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin's lymphoma (CHL), primary longitudinal myeloma, mycosis fungoides, mycosis fungoides, mycosis fungoides, mycosis fungoides and other hematologic malignancies, such as primary longitudinal myeloma, mycosis fungoides, mycosis fungoides and mycosis fungoides. Septal large B-cell lymphoma, T cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, central nervous system (CNS) neoplasms such as primary CNS lymphoma, spinal cord tumors, brainstem gliomas, Anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer In some embodiments, the present invention relates to a cancer cell line comprising at least one of: ...

[0397] In certain embodiments, the disease is cancer. In certain embodiments, the cancer is selected from the group consisting of adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioalveolar lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.

[0398] In some embodiments, the cancer is a GUCY2C-positive cancer. In some embodiments, the subject to be treated has been identified as having a GUCY2C-positive cancer. As used herein, a "GUCY2C-positive" cancer refers to a cancer characterized by expression of GUCY2C in cancer cells or expression of GUCY2C in cancer cells at a level significantly higher than expected in normal cells.

[0399] The presence and / or amount of GUCY2C in a biological sample of interest can be determined in a test biological sample from a subject using various suitable methods. For example, the test biological sample can be exposed to an anti-GUCY2C antibody or antigen-binding fragment thereof, which binds to and detects the expressed GUCY2C protein. Alternatively, GUCY2C can be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase-PCR, microarrays, SAGE, FISH, etc. In some embodiments, the test sample is derived from cancer cells or tissues or tumor-infiltrating immune cells. In certain embodiments, the presence or upregulated level of GUCY2C in the test biological sample indicates the likelihood of a response. As used herein, the term "upregulated" refers to an overall increase in the expression level of GUCY2C in the test sample by no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or more, compared to a reference expression level of GUCY2C. The reference level can be the level of CD47 or target antigen expression found in normal cells of the same tissue type, optionally normalized to the expression level of another gene (e.g., a housekeeping gene). Alternatively, the reference level can be the level of GUCY2C expression found in healthy subjects. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from which the test sample is obtained. For example, the reference sample can be a non-diseased sample adjacent to or near the test sample (e.g., a tumor). In some embodiments, the test and / or determination of the reference is performed substantially simultaneously with the test or determination of interest. In some embodiments, the reference is a historical reference optionally embodied in a tangible medium. Typically, as will be appreciated by those skilled in the art, the reference is determined or characterized under conditions or environments comparable to those under evaluation.

[0400] In certain embodiments, the tumors and cancers are metastatic, particularly metastatic tumors that express GUCY2C.

[0401] The therapeutically effective amount of an antibody or antigen-binding fragment thereof as provided herein will depend on various factors known in the art, such as body weight, age, past medical history, current medications, the health status of the subject and the potential for cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease progression. As indicated by these and other circumstances or requirements, one of ordinary skill in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage.

[0402] In certain embodiments, antibody or its Fab as provided herein can be used with about 0.01mg / kg to the treatment effective dose of about 100mg / kg.In certain embodiments in these embodiments, antibody or its Fab is used with about 50mg / kg or dosage still less, and in certain embodiments in these embodiments, dosage is 10mg / kg or still less, 5mg / kg or still less, 3mg / kg or still less, 1mg / kg or still less, 0.5mg / kg or still less or 0.1mg / kg or still less.In certain embodiments, administration dosage can change in therapeutic process.For example, in certain embodiments, initial administration dosage can be higher than administration dosage subsequently.In certain embodiments, administration dosage can change according to experimenter's reaction in therapeutic process.

[0403] Dosage regimens may be adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0404] The antibodies or antigen-binding fragments thereof provided herein can be administered by any route known in the art, e.g., parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal or topical) routes.

[0405] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein can be administered alone or in combination with one or more additional therapeutic modalities or agents. For example, the antibodies or antigen-binding fragments thereof disclosed herein can be administered in combination with another therapeutic agent, such as a chemotherapeutic agent or an anticancer drug.

[0406] In some of these embodiments, an antibody or antigen-binding fragment thereof as disclosed herein, administered in combination with one or more additional therapeutic agents, can be administered concurrently with the one or more additional therapeutic agents, and in some of these embodiments, the antibody or antigen-binding fragment thereof and the additional therapeutic agent can be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment thereof administered "in combination" with another therapeutic agent need not be administered concurrently with the agent or in the same composition as the agent. An antibody or antigen-binding fragment thereof administered before or after another agent is considered to be administered "in combination" with the agent, as the phrase is used herein, even if the antibody or antigen-binding fragment thereof and the second agent are administered by different routes. Where possible, additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments thereof disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)) or protocols well known in the art.

[0407] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the present invention. All specific compositions, materials and methods described below fall within the scope of the present invention in whole or in part. These specific compositions, materials and methods are not intended to limit the present invention, but are only used to illustrate specific embodiments falling within the scope of the present invention. Those skilled in the art can develop equivalent compositions, materials and methods without exercising inventive ability and without departing from the scope of the present invention. It should be understood that many changes can be made in the procedures described herein while still remaining within the scope of the present invention. It is the intention of the inventors of the present invention that such changes are all included within the scope of the present invention.

[0408] Example

[0409] Example 1 Screening and Identification of Anti-huGUCY2C Murine Monoclonal Antibodies

[0410] 1.1hu / cy GUCY2C antigen preparation

[0411] The huGUCY2C extracellular domain (ECD) was fused with mouse IgG2a Fc and His tag for mouse immunization; the hu / cy GUCY2C extracellular domain (ECD) was fused with Avi-His tag for biotinylation modification and antibody screening.

[0412] The corresponding protein sequences are as follows:

[0413] huGUCY2C ECD-mIgG2a Fc-His:

[0414] huGUCY2C ECD-Avi-His:

[0415] cyGUCY2C ECD-Avi-His:

[0416] Table 9: CD3 and GUCY2C antigen sequences

[0417] 1.2 Immunization of mice with huGUCY2C

[0418] Ten Balb / C mice were immunized with 50 μg of huGUCY2C ECD-mIgG2a Fc-His fusion protein per immunization, followed by a weekly booster immunization with 25 μg of huGUCY2C ECD-mIgG2a Fc-His. Serum titers were measured 3-5 times after immunization. Serum from mice with acceptable titers was analyzed by flow cytometry. Combining the results of ELISA and flow cytometry, mice with high titers were selected for Beacon monoclonal antibody production. Phage libraries were constructed from the remaining mice with low titers.

[0419] 1.3 Screening of anti-huGUCY2C mouse monoclonal antibodies based on Beacon single B cell technology

[0420] Prepare antigen microspheres: Take 10-50 μg of biotin-labeled hu / cyGUCY2C ECD-Avi-His and incubate them with the microspheres. Then use positive antibodies or positive serum to perform quality control on the antigen microspheres.

[0421] Plasma cell isolation: spleens of two mice with qualified titers were collected to prepare single-cell suspensions, which were then sorted using magnetic beads to enrich plasma cells.

[0422] Plasma cell introduction chip: Plasma cells are introduced into the beacon system using a 14K chip containing 14,000 chambers.

[0423] Antigen microsphere-based binding assay: hu / cyGUCY2C ECD-Avi-His microspheres and fluorescently labeled secondary antibodies are introduced into the chip. By observing the formation of antigen microsphere fluorescence, it is determined whether the corresponding plasma cells secrete antibodies that specifically bind to hu / cyGUCY2C.

[0424] Cell export: Select target cells and export them to a well plate containing cell lysate for sequencing and analysis of antibody sequences.

[0425] 1.4 Screening of anti-huGUCY2C mouse monoclonal antibodies based on phage display technology

[0426] Total RNA was extracted from single mouse spleen cells using Trizol lysis and reverse transcribed into cDNA. PCR-amplified VH and VL fragments were then inserted into phagemid vectors. The constructed phagemids were packaged with helper phage into a phage library with a capacity of approximately 3.0E+8. After three rounds of phage library panning, clones positive for ELISA were selected and sequenced.

[0427] 1.5 Identification of cross-reactivity of anti-huGUCY2C chimeric antibodies

[0428] In this experiment, 96-well plates were coated with huGUCY2C ECD-Avi-His, cyGUCY2C ECD-Avi-His, and mouse GUCY2C ECD-His (purchased from ACRO, Catalog No. GUC-M52H3) as antigens. A screened anti-huGUCY2C mouse monoclonal antibody was used as the primary antibody, diluted to 20 nM in 5% skim milk, and incubated for 1 hour. After three washes with PBST, anti-mouse F(ab)2-HRP (purchased from Jackson, Catalog No. 115-006-071) diluted 5000-fold in 5% skim milk was added as the secondary antibody and incubated for 45 minutes. TMB staining was performed and the results, as shown in Table 10, showed that M0047, GCAb0002, and GCAb0005 specifically bound to hu / cy / mouseGUCY2C ECD, while M0055 specifically bound to hu / cyGUCY2C.

[0429] Table 10: Cross-reactivity of anti-huGUCY2C chimeric antibodies

[0430] 1.6 Flow cytometry detection of the binding activity of anti-huGUCY2C chimeric antibody to tumor cells

[0431] The experiment used T84 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) with high expression of GUCY2C on the cell surface and GUCY2C-HEK293 cells (purchased from Jiman Biotechnology (Shanghai) Co., Ltd.) as target cells, and HEK293 cells (donated by Jiman Biotechnology (Shanghai) Co., Ltd.) as negative control. The cells were washed three times with staining buffer (PBS + 2% FBS (Gibco, Cat#10091148) + 5mM EDTA (Gibco, Cat#15575020)), centrifuged at 450g for 5 minutes each time, and the supernatant was discarded. The cells were resuspended in staining buffer at a cell density of 1×10 6 Cells were diluted to 400 nM at 100 μL / well in a 96-well V-bottom plate (Beyotime, Catalog #FPT019). Anti-huGUCY2C chimeric antibodies GCAb0002, GCAb0005, M0055, and M0047, the positive control PF-1608 monoclonal antibody (Pfizer's anti-GUCY2C antibody PF1608 was used as a control), and the negative control huIgG1 antibody (Anti-HEL Human IgG1-Kappa Isotype control, abinvivo, Catalog #:B117901) were diluted to 400 nM and added to 100 μL / well in a 96-well V-bottom plate. The cells were then mixed evenly with T84, GUCY2C-HEK293, and HEK293 cells, respectively. The cells were incubated at 4°C for 30 min, and the cells were washed three times with PBS to remove unbound antibody. Then, 100 μL / well of a 1:1000 dilution of goat anti-human IgG-PE (Southern Biotech, Cat# 2040-09) secondary antibody was added and incubated at 4°C for 30 minutes. The cells were centrifuged at 450 g for 5 minutes and washed three times with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 100 μL of PBS, and the binding affinity of the monoclonal antibodies to T84 and GUCY2C-HEK293 cells was measured using a Beckman Coulter CytoFLEX flow cytometer. Data were analyzed using FlowJo V10.8.1 software.

[0432] The experimental results are shown in Figure 1. M0055 has a higher MFI value on tumor cells T84. GCAb0002, GCAb0005, M0055, and M0047 all have higher MFI values ​​on GUCY2C-HEK293 and show no nonspecific binding on HEK293.

[0433] 1.7 Fortebio determination of affinity of anti-huGUCY2C chimeric antibody

[0434] The Fortebio Octet R8 molecular interaction instrument was used, and The AHC2 Biosensors probe capture assay was used to determine the kinetic parameters of binding between an anti-huGUCY2C antibody and the hu / cyGUCY2C ECD-Avi-His antigen. The AHC2 probe was activated by soaking it in 1× PBS for 20 minutes. The huGUCY2C antibody was diluted to 10 μg / ml in 1× PBS and the probe was soaked in PBS for 120 seconds to allow binding. The probe was then soaked in 1× PBS for another 120 seconds. The hu / cyGUCY2C ECD-Avi-His antigen was diluted two-fold downwards from 400 nM in 1× PBS, creating three concentration gradients. The probe was soaked in PBS for 180 seconds to measure the association rate. The probe was then soaked in 1× PBS for 360 seconds to measure the dissociation rate.

[0435] Both M0055 and GCAb0002 had good affinity for the hu / cyGUCY2C ECD, and the binding kinetic parameters are shown in Table 11. The KD values ​​for M0055 with huGUCY2C were 4.16E-8M and 1.36E-8M for cyGUCY2C; the KD values ​​for GCAb0002 with huGUCY2C were 7.35E-9M and 1.47E-8M for cyGUCY2C.

[0436] Table 11: Affinity of anti-huGUCY2C chimeric antibodies

[0437] 1.8 Fortebio analysis of anti-huGUCY2C chimeric antibody epitopes

[0438] The experiment used human IgG1 monoclonal antibodies expressed by the publicly published international patent sequence No. WO2017136693A1 5F9 and No. WO2019224716A2 PF-1608 as controls.

[0439] >5F9-VH:

[0440] >5F9-VL:

[0441] >PF-1608-VH:

[0442] >PF-1608-VL:

[0443] The Fortebio Octet R8 molecular interaction instrument was used, and The kinetic parameters of the binding between the anti-huGUCY2C specific antibody and the hu / cyGUCY2C ECD-Avi-His antigen were determined by the HIS1K Biosensors probe capture method. The HIS1K probe was immersed in 1×PBS working solution for 20 minutes to activate the probe. The huGUCY2C ECD-Avi-His antigen was diluted to 40μg / ml with 1×PBS working solution, and the probe was immersed in it for 120s to allow the probe to bind to the antibody. The probe was immersed in 1×PBS working solution for another 120s. The anti-GUCY2C monoclonal antibody was diluted with 1×PBS working solution to 200nM for the first antibody and 800nM for the second antibody, as shown in Figure 2. Epitope analysis was performed using the In-tandem method of the Binning epitope analysis guide.

[0444] The results in Table 12 show that GCAb0002 competes with the PF-1608 epitope, while M0055 and 5F9 are separate epitopes.

[0445] Table 12: Epitope analysis of anti-huGUCY2C chimeric antibodies

[0446] 1.9 Flow cytometry detection of the binding activity of huGUCY2C bi-epitope antibody on tumor cells

[0447] The target cells were T84 cells that highly expressed GUCY2C on their cell surface. The cells were washed three times with staining buffer, centrifuged at 450 g for 5 min each time, and the supernatant was discarded. The cells were resuspended in staining buffer at a cell density of 1×10 6100 μL / well of the 96-well plate was added to 100 cells / mL. Anti-huGUCY2C chimeric antibodies GCAb0002, M0055, and M0055 + GCAb0002, the positive control PF-07062119 antibody, and the negative control huIgG1 antibody (Anti-HEL Human IgG1-Kappa Isotype control, abinvivo, Catalog #: B117901) were diluted to 500 nM and serially diluted 3-fold over 10 steps (maximum concentration 500 nM). 100 μL / well of the 96-well V-bottom plate was added and mixed evenly with the T84 cells. Incubate at 4°C for 30 minutes, then wash the cells three times with staining buffer to remove unbound antibody. Then, 100 μL / well of a 1:1000 dilution of goat anti-human secondary antibody was added and incubated at 4°C for 30 minutes. Centrifuge at 450 g for 5 minutes, then wash the cells three times with staining buffer to remove unbound secondary antibody. Finally, the cells were resuspended in 100 μL of staining buffer, and the binding affinity of the monoclonal antibody to T84 was measured using a Beckman Coulter CytoFLEX flow cytometer. The data were analyzed using FlowJo_V10.8.1 software and fitted using GraphPad Prism software.

[0448] The experimental results are shown in Figure 2. Starting from 18nM, the MFI value of M0055+GCAb0002 binding to T84 cells is higher than the MFI value of T84 binding to M0055 and GCAb0002 respectively. At the same time, as shown in Table 13, the EC50 value of M0055+GCAb0002 is about 1.46 times lower than that of M0055. 50 The values ​​were 7.44 nM and 10.84 nM, respectively.

[0449] Table 13: EC of bi-epitope antibodies on T84 cells 50 and MFI max

[0450] Example 2 Identification of Anti-huGUCY2C Antibody Binding Epitope

[0451] 2.1 Prediction of huGUCY2C antigen immunogenicity based on AI model

[0452] An AI-powered immunogenicity prediction model was used to estimate the confidence level of potential anti-huGUCY2C antibody binding epitopes. Using the huGUCY2C ECD structure as input, the model assigned a probability score to each residue position, with higher scores indicating a higher probability of binding epitopes. Epitopes with higher binding probabilities are highlighted, as shown by the darker markers in Figure 3.

[0453] 2.2 Anti-huGUCY2C antibody binding epitope prediction

[0454] Using random sampling techniques from an AI-based structure prediction model based on antigen-antibody pairings, we predicted binding epitopes for all anti-huGUCY2C antibodies screened. For epitope prediction, we generated multiple candidate positions for each antibody, ranked them from highest to lowest probability, and grouped the epitopes for each possible antigen-antibody combination. This was further validated through experimental antibody grouping experiments, eliminating several candidate positions for each antibody, ultimately resulting in a more streamlined set of candidate epitopes.

[0455] 2.3 Prediction and verification of huGUCY2CECD epitope mutations

[0456] Combining the probability scores of candidate epitopes obtained in Examples 2.1 and 2.2, all anti-huGUCY2C antibodies were classified according to the antigen-binding epitopes, and finally the possible binding sites were obtained. As shown in Figure 4 (left and right), the structural diagrams of M0055 and GCAb0002 predicted to bind to huGUCY2CECD from different perspectives are shown. M0055 is predicted to bind to 3 epitopes, and GCAb0002 is predicted to bind to 1 epitope.

[0457] Key residues on the candidate epitope were point mutated, and the mutant plasmids were transfected into HEK293 cells. The mutant strains were screened for Zeocin resistance. The huGUCY2C ECD mutant was used as the target cell line. The cells were washed three times with staining buffer, centrifuged at 450 g for 5 minutes each time, and the supernatant was discarded. The cells were resuspended in staining buffer at a cell density of 1×10 6 Cells were diluted to 100 μL / well in a 96-well plate. M0055, GCAb0002, control antibodies 5F9 and PF-1608, and a negative control huIgG1 antibody (Anti-HEL Human IgG1-Kappa Isotype control, abinvivo, Catalog #: B117901) were diluted to 100 nM and added to the 96-well plate at 100 μL / well. The cells were mixed thoroughly. Incubated at 4°C for 30 minutes. The cells were washed three times with staining buffer to remove unbound antibody. Then, 100 μL / well of a 1:1000 dilution of goat anti-human secondary antibody was added and incubated at 4°C for 30 minutes. Centrifuged at 450 g for 5 minutes, the cells were washed three times with staining buffer to remove unbound secondary antibody. Finally, the cells were resuspended in 100 μL of staining buffer, and binding activity was measured using a Beckman Coulter CytoFLEX flow cytometer. The data were analyzed using FlowJo v10.8.1 software and fitted using GraphPad Prism software. Multiple mutant display cell lines were constructed, and antibody binding to the mutants was analyzed by flow cytometry. Mutants that ultimately lost antibody binding were identified as the precise sites of antigen-antibody binding.

[0458] Based on the antigen-binding site disclosed by PF-1608, huGUCY2C ECD multi-site mutants (S15N, S62F, I66L, and L80V) were constructed. Flow cytometry results are shown in Figure 5. HuGUCY2C ECD mutants (S15N, S62F, I66L, and L80V) do not bind to PF-1608, but still bind to 5F9, M0055, and GCAb0002.

[0459] Based on AI-predicted binding sites for M0055 and the antigen, single-point mutations were selected to construct huGUCY2CECD mutants (E55A, D56A, N59A, E63A, Q70A, K403D, Y405T, and M409T). The flow cytometry results are shown in Figure 6. The single-point mutants (E55A, D56A) do not bind to M0055, but still bind to PF-1608, 5F9, and GCAb0002. Based on the binding results of the mutants with the antibodies, the precise epitopes of M0055 were determined to be E55 and D56.

[0460] Based on the prediction of the GCAb0002 antigen binding site by AI, single-point mutations were selected to construct huGUCY2C ECD mutants (E101A, D104A, L105E, R107A, Q130A, D134A, and K154A). The flow cytometry results are shown in Figure 7. The single-point mutants (E101A, L105E, and R107A) did not bind to GCAb0002, but still bound to PF-1608, 5F9, and M0055. Based on the results of the mutant binding to the antibody, the precise epitopes of GCAb0002 were E101, L105, and R107.

[0461] 2.4 Precision structure prediction of anti-huGUCY2C antibody-huGUCY2C complex

[0462] After obtaining the precise antigen epitope in Example 2.3, the position of the precise antigen epitope mutation was predicted using an antibody-based AI structure prediction model and additional information enhancement technology for the model. First, a fast empirical force field function was used to search for important binding positions on the antigen to obtain potential amino acid side chain binding conformations with lower energy in three-dimensional space. This was used as additional information to re-predict the polar structure, and finally the precise complex structure was obtained as shown in Figure 8. Figure 8 (left and right) shows different perspectives of the M0055@huGUCY2C ECD@GCAb0002 complex. As shown in the left figure, the middle is the huGUCY2C ECD structure, the upper left corner is the scFv structure of M0055 bound to huGUCY2C ECD, and the upper right corner is the scFv structure of GCAb0002 bound to huGUCY2C ECD. As shown in the figure on the right, from top to bottom are the scFv structure of M0055 bound to huGUCY2C ECD, the huGUCY2C ECD structure, and the scFv structure of GCAb0002 bound to huGUCY2C ECD.

[0463] Example 3: Configuration Optimization and Activity Testing of Anti-huCD3 Humanized Antibody and Anti-huGUCY2C Murine Chimeric Bispecific Antibody

[0464] 3.1 GUCY2C chimeric multispecific antibody configuration design

[0465] Using the VH, VL, and scFv sequences of the anti-GUCY2C murine antibodies M0055 and GCAb0002, and the VH, VL, and scFv sequences of the anti-huCD3ed antibody CD3-002 / 007, we designed antibodies with bispecific binding activity: GCbi0007 ( FIG. 9A ), GCbi0019 ( FIG. 9B ), GCbi0021 ( FIG. 9C ), GCbi0023 ( FIG. 9D ), GCbi0025 ( FIG. 9E ), and GCbi0026 ( FIG. 9F ).

[0466] Using the VH and VL sequences of the anti-GUCY2C murine antibodies M0055 and GCAb0002, the VH and VL and scFv sequences of the anti-huCD3ed antibody CD3-002 / 007, and the VH and VL and scFv sequences of the anti-CD28 antibody, the antibodies GCTr0001 (Figure 9G), GCTr0002 (Figure 9H), GCTr0006 (Figure 9I), GCTr0009 (Figure 9J), GCTr0010 (Figure 9K), GCTr0011 (Figure 9L) and GCTr0012 (Figure 9M) with trispecific binding activity were designed.

[0467] At the same time, the GUCY2C negative bispecific antibody IsoB0001 was designed using the VH and VL sequences of the anti-HEL and the scFv sequences of the anti-huCD3ed antibody CD3-002 ( FIG. 9N ).

[0468] The sequences of the above multispecific antibodies are shown in Table 7, and the configurations are shown in Table 8.

[0469] 3.2 Fortebio Determines the Affinity of the GUCY2C×CD3 Chimeric Bispecific Antibody

[0470] Using the Fortebio Octet R8 molecular interaction instrument, The AHC2 Biosensors probe capture assay was used to determine the kinetic parameters of binding between a chimeric bispecific antibody and the huGUCY2C ECD-Avi-His antigen. The AHC2 probe was activated by soaking it in 1× PBS for 20 minutes. GCbi0007, GCbi0019, GCbi0021, GCbi0023, GCbi0026, and PF-07062119 were diluted to 10 μg / ml in 1× PBS and the probes were soaked for 120 seconds to allow binding. The probes were then soaked in 1× PBS for another 120 seconds. The huGUCY2C ECD-Avi-His antigen was diluted two-fold downwards in 1× PBS from 400 nM to form three concentration gradients. The probes were soaked for 180 seconds to measure the association rate of the antigen-antibody. The probes were then soaked in 1× PBS for 360 seconds to measure the dissociation rate of the antigen-antibody. The results in Table 14 and Figure 10 (A, C) show that the affinities of GCbi0007 and GCbi0021 are close to those of the corresponding monoclonal antibodies M0055 and GCAb0002. The results in Table 14 and Figure 10 (B, D, E) show that the binding affinities of GCbi0019, GCbi0023, and GCbi0026 to huGUCY2C are significantly improved, with KD values ​​of 5.06E-12M, 5.63E-12M, and 3.76E-12M, respectively; the KD value of PF-07062119 (Figure 10F) is 3.60E-09M.

[0471] Table 14: Affinity of GUCY2C×CD3 chimeric bispecific antibodies binding to huGUCY2C

[0472] At the same time, the experiment used the Fortebio Octet R8 molecular interaction instrument, The HIS1K Biosensor probe capture assay was used to determine the kinetic parameters of binding of GCbi0007, GCbi0019, and GCbi0021 to the huGUCY2C ECD-Avi-His antigen. The HIS1K probe was activated by soaking in 1× PBS for 20 minutes. huGUCY2C ECD-Avi-His was diluted to 10 μg / ml in 1× PBS and the probe was soaked in PBS for 120 seconds to allow binding. The probe was then soaked in 1× PBS for another 120 seconds. GCbi0007, GCbi0019, and GCbi0021 were diluted two-fold downwards from 200 nM in 1× PBS to create three concentration gradients. The probes were soaked in PBS for 180 seconds to measure the antigen-antibody association rate. The probes were then soaked in 1× PBS for 360 seconds to measure the antigen-antibody dissociation rate. The results in Table 15 and Figure 10 (G, H, I) show that the KD values ​​of huGUCY2C ECD-Avi-His binding to GCbi0007, GCbi0019, and GCbi0021 were 2.23E-12M, 1.48E-12M, and 2.18E-12M, respectively.

[0473] Table 15: Avidity of GUCY2C×CD3 chimeric bispecific antibodies binding to huGUCY2C

[0474] The experiment used the Fortebio Octet R8 molecular interaction instrument, The AHC2 Biosensors probe capture assay was used to determine the kinetic parameters of binding between a GUCY2C×CD3 chimeric bispecific antibody and human CD3 epsilon and CD3 delta-His antigens. The AHC2 probe was activated by soaking it in 1× PBS for 20 minutes. The GUCY2C×CD3 chimeric bispecific antibody was diluted to 20 μg / ml in 1× PBS and the probe was soaked in PBS for 120 seconds to allow binding. The probe was then soaked in 1× PBS for another 120 seconds. Human CD3 epsilon and CD3 delta-His (purchased from ACRO, Catalog No. CDD-H52W1) was diluted two-fold downwards in 1× PBS from 400 nM. Three concentration gradients were established, and the probe was soaked in PBS for 180 seconds to measure the antigen-antibody association rate. The probe was then soaked in 1× PBS for 360 seconds to measure the antigen-antibody dissociation rate. The results in Table 16 and Figure 11 show that compared to IsoB0001 (Figure 11G, KD value of 6.33E-08M), GCbi0007 (Figure 11A), GCbi0019 (Figure 11B), and GCbi0021 (Figure 11C) have weakened affinities, with KD values ​​of 1.3E-07M, 1.5E-07M, and 2.0E-07M, respectively. The KD values ​​of conformations GCbi0023 (Figure 11D), GCbi0025 (Figure 11E), and GCbi0026 (Figure 11F) are 4.02E-08M and 3.83E-08M, respectively.

[0475] Table 16: Affinity of GUCY2C×CD3 chimeric bispecific antibodies binding to huCD3ed

[0476] 3.3 Flow cytometry detection of tumor cell binding activity of GUCY2C×CD3 chimeric bispecific antibody

[0477] The target cells were T84 cells that highly expressed GUCY2C on their cell surface. The cells were washed three times with staining buffer, centrifuged at 450 g for 5 min each time, and the supernatant was discarded. The cells were resuspended in staining buffer at a cell density of 1×10 6100 μL / well of the 96-well plate was added to 100 cells / mL. GUCY2C×CD3 chimeric bispecific antibodies GCbi0007, GCbi0019, and GCbi0021, the positive control PF-07062119 antibody, and the negative control huIgG1 antibody (Anti-HEL Human IgG1-Kappa Isotype control, abinvivo, Catalog #: B117901) were diluted to 500 nM and serially diluted 3-fold over 10 steps (maximum concentration 500 nM). 100 μL / well of the 96-well plate was added and mixed evenly with the T84 cells. The cells were incubated at 4°C for 30 minutes, and the cells were washed three times with staining buffer to remove unbound antibody. Then, 100 μL / well of a 1:1000 dilution of goat anti-human secondary antibody was added and incubated at 4°C for 30 minutes. The cells were centrifuged at 450 g for 5 minutes, and the cells were washed three times with staining buffer to remove unbound secondary antibody. Finally, the cells were resuspended in 100 μL of staining buffer, and the binding activity of the GUCY2C×CD3 chimeric bispecific antibody to T84 was measured using a Beckman Coulter CytoFLEX flow cytometer. The data were analyzed using FlowJo_V10.8.1 software and fitted using GraphPad Prism software.

[0478] The experimental results are shown in Table 17 and Figure 12. The binding activities of GCbi0007, GCbi0019 and GCbi0021 are all higher than those of PF-07062119. As shown in Table 17, the EC values ​​of GCbi0007, GCbi0019 and GCbi0021 and PF-07062119 are 50 They are 7.65nM, 4.58nM, 1.38nM and 32.44nM respectively.

[0479] Table 17: EC of GUCY2C×CD3 chimeric bispecific antibodies on T84 cells 50 and MFI max

[0480] 3.4 Flow cytometry detection of pan T cell binding activity of GUCY2C×CD3 chimeric bispecific antibody

[0481] The experiment used pan T cells with high expression of human CD3 on the cell surface as target cells. TMHuman T Cell Isolation Kit (Stemcell, Cat#17951) was used to isolate cells from commercial PBMCs (Aoneng Biotech, Donor ID#Y1584) according to the kit instructions. The cells were washed three times with staining buffer, centrifuged at 450 g for 5 min each time, and the supernatant was discarded. The cells were resuspended in staining buffer at a cell density of 1×10 6 100 μL / well of the 96-well V-bottom plate was added. GUCY2C × CD3 chimeric bispecific antibodies GCbi0007, GCbi0019, and GCbi0021, the positive control PF-07062119 antibody, and the negative control huIgG1 antibody (Anti-HEL Human IgG1-Kappa Isotype control, abinvivo, Catalog #: B117901) were diluted to 500 nM and serially diluted 3-fold over 10 steps (maximum concentration 500 nM). 100 μL / well of the 96-well plate was added and mixed evenly with the pan T cells. The cells were incubated at 4°C for 30 minutes. The cells were then washed three times with staining buffer to remove unbound antibody. Then, 100 μL / well of a 1:1000 dilution of goat anti-human secondary antibody was added and incubated at 4°C for 30 minutes. The cells were centrifuged at 450 g for 5 minutes and washed three times with staining buffer to remove unbound secondary antibody. Finally, the cells were resuspended in 100 μL of staining buffer, and the binding activity of the GUCY2C×CD3 chimeric bispecific antibody to pan T was measured using a Beckman Coulter CytoFLEX flow cytometer. The data were analyzed using FlowJo_V10.8.1 software and fitted using GraphPad Prism software.

[0482] The experimental results are shown in Table 18 and Figure 13. At a concentration of 100 nM, the maximum MFIs of GCbi0007, GCbi0019, GCbi0021, and PF-07062119 binding to pan T cells were 9179, 8838, 12041, and 9092, respectively.

[0483] Table 18: EC of GUCY2C×CD3 chimeric bispecific antibodies on panT cells 50 and MFI max

[0484] 3.5 Study on the TDCC-mediated killing activity of GUCY2C×CD3 chimeric bispecific antibody against T84, HT55, and HCT15 tumor cells

[0485] This experiment used GUCY2C high-expressing T84 cells, medium-expressing HT55 cells, and negative HCT15 cells as target cells. Tumor cells in the logarithmic growth phase were digested with trypsin (Source Culture, Cat#S310KJ) and resuspended in DMEM medium (Gibco, Cat#11965092) containing 10% FBS (Gibco, Cat#10091148) and the cell density was adjusted to 1×10 5 cells / mL, and tumor cells were added to a 96-well U-bottom plate (NEST, Cat#701101), with 100 μL added to each well, i.e., 1×10 4 Cells / well were grown overnight. Then different concentrations of the antibody to be tested were added, diluted with DMEM medium containing 10% FBS. The starting working concentration of the antibody to be tested was 400nM (4X working concentration), and 5-fold serial dilution was performed, with a total of 9 different concentrations, 50μL per well. TM Human T Cell Isolation Kit instructions: Effector cells (pan T) were isolated from commercial PBMCs (Sai ​​Li Biotechnology, Donor ID# XW0801211W). The cells were resuspended in DMEM medium containing 10% FBS and the cell concentration was adjusted to 1×10 cells / mL according to an E:T ratio of 5:1. 6 mL, add 5×10 4 Cells / 50μL. Note: Frozen PBMCs can be revived one day in advance and cultured overnight with RPMI 1640 (Source Culture, Cat#L210KJ) + 10% FBS medium. Add appropriate amount of DNase to prevent DNA entanglement of dead cells. Place the 96-well U-bottom plate in a 37°C, 5% CO2 incubator and incubate for 72 hours. 2 hours before the end of incubation, add 10×Lysis buffer (Promega, Cat#G182A) to the wells containing only target cells and continue incubation for 1 hour. Centrifuge the culture plate at 450g for 5 minutes and transfer 50μL of supernatant to a 96-well flat-bottom plate (NEST, Cat#701301). Press The substrate solution was prepared according to the instructions of the Non-Radioactive Cytotoxicity Assay (Promega, Cat# G1780) kit, equilibrated to room temperature, and 50 μL was added to each well. The cells were incubated at room temperature in the dark for approximately 30 minutes. The reaction was terminated by adding 50 μL of Stop Solution to each well. The absorbance at 490 nm or 492 nm was measured using a microplate reader (TECAN, Spark). The logarithm of the antibody concentration was plotted against the killing ratio using GraphPad Prism, and the EC was calculated. 50 .

[0486] The experimental results are shown in Table 19 and Figure 14. In the TDCC experiment, the killing activities of GCbi0007 and GCbi0021 in T84 and HT55 cells were weaker than those of GCbi0019. 50 The activity was similar to that of PF-07062119 (Figure 14A), but superior to that of PF-07062119 in HT55 (Figure 14B). GCbi0007, GCbi0019, and GCbi0021 had no nonspecific T cell killing in negative HCT15 cells; IsoB0001 had no nonspecific killing activity in all tumor cells.

[0487] Table 19: TDCC EC of GUCY2C×CD3 chimeric bispecific antibodies against HT55, T84, and HCT15 50 and Emax

[0488] 3.6 Study on the activation effect of GUCY2C×CD3 chimeric bispecific antibody on T cells in TDCC killing assay of HT55, T84 and HCT15 tumor cells

[0489] In this study, in the TDCC experiment of Example 3.5, after the killing experiment incubation, the 96-well U-bottom plate was removed, centrifuged at 450g for 5 minutes, and the supernatant was discarded; then the staining solution was prepared with staining buffer, including Brilliant Violet 785 TM anti-human CD3 Antibody (BioLegend, Cat#344842), Brilliant Violet 605 TM anti-human CD4 Antibody (BioLegend, Cat#344646), Brilliant Violet 421 TM anti-human CD8 Antibody (BioLegend, Cat#344748), BD Pharmingen TM PE Mouse Anti-Human CD25 (BD, Cat#555432) and BD Pharmingen TM APC Mouse Anti-Human CD69 (BD, Cat#555533) and other flow cytometry antibody solutions (panel shown in Table 20) were prepared. The cells were resuspended with the prepared flow cytometry antibody solution and incubated at 4°C in the dark for 30 minutes.

[0490] Table 20: Flow pannel

[0491] After incubation, centrifuge at 450g for 5 minutes, discard the supernatant, wash twice with staining buffer, centrifuge, discard the supernatant, add 100 μL of staining buffer to each well of a 96-well cell plate, and collect cell detection signals using a flow cytometer CytoFLEX (Beckman). After data collection, FlowJo software was used to export CD4 cells and CD8 cells CD25 + CD69 + The percentage of the population was calculated and the logarithm of the sample concentration was taken using Graphpad and compared with CD25 + CD69 + The percentage of double positive samples was plotted using four-parameter fitting.

[0492] The experimental results are shown in Figure 15. In the T cell activation assay accompanying the TDCC assay, the GUCY2C×CD3 chimeric bispecific antibodies GCbi0007, GCbi0019, and GCbi0021 demonstrated overall lower activity than PF-07062119. GCbi0007, GCbi0019, and GCbi0021 showed no nonspecific T cell activation in negative HCT15 cells; IsoB0001 also showed no nonspecific T cell activation in tumor cells.

[0493] 3.7 Study on the release levels of cytokines IL-2, IFN-γ, IL-6, and TNF-α during the killing effect of GUCY2C×CD3 chimeric bispecific antibody on HT55, T84, and HCT15 TDCC

[0494] In this study, in the TDCC experiment of Example 3.5, after the incubation of the killing experiment, the 96-well U-bottom plate was removed and centrifuged at 450g for 5 minutes to collect the cell supernatant. The release levels of various cytokines in the TDCC reaction supernatant were then measured according to the CBA reagent instructions. First, according to the instructions, the standard pellet was transferred to a centrifuge tube, diluted with diluent, and allowed to stand for 15 minutes. Human IL-2 (BD TM Cytometric Bead Array(CBA)Human IL-2Flex Set,BD,Cat#558270), human TNF-α(BD TM Cytometric Bead Array(CBA)Human TNF Flex Set,BD,Cat#560112), human IFN-γ(BD TM Cytometric Bead Array (CBA) Human IFN-γFlex Set, BD, Cat#558269) and human IL-6 (BD TMCapture microspheres from the Cytometric Bead Array (CBA) Human IL-6 Flex Set (BD, Cat# 558276) were mixed in a 1:1 ratio, vortexed thoroughly before mixing, and then aliquoted into a 96-well plate, 50 μL per well. Dilute the supernatant sample as needed and prepare the standard according to the kit instructions. Add the diluted standard and sample to the 96-well plate, mix thoroughly with the previously added microsphere mixture, and incubate on a shaker at 500 rpm for 5 minutes before incubating in the dark for 1 hour. Detection antibodies for IL-2, TNF-α, IFN-γ, and IL-6 were mixed in a 1:1 ratio and aliquoted into a 96-well plate, 50 μL per well. Mix on a shaker at 500 rpm for 5 minutes before incubating in the dark for 2 hours. After the reaction, add 150 μL of wash buffer, centrifuge at 500 g for 5 minutes, discard the supernatant, resuspend in 100 μL of wash buffer, and analyze on a flow cytometer. Finally, FlowJo was used to analyze the data and export the MFI value of each cytokine in each well. The cytokine concentration of the sample well was calculated based on the standard well. Then, Graphpad was used to take the logarithm of the antibody concentration and perform a four-parameter fitting graph with the cytokine concentration.

[0495] The experimental results are shown in Figures 16, 17, and 18. In the cytokine (IL-2, IFN-γ, IL-6, and TNF-α) release study accompanying the TDCC experiment, the cytokine release levels of GUCY2C×CD3 chimeric bispecific antibodies GCbi0007, GCbi0019, and GCbi0021 were generally lower than those of PF-07062119, and there was no non-specific cytokine release in the negative cell HCT15 cells.

[0496] 3.8 Study on the nonspecific release of cytokines IL-2, IFN-γ, IL-6, and TNF-α by GUCY2C×CD3 chimeric bispecific antibody in PBMCs

[0497] The negative HEK293 cells were used as target cells. Tumor cells in the logarithmic growth phase were digested with trypsin, resuspended in 1640 medium (Gibco, Cat#11875093) containing 10% FBS, and the cell density was adjusted to 1×10 5 cells / mL, tumor cells were added to a 96-well U-bottom plate, 100 μL was added to each well, i.e. 1×10 4 In this experiment, commercial PBMC (Aoneng Biotech, Donor ID#Z0315) were used as effector cells. The cells were resuspended in 1640 medium containing 10% FBS and the cell density was adjusted to 2×10 6 cells / mL, PBMCs were added to a 96-well U-bottom plate, 50 μL was added to each well, i.e. 1×10 5cells / well. Then add different concentrations of the antibody to be tested diluted with 1640 culture medium containing 10% FBS. The starting working concentration of the antibody to be tested is 400nM (4X working concentration), 5-fold gradient dilution, a total of 10 different concentrations, 50μL per well. This experiment uses anti-CD3 monoclonal antibody OKT3 (Purified anti-human CD3 Antibody, BioLegend, Cat#317302) as a positive control antibody. Then place the 96-well U-bottom plate in a 37°C, 5% CO2 incubator and incubate for 24h and 72h respectively. After the incubation is completed, remove the 96-well U-bottom plate, centrifuge at 450g for 5min, and collect the cell supernatant. Then detect the release level of each cytokine in the supernatant according to the CBA reagent instructions. First, according to the manufacturer's instructions, transfer the standard beads to a centrifuge tube and dissolve in diluent. After allowing to stand for 15 minutes, mix the capture beads for human IL-2, human TNF-α, human IFN-γ, and human IL-6 in a 1:1 ratio. Vortex the beads thoroughly before mixing and then aliquot 50 μL into a 96-well plate. Dilute the supernatant sample according to experimental requirements and prepare the standard according to the kit instructions. Add the diluted standard and sample to the 96-well plate, mix thoroughly with the previously added bead mixture, and incubate on a shaker at 500 rpm for 5 minutes. Then, incubate in the dark for 1 hour. Detection antibodies for IL-2, TNF-α, IFN-γ, and IL-6 are mixed in a 1:1 ratio and aliquot 50 μL into a 96-well plate. Then, incubate on a shaker at 500 rpm for 5 minutes and incubate in the dark for 2 hours. After the reaction, 150 μL of wash buffer was added, and the cells were centrifuged at 500 g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 100 μL of wash buffer before analysis on a flow cytometer. FlowJo was used to analyze the data, and the MFI values ​​of each cytokine in each well were derived. The cytokine concentrations in the sample wells were calculated based on the standard wells. Graphpad was then used to perform a four-parameter fitting of the antibody concentrations and the cytokine concentrations.

[0498] The experimental results are shown in Figures 19 and 20. In negative HEK293 cells, there was no high nonspecific cytokine release at 24 h and 72 h.

[0499] 3.9 Study on the TDCC killing activity of GUCY2C×CD3 optimized configuration against T84 tumor cells

[0500] In this experiment, cells expressing different levels of GUCY2C were used as target cells. Tumor cells in the logarithmic growth phase were digested with trypsin (Source Culture, Cat#S310KJ) and resuspended in DMEM medium (Gibco, Cat#11965092) containing 10% FBS (Gibco, Cat#10091148) and the cell density was adjusted to 1×10 5 cells / mL, and tumor cells were added to a 96-well U-bottom plate (NEST, Cat#701101), with 100 μL added to each well, i.e., 1×10 4 Cells / well were grown overnight. Then different concentrations of the antibody to be tested were added, diluted with DMEM medium containing 10% FBS. The starting working concentration of the antibody to be tested was 400nM (4X working concentration), and 5-fold serial dilution was performed, with a total of 9 different concentrations, 50μL per well. TM Human T Cell Isolation Kit instructions: Effector cells pan T were isolated from commercial PBMC (Sai ​​Li Biotechnology) and resuspended in DMEM medium containing 10% FBS. The cell concentration was adjusted to 1×10 based on an E:T ratio of 5:1. 6 mL, add 5×10 4 Cells / 50μL. Note: Frozen PBMCs can be revived one day in advance and cultured overnight with RPMI 1640 (Source Culture, Cat#L210KJ) + 10% FBS medium. Add appropriate amount of DNase to prevent DNA entanglement of dead cells. Place the 96-well U-bottom plate in a 37°C, 5% CO2 incubator and incubate for 72 hours. 2 hours before the end of incubation, add 10×Lysis buffer (Promega, Cat#G182A) to the wells containing only target cells and continue incubation for 1 hour. Centrifuge the culture plate at 450g for 5 minutes and transfer 50μL of supernatant to a 96-well flat-bottom plate (NEST, Cat#701301). Press CytoTox The substrate solution was prepared according to the instructions of the Non-Radioactive Cytotoxicity Assay (Promega, Cat# G1780) kit, equilibrated to room temperature, and 50 μL was added to each well. The cells were incubated at room temperature in the dark for approximately 30 minutes. The reaction was terminated by adding 50 μL of Stop Solution to each well. The absorbance at 490 nm or 492 nm was measured using a microplate reader (TECAN, Spark). The logarithm of the antibody concentration was plotted against the killing ratio using GraphPad Prism, and the EC was calculated. 50 .

[0501] Donor #XW081187W, high-expressing tumor cells T84, medium-expressing cells HT55 and LS1034, and negative cells HEK293 were used as target cells to compare the TDCC activities of GCbi0007, GCbi0019, GCbi0021, GCbi0023, and PF-07062119. The experimental results are shown in Table 21 and Figures 21A to 21D. The EC of GCbi0023 was 50 It has an advantage on T84, HT55 and LS1034, but is inferior to Iamx. Compared with PF-07062119, GCbi0019 has an EC of 50 It has more advantages, with Emax weaker than PF-07062119 on T84 and LS1034, and comparable to PF-07062119Iamx on HT55; all antibodies have no nonspecific killing on HEK293 cells.

[0502] Table 21: TDCC EC of GCbi0007, GCbi0019, GCbi0021, and GCbi0023 50 and Emax

[0503] Using Donor#Z0315 and low-expressing tumor cells SW403 as target cells, we compared the TDCC activities of GCbi0007, GCbi0019, GCbi0021, GCbi0025, and PF-07062119. The results are shown in Table 22 and Figure 22. In low-expressing tumor cells SW403, GCbi0019 and GCbi0025 exhibited comparable TDCC activity, while PF-07062119 exhibited the highest TDCC activity.

[0504] Table 22: TDCC EC of GCbi0007, GCbi0019, GCbi0021, and GCbi0025 50 and Emax

[0505] Using Donor #Y1584 and high-expressing tumor cells T84 as target cells, we compared the TDCC activity of GCbi0019, GCbi0026, and PF-07062119. The results are shown in Table 23 and Figure 23. PF-07062119 exhibited the best TDCC activity on T84 cells; GCbi0026 exhibited poor activity, while GCbi0019 exhibited superior TDCC activity to GCbi0025.

[0506] Table 23: TDCC EC of GCbi0019 and GCbi0026 50 and Emax

[0507] 3.10 Study on the TDCC-mediated cytotoxicity of GUCY2C×CD3×CD28 chimeric trispecific antibody against T84, HT55, and LS1034 tumor cells

[0508] In this experiment, GUCY2C high-expressing T84 cells, medium-expressing HT55 and LS1034 cells were used as target cells. Tumor cells in the logarithmic growth phase were digested with trypsin (Source Culture, Cat#S310KJ) and resuspended in DMEM medium (Gibco, Cat#11965092) containing 10% FBS (Gibco, Cat#10091148) and the cell density was adjusted to 1×10 5 cells / mL, and tumor cells were added to a 96-well U-bottom plate (NEST, Cat#701101), with 100 μL added to each well, i.e., 1×10 4 Cells / well were grown overnight. Then different concentrations of the antibody to be tested were added, diluted with DMEM medium containing 10% FBS. The starting working concentration of the antibody to be tested was 400nM (4X working concentration), and 5-fold serial dilution was performed, with a total of 9 different concentrations, 50μL per well. TM Human T Cell Isolation Kit instructions: Effector cells (pan T) were isolated from commercial PBMCs (Sai ​​Li Biotechnology, Donor ID# Y1652). The cells were resuspended in DMEM medium containing 10% FBS and the cell concentration was adjusted to 1×10 cells / mL according to an E:T ratio of 5:1. 6 mL, add 5×10 4 Cells / 50μL. Note: Frozen PBMCs can be revived one day in advance and cultured overnight with RPMI 1640 (Source Culture, Cat#L210KJ) + 10% FBS medium. Add appropriate amount of DNase to prevent DNA entanglement of dead cells. Place the 96-well U-bottom plate in a 37°C, 5% CO2 incubator and incubate for 72 hours. 2 hours before the end of incubation, add 10×Lysis buffer (Promega, Cat#G182A) to the wells containing only target cells and continue incubation for 1 hour. Centrifuge the culture plate at 450g for 5 minutes and transfer 50μL of supernatant to a 96-well flat-bottom plate (NEST, Cat#701301). Press The substrate solution was prepared according to the instructions of the Non-Radioactive Cytotoxicity Assay (Promega, Cat# G1780) kit, equilibrated to room temperature, and 50 μL was added to each well. The cells were incubated at room temperature in the dark for approximately 30 minutes. The reaction was terminated by adding 50 μL of Stop Solution to each well. The absorbance at 490 nm or 492 nm was measured using a microplate reader (TECAN, Spark). The logarithm of the antibody concentration was plotted against the killing ratio using GraphPad Prism, and the EC was calculated. 50 .

[0509] The experimental results are shown in Table 24 and Figure 24. In the TDCC assay on T84 cells, GCTr0001, GCTr0002, and GCTr0006 showed superior cytotoxicity to the control antibody PF-07062119, while the cytotoxicity of GCTr0006 was comparable to that of the control antibody PF-07062119. In the TDCC assay on HT55 cells, GCTr0001, GCTr0002, GCTr0006, and GCTr0011 showed superior cytotoxicity to the control antibody PF-07062119, while the cytotoxicity of GCTr0009 was comparable to that of the control antibody PF-07062119. In the TDCC assay on LS1034 cells, GCTr0001, GCTr0002, and GCTr0006 showed superior cytotoxicity to the control antibody PF-07062119.

[0510] Table 24: TDCC EC of GUCY2C×CD3×CD28 chimeric trispecific antibodies against T84, HT55 and LS1034 50 and Emax

[0511] 3.11 Study on the Antitumor Efficacy of GUCY2C×CD3 Chimeric Bispecific Antibody in the HT55 Mouse Xenograft Tumor Model

[0512] The experiment used SPF-grade female NCG mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), and the animal certificate number was NO.A202312280149.

[0513] After the animals were released from quarantine, each animal was injected with 1×10 7 PBMC (Sai ​​Li Biotechnology, donor: #XW0801187W) were used to construct a humanized animal model. Three days after PBMC inoculation, 5×10 6 HT55 cells. Ten days after PBMC inoculation, the average tumor volume reached 200 mm 3About 20 tumor-bearing mice were randomly divided into 4 groups of 5 mice each based on tumor volume and body weight. The day of grouping was defined as Day 0. Dosing began on the day of grouping. The grouping and dosing schedule are shown in Table 25.

[0514] Tumor volume and mouse body weight were measured twice a week. The results are shown in FIG25 . The relative tumor inhibition rate (TGI%) was calculated from monitoring to Day 24. The calculation formula is as follows:

[0515] Relative tumor growth inhibition rate (TGI): TGI% = (1-T / C) × 100%. T / C = T RTV / C RTV ×100(T RTV : Average RTV of the experimental group; C RTV : Average RTV of negative control group; RTV=V t / V0, V0 is the tumor volume of the animal when grouped, V t is the tumor volume of the animal after treatment).

[0516] Tumor volume (TV): TV = (length × width 2 ) / 2.

[0517] Table 25: Grouping and Dosing Regimen

[0518] N: Number of animals in each group

[0519] Dosing volume: The dosing volume for animals was adjusted to 10 μL / g body weight.

[0520] Tumor inhibition results are shown in Table 26. On day 24 after grouping, PF-07062119 (0.5 mg / kg), GCbi0007 (0.5 mg / kg), and GCbi0019 (0.5 mg / kg) all significantly inhibited tumor growth relative to the PBS group (P < 0.01), with tumor inhibition rates (TGI) reaching 98.91%, 76.03%, and 98.86%, respectively. Furthermore, there was no significant difference in tumor inhibition between GCbi0019 (0.5 mg / kg) and the positive control, PF-07062119 (0.5 mg / kg) (P > 0.05), demonstrating comparable efficacy. Mouse body weights were also measured, as shown in Figures 25A and 25B. Except for the PBS group, where body weight decreased with increasing tumor volume, the other groups showed no abnormal body weights.

[0521] Table 26: Average tumor volume and tumor inhibition rate on day 24

[0522] Example 4 Humanization and Identification of Anti-huGUCY2C Murine Monoclonal Antibody

[0523] 4.1 Humanization and Characterization of M0055 Antibody

[0524] The humanization method of the M0055 antibody is mainly based on the CDR structure of mouse VH / VL. The IGHV3-48 / IGKV1-27 / IGKV1-16 / IGKV2-40 / IGKV1-9 sequences with the highest homology in the human germline database are selected as templates, and the CDR region of the mouse antibody is transplanted onto the corresponding human template to obtain the humanized sequence.

[0525] M0055 humanized VH sequence:

[0526] M0055 humanized VL sequence:

[0527] The humanized VH / VL 4×4 combination was combined with the huIgG1 light and heavy chain constant regions and Fc to express antibodies. Fortebio measured the affinity of the humanized antibodies, among which the affinity of huM0055-15 was comparable to that of the original M0055 (KD value of 4.16E-8M), with a KD value of 3.93E-8M.

[0528] Table 27: Expression and affinity analysis of M0055 humanized antibody

[0529] 4.2 Humanization and identification of GCAb0002 antibody

[0530] The humanization method of GCAb0002 antibody is mainly based on the CDR structure of mouse VH / VL. The IGHV3-21 / IGKV1-33 / IGKV1-39 sequences with the highest homology in the human germline database are selected as templates, and the CDR region of the mouse antibody is transplanted onto the corresponding human template to obtain the humanized sequence.

[0531] GCAb0002 humanized VH sequence:

[0532] GCAb0002 humanized VL sequence:

[0533] Humanized GCAb0002 VH / VL 2×2 was combined with the huIgG1 light and heavy chain constant regions and Fc to express antibodies. Fortebio measured the affinity of the humanized antibodies, among which the affinity of huGCAb0002-1 was closest to the initial GCAb0002 affinity (KD value of 7.35E-9M), with a KD value of 7.72E-9M.

[0534] Table 28: Expression and affinity analysis of GCAb0002 humanized antibody

[0535] Table 29: Humanized sequences

[0536] Example 5: Configuration and activity detection of humanized GUCY2C×CD3 antibody

[0537] 5.1 GUCY2C×CD3 Antibody Conformation Design

[0538] Based on the humanized sequence of Example 4, a humanized GUCY2C×CD3 bispecific antibody GCbi0031 ( FIG. 26A ) based on GCbi0019 was designed. At the same time, a bispecific antibody GCbi0032 ( FIG. 26B ) with increased affinity for huCD3ed was designed to enhance TDCC activity.

[0539] Table 30: Humanized GUCY2C×CD3 bispecific antibody sequences

[0540] 5.2 Fortebio Determines the Affinity of the GUCY2C×CD3 Antibody

[0541] The experiment used the Fortebio Octet R8 molecular interaction instrument, and The AHC2 Biosensors probe capture assay was used to determine the kinetic parameters of GCbi0031 and GCbi0032 binding to huGUCY2C ECD-Avi-His or Human CD3 epsilon & CD3delta-His antigens. The AHC2 probes were activated by soaking in 1× PBS for 20 minutes. GCbi0031 and GCbi0032 were each diluted to 20 μg / ml in 1× PBS and soaked for 120 seconds to allow binding. The probes were then soaked in 1× PBS for another 120 seconds. The antigen huGUCY2CECD-Avi-His or Human CD3 epsilon & CD3 delta-His (purchased from ACRO, catalog number: CDD-H52W1) was diluted 2-fold from 400 nM with 1× PBS working solution. Three concentration gradients were set up and the probe was immersed in it for 180 s to measure the antigen-antibody binding rate. The probe was then immersed in 1× PBS working solution for 360 s to measure the antigen-antibody dissociation rate.

[0542] The experimental results are shown in Table 31 and Figure 27 (A, B). The humanized GUCY2C×CD3 antibodies GCbi0031 and GCbi0032 have affinities for huGUCY2C ECD-Avi-His that are comparable to that of the pre-humanized GCbi0019.

[0543] The experimental results are shown in Table 31 and Figure 27 (C, D). The affinity of GCbi0032 to huCD3ed is about 2.5 times higher than that of GCbi0031, with KD values ​​of 1.23E-07M and 4.88E-08M, respectively.

[0544] Table 31: Affinity of GUCY2C×CD3 Antibodies

[0545] 5.3 Flow cytometry detection of the binding activity of GUCY2C×CD3 antibody to tumor cells

[0546] The target cells were T84 cells that highly expressed GUCY2C on their cell surface. The cells were washed three times with staining buffer, centrifuged at 450 g for 5 min each time, and the supernatant was discarded. The cells were resuspended in staining buffer at a cell density of 1×10 6100 μL / well of the 96-well plate was added to 100 cells / mL. GUCY2C×CD3 antibodies GCbi0031 and GCbi0032, the positive control PF-07062119 antibody, and the negative control IsoB0001 were diluted to 500 nM and serially diluted 3-fold over 10 steps (maximum concentration 500 nM). 100 μL / well of the 96-well plate was then added and mixed evenly with the T84 cells. The cells were incubated at 4°C for 30 minutes. The cells were then washed three times with staining buffer to remove unbound antibody. Then, 100 μL / well of a 1:1000 dilution of goat anti-human secondary antibody was added and incubated at 4°C for 30 minutes. The cells were centrifuged at 450 g for 5 minutes and washed three times with staining buffer to remove unbound secondary antibody. Finally, the cells were resuspended in 100 μL of staining buffer, and the binding activity of the GUCY2C×CD3 antibody to T84 cells was measured using a Beckman Coulter CytoFLEX flow cytometer. The data were analyzed by FlowJo_V10.8.1 software and fitted by GraphPad Prism software.

[0547] The experimental results are shown in Table 32 and Figure 28. The binding activity of GCbi0031 and GCbi0032 did not decrease significantly, and the MFI max was higher than that of PF-07062119.

[0548] Table 32: EC of GUCY2C×CD3 antibody on T84 cells 50 and MFI max

[0549] 5.4 Flow cytometry detection of GUCY2C×CD3 antibody binding activity to T cells

[0550] The experiment used pan T cells with high expression of human CD3 on the cell surface as target cells. TM Human T Cell Isolation Kit (Stemcell, Cat#17951) was used to isolate cells from commercial PBMCs (Aoneng Biotech, Donor ID#XW081187W) according to the kit instructions. The cells were washed three times with staining buffer, centrifuged at 450 g for 5 min each time, and the supernatant was discarded. The cells were resuspended in staining buffer at a cell density of 1×10 6100 μL / well of a 96-well V-bottom plate was added. GUCY2C×CD3 antibodies GCbi0031 and GCbi0032, the positive control PF-07062119 antibody, and IsoB0001 were diluted to 500 nM and serially diluted 3-fold through 10 steps (maximum concentration 500 nM). 100 μL / well of the 96-well plate was then added and mixed evenly with the pan T cells. The cells were incubated at 4°C for 30 minutes. The cells were then washed three times with staining buffer to remove unbound antibody. Then, 100 μL / well of a 1:1000 dilution of goat anti-human secondary antibody was added and incubated at 4°C for 30 minutes. The cells were centrifuged at 450 g for 5 minutes and washed three times with staining buffer to remove unbound secondary antibody. Finally, the cells were resuspended in 100 μL of staining buffer, and the binding activity of the GUCY2C×CD3 antibody to pan T cells was measured using a Beckman Coulter CytoFLEX flow cytometer. The data were analyzed by FlowJo_V10.8.1 software and fitted by GraphPad Prism software.

[0551] The experimental results are shown in Table 33 and Figure 29. At a concentration of 100 nM, the maximum MFIs of GCbi0031, GCbi0032, and PF-07062119 binding to pan T cells were 170406, 273785, and 80106, respectively.

[0552] Table 33: EC of GUCY2C×CD3 antibodies on pan T cells 50 and MFI max

[0553] 5.5 Study on the TDCC-mediated cytotoxicity of GUCY2C×CD3 antibody against T84 and HT55 tumor cells

[0554] In this experiment, GUCY2C high-expressing T84 cells and medium-expressing HT55 cells were used as target cells. Tumor cells in the logarithmic growth phase were digested with trypsin (Source Culture, Cat#S310KJ) and resuspended in DMEM medium (Gibco, Cat#11965092) containing 10% FBS (Gibco, Cat#10091148) and the cell density was adjusted to 1×10 5 cells / mL, and tumor cells were added to a 96-well U-bottom plate (NEST, Cat#701101), with 100 μL added to each well, i.e., 1×10 4 Cells / well were grown overnight. Then different concentrations of the antibody to be tested were added, diluted with DMEM medium containing 10% FBS. The starting working concentration of the antibody to be tested was 400nM (4X working concentration), and 5-fold serial dilution was performed, with a total of 9 different concentrations, 50μL per well. TMHuman T Cell Isolation Kit instructions: Effector cells (pan T) were isolated from commercial PBMCs (Sai ​​Li Biotechnology, Donor ID#Y1584). The cells were resuspended in DMEM medium containing 10% FBS and the cell concentration was adjusted to 1×10 cells / mL according to the E:T ratio of 5:1. 6 mL, add 5×10 4 Cells / 50μL. Note: Frozen PBMCs can be revived one day in advance and cultured overnight with RPMI 1640 (Source Culture, Cat#L210KJ) + 10% FBS medium. Add appropriate amount of DNase to prevent DNA entanglement of dead cells. Place the 96-well U-bottom plate in a 37°C, 5% CO2 incubator and incubate for 72 hours. 2 hours before the end of incubation, add 10×Lysis buffer (Promega, Cat#G182A) to the wells containing only target cells and continue incubation for 1 hour. Centrifuge the culture plate at 450g for 5 minutes and transfer 50μL of supernatant to a 96-well flat-bottom plate (NEST, Cat#701301). Press CytoTox The substrate solution was prepared according to the instructions of the Non-Radioactive Cytotoxicity Assay (Promega, Cat# G1780) kit, equilibrated to room temperature, and 50 μL was added to each well. The cells were incubated at room temperature in the dark for approximately 30 minutes. The reaction was terminated by adding 50 μL of Stop Solution to each well. The absorbance at 490 nm or 492 nm was measured using a microplate reader (TECAN, Spark). The logarithm of the antibody concentration was plotted against the killing ratio using GraphPad Prism, and the EC was calculated. 50 .

[0555] The experimental results are shown in Table 34 and Figure 30. In the TDCC experiment, the killing activities of GCbi0031 and GCbi0032 in T84 and HT55 cells were not much different, but both were weaker than PF-07062119.

[0556] Table 34: TDCC EC of GUCY2C×CD3 antibodies against T84 and HT55 50 and Emax

[0557] 5.6 Study on the activation effect of GUCY2C×CD3 antibody on T cells in TDCC killing assay of HT55 and T84 tumor cells

[0558] In this study, in the TDCC experiment of Example 5.5, after the killing experiment incubation, the 96-well U-bottom plate was removed, centrifuged at 450g for 5 minutes, and the supernatant was discarded; then the staining solution was prepared with staining buffer, including Brilliant Violet 785 TM anti-human CD3 Antibody (BioLegend, Cat#344842), Brilliant Violet 605 TM anti-human CD4 Antibody (BioLegend, Cat#344646), Brilliant Violet 421 TM anti-human CD8 Antibody (BioLegend, Cat#344748), BD Pharmingen TM PE Mouse Anti-Human CD25 (BD, Cat#555432) and BD Pharmingen TM APC Mouse Anti-Human CD69 (BD, Cat#555533) and other flow cytometry antibody solutions (panel shown in Table 20) were prepared. The cells were resuspended with the prepared flow cytometry antibody solution and incubated at 4°C in the dark for 30 minutes.

[0559] After incubation, the cells were centrifuged at 450 g for 5 min, the supernatant was discarded, and the cells were washed twice with staining buffer, centrifuged, and the supernatant was removed. 100 μL of staining buffer was added to each well of a 96-well cell plate, and the cell detection signal was collected using a flow cytometer CytoFLEX (Beckman). After data collection, the percentage of CD25+CD69+ populations in CD4 cells and CD8 cells was exported using FlowJo software, and then the logarithm of the sample concentration was taken and compared with CD25 using Graphpad. + CD69 + The percentage of double positive samples was plotted using four-parameter fitting.

[0560] The experimental results are shown in Figure 31. In the T cell activation experiment accompanied by the TDCC experiment, the activities of the GUCY2C×CD3 antibodies GCbi0031 and GCbi0032 were generally lower than those of PF-07062119, and the T cell activation activity of GCbi0032 was stronger than that of GCbi0031.

[0561] 5.7 Study on the Release of Cytokines IL-2, IFN-γ, IL-6, and TNF-α During the Killing Effect of GUCY2C×CD3 Antibody on T84 and HT55 TDCC

[0562] In this study, in the TDCC experiment of Example 5.5, after the incubation of the killing experiment, the 96-well U-bottom plate was removed and centrifuged at 450g for 5 minutes to collect the cell supernatant. The release levels of various cytokines in the TDCC reaction supernatant were then measured according to the CBA reagent instructions. First, according to the instructions, the standard pellet was transferred to a centrifuge tube, diluted with diluent, and allowed to stand for 15 minutes. Human IL-2 (BD TM Cytometric Bead Array(CBA)Human IL-2Flex Set,BD,Cat#558270), human TNF-α(BD TM Cytometric Bead Array(CBA)Human TNF Flex Set,BD,Cat#560112), human IFN-γ(BD TM Cytometric Bead Array (CBA) Human IFN-γFlex Set, BD, Cat#558269) and human IL-6 (BD TM Capture microspheres from the Cytometric Bead Array (CBA) Human IL-6 Flex Set (BD, Cat# 558276) were mixed in a 1:1 ratio, vortexed thoroughly before mixing, and then aliquoted into a 96-well plate, 50 μL per well. Dilute the supernatant sample as needed and prepare the standard according to the kit instructions. Add the diluted standard and sample to the 96-well plate, mix thoroughly with the previously added microsphere mixture, and incubate on a shaker at 500 rpm for 5 minutes before incubating in the dark for 1 hour. Detection antibodies for IL-2, TNF-α, IFN-γ, and IL-6 were mixed in a 1:1 ratio and aliquoted into a 96-well plate, 50 μL per well. Mix on a shaker at 500 rpm for 5 minutes before incubating in the dark for 2 hours. After the reaction, add 150 μL of wash buffer, centrifuge at 500 g for 5 minutes, discard the supernatant, resuspend in 100 μL of wash buffer, and analyze on a flow cytometer. Finally, FlowJo was used to analyze the data and export the MFI value of each cytokine in each well. The cytokine concentration of the sample well was calculated based on the standard well. Then, Graphpad was used to take the logarithm of the antibody concentration and perform a four-parameter fitting graph with the cytokine concentration.

[0563] The experimental results are shown in Figures 32 and 33. In the cytokine (IL-2, IFN-γ, IL-6 and TNF-α) release study accompanying the TDCC experiment, the cytokine release levels of GUCY2C×CD3 antibodies GCbi0031 and GCbi0032 were generally lower than those of PF-07062119.

[0564] 5.8 Antitumor efficacy of GUCY2C×CD3 antibody in the HT55 mouse transplanted tumor model

[0565] The experiment used SPF-grade female NCG mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), and the animal certificate number was NO.A202404110221.

[0566] After the animals were released from quarantine, each animal was injected with 1×10 7 PBMC (Miaoshun Biotechnology, donor: P123041110C) were used to construct a humanized animal model. Seven days after PBMC inoculation, 3×10 6 HT55 cells. Eleven days after PBMC inoculation, the average tumor volume reached 80 mm 3 About 35 tumor-bearing mice were randomly divided into 7 groups based on tumor volume and body weight, with 5 mice in each group. The day of grouping was defined as Day 0. Dosing began on the day of grouping. The grouping and dosing schedule are shown in Table 35.

[0567] Tumor volume and mouse body weight were measured twice a week, and the results are shown in Figure 34. The data on Day 21 were used to calculate the relative tumor inhibition rate (TGI%), and the calculation formula is as follows:

[0568] Relative tumor growth inhibition rate (TGI): TGI% = (1-T / C) × 100%. T / C = T RTV / C RTV ×100(T RTV : Average RTV of the experimental group; C RTV : Average RTV of negative control group; RTV=V t / V0, V0 is the tumor volume of the animal when grouped, V t is the tumor volume of the animal after treatment).

[0569] Tumor volume (TV): TV = (length × width 2 ) / 2.

[0570] Table 35: Grouping and Dosing Regimen

[0571] The results of tumor inhibition rates are shown in Table 36 and Figure 34A. On day 21 after grouping, PF-07062119 (0.1 mg / kg), PF-07062119 (0.3 mg / kg), GCbi0031 (0.1 mg / kg), GCbi0031 (0.3 mg / kg), GCbi0032 (0.1 mg / kg), and GCbi0032 (0.3 mg / kg) all significantly inhibited tumor growth relative to the Vehicle group (P < 0.01), with tumor inhibition rates (TGI) (%) reaching 99.46%, 99.52%, 96.66%, 99.32%, 85.43%, and 95.09%, respectively. GCbi0031 and GCbi0032 antibodies had comparable efficacy to PF-07062119. At the same time, the body weight of the mice was monitored. As shown in FIG34B , the body weight of the Vehicle group decreased as the tumor volume increased, while the body weights of the mice in the other groups were normal.

[0572] Table 36: Day 21 Average Tumor Volume and Tumor Inhibition Rate

[0573] Example 6: Configuration and Activity Detection of Humanized GUCY2C×CD3 Antibody

[0574] 6.1 GUCY2C×CD3 Antibody Conformation Optimization and Binding Activity Analysis

[0575] Based on the humanized sequence of Example 4, a humanized GUCY2C×CD3 bispecific antibody GCbi0043 ( FIG. 35 ) based on GCbi0031 was designed to enhance TDCC activity.

[0576] Table 37: Humanized GUCY2C×CD3 bispecific antibody sequences

[0577] 6.2 Biacore determination of affinity of GUCY2C×CD3 antibody - huGUCY2C ECD

[0578] The binding affinity of GCbi0031 and GCbi0043 to huGUCY2C ECD was analyzed using channels 1 and 2 of a Biacore 1K instrument and a Protein A chip.

[0579] A 2 μg / mL sample of GCbi0031 was flowed through channel 2 at 10 μL / min for 60 seconds. The analyte was then run using a multi-cycle method. The antigen huGUCY2C ECD-Avi-His was diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 0 nM in 1× HBS-EP. The protein was injected into channels 1 and 2 at a flow rate of 30 μL / min, with binding for 180 seconds and dissociation for 450 seconds, all in buffer. Seven analyte cycles were repeated in order of increasing analyte concentration. After each cycle, the sensor chip surface was fully regenerated for 30 seconds using Glycine (pH 1.50) at a flow rate of 30 μL / min to remove both the ligand and analyte. The ligand capture, wash, analyte injection, and regeneration steps were then repeated for the next cycle.

[0580] A 2 μg / mL sample of GCbi0043 was flowed through channel 2 at 10 μL / min for 30 seconds. The analyte was then run using a multi-cycle method. The antigen huGUCY2C ECD-Avi-His was diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, and 0 nM in 1× HBS-EP. The protein was injected into channels 1 and 2 at a flow rate of 30 μL / min, with binding for 300 seconds and dissociation for 600 seconds, all in buffer. Seven analyte cycles were repeated in order of increasing analyte concentration. After each cycle, the sensor chip surface was fully regenerated for 30 seconds using Glycine (pH 1.50) at a flow rate of 30 μL / min to remove both the ligand and analyte. The ligand capture, wash, analyte injection, and regeneration steps were then repeated for the next cycle.

[0581] The experimental results were analyzed using a 1:1 fitting method. The data are shown in Table 38 and Figure 36 (A, B). The binding affinity of GCbi0031 to huGUCY2C ECD is 1.07E-08M, and the binding affinity of GCbi0043 to huGUCY2C ECD is 1.43E-08M.

[0582] Table 38: SPR analysis of the binding affinity of GCbi0031 and GCbi0043 to huGUCY2C ECD

[0583] 6.3 Biacore determination of affinity of GUCY2C×CD3 antibodies - huGUCY2C ECD and cyGUCY2C ECD

[0584] The binding affinity of GCbi0031 and GCbi0043 to huGUCY2C ECD and cyGUCY2C ECD was analyzed using channels 3 and 4 of a Biacore 1K instrument and a CM5 chip.

[0585] 1 μg / mL of huGUCY2C ECD-Avi-His antigen was flowed through four channels at 10 μL / min for 30 seconds. Analytes were then run using a multi-cycle method. Antibody GCbi0031 was diluted to (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM, 0 nM), and antibody GCbi0043 was diluted to (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0 nM). Protein was injected into channels 3 and 4 at a flow rate of 30 μL / min, with association for 200 seconds and dissociation for 600 seconds, all run in buffer. Seven cycles of analytes were repeated, starting from the lowest analyte concentration and ending at the highest. After each cycle, the sensor chip surface was fully regenerated for 60 seconds using Glycine (pH 1.50) at a flow rate of 30 μL / min to remove the ligand and analyte. The ligand capture, wash, analyte injection, and regeneration steps were then repeated for the next cycle. The experimental results were analyzed using a 1:1 fit. The data are shown in Table 39, Figures 37A, and 37B. The binding affinity of GCbi0031 for huGUCY2C ECD was 8.57E-10M, and the binding affinity of GCbi0043 for huGUCY2C ECD was 8.36E-10M.

[0586] 1 μg / mL of the antigen cyGUCY2C ECD-Avi-His was flowed through four channels at 10 μL / min for 60 seconds. Analytes were then run using a multi-cycle method. Antibodies GCbi0043 and GCbi0031 were diluted to 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, and 0 nM in 1× HBS-EP. Proteins were injected into channels 3 and 4 at a flow rate of 30 μL / min, with binding for 200 seconds and dissociation for 600 seconds, all in buffer. Seven analyte cycles were repeated, starting with the lowest analyte concentration and ending with the highest. After each cycle, the sensor chip surface was fully regenerated using Glycine (pH 1.50) at a flow rate of 30 μL / min for 60 seconds to remove the ligand and analyte. The ligand capture, wash, analyte injection, and regeneration steps were then repeated for the next cycle. The experimental results were analyzed using a 1:1 fitting method. The data are shown in Table 39, Figure 37C, and Figure 37D. The binding affinity of GCbi0031 to cyGUCY2C ECD is 2.17E-09M, and the binding affinity of GCbi0043 to cyGUCY2C ECD is 2.60E-09M.

[0587] Table 39: SPR analysis of the binding affinity of GCbi0031 and GCbi0043 to huGUCY2C ECD and cyGUCY2C ECD, respectively

[0588] 6.4 Biacore determination of affinity of GUCY2C×CD3 antibodies - huGUCY2C_D56A and huGUCY2C_E101A

[0589] The binding affinity of GCbi0043 to huGUCY2C_D56A and huGUCY2C_E101A was analyzed using channels 1 and 2 of a Biacore 1K instrument and a Protein A chip. The 2 μg / mL sample flowed through 4 channels at 10 μL / min for 30 s. During the cleaning stage, the pipelines except the chip were cleaned with Glycine at pH 1.50. Then, the analytes were run using a multi-cycle method. The antigen huGUCY2C_D56A-His was diluted to (400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 0 nM) and the antigen huGUCY2C_E101A-His was diluted to (800 nM, 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 0 nM) with 1×HBS-EP. Protein was injected into channels 3 and 4 at a flow rate of 30 μL / min, with binding for 120 s and dissociation for 240 s, all performed in buffer. Nine analyte cycles were repeated, starting from low to high analyte concentration. After each cycle, the sensor chip surface was fully regenerated for 30 s using Glycine (pH 1.50) at a flow rate of 30 μL / min to remove both ligand and analyte. The ligand capture, wash, analyte injection, and regeneration steps were then repeated in the next cycle. The results were analyzed using a 1:1 fit. The data are shown in Table 40 and Figure 38. The binding affinity of GCbi0043 for huGUCY2C_D56A was 3.24E-08 M, and the binding affinity for GCbi0043 for huGUCY2C_E101A was 1.01E-07 M.

[0590] Table 40: SPR analysis of the binding affinity of GCbi0043 to huGUCY2C_D56A and huGUCY2C_E101A

[0591] Study on the TDCC-mediated cytotoxicity of 6.5GUCY2C×CD3 antibody against HT55, SW403, LS174T, and AGS tumor cells

[0592] This experiment used GUCY2C high-expressing cell line HT55, medium-expressing cell line SW403, low-expressing cell line LS174T, and negative cell line AGS as target cells. Tumor cells in the logarithmic growth phase were digested with trypsin (Source Culture, Cat#S310KJ) and resuspended in DMEM medium (Gibco, Cat#11965092) containing 10% FBS (Gibco, Cat#10091148) and the cell density was adjusted to 1×10 5cells / mL, and tumor cells were added to a 96-well U-bottom plate (NEST, Cat#701101), with 100 μL added to each well, i.e., 1×10 4 cells / well, adhere to the wall and grow overnight. Then add different concentrations of the antibody to be tested diluted in serum-free DMEM medium. The starting working concentration of the antibody to be tested is 400nM (4X working concentration), and 5-fold gradient dilution is used for a total of 9 different concentrations, with 50μL per well. TM Human T Cell Isolation Kit instructions: Pan T effector cells were isolated from commercial PBMC (Aoneng Biotechnology, Donor ID#Y1584), resuspended in serum-free DMEM medium, and the cell concentration was adjusted to 1×10 cells according to the E:T ratio of 5:1. 6 mL, add 5×10 4 cells / 50μL. Note: Frozen PBMCs can be revived one day in advance and cultured overnight with RPMI 1640 (Source Culture, Cat#L210KJ) + 10% FBS medium. Add appropriate amount of DNase to prevent DNA entanglement of dead cells. Place the 96-well U-bottom plate in a 37°C, 5% CO2 incubator and incubate for 72 hours. 2 hours before the end of incubation, add 10×Lysis buffer (Promega, Cat#G182A) to the wells containing only target cells and continue incubation for 1 hour. Centrifuge the culture plate at 450g for 5 minutes and transfer 50μL of supernatant to a 96-well flat-bottom plate (NEST, Cat#701301). Press CytoTox The substrate solution was prepared according to the instructions of the Non-Radioactive Cytotoxicity Assay (Promega, Cat# G1780) kit, equilibrated to room temperature, and 50 μL was added to each well. The cells were incubated at room temperature in the dark for approximately 30 minutes. The reaction was terminated by adding 50 μL of Stop Solution to each well. The absorbance at 490 nm or 492 nm was measured using a microplate reader (TECAN, Spark). The logarithm of the antibody concentration was plotted against the killing ratio using GraphPad Prism, and the EC was calculated. 50 .

[0593] The results of the TDCC assay are shown in Table 41 and Figures 39A, 39B, 39C, and 39D. GCbi0043 had the best EC50 in TDCC, and its maximum killing was similar to that of PF-07062119 and GCbi0031. There was no nonspecific T cell killing in negative AGS cells.

[0594] Table 41: TDCC EC50 and Emax of GCbi0031 and GCbi0043

[0595] 6.6 Study on the activation of T cells by GUCY2C×CD3 antibody in the TDCC killing activity of HT55, SW403, LS174T and AGS tumor cells

[0596] In this study, in the TDCC experiment of Example 6.5, after the killing experiment incubation was completed, the supernatant of the 96-well U-bottom plate with 50 μL of the above-mentioned aspirated liquid was discarded; then the staining buffer was used to prepare the staining solution, which included Brilliant Violet 785 TM anti-human CD3 Antibody (BioLegend, Cat#344842), Brilliant Violet 605 TM anti-human CD4 Antibody (BioLegend, Cat#344646), Brilliant Violet 421 TM anti-human CD8 Antibody (BioLegend, Cat#344748), BD Pharmingen TM PE Mouse Anti-Human CD25 (BD, Cat#555432) and BD Pharmingen TM APC Mouse Anti-Human CD69 (BD, Cat#555533) and other flow cytometry antibody solutions were prepared. The cells were resuspended with the prepared flow cytometry antibody solution and incubated at 4°C in the dark for 30 minutes.

[0597] After incubation, cells were centrifuged at 450 g for 5 minutes, the supernatant discarded, and washed twice with staining buffer. The cells were centrifuged again, and the supernatant discarded. 100 μL of staining buffer was added to each well of a 96-well plate. Cellular signals were collected using a CytoFLEX flow cytometer (Beckman, CytoFLEX). After data collection, the percentages of CD25+CD69+ populations in CD4 and CD8 cells were derived using FlowJo software. Graphpad was then used to perform a four-parameter fitting of the logarithm of the sample concentration and the percentage of CD25+CD69+ double-positive cells.

[0598] The results of the TDCC-induced T cell activation experiment are shown in Figures 39E, 39F, 39G, 39H, 39I, 39J, 39K, and 39L. GCbi0031 and GCbi0043 showed similar activity to PF-07062119. There was no nonspecific T cell activation in negative AGS cells.

[0599] Study on the TDCC-mediated cytotoxicity of 6.7GUCY2C×CD3 antibody against HT55 tumor cells

[0600] In this experiment, the GUCY2C high-expressing cell line HT55 was used as the target cell. After digestion, the cells were resuspended and the cell density was adjusted to 1×10 5 cells / mL, and tumor cells were added to a 96-well U-bottom plate, with 100 μL added to each well, i.e., 1×10 4 cells / well, adhere to the wall and grow overnight. Then add different concentrations of the antibody to be tested diluted with serum-free DMEM medium. The starting working concentration of the antibody to be tested is 400nM (4× working concentration), 5-fold gradient dilution, a total of 9 different concentrations, 50μL per well. PBMCs from different donors (Aoneng Bio, Donor ID#Y1584,#NF0035; Sai Li, Donor ID#XW0801211W) were resuspended in serum-free DMEM medium, and the cell concentration was adjusted to 2×10 based on the E:T=10:1 ratio. 6 mL, add 1×10 5 cells / 50μL. Place the 96-well U-bottom plate in a 37°C, 5% CO2 incubator for 72 hours. 2 hours before the end of incubation, add 10× Lysis buffer to the wells containing only target cells and continue incubation for 1 hour. Centrifuge the culture plate at 450g for 5 minutes and transfer 50μL of the supernatant to a 96-well flat-bottom plate. Press CytoTox Prepare the substrate solution according to the Non-Radioactive Cytotoxicity Assay Kit instructions, equilibrate to room temperature, add 50 μL to each well, and incubate at room temperature in the dark for approximately 30 minutes. Terminate the reaction by adding 50 μL of Stop Solution to each well, and measure absorbance at 490 nm or 492 nm using an enzyme-labeled assay. GraphPad Prism was used to perform a four-parameter fitting of the logarithm of the antibody concentration and the killing ratio, and calculate the EC. 50 .

[0601] The experimental results of TDCC killing activity are shown in Table 42, Figure 40A, Figure 41A and Figure 42A. In different donors, the EC of GCbi0043 in TDCC 50 It is similar to PF-07062119 and better than GCbi0031.

[0602] Table 42: TDCC EC50 and Emax of GCbi0031 and GCbi0043 against HT55 in different donor PBMCs

[0603] 6.8 Study on Cytokine Release in TDCC Killing Assay of HT55 Tumor Cells by GUCY2C×CD3 Antibody

[0604] This study accompanied the TDCC experiment described in Example 6.7. After centrifugation of the 96-well U-bottom plate, 50 μL of cell supernatant was collected and transferred to a 96-well conical-bottom plate. The TDCC reaction supernatant was then assayed for cytokine release levels according to the Cytometric Bead Array (CBA) Human Th1 / Th2 Cytokine Cytometric Bead Array (CBA) Kit II (BD, Cat# 551809) reagent instructions. The supernatant sample was diluted according to experimental requirements, and the standard was prepared according to the kit instructions. The diluted standard and sample were then added to the 96-well plate. The capture microspheres were mixed in a 1:1 ratio, vortexed thoroughly before mixing, and then aliquoted into the 96-well plate. After thoroughly vortexing the detection microspheres, 12.5 μL was added to each well, mixed with the previously added microsphere mixture, and then placed on a shaker at 500 rpm for 5 minutes. The mixture was then incubated in the dark for 3 hours. After the reaction, 150 μL of wash buffer was added, and the cells were centrifuged at 500 g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 100 μL of wash buffer before analysis on a flow cytometer. Finally, the data were analyzed using FCAP to export the concentrations of each cytokine in each well. Graphpad was then used to perform a four-parameter fitting of the logarithm of the antibody concentration and the cytokine concentration.

[0605] The experimental results of cytokine release are shown in Figures 40B to 40G (Donor Y1584), Figures 41B to 41G (Donor NF0035), and Figures 42B to 42G (Donor XW0801211W). In the cytokine release studies accompanying different donor TDCC experiments, the level of PF-07062119 mediated by Donor Y1584 was comparable to that of GCbi0043, while GCbi0031 was relatively low. In Donors NF0035 and XW0801211W, the levels of IL-2 and TNFα released by PF-07062119 were significantly higher than those of GCbi0043 and GCbi0031.

[0606] Study on TDCC activity of 6.9GUCY2C×CD3 antibody on negative expression cells

[0607] In this experiment, GUCY2C-negative cell lines A431, APRE-19, BEAS-2β, HACAT, HFL-1, HK-2, IMR-90, MRC-5, Nthy-ori3, PNT1A, RWPE-1, and WI38 / VA13 were used as target cells. After cell digestion, the cells were resuspended in DMEM medium containing 10% FBS and the cell density was adjusted to 1×10 5 cells / mL, added to a 96-well U-bottom plate, 100 μL was added to each well, i.e. 1×104 cells / well, and grown overnight. Then different concentrations of the antibody to be tested were added with serum-free DMEM medium. The starting working concentration of the antibody to be tested was 400 nM (4X working concentration), 5-fold gradient dilution, a total of 9 different concentrations, 50 μL per well. The PBMC (Aoneng Bio, Donor ID#Y1584) was adjusted to a cell concentration of 2×10 according to the E:T=10:1 ratio. 6 mL, add 1×105 cells / 50μL to each well. Place the 96-well U-bottom plate in a 37°C, 5% CO2 incubator and incubate for 72 hours. 2 hours before the end of incubation, add 10× Lysis buffer to the wells containing only target cells and continue incubation for 1 hour. Centrifuge the culture plate at 450g for 5 minutes and transfer 50μL of the supernatant to a 96-well flat-bottom plate. Press CytoTox Prepare substrate solution according to the Non-Radioactive Cytotoxicity Assay Kit instructions, equilibrate to room temperature, add 50 μL to each well, and incubate at room temperature in the dark for approximately 30 minutes. Terminate the reaction by adding 50 μL of Stop Solution to each well. Measure absorbance at 490 nm or 492 nm using a microplate reader. Use GraphPad Prism to plot the logarithm of the antibody concentration against the cytotoxicity ratio for four-parameter fitting.

[0608] The experimental results are shown in Figures 43A to 43L. GCbi0043 did not cause nonspecific killing in cells negative for GUCY2C expression.

[0609] 6.10 Study on the Antitumor Efficacy of GUCY2C×CD3 Antibody in the HT55 Mouse Transplanted Tumor Model

[0610] The experiment used SPF-grade female NCG mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), and the animal certificate number was NO.A202502170390.

[0611] After the animals were released from quarantine, each animal was injected with 1×10 7PBMC (Shanghai Saili Biopharmaceutical Co., Ltd., donor: XW0102238W) were used to construct a humanized animal model. Five days after PBMC inoculation, 3×10 6 HT55 cells. Six days after tumor inoculation, the average tumor volume reached 100 mm 3 Around 20 tumor-bearing mice were randomly divided into four groups of five mice each based on tumor volume and body weight. The day of grouping was defined as Day 0. Dosing began on the day of grouping. The grouping and dosing schedule are shown in Table 1.

[0612] Tumor volume and mouse body weight were measured twice a week, and the results are shown in Figure 44. The data on Day 21 were used to calculate the relative tumor inhibition rate (TGI%), and the calculation formula is as follows:

[0613] Relative tumor growth inhibition rate (TGI): TGI% = (1-T / C) × 100%. T / C = T RTV / C RTV ×100(T RTV : Average RTV of the experimental group; C RTV : Average RTV of negative control group; RTV=V t / V0, V0 is the tumor volume of the animal when grouped, V t is the tumor volume of the animal after treatment).

[0614] Tumor volume (TV): TV = (length × width 2 ) / 2.

[0615] Table 43: Grouping and Dosing Regimen

[0616] Tumor inhibition results are shown in Figure 44A and Table 44. On day 21 after grouping, PF-07062119 (0.059 mg / kg) (TGI = 99.36%), GCbi0031 (0.1 mg / kg) (TGI = 91.00%), and GCbi0043 (0.089 mg / kg) (TGI = 99.10%) all significantly inhibited tumor growth relative to the Vehicle group (P < 0.01). GCbi0043 (0.089 mg / kg) (TGI = 99.10%) and PF-07062119 (0.059 mg / kg) (TGI = 99.36%) showed comparable efficacy. Mouse body weights were also monitored, as shown in Figure 44B. During the dosing period, some mice experienced slight weight loss, likely due to GVHD, while the majority of mice showed no significant abnormalities in weight or condition.

[0617] Table 44: Day 21 Average Tumor Volume and Tumor Inhibition Rate

Claims

1. An antibody or an antigen-binding fragment thereof, comprising: (a) a GUCY2C binding domain, wherein the GUCY2C binding domain comprises GUCY2C-LCDR1, GUCY2C-LCDR2 and GUCY2C-LCDR3 contained in the VL of the GUCY2C antibody shown in Table 1 or Table 3, and GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 contained in the VH of the GUCY2C antibody shown in Table 1 or Table 3; and (b) one or more T cell antigen binding domains.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the GUCY2C-LCDR1 comprises the GUCY2C-LCDR1 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, the GUCY2C-LCDR2 comprises the GUCY2C-LCDR2 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, and the GUCY2C-LCDR3 comprises the GUCY2C-LCDR1 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4. Y2C-LCDR3 sequence, the GUCY2C-HCDR1 includes the GUCY2C-HCDR1 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, the GUCY2C-HCDR2 includes the GUCY2C-HCDR2 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, and the GUCY2C-HCDR3 includes the GUCY2C-HCDR2 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the GUCY2C-LCDR1 comprises SASQGISNYLN (SEQ ID NO: 116) and / or RASQDINNYLN (SEQ ID NO: 292).

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the GUCY2C-LCDR2 comprises YTSTLHS (SEQ ID NO: 117) and / or YTSRLHS (SEQ ID NO: 293).

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the GUCY2C-LCDR3 comprises LQYRKFPYT (SEQ ID NO: 118) and / or QQTKMMYT (SEQ ID NO: 294). 6 . The antibody or antigen-binding fragment thereof according to claim 1 , wherein the GUCY2C-HCDR1 comprises NFGMH (SEQ ID NO: 128) and / or SYAMS (SEQ ID NO: 304).

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the GUCY2C-HCDR2 comprises YISSGSGTIYYADTVKG (SEQ ID NO: 129) and / or TISSGGSYIYYSDSVKG (SEQ ID NO: 305).

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the GUCY2C-HCDR3 comprises QRVLTGTLFDY (SEQ ID NO: 130) and / or HDSGDYAMDY (SEQ ID NO: 306).

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein the GUCY2C binding domain comprises a combination of 6 CDRs shown in Table 2 or Table 4.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH and VL of the GUCY2C binding domain comprise the VH and VL pairs of the M0047, M0055, GCAb0002 and GCAb0005 antibodies in Table 1 or Table 3, respectively.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein the GUCY2C binding domain is a fragment antigen-binding domain (Fab).

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein the GUCY2C binding domain is a variable region domain (Fv).

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the GUCY2C binding domain is a single-chain variable region domain (scFv). 14 . The antibody or antigen-binding fragment thereof according to claim 13 , wherein the amino acid sequence of the GUCY2C binding domain comprises the scFv sequence shown in Table 1 .

15. The antibody or antigen-binding fragment thereof according to claim 1, wherein the one or more T cell antigens are selected from CD3 or CD28 or both.

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein the CD3 binding domain comprises the CDR sequence of the CD3 antibody shown in Table 5.

17. The antibody or antigen-binding fragment thereof according to claim 15, wherein the CD3 binding domain comprises the VH and VL sequences of the CD3 antibody shown in Table 5.

18. The antibody or antigen-binding fragment thereof according to claim 15, wherein the CD3 binding domain is a scFv.

19. The antibody or antigen-binding fragment thereof according to claim 18, wherein the CD3 binding domain comprises the scFv sequence of the CD3 antibody shown in Table 5.

20. The antibody or antigen-binding fragment thereof according to claim 15, wherein the CD3 binding domain is in Fab format or IgG format.

21. The antibody or antigen-binding fragment thereof according to claim 20, wherein the CD3 binding domain comprises the VH and VL sequences of the CD3 antibody shown in Table 5.

22. The antibody or antigen-binding fragment thereof of claim 15, wherein the antibody comprises a CD28 binding domain.

23. The antibody or antigen-binding fragment thereof according to claim 15, wherein the CD28 binding domain comprises the CDR sequence of the CD28 antibody shown in Table 6.

24. The antibody or antigen-binding fragment thereof of claim 15, wherein the CD28 binding domain comprises the VH and VL sequences of the CD28 antibody shown in Table 6.

25. The antibody or antigen-binding fragment thereof of claim 15, wherein the CD28 binding domain is a scFv.

26. The antibody or antigen-binding fragment thereof according to claim 25, wherein the CD28 binding domain comprises the scFv sequence of the CD28 antibody shown in Table 6.

27. The antibody or antigen-binding fragment thereof according to claim 15, wherein the CD28 binding domain is in the form of Fab or IgG.

28. The antibody or antigen-binding fragment thereof according to claim 27, wherein the CD28 binding domain comprises the VH and VL sequences of the CD28 antibody shown in Table 6.

29. A multispecific antibody or antigen-binding fragment thereof, comprising: (a) a GUCY2C binding domain, wherein the GUCY2C binding domain comprises GUCY2C-LCDR1, GUCY2C-LCDR2, GUCY2C-LCDR3, GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 of a GUCY2C antibody shown in Table 2 or Table 4; and (b) a CD3 binding domain, wherein the CD3 binding domain comprises the CDR sequence of the CD3 antibody shown in Table 5.

30. A multispecific antibody or antigen-binding fragment thereof, comprising: (a) a GUCY2C binding domain, wherein the GUCY2C binding domain comprises GUCY2C-LCDR1, GUCY2C-LCDR2, GUCY2C-LCDR3, GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 of a GUCY2C antibody shown in Table 2 or Table 4; and (b) a CD28 binding domain, wherein the CD28 binding domain comprises the CDR sequence of the CD28 antibody shown in Table 6.

31. A multispecific antibody or antigen-binding fragment thereof, comprising: (a) a GUCY2C binding domain, wherein the GUCY2C binding domain comprises GUCY2C-LCDR1, GUCY2C-LCDR2, GUCY2C-LCDR3, GUCY2C-HCDR1, GUCY2C-HCDR2 and GUCY2C-HCDR3 of a GUCY2C antibody shown in Table 2 or Table 4; (b) a CD3 binding domain, wherein the CD3 binding domain comprises the CDR sequence of the CD3 antibody shown in Table 5; and (c) a CD28 binding domain, wherein the CD28 binding domain comprises the CDR sequence of the CD28 antibody shown in Table 6.

32. The antibody or antigen-binding fragment thereof of any preceding claim, having the structure shown in Figures 9A to 9F or Figures 9G to 9M.

33. The antibody or antigen-binding fragment thereof of claim 32, comprising the sequence shown in Table 7.

34. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, further comprising an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG.

35. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a murine, rodent, rabbit, chimeric, humanized or human antibody.

36. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, linked to one or more conjugate moieties.

37. The antibody or antigen-binding fragment thereof of claim 36, wherein the conjugate moiety comprises an immunomodulator, an anti-tumor drug, a radioisotope, a clearance regulator, a toxin, a detectable label, RNA, DNA, a cytokine, or a purification moiety.

38. A GUCY2C antibody or an antigen-binding fragment thereof, wherein the GUCY2C antibody comprises GUCY2C-LCDR1, GUCY2C-LCDR2, and GUCY2C-LCDR3 contained in the VL of the GUCY2C antibody shown in Table 1 or Table 3, and GUCY2C-HCDR1, GUCY2C-HCDR2, and GUCY2C-HCDR3 contained in the VH of the GUCY2C antibody shown in Table 1 or Table 3.

39. The antibody or antigen-binding fragment thereof of claim 38, wherein the GUCY2C-LCDR1 comprises the GUCY2C-LCDR1 sequence of M0047, M0055, GCAb0002, and GCAb0005 in Table 2 or Table 4, the GUCY2C-LCDR2 comprises the GUCY2C-LCDR2 sequence of M0047, M0055, GCAb0002, and GCAb0005 in Table 2 or Table 4, and the GUCY2C-LCDR3 comprises the GUCY2C-LCDR1 sequence of M0047, M0055, GCAb0002, and GCAb0005 in Table 2 or Table 4. CY2C-LCDR3 sequence, the GUCY2C-HCDR1 includes the GUCY2C-HCDR1 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, the GUCY2C-HCDR2 includes the GUCY2C-HCDR2 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4, and the GUCY2C-HCDR3 includes the GUCY2C-HCDR2 sequence of M0047, M0055, GCAb0002 and GCAb0005 in Table 2 or Table 4.

40. The antibody or antigen-binding fragment thereof according to claim 38, wherein the GUCY2C antibody comprises the sequence of the VH and VL pairings of the GUCY2C antibody in Table 1 or Table 3.

41. The antibody or antigen-binding fragment thereof according to claim 38, wherein the GUCY2C antibody comprises the sequences of the VH and VL pairings of the GUCY2C antibody in Table 29.

42. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of the preceding claims, and a pharmaceutically acceptable carrier.

43. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-41.

44. A vector comprising the isolated polynucleotide of claim 43. A host cell comprising the vector of claim 44 .

46. ​​A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell of claim 45 under conditions whereby the antibody or antigen-binding fragment thereof is expressed, and recovering the antibody or antigen-binding fragment thereof.

47. A method for treating or ameliorating a disease that benefits from T lymphocyte killing and clearance or a GUCY2C-related disease in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-41, or the pharmaceutical composition according to claim 42.

48. The method of claim 47, comprising administering the antibody or antigen-binding fragment thereof according to claim 29 in combination with claim 30.

49. The method of claim 47, wherein the disease is cancer.

50. The method of claim 49, wherein the cancer is selected from the group consisting of adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioalveolar lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.

51. The method of claim 47, wherein the subject is a human.

52. The method of claim 47, wherein the antibody or antigen-binding fragment thereof or the pharmaceutical composition is administered intravenously, intraarterially, intratumorally, intramuscularly or subcutaneously.

53. The method of claim 47, further comprising administering one or more additional therapeutic agents to the subject.

54. The method of claim 53, wherein the additional therapeutic agent is selected from a chemotherapeutic agent, an anticancer drug, a radiotherapeutic agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic agent, an antioxidant, a metal chelator, a cytokine, an anti-infective agent, an anti-inflammatory agent.

55. The method of claim 53, wherein the additional therapeutic agent is selected from a monospecific antibody, a bispecific antibody, a multispecific antibody, a fusion protein, an ADC, an LDC, an RDC, a cell therapy, a small molecule drug, an antisense nucleic acid, an siRNA, an mRNA, a PROTAC.

56. The method of claim 53, wherein the additional therapeutic agent acts directly on GUCY2C or a variant thereof, such as a monospecific antibody targeting GUCY2C or a variant thereof, a bispecific antibody targeting GUCY2C or a variant thereof, a multispecific antibody targeting GUCY2C or a variant thereof, a fusion protein targeting GUCY2C or a variant thereof, an ADC targeting GUCY2C or a variant thereof, an LDC targeting GUCY2C or a variant thereof, an RDC targeting GUCY2C or a variant thereof, a cell therapy targeting GUCY2C or a variant thereof, a small molecule drug targeting GUCY2C or a variant thereof, an antisense nucleic acid targeting GUCY2C or a variant thereof, an siRNA targeting GUCY2C or a variant thereof, an mRNA expressing GUCY2C or a variant thereof, or a PROTAC targeting GUCY2C or a variant thereof.

57. The method of claim 53, wherein the one or more additional therapeutic agents are administered concurrently or sequentially with the antibody or antigen-binding fragment thereof.

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