WO2025209593A1 - Multi-specific antibody t cell engager - Google Patents

Abstract

Provided are a multispecific antibody 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 the use thereof.

Description

A multispecific antibody T cell engager Technical Field

The present disclosure generally relates to multispecific T cell engagers. Specifically, the present disclosure relates to multispecific antibodies and antigen-binding fragments directed against CD3 and/or CD28.

Background Art

Multispecific antibodies are engineered antibodies that can simultaneously bind to different epitopes of one or more antigens. In immunotherapy, multispecific antibodies that target T cell surface antigens (such as CD3, CD28) and another target antigen (such as a tumor antigen) are used as T cell engagers to recruit and activate T cells in a specific area.

CD3 is a conserved component of the T cell receptor (TCR) complex that has signal transduction capabilities. The binding of the major histocompatibility complex (pMHC) to the TCR conducts signal transduction through the CD3 complex, which transmits signals inside the cell to activate T cells. By binding a CD3-binding antibody to the CD3 complex, it is possible to bypass the restrictions of the pMHC and stimulate immunity (for example, activating the proliferation of cytotoxic lymphocytes (CTLs)). When a CD3-binding antibody and a tumor antigen-binding antibody are constructed into a multispecific antibody, the multispecific antibody can simultaneously bind to CD3 and tumor-associated antigens, redirecting the activation of CTLs to the vicinity of cancer cells to kill cancer cells.

T cell activation and proliferation can also be enhanced by specific binding to co-stimulatory receptors (such as CD28 or 4-1BB). Multispecific antibodies containing T cell co-stimulatory receptor targeting domains can regulate and redirect immune activation, such as multispecific antibodies that connect CD3 and co-stimulatory receptors (such as CD28 or 4-1BB), or multispecific antibodies that simultaneously target co-stimulatory receptors (such as CD28 or 4-1BB) and TAAs.

Tumor antigens include tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). TAAs are present in both cancer cells and normal somatic cells, but are overexpressed on the surface of cancer cells. Therefore, appropriate targets can be selected based on the type of cancer, using multispecific antibodies that simultaneously target cancer antigens and T cells to treat cancer.

However, CD3 antibodies may mediate overactivation of T cells, leading to adverse reactions such as cytokine storms. Furthermore, expressing multispecific CD3 antibodies at high levels often presents a technical challenge. Therefore, there is a need to establish a multispecific antibody T cell engager platform to develop more effective multispecific antibodies with enhanced therapeutic efficacy and expression levels.

Summary of the Invention

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.

The present disclosure provides a multispecific binding antibody or antigen-binding fragment thereof, an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof, a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, and uses thereof.

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

(a) a CD3 binding domain comprising:

(i) CD3-LCDR1, CD3-LCDR2, CD3-LCDR3, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, wherein the CD3-LCDR1 comprises SEQ ID NO: 113, the CD3-LCDR2 comprises SEQ ID NO: 114, the CD3-LCDR3 comprises SEQ ID NO: 110, 115, 116 or 126, the CD3-HCDR1 comprises SEQ ID NO: 111 or 127, the CD3-HCDR2 comprises SEQ ID NO: 106 and the CD3-HCDR3 comprises SEQ ID NO: 33, 112, 120, 122, 123 or 124, and when the CD3-HCDR1 comprises GFTFSTYA, the CD3-LCDR3 does not comprise ALWYSNHWV or the CD3-HCDR3 does not comprise VRHGNFGDSYVSWFAY; or

(ii) CD3-VL and CD3-VH, wherein the CD3-VL comprises SEQ ID NO: 133 and the CD3-VH comprises SEQ ID NO: 134; and

(b) Target antigen binding domain.

In some embodiments, the CD3-LCDR3 comprises SEQ ID NO:115 or 116.

In some embodiments, the CD3-HCDR1 comprises SEQ ID NO:111.

In some embodiments, the CD3-HCDR3 comprises SEQ ID NO:112, 122 or 123.

In some embodiments, the CD3-HCDR3 comprises SEQ ID NO:112.

In certain embodiments, the CD3 binding domain comprises a combination of 6 CDRs as shown in Table 6.

In certain embodiments, _XL1 is A, _XL2 is P, _XL3 is K, XL4 is S, _XL5 is K, _XL6 is R or L, _XL7 is I, _XL8 is V or A, _XL9 is D or E, _XL10 is _{N, and XL11 is} L or H.

In certain embodiments, X _H1 is Q, X _H2 is T, X _H3 is N, X _H4 is G, X _H5 is G, X _H6 is G, X _H7 is N, X _H8 is V, X _H9 is G, X _H10 is D, X _H11 is S, G or Q, X _H12 is V and X _H13 is W.

In certain embodiments, the VH and VL of the CD3 binding domain comprise the paired VH and VL shown in Tables 2-4, respectively.

In some embodiments, the VL and VH of the CD3 binding domain comprise the following sequence pairs: SEQ ID NO: 58 and 59, respectively.

In certain embodiments, the CD3 binding domain is a fragment antigen binding domain (Fab).

In certain embodiments, the CD3 binding domain is a variable region domain (Fv).

In certain embodiments, the CD3 binding domain is a single-chain variable domain (scFv).

In certain embodiments, the amino acid sequence of the CD3 binding domain comprises the amino acid sequence in Table 5.

In certain embodiments, the target antigen is selected from CD28 or a cancer-associated antigen.

In certain embodiments, the cancer-associated antigen is selected from TROP2 or CUCY2C.

In another aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, comprising:

(a) a CD28 binding domain comprising:

(i) CD28-LCDR1, CD28-LCDR2, CD28-LCDR3, CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3, wherein the CD28-LCDR1 comprises SEQ ID NO: 187, 192 or 195, the CD28-LCDR2 comprises SEQ ID NO: 121, the CD28-LCDR3 comprises SEQ ID NO: 188 or 193, the CD28-HCDR1 comprises SEQ ID NO: 189, the CD28-HCDR2 comprises SEQ ID NO: 190 or 194 and the CD28-HCDR3 comprises SEQ ID NO: 191; or

(ii) CD28-VL and CD28-VH, wherein the CD28-VL comprises a light chain variable region sequence as shown in Tables 7-8, and the CD28-VH comprises a heavy chain variable region sequence as shown in Tables 7-8; and

(b) Target antigen binding domain.

In some embodiments, the CD28-LCDR1 comprises SEQ ID NO:195.

In certain embodiments, the 1st, 2nd, 4th or 5th amino acid (corresponding to Q, N, Y or V, respectively) of the CD28 LCDR1 sequence QNIYVW can be replaced by alanine (A).

In some embodiments, the CD28-HCDR1 comprises SEQ ID NO:189.

In certain embodiments, the first amino acid (G) of the CD28-HCDR1 sequence GYTFTSYY may be substituted with alanine (A).

In certain embodiments, the CD28 binding domain comprises a combination of the six CDR sequences shown in Table 10.

In certain embodiments, the VH and VL of the CD28 binding domain comprise the paired VH and VL shown in Tables 7-8, respectively.

In some embodiments, the VH and VL of the CD28 binding domain comprise the following sequence pairs, respectively: SEQ ID NO: 173 and 174.

In certain embodiments, the CD28 binding domain is a fragment antigen binding domain (Fab).

In certain embodiments, the CD28 binding domain is a variable region domain (Fv).

In certain embodiments, the CD28 binding domain is a single-chain variable domain (scFv).

In certain embodiments, the amino acid sequence of the CD28 binding domain comprises the sequence shown in Tables 7-9.

In certain embodiments, the target antigen is selected from CD28, CD3, or a cancer-associated antigen.

In certain embodiments, the cancer-associated antigen is selected from TROP2 or CUCY2C.

In certain embodiments, the antibody or antigen-binding fragment thereof further comprises an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG.

In certain embodiments, the Fc region comprises a knob-in-hole mutation.

In certain embodiments, the Fc region comprises KIH1 and KIH2 as shown in Table 31.

In certain embodiments, the antigen binding domain is selected from: bifunctional Fab, bifunctional Fab', F(ab')2, bifunctional Fd, bispecific dsFv (dsFv-dsFv'), scFv dimer (bivalent bifunctional antibody), bivalent camelized single domain antibody, bivalent nanobody, and bivalent domain antibody.

In some embodiments, the target antigen is selected from the group consisting of: BCMA, CS1, CD123, CD38, CD22, CD33, CD138, DLL3, FLT3, FLT3 Ligand, CD30, CD30 Ligand, CD27, BAFF, SIRPα, CD47, BAFF-R, EPHA3, PD-1, PD-L1, PDL2, CTLA-4, B7-1, B7-2, CD28, TYRO3, CD81, CD96, CD155, DNAM-1, TIM3, VWF, FGFR4, B7-H3, B7-H4, GITR, GITR Ligand, ICOS, B7-H2, 4-1BB, 4-1BB Ligand, OX40, OX40 Ligand, 2B4, CD48, TRPV1, CD40, BTN3A1, SLAMF5, NTB-A, SLAMF1, Mesothelin, IL6, ACE2, CD70, TROP2, NKP30, GPRC5D, PSCA, IL5, TweakR, TIGIT, CSF1R, TNFRSF10B, CD37, CD7, FCGR3A, EPCAM, CLDN6, CD200, AXL, TGFBR1, CD 40 Ligand, TACI, CB1, NKG2D, CD5, IL17RA, IL2RA, CD34, CEACAM5, EGFR, CD46, CLEC12A, ROR2, HVEM, 5T4, IL6R, CD171, CA9, CD52, FAP, TNFSF12, SELP, ROR1, B7-H6, EPHA2, MICA, TNFSF11, VE GFA, MICB, LAG3, Her3, CD10, CD114, CD117, LIGHT, CD24, KLRG1, TM4SF1, CD56, CD44, CD160, IL13RA1, BTLA, VEGFR2, ADAM9, GM-CSF, BCL2L1, ADORA2A, CCR4, ALB, AFP, AMHR2, CB2, DKK1, IL1 B, IL2, B7-H5, AFP(TCR), NY-ESO-1(TCR), MAGE-A4(TCR), WT1(TCR), IL18RA, CXCR3, CCR8, CFB , CD19, NEFL, FCRL5, CD99, CCR2, NRG1, PGF, SCF, CD43, ANGPTL3, CD112, CLDN18.2, IL5RA, CD26 , CD45, IL15RA, UCHL1, CD73, GFAP, TREM2, PCSK9, MDR-1, IL21, IL21R, PROM1, IL7RA, VSIG4, IL4RA, LGALS1, JAM-A, Galectin-9, SLC7A11, CD36, NKG2A, CD21, IFNAR1, IL11RA, THEM, GPC3, P2 RX7, ICAM-1, SELPLG, CD2, FOLR1, PMEL, B4GALT1, GPR75, MUC1, CLEC2D, HER2, EDA, IGFBP7, DDR1, CD93, FOLR2, GUCY2C, CLEC14A, PSMA, M-CSF, CD83, CLU, CD62L, UPA, ADGRE2, Nectin-4, CD5L , CRTAM, EFNA3, CD69, CHODL, EREG, CD63, CXCR7, GFRA3, EPHA4, GP6, TSLP, BST1, ANXA1, RNF43, SSTR2, PRLR, CD142, TNFSF15, CCR1, EPHA5, GHR, FZD4, CDCP1, CSPG4, GPR55, SPARC, IFNAR2, T GFBR2, CLEC1A, CLEC9A, PVRIG, MMP9, GAS6, ADGRE1, ANTXR1, FGF21, NPR1, CXCR1, CD74, IL22, G RP, EMCN, NOTCH3, GDF15, SIGLEC7, CD164, CCR6, GPR87, KCNK9, TPSAB1, SLC4A7, GPR77, CD32a, CHI3L1, PTPRG, CXCL4, CDH1, YAP1, CDH17, LILRB2, APCDD1, CD23, FZD10, GPA33, GDNF, TENM4, S EMA4D, CXCL1, HBEGF, CCR9, LIV-1, CDH6, CXCR5, ASGR1, ENPP3, CXCR4, CXCR2, CD166, EGFP, RSP O3, PGLYRP1, GNRHR, CD147, CCR5, CD79B, IGF-1R, GPC1, CD6, IL1A, NCR1, LGALS3, SIGLEC9, CEACAM6, IFNB1, SLC2A4, AGTR1, CDH3, IL18BP, AREG, CCR7, LGR4, MMP14, RNASE4, CXCL10, IL23 (I L23A&IL12B), CD14, ACVR1C, ACVRL1, BTC, CEACAM1, APLP2, GPR56, ALPP, CXADR, IL20RA, IL19, LAIR1, CD9, GAST, GPNMB, MRGPRX2, TRPA1, TNFRSF1B, TIM1, GIPR, CALCA, A35R, A29L, ADAM8, C D205, LRP10, GRPR, GLP1R, HAMP, GPR20, AGR2, BTN3A2, BTN3A3, ADAMTS1, CPM, FCGR3B, TAFA5, E CSCR, PF4V1, CHRM2, BCAM, CALR, ITGA2&ITGB1, B7-H7, FGF19, CXCL5, CD200R1, CD304, CD98, MS T1R, BRD4, NLRP3, FSTL1, S100A9, KIR2DL1, CLEC4C, EGFRVIII, TFRC, SEZ6, CD72, IGF1, ANPEP, OR2H1, MUC16, IL12RB1, IFNA2, TSHR, STEAP1, CD20, FGFR2IIIb, SIGLEC15, ZNRF3, LY6E, DLK1, IL31RA, ALK, cMET, ROS1, KRAS, CTLA4, LAG-3, TIM-3, CD2, PD-L1, STING, WNT, VISITA, TLR receptor, IL receptor, GMCSFR, CD25, CEA, PSA, NY-ESO-1, GD2, WT1, MAGE-A3, PRAME, Globo H, SP, Sca-1 and CD133.

In certain embodiments, the target antigen is TROP2 or CUCY2C.

In some embodiments, the TROP2 binding domain comprises TROP2-LCDR1, TROP2-LCDR2, TROP2-LCDR3, TROP2-HCDR1, TROP2-HCDR2 and TROP2-HCDR3, wherein the TROP2-LCDR1 comprises SEQ ID NO: 217, the TROP2-LCDR2 comprises SEQ ID NO: 218, the TROP2-LCDR3 comprises SEQ ID NO: 219 or 221, the TROP2-HCDR1 comprises SEQ ID NO: 214, the TROP2-HCDR2 comprises SEQ ID NO: 215 and the TROP2-HCDR3 comprises SEQ ID NO: 216 or 220.

In certain embodiments, the TROP2 binding domain comprises a combination of 6 CDRs as shown in Table 13.

In certain embodiments, the TROP2 binding domain comprises the paired VH and VL shown in Table 11.

In some embodiments, the VL and VH of the TROP2 binding domain respectively comprise the following sequence pairs: SEQ ID NO: 200 and 201, or SEQ ID NO: 204 and 205.

In some embodiments, the VL and VH of the TROP2 binding domain respectively comprise the following sequence pairs: SEQ ID NO: 204 and 205.

In certain embodiments, the TROP2 binding domain is in Fab or IgG form.

In certain embodiments, the TROP2 binding domain comprises the sequence shown in Table 11.

In certain embodiments, the antibody or antigen-binding fragment thereof has the structure shown in FIG11 .

In certain embodiments, the antibody or antigen-binding fragment thereof comprises three polypeptides:

(i) a first polypeptide comprising the VL and light chain constant regions of a TROP2 binding domain;

(ii) a second polypeptide comprising the VH and heavy chain constant regions of the TROP2 binding domain; and

(iii) a third polypeptide comprising the CD3 binding domain and a heavy chain constant region, wherein the CD3 binding domain is a scFv.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises the sequence shown in Table 15.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises four polypeptides:

(i) a first polypeptide comprising the VL and light chain constant regions of a TROP2 binding domain;

(ii) a second polypeptide comprising the VH and heavy chain constant regions of the TROP2 binding domain; and

(iii) a third polypeptide comprising (a) a VH of a TROP2 binding domain, (b) a heavy chain constant region, (c) the CD3 binding domain, and (d) a heavy chain constant region, wherein the CD3 binding domain is a scFv; and

(iv) a fourth polypeptide comprising the VL and light chain constant regions of the TROP2 binding domain.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises the sequence shown in Table 15.

In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising:

CD3-LCDR1, CD3-LCDR2, CD3-LCDR3, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, wherein the CD3-LCDR1 comprises SEQ ID NO: 113, the CD3-LCDR2 comprises SEQ ID NO: 114, the CD3-LCDR3 comprises SEQ ID NO: 110, 115, 116 or 126, the CD3-HCDR1 comprises SEQ ID NO: 111 or 127, the CD3-HCDR2 comprises SEQ ID NO: 106 and the CD3-HCDR3 comprises SEQ ID NO: 33, 112, 120, 122, 123 or 124, and when the CD3-HCDR1 comprises GFTFSTYA, the CD3-LCDR3 does not comprise ALWYSNHWV or the CD3-HCDR3 does not comprise VRHGNFGDSYVSWFAY; or

CD3-VL and CD3-VH, wherein the CD3-VL comprises SEQ ID NO:133 and the CD3-VH comprises SEQ ID NO:134.

In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising:

CD28-LCDR1, CD28-LCDR2, CD28-LCDR3, CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3, wherein the CD28-LCDR1 comprises 187, 192 or 195, the CD28-LCDR2 comprises SEQ ID NO: 121, the CD28-LCDR3 comprises SEQ ID NO: 188 or 193, the CD28-HCDR1 comprises SEQ ID NO: 189, the CD28-HCDR2 comprises SEQ ID NO: 190 or 194 and the CD28-HCDR3 comprises SEQ ID NO: 191; or

CD28-VL and CD28-VH, wherein the CD28-VL comprises the light chain variable region sequence shown in Table 7-8, and the CD28-VH comprises the heavy chain variable region sequence shown in Table 7-8.

In another aspect, the present disclosure provides a TROP2 antibody or an antigen-binding fragment thereof, wherein the TROP2 antibody comprises:

TROP2-LCDR1, TROP2-LCDR2, TROP2-LCDR3, TROP2-HCDR1, TROP2-HCDR2 and TROP2-HCDR3, wherein the TROP2-LCDR1 comprises SEQ ID NO:217, the TROP2-LCDR2 comprises SEQ ID NO:218, the TROP2-LCDR3 comprises SEQ ID NO:219 or 221, the TROP2-HCDR1 comprises SEQ ID NO:214, the TROP2-HCDR2 comprises SEQ ID NO:215 and the TROP2-HCDR3 comprises SEQ ID NO:216 or 220.

In another aspect, the present disclosure provides a TROP2 antibody or an antigen-binding fragment thereof, wherein the TROP2 antibody comprises the paired VH and VL shown in Table 11.

In certain embodiments, the antibody or antigen-binding fragment thereof is linked to one or more conjugate moieties.

In certain embodiments, the antibody or antigen-binding fragment thereof, wherein the conjugate portion comprises an immunomodulatory agent, an anti-tumor drug, a radioisotope, a clearance regulator, a toxin, a detectable label, RNA, DNA, a cytokine or a purification moiety.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides an isolated polynucleotide encoding the aforementioned antibody or antigen-binding fragment thereof.

In another aspect, the present disclosure provides a vector comprising the aforementioned polynucleotide.

In another aspect, the present disclosure provides a host cell comprising the aforementioned vector.

In another aspect, the present disclosure provides a method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the aforementioned host cell under conditions where the antibody or antigen-binding fragment thereof is expressed, and recovering the antibody or antigen-binding fragment thereof.

In another aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, comprising:

(a) a CD3 binding domain comprising:

CD3-LCDR1, CD3-LCDR2, CD3-LCDR3, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, wherein the CD3-LCDR1 comprises SEQ ID NO: 113, the CD3-LCDR2 comprises SEQ ID NO: 114, the CD3-LCDR3 comprises SEQ ID NO: 110, 115, 116 or 126, the CD3-HCDR1 comprises SEQ ID NO: 111 or 127, the CD3-HCDR2 comprises SEQ ID NO: 106 and the CD3-HCDR3 comprises SEQ ID NO: 33, 112, 120, 122, 123 or 124, and when the CD3-HCDR1 comprises GFTFSTYA, the CD3-LCDR3 does not comprise ALWYSNHWV or the CD3-HCDR3 does not comprise VRHGNFGDSYVSWFAY; or

CD3-VL and CD3-VH, wherein the CD3-VL comprises SEQ ID NO: 133 and the CD3-VH comprises SEQ ID NO: 134;

(b) a CD28 binding domain comprising:

CD28-LCDR1, CD28-LCDR2, CD28-LCDR3, CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3, wherein the CD28-LCDR1 comprises 187, 192 or 195, the CD28-LCDR2 comprises SEQ ID NO: 121, the CD28-LCDR3 comprises SEQ ID NO: 188 or 193, the CD28-HCDR1 comprises SEQ ID NO: 189, the CD28-HCDR2 comprises SEQ ID NO: 190 or 194 and the CD28-HCDR3 comprises SEQ ID NO: 191; or

CD28-VL and CD28-VH, wherein the CD28-VL comprises a light chain variable region sequence as shown in Tables 7-8, and the CD28-VH comprises a heavy chain variable region sequence as shown in Tables 7-8; and

(c) Target antigen binding domain.

In certain embodiments, the CD3 binding domain comprises a combination of 6 CDRs as shown in Table 6.

In certain embodiments, the CD28 binding domain comprises a combination of the six CDR sequences shown in Table 10.

In certain embodiments, the target antigen is a cancer-associated antigen.

In certain embodiments, the cancer-associated antigen is selected from TROP2 or CUCY2C.

In certain embodiments, the cancer-associated antigen is TROP2.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a TROP2 binding domain, wherein the TROP2 binding domain comprises:

TROP2-LCDR1, TROP2-LCDR2, TROP2-LCDR3, TROP2-HCDR1, TROP2-HCDR2 and TROP2-HCDR3, wherein the TROP2-LCDR1 comprises SEQ ID NO:217, the TROP2-LCDR2 comprises SEQ ID NO:218, the TROP2-LCDR3 comprises SEQ ID NO:219 or 221, the TROP2-HCDR1 comprises SEQ ID NO:214, the TROP2-HCDR2 comprises SEQ ID NO:215 and the TROP2-HCDR3 comprises SEQ ID NO:220; or

TROP2-VL and TROP2-VH, wherein the TROP2-VL comprises the light chain variable region sequence shown in Table 11, and the TROP2-VH comprises the heavy chain variable region sequence shown in Table 11.

In certain embodiments, the CD3 binding domain, CD28 binding domain, or target antigen binding domain is a fragment antigen binding domain (Fab).

In certain embodiments, the CD3 binding domain, CD28 binding domain, or target antigen binding domain is a variable region domain (Fv).

In certain embodiments, the CD3 binding domain, CD3 binding domain, or target antigen binding domain is a single chain variable domain (scFv).

In certain embodiments, the aforementioned antibody or antigen-binding fragment thereof has the structure shown in FIG11 .

On the other hand, the present disclosure provides a method for treating or ameliorating a disease that benefits from T lymphocyte killing and clearance or a disease associated with tumor-associated antigens in a subject, comprising administering to the subject a therapeutically effective amount of the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned pharmaceutical composition.

In certain embodiments, the disease is cancer or a disease of the immune system.

In certain embodiments, the cancer is selected from 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 cell 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, vaginal cancer, neural or neuroendocrine tumors, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal neuroendocrine tumor (GI-NEC), small cell bladder cancer (SCBC), glioblastoma multiforme, metastatic castration-resistant pulmonary neuroendocrine tumor, neuroblastoma, metastatic carcinoma, diffuse intrinsic pontine glioma, peritoneal cancer, central nervous system tumor, prostate Tumors, epithelial ovarian cancer, renal cell carcinoma, solid tumors, pancreatic ductal carcinoma, abdominal tumors, fallopian tube cancer, desmoplastic small round cell tumor, osteosarcoma, rhabdomyosarcoma, synovial sarcoma, neurofibrosarcoma, Wilms' tumor, bladder cancer, thyroid cancer, glioblastoma, urothelial carcinoma, triple-negative breast cancer, Hodgkin's lymphoma, anaplastic large cell lymphoma, diffuse large B-cell lymphoma, peripheral T-cell lymphoma, adult T-cell lymphoma/leukemia, mediastinal B-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal NK/T-cell lymphoma, enteropathy-associated T-cell lymphoma, cutaneous T-cell lymphoma, large B-cell non-Hodgkin's lymphoma subtypes, primary mediastinal large B-cell lymphoma, gray zone lymphoma, Epstein-Barr virus-positive diffuse large B-cell lymphoma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma.

In certain embodiments, the immune system disease is selected from Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, undifferentiated spondyloarthropathy, juvenile spondyloarthropathy, Behcet's disease, enthesitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, fibromyalgia, chronic fatigue syndrome, pain conditions associated with systemic inflammatory diseases, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile-onset diabetes mellitus (also known as type 1 diabetes), Wegener's granulomatosis, polymyositis, dermatomyositis, inclusion body myositis, multiple endocrine failure, Schmidt's syndrome. syndrome), autoimmune uveitis, Addison's disease, Grave's disease, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, multiple sclerosis, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler's syndrome, myasthenia gravis, Eaton-Lambert syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia, scleroderma, progressive systemic sclerosis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), adult-onset diabetes mellitus (also known as type 2 diabetes), mixed connective tissue disease tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, antiphospholipid syndrome, erythema multiforme, Cushing's syndrome, autoimmune chronic active hepatitis, allergic diseases, allergic encephalomyelitis, transfusion reaction, leprosy , malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, eczema, lymphomatoid granulomatosis, Kawasaki's disease, endophthalmitis, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, Fuchs' cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft-versus-host disease, transplant rejection, tularemia, periodic fever syndrome, suppurative arthritis, familial Mediterranean fever, TNF receptor-associated periodic syndrome (TRAPS), Muckle-Wells syndrome, hyper-IgD syndrome, central nervous system tetradyarrhythmia, celiac disease, Type 2 diabetes mellitus, diffuse toxic goiter (also known as Graves' disease), inflammatory bowel disease, psoriasis (also known as psoriasis, psoriasis), lupus nephritis, skin inflammation, immune thrombocytopenic purpura, thrombotic thrombocytopenic purpura, antiphospholipid syndrome, autoimmune hemolytic anemia, myasthenia gravis, neuromyelitis optica, CIDP (chronic inflammatory demyelinating polyneuropathy), anti-NMDAR encephalitis, Lambert-Eaton syndrome, pemphigus foliaceus, epidermolysis bullosa, and bullous pemphigoid.

In certain embodiments, the subject is a human.

In certain embodiments, the administration is performed by parenteral routes, including subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection; or non-parenteral routes, including transdermal, oral, intranasal, intraocular, sublingual, rectal, or topical.

In certain embodiments, the method further comprises administering an additional therapeutic agent to the subject in need thereof.

In certain embodiments, the additional therapeutic agent is selected from the group consisting of: 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 hormonal therapy agent, an antiviral agent, an antibiotic, an analgesic agent, an antioxidant, a metal chelator, a cytokine, an anti-infective agent, and an anti-inflammatory agent.

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, or a PROTAC.

In certain embodiments, the additional therapeutic agent directly acts on CD3, CD28, tumor-associated antigens or variants thereof, such as a monospecific antibody targeting CD3, CD28, tumor-associated antigens or variants thereof, a bispecific antibody targeting CD3, CD28, tumor-associated antigens or variants thereof, a multispecific antibody targeting CD3, CD28, tumor-associated antigens or variants thereof, a fusion protein targeting CD3, CD28, tumor-associated antigens or variants thereof, an ADC targeting CD3, CD28, tumor-associated antigens or variants thereof, or a cytokine targeting CD3, CD28, tumor-associated antigens or variants thereof. or variants thereof, LDC targeting CD3, CD28, tumor-associated antigens or variants thereof, RDC targeting CD3, CD28, tumor-associated antigens or variants thereof, cell therapy targeting CD3, CD28, tumor-associated antigens or variants thereof, small molecule drugs targeting CD3, CD28, tumor-associated antigens or variants thereof, antisense nucleic acids targeting CD3, CD28, tumor-associated antigens or variants thereof, siRNA targeting CD3, CD28, tumor-associated antigens or variants thereof, mRNA expressing CD3, CD28, tumor-associated antigens or variants thereof, PROTAC targeting CD3, CD28, tumor-associated antigens or variants thereof.

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

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

FIG1 shows a schematic diagram of the structure of an antibody-antigen complex.

FIG2A shows the results of light chain mutation energy analysis of antibody CDR regions (Chothia encoding).

FIG2B shows the results of heavy chain mutation energy analysis of antibody CDR regions (Chothia encoding).

FIG3 shows that the antibody-based AI structure prediction model re-predicts the optimized sequence CD3-002IgG and examines the intramolecular interactions of the mutation sites.

FIG4A shows the HPLC-SEC detection profiles of the expression and purification of the purified anti-CD3, CD28 and TROP2 primary antibodies.

Figure 4B shows the SDS-PAGE analysis of the expression and purification of the purified anti-CD3, CD28, and TROP2 primary antibodies. M: protein marker, R: reducing SDS-PAGE, N-R: non-reducing SDS-PAGE.

FIG5 shows the HPLC-SEC detection profiles of the stability of the main anti-CD3, CD28 and TROP2 antibodies.

FIG6 shows FACS analysis of the binding of anti-TROP2 antibodies to the extracellular regions of human and monkey TROP2 on the cell surface.

FIG. 7 shows FACS analysis of the binding of anti-CD3 antibodies to CD3 on the surface of Jurkat cells.

FIG8A shows the ELISA determination of the affinity of anti-CD3 mAbs to CD3ed-HIS.

FIG8B shows an ELISA assay of the affinity of anti-CD3 single-chain antibody (scFv-Fc) to CD3ed-HIS.

FIG9 shows the results of determining the kinetic characteristic parameters of the binding of anti-CD3 specific antibodies to CD3ed-HIS.

FIG10 shows the results of determining the kinetic characteristic parameters of the binding of anti-TROP2 specific antibodies to TROP2-HIS.

11A to 11G show the configurational designs of multispecific antibodies.

FIG12 shows the results of determining the kinetic characteristic parameters of the binding of TPt0025 alanine scanning antibody to CD28-HIS.

FIG13 shows the results of determining the kinetic characteristic parameters of the binding of TP-023 alanine scanning antibody to huTROP2-HIS.

FIG14A shows the TPb0043 structural model.

FIG14B shows the TPt0025 structural model.

FIG14C shows a TPt0042 structural model.

Figure 15 shows the HPLC-SEC detection profile and SDS-PAGE detection results of the expression and purification of anti-TROP2×CD3 dual antibody and TROP2×CD3×CD28 triple antibody, M: protein marker, R: reducing SDS-PAGE, N-R: non-reducing SDS-PAGE.

Figure 16 shows the HPLC-SEC detection profile and SDS-PAGE detection results of the expression and purification of the anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 triple antibody with weakened affinity, M: protein marker, R: reducing SDS-PAGE, N-R: non-reducing SDS-PAGE.

FIG17A to FIG17C show the detection results of kinetic characteristic parameters of the binding of double antibodies and triple antibodies to TROP2, CD28-HIS, and CD3ed-HIS.

FIG18A to FIG18C show the detection results of kinetic characteristic parameters of the binding of TROP2-Biotin, CD28-HIS and CD3ed-HIS to double antibodies and triple antibodies.

FIG19 shows the results of ELISA assays of the affinity of anti-TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies for TROP2.

FIG20 shows the results of ELISA assays of the affinity of anti-TROP2×CD3 dual antibodies and TROP2×CD3×CD28 triple antibodies to CD28-HIS.

FIG21 shows the results of ELISA assays of the affinity of anti-TROP2×CD3 dual antibodies and anti-TROP2×CD3×CD28 triple antibodies for CD3ed-HIS.

FIG22 shows the results of FACS measurement of the binding affinities of anti-TROP2×CD3 dual antibodies and TROP2×CD3×CD28 triple antibodies to 293-huTROP2 target cells.

FIG23 shows the results of FACS measurement of the binding affinities of anti-TROP2×CD3 dual antibodies and TROP2×CD3×CD28 triple antibodies to 293-TCR target cells.

FIG24 shows the results of FACS measurement of the binding affinities of anti-TROP2×CD3 dual antibodies and TROP2×CD3×CD28 triple antibodies to Jurkat target cells.

FIG25 shows the results of FACS measurement of the binding affinity of the TROP2 antibody-weakened anti-TROP2×CD3 bispecific antibody and the TROP2×CD3×CD28 tertiary antibody to 293-huTROP2 target cells.

FIG26 shows the results of FACS measurement of the binding affinities of the TROP2 antibody-weakened anti-TROP2×CD3 bispecific antibody and the TROP2×CD3×CD28 tertiary antibody to BxPC3, COLO205, and SW403 target cells.

Figures 27 to 41 show the SDS-PAGE determination results of the stability of TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody in different buffer systems, wherein Figure 27A, Figure 27B, Figure 30A, Figure 30B, Figure 33A, Figure 33B, Figure 36A, Figure 36B, Figure 39A, and Figure 39B are blank controls, Figures 28 to 29 represent TP0019, Figures 31 to 32 represent TP0025, Figures 34 to 35 represent TP0042, Figures 37 to 38 represent TPb0043, and Figures 40 to 41 represent TPb0059.

Figures 42 to 51 show the SDS-PAGE test results of the stability of TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody after repeated freeze-thaw cycles, wherein Figures 42 to 43 represent TPt0019, Figures 44 to 45 represent TPt0025, Figures 46 to 47 represent TPt0042, Figures 48 to 49 represent TPt0043, and Figures 50 to 51 represent TPb0059.

Figures 52-61 show the results of TDCC killing activity assays of TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies against 293-huTROP2 (Figures 52A and 52B), BxPC3 (Figures 53A and 53B), MDA-MB-468 (Figures 54A to 54E), NCI-N87 (Figures 55A and 55B), MDA-MB-231 (Figure 56), DLD-1 (Figures 57A to 57D), COLO205 (Figures 58A and 58B), SW403 (Figure 59), T84 (Figure 60) and HEK293 (Figure 61) tumor cells.

Figures 62-68 show the results of the activation effect determination of anti-TROP2×CD3 dual antibodies and TROP2×CD3×CD28 triple antibodies on T cells in TDCC killing experiments of BxPC3 (Figures 62A and 62B), MDA-MB-468 (Figures 63A and 63B), NCI-N87 (Figures 64A and 64B), MDA-MB-231 (Figures 65A and 65B), COLO205 (Figures 66A and 66B), SW403 (Figures 67A and 67B) and HEK293 (Figures 68A and 68B).

Figures 69-73 show the results of measuring the release levels of cytokines IL-2, IFN-r, IL6 and TNFa by anti-TROP2×CD3 dual antibody and TROP2×CD3×CD28 triple antibody in the TDCC killing effect of 293-huTROP2 (Figures 69A and 69B), BxPC3 (Figure 70), MDA-MB-468 (Figures 71A to 71E), DLD-1 (Figure 72), and HEK293 (Figure 73).

Figures 74 to 85 show the results of the killing effect test of TPt0042 and TPb0043 on TROP2 high-expressing, medium-expressing and low-expressing cells in the TDCC experiment.

Figures 86 and 87 show the results of the TDCC killing activity assay of TROP2×CD3×CD28 triple antibody weakened by CD28 antibody against BxPC3 and HEK293 cells.

FIG88A and FIG88B show the results of measuring the activation effect of CD28 antibody-weakened TROP2×CD3×CD28 triple antibody on T cells in a TDCC killing experiment on HEK293 cells.

Figures 89 and 90 show the results of TDCC killing activity assays against BxPC3 and SW403 tumor cells using TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 triple antibody attenuated by CD28 and TROP2 antibodies.

Figures 91-93 show the results of TDCC killing activity assays of TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 triple antibodies attenuated by CD28 and TROP2 antibodies against BxPC3 (Figure 91), SW403 (Figure 92) and Colo205 (Figure 93) tumor cells at 24h, 48h and 72h.

Figures 94-96 show the results of measuring the release levels of cytokines IFNr, TNFa, IL-2 and IL10 during the TDCC killing effect of TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 triple antibody weakened by CD28 and TROP2 antibodies on BxPC3, SW403 and Colo205 tumor cells at 24h, 48h and 72h.

Figures 97 and 98 show the anti-tumor efficacy test results of the anti-TROP2×CD3 dual antibody in the BxPC3 mouse transplanted tumor model.

Figures 99 and 100 show the anti-tumor efficacy test results of the anti-TROP2×CD3 dual antibody in the Colo-205 mouse transplanted tumor model.

Figures 101 and 102 show the anti-tumor efficacy test results of the anti-TROP2×CD3 dual antibody in the MDA-MB-231 mouse transplanted tumor model.

Figures 103A and 103B show the anti-tumor efficacy test results of the anti-TROP2×CD3 dual antibody in the NCI-H292 mouse transplanted tumor model.

Figures 104A and 104B show the anti-tumor efficacy test results of the anti-TROP2×CD3 dual antibody in the NCI-N87 mouse transplanted tumor model.

Figure 105 shows the results of PK assay of TPt0042 molecule in Balb/c mice.

Figures 106A and 106B show the anti-tumor efficacy test results of the CD28 antibody-weakened anti-TROP2×CD3×CD28 triple antibody in the BxPC3 mouse transplanted tumor model.

Figures 107A and 107B show the anti-tumor efficacy test results of anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 triple antibody with weakened CD28 and TROP2 antibodies in the BxPC3 mouse transplanted tumor model.

FIG108 shows the results of toxicity testing of TROP2×CD3 dual antibodies TPt0042 and TPt0047 in hTrop2/hCD3E humanized mice.

Figure 109 shows exemplary configurations of multispecific antibodies.

DETAILED DESCRIPTION

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.

definition

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. A natural, intact IgG antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, each consisting of a variable region ( _VH ) and a first constant region, a second constant region, and a third constant region ( _CH1 , _CH2 , _CH3 , respectively); mammalian light chains are classified as λ or κ, each consisting of a variable region ( _VL ) and a constant region. Antibodies are "Y"-shaped, with the stem of the Y consisting of the second constant region and the third constant region of two heavy chains bound together by disulfide bonds. Each arm of the Y comprises the variable region and the first constant region of a single heavy chain, bound to the variable region and constant region 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, termed complementarity determining regions (CDRs) (the light chain CDRs include LCDR1, LCDR2, LCDR3 and the heavy chain CDRs include HCDR1, HCDR2, HCDR3). The CDR boundaries of the antibodies and antigen binding domains disclosed herein can be defined or identified by 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., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., 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 inserted between flanking extensions called framework regions (FRs), which are more highly conserved than 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 main 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).

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. Patent No. 6,005,079). Heavy-chain antibodies are 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)).

As used herein, the term "target antigen binding domain" refers to an antigen binding domain that targets a target antigen. The target antigen binding domain of the antibody or antigen binding fragment thereof provided herein may be a tumor antigen binding domain. In certain embodiments, the target antigen includes a tumor surface antigen. As used herein, the term "tumor surface antigen" refers to an antigen that is primarily presented by tumor cells to distinguish it from non-malignant tissue, and is preferably located on the cell membrane of a tumor cell. Tumor surface antigens can be in various forms, such as polypeptides (specifically glycosylated proteins) or polypeptides, glycosylation patterns, glycolipids (e.g., gangliosides, such as GM2), or even changes in the composition of lipids of the cell membrane, which may be characteristics of cancer cells. Tumor surface antigens can be antigens that specifically express on cancer cells that trigger an immune response; and/or bind to T cell receptors (e.g., when presented by MHC molecules) or bind to antibodies. In some embodiments, tumor surface antigens trigger a humoral response (e.g., comprising the production of antigen-specific antibodies). In some embodiments, tumor surface antigens trigger a cellular response (e.g., involving T cells whose receptors specifically interact with tumor surface antigens). In some embodiments, the tumor surface antigen binds to the antibody and may or may not induce a specific physiological response in the organism.

As used herein, the term "multispecific antibody" refers to an antibody or antigen-binding fragment thereof that comprises at least two different antigen-binding domains. The at least two different antigen-binding domains target different antigens, or different epitopes of the same antigen. A multispecific antibody can be configured as needed. In certain embodiments, the configuration of a multispecific antibody is shown in Figure 11.

As used herein, the term "antigen binding fragment" refers to an antibody fragment formed by a part of an antibody including one or more CDRs or any other antibody fragment that binds to an antigen but does not include a complete native antibody structure. Examples of antigen binding fragments 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 fragment can bind to the same antigen as the antigen bound by the parent antibody. In certain embodiments, an antigen binding fragment can include one or more CDRs from a specific human antibody that is transplanted to the framework region from one or more different human antibodies. Further and detailed forms of antigen-binding fragments 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.

As used herein, the term "antigen binding domain" refers to an antibody fragment formed by a portion of an antibody including one or more CDRs or any other antibody fragment that binds to an antigen but does not include a complete native antibody structure. Examples of antigen binding fragments 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. In certain embodiments, an antigen binding domain may include 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.

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).

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

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

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

The term "fragment difficult" (Fd) in the context of 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.

"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 bound to the variable region of a single heavy chain. Many Fv designs have been proposed, including dsFv, in which the association between the two domains is enhanced by an introduced disulfide bond, and scFv, which can be formed by joining the two domains together as a single polypeptide using a peptide linker. Fv constructs containing the variable domains of an immunoglobulin heavy or light chain associated with the variable and constant domains of the corresponding immunoglobulin heavy or light chain have also been produced. Fvs have also been multimerized to form bi- and tri-functional antibodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

A "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 linked to each other directly or through a peptide linker sequence (Huston JS et al., Proc. Natl. Acad. Sci. USA, 85:5879 (1988)). ScFv can also be used as a building block for developing multimeric structures (dimer: "diabody"; trimer: "tribody"; tetramer: "tetrabody").

"Diabodies" or "dAbs" include small antibody fragments with two antigen-binding sites, wherein the fragments include a _VH domain connected to a _VL domain in the same polypeptide chain ( _VH - _VL or VL- _VH ) (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA Jul 15;90(14):6444-8 (1993); EP404097; WO93 _/ 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 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 dsdiabody" is a bifunctional antibody that targets two different antigens (or epitopes).

"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 _VH portions are connected by a peptide linker (e.g., a long flexible linker), and are each bound to two _VL portions by a disulfide bridge. In some embodiments, dsFv-dsFv' has bispecificity, wherein each pair of heavy and light chains paired by disulfide bonds has a different antigenic specificity.

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.

"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.

"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.

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.

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).

The term "operably linked" or "operably linked" refers to the juxtaposition of two or more biological sequences of interest, with or without a spacer, linker, or intervening sequence, in a manner that places the biological sequences of interest in a relationship that allows them to function in their 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 for regulated expression of the polypeptide from the polynucleotide.

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.

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 containing mutations (e.g., point mutations), fragments, insertions, deletions, and splice variants. In certain embodiments, the human CD3 protein comprises the amino acid sequence shown in Table 1.

As used herein, "CD28" refers to cluster of differentiation 28, which is expressed on T cells and is used to provide co-stimulatory signals in the T cell activation and survival pathway. Through the joint action of CD28 and the T cell receptor, T cells can be stimulated and effectively activated, thereby producing a large amount of cytokines (such as IL-6). The amino acid and nucleic acid sequences of CD28 of humans, mice and crab-eating 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 proteins thereof containing mutations (e.g., point mutations), fragments, insertions, deletions and splice variants. In certain embodiments, the human CD28 protein comprises the amino acid sequence shown in Table 1.

As used herein, "TROP2", also known as "TACSTD2" or "EGP-1", refers to tumor-associated calcium signal sensor 2 or epidermal glycoprotein-1. TROP2 is associated with the occurrence and progression of cancer. It can interact with key molecular signaling pathways and play a role in tumor progression. TROP2 has been found to be abnormally overexpressed in some solid cancers, such as colorectal cancer, renal cancer, lung cancer and breast cancer. The amino acid and nucleic acid sequences of TROP2 in humans, mice and crab-eating macaques can be found in public databases such as GenBank, UniProt and Swiss-Prot. As used herein, the term TROP2 includes full-length wild-type TROP2 and proteins thereof containing mutations (e.g., point mutations), fragments, insertions, deletions and splice variants. In certain embodiments, the human TROP2 protein comprises the amino acid sequence shown in Table 1.

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 crab-eating macaques 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 comprising mutations (e.g., point mutations), fragments, insertions, deletions and splice variants. In certain embodiments, the human GUCY2C protein comprises the amino acid sequence shown in Table 1.

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 tumor-associated antigens with a binding affinity ( _KD ) of ^≤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). _KD as used herein refers to the ratio of the dissociation rate to the association rate ( _koff / _kon ), which 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, _KD values can be suitably determined using flow cytometry.

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.

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 by standard single-letter or three-letter codes, which are summarized below.

"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.

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.

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.

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

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.

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.

A. Antibodies or Antigen-Binding Fragments thereof

On the one hand, the present disclosure provides multispecific antibodies or antigen-binding fragments thereof that bind to T cell surface antigens (e.g., CD3, CD28) and another target antigen (e.g., tumor antigen), which can serve as T cell engagers in immunotherapy to recruit and activate T cells in designated areas.

In certain embodiments, the multispecific antibodies or antigen-binding fragments thereof provided herein comprise a CD3 binding domain and a target antigen binding domain. In certain embodiments, the multispecific antibodies or antigen-binding fragments thereof provided herein comprise a CD28 binding domain and a target antigen binding domain. In certain embodiments, the multispecific antibodies or antigen-binding fragments thereof provided herein comprise a CD3 binding domain, a CD28 binding domain, and a target antigen binding domain.

i. Antigen

The sequences of T cell surface antigens and target antigens involved in the present disclosure are well known in the art. Table 1 provides the sequences of exemplary T cell surface antigens and target antigens.

Table 1: CD3, TROP2 and TCR antigen sequences

ii. CD3 binding domain

In certain embodiments, the present invention provides a series of anti-CD3 antibodies engineered based on the tidutamab antibody using an AI structure prediction model and an empirical force field energy function. The variable region amino acid sequences of these anti-CD3 antibodies are shown in Table 2.

Table 2: Amino acid sequences of variable regions of energetically advantageous anti-CD3 antibodies based on tidutamab (CDRs classified according to IMGT rules are underlined)

In certain embodiments, provided herein are anti-CD3 antibodies engineered based on the huSP34 antibody using an AI structure prediction model and empirical force field energy functions. The variable region amino acid sequences of these anti-CD3 antibodies are shown in Table 3.

Table 3: Amino acid sequences of variable regions of energetically advantageous anti-CD3 antibodies based on huSP34 (CDRs are underlined)

Design and selection of huSP34 mutants

In certain embodiments, the present disclosure provides a series of optimized CD3 binding domains based on the AI structure prediction model and the empirical force field energy function, and the amino acid sequences of the variable regions thereof are shown in Table 4.

Table 4: Amino acid sequences of the variable regions of the CD3 binding domain (CDRs are underlined)

In certain embodiments, the optimized CD3 binding domain provided herein is a single-chain antibody (scFv), and the amino acid sequence of its variable region is shown in Table 5.

Table 5: CD3 single-chain antibody amino acid sequence (CDRs are underlined)

In certain embodiments, the optimized CD3 binding domains provided herein have the CDR sequences shown in Table 6.

Table 6: CDRs of CD3 binding domains provided herein

In certain embodiments, the optimized CD3 binding domains provided herein comprise a VL and a VH having the following general formula:

CD3-VL：SEQ ID NO:133

QX _L1 VVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYX _L2 NWVQQKPGX _L3 X _L4 PRGLIGGTNX _L5 X _L6 APGVPARFSGSLLGGKAALTX _L7 GX _{L8 QPEDEAX L9 YYCALWYSX L10}

Among them, X _L1 is A or T; X _L2 is P or A; X _L3 is K or Q; X _L4 is S or A; X _L5 is K or F; X _L6 is R or L; X _L7 is I or L; X _L8 is V or A; X _L9 is D, I or E; X _L10 is N or D; X _L11 is L, H or R; X _L12 is G or C.

CD3-VH：SEQ ID NO:134

EVX _H1 LVESGGGLVQPGGSLRLSCAASGFX _H2 FSTYAMX _H3 WVRQAPGKX _H4 LEWVX _H5 RIRSKYNNYATYYADSVKX _H6 RFTISRDDSKNTLYLQMX _H7 SLLAEDTAVYYCX _H8 RHX _H9 NFGXH ₁₀ X _H11 YX _H12 SX _H13 FAYWGQGTLVTVSS

wherein X _H1 is Q or K; X _H2 is T or I; X _H3 is N or S; X _H4 is G or C; X _H5 is G or S; X _H6 is G or D; X _H7 is N or E; X _H8 is V or A; X _H9 is G or D; X _H10 is D, G or N; X _H11 is S, N, G, Q, E or P; X _H12 is V or I; and X _H13 is W or Y.

iii. CD28 binding domain

In certain embodiments, the present disclosure provides a series of optimized CD28 antibodies based on the AI structure prediction model and the empirical force field energy function, and the amino acid sequences of their variable regions are shown in Table 7.

Table 7: Variable region amino acid sequences of anti-CD28 antibodies with energy advantages (CDRs divided according to IMGT rules are underlined)

In certain embodiments, the present disclosure provides a series of optimized CD28 binding domains based on the AI structure prediction model and the empirical force field energy function, and the amino acid sequences of their variable regions are shown in Table 8.

Table 8: Preferred amino acid sequences of the variable regions of the CD28 binding domain (CDRs divided according to the IMGT rules are underlined)

In certain embodiments, the optimized CD28 binding domain provided herein is a single-chain antibody (scFv), and the amino acid sequence of its variable region is shown in Table 9.

Table 9: CD28 single-chain antibody amino acid sequence (CDRs divided according to IMGT rules are underlined)

Table 10: CDRs of CD28 binding domains provided herein

iv. TROP2 binding domain

In certain embodiments, provided herein are a series of TROP2 binding domains optimized based on the huE11 antibody using an AI structure prediction model and empirical force field energy functions. The variable region amino acid sequences of these TROP2 binding domains are shown in Table 11.

Table 11: Variable region amino acid sequences of TROP2 binding domain (CDRs are underlined)

In certain embodiments, the optimized TROP2 binding domain provided herein is a single-chain antibody (scFv), and the amino acid sequence of its variable region is shown in Table 12.

Table 12: TROP2 single-chain antibody amino acid sequence (CDRs are underlined)

In certain embodiments, the optimized TROP2 binding domains provided herein have the CDR sequences shown in Table 13.

Table 13: CDRs of anti-TROP2 binding domains provided herein

v. Anti-TROP2×CD3 dual antibody

In certain embodiments, provided herein are TROP2×CD3 bispecific antibodies designed based on huSP34, CD3-002IgG, CD3-002scFv, huE11, and TP-023IgG.

Table 14: huSP34, CD3-002 IgG, CD3-002scFv, huE11, TP-023 IgG antibody amino acid sequences

In certain embodiments, provided herein are TROP2×CD3 bispecific antibodies TPb0043 and TPt0042 constructed based on CD3-002 scFv and TP-023 IgG, with bispecific antibody amino acid sequences shown in Table 15 and configurations shown in Figure 11. In TPt0042, H44/L100 of the CD3-002 scFv are mutated to Cysteine to form an intramolecular disulfide bond to stabilize the antibody.

Table 15: Amino acid sequences of anti-TROP2×CD3 dual antibodies

vi. Anti-CD3×CD28 dual antibody

Based on the optimization results of CD3 and CD28 antibodies, CD3xCD28 bispecific antibodies or TROP2×CD3×CD28 trispecific antibodies were designed, as shown in Figure 11. The key antibody sequences are shown in Tables 11, 31, and 39.

vii. Humanization of Antibodies

It is known that CDRs are responsible for antigen binding, however, it has been found that not all 6 CDRs are essential or unchangeable. In other words, one or more CDRs provided herein for use in a CD3 binding domain can be replaced or changed or modified while still substantially retaining specific binding affinity for CD3.

In certain embodiments, the antibody or its Fab provided herein is humanized.Humanized antigen-binding domains are desirable in terms of their immunogenicity in reducing people.Humanized antigen-binding domains are chimeric in their variable regions because non-human CDR sequences are transplanted to people or substantially people's FR sequences. The humanization of antigen-binding domains can be carried out substantially by replacing the corresponding people's CDR genes in human immunoglobulin genes with non-human (such as mouse) CDR genes (see, for example, Jones et al. (1986), < Nature > 321:522-525; Riechmann et al. (1988), < Nature > 332:323-327; Verhoeyen et al. (1988), < Science > 239:1534-1536).

Suitable human heavy chain and light chain variable domains can be selected using methods known in the art to achieve this purpose.In an illustrative example, "best fit" method can be used, wherein for the database screening of known human variable domain sequences or BLAST non-human (for example, rodent) antibody variable domain sequences, and identify the human sequence closest to the non-human query sequence and use it as the people's support for transplanting non-human CDR sequences (see, for example, Sims et al., (1993) "Journal of Immunology (J.Immunol.)" 151:2296; Chothia et al. (1987) "Journal of Molecular Biology" 196:901). Alternatively, the framework derived from the consensus sequence of all human antibodies can be used for the transplantation of non-human CDR (see, for example, Carter et al. (1992) "Proceedings of the National Academy of Sciences of the United States of America", 89:4285; Presta et al. (1993) "Journal of Immunology", 151:2623).

viii. Antibody Production

Various techniques can be used to produce such antigen-binding fragments. Illustrative methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods, 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression in host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), and screening from phage display libraries as discussed above (e.g., for ScFv). Other techniques for producing antibody fragments will be apparent to the skilled practitioner.

ix. Conjugates

In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are linked to one or more conjugate moieties. A conjugate moiety is a non-protein moiety that can be linked to an antibody or antigen-binding fragment thereof. It is envisioned that a variety of conjugate moieties can be linked to an antibody or antigen-binding fragment thereof provided herein (see, for example, "Conjugate Vaccines," in Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds.), Carger Press, New York (1989)). These conjugate moieties can be linked to an antibody or antigen-binding fragment thereof by covalent bonding, affinity bonding, embedding, coordination bonding, complexation, association, blending, or addition.

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.

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.

A "toxin" can be any agent that is detrimental to cells or that 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, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6 6-thioguanine, cytarabine, 5-fluorouracil (dacarbazine), alkylating agents (e.g., mechlorethamine, 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.

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 Bi, and ³² P, other lanthanides), luminescent labels, chromophore moieties, digoxigenin, biotin/avidin, DNA molecules, or gold for detection.

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.

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

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

B. Pharmaceutical Compositions

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

The pharmaceutical compositions disclosed herein may 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.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants may 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 antigen-binding fragment thereof and a conjugate as provided herein reduces oxidation of the antibody or antigen-binding fragment thereof. This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Therefore, in certain embodiments, a composition comprising one or more antibodies or antigen-binding fragments thereof as disclosed herein and one or more antioxidants such as methionine is provided. 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.

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 plant 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.

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.

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.

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.

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.

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.

C. Host cell (containing a vector containing a polynucleotide)

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

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)).

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.

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 lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda 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, pFUS, E. pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-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.

The carrier that comprises the polynucleotide of the separation of antibody or its Fab that coding this paper provides is 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 (e.g., Serratia marcescans), and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are 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 are suitable for use herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as 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 (ATCC 16,045). arxianus); Yarrowia (EP 402,226); Pichia pastoris (EP 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.

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, such as 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.

However, vertebrate cells are of greatest interest, and propagation of vertebrate cells in culture (tissue culture) has become 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., PNAS 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 cancer 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 N.Y. 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.

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.

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.

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 culture medium 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. Reissue Patent 30,985 can be used as culture medium for the host cells. Any of these culture media can be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN ^™ drugs), trace elements (defined as inorganic compounds whose final concentrations are generally in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. The culture conditions (such as temperature, pH, etc.) are those previously used with the selected host cell for expression and will be apparent to those of ordinary skill in the art.

When recombinant techniques are used, the antibody or antigen-binding fragment thereof can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris of the host cells or lysed fragments can be removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of Escherichia coli. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. When the antibody or antigen-binding fragment thereof is secreted into the culture medium, the supernatant from such expression systems is typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF can be included in any of the preceding steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

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.

In some embodiments, the protein A fixed on the solid phase is used for the 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 antibodies or their antigen-binding fragments. Protein A can be used for purifying antibodies based on people's 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 people's 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 rate and shorter processing time. Where the antibody or antigen-binding fragment thereof comprises a CH3 domain, Bakerbond ABX ^™ resin (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 an ion exchange column, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE ^™ , chromatography on anion or cation exchange resins (e.g., a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

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).

D. Treatment methods

The present disclosure further provides a method for treating or improving a disease that benefits from T lymphocyte killing and clearance or a TROP2-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.

In certain embodiments, the subject is a human.

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

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.

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

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.

In some embodiments, the condition or illness that can be treated by the methods provided herein can be an immune-related disease or illness, tumor and cancer, autoimmune disease or infectious disease. In some embodiments, the immune-related disease or illness is selected from the group consisting of: systemic lupus erythematosus, acute respiratory distress syndrome (ARDS), vasculitis, myasthenia gravis, idiopathic pulmonary fibrosis, Crohn's disease, asthma, rheumatoid arthritis, graft-versus-host disease, spondyloarthropathy (e.g., ankylosing spondylitis, psoriatic arthritis, isolated acute enteropathic arthritis associated with inflammatory bowel disease, reactive arthritis, Behcet's syndrome, undifferentiated spondyloarthropathy, anterior uveitis and juvenile idiopathic arthritis), multiple sclerosis, endometriosis, glomerulonephritis, sepsis, diabetes, acute coronary syndrome, ischemia-reperfusion, psoriasis, progressive systemic sclerosis, atherosclerosis, Sjögren's syndrome, scleroderma or inflammatory autoimmune myositis.

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: ...

In certain embodiments, the disease is cancer.

In certain embodiments, the cancer is selected from 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.

In some embodiments, the cancer is a TROP2-positive cancer. In some embodiments, the cancer is a TROP2-positive cancer and a target antigen-positive cancer. In some embodiments, the subject to be treated has been identified as suffering from a TROP2-positive cancer, or a TROP2-positive cancer and a target antigen-positive cancer. As used herein, a "TROP2-positive" cancer refers to a cancer characterized by expressing TROP2 in cancer cells or expressing TROP2 in cancer cells at a level significantly higher than that expected from normal cells. As used herein, a "target antigen-positive" cancer refers to a cancer characterized by expressing a target antigen in cancer cells or expressing a target antigen in cancer cells at a level significantly higher than that expected from normal cells.

In some embodiments, the cancer is a GUCY2C-positive cancer. In some embodiments, the cancer is a GUCY2C-positive cancer and a target antigen-positive cancer. In some embodiments, the subject to be treated has been identified as having a GUCY2C-positive cancer, or a GUCY2C-positive cancer and a target antigen-positive cancer. As used herein, a "GUCY2C-positive" cancer refers to a cancer characterized by expressing GUCY2C in cancer cells or expressing GUCY2C in cancer cells at a level significantly higher than that expected in normal cells. As used herein, a "target antigen-positive" cancer refers to a cancer characterized by expressing a target antigen in cancer cells or expressing a target antigen in cancer cells at a level significantly higher than that expected in normal cells.

The presence and/or amount of the target antigen in the 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-target antigen antibody or its antigen-binding fragment, which binds to and detects the expressed target antigen protein. Alternatively, methods such as qPCR, reverse transcriptase PCR, microarrays, SAGE, FISH, etc. can also be used to detect the target antigen protein at the nucleic acid expression level. In some embodiments, the test sample is derived from cancer cells or tissues or tumor-infiltrating immune cells. In certain embodiments, the presence or upregulation level of the target antigen protein in the test biological sample indicates the possibility of a response. As used herein, the term "upregulation" refers to an overall increase in the expression level of the target antigen protein in the test sample compared to the reference expression level of the target antigen by no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more. Reference level can be the level of target antigen expression found in normal cells of the same tissue type, optionally normalized relative to the expression level of another gene (e.g., housekeeping gene). Alternatively, reference level can be the level of target antigen expression found in healthy subjects. Reference sample can be a control sample obtained from healthy or non-diseased individuals, 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., tumor). In some embodiments, the test and/or reference are tested and/or determined substantially simultaneously with the test being paid attention to. In some embodiments, reference is a historical reference optionally embodied in a tangible medium. Typically, as will be appreciated by those skilled in the art, determine or characterize a reference under conditions or environments comparable to the conditions or environments in the assessment.

In certain embodiments, the tumors and cancers are metastatic, particularly metastatic tumors that express TROP2 or GUCY2C.

In certain embodiments, the tumor or 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, bronchioloalveolar cell 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, vaginal cancer, neural or neuroendocrine tumors, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal neuroendocrine tumor (GI-NEC), small cell bladder cancer (SCBC), glioblastoma multiforme, metastatic castration-resistant neuroendocrine tumor, neuroblastoma, central nervous system dysregulation, metastatic carcinoma, diffuse intrinsic pontine glioma, peritoneal cancer, central nervous system tumor , prostate tumors, ovarian epithelial cancer, renal cell carcinoma, solid tumors, pancreatic ductal carcinoma, abdominal tumors, fallopian tube cancer, desmoplastic small round cell tumors, osteosarcoma, rhabdomyosarcoma, synovial sarcoma, neurofibrosarcoma, Wilms tumor, bladder cancer, thyroid tumor, glioblastoma, urothelial carcinoma, triple-negative breast cancer, Hodgkin lymphoma, anaplastic large cell lymphoma, diffuse large B-cell lymphoma, peripheral T-cell lymphoma, adult T leukemia, mediastinal B-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal NK/T-cell lymphoma, enteropathy-associated T-cell lymphoma, cutaneous T-cell lymphoma, mycosis fungoides (MF), large B-cell non-Hodgkin lymphoma subtypes, primary mediastinal large B-cell lymphoma, gray zone lymphoma, Epstein-Barr virus-positive diffuse large B-cell lymphoma, diffuse large B-cell lymphoma, and non-Hodgkin lymphoma.

In certain embodiments, the condition or disorder treatable by the methods provided herein comprises an autoimmune disease. Autoimmune diseases include, but are not limited to, acquired immunodeficiency syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diabetes, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, sprue sprue-dermatitis herpetiformis; chronic fatigue immune dysfunction syndrome (CFS); CFIDS, chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis s thyroiditis), idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, severe anemia (pemacious anemia), polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, essential agammaglobulinemia, primary biliary cirrhosis, Psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjögren's syndrome, stiff-person syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

In certain embodiments, the conditions or disorders treatable by the methods provided herein include infectious diseases. Infectious diseases include, for example, chronic viral infections, e.g., fungal infections, parasitic/protozoal infections, or chronic viral infections, e.g., malaria, coccidioidomycosis immitis, histoplasmosis, onychomycosis, aspergillosis, blastomycosis, candidiasis albicans, paracoccidiomycosis, microsporidiosis, Acanthamoeba keratitis, amoebiasis, ascariasis, babesiosis, esiosis), Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis , Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis plasmosis, trichinosis, trichuriasis, trypanosomiasis, helminth infection, hepatitis B (HBV) infection, hepatitis C (HCV) infection, herpes virus infection, Epstein-Barr virus infection, HIV-1 infection, HIV-2 infection, cytomegalovirus infection, herpes simplex virus type 1 infection, herpes simplex virus type 2 infection, human papillomavirus infection, adenovirus infection, Kaposi's sarcoma-associated herpesvirus-epidemic infection, parvovirus (Torquetenovirus) infection, human T-lymphotropic virus I infection, human T-lymphotropic virus II infection, varicella-zoster virus infection, JC virus infection, or BK virus infection.

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.

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.

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.

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.

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.

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, the additional therapeutic agent administered in combination with the antibodies or antigen-binding fragments thereof disclosed herein is 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 regimens well known in the art.

E. Multispecific molecules

In certain embodiments, the multispecific molecules provided herein include an antigen binding domain provided herein and a target antigen binding domain provided herein. In certain embodiments, the multispecific molecules provided herein are designed as shown in FIG. 11 .

F. Characterization of Multispecific Molecules

In certain embodiments, the multispecific molecules provided herein are capable of specifically binding to one, two, or three of the cell surface markers of human CD3, TROP2, and CD28. The multispecific molecules provided herein retain specific binding affinity for one, two, or three of human CD3, TROP2, and CD28, and in certain embodiments, are at least comparable to or even better than the parent antibody in these aspects.

The binding of a multispecific molecule can be expressed as a "half maximal effective concentration" ( _EC50 ) value, which refers to the concentration of the antibody at which 50% of the maximal effect (e.g., binding or inhibition, etc.) of the antibody is observed. _EC50 values can be measured by methods known in the art, such as sandwich assays, such as ELISA, Western blots, flow cytometry assays, and other binding assays.

The binding affinity of the antigen-binding domains provided herein can also be expressed by a _K value, which 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 / _kon ). Antigen binding affinity (e.g., K _D ) can be suitably determined using suitable methods known in the art (including, for example, flow cytometry). In some embodiments, the 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 be first plotted against the concentration of the antigen-binding domain, and then the K _D value can be calculated by fitting the dependence of the specific binding fluorescence intensity (Y) and the antibody concentration (X) to a site saturation equation: Y = B _max * X / (K _D + X), using Prism version 5 (GraphPad Software, San Diego, CA), wherein B _max refers to the maximum specific binding of the tested antigen-binding domain to the antigen.

In certain embodiments, the multispecific molecules provided herein specifically bind to human CD3, TROP2, or CD28 with a binding affinity ( _KD ) as measured by an AI assay.

In certain embodiments, the multispecific molecules provided herein specifically bind to human CD3, TROP2, or CD28 with a binding affinity ( _KD ) as measured by an Octet assay.

In certain embodiments, the multispecific molecules provided herein specifically bind to human CD3, TROP2, or CD28 with a binding affinity ( _KD ) as measured by an ELISA assay.

In certain embodiments, the multispecific molecules provided herein specifically bind to human CD3, TROP2, or CD28 with a binding affinity ( _KD ) as measured by FACS assay.

G. Variants

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, TROP2 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).

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.).

In certain embodiments, the CD3 binding domain, CD28 binding domain, and/or TROP2 binding domain 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 substitutions in a total of CDR sequences and/or FR sequences and/or one or more variable region sequences.

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 selected from Table 6, while retaining binding affinity for CD3 at a similar or even higher level relative to its parent antibody.

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 selected from Table 10, while retaining binding affinity for CD28 at a similar or even higher level relative to its parent antibody.

In certain embodiments, the TROP2 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 selected from Table 13, and at the same time retains binding affinity for TROP2 at a similar or even higher level relative to its parent antibody.

i. Glycosylation variants

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.

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.

ii. Cysteine engineered variants

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

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.

iii. Fc variants

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, for example, 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. et al., Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(11):4005-4010; Ryan MC. et al., Mol. Cancer Ther., 2007, 6:3009-3018; Richards JO, et al., Mol. Cancer Ther., 2008, 7(8):2517-27; Shields R.L. et al., Journal of Biological Chemistry, 2002, 277:26733-26740; Shinkawa T. et al., Journal of Biological Chemistry, 2003, 278:3466-3473.

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 the antibody to fix complement (see PCT Publication WO 94/29351 to Bodmer et al.).

The terms "antibody-dependent cellular phagocytosis" and "ADCP" refer to a process by which antibody-coated cells or particles are fully or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of an immunoglobulin. Methods for altering the ADCP activity of antibodies by antibody engineering are known in the art, see, for example, Kellner C et al., Transfus Med Hemother, (2017) 44:327-336 and Chung AW et al., 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., Exp&Mol., Med. (2019) 51:138, which are incorporated herein in their entirety.

i) Fc variants with enhanced effector function

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., Fc of IgG1). In certain embodiments, the Fc variants comprise 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, 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 35, 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 WO 00/42072 to Presta, WO 2006/019447 and WO 2016/196228 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 E.A. 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, 305 I, 324T, 326A, 326D, 326W, 330L, 330M, 333S, 332D, 332E, 298A, 333A, 334A, 334E , 326A, 247I, 339D, 339Q, 345R, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3 05I, 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, WO2016/196228; 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).

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.

ii) Fc with reduced effector function

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).

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.

iii) Fc with altered binding to FcRn

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 can 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 Research, 70:3269-3277 (2010); Hinton, P. et al., Immunofluorescence, 2012; Journal of Epidemiology, 176:346-356 (2006); Petkova et al. (2006) Int. Immunol. 18:1759; Ball Acqua et al. Journal of Immunology 2002, 169:5171-5180; Dall'Acqua WF. et al., Journal of Biochemistry 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.

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., with A or R), 312 (e.g., with F, C or P), 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.

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.

H. How to use

In certain embodiments, the target cells co-express the target antigen and CD3. In some embodiments, the target cells include cancer cells, inflammatory cells and/or chronically infected cells. In some embodiments, the target antigen is a tumor surface antigen, an inflammatory antigen, or an antigen of an infectious microorganism. In some embodiments, the target antigen can be a tumor antigen (e.g., a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), such as a neoantigen) or an antigen presented on an infected cell (e.g., hepatitis B surface antigen (HBsAg)).

On the other hand, the present disclosure also provides a method for treating a target antigen-related disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein. For example, if the target antigen comprises a tumor antigen, the target antigen-related disease may comprise a tumor or cancer. For example, if the target antigen comprises an antigen presented on an infected cell, the target antigen-related disease may comprise a related infectious disease. In certain embodiments, the target antigen comprises CD28. In certain embodiments, the target antigen comprises TROP2. In certain embodiments, the target antigen comprises GUCY2C.

In another aspect, the present disclosure also provides methods of treating a CD3-related disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein.

In another aspect, the present disclosure also provides methods of treating a CD28-associated disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein.

In another aspect, the present disclosure also provides methods of treating a TROP2-associated disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein.

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.

Example

Example 1 Energy calculation of anti-CD3 monoclonal antibody

Sequence stability was assessed for all SP34-based engineered anti-CD3 antibodies using an AI structure prediction model and empirical force field energy functions to generate stability scores. To calculate the stability score, a stable antibody structure was first determined using the sequence-to-structure AI structure prediction model. Using the AI-predicted antibody structure as the starting point for energy optimization, the energy was optimized within 10 iterations using a greedy algorithm within the force field. Finally, all values were averaged. The prediction results are shown in Table 16.

Table 16: Anti-CD3 antibody energy obtained based on AI model and empirical force field

Using HuSP34 and tidutamab (sequences shown in Tables 2 and 3), respectively, as starting points, dominant mutation enrichment was performed, and energy stability calculations were re-performed to obtain complete test mutation sequences. The dominant mutations were evaluated separately, and when calculating the effects of multiple mutations, the empirical force field was used to evaluate the superposition of the effects of single mutations. The different results obtained from the two dominant mutation enrichments are shown in Tables 2 and 3. Candidate antibodies are shown in Table 4 for antibody variable region (VL and VH) sequences and Table 5 for single-chain antibody (scFv) sequences.

Example 2 Multi-objective optimization of anti-CD28 antibodies

All anti-CD28 antibodies based on TGN1412 were re-optimized using the antibody AI structure prediction model and empirical force field energy function. The public protein structure database RCSB PDB structure 1YJD was used as a sequence and structural template to perform back mutation energy analysis on all sequences modified based on the TGN1412 sequence. The mutation energy was analyzed to obtain multiple humanized and monomer stability multi-optimized precursors and reselect the starting point for the downstream algorithm. Anti-CD28 antibody energy calculation results (see Table 17) and optimized sequences selected based on energy analysis (see Table 18)

Table 17: Anti-CD28 antibody energy based on protein structure and empirical force field

Table 18: Optimized sequences selected based on energy analysis

Example 3 Multi-objective optimization of anti-CD28 antibodies

We used the fully human Germline sequence and AI-guided multi-objective optimization of the above-mentioned optimized No. 3 antibody for humanization and stability. The antibody AI structure prediction model was used to predict the dynamic effects of changes in the antibody framework region on the CDR region, and an empirical force field was used to perform additional overall energy evaluation of the complete structure. The results of the multiple optimization evaluation of the anti-CD28 antibody using the empirical force field are shown in Table 19. The candidate antibodies selected based on the evaluation results are shown in Table 8 for the antibody variable region (VL and VH) sequences and Table 9 for the single-chain antibody (scFv) sequences.

Table 19: Multiple optimization of anti-CD28 antibodies based on AI and empirical force fields

Example 4 Humanization of anti-TROP2 monoclonal antibody

The original sequence Pr1E11-Chi (sequence shown in Table 20) was humanized using a CDR grafting approach. The human germline sequences IGHV1-46 and IGKV4-1, which share the highest homology with the original heavy and light chains, respectively, were selected as target germline sequences. The original amino acids in all CDR regions were retained, and some amino acids in the FR regions were backmutated to obtain three humanized sequences (see Table 20, sequences huE11, huE11-2, and huE11-3). Sequence huE11 was selected as the starting antibody, and the amino acid sequence of the huE11 variable region is shown in Tables 11 and 20.

Table 20: Pr1E11-Chi amino acid sequence

Example 5 Structural prediction and energy calculation of the huE11 mAb-TROP2 complex

An AI-based antigen-antibody complex structure prediction model was used to predict the complex structure of the HuE11 sequence and the TROP2 crystal structure, resulting in a high-quality complex structure. The antibody-antigen complex structure is shown in Figure 1. An empirical force field function was used to fine-tune the complex structure. After fine-tuning, a full mutation energy analysis was performed on the antibody CDR region (Chothia encoding). The energy analysis results are shown in Figures 2A and 2B.

Example 6 Design and selection of huE11 mutants

Based on AI prediction and calculation results, we selected a series of advantageous mutations to design candidate TROP2 antibodies. The candidate antibodies are shown in Table 11 for the antibody variable region (VL and VH) sequences and Table 12 for the single-chain antibody (scFv) sequences. We also selected another TROP2 antibody, huRS7 (derived from patent WO2003074566A3), as one of the candidate antibodies. The huRS7 sequence is shown in Tables 11 and 12.

Example 7 Expression and Detection of Anti-CD3 Optimized Candidate Monoclonal Antibodies

The heavy chain and light chain DNA fragments of the anti-CD3 monoclonal antibody designed by AI were subcloned into the pcDNA3.4 vector respectively, and the recombinant plasmids were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer at pH 3.4 and dialyzed to PBS at pH 7.4. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.

The protein yield, purity, Jurkat cell binding, CD3ed-HIS ELISA binding, Tm value, accelerated stability (incubation at 40°C for 7 days), DLS, and SEC data of different CD3 monoclonal antibodies after AI optimization are shown in Table 21. Among them, CD3-002IgG, CD3-006IgG, CD3-007IgG, and CD3-008IgG exhibited excellent physical and chemical properties.

Table 21: Optimization results of anti-CD3 mAbs based on huSP34

(Note: DNT stands for did not test. / indicates no results were found for that item)

Example 8 Expression and Detection of Anti-CD3 Optimized Candidate Single-Chain Antibody (scFv-Fc)

The anti-CD3 scFv-Fc DNA fragment designed by AI was cloned into the pcDNA3.4 vector, and the recombinant plasmid was extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with pH 3.4 sodium acetate buffer and dialyzed to pH 7.4 PBS. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.

Table 22 shows the DLS and SEC data for the yield, purity, Jurkat cell binding, CD3ed-HIS binding (ELISA), Tm values, and accelerated stability (7 days incubation at 40°C) of different anti-CD3 scFv-Fc proteins after AI optimization. A comprehensive comparison of cell binding, ELISA binding, Tm values, and accelerated stability of each CD3 scFv-Fc antibody revealed that CD3-002scFv, CD3-006scFv, and CD3-007scFv exhibited excellent physical and chemical properties. The expression level of CD3-002scFv was significantly higher than that of CD3-006scFv and CD3-007scFv.

Table 22: Optimization results of anti-CD3 single-chain antibody (scFv-Fc) based on huSP34

(Note: DNT stands for did not test, NE stands for no expression, and / stands for no result)

Example 9 Interpretation of AI structure prediction of anti-CD3 monoclonal antibody experimental results

Taking CD3-002IgG (sequence shown in Table 4) as an example, the optimized sequence has good physicochemical properties. Using an antibody-based AI structure prediction model, CD3-002 IgG was re-predicted and the intramolecular interactions at the mutation sites were examined. As shown in Figure 3, 244G245G-309V forms two small polar interactions that stabilize the two G loops, 264P forms a pair of polar interactions with 282T, and 323Y and 326L form a pair of polar interactions. These polar interactions can enhance the stability of the antibody.

Example 10 Expression and Detection of Anti-CD28 Optimized Candidate Monoclonal Antibodies

The heavy chain and light chain DNA fragments of the anti-CD28 monoclonal antibody designed by AI were subcloned into the pcDNA3.4 vector respectively, and the recombinant plasmids were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer at pH 3.4 and dialyzed to PBS at pH 7.4. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.

The yield, purity, Jurkat cell binding, CD28-HIS binding (ELISA, KD), accelerated stability (incubation at 40°C for 7 days) DLS and SEC data of different CD28 monoclonal antibodies after AI optimization are shown in Table 23. Among them, CD28-041IgG and CD28-065IgG have good various physical and chemical properties.

Table 23: Optimization results of anti-CD28 mAbs based on TGN1412

(Note: DNT stands for did not tested, and NE stands for no expression)

Example 11 Expression and Detection of Anti-CD28 Optimized Candidate Single-Chain Antibody (scFv-Fc)

The anti-CD28 scFv-Fc DNA fragment designed by AI was cloned into the pcDNA3.4 vector, and the recombinant plasmid was extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer at pH 3.4 and dialyzed to PBS at pH 7.4. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.

Table 24 shows the yield, purity, Jurkat cell binding, CD28-HIS binding (ELISA, KD), and accelerated stability (7 days incubation at 40°C) of different AI-optimized CD28 scFv-Fc proteins using DLS and SEC. CD28-041scFv exhibited favorable physicochemical properties. CD28-065scFv was not tested.

Table 24: Optimization results of anti-CD28 single-chain antibody (scFv-Fc) based on TGN1412

(Note: DNT stands for did not tested, and NE stands for no expression)

Example 12 Design Optimization of Anti-TROP2 Monoclonal Antibodies

The heavy chain and light chain DNA fragments of the anti-TROP2 monoclonal antibody designed by AI were subcloned into the pcDNA3.4 vector respectively, and the recombinant plasmids were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer at pH 3.4 and dialyzed to PBS at pH 7.4. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.

The protein yield, purity, 293-huTROP2 cell binding, Tm value, accelerated stability (incubated at 40°C for 7 days), DLS, and SEC data of different TROP2 monoclonal antibodies after AI optimization are shown in Table 25. TP-021IgG and TP-023IgG exhibited excellent physical and chemical properties.

Table 25: Optimization results of anti-TROP2 mAbs based on huE11

(Note: DNT stands for did not test, NE stands for no expression, and / stands for no result)

Example 13 Design Optimization Results of Anti-TROP2 Single-Chain Antibody (scFv-Fc)

The AI-designed anti-TROP2 scFv-Fc DNA fragments were subcloned into the pcDNA3.4 vector. The recombinant plasmids were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was centrifuged at high speed and vacuum filtered through a microporous filter membrane. The sample was then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer (pH 3.4) and dialyzed into PBS (pH 7.4). Protein concentration was determined by measuring absorbance at 280 nm using a NanoDrop instrument.

The yield, purity, 293-huTROP2 cell binding, Tm value, accelerated stability (incubation at 40°C for 7 days) DLS and SEC data of different TROP2 scFv-Fc proteins after AI optimization are shown in Table 26.

Table 26: Optimization results of anti-TROP2 single-chain antibody (scFv-Fc) based on huE11

(Note: DNT stands for did not test, NE stands for no expression, and / stands for no result)

According to the optimization results of CD3, CD28 and TROP2 antibodies in Examples 7-13, huSP34, CD3-002IgG, CD3-006IgG, CD3-007IgG, CD28-065IgG, huRS7, huE11, TP-021IgG, TP-023IgG, huSP34-scFv, CD3-002scFv, CD3-006scFv, CD3-007scFv and CD28-065scFv (sequences are shown in Table 27) were selected as the main antibodies, and TROP2×CD3 bispecific antibodies or TROP2×CD3×CD28 trispecific antibodies were designed, as shown in Figure 11.

Table 27: Amino acid sequences of major antibodies against CD3, CD28 and TROP2

Example 14 Expression and purification results of anti-CD3, CD28 and TROP2 antibodies

CHO cells were co-transfected with recombinant plasmids encoding the heavy and light chains or scFvs of anti-CD3, CD28, and TROP2 antibodies and Fc. After 7 days of culture, the culture medium was centrifuged at high speed and vacuum filtered through a microporous filter membrane. The sample was then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer (pH 3.4) and dialyzed into PBS (pH 7.4). Protein concentration was determined by measuring absorbance at 280 nm using a NanoDrop instrument. Expression results are shown in Table 28.

The purified proteins were analyzed by HPLC. The HPLC-SEC chromatogram is shown in Figure 4A , demonstrating that the purity of the monoclonal antibody monomers exceeded 98%, the purity of the CD3-002scFv single-chain antibody monomers exceeded 90%, and the purity of the remaining single-chain antibody monomers exceeded 95%. SDS-PAGE analysis results are shown in Figure 4B , showing that the reduced purity of all antibodies was greater than 95%. M: protein marker, R: reduced SDS-PAGE, N-R: non-reduced SDS-PAGE. The theoretical molecular weights of each protein are shown in Table 28.

Table 28: Expression and purification results of primary antibodies against CD3, CD28 and TROP2

(Note: DNT stands for did not test)

Example 15 Stability results of anti-CD3, CD28 and TROP2 primary antibodies

Anti-CD3, CD28, and TROP2 antibodies were placed in a 40°C incubator and incubated for 7 days before analysis by HPLC-SEC. The HPLC-SEC analysis profile is shown in Figure 5. No aggregation peaks were observed for CD3-002IgG, CD3-006IgG, CD3-007IgG, TP-021IgG, TP-023IgG, CD3-002scFv, CD3-006scFv, and CD3-007scFv. However, significant precipitation was observed for huSP34, huRS7, huE11, and huRS7-scFv, which were not detected by SEC (ND).

Example 16 FACS detection of the binding of huRS7, huE11, TP-021 IgG, and TP-023 IgG to 293-huTROP2 and 293-cyTROP2 target cells

The DNA fragments encoding the extracellular domain (ECD) of human and monkey TROP2 (huTROP2, cyTROP2 in Table 1) were cloned into the mDeZ-TM (the company's own vector, with a secretion signal peptide, a transmembrane sequence and a Zeocin resistance gene) vector and transiently transfected into Expi293 cells. After 24 hours, 700ug/mL Zeocin (final concentration) was added and screened for 7 days to obtain Expi293 cells (293-huTROP2, 293-cyTROP2) with high display of the extracellular domain of human and monkey TROP2 on the cell surface.

293-huTROP2 and 293-cyTROP2, which highly display the extracellular domain of human and monkey TROP2 on their cell surfaces, were used as target cells, and Expi293 cells (293-Ctl) were used as a negative control. The cells were washed three times with PBS, centrifuged at 300 g for 5 minutes each time, and the supernatant discarded. The cells were resuspended in PBS, diluted to a density of 1 × ¹⁰⁶ cells/mL, and 100 μL/well was added to a 96-well plate. The monoclonal antibody was diluted to 200 nM, and 100 μL/well was added to a 96-well plate. The cells were mixed evenly with 293-huTROP2, 293-cyTROP2, and Expi293 cells. The cells were incubated at 4°C for 30 minutes. The cells were washed twice with PBS to remove unbound antibody. Then, 100 μL/well of goat anti-human IgG-PE (1:200 dilution) was added and incubated at 4°C for 30 minutes. The cells were centrifuged at 300 g for 5 minutes and washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200 μl of PBS, and antibody binding to the cells was measured using a Beckman Coulter CytoFLEX flow cytometer. The data were analyzed using GraphPad Prism software. As shown in Figure 6, huRS7, huE11, TP-021 IgG, and TP-023 IgG all bound to both huTROP2 and cyTROP2 displayed on the surface of 293 cells.

Example 17 FACS detection of the binding of CD3-002IgG, CD3-006IgG, CD3-007IgG, CD3-002scFv-Fc, CD3-006scFv-Fc and CD3-007scFv-Fc to Jurkat cells

Jurkat cells, which display high CD3 expression on their cell surface, were used as target cells. The cells were washed three times with PBS, centrifuged at 300 g for 5 minutes each time, and the supernatant discarded. The cells were resuspended in PBS, diluted to a density of 1 × ¹⁰⁶ cells/mL, and 100 μL/well of the solution was added to a 96-well plate. Each antibody was diluted to 200 nM, 100 μL/well of the solution was added to a 96-well plate, and the cells were mixed evenly (no antibody was added to the negative control). The cells were incubated at 4°C for 30 minutes. The cells were washed twice with PBS to remove unbound antibody. 100 μL/well of goat anti-human IgG-PE was then added and incubated at 4°C for 30 minutes. The cells were centrifuged at 300 g for 5 minutes and washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200 μL of PBS, and antibody binding to the cells was detected using a Beckman Coulter CytoFLEX flow cytometer. The data were fitted and analyzed using GraphPad Prism software. The results are shown in Figure 7, showing that all antibodies bound to Jurkat cells.

Example 18 ELISA to detect the affinity of anti-CD3 antibodies to CD3ed-HIS

Recombinant CD3ed-HIS protein was diluted to 2 μg/ml with PBS and added to an ELISA plate at 100 μl/well (coated with an equal amount of BSA as a control) and incubated at 4°C overnight. The coating solution was removed, and blocking solution was added at 200 μl/well and incubated at room temperature for 2 hours. The blocking solution was removed, and the plates were washed three times with 250 μl/well of 0.5‰ PBST. CD3-002 IgG, CD3-006 IgG, CD3-007 IgG, CD3-002 scFv-Fc, CD3-006 scFv-Fc, CD3-007 scFv-Fc, and the positive control antibody huSP34 were diluted to 1 μM in blocking solution. The plates were diluted fivefold to form an 8-point concentration gradient (maximum concentration 1 μM) and added sequentially to the blocked ELISA plate at 100 μl/well and incubated at room temperature for 1 hour. Wash the plate three times with 0.5‰ PBST (remove any remaining droplets with absorbent paper), add 100 μl/well of HRP-labeled goat anti-human IgG antibody, and incubate at room temperature for 45 minutes. Wash the plate five times with 0.5‰ PBST, add 100 μl/well of TMB, incubate at room temperature in the dark for 5 minutes, and add 100 μl/well of stop solution to terminate the substrate color development reaction. Read the OD value at 450 nm using a microplate reader. Analyze the data using GraphPad, plot, and calculate the EC50.

The test results are shown in Figure 8. The EC50 (unit: nM) of CD3-002IgG, CD3-006IgG, CD3-007IgG, huSP34 and CD3-002scFv-Fc, CD3-006scFv-Fc, CD3-007scFv-Fc binding to CD3ed-HIS were 0.14, 0.16, 0.15, 0.17, 1.41, 1.36 and 1.00, respectively.

Example 19 Fortibio determines the affinity of anti-CD3 antibodies for CD3ed-HIS

The Fortebio Octet R8 molecular interaction instrument was used, and The AHC2 Biosensor probe capture method was used to determine the kinetic parameters of binding between anti-CD3-specific antibodies and CD3ed-HIS antigens. The AHC2 probe was activated by soaking it in 1× PBS for 20 minutes. CD3-specific antibodies CD3-002IgG, CD3-006IgG, CD3-007IgG, CD3-002scFv-Fc, CD3-006scFv-Fc, CD3-007scFv-Fc, and huSP34 were diluted to 30 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 CD3ed-HIS antigen was diluted two-fold downwards in 1× PBS to create three concentration gradients, and 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.

The kinetic parameters for the binding of anti-CD3-specific antibodies CD3-002IgG, CD3-006IgG, CD3-007IgG, huSP34, CD3-002scFv-Fc, CD3-006scFv-Fc, CD3-007scFv-Fc, and huSP34-scFv-Fc to CD3ed-HIS are shown in Table 29, and the kinetic characteristic parameter detection results are shown in Figure 9. The results show that all antibodies have good affinity for CD3ed-HIS. Compared with huSP34, CD3-002IgG, CD3-002scFv-Fc, CD3-006IgG, CD3-006scFv-Fc, and huSP34-scFv-Fc have approximately 3- to 4-fold lower affinity. CD3-007IgG and CD3-007scFv-Fc showed no significant difference in affinity compared with huSP34.

Table 29: Kinetic characteristic parameters of anti-CD3 antibodies binding to CD3ed-HIS

Example 20 Fortibio determines the affinity of anti-TROP2 antibodies for TROP2-HIS

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 anti-TROP2 antibodies and the TROP2-HIS antigen. The AHC2 probe was activated by soaking it in 1× PBS for 20 minutes. TROP2-specific antibodies huRS7, huE11, TP-021 IgG, and TP-023 IgG were diluted to 30 μg/ml in 1× PBS and the probe was soaked for 120 seconds to allow binding. The probe was then soaked in 1× PBS for another 120 seconds. The TROP2-HIS antigen was diluted two-fold downwards from 400 nM in 1× PBS to create three concentration gradients. The probe was soaked for 120 seconds to measure the association rate of the antigen and antibody, and the dissociation rate of the antigen and antibody was measured by soaking the probe in 1× PBS for 240 seconds.

The kinetic parameters for the binding of anti-TROP2-specific antibodies huRS7, huE11, TP-021IgG, and TP-023IgG to TROP2-HIS are shown in Table 30, and the results of the kinetic characteristic parameter testing are shown in Figure 10. The results showed that TP-021IgG, TP-023IgG, and huE11 all had good affinity for TROP2-HIS. TP-023IgG had an approximately 2.5-fold higher affinity than huE11. TP-021IgG showed no significant change in affinity compared to HuE11.

Table 30: Kinetic characteristic parameters of huRS7, huE11, TP-021IgG, and TP-023IgG binding to TROP2-HIS

Example 21 Design of anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 tertiary antibody

Based on the results of monoclonal antibody experiments, CD3, CD28, and TROP2 primary antibodies were selected as the basis for constructing TROP2×CD3 bispecific and TROP2×CD3×CD28 trispecific antibodies. The configurations are shown in Figure 11. In some configurations, the H44/L100 positions of the CD3 single-chain antibody (scFv) were mutated to Cysteine to form an intramolecular disulfide bond to stabilize the antibody. Knob-into-Hole (KIH) technology was also used to achieve recombinant heavy chain heterodimers. The sequences are shown in Table 31.

Table 31: Amino acid sequences of candidate anti-TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies

Example 22 Expression and Detection of Anti-TROP2×CD3 Dual Antibodies and TROP2×CD3×CD28 Triple Antibodies

The designed double-antibody and triple-antibody DNA fragments were cloned into the pcDNA3.4 vector, and the recombinant plasmids were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer at pH 3.4 and dialyzed to PBS at pH 7.4. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.

The expressed bispecific and tertiary antibodies were tested for protein yield, purity, cell binding (Jurkat, 293-CD28, and 293-huTROP2), antigen binding ELISA (TROP2-HIS, CD3ed-HIS, and CD28-HIS), accelerated stability (aggregation assayed by DLS and SEC after incubation at 40°C for 7 days), and antigen binding affinity (KD value). The data are shown in Table 32. TPt0019, TPt0025, TPt0042, TPb043, and TPb059 exhibited favorable physical and chemical properties and were selected for further testing.

Table 32: Expression and properties of TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 tertiary antibodies


(Note: DNT stands for did not test, and nonspecific means nonspecific binding)

Example 23 Alanine scanning (ALA scanning) of anti-CD28 antibody CD28-065IgG

In order to obtain CD28 antibodies with different affinities, we performed alanine scanning based on TPt0025, and replaced each amino group of CDRL1, CDRL3, CDRH1, CDRH2 and CDRH3 of the CD28 antibody in the TPt0025 tri-antibody (based on the CD28-065IgG variable region sequence) with alanine. The sequences after replacement are shown in Tables 33 and 34.

Table 33: Anti-CD28 Antibody Light Chain Alanine Scanning CDR Sequences

Table 34: Anti-CD28 Antibody Heavy Chain Alanine Scanning CDR Sequences

Example 24 Affinity Detection of the Anti-TROP2×CD3×CD28 Triple Antibody Alanine Scanning Antibody

The recombinant plasmids against heavy chain and light chain were co-transfected into Expi293 cells. After 3 days of cell culture, the culture medium was centrifuged at high speed and vacuum filtered through a microporous filter membrane. The culture medium was then analyzed using a Fortebio Octet R8 molecular interaction instrument and a The AHC2 Biosensors probe capture assay was used to determine the kinetic parameters of binding between an alanine-scanning anti-TROP2×CD3×CD28 trispecific antibody and the CD28-HIS antigen. The AHC2 probe was activated by soaking it in 1× PBS for 20 minutes. The probe was then soaked in the antibody expression supernatant for 120 seconds to allow binding. The probe was then soaked in 1× PBS for another 120 seconds. The CD28-HIS antigen was diluted to 200 nM in 1× PBS and the probe was soaked for 120 seconds to measure the binding rate. The probe was then soaked in 1× PBS for 240 seconds to measure the dissociation rate.

The affinity (KD) of the alanine scanning antibody to CD28-HIS is shown in Table 35. The results of the kinetic characteristic parameter detection of the binding of the alanine scanning antibody to CD28-HIS are shown in Figure 12.

Table 35: Affinity of alanine scanning antibodies binding to CD28-HIS

(Note: LS means the detection signal is very low (Low signal))

Example 25 Alanine scanning (ALA scanning) of anti-TROP2 antibody TP-023

In order to obtain TROP2 antibodies with different affinities, we performed alanine scanning based on TP-023, replacing each amino group of CDRL1, CDRL3, CDRH1, CDRH2 and CDRH3 of TP-023 monoclonal antibody with alanine. The sequences after replacement are shown in Tables 36 and 37.

Table 36: TP-023 light chain alanine scanning CDR sequences

Table 37: TP-023 Heavy Chain Alanine Scanning CDR Sequences

Example 26 Alanine Scanning Antibody Affinity Detection of Anti-TROP2 Antibody TP-023

The recombinant plasmids against heavy chain and light chain were co-transfected into Expi293 cells. After 3 days of cell culture, the culture medium was centrifuged at high speed and vacuum filtered through a microporous filter membrane. The culture medium was then analyzed using a Fortebio Octet R8 molecular interaction instrument and a The ProA Biosensors probe capture assay was used to determine the kinetic parameters of binding between the alanine-scanning TP-023 antibody and the huTROP2-HIS antigen. The ProA probe was activated by soaking it in 1× PBS for 20 minutes. The probe was then soaked in the antibody expression supernatant for 120 seconds to allow binding. The probe was then soaked in 1× PBS for another 120 seconds. The huTROP2-HIS antigen was diluted to 200 nM in 1× PBS and the probe was soaked for 100 seconds to measure the binding rate. The probe was then soaked in 1× PBS for 200 seconds to measure the dissociation rate.

The affinity (KD) of the alanine scanning antibody to huTROP2-HIS is shown in Table 38. The results of the kinetic characteristic parameter detection of the binding of the alanine scanning antibody to huTROP2-HIS are shown in Figure 13.

Table 38: Affinity of TP-023 Alanine Scanning Antibodies Binding to huTROP2-HIS

Example 27 Design of anti-TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 tertiary antibodies with weakened affinity

Based on the results of alanine scanning experiments, the anti-CD28 affinity-weakened antibody mutants H1A8, H3A2, and H3A9, and the TP-023 affinity-weakened mutants L1A3, L1A12, and L3A3 were selected as the basis for constructing TROP2×CD3 bispecific and TROP2×CD3×CD28 trispecific antibodies. The configurations are shown in Figure 11. All antibodies were constructed using Knob-into-Hole (KIH2) technology to create recombinant heavy chain heterodimers. Sequences are shown in Table 39.

Table 39: Amino acid sequences of candidate anti-TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies with weakened affinity

Example 28: Complete structural analysis of anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody

An AI-based complex structure prediction model was used to analyze the dominant sequences in the experiment and observe the overall structural stability. The structural models of TPb0043, TPt0025, and TPt0042 are shown in Figures 14A, 14B, and 14C.

Example 29 Optimized expression and purification of anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody

The TROP2×CD3 dual antibody and TROP2×CD3×CD28 triple antibody expression vectors were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was centrifuged at high speed and vacuum filtered through a microporous filter membrane. The sample was then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer (pH 3.4) and dialyzed into PBS (pH 7.4). Protein concentration was determined by measuring absorbance at 280 nm using a NanoDrop instrument. Expression results are shown in Table 40.

The purified proteins were analyzed by HPLC. HPLC-SEC purity data for the anti-TROP2×CD3 and TROP2×CD3×CD28 triple antibodies are shown in Table 40 and Figure 15 . The purity of the monoclonal antibodies, purified using a Protein A affinity column, was >88%. SDS-PAGE analysis results are shown in Figure 15 , demonstrating that the reduced purity of the antibodies was >95%. M: protein marker; R: reduced SDS-PAGE; N-R: non-reduced SDS-PAGE.

Table 40: Optimized expression and purification results of TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody

Example 30 Optimized Expression and Purification of Anti-TROP2×CD3 Bispecific Antibodies and TROP2×CD3×CD28 Trispecific Antibodies with Weakened Affinity

The affinity-weakened TROP2×CD3 dual antibody and TROP2×CD3×CD28 triple antibody expression vectors were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was centrifuged at high speed and vacuum-filtered through a microporous filter membrane. The sample was then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer (pH 3.4) and dialyzed into PBS (pH 7.4). Protein concentration was determined by measuring absorbance at 280 nm using a NanoDrop instrument. Expression results are shown in Table 41.

The purified proteins were analyzed by HPLC. HPLC-SEC purity data for the anti-TROP2×CD3 and TROP2×CD3×CD28 triple antibodies are shown in Table 41 and Figure 16. The purity of the monoclonal antibodies, both purified using a Protein A affinity column, was >64%. SDS-PAGE analysis results are shown in Figure 16, demonstrating that the reduced purity of the antibodies was >95%. M: protein marker; R: reduced SDS-PAGE; N-R: non-reduced SDS-PAGE.

Table 41: Optimized expression and purification results of TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies with weakened affinity

Example 31 Fortibio determined the affinity of anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody for the antigens TROP2-HIS, CD28-HIS, and CD3ed-HIS

The Fortebio Octet R8 molecular interaction instrument was used, and The AHC2 Biosensor probe capture assay measured the kinetic parameters of binding between anti-TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies and the antigens TROP2-HIS, CD28-HIS, and CD3ed-HIS. The AHC2 probe was activated by soaking in 1× PBS for 20 minutes. The antibodies were diluted to 30 μg/ml in 1× PBS, and the probe was soaked in this solution for 120 seconds to allow binding. The probe was then soaked in 1× PBS for another 120 seconds. Six to seven concentration gradients of TROP2-HIS, CD28-HIS, and CD3ed-HIS were diluted two-fold in 1× PBS from 400 nM. The probe was soaked in this solution for 120 seconds to measure the association rate of the antigen and antibody. The probe was then soaked in 1× PBS for 240 seconds to measure the dissociation rate of the antigen and antibody.

The kinetic parameters for the binding of the bispecific and tertiary antibodies to TROP2-HIS, CD28-HIS, and D3ed-HIS are shown in Table 42. The kinetic characteristic parameters for the binding of the bispecific and tertiary antibodies to TROP2, CD28-HIS, and CD3ed-HIS are shown in Figure 17. The results showed that TPb0043, TPb0059, TPt0019, TPt0025, and TPt0042 had good affinity for TROP2; TPb0059, TPt0019, and TPt0025 had good affinity for CD28-HIS. TPb0043, TPt0019, TPt0025, and TPt0042 had weaker affinity for CD3ed-HIS, with KD values of ^3.00x10-8 M, ^1.17x10-8 M, ^1.25x10-7 M, and ^1.29x10-7 M, respectively.

Table 42: Kinetic characteristic parameters of the binding of TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies to TROP2-His, CD28-HIS and CD3ed-HIS

Example 32 Fortibio determines the affinity (combination) of TROP2-biotin, CD28-biotin, and CD3ed-biotin antigens against the TROP2×CD3 bispecific antibody and the TROP2×CD3×CD28 trispecific antibody

The Fortebio Octet R8 molecular interaction instrument was used, and The streptavidin (SA) probe capture assay was used to determine the kinetic parameters of binding of TROP2-biotin, CD28-biotin, and CD3ed-biotin antigens to TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 tertiary antibodies. The SA probes were activated by soaking in 1× PBS for 20 minutes. TROP2-biotin, CD28-biotin, and CD3ed-biotin were diluted to 10 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 bispecific and tertiary antibodies were diluted two-fold in 1× PBS from 400 nM to a gradient of 4-7 concentrations. The probes were soaked in 1× PBS for 120 seconds to measure the association rate. The probes were then soaked in 1× PBS for 240 seconds to measure the dissociation rate.

The kinetic parameters for the binding of TROP2-Biotin, CD28-Biotin, and CD3ed-Biotin to the bispecific and tertiary antibodies are shown in Table 43, and the kinetic characteristic parameter detection results are shown in Figure 18. The results showed that TROP2-Biotin had good affinity for both the bispecific and tertiary antibodies, CD28-HIS had good binding to TPb0059, TPt0019, and TPt0025, CD3ed-Biotin had moderate binding to TPb0043 and TPt0019, and weaker binding to TPt0025 and TPt0042.

Table 43: Kinetic characteristic parameters of the binding of TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies to TROP2-biotin, CD28-biotin and CD3ed-biotin

Example 33 ELISA detection of the affinity (combination) of anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody to TROP2

Recombinant TROP2-HIS protein was diluted to 2 μg/ml with PBS and added to an ELISA plate at 100 μl/well (coated with an equal amount of BSA as a control) and incubated at 4°C overnight. The coating solution was removed, and blocking solution was added at 200 μl/well and incubated at room temperature for 2 hours. The blocking solution was removed, and the plate was washed three times with 250 μl/well of 0.5‰ PBST. The antibody was then diluted to 100 nM in blocking solution, and a five-fold dilution was used to form 12 concentration gradients (maximum concentration 100 nM). 100 μl/well was added to the blocked ELISA plate and incubated at room temperature for 1 hour. The plate was washed three times with PBST (removing any remaining droplets with absorbent paper), and HRP-labeled goat anti-human IgG antibody was added at 100 μl/well and incubated at room temperature for 45 minutes. The plate was washed five times with 0.5‰ PBST, TMB was added at 100 μl/well, and the plate was placed in the dark at room temperature for 5 minutes. Stop solution was added at 100 μl/well to stop the substrate color development reaction, and the OD value at 450 nm was read with a microplate reader. The data were analyzed with GraphPad, and graphs were drawn and EC50 was calculated.

The detection results are shown in Figure 19. The EC50 (unit: nM) of anti-TROP2×CD3 bispecific antibodies TPb0043 and TPb0059, TROP2×CD3×CD28 triple antibodies TPt0019, TPt0025, TPt0042 and positive control huRS7 and huE11 monoclonal antibodies binding to TROP2-HIS were 0.049, 0.043, 0.12, 0.15, 0.012, 0.022 and 0.027, respectively.

Example 34 Detection of the affinity (combination) of anti-TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies to CD28-HIS

Recombinant CD28-HIS protein was diluted to 2 μg/ml with PBS and added to the ELISA plate at 100 μl/well (coated with an equal amount of BSA as a control) and incubated at 4°C overnight. The coating solution was removed and blocking solution was added at 200 μl/well and incubated at room temperature for 2 hours. The blocking solution was removed and the plates were washed three times with 250 μl/well of 0.5‰ PBST. The antibody was then diluted to 2 μM with blocking solution and diluted fivefold to form 12 concentration gradients (highest concentration 2 μM). The plates were then added to the blocked ELISA plate at 100 μl/well and incubated at room temperature for 1 hour. The plates were washed three times with 0.5‰ PBST (removing any remaining droplets with absorbent paper) and HRP-labeled goat anti-human IgG antibody was added at 100 μl/well and incubated at room temperature for 45 minutes. The plate was washed five times with 0.5‰ PBST, TMB was added at 100 μl/well, and the plate was placed in the dark at room temperature for 5 minutes. Stop solution was added at 100 μl/well to stop the substrate color development reaction, and the OD value at 450 nm was read with a microplate reader. The data were analyzed with GraphPad, and graphs were drawn and EC50 was calculated.

The test results are shown in FIG20 . The EC50 (unit: nM) of the anti-TPb0059, TPt0019 and TPt0025 antibody molecules binding to CD28-HIS are 5.40, 2.68 and 1.03, respectively.

Example 35 ELISA detection of the affinity of anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody to CD3ed-HIS

Recombinant CD3ed-HIS protein was diluted to 2 μg/ml in PBS and added to an ELISA plate at 100 μl/well (coated with an equal amount of BSA as a control) and incubated at 4°C overnight. The coating solution was removed, and blocking solution was added at 200 μl/well. The plate was incubated at room temperature for 2 hours. The blocking solution was removed, and the plate was washed three times with 250 μl/well of 0.5‰ PBST. The antibody was then diluted to 2 μM in blocking solution. A five-fold dilution series (maximum concentration 2 μM) was created, and 12 concentrations were added to the blocked plate at 100 μl/well. The plate was incubated at room temperature for 1 hour. The plate was washed three times with 0.5‰ PBST (removing any remaining droplets with absorbent paper). HRP-conjugated goat anti-human IgG antibody was added at 100 μl/well and incubated at room temperature for 45 minutes. The plate was washed five times with 0.5‰ PBST, TMB was added at 100 μl/well, and the plate was placed in the dark at room temperature for 5 minutes. Stop solution was added at 100 μl/well to stop the substrate color development reaction, and the OD value at 450 nm was read with a microplate reader. The data were analyzed with GraphPad, and graphs were drawn and EC50 was calculated.

The test results are shown in FIG21 . The EC50 (unit: nM) of TPb0043, TPt0019, TPt0025, TPt0042 and the positive control huSP34 monoclonal antibody binding to CD3ed-HIS were 0.58, 0.79, 3.74, 7.00 and 0.035, respectively.

Example 36 FACS detection of the binding affinity of anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody to target cells

In this experiment, Expi293 cells, which highly display the extracellular domain of human TROP2 on their cell surface, were used as target cells (293-huTROP2). Expi293 cells served as negative controls. Wash the cells three times with PBS, centrifuging at 300g for 5 minutes each time, and discarding the supernatant. Resuspend the cells in PBS, dilute to a density of 1× ¹⁰⁶ cells/mL, and add 100 μL/well to a 96-well plate. Antibodies and positive controls, huRS7 and huE11 monoclonal antibodies, were diluted to 1 μM and serially diluted 5-fold over eight steps (maximum concentration 500 nM). 100 μL/well was added to a 96-well plate and mixed evenly with the 293-huTROP2 cells. Incubate at 4°C for 30 minutes. Wash the cells twice with PBS to remove unbound antibody. Then, add 100 μL/well of goat anti-human IgG-PE and incubate at 4°C for 30 minutes. Centrifuge at 300g for 5 minutes, and wash the cells twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200 μl of PBS, and the binding of the antibodies to the cells was measured using a Beckman Coulter CytoFLEX flow cytometer. The data were fitted and analyzed using GraphPad Prism software.

The test results are shown in Figure 22. All antibodies bound well to 293-huTROP2 cells. The EC50 (unit: nM) of TPb0043, TPb0059, TPt0019, TPt0025, TPt0042, huRS7 and huE11 were 6.48, 4.55, 11.0, 13.8, 7.95, 6.75 and 2.15, respectively; TPb0059 had a higher saturation MFI value.

Example 37 FACS detection of the binding affinity of anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody to 293-TCR target cells

The DNA fragments encoding human CD3 and 1G4 TCR (Table 1) were cloned into mDeZ-HIS (the company's own vector, with a secretion signal peptide and a Zeocin resistance gene) and mDeH-HIS (the company's own vector, with a secretion signal peptide and a Hygromycin resistance gene) vectors, respectively. The dual plasmids were transiently transfected into Expi293 cells. After 24 hours, 700ug/mL Zeocin (final concentration) and 500ug/ml Hygromycin were added and screened for 7 days to obtain Expi293 cells (293-TCR) with high display of human TCR on the cell surface.

In this experiment, Expi293 cells, which display a highly expressed TCR on their cell surface, were used as target cells (293-TCR). Expi293 cells were used as negative cells. Wash the cells three times with PBS, centrifuging at 300g for 5 minutes each time, and discarding the supernatant. Resuspend the cells in PBS and dilute them to a density of 1× ^10⁶ cells/mL. 100 μL/well of the cells were added to a 96-well plate. Antibody was diluted to 2 μM and serially diluted 5-fold over eight steps (maximum concentration: 1 μM). 100 μL/well of the antibody was added to a 96-well plate and mixed with the 293-huTROP2 cells. Incubate at 4°C for 30 minutes. Wash the cells twice with PBS to remove unbound antibody. Then, add 100 μL/well of goat anti-human IgG-PE and incubate at 4°C for 30 minutes. Centrifuge at 300g for 5 minutes, then wash the cells twice with PBS to remove unbound secondary antibody. Finally, resuspend the cells in 200 μL of PBS, and antibody binding to the cells was detected using a Beckman Coulter CytoFLEX flow cytometer. The obtained data were fitted and analyzed by GraphPad Prism software.

The test results are shown in Figure 23. The EC50 (nM) of TPt0019, TPt0025, TPt0042, TPb0043 and huSP34 monoclonal antibodies binding to 293-TCR cells were 25.9, 240.6, 236.0, 37.4 and 3.09, respectively.

Example 38 FACS detection of the binding affinity of anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody to Jurkat target cells

In this experiment, Jurkat cells, which display a high TCR on their cell surface, were used as target cells. The cells were washed three times with PBS, centrifuged at 300 g for 5 minutes each time, and the supernatant discarded. The cells were resuspended in PBS and diluted to a density of 1 × ¹⁰⁶ cells/mL. 100 μL/well of the solution was added to a 96-well plate. The antibody was diluted to 1 μM and serially diluted 5-fold over eight steps (maximum concentration 500 nM). 100 μL/well of the solution was added to a 96-well plate and mixed thoroughly with the Jurkat cells. The cells were incubated at 4°C for 30 minutes. The cells were washed twice with PBS to remove unbound antibody. Then, 100 μL/well of goat anti-human IgG-PE was added and incubated at 4°C for 30 minutes. The cells were centrifuged at 300 g for 5 minutes and washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200 μL of PBS, and antibody binding to the cells was detected using a Beckman Coulter CytoFLEX flow cytometer. The data were analyzed using GraphPad Prism software.

The experimental results are shown in FIG24 . The EC50 (nM) of TPt0019, TPt0025, TPt0042 and TPb0043 binding to Jurkat cells were 6.15, 7.08, 445.6 and 35.8, respectively.

Example 39 FACS detection of the binding of TROP2 antibody-weakened anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody to 293-huTROP2 target cells

293-huTROP2 cells, which display the extracellular domain of human TROP2 on their cell surface, were used as target cells. The cells were washed three times with PBS, centrifuged at 300 g for 5 minutes each time, and the supernatant discarded. The cells were resuspended in PBS and diluted to a density of 1 × ¹⁰⁶ cells/mL. 100 μL/well of the solution was added to a 96-well plate. Each antibody was serially diluted fivefold starting at 200 nM, and 100 μL/well of the solution was added to a 96-well plate and mixed evenly with the 293-huTROP2 cells. The cells were incubated at 4°C for 30 minutes. The cells were washed twice with PBS to remove unbound antibody. Then, 100 μL/well of goat anti-human IgG-PE (1:200 dilution) was added and incubated at 4°C for 30 minutes. The cells were centrifuged at 300 g for 5 minutes and washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200 μL of PBS, and antibody binding to the cells was detected using a Beckman Coulter CytoFLEX flow cytometer. The data were analyzed using GraphPad Prism software. The experimental results are shown in Figure 25. All candidate antibodies bound to huTROP2 displayed on the surface of 293 cells. Flow cytometry detection showed that the binding order was TPt0042≈TPa0001>TPt0047>TPb0043≈TPt0045≈TPt0046>TPb0072.

Example 40 FACS detection of the binding of anti-TROP2×CD3 bispecific antibody and TROP2×CD3×CD28 trispecific antibody weakened by TROP2 antibody to tumor target cells

BxPC3, SW403, and COLO 205 tumor cells were used as target cells. They were washed three times with PBS, centrifuged at 300 g for 5 minutes each time, and the supernatant discarded. The cells were resuspended in PBS and diluted to a density of 1 × ¹⁰⁶ cells/mL. 100 μL/well of each solution was added to a 96-well plate. Each antibody was serially diluted fivefold starting at 800 nM, and 100 μL/well of each solution was added to the 96-well plate and mixed thoroughly. The cells were incubated at 4°C for 30 minutes. The cells were washed twice with PBS to remove unbound antibody. 100 μL/well of goat anti-human IgG-PE was then added and incubated at 4°C for 30 minutes. The cells were centrifuged at 300 g for 5 minutes and washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200 μL of PBS, and antibody binding to the cells was detected using a Beckman Coulter CytoFLEX flow cytometer. Data were analyzed using GraphPad Prism software. The experimental results are shown in Figure 26. All candidate antibodies bound to tumor cells. Flow cytometry showed that the binding order was TPt0042 > TPb0043 > TPt0047 > TPb0072.

Example 41 Stability Evaluation of Anti-TROP2×CD3 Bispecific Antibodies and TROP2×CD3×CD28 Trispecific Antibodies in Different Buffer Systems

As shown in Table 44, stability tests were conducted using a histidine buffer system (pH 5.5/pH 6.0/pH 6.5), a citric acid buffer system (pH 5.0/pH 5.5), and a PBS buffer system (pH 7.2). Samples were diluted to a concentration of 5 mg/mL. Sample incubation conditions, test time points, and test items are shown in Table 45.

Table 44: Buffer systems and corresponding numbers

Table 45: Storage temperature, test items and test time points


X=DLS,DSF,SEC,SDS-PAGE

SEC test results

Table 46: SEC-HPLC test data

As shown in Table 46, the purity of TPt0019/TPt0042/TPt0025 in the histidine system (pH 5.5-6.5), the citric acid system (pH 5.0-5.5), and PBS at 4°C for 28 days was above 97%. After 14 days at 40°C, the purity of the PBS group decreased more rapidly than that of the other groups, but the overall value was above 95%. At T0, the overall purity of TPb0043 samples decreased significantly after the solution change, except for the original PBS system, indicating poor stability. The purity of TPb0059 decreased to below 90% after 28 days at 4°C, and there was no significant change in purity after 14 days at 40°C.

In the repeated freeze-thaw experiment, the purity of TPb0043 dropped below 90% after one freeze-thaw cycle, while the purity of the other groups showed no significant changes after five freeze-thaw cycles.

Tm(DSF) test results

Table 47 Tm detection data

Note: Samples used for Tm detection are T0 samples stored at -80℃ and thawed for use in detection.

Table 47 Tm value test results show that the thermal stability from high to low is: TPt0042＞TPt0019＝TPt0025＞TPb0059＞TPb0043.

DLS test results

Table 48 DLS test data

The statistical results in Table 48 show that after 28 days at 4°C, the average particle size and PI (dispersity index) of TPt0019 in each buffer system gradually increased. The average particle size of TPt0042 in each buffer system did not increase significantly, with most PIs <0.3. TPt0025 had a PI <0.3 in all buffer systems, indicating good particle size uniformity. The average particle size of TPb0043 increased significantly, with two groups of PIs >0.3, indicating a polydisperse state with aggregates. TPb0059 had a PI <0.2, indicating good particle size uniformity. After 14 days at 40°C, TPb0043 and TPb0059 showed a clear tendency to aggregate.

After 1/3/5 freeze-thaw cycles, except for the PBS system, the particle size trend and PI of each molecule showed an increasing trend, and the aggregation tendency was obvious.

Figures 27 to 41 show that after 7 and 14 days at 40°C, no obvious degradation bands were observed for TPt0019, TPt0025, TPt0042, TPb0043, and TPb0059 in various buffer systems. Figures 42 to 51 show that after five freeze-thaw cycles, no obvious degradation bands were observed for TPt0019, TPt0025, TPt0042, TPb0043, and TPb0059. SDS-PAGE revealed a band slightly larger than the monomer for purified TPt0025 and TPb0043, which was confirmed by mass spectrometry to be a hole-hole dimer.

Based on the above results, Tpt0019 and TPt0042 performed better than TPb0043 in terms of purity, thermal stability, and colloidal stability.

Example 42 Study on the TDCC killing activity of anti-TROP2×CD3 dual antibody and TROP2×CD3×CD28 triple antibody against BxPC3, MDA-MB-468, NCI-N87 and other tumor cells and negative HEK293 cells

TROP2 is expressed on the surface of tumor cells. Anti-TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies can exert strong T-cell dependent cellular cytotoxicity (TDCC), thereby specifically killing tumor cells. This experiment used TROP2-high-expressing cells 293-huTROP2, BxPC3, MDA-MB-468, and NCI-N87; TROP2-intermediate-expressing MDA-MB-231; TROP2-low-expressing cells DLD-1, Colo-205, SW403, and T84; and negative cells HEK293 as target cells. Tumor cells in the logarithmic growth phase were digested with trypsin (Source Culture, Cat#S310KJ) and resuspended in phenol red-free 1640 medium (Source Culture, Cat#L230KJ) supplemented with 2% FBS (Gibco, Cat#10091148). The cell density was adjusted to 2× ^10⁵ cells/mL. Tumor cells were added to a 96-well U-bottom plate (NEST, Cat#701101) at a density of 1× ^10⁴ cells/well, with 50 μL added to each well. The cells were allowed to adhere overnight. Test antibodies were then added at varying concentrations diluted in phenol red-free 1640 medium supplemented with 2% FBS. The starting working concentration of the test antibody was 30 nM or 300 nM (3X working concentration), and a ten-fold serial dilution was performed, totaling 7-9 different concentrations, with 50 μL added to each well. Effector Pan T cells were isolated from commercially purchased PBMCs (Sai Li Biotechnology, Donor ID#XW0801211W) using the EasySep ^™ Human T Cell Isolation Kit (Stemcell, Cat#17951) according to the kit's instructions. Cells were resuspended in phenol red-free 1640 medium supplemented with 2% FBS, and the cell concentration was adjusted to 1× ^10⁶ cells/mL using an E:T ratio of 5:1. 5× ^10⁴ cells/50 μL were added to each well. Note: Frozen PBMCs can be thawed one day in advance and cultured overnight in RPMI 1640 (Source Cell, Cat#L210KJ) supplemented with 10% FBS. DNase was added as appropriate to prevent DNA entanglement in dead cells. The 96-well U-bottom plate was then incubated in a 37°C, 5% _CO₂ incubator for 24 hours. Two hours before the end of the incubation period, 10× Lysis buffer was added to the target cell-only wells and incubated for an additional hour. Centrifuge the culture plate at 300g for 5 minutes and transfer 50 μL of supernatant to a 96-well plate. Prepare substrate solution according to the Non-Radioactive Cytotoxicity Assay (Promega, Cat# G1780) 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 (TECAN, Spark). GraphPad Prism was used to calculate the _EC50 by plotting the logarithm of the antibody concentration against the killing ratio using a four-parameter fitting.

The experimental results are shown in Figures 52 to 61. In the TDCC assay, TPt0019, TPt0025, TPt0042, and TPb0043 all demonstrated good cytotoxicity against TROP2-positive cells, while no nonspecific T cell killing was observed in negative HEK293 cells.

Example 43: Study on the Activation Effect of Anti-TROP2×CD3 Bispecific Antibodies and TROP2×CD3×CD28 Trispecific Antibodies on T Cells in the TDCC Killing Assay of Tumor Cells Such as BxPC3, MDA-MB-468, NCI-N87, and MDA-MB-231 and Negative Cells HEK293

In this study, after the killing assay in Example 42, the 96-well U-bottom plate (NEST, Cat#701101) was removed and centrifuged at 450 g for 5 minutes, and the supernatant was discarded. Then, a staining buffer (PBS + 2% FBS + 5mM EDTA) was used to prepare a staining solution containing Brilliant Violet 785 ^™ anti-human CD3 Antibody (BioLegend, Cat#344842), Brilliant Violet 605 ^™ anti-human CD4 Antibody (BioLegend, Cat#344646), Brilliant Violet 421 ^™ anti-human CD8 Antibody (BioLegend, Cat#344748), BD Pharmingen ^™ PE Mouse Anti-Human CD25 (BD, Cat#555432), and BD Pharmingen ^™ APC Mouse Anti-Human CD69 (BD, Cat#555533) and other flow cytometry antibody solutions (panel as shown in Table 49) were prepared. The cells were resuspended with the prepared flow cytometry antibody solution and incubated at 4°C in the dark for 30 minutes.

Table 49: Streaming panel

After incubation, cells were centrifuged at 450 g for 5 minutes, the supernatant discarded, and washed twice with staining buffer. The supernatant was removed and 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). After data collection, the percentages of CD25+CD69+ populations in CD4 and CD8 cells were derived using FlowJo software. Four-parameter fitting was then performed using GraphPad to plot the logarithm of sample concentration against the percentage of CD25+CD69+ double-positive cells.

The experimental results are shown in Figures 62 to 68. In T cell activation experiments accompanied by TDCC experiments, T cell activation in the TROP2-positive cell killing assay was TPt0019≈TPt0025>TPb0043+TPt0059>TPt0042≈TPb0043. In TROP2-negative HEK293 cells, TPt0019>TPt0025 resulted in a small amount of non-specific T cell activation, while no T cell activation was detected with TPt0042, TPb0043, or TPb0043+TPt0059.

Example 44 Study on the Release Levels of Cytokines IL-2, IFNr, IL6, and TNFa in the TDCC Killing Effects of Anti-TROP2×CD3 Dual Antibody and TROP2×CD3×CD28 Triple Antibody on BxPC3, MDA-MB-468, and Negative HEK293 Cells

In this study, following the incubation period in Example 42, the 96-well U-bottom plate (NEST, Cat# 701101) was removed and centrifuged at 450 g 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 beads were transferred to a centrifuge tube and dissolved in diluent. After standing for 15 minutes, the capture microspheres of IL-2 (BD ^™ Cytometric Bead Array (CBA) Human IL-2 Flex Set, BD, Cat#558270), TNF-a (BD ^™ Cytometric Bead Array (CBA) Human TNF Flex Set, BD, Cat#560112), IFN-γ (BD ^™ Cytometric Bead Array (CBA) Human IFN-γ Flex Set, BD, 558269), and IL-6 (BD ^™ Cytometric Bead Array (CBA) Human IL-6 Flex Set, BD 558276) were mixed in a 1:1 ratio. The microspheres were vortexed thoroughly before mixing and then aliquoted into a 96-well plate, with 50 μl per well. Dilute the supernatant sample according to the experimental requirements and prepare the standard according to the kit instructions. Add the diluted standard and sample to a 96-well plate, mix thoroughly with the previously added microsphere mixture, and shake on a shaker at 500 rpm for 5 minutes. Incubate in the dark for 2 hours. 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. Shake on a shaker at 500 rpm for 5 minutes, and incubate in the dark for 1 hour. After the reaction, add 100 μl of wash buffer, centrifuge at 500 g for 5 minutes, discard the supernatant, and resuspend in 100 μl of FACS staining buffer (PBS + 2% FBS + 5 mM EDTA) before analysis on a flow cytometer. FlowJo was used to analyze the data, and the MFI values for each cytokine were derived for each well. Cytokine concentrations in the sample wells were calculated based on the standard wells. Four-parameter fitting was performed using GraphPad to plot the logarithm of the antibody concentration against the cytokine concentration.

The experimental results are shown in Figures 69 to 73. In the Th1/Th2 cytokine (IL-2, IFNγ, IL-6, and TNF-α) release studies accompanying the TDCC experiment, TPt0042 and TPb0043 showed low cytokine release. No nonspecific cytokine release was observed in negative HEK293 cells.

Example 45 Study on the TDCC killing activity of anti-TROP2×CD3 dual antibodies TPt0042 and TPb0043 against non-tumor cells

In order to detect the specific and nonspecific killing effects of TROP2×CD3 bispecific antibodies and TPt0042 and TPb0043 on non-tumor cells based on the TROP2 target, this experiment used TROP2 high-expressing cells HACAT, TROP2 medium-expressing cells RWPE, and low-expressing and negative cells HUVEC, PNT1A, BEAS-2B, Nthy-Ori3-1, HK-2, MRC5, IMR-90, HFL-1, W138-VA13 and ARPE-19 as target cells. Tumor cells in the logarithmic growth phase were digested with trypsin (Source Culture, Cat#S310KJ) and resuspended in phenol red-free 1640 medium (Source Culture, Cat#L230KJ) supplemented with 2% FBS (Gibco, Cat#10091148). The cell density was adjusted to 2× ^10⁵ cells/mL. Tumor cells were added to a 96-well U-bottom plate (NEST, Cat#701101) at a density of 1× ^10⁴ cells/well, with 50 μL added to each well. The cells were allowed to adhere overnight. The test antibody was then added at various concentrations diluted in phenol red-free 1640 medium supplemented with 2% FBS. The starting working concentration of the test antibody was 300 nM (3X the working concentration), and a ten-fold serial dilution was performed, resulting in nine different concentrations. 50 μL was added to each well. Effector Pan T cells were isolated from commercially purchased PBMCs (Sai Li Biotechnology, Donor ID#XW0801211W) using the EasySep ^™ Human T Cell Isolation Kit (Stemcell, Cat#17951) according to the kit's instructions. Cells were resuspended in phenol red-free 1640 medium supplemented with 2% FBS, and the cell concentration was adjusted to 1× ^10⁶ mL using an E:T ratio of 5:1. 5× ^10⁴ cells/50 μL were added to each well. Note: Frozen PBMCs can be thawed one day in advance and cultured overnight in RPMI 1640 (Source Cell, Cat#L210KJ) supplemented with 10% FBS. DNase was added as appropriate to prevent DNA entanglement in dead cells. The 96-well U-bottom plate was then incubated in a 37°C, 5% _CO₂ incubator for 24 hours. Two hours before the end of the incubation period, 10× Lysis buffer was added to the target cell-only wells and incubated for an additional hour. Centrifuge the culture plate at 300g for 5 minutes and transfer 50 μL of supernatant to a 96-well plate. Prepare the substrate solution according to the Non-Radioactive Cytotoxicity Assay (Promega, Cat# G1780) 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 (TECAN, Spark). GraphPad Prism was used to calculate the EC50 by plotting the logarithm of the antibody concentration against the cytotoxicity ratio using a four-parameter fitting.

The experimental results are shown in Figures 74 to 85. In the TDCC assay, TPt0042 and TPb0043 demonstrated strong cytotoxicity against both high- and medium-expressing TROP2 cells, but exhibited less cytotoxicity against low-expressing TROP2 cells (HUVEC, PNT1A, and BEAS-2B). TPt0042 showed no cytotoxicity against very low-expressing and negative TROP2 cells, and TPb0043 demonstrated low cytotoxicity against IMR-90 and HFL-1 cells at high concentrations.

Example 46 Study on the TDCC killing activity of CD28-weakened anti-TROP2×CD3×CD28 triple antibody against BxPC3 tumor cells and negative HEK293 cells

Tumor cells express TROP2 on their surface. The anti-TROP2×CD3×CD28 triple antibody can exert potent T-cell-dependent cellular cytotoxicity (TDCC), thereby specifically killing tumor cells. In this experiment, BxPC3 cells, which overexpress TROP2, and HEK293 cells, which negatively express TROP2, were used as target cells. Tumor cells in the logarithmic growth phase were digested with trypsin (Source Culture, Cat#S310KJ) and resuspended in phenol red-free 1640 medium (Source Culture, Cat#L230KJ) supplemented with 4% FBS (Gibco, Cat#A5669701) to a cell density of 2× ^10⁵ cells/mL. Tumor cells were then plated in 96-well U-bottom plates (NEST, Cat#701101) at a density of 1× ^10⁴ cells/well (50 μL) and allowed to adhere and grow overnight. Then, different concentrations of the test antibody diluted in phenol red-free 1640 medium containing 4% FBS were added. The starting working concentration of the test antibody was 400 nM (4X working concentration), and a ten-fold serial dilution was performed, resulting in nine different concentrations. 50 μL was added to each well. Pan T effector cells were isolated from commercially purchased PBMCs (Sai Li Bio, Donor ID #XW0301032W) according to the EasySep ^™ Human T Cell Isolation Kit (Stemcell, Cat#17951). The cells were resuspended in phenol red-free 1640 medium containing 4% FBS, and the cell concentration was adjusted to 5 × 10 ⁵ cells/mL using an E:T ratio of 5:1. 5 × 10 ⁴ cells/100 μL were added to each well. Note: Frozen PBMC 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 dead cell DNA from entanglement. Then place the 96-well U-bottom plate in a 37°C, 5% _CO2 incubator and incubate for 24h or 48h. 2h before the end of incubation, add 10× Lysis buffer to the target cell wells and continue incubation for 1h. Centrifuge the culture plate at 300g for 5min and transfer 50μL of supernatant to the 96-well plate. Press CytoTox Prepare substrate solution according to the Non-Radioactive Cytotoxicity Assay (Promega, Cat# G1780) 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 (TECAN, Spark). GraphPad Prism was used to calculate the _EC50 by plotting the logarithm of the antibody concentration against the killing ratio using a four-parameter fitting.

The experimental results are shown in Figures 86 and 87. In the TDCC experiment, TPt0050, TPt0051, TPt0052, TPt0053, TPt0054, and TPb0055 all showed good killing effects in TROP2-high-expressing BxPC3 cells. There was no nonspecific T cell killing in negative HEK293 cells.

Example 47 Study on the activation effect of CD28 antibody-weakened anti-TROP2×CD3×CD28 triple antibody on T cells in the killing experiment of negative HEK293 cells TDCC

In this study, after the killing assay in Example 46, the 96-well U-bottom plate (NEST, Cat#701101) was removed and centrifuged at 450 g for 5 minutes, and the supernatant was discarded. Then, a staining buffer (PBS + 2% FBS + 5mM EDTA) was used to prepare a staining solution containing BD Pharmingen ^™ PerCP-Cy ^™ 5.5 Mouse Anti-Human CD4 Antibody (BioLegend, Cat#552838), Brilliant Violet 421 ^™ anti-human CD8 Antibody (BioLegend, Cat#344748), BD Pharmingen ^™ PE Mouse Anti-Human CD25 (BD, Cat#555432), and BD Pharmingen ^™ APC Mouse Anti-Human CD69 (BD, Cat#555533) and other flow cytometry antibody solutions (panel as Table 50) were prepared, and the cells were resuspended with the prepared flow cytometry antibody solution and incubated at 4°C in the dark for 30 minutes.

Table 50: Streaming panel

After incubation, cells were centrifuged at 450 g for 5 minutes, the supernatant discarded, and washed twice with staining buffer. The supernatant was removed and 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). After data collection, the percentages of CD25+CD69+ populations in CD4 and CD8 cells were derived using FlowJo software. Four-parameter fitting was then performed using GraphPad to plot the logarithm of sample concentration against the percentage of CD25+CD69+ double-positive cells.

The experimental results are shown in Figure 88. In the T cell activation experiment accompanied by the TDCC experiment, a small number of T cells were non-specifically activated at high concentrations of TPt0050 and TPt0051 in TROP2-negative HEK293 cells, while no T cell activation was detected with TPt0052, TPb0053, TPb0054, and TPb0055.

Example 48 Study on the TDCC Cytotoxicity of Anti-TROP2×CD3 Bispecific Antibodies and TROP2×CD3×CD28 Trispecific Antibodies Against BxPC3 and SW403 Tumor Cells

Since tumor cells express TROP2 on their surface, anti-TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies can exert potent T-cell-dependent cellular cytotoxicity (TDCC), thereby specifically killing tumor cells. In this experiment, BxPC3 cells with high TROP2 expression and SW403 cells with low TROP2 expression were used as target cells. Tumor cells in the logarithmic growth phase were digested with trypsin (Source Culture, Cat#S310KJ) and resuspended in phenol red-free 1640 medium (Source Culture, Cat#L230KJ) supplemented with 4% FBS (Gibco, Cat#A5669701) to a cell density of 2× ^10⁵ cells/mL. Tumor cells were then added to a 96-well U-bottom plate (NEST, Cat#701101) at a density of 1× ^10⁴ cells/well (50 μL) and allowed to adhere and grow overnight. Then, different concentrations of the test antibody diluted in phenol red-free 1640 medium containing 4% FBS were added. The starting working concentration of the test antibody was 400 nM (4X working concentration), and a ten-fold serial dilution was performed, resulting in nine different concentrations. 50 μL was added to each well. Pan T effector cells were isolated from commercially purchased PBMCs (Sai Li Bio, Donor ID #XW0801211W) according to the EasySep ^™ Human T Cell Isolation Kit (Stemcell, Cat#17951) instructions. The cells were resuspended in phenol red-free 1640 medium containing 4% FBS, and the cell concentration was adjusted to 5 × 10 ⁵ cells/mL using an E:T ratio of 5:1. 5 × 10 ⁴ cells/100 μL were added to each well. Note: Frozen PBMCs can be revived one day in advance and cultured overnight with RPMI 1640 (source culture, Cat#L210KJ) + 10% FBS medium. An appropriate amount of DNase is added to prevent DNA entanglement of dead cells. Then the 96-well U-bottom plate is placed in a 37°C, 5% CO ₂ incubator for 24h, 48h and 72h. 2h before the end of incubation, 10× Lysis buffer is added to the target cell wells and incubated for another 1h. Centrifuge the culture plate at 300g for 5min and transfer 50μL of supernatant to the 96-well plate. Press CytoTox Prepare substrate solution according to the Non-Radioactive Cytotoxicity Assay (Promega, Cat# G1780) 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 (TECAN, Spark). GraphPad Prism was used to calculate the _EC50 by plotting the logarithm of the antibody concentration against the killing ratio using a four-parameter fitting.

The experimental results are shown in Figures 89 and 90. In the TDCC assay, TPt0042, TPt0045, TPt0046, TPt0052, and TPb0043 showed strong cytotoxicity against TROP2-positive cells, including BxPC3 and SW403. TPt0047 showed the second strongest cytotoxicity, while TPb0072 had a weaker cytotoxicity against TROP2-positive cells.

Example 49: Study on the TDCC Cytotoxicity of Anti-TROP2×CD3 Bispecific Antibodies and TROP2×CD3×CD28 Trispecific Antibodies Against BxPC3, SW403, and Colo205 Tumor Cells

Tumor cells express TROP2 on their surface. Anti-TROP2×CD3 bispecific antibodies and TROP2×CD3×CD28 trispecific antibodies can exert potent T-cell-dependent cellular cytotoxicity (TDCC), thereby specifically killing tumor cells. In this experiment, TROP2-high-expressing BxPC3 cells and TROP2-low-expressing Colo205 and SW403 cells were used as target cells. Tumor cells in the logarithmic growth phase were digested with trypsin (Source Culture, Cat#S310KJ) and resuspended in phenol red-free 1640 medium (Source Culture, Cat#L230KJ) supplemented with 4% FBS (Gibco, Cat#A5669701) to a cell density of 2× ^10⁵ cells/mL. Tumor cells were then plated in 96-well U-bottom plates (NEST, Cat#701101) at a density of 1× ^10⁴ cells/well (50 μL) and allowed to adhere and grow overnight. Then, different concentrations of the test antibody diluted in phenol red-free 1640 medium containing 4% FBS were added. The starting working concentration of the test antibody was 400 nM (4X working concentration), and a ten-fold serial dilution was performed, resulting in nine different concentrations. 50 μL was added to each well. Pan T effector cells were isolated from commercially purchased PBMCs (Sai Li Bio, Donor ID #XW0801211W) according to the EasySep ^™ Human T Cell Isolation Kit (Stemcell, Cat#17951) instructions. The cells were resuspended in phenol red-free 1640 medium containing 4% FBS, and the cell concentration was adjusted to 5 × 10 ⁵ cells/mL using an E:T ratio of 5:1. 5 × 10 ⁴ cells/100 μL were added to each well. Note: Frozen PBMCs can be revived one day in advance and cultured overnight with RPMI 1640 (source culture, Cat#L210KJ) + 10% FBS medium. An appropriate amount of DNase is added to prevent DNA entanglement of dead cells. Then the 96-well U-bottom plate is placed in a 37°C, 5% CO ₂ incubator for 24h, 48h and 72h. 2h before the end of incubation, 10× Lysis buffer is added to the target cell wells and incubated for another 1h. Centrifuge the culture plate at 300g for 5min and transfer 50μL of supernatant to the 96-well plate. Press CytoTox Prepare substrate solution according to the Non-Radioactive Cytotoxicity Assay (Promega, Cat# G1780) 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 (TECAN, Spark). GraphPad Prism was used to calculate the _EC50 by plotting the logarithm of the antibody concentration against the killing ratio using a four-parameter fitting.

The experimental results, shown in Figures 91 to 93, show that in the TDCC assay, the cytotoxic effects of TPt0042, TPt0047, and TPt0052 on TROP2-positive cells increased over time. They demonstrated strong cytotoxicity against TROP2-overexpressing BxPC3 cells at 24, 48, and 72 hours. TPt0042, TPt0047, and TPt0052 had weaker cytotoxicity against TROP2-low expressing SW403 and Colo205 cells at 24 hours, but demonstrated strong cytotoxicity at 48 and 72 hours. The cytotoxicity trend was TPt0042 > TPt0052 > TPt0047.

Example 50 Study on the Release Levels of Cytokines IL-2, IL-4, IL-6, IL-10, IFNr, and TNFa in the Cytotoxic Effects of Anti-TROP2×CD3 Bispecific Antibodies and TROP2×CD3×CD28 Trispecific Antibodies on BxPC3, SW403, and Colo205 TDCC

In this study, following the incubation period in Example 49, the 96-well U-bottom plates (NEST, Cat#701101) were removed and centrifuged at 450 g for 5 minutes, and the cell supernatant was collected. The BD ^™ Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Cytometric Bead Array (CBA) Kit II (BD, Cat#551809) was used to simultaneously detect IL-2, IL-4, IL-6, IL-10, IFNr, and TNF cytokine levels. First, according to the manufacturer's instructions, the capture microspheres in the kit were mixed in a 1:1 ratio. The microspheres were thoroughly vortexed before mixing, and then aliquoted into a 96-well plate, 50 μl per well. The standard beads were transferred to a centrifuge tube, dissolved in diluent, and allowed to stand for 15 minutes. The standard was then prepared according to the manufacturer's instructions and the supernatant sample was diluted according to experimental requirements. The diluted standard and sample were then added to the 96-well plate, 50 μl per well. Detection antibodies for IL-2, IL-4, IL-6, IL-10, IFNr, and IL-6 were mixed in a 1:1 ratio and aliquoted into a 96-well plate, 50 μl per well. The plate was then incubated in the dark for 3 hours. After the reaction, 100 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 FACS staining buffer (PBS + 2% FBS + 5 mM EDTA) before flow cytometry analysis. Data were analyzed using FCAP array software. Cytokine concentrations in the sample wells were calculated based on the standard wells. Four-parameter fitting was then performed using GraphPad to plot the logarithms of the antibody concentrations against the cytokine concentrations.

The experimental results are shown in Figures 94 to 96. In the study of Th1/Th2 cytokine release (IL-2, IL-10, IFNγ, and TNF-α) during the TDCC experiment, TPt0052 released the highest levels of cytokines, TPb0042 released intermediate levels, and TPt0047 released relatively low levels. IL-4 and IL-6 cytokine release levels were below the detection limit.

Example 51 Antitumor efficacy of anti-TROP2×CD3 dual antibody in BxPC3 mouse transplanted tumor model

The experiment used SPF-grade female NCG mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with the certificate number NO.A202311090101.

BxPC3 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS. 5×10 ⁶ BxPC3 cells in 100 μL of PBS were mixed with an equal volume of Matrigel and inoculated subcutaneously in the axilla of the right forelimb of mice in a 0.2 ml inoculation volume.

When tumors grew to an average of approximately 250 ^mm³ , 15 tumor-bearing mice were randomly divided into three groups of five mice each based on tumor volume and body weight. Ten days prior to grouping, 1× ^10⁷ PBMCs were injected via the tail vein. The day of grouping was designated Day 0, and dosing began. The groupings and dosing schedule are shown in Table 51.

The body weight and tumor volume of mice were measured twice a week, as shown in Figures 97A and 97B. The relative tumor inhibition rate (TGI%) was calculated on Day 28 using the following formula:

Relative tumor growth inhibition rate (TGI): TGI% = (1-T/C) × 100%. Calculation formula: T/C% = _TRTV / _CRTV × 100% ( _TRTV : mean RTV of the experimental group; _CRTV : mean RTV of the negative control group; RTV = Vt/V0, where V0 is the tumor volume at the time of grouping and Vt is the tumor volume after treatment).

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

Table 51 Grouping and Dosage Regimen

N: Number of animals in each group. Dosage volume: The administration volume for animals was adjusted to 10 μL/g body weight.

The tumor inhibition rate results are shown in Table 52. On day 28 after grouping, the TPt0042 and TPb0043 groups significantly inhibited tumor growth compared to the vehicle group, with tumor inhibition rates of 99.71% and 99.95%, respectively. Simultaneously, we measured the body weight of the mice, and the results, as shown in Figure 97B, showed no significant differences in mouse body weight.

Table 52 Tumor inhibition rate on Day 28

Example 52 Antitumor efficacy of anti-TROP2×CD3 dual antibody in BxPC3 mouse transplanted tumor model

The experiment used SPF-grade female NCG mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with the certificate number NO.A202312140110.

BxPC3 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS. 5 × 10 ⁶ BxPC3 cells in 100 μL of PBS were mixed with an equal volume of Matrigel and inoculated subcutaneously in the axilla of the right forelimb of mice in a 0.2 ml inoculation volume.

When tumors grew to an average size of approximately 100-200 ^mm³ , 35 tumor-bearing mice were randomly divided into seven groups of five mice based on tumor volume and body weight. Ten days prior to grouping, 1× ^10⁷ PBMCs were injected via the tail vein. The day of grouping was designated Day 0, and dosing began. The groupings and dosing schedule are shown in Table 53.

The body weight and tumor volume of mice were measured twice a week, as shown in Figures 98A and 98B. The relative tumor inhibition rate (TGI%) was calculated on Day 31 using the following formula:

Relative tumor growth inhibition rate (TGI): TGI% = (1-T/C) × 100%. Calculation formula: T/C% = _TRTV / _CRTV × 100% ( _TRTV : mean RTV of the experimental group; _CRTV : mean RTV of the negative control group; RTV = Vt/V0, where V0 is the tumor volume at the time of grouping and Vt is the tumor volume after treatment).

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

Table 53 Grouping and Dosage Regimen

N: Number of animals in each group. Dosage volume: The administration volume for animals was adjusted to 10 μL/g body weight.

The results of tumor inhibition rates are shown in Table 54. On day 31 after grouping, TPt0042 and TPb0043 antibodies significantly inhibited tumor growth at doses of 0.03 mg/kg, 0.1 mg/kg, and 0.3 mg/kg, compared to the vehicle group. Mouse body weights were also measured, and the results, as shown in Figure 98B, showed a downward trend in mouse body weight on Day 31, but no significant differences were observed between groups.

Table 54 Tumor inhibition rate on Day 31

Example 53 Antitumor efficacy of anti-TROP2×CD3 dual antibody in Colo-205 mouse transplanted tumor model

The experiment used SPF-grade female NCG mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with the certificate number NO.A202312210099.

After the experimental mice were released from quarantine, all animals were intravenously injected with 1×10 ⁷ PBMCs, and the day of inoculation was defined as Day-0.

Colo-205 cells were routinely subcultured for subsequent in vivo experiments. On Day 8, cells were harvested by centrifugation and resuspended in PBS. 5×10 ⁶ Colo-205 cells in 100 μL of PBS were mixed with an equal volume of Matrigel and inoculated subcutaneously in the axilla of the right forelimb of mice in a 0.2 ml inoculation volume.

On Day 0, 15 mice with tumor volumes ranging from 192.34 to 399.81 ^mm³ were randomly divided into three groups of 5 mice each based on tumor volume and body weight for the regimen described in Table 55. Fifteen mice with tumor volumes ranging from 411.35 to 592.92 ^mm³ were randomly divided into three groups of 5 mice each based on tumor volume and body weight for the regimen described in Table 56. Dosing began on Day 0.

The body weight and tumor volume of mice were measured twice a week, as shown in Figures 99 and 100. The relative tumor inhibition rate (TGI%) was calculated on Day 17 and Day 14 using the following formula:

Relative tumor growth inhibition rate (TGI): TGI% = (1-T/C) × 100%. Calculation formula: T/C% = _TRTV / _CRTV × 100% ( _TRTV : mean RTV of the experimental group; _CRTV : mean RTV of the negative control group; RTV = Vt/V0, where V0 is the tumor volume at the time of grouping and Vt is the tumor volume after treatment).

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

Table 55 Grouping and Dosage Regimen

Table 56 Grouping and Dosage Regimen

N: Number of animals in each group. Dosage volume: The administration volume for animals was adjusted to 10 μL/g body weight.

Table 55: Tumor inhibition rates. Results for the vehicle group are shown in Table 57. On day 17 after grouping, the average tumor volume in the vehicle group exceeded 2000 mm ³ , and mice in this group were euthanized. Relative to the vehicle group, both the TPt0042 0.1 mg/kg and TPb0043 0.1 mg/kg treatment groups significantly inhibited tumor growth, with tumor inhibition rates of 74.53% and 99.13%, respectively.

Table 56: Tumor inhibition rate results, as shown in Table 58, indicate that on day 14 after grouping, the average tumor volume in the vehicle group exceeded 2000 ^mm³ , and mice in this group were euthanized. Compared to the vehicle group, the TPt0042 0.3 mg/kg and TPb0043 0.3 mg/kg treatment groups also significantly inhibited tumor growth, with tumor inhibition rates of 88.67% and 99.97%, respectively. With prolonged dosing, tumor volume continued to decrease in the TPt0042 0.3 mg/kg and TPb0043 0.3 mg/kg treatment groups, as shown in Table 58. We also measured mouse body weight. Since Colo-205 is a cachexia model, mouse body weight decreased as tumor volume increased, as shown in Figures 99B and 100B.

Table 57 Day 17 tumor inhibition rate

Table 58 Tumor inhibition rate on Day 14

Example 54 Anti-tumor efficacy of anti-TROP2×CD3 dual antibody in MDA-MB-231 mouse transplanted tumor model

The experiment used SPF-grade female NCG mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with the certificate number NO.A202312280152.

MDA-MB-231 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS. 5 × 10 ⁶ MDA-MB-231 cells (100 μL) of PBS were mixed with an equal volume of Matrigel and inoculated subcutaneously in the axilla of the right forelimb of mice in a 0.2 ml inoculation volume.

When tumors grew to an average size of approximately 100-200 ^mm³ , 15 tumor-bearing mice were randomly divided into three groups of five mice each based on tumor volume and body weight. Ten days before grouping, 1× ^10⁷ PBMCs were injected via the tail vein. The day of grouping was designated Day 0, and dosing began. The groupings and dosing schedule are shown in Table 59.

The body weight and tumor volume of mice were measured twice a week, as shown in Figures 101A and 101B. The relative tumor inhibition rate (TGI%) was calculated on Day 27 using the following formula:

Relative tumor growth inhibition rate (TGI): TGI% = (1-T/C) × 100%. Calculation formula: T/C% = _TRTV / _CRTV × 100% ( _TRTV : mean RTV of the experimental group; _CRTV : mean RTV of the negative control group; RTV = Vt/V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

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

Table 59 Grouping and Dosage Regimen

N: Number of animals in each group. Dosage volume: The administration volume for animals was adjusted to 10 μL/g body weight.

The tumor inhibition rate results are shown in Table 60. On day 27 after grouping, tumor growth was significantly inhibited compared to the vehicle, TPt0042, and TPb0043 groups, with tumor inhibition rates of 100.00% and 100.00%, respectively. Simultaneously, we measured the body weight of the mice, and as shown in Figure 101B, no significant differences in mouse body weight were observed.

Table 60 Tumor inhibition rate on Day 27

Example 55 Anti-tumor efficacy of anti-TROP2×CD3 dual antibody in MDA-MB-231 mouse transplanted tumor model

The experiment used SPF-grade female NPG mice (18-25 g, purchased from Beijing Weitonglihua Laboratory Animal Counting Co., Ltd.), and the animal certificate number was NO.110341231100152781.

After the animals were released from quarantine, each mouse was inoculated subcutaneously with 5×106 MDA-MB-231 cells on the right flank. On the day of tumor inoculation, each mouse received a tail vein injection of 1× ¹⁰⁷ PBMCs. When tumors grew to an average size of 80-100 ^mm3 , 15 tumor-bearing mice were randomly divided into three groups of five mice each based on tumor volume and body weight. The day of grouping was designated Day 0. Dosing began on Day 0. The groupings and dosing schedule are shown in Table 61.

Tumor volume and mouse body weight were measured twice weekly. The results are shown in Figures 102A and 102B. The relative tumor inhibition rate (TGI%) was calculated on Day 42 using the following formula:

Relative tumor growth inhibition rate (TGI): TGI% = (1-T/C) × 100%. T/C% = _TRTV / _CRTV × 100% ( _TRTV : mean RTV of the experimental group; _CRTV : mean RTV of the negative control group; RTV = Vt/V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

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

Table 61 Grouping and Dosage Regimen

N: Number of animals in each group. Dosage volume: The administration volume for animals was adjusted to 10 μL/g body weight.

The tumor inhibition rate results are shown in Table 62. On day 42 after grouping, both TPt0042 (1 mg/kg) and TPb0043 (1 mg/kg) significantly inhibited tumor growth compared to the PBS group (P < 0.01), with tumor inhibition rates (TGI) reaching 99.19% and 100%, respectively. Simultaneously, we measured the body weight of the mice, as shown in Figure 102B. Except for one mouse in the PBS group and two mice in the TPb0043 (1 mg/kg) group that were euthanized on Day 31 due to weight loss exceeding 20%, the weight of the remaining mice remained normal.

Table 62 Average tumor volume and tumor inhibition rate on Day 42

Example 56 Antitumor efficacy of TROP2×CD3 dual antibody in the NCI-H292 mouse transplant tumor model

The experiment used SPF-grade female NCG mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with the certificate number NO.A202312280151.

NCI-H292 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS. 5 × 10 ⁶ NCI-H292 cells in 100 μL of PBS were mixed with an equal volume of Matrigel and inoculated subcutaneously in the axilla of the right forelimb of mice in a volume of 0.2 ml.

When tumors grew to an average size of approximately 100-200 ^mm³ , 15 tumor-bearing mice were randomly divided into three groups of five mice each based on tumor volume and body weight. Ten days before grouping, 1× ^10⁷ PBMCs were injected via the tail vein. The day of grouping was designated Day 0, and dosing began. The groupings and dosing schedule are shown in Table 63.

The body weight and tumor volume of mice were measured twice a week, as shown in Figures 103A and 103B. The relative tumor inhibition rate (TGI%) was calculated on Day 28 using the following formula:

Relative tumor growth inhibition rate (TGI): TGI% = (1-T/C) × 100%. Calculation formula: T/C% = _TRTV / _CRTV × 100% ( _TRTV : mean RTV of the experimental group; _CRTV : mean RTV of the negative control group; RTV = Vt/V0, where V0 is the tumor volume at the time of grouping and Vt is the tumor volume after treatment).

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

Table 63 Grouping and Dosage Regimen

N: Number of animals in each group. Dosage volume: The administration volume for animals was adjusted to 10 μL/g body weight.

The tumor inhibition rate results are shown in Table 64. On day 28 after grouping, the TPt0042 and TPb0043 groups significantly inhibited tumor growth compared to the vehicle group, with tumor inhibition rates of 100.00% and 100.00%, respectively. We also measured the body weight of the mice, and as shown in Figure 103B, no significant differences in mouse body weight were observed.

Table 64 Tumor inhibition rate on Day 28

Example 57 Anti-tumor efficacy of anti-TROP2×CD3 dual antibody in the NCI-N87 mouse transplanted tumor model

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.B202310070147.

After the animals were released from quarantine, each mouse was inoculated subcutaneously with 5×10⁶ NCI-N87 cells on the right flank. When tumors grew to 80-100 ^mm⁻³ , 16 tumor-bearing mice were randomly divided into two groups of 8 mice each based on tumor volume and body weight. On the day of grouping, 5× ^10⁶ PBMCs were injected via the tail vein. This grouping date was designated Day 0. Dosing began on Day 1, the second day of grouping. The grouping and dosing schedule are shown in Table 65.

Tumor volume and mouse body weight were measured twice weekly. The results are shown in Figures 104A and 104B. The relative tumor inhibition rate (TGI%) was calculated on Day 28 using the following formula:

Relative tumor growth inhibition rate (TGI): TGI% = (1-T/C) × 100%. T/C% = _TRTV / _CRTV × 100% ( _TRTV : mean RTV of the experimental group; _CRTV : mean RTV of the negative control group; RTV = Vt/V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

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

Table 65 Grouping and Dosage Regimen

N: Number of animals in each group. Dosage volume: The administration volume for animals was adjusted to 10 μL/g body weight.

The tumor inhibition rate results are shown in Table 66. On day 28 after grouping, TPt0042 (1 mg/kg) significantly inhibited tumor growth compared to the PBS group (P < 0.01), with a tumor inhibition rate (TGI) of 94.41%. Simultaneously, we measured the body weight of the mice, and the results, as shown in Figure 104B, showed no abnormalities in the weight of the mice.

Table 66 Average tumor volume and tumor inhibition rate on Day 28

Example 58 PK of TPt0042 in Balb/c mice

The experiment used SPF-grade female Balb/c mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with the certificate number NO.A202312070173.

After the animals were released from quarantine, they were divided into two groups of three mice each. Each mouse was intravenously injected with 1 mg/kg TPt0042, and then blood was collected at the time points shown in Table 67.

Recombinant TROP2-HIS protein was diluted to 2 μg/ml in PBS and added to an ELISA plate at 100 μl/well (coated with an equal amount of BSA as a control) and incubated at 4°C overnight. The coating solution was removed, and blocking solution was added at 200 μl/well. The plates were incubated at room temperature for 2 hours. The blocking solution was removed, and the plates were washed three times with 250 μl/well of 0.5‰ PBST. Serum samples were then diluted in blocking solution. Three dilutions were set: 800x, 1600x, and 3200x at 5 min, 30 min, 1 hour, 3 hours, and 6 hours, D1, D2, D3, D4, and D5; and three dilutions were set: 200x, 400x, and 800x at D7, D14, and D25. The positive control, TPt0042, was diluted two-fold starting at 1 nM to form a six-point concentration gradient (maximum concentration 1 nM). The plate was then added to the blocked ELISA plate at 100 μl/well and incubated at room temperature for 1 hour. The plate was washed three times with 0.5‰ PBST, and HRP-labeled goat anti-human IgG antibody was added at 100 μl/well and incubated at room temperature for 45 minutes. The plate was washed five times with 0.5‰ PBST, and TMB was added at 100 μl/well. The plate was incubated at room temperature in the dark for 5 minutes. The substrate color development reaction was terminated by adding 100 μl/well stop solution. The OD value at 450 nm was read using a microplate reader. The data were analyzed using GraphPad, and graphs were constructed and half-life (T1/2) was calculated.

The PK results are shown in Figure 105. The half-life T1/2 of TPt0042 is 152.2h.

Table 67 PK blood collection time points

Blood collection time: min (minutes); h (hours)

Example 59 Antitumor efficacy of antibodies with weakened affinity in the BxPC3 mouse transplant tumor model

The experiment used SPF-grade female N PG mice (18-25g, purchased from Beijing Weitongda Biotechnology Co., Ltd.). After the animals were out of quarantine, each animal was injected with 1×10 ⁷ PBMC (Sai Li Bio, donor: XW0801187W) through the tail vein to construct a humanized animal model. Three days after PBMC inoculation, each mouse was subcutaneously inoculated with 5×10 ⁶ BxPC3 cells on the right side. Seven days after tumor inoculation, the average tumor volume reached about 170 mm ^3. 40 tumor-bearing mice were randomly divided into 8 groups based on tumor volume and body weight, with 5 mice in each group. The day of grouping was defined as Day 0. Drug administration began on the day of grouping. The grouping situation and dosing schedule are shown in Table 68.

Tumor volume and mouse body weight were measured twice weekly. The results are shown in Figures 106A and 106B. The tumor growth inhibition rate (TGI%) was calculated using the following formula:

Relative tumor growth inhibition rate (TGI): TGI% = (1-T/C) × 100%. T/C = _TRTV / _CRTV × 100 ( _TRTV : average RTV of the experimental group; _CRTV : average RTV of the negative control group; RTV = _Vt / _V0 , where _V0 is the tumor volume of the animal at the time of grouping and _Vt is the tumor volume of the animal after treatment).

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

Table 68 Grouping and Dosage Regimen

The results of tumor growth inhibition are shown in Table 69. On day 21 after grouping, both TPt0051 0.03 mg/kg and TPt0052 0.03 mg/kg significantly inhibited tumor growth compared to the Vehicle group (P < 0.05), with tumor growth inhibition rates (TGI) reaching 73.57% and 97.75%, respectively. Simultaneously, mouse body weight was monitored, and the results are shown in Figure 106B. The body weight of mice in the TPt0051 0.03 mg/kg and TPt0052 0.03 mg/kg groups showed a downward trend, which may be related to the activation and expansion of T cells.

Table 69 Day 21 average tumor volume and tumor growth inhibition rate

Example 60 Antitumor efficacy of antibodies with weakened affinity in the BxPC3 mouse transplanted tumor model

The experiment used SPF-grade female N PG mice (18-25g, purchased from Beijing Weitongda Biotechnology Co., Ltd.). After the animals were released from quarantine, each animal was injected with 1×10 ⁷ PBMC (Miaoshun Bio, donor: P123041110C) through the tail vein to construct a humanized animal model. Six days after PBMC inoculation, each mouse was subcutaneously inoculated with 5×10 ⁶ BxPC3 cells on the right side. Four days after tumor inoculation, the average tumor volume reached about 85 mm ^3. 25 tumor-bearing mice were randomly divided into 5 groups based on tumor volume and body weight, with 5 mice in each group. The day of grouping was defined as Day 0. Drug administration began on the day of grouping. The grouping situation and dosing schedule are shown in Table 70.

Tumor volume and mouse body weight were measured twice weekly. The results are shown in Figures 107A and 107B. The relative tumor inhibition rate (TGI%) was calculated using the Day 25 data as follows:

Relative tumor growth inhibition rate (TGI): TGI% = (1-T/C) × 100%. T/C = _TRTV / _CRTV × 100 ( _TRTV : average RTV of the experimental group; _CRTV : average RTV of the negative control group; RTV = _Vt / _V0 , where _V0 is the tumor volume of the animal at the time of grouping and _Vt is the tumor volume of the animal after treatment).

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

Table 70 Grouping and Dosage Regimen

The results of tumor inhibition rates are shown in Table 71. On day 25 after grouping, TPt0052 0.1 mg/kg, TPt0042 0.1 mg/kg, and TPt0047 0.1 mg/kg significantly inhibited tumor growth compared to the Vehicle group (P < 0.01), with tumor growth inhibition rates (TGI) reaching 99.81%, 99.79%, and 99.04%, respectively. Simultaneously, mouse body weights were monitored, and the results are shown in Figure 107B: The weight of mice in each group showed a downward trend, which may be related to GVHD after PBMC reconstitution.

Table 71 Day 25 average tumor volume and tumor inhibition rate

Example 61 Antibody tolerance experiment in mice

To verify the toxicity of different antibodies in mice, SPF-grade female hTrop2/hCD3E humanized mice (18-25 g, purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.) were used. After the animals were released from quarantine, eight mice were randomly divided into four groups based on body weight, with two mice in each group. The day of grouping was defined as Day 0. Dosing began on the same day of grouping. The grouping and dosing schedule are shown in Table 72.

The weight and status of the mice were monitored regularly after administration, and the results are shown in Figure 108: at a dose of 0.1 mg/kg for antibody TPt0042, the weight of the mice showed a downward trend from Day 1 to Day 5, and then the weight recovered; at a dose of 1 mg/kg for antibody TPt0042, the weight of the mice showed a downward trend from Day 1 until all died on Day 4; at a dose of 1 mg/kg for antibody TPt0047, the weight of the mice showed a downward trend from Day 0 to Day 2, and then the weight recovered; at a dose of 10 mg/kg for antibody TPt0047, the weight of the mice showed a downward trend from Day 1 to Day 5, and then the weight recovered, which demonstrated that the mouse tolerance dose of TPt0047 antibody was higher than that of TPt0042 antibody.

Table 72 Grouping and Dosage Regimen

Example 62 Exploratory Toxicology Study in Rhesus Monkeys

In this exploratory toxicology study, a single male rhesus monkey received repeated intravenous injections of the TROP2xCD3 bispecific antibody TPt0047. Doses were administered four times on Days 1, 8, 15, and 22 (doses of 0.05 mg/kg, 0.15 mg/kg, 0.5 mg/kg, and 3 mg/kg, respectively). No abnormal clinical symptoms or weight changes were observed. However, increases in basophil percentage (BASO%), absolute eosinophil count (EO#), reticulocyte count (RET%/RET#), lactate dehydrogenase (LDH), and gamma-glutamyl transpeptidase (GGT) were observed. Increases in CD3+CD4+CD69+ T lymphocyte surface antigen and CD3+CD8+CD69+ T lymphocyte surface antigen were observed on Days 14, 22, and 28 (maximum increases of 227.8% and 281.2%, respectively). In addition, blood samples were collected and tested for drug toxicity after administration on days 1, 8, 15, and 22. No drug was detected in samples collected on days 15 and 22, presumably due to the production of ADA, which affected drug exposure. In summary, under the conditions of this study, rhesus monkeys were able to tolerate at least 0.15 mg/kg of the TPt0047 bispecific antibody after repeated intravenous administration of the TROP2xCD3 bispecific antibody.

Claims (81)

A multispecific antibody or antigen-binding fragment thereof, comprising: (a) a CD3 binding domain comprising: (i) CD3-LCDR1, CD3-LCDR2, CD3-LCDR3, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, wherein the CD3-LCDR1 comprises SEQ ID NO: 113, the CD3-LCDR2 comprises SEQ ID NO: 114, the CD3-LCDR3 comprises SEQ ID NO: 110, 115, 116 or 126, and the CD3-HCDR1 comprises SEQ ID NO: 1 D NO:111 or 127, the CD3-HCDR2 comprises SEQ ID NO:106 and the CD3-HCDR3 comprises SEQ ID NO:33, 112, 120, 122, 123 or 124, and when the CD3-HCDR1 comprises SEQ ID NO:111, the CD3-LCDR3 does not comprise SEQ ID NO:115 or the CD3-HCDR3 does not comprise SEQ ID NO:112; or (ii) CD3-VL and CD3-VH, wherein the CD3-VL comprises SEQ ID NO: 133 and the CD3-VH comprises SEQ ID NO: 134; and (b) Target antigen binding domain. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CD3-LCDR3 comprises SEQ ID NO: 115 or 116. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CD3-HCDR1 comprises SEQ ID NO:111. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CD3-HCDR3 comprises SEQ ID NO: 112, 122 or 123. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CD3-HCDR3 comprises SEQ ID NO:112. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CD3 binding domain comprises a combination of 6 CDRs as shown in Table 6. The antibody or antigen-binding fragment thereof according to claim 1, wherein _XL1 is A, _XL2 is P, _XL3 is K, _XL4 is S, _XL5 is K, _XL6 is R or L, _XL7 is I, _XL8 is V or A, _XL9 is D or E, _XL10 is N, and _XL11 is L or H. The antibody or antigen-binding fragment thereof according to claim 1, wherein X _H1 is Q, X _H2 is T, X _H3 is N, X _H4 is G, X _H5 is G, X _H6 is G, X _H7 is N, X _H8 is V, X _H9 is G, X _H10 is D, X _H11 is S, G or Q, X _H12 is V and X _H13 is W. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH and VL of the CD3 binding domain comprise the paired VH and VL shown in Tables 2-4, respectively. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VL and VH of the CD3 binding domain respectively comprise the following sequence pairs: SEQ ID NO: 58 and 59. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CD3 binding domain is a fragment antigen-binding domain (Fab). The antibody or antigen-binding fragment thereof according to claim 1, wherein the CD3 binding domain is a variable region domain (Fv). The antibody or antigen-binding fragment thereof according to claim 12, wherein the CD3 binding domain is a single-chain variable region domain (scFv). The antibody or antigen-binding fragment thereof according to claim 13, wherein the amino acid sequence of the CD3 binding domain comprises the amino acid sequence in Table 5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the target antigen is selected from cancer-associated antigens. The antibody or antigen-binding fragment thereof according to claim 15, wherein the cancer-associated antigen is selected from TROP2 or CUCY2C. A multispecific antibody or antigen-binding fragment thereof, comprising: (a) a CD28 binding domain comprising: (i) CD28-LCDR1, CD28-LCDR2, CD28-LCDR3, CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3, wherein the CD28-LCDR1 comprises SEQ ID NO: 187, 192 or 195, the CD28-LCDR2 comprises SEQ ID NO: 121, the CD28-LCDR3 comprises SEQ ID NO: 188 or 193, the CD28-HCDR1 comprises SEQ ID NO: 189, the CD28-HCDR2 comprises SEQ ID NO: 190 or 194 and the CD28-HCDR3 comprises SEQ ID NO: 191; or (ii) CD28-VL and CD28-VH, wherein the CD28-VL comprises a light chain variable region sequence as shown in Tables 7-8, and the CD28-VH comprises a heavy chain variable region sequence as shown in Tables 7-8; and (b) Target antigen binding domain. The antibody or antigen-binding fragment thereof according to claim 17, wherein the CD28-LCDR1 comprises SEQ ID NO:195. The antibody or antigen-binding fragment thereof according to claim 18, wherein the 1st, 2nd, 4th or 5th amino acid (corresponding to Q, N, Y or V, respectively) of the CD28 LCDR1 sequence QNIYVW can be replaced by alanine (A). The antibody or antigen-binding fragment thereof according to claim 17, wherein the CD28-HCDR1 comprises SEQ ID NO: 189. The antibody or antigen-binding fragment thereof according to claim 20, wherein the first amino acid (G) of the CD28-HCDR1 sequence GYTFTSYY can be substituted by alanine (A). The antibody or antigen-binding fragment thereof according to claim 17, wherein the CD28 binding domain comprises a combination of 6 CDR sequences as shown in Table 10. The antibody or antigen-binding fragment thereof according to claim 17, wherein the VH and VL of the CD28 binding domain respectively comprise the paired VH and VL shown in Tables 7-8. The antibody or antigen-binding fragment thereof according to claim 17, wherein the VH and VL of the CD28 binding domain respectively comprise the following sequence pairs: SEQ ID NO: 173 and 174. The antibody or antigen-binding fragment thereof according to claim 17, wherein the CD28 binding domain is a fragment antigen-binding domain (Fab). The antibody or antigen-binding fragment thereof according to claim 17, wherein the CD28 binding domain is a variable region domain (Fv). The antibody or antigen-binding fragment thereof according to claim 26, wherein the CD28 binding domain is a single-chain variable region domain (scFv). The antibody or antigen-binding fragment thereof according to claim 27, wherein the amino acid sequence of the CD28 binding domain comprises the sequence shown in Tables 7-9. The antibody or antigen-binding fragment thereof according to claim 17, wherein the target antigen is selected from CD28, CD3 or a cancer-associated antigen. The antibody or antigen-binding fragment thereof according to claim 29, wherein the cancer-associated antigen is selected from TROP2 or CUCY2C. A multispecific antibody or antigen-binding fragment thereof, comprising: (a) a CD3 binding domain comprising: CD3-LCDR1, CD3-LCDR2, CD3-LCDR3, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, wherein the CD3-LCDR1 comprises SEQ ID NO: 113, the CD3-LCDR2 comprises SEQ ID NO: 114, the CD3-LCDR3 comprises SEQ ID NO: 110, 115, 116 or 126, the CD3-HCDR1 comprises SEQ ID NO: 111 or 127, the CD3-HCDR2 comprises SEQ ID NO: 106 and the CD3-HCDR3 comprises SEQ ID NO: 33, 112, 120, 122, 123 or 124, and when the CD3-HCDR1 comprises SEQ ID NO: 111, the CD3-LCDR3 does not comprise SEQ ID NO: 115 or the CD3-HCDR3 does not comprise SEQ ID NO: 112; or CD3-VL and CD3-VH, wherein the CD3-VL comprises SEQ ID NO: 133 and the CD3-VH comprises SEQ ID NO: 134; (b) a CD28 binding domain comprising: CD28-LCDR1, CD28-LCDR2, CD28-LCDR3, CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3, wherein the CD28-LCDR1 comprises SEQ ID NO: 187, 192 or 195, the CD28-LCDR2 comprises SEQ ID NO: 121, the CD28-LCDR3 comprises SEQ ID NO: 188 or 193, the CD28-HCDR1 comprises SEQ ID NO: 189, the CD28-HCDR2 comprises SEQ ID NO: 190 or 194 and the CD28-HCDR3 comprises SEQ ID NO: 191; or CD28-VL and CD28-VH, wherein the CD28-VL comprises a light chain variable region sequence as shown in Tables 7-8, and the CD28-VH comprises a heavy chain variable region sequence as shown in Tables 7-8; and (c) Target antigen binding domain. The antibody or antigen-binding fragment thereof according to claim 31, wherein the CD3 binding domain comprises a combination of 6 CDRs as shown in Table 6. The antibody or antigen-binding fragment thereof according to claim 31, wherein the CD28 binding domain comprises a combination of 6 CDR sequences as shown in Table 10. The antibody or antigen-binding fragment thereof according to claim 31, wherein the target antigen is selected from cancer-associated antigens. The antibody or antigen-binding fragment thereof according to claim 34, wherein the cancer-associated antigen is selected from TROP2 or CUCY2C. The antibody or antigen-binding fragment thereof according to claim 35, wherein the cancer-associated antigen is TROP2. The antibody or antigen-binding fragment thereof according to claim 36, comprising a TROP2 binding domain, wherein the TROP2 binding domain comprises: TROP2-LCDR1, TROP2-LCDR2, TROP2-LCDR3, TROP2-HCDR1, TROP2-HCDR2 and TROP2-HCDR3, wherein the TROP2-LCDR1 comprises SEQ ID NO:217, the TROP2-LCDR2 comprises SEQ ID NO:218, the TROP2-LCDR3 comprises SEQ ID NO:219 or 221, the TROP2-HCDR1 comprises SEQ ID NO:214, the TROP2-HCDR2 comprises SEQ ID NO:215 and the TROP2-HCDR3 comprises SEQ ID NO:216 or SEQ ID NO:220; or TROP2-VL and TROP2-VH, wherein the TROP2-VL comprises the light chain variable region sequence shown in Table 11, and the TROP2-VH comprises the heavy chain variable region sequence shown in Table 11. The antibody or antigen-binding fragment thereof according to claims 31-37, wherein the CD3 binding domain, CD28 binding domain, or target antigen binding domain is a fragment antigen binding domain (Fab). The antibody or antigen-binding fragment thereof according to claims 31-37, wherein the CD3 binding domain, CD28 binding domain, or target antigen binding domain is a variable region domain (Fv). The antibody or antigen-binding fragment thereof of claim 39, wherein the CD3 binding domain, CD3 binding domain, or target antigen binding domain is a single-chain variable region domain (scFv). The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antigen-binding domain is selected from the group consisting of: bifunctional Fab, bifunctional Fab', F(ab') ₂ , bifunctional Fd, bispecific dsFv (dsFv-dsFv'), scFv dimer (bivalent diabody), bivalent camelized single domain antibody, bivalent nanobody, and bivalent domain antibody. 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. The antibody or antigen-binding fragment thereof according to claim 42, wherein the Fc region comprises a knob-in-hole mutation. The antibody or antigen-binding fragment thereof according to claim 42, wherein the Fc region comprises KIH1 and KIH2 shown in Table 31. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which has the structure shown in Figure 11. The antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the target antigen is selected from the group consisting of: BCMA, CS1, CD123, CD38, CD22, CD33, CD138, DLL3, FLT3, FLT3 Ligand, CD30, CD30 Ligand, CD27, BAFF, SIRPα, CD47, BAFF-R, EPHA3, PD-1, PD-L1, PDL2, CTLA-4, B7-1, B7-2, CD28, TYRO3, CD81, CD96, CD155, DNAM-1, TIM3, VWF, FGFR4, B7-H3, B7-H4, GITR, GITR Ligand, ICOS, B7-H2, 4-1BB, 4-1BB Ligand, OX40, OX40 Ligand, 2B4, C D48, TRPV1, CD40, BTN3A1, SLAMF5, NTB-A, SLAMF1, Mesothelin, IL6, ACE2, CD70, TROP2, NKP30, GPRC5D, PSCA, IL5, TweakR, TIGIT, CSF1R, TNFRSF10B, CD37, CD7, FCGR3A, EPCAM, CLDN6, CD200 , AXL, TGFBR1, CD40 Ligand, TACI, CB1, NKG2D, CD5, IL17RA, IL2RA, CD34, CEACAM5, EGFR, CD46 , CLEC12A, ROR2, HVEM, 5T4, IL6R, CD171, CA9, CD52, FAP, TNFSF12, SELP, ROR1, B7-H6, EPHA2, MI CA, TNFSF11, VEGFA, MICB, LAG3, Her3, CD10, CD114, CD117, LIGHT, CD24, KLRG1, TM4SF1, CD56, CD44, CD160, IL13RA1, BTLA, VEGFR2, ADAM9, GM-CSF, BCL2L1, ADORA2A, CCR4, ALB, AFP, AMHR2, CB2, DKK1, IL1B, IL2, B7-H5, AFP(TCR), NY-ESO-1(TCR), MAGE-A4(TCR), WT1(TCR), IL18RA, CX CR3, CCR8, CFB, CD19, NEFL, FCRL5, CD99, CCR2, NRG1, PGF, SCF, CD43, ANGPTL3, CD112, CLDN18.2 , IL5RA, CD26, CD45, IL15RA, UCHL1, CD73, GFAP, TREM2, PCSK9, MDR-1, IL21, IL21R, PROM1, IL7RA, VSIG4, IL4RA, LGALS1, JAM-A, Galectin-9, SLC7A11, CD36, NKG2A, CD21, IFNAR1, IL11RA, TH EM, GPC3, P2RX7, ICAM-1, SELPLG, CD2, FOLR1, PMEL, B4GALT1, GPR75, MUC1, CLEC2D, HER2, EDA, IGFBP7, DDR1, CD93, FOLR2, GUCY2C, CLEC14A, PSMA, M-CSF, CD83, CLU, CD62L, UPA, ADGRE2, Nect in-4, CD5L, CRTAM, EFNA3, CD69, CHODL, EREG, CD63, CXCR7, GFRA3, EPHA4, GP6, TSLP, BST1, ANXA1, RNF43, SSTR2, PRLR, CD142, TNFSF15, CCR1, EPHA5, GHR, FZD4, CDCP1, CSPG4, GPR55, SPARC, IFNAR2, TGFBR2, CLEC1A, CLEC9A, PVRIG, MMP9, GAS6, ADGRE1, ANTXR1, FGF21, NPR1, CXCR1, CD7 4. IL22, GRP, EMCN, NOTCH3, GDF15, SIGLEC7, CD164, CCR6, GPR87, KCNK9, TPSAB1, SLC4A7, GPR77 , CD32a, CHI3L1, PTPRG, CXCL4, CDH1, YAP1, CDH17, LILRB2, APCDD1, CD23, FZD10, GPA33, GDNF, TENM4, SEMA4D, CXCL1, HBEGF, CCR9, LIV-1, CDH6, CXCR5, ASGR1, ENPP3, CXCR4, CXCR2, CD166, EG FP, RSPO3, PGLYRP1, GNRHR, CD147, CCR5, CD79B, IGF-1R, GPC1, CD6, IL1A, NCR1, LGALS3, SIGLEC9, CEACAM6, IFNB1, SLC2A4, AGTR1, CDH3, IL18BP, AREG, CCR7, LGR4, MMP14, RNASE4, CXCL10, IL 23(IL23A&IL12B), CD14, ACVR1C, ACVRL1, BTC, CEACAM1, APLP2, GPR56, ALPP, CXADR, IL20RA, IL19, LAIR1, CD9, GAST, GPNMB, MRGPRX2, TRPA1, TNFRSF1B, TIM1, GIPR, CALCA, A35R, A29L, ADAM 8. CD205, LRP10, GRPR, GLP1R, HAMP, GPR20, AGR2, BTN3A2, BTN3A3, ADAMTS1, CPM, FCGR3B, TAFA 5. ECSCR, PF4V1, CHRM2, BCAM, CALR, ITGA2&ITGB1, B7-H7, FGF19, CXCL5, CD200R1, CD304, CD98, MST1R, BRD4, NLRP3, FSTL1, S100A9, KIR2DL1, CLEC4C, EGFRVIII, TFRC, SEZ6, CD72, IGF1, ANPEP, OR2H1, MUC16, IL12RB1, IFNA2, TSHR, STEAP1, CD20, FGFR2IIIb, SIGLEC15, ZNRF3, LY6E, DLK1 , IL31RA, ALK, cMET, ROS1, KRAS, CTLA4, LAG-3, TIM-3, CD2, PD-L1, STING, WNT, VISITA, TLR receptor, IL receptor, GMCSFR, CD25, CEA, PSA, NY-ESO-1, GD2, WT1, MAGE-A3, PRAME, Globo H, SP, Sca-1 and CD133. The antibody or antigen-binding fragment thereof according to claim 46, wherein the target antigen is TROP2 or CUCY2C. The antibody or antigen-binding fragment thereof according to claim 47, wherein the TROP2 binding domain comprises TROP2-LCDR1, TROP2-LCDR2, TROP2-LCDR3, TROP2-HCDR1, TROP2-HCDR2 and TROP2-HCDR3, wherein the TROP2-LCDR1 comprises SEQ ID NO: 217, the TROP2-LCDR2 comprises SEQ ID NO: 218, the TROP2-LCDR3 comprises SEQ ID NO: 219 or 221, the TROP2-HCDR1 comprises SEQ ID NO: 214, the TROP2-HCDR2 comprises SEQ ID NO: 215 and the TROP2-HCDR3 comprises SEQ ID NO: 216 or 220. The antibody or antigen-binding fragment thereof according to claim 48, wherein the TROP2 binding domain comprises a combination of 6 CDRs as shown in Table 13. The antibody or antigen-binding fragment thereof according to claim 48, wherein the TROP2 binding domain comprises the paired VH and VL shown in Table 11. The antibody or antigen-binding fragment thereof according to claim 48, wherein the VL and VH of the TROP2 binding domain respectively comprise the following sequence pairs: SEQ ID NO: 200 and 201, or SEQ ID NO: 204 and 205. The antibody or antigen-binding fragment thereof according to claim 51, wherein the VL and VH of the TROP2 binding domain respectively comprise the following sequence pairs: SEQ ID NO: 204 and 205. The antibody or antigen-binding fragment thereof according to claim 48, wherein the TROP2 binding domain is in the form of Fab or IgG. The antibody or antigen-binding fragment thereof according to claim 53, wherein the TROP2 binding domain comprises the sequence shown in Table 11. The antibody or antigen-binding fragment thereof according to claim 54, comprising three polypeptides: (i) a first polypeptide comprising the VL and light chain constant regions of a TROP2 binding domain; (ii) a second polypeptide comprising the VH and heavy chain constant regions of the TROP2 binding domain; and (iii) a third polypeptide comprising the CD3 binding domain and a heavy chain constant region, wherein the CD3 binding domain is a scFv. The antibody or antigen-binding fragment thereof according to claim 55, comprising the sequence shown in Table 15. The antibody or antigen-binding fragment thereof according to claim 54, comprising four polypeptides: (i) a first polypeptide comprising the VL and light chain constant regions of a TROP2 binding domain; (ii) a second polypeptide comprising the VH and heavy chain constant regions of the TROP2 binding domain; and (iii) a third polypeptide comprising (a) a VH of a TROP2 binding domain, (b) a heavy chain constant region, (c) the CD3 binding domain, and (d) a heavy chain constant region, wherein the CD3 binding domain is a scFv; and (iv) a fourth polypeptide comprising the VL and light chain constant regions of the TROP2 binding domain. The antibody or antigen-binding fragment thereof according to claim 57, comprising the sequence shown in Table 15. An antibody or antigen-binding fragment thereof comprising: CD3-LCDR1, CD3-LCDR2, CD3-LCDR3, CD3-HCDR1, CD3-HCDR2, and CD3-HCDR3, wherein the CD3-LCDR1 comprises SEQ ID NO: 113, the CD3-LCDR2 comprises SEQ ID NO: 114, the CD3-LCDR3 comprises SEQ ID NO: 110, 115, 116, or 126, the CD3-HCDR1 comprises SEQ ID NO: 111 or 127, the CD3-HCDR2 comprises SEQ ID NO: 106, and the CD3-HCDR3 comprises SEQ ID NO: 33, 112, 120, 122, 123, or 124, and when the CD3-HCDR1 comprises SEQ ID NO: 111, the CD3-LCDR3 does not comprise SEQ ID NO: 115 or the CD3-HCDR3 does not comprise SEQ ID NO: 112; or CD3-VL and CD3-VH, wherein the CD3-VL comprises SEQ ID NO:133 and the CD3-VH comprises SEQ ID NO:134. An antibody or antigen-binding fragment thereof comprising: CD28-LCDR1, CD28-LCDR2, CD28-LCDR3, CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3, wherein the CD28-LCDR1 comprises SEQ ID NO: 187, 192 or 195, the CD28-LCDR2 comprises SEQ ID NO: 121, the CD28-LCDR3 comprises SEQ ID NO: 188 or 193, the CD28-HCDR1 comprises SEQ ID NO: 189, the CD28-HCDR2 comprises SEQ ID NO: 190 or 194 and the CD28-HCDR3 comprises SEQ ID NO: 191; or CD28-VL and CD28-VH, wherein the CD28-VL comprises the light chain variable region sequence shown in Table 7-8, and the CD28-VH comprises the heavy chain variable region sequence shown in Table 7-8. A TROP2 antibody or an antigen-binding fragment thereof, wherein the TROP2 antibody comprises TROP2-LCDR1, TROP2-LCDR2, TROP2-LCDR3, TROP2-HCDR1, TROP2-HCDR2 and TROP2-HCDR3, wherein the TROP2-LCDR1 comprises SEQ ID NO:217, the TROP2-LCDR2 comprises SEQ ID NO:218, the TROP2-LCDR3 comprises SEQ ID NO:219 or 221, the TROP2-HCDR1 comprises SEQ ID NO:214, the TROP2-HCDR2 comprises SEQ ID NO:215 and the TROP2-HCDR3 comprises SEQ ID NO:216 or SEQ ID NO:220. A TROP2 antibody or an antigen-binding fragment thereof, comprising the paired VH and VL shown in Table 11. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is linked to one or more conjugate moieties. The antibody or antigen-binding fragment thereof of claim 63, wherein the conjugate moiety comprises an immunomodulatory agent, an anti-tumor drug, a radioisotope, a clearance regulator, a toxin, a detectable label, RNA, DNA, a cytokine, or a purification moiety. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of the preceding claims, and a pharmaceutically acceptable carrier. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 62. A vector comprising the isolated polynucleotide of claim 66. A host cell comprising the vector of claim 78. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell of claim 68 under conditions where the antibody or antigen-binding fragment thereof is expressed, and recovering the antibody or antigen-binding fragment thereof. A method for treating or ameliorating a disease that benefits from T lymphocyte killing and clearance or a disease associated with tumor-associated antigens 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 to 64, or the pharmaceutical composition according to claim 65. The method of claim 70, wherein the disease is cancer or an immune system disorder. The method of claim 71, 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 cell lung cancer, mesothelioma, 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, vaginal cancer, neural or neuroendocrine tumors, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal neuroendocrine tumor (GI-NEC), small cell bladder cancer (SCBC), glioblastoma multiforme, metastatic castration-resistant pulmonary neuroendocrine tumor, neuroblastoma, metastatic carcinoma, diffuse intrinsic pontine glioma, peritoneal cancer, central nervous system tumors, Prostate cancer, ovarian epithelial cancer, renal cell carcinoma, pancreatic ductal carcinoma, abdominal cancer, fallopian tube cancer, desmoplastic small round cell tumor, osteosarcoma, rhabdomyosarcoma, synovial sarcoma, neurofibrosarcoma, Wilms tumor, bladder cancer, thyroid cancer, glioblastoma, urothelial carcinoma, triple-negative breast cancer, Hodgkin lymphoma, anaplastic large cell lymphoma, diffuse large B-cell lymphoma, peripheral T-cell lymphoma, adult T-cell lymphoma/leukemia, mediastinal B-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal NK/T-cell lymphoma, enteropathy-associated T-cell lymphoma, cutaneous T-cell lymphoma, large B-cell non-Hodgkin lymphoma subtypes, primary mediastinal large B-cell lymphoma, gray zone lymphoma, Epstein-Barr virus-positive diffuse large B-cell lymphoma, diffuse large B-cell lymphoma, and non-Hodgkin lymphoma. According to the method of claim 71, the immune system disease is selected from Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, undifferentiated spondyloarthropathy, juvenile spondyloarthropathy, Behcet's disease, enthesitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, fibromyalgia, chronic fatigue syndrome, pain conditions associated with systemic inflammatory diseases, systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile-onset diabetes mellitus (also known as type 1 diabetes), Wegener's granulomatosis, polymyositis, dermatomyositis, inclusion body myositis, multiple endocrine failure, Schmidt's syndrome, t's syndrome), autoimmune uveitis, Addison's disease, Grave's disease, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, multiple sclerosis, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler syndrome Ssler's syndrome), myasthenia gravis, Eaton-Lambert syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia, scleroderma, progressive systemic sclerosis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), adult-onset diabetes mellitus (also known as type 2 diabetes), mixed Connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, antiphospholipid syndrome, erythema multiforme, Cushing's syndrome, autoimmune chronic active hepatitis, allergic diseases, allergic encephalomyelitis, transfusion reaction leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, eczema, lymphomatoid granulomatosis, Kawasaki's disease, endophthalmitis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, Fuchs' cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft-versus-host disease, transplant rejection, tularemia, periodic fever syndrome, suppurative arthritis, familial Mediterranean fever, TNF receptor-associated periodic syndrome (TRAPS), Muckle-Wells syndrome, hyper-IgD syndrome, central nervous system tetradyarrhythmia, celiac disease, Type 2 diabetes mellitus, diffuse toxic goiter (also known as Graves' disease), inflammatory bowel disease, psoriasis (also known as psoriasis, psoriasis), lupus nephritis, skin inflammation, immune thrombocytopenic purpura, thrombotic thrombocytopenic purpura, antiphospholipid syndrome, autoimmune hemolytic anemia, myasthenia gravis, neuromyelitis optica, CIDP (chronic inflammatory demyelinating polyneuropathy), anti-NMDAR encephalitis, Lambert-Eaton syndrome, pemphigus foliaceus, epidermolysis bullosa, and bullous pemphigoid. The method of claim 70, wherein the subject is human. The method of claim 70, wherein the administration is performed by: a parenteral route including subcutaneous, intraperitoneal, intravenous, intramuscular or intradermal injection; or a non-parenteral route including transdermal, oral, intranasal, intraocular, sublingual, rectal or topical. The method of claim 70, wherein the method further comprises administering an additional therapeutic agent to a subject in need thereof. The method of claim 76, wherein the additional therapeutic agent is selected from the group consisting of: 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 hormonal therapy agent, an antiviral agent, an antibiotic, an analgesic agent, an antioxidant, a metal chelator, a cytokine, an anti-infective agent, and an anti-inflammatory agent. The method of claim 76, 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. The method of claim 76, wherein the additional therapeutic agent directly acts on CD3, CD28, a tumor-associated antigen, or a variant thereof, such as a monospecific antibody targeting CD3, CD28, a tumor-associated antigen, or a variant thereof, a bispecific antibody targeting CD3, CD28, a tumor-associated antigen, or a variant thereof, a multispecific antibody targeting CD3, CD28, a tumor-associated antigen, or a variant thereof, a fusion protein targeting CD3, CD28, a tumor-associated antigen, or a variant thereof, an ADC targeting CD3, CD28, a tumor-associated antigen, or a variant thereof. LDC targeting CD3, CD28, tumor-associated antigens or variants thereof, RDC targeting CD3, CD28, tumor-associated antigens or variants thereof, cell therapy targeting CD3, CD28, tumor-associated antigens or variants thereof, small molecule drugs targeting CD3, CD28, tumor-associated antigens or variants thereof, antisense nucleic acids targeting CD3, CD28, tumor-associated antigens or variants thereof, siRNA targeting CD3, CD28, tumor-associated antigens or variants thereof, mRNA expressing CD3, CD28, tumor-associated antigens or variants thereof, PROTAC targeting CD3, CD28, tumor-associated antigens or variants thereof. The method of claim 76, wherein the one or more additional therapeutic agents are administered concurrently or sequentially with the antibody or antigen-binding fragment thereof. The method of claim 70, wherein the method comprises administering the antibody or antigen-binding fragment thereof according to claim 1 in combination with the antibody or antigen-binding fragment thereof according to claim 17.