Antibody having t-cell activating function

Abstract

The present disclosure relates to T cell activated anti-CD3 antibodies and multispecific antibodies comprising the same, in particular T cell adapters. The present disclosure also relates to compositions containing said antibodies or multispecific antibodies and therapeutic applications thereof.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511918161.3 (22) Application Date 2025.12.18 (66) Domestic Priority Data PCT / CN2024 / 140604 2024.12.19 CN (71) Applicant Shanghai Chengfan Pharmaceutical Co., Ltd. Address Building 1, No. 215, Fute South Road, Waigaoqiao Free Trade Zone, Pudong New Area, Shanghai 200131 (72) Inventors Xu Man, Li Jing, Zhang Xuekun, Wang Wei, Yuan Mei (74) Patent Agency Beijing Zhongzi Law Firm 11247 Patent Attorneys Zhang Li, Huang Gesheng (51) Int.Cl. C07K 16 / 28 (2006.01) C07K 16 / 46 (2006.01) C12N 15 / 13 (2006.01) A61K 39 / 395 (2006.01) A61P 35 / 00 (2006.01) (54) Invention Title: Antibody with T-cell Activation Function (57) Abstract: This disclosure relates to T-cell activating anti-CD3 antibodies and multispecific antibodies containing the same, particularly T-cell connectors. This disclosure also relates to compositions containing said antibodies or multispecific antibodies and their therapeutic applications. Claims (7 pages), Description (51 pages), Sequence Listing (electronic publication), Drawings (31 pages), CN 122255272 A, 2026.06.23, CN 1 22 25 52 72 A 1. An anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: - three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained in the heavy chain variable region (VH) sequence selected from SEQ ID NOs: 31 and 67-69, and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained in the light chain variable region (VL) sequence selected from SEQ ID NOs: 32 and 70-71, and optionally comprising the following amino acid substitutions: LCDR1 is selected from S27eT Amino acid substitutions in S27eV and S27eI, and / or amino acid substitutions in LCDR2 selected from V51I, S52Q, V55R, and S56I, wherein the variable region amino acid residues are numbered according to the Kabat numbering system; - HCDR1-3 contained in the VH sequence of SEQ ID NO: 7, and LCDR1-3 contained in the VL sequence of SEQ ID NO: 8; - HCDR1-3 contained in the VH sequence of SEQ ID NO: 15, and LCDR1-3 contained in the VL sequence of SEQ ID NO: 16; - In SEQ ID NO:The HCDR1-3 contained in the VH sequence of SEQ ID NO: 23, and the LCDR1-3 contained in the VL sequence of SEQ ID NO: 24; - The HCDR1-3 contained in the VH sequence of SEQ ID NO: 39, and the LCDR1-3 contained in the VL sequence of SEQ ID NO: 40; or - The HCDR1-3 contained in the VH sequence of SEQ ID NO: 47, and the LCDR1-3 contained in the VL sequence of SEQ ID NO: 48; Preferably, the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3 contained in the following VH and VL sequence pairs: (a) The VH sequence of SEQ ID NO: 31 and the VL sequence of one of SEQ ID NOs: 32 and 72-74; (b) The VH sequence of one of SEQ ID NOs: 67-69 and the VL sequence of one of SEQ ID NOs: 70-71; (c) SEQ ID NO: The VH sequence of SEQ ID NO: 68 and the VL sequence of one of SEQ ID NOs: 78-82; or (d) the VH sequence of SEQ ID NO: 68 and the VL sequence of SEQ ID NO: 87. 2. The anti-CD3 antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3, wherein: - HCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively; and LCDR1 comprises or is composed of the amino acid sequences selected from SEQ ID NOs: 28 and 75-77, LCDR2 comprises or is composed of the amino acid sequences selected from SEQ ID NOs: 29 and 83-86, and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 30; - HCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and LCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; - HCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs: 9, 10, and 11, respectively; and LCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs: 1, 2, and 11, respectively. The amino acid sequences of SEQ ID NOs 12, 13, and 14 are used to form HCDR1-3; HCDR1-3 contains or is composed of the amino acid sequences of SEQ ID NOs 17, 18, and 19, respectively, and LCDR1-3 contains or is composed of the amino acid sequences of SEQ ID NOs 20, 21, and 22, respectively; HCDR1-3 contains or is composed of the amino acid sequences of SEQ ID NOs 33, 34, and 35, respectively, and LCDR1-3 contains or is composed of the amino acid sequences of SEQ ID NOs 17, 18, and 19, respectively.The antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3, wherein: (i) HCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs: 25, 26, and 27, and LCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively; (ii) HCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively, and LCDR1-3 comprises or is composed of the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively; (iii) HCDR1-3 containing or consisting of the amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively, and LCDR1 and LCDR3 containing or consisting of the amino acid sequences of SEQ ID NOs: 28 and 30, respectively, and LCDR2 containing or consisting of the amino acid sequences of SEQ ID NOs: 83, 84, 85, or 86, respectively; or (iv) HCDR1-3 containing or consisting of the amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively, and LCDR1-3 containing or consisting of the amino acid sequences of SEQ ID NOs: 75, 86, and 30, respectively. 3. The anti-CD3 antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 31, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or is composed thereof; and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 32 and 72-74, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or is composed thereof; (b) the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs:The amino acid sequence of SEQ ID NOs: 67-69, or having at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or consisting thereof; and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 70-71 and SEQ ID NOs: 78-82 and 87, or having at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or consisting thereof; (c) the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 7, or having at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions; The amino acid sequence comprising, or consisting of, the addition, deletion, and / or substitution of amino acids; and / or the light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 8, or having at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or having, or consisting of, one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions; and / or the heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 15, or having at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or having, or consisting of, one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions; and / or the light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 15. (e) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 23, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or is composed of the amino acid sequence of SEQ ID NO: 23; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or is composed of the amino acid sequence of SEQ ID NO: 24; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence. (Claims 2 / 7, Page 3, CN 122255272 A)(f) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39, or has at least 85%, 90%, 95% or 99% identity with the amino acid sequence, or has at least one (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed of the amino acid sequence of SEQ ID NO: 40; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 40, or has at least 85%, 90%, 95% or 99% identity with the amino acid sequence, or has at least one (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed of the amino acid sequence of SEQ ID NO: 40; or (g) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 40. The amino acid sequence of SEQ ID NO: 47, or having at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or consisting thereof; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48, or having at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or consisting thereof. 4. An anti-CD3 antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 31; and the light chain variable region comprises or is composed of the amino acid sequence selected from SEQ ID NOs: 32 and 72-74; (b) the heavy chain variable region comprises or is composed of the amino acid sequence selected from SEQ ID NOs: 67-69; and the light chain variable region comprises or is composed of the amino acid sequence selected from SEQ ID NOs: 70-71, 78-82 and 87; (c) the heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 7; and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 8; (d) the heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 70-71, 78-82 and 87. The amino acid sequence of SEQ ID NO: 15, or composed thereof; and the light chain variable region comprises, or is composed of, the amino acid sequence of SEQ ID NO: 16; (e) the heavy chain variable region comprises, SEQ ID NO:The amino acid sequence of SEQ ID NO: 23, or composed thereof; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24, or composed thereof; (f) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39, or composed thereof; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 40, or composed thereof; or (g) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 47, or composed thereof; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48, or composed thereof. Preferably, the antibody or antigen binding fragment comprises: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, or composed thereof, and a light chain variable region comprising the amino acid sequence of one of SEQ ID NO: 32 and 72-74, or composed thereof; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 68, or composed thereof, and comprising a region selected from SEQ ID NOs: 70-71, The amino acid sequences 78-82 and 87 or the light chain variable region thereof; or (iii) the amino acid sequences SEQ ID NO: 67 or 69 or the heavy chain variable region thereof, and the amino acid sequences SEQ ID NO: 70 or 71 or the light chain variable region thereof. 5. An anti-CD3 antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein said antibody or antigen-binding fragment has one or more of the following characteristics: Claims 3 / 7 Page 4 CN 122255272 A (a) specifically binds to human CD3 antigen, preferably wherein, as measured by in vitro surface plasmon resonance (SPR) binding analysis, the binding affinity KD value is about 10 x 10⁻⁷ M to about 1 x 10⁻⁸ M, preferably about 5 x 10⁻⁷ M to about 5 x 10⁻⁸ M; (b) has immune cross-reactivity with human and monkey CD3 antigens, preferably wherein, as measured by in vitro surface plasmon resonance (SPR) binding analysis, the binding affinity KD values ​​for human and monkey CD3 antigens differ by no more than about 5 times, preferably no more than about 3 times; (c) has substantially no nonspecific binding to surface CD3 antigen-negative cells; and (d) activates CD4+ and CD8+ T cells. 6. An anti-CD3 antibody or an antigen-binding fragment thereof according to any one of claims 1-5, wherein: - the antibody or the antigen-binding fragment thereof is murine, chimeric, or humanized; and / or - the antibody or the antigen-binding fragment thereof is selected from full-length antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv and scFab), crossFab, F(ab')2, or linear antibodies.7. An anti-CD3 antibody or an antigen-binding fragment thereof according to any one of claims 1-6, wherein the antibody comprises an immunoglobulin Fc region, and preferably the Fc region is an IgG isotype, such as the human IgG1 or IgG4 isotype Fc region. 8. A multispecific antibody comprising at least two different antigen-binding specificities, wherein a first binding specificity is provided by a first antigen-binding domain that specifically binds to CD3, and wherein the CD3 antigen-binding domain comprises or is composed of an anti-CD3 antibody or an antigen-binding fragment thereof according to any one of claims 1-7. 9. A multispecific antibody according to claim 8, wherein, in addition to the CD3 binding specificity, the multispecific antibody further comprises at least one, two, three, four, or five different antigen-binding specificities, optionally the antigens being independently selected from tumor-associated antigens (TAAs), other immune-associated molecules, and co-stimulatory molecules. 10. A multispecific antibody according to claim 9, wherein the multispecific antibody has binding specificity for binding to CD3 and at least one (e.g., 1-3 different) TAAs, and optionally also has binding specificity for binding to at least one (e.g., 1) co-stimulatory molecule. 11. The multispecific antibody according to any one of claims 9-10, wherein the TAA is a solid tumor cell surface antigen or a hematologic tumor cell surface antigen, and is optionally selected from CD19, BCMA, TSHR, CD171, CS-1, CLL-1, GD3, TnAg, FLT3, CD38, CD123, CD44v6, B7H3, B7H4, KIT, IL-13Ra2, IL-11Ra, PSCA, PSMA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, sLe, GM3, TGS5, HMWMAA, GD2, FOLR1, FOLR2, TEM1 / CD248, TEM7R , CLDN6 , GPRC5D, CXORF61 , CD97 , CD179a , ALK , PLAC1 , GloboH , NY‑BR‑1 , UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TAARP, WT1, ETV6‑AML, SPA17, XAGE1, Tie 2, MAD‑CT‑1, MAD‑CT‑2, FOSL1, hTERT, ML‑IAP, ERG, NA17, PAX3, AR,Cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY‑TES1, LCK, AKAP‑4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD20, CD30, HER2, ROR1, FLT3, TAAG72, CD22, CD33, GD2, gp100Tn, FAP, TYR, EPCAM, CEA, IGF‑1R, EphB2, MSLN, Claudin18.2, CDH17, CD32b, EGFRvIII, GPNMB, GPR64 ,HER3, LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C, and TACSTD2. 12. The multispecific antibody according to any one of claims 9-11, wherein the co-stimulatory molecule is selected from CD28, OX40, CD137, CD8, ICOS, CD27, GITR, CD2, IL-2RP, and MyD88 / CD40. Claims 4 / 7, page 5, CN 122255272, A 13. A multispecific antibody according to any one of claims 8-12, wherein the multispecific antibody comprises a second antigen-binding domain that specifically binds to a tumor-associated antigen (TAA), preferably, the TAA is a solid tumor cell surface antigen selected from, for example, MSLN, CEA, EpCAM, HER2, PSMA, EGFR, Claudin18.2 and CDH17, or the TAA is a hematologic tumor cell surface antigen selected from, for example, CD19, CD20, CD79b, CD33, BCMA and GPRC5D. 14. The multispecific antibody according to any one of claims 8-13, wherein the antibody comprises a CD3 antigen-binding domain and a TAA antigen-binding domain, and has one or more of the following features: (a) the valence ratio of the TAA antigen-binding domain to the CD3 antigen-binding domain is 1:1 or 2:1, preferably 2:1; (b) the antibody is a bivalent, trivalent, or tetravalent bispecific antibody, preferably a trivalent bispecific antibody; and (c) the antibody is a T-cell adaptor (TCE). 15. The multispecific antibody according to any one of claims 8-14, wherein the antibody further comprises an Fc dimer having first and second immunoglobulin Fc regions, and optionally wherein:(i) The first Fc region and the second Fc region contain amino acid mutations that promote the formation of the Fc dimer; (ii) The first Fc region is a human IgG Fc region containing T336W and S354C, and the second Fc region is a human IgG Fc region containing T366S, L368A, Y407V, and Y349C; (iii) The first Fc region and the second Fc region each contain mutations that reduce or eliminate the binding of the Fc region to FcγR, for example, L234A and L235A mutations; (iv) The first Fc region and the second Fc region are IgG1 or IgG4 isotypes; and / or (v) The first and second Fc regions each contain the amino acid sequences of SEQ ID NO: 108 and SEQ ID NO: 107, or amino acid sequences that are at least 95%, 96%, 98%, or 99% identical to them. 16. The multispecific antibody according to any one of claims 8-15, wherein the antibody comprises: - a first structural portion comprising, from the N-terminus to the C-terminus, a first antigen-binding domain, optionally a linker, and a first immunoglobulin Fc region; - a second structural portion comprising, from the N-terminus to the C-terminus, a second antigen-binding domain, optionally a linker, and a second immunoglobulin Fc region; and - a third antigen-binding domain optionally connected to the N-terminus or C-terminus of the first or second structural portion via the linker; wherein the first and second immunoglobulin Fc regions dimerize to form an Fc dimer, preferably, the linker is 5-25 amino acids in length, or comprises an amino acid sequence of SEQ ID NO: 106, 136, or 112. 17. The multispecific antibody according to any one of claims 8-16, wherein: - the first antigen-binding domain binds CD3 and comprises or is composed of Fab, scFab, or scFv domains; and / or - the second and third antigen-binding domains bind TAA and comprise or are composed of Fab, scFab, scFv, or VHH domains, preferably, wherein the first antigen-binding domain is a Fab domain that binds CD3, and the second and third antigen-binding domains are VHH domains that bind TAA. 18. A multispecific antibody according to any one of claims 8-17, wherein the antibody comprises a TAA antigen-binding domain, and wherein the TAA antigen-binding domain specifically binds to CD20, preferably, the CD20 antigen-binding domain comprises or is composed of a VHH domain, more preferably, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of one of the amino acid sequences in claim 5 / 7 of SEQ ID NO: 102 and 115-124; more preferably, the CDR1, CDR2, and CDR3 sequences respectively comprise SEQ ID NOs: 103,The amino acid sequences 104 and 105 are either composed of or comprise the amino acid sequences of SEQ ID NOs: 137, 104, and 105, respectively; more preferably, the VHH domain comprises amino acid sequences selected from SEQ ID NOs: 102 and 115-124, or has at least 85%, 90%, 95%, or 99% identity with the amino acid sequences, or has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or is composed of, or wherein the TAA antigen-binding domain specifically binds to MSLN, preferably, the MSLN antigen-binding domain comprises or is composed of the VHH domain, more preferably, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of the amino acid sequence of SEQ ID NO: 97; more preferably, the CDR1, CDR2, and CDR3 sequences respectively comprise SEQ ID NOs: 98, The amino acid sequences of 99 and 100 or thereof; more preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 97, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of thereof. 19. A multispecific antibody according to any one of claims 8-18, wherein the antibody is a bispecific antibody against CD3 and CD20, and comprises: (i) first, second, and third polypeptide chains comprising SEQ ID NOs: 88, 89, and 101, respectively, or having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them; (ii) first, second, and third polypeptide chains comprising SEQ ID NOs: 90, 91, and 101, respectively, or having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them; (iii) first, second, and third polypeptide chains comprising SEQ ID NOs: 130, 131, and 132, respectively, or having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them; or (iv) comprising SEQ ID NOs: 88, 89, and 101, respectively. NOs: 133, 134, and 135, or the first, second, and third polypeptide chains having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them, or said antibody is a bispecific antibody against CD3 and MSLN, and comprises: (v) comprising SEQ ID NOs: 88, 89, and 96, or the first, second, and third polypeptide chains having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them, or (vi ...NOs: 90, 91 and 96, or the first, second and third polypeptide chains having an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity with them, preferably, the first, second and third polypeptide chains comprising the amino acid sequence of SEQ ID NOs: 88, 89 and 96, or the amino acid sequence of SEQ ID NOs: 88, 89 and 101, respectively. 20. An anti-CD20 antibody or an antigen-binding fragment thereof, comprising a VHH domain specifically binding to CD20, wherein the VHH domain comprises CDR1, CDR2, and CDR3 sequences of one of the amino acid sequences of SEQ ID NOs: 102, 115-124; preferably, the CDR1, CDR2, and CDR3 sequences comprise or consist of the amino acid sequences of SEQ ID NOs: 103, 104, and 105, respectively, or comprise or consist of the amino acid sequences of SEQ ID NOs: 137, 104, and 105, respectively; more preferably, the VHH domain comprises an amino acid sequence of one of SEQ ID NOs: 102, 115-124, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has one or more (preferably 1-10). (Claims 6 / 7, page 7, CN 122255272 A) The VHH domain comprises, more preferably, 1-5 amino acid sequences with added, deleted, and / or substituted amino acids. Most preferably, the VHH domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 102. 21. A polynucleotide encoding an anti-CD3 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-7, a multispecific antibody as claimed in any one of claims 8-19, or an anti-CD20 antibody or an antigen-binding fragment thereof as claimed in claim 20. 22. A vector, preferably an expression vector, comprising the polynucleotide of claim 21. 23. A host cell comprising the polynucleotide of claim 21 or the vector of claim 22, optionally the host cell being a mammalian cell. 24. A method for producing an anti-CD3 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-7, a multispecific antibody as claimed in any one of claims 8-19, or an anti-CD20 antibody or an antigen-binding fragment thereof as claimed in claim 20, the method comprising: culturing a host cell comprising a polynucleotide encoding the polypeptide chain under conditions suitable for producing the polypeptide chain of the antibody or an antigen-binding fragment thereof or the multispecific antibody. 25. An antigen-binding molecule comprising the anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-7, the multispecific antibody according to any one of claims 8-19, or the anti-CD20 antibody or its antigen-binding fragment according to claim 20, for example...26. An immune conjugate or immune fusion compound. 27. A pharmaceutical composition comprising an anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-7, a multispecific antibody according to any one of claims 8-19, an anti-CD20 antibody or its antigen-binding fragment according to claim 20, or an antigen-binding molecule according to claim 25, and a pharmaceutically acceptable carrier. 28. Use as a pharmaceutical or for the preparation of a pharmaceutical by means of the anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-7, a multispecific antibody according to any one of claims 8-19, an anti-CD20 antibody or its antigen-binding fragment according to claim 20, or an antigen-binding molecule according to claim 25. 29. The use of claim 27, wherein the pharmaceutical is used to treat and / or prevent cancer in an individual. 20. A method of treating or preventing cancer, comprising administering to an individual in need an effective amount of the anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-7, a multispecific antibody according to any one of claims 8-19, an anti-CD20 antibody or its antigen-binding fragment according to claim 20, an antigen-binding molecule according to claim 25, or the pharmaceutical composition of claim 26. 30. The use or method of claim 28 or 29, wherein the cancer is a solid tumor or hematologic malignancy, preferably wherein: the solid tumor is an MSLN-positive solid tumor selected, for example, mesothelioma (such as malignant mesothelioma), pancreatic cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), and colorectal cancer; the hematologic malignancy is a CD20-positive hematologic malignancy selected, for example, B-cell lymphoma (such as DLBCL and LBCL). Claims 7 / 7 Page 8 CN 122255272 A Antibody with T-cell activation function

[0001] Cross-reference to related applications

[0002] This application was filed on December 18, 2025 as a PCT international patent application, claiming priority to PCT international patent application No. PCT / CN2024 / 140604, filed on December 19, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of antibody technology, and more specifically to T-cell activating anti-CD3 antibodies and multispecific antibodies containing them, especially T-cell engagers. This invention also relates to compositions containing said antibodies or multispecific antibodies and their therapeutic applications. Background Art

[0004] T-cell engagers (TCEs) are an important molecular form in the field of antibody therapy. Several TCE drugs have already been approved for marketing. For example, the 2:1 type anti-CD3xCD20 monoclonal antibody Glofitamab has received accelerated approval for the treatment of relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma.Effective drugs for adult patients with large B-cell lymphoma (LBCL) caused by T-cell activation. The core mechanism of TCE is to use T-cell activated anti-CD3 antibodies to recruit human T cells to target cells (e.g., tumor cells) that express target antigens (e.g., tumor-associated antigens) on their surface, thereby selectively killing the target cells. Therefore, providing CD3 antibodies with good performance is an important part of TCE development.

[0005] Currently, the number of commercially available anti-CD3 antibodies is limited. Most of the non-patented anti-CD3 antibodies that are freely available to the public have their own drawbacks. The most common one, OKT3, has no immune cross-reactivity with monkey CD3, which increases the difficulty of toxicological experiments for drugs based on this molecule. SP34, as another commonly used anti-CD3 molecule, not only needs to be humanized when used to construct human drugs, but its physicochemical properties have also been found to be defective, with weak non-specific binding and poor molecular stability, and there are CMC (Chemical Manufacturing and Control) problems in the construction of polyclonal antibodies. On the other hand, existing research and development of CD3-targeted drugs has revealed that most traditional anti-CD3 antibodies, while recruiting T cells to kill tumor cells, can also cause potentially serious toxicities related to T cell activation, especially cytokine release syndrome (CRS) and neurotoxicity, thus limiting the clinical use of the drugs. In addition, there are very few successful examples of anti-CD3 drugs in solid tumors, and they face multiple challenges. See, Overcoming Challenges for CD3-Bispecific Antibody Therapy in Solid Tumors, Cancers (Basel). 2021 Jan; 13(2): 287, doi: 10.3390 / cancers13020287.

[0006] Therefore, there is still a need in the art to develop new anti-CD3 antibodies and T cell activating antibodies and TCE molecules based on them. In the development of CD3-based multi-(bi)specific antibody drugs, optimized solutions are achieved by appropriately selecting targets and designing and matching binding sites in terms of epitope position, affinity range, molecular conformation, and drugability. Summary of the Invention

[0008] The inventors have obtained a novel functional sequence of an anti-CD3 antibody de novo using hybridoma technology. The CD3 sequence exhibits unique behavior. When applied alone, especially in monovalent form, its binding to cell surface CD3 is very weak. However, when spliced ​​into a TCE, it demonstrates very strong T cell activation and killing capabilities, and this ability…Its efficacy is comparable to that of commercially available main competitors. This characteristic of the anti-CD3 sequence of the present invention potentially provides a better efficacy window; the weak binding to CD3 can prevent T cell exhaustion, while the strong killing effect on target cells ensures good anti-tumor efficacy.

[0009] Furthermore, the inventors have also obtained a novel CD20 single-domain antibody de novo. The anti-CD20 single-domain antibody of the present invention has excellent CD20 binding properties and is suitable for tumor treatment applications.

[0010] Therefore, in a first aspect, the present disclosure provides an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody of the present invention comprises three CDRs (HCDR1, HCDR2, and HCDR3) or variants thereof contained in the heavy chain variable region selected from SEQ ID NOs: 7, 15, 23, 31, 39, 47, and 67-69; and three CDRs (LCDR1, LCDR2, and LCDR3) or variants thereof contained in the light chain variable region selected from SEQ ID NOs: 8, 16, 24, 32, 40, 48, 70-74, 78-82, and 87.

[0011] In a second aspect, the present disclosure provides an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody of the present invention comprises three CDRs (CDR1, CDR2, and CDR3) or variants thereof contained in the heavy chain variable region (VHH) of one of SEQ ID NOs: 102 or 115-124.

[0012] In a third aspect, this disclosure provides a multispecific antibody comprising a CD3 antigen-binding domain, particularly a T-cell connector comprising a CD3 antigen-binding domain and a tumor-associated antigen (TAA) binding domain. In some embodiments, the multispecific antibody according to the invention is a multispecific antibody comprising CD3 and CD20 binding specificity, particularly a bispecific antibody. In other embodiments, the multispecific antibody according to the invention is a multispecific antibody comprising CD3 and MSLN binding specificity, particularly a bispecific antibody.

[0013] In a fourth aspect, this disclosure provides a nucleic acid encoding the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the invention, a host cell comprising the same, and a method for generating the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the invention.

[0014] In a fifth aspect, this disclosure provides an antigen-binding molecule comprising the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the invention, such as an immunoconjugate and an immunofusion.

[0015] In a sixth aspect, this disclosure provides a pharmaceutical composition comprising the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the invention.

[0016] In a seventh aspect, this disclosure provides that the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the present invention can be used as...The invention relates to pharmaceuticals or their use in the preparation of pharmaceuticals, and to methods of treating or preventing cancer (including solid tumors and hematologic malignancies) using the anti-CD3 antibody, anti-CD20 antibody, or multispecific antibody of the present invention.

[0017] The invention is further illustrated in the following figures and specific embodiments. However, these figures and specific embodiments should not be considered as limiting the scope of the invention, and modifications readily conceived by those skilled in the art will be included within the spirit of the invention and the scope of protection of the appended claims. Brief Description of the Figures

[0019] Figure 1 shows the binding of the hybridoma chimeric antibody to Jurkat cells.

[0020] Figure 2 shows the binding of the hybridoma chimeric antibody to Jurkat-CD3KO cells.

[0021] Figure 3 shows the binding of the hybridoma chimeric antibody to cynomolgus monkey CD4+ T cells (cyno CD4+ T cells).

[0022] Figure 4 shows the binding of the hybridoma chimeric antibody to cynomolgus monkey CD8+ T cells (cyno CD8+ T cells).

[0023] Figure 5 shows the activation of CD4+ T cells from donor Donor1 by the hybridoma chimeric antibody. Instructions for Use, Page 2 / 51, CN 122255272 A

[0024] Figure 6 shows the activation of CD8+ T cells from donor Donor1 by the hybridoma chimeric antibody.

[0025] Figure 7 shows the activation of CD4+ T cells from donor Donor2 by the hybridoma chimeric antibody.

[0026] Figure 8 shows the activation of CD8+ T cells from donor Donor2 by the hybridoma chimeric antibody.

[0027] Figure 9 shows the binding of the humanized variant of 136G6.3 to Jurkat cells.

[0028] Figure 10 shows the binding of the humanized variant of 136G6.3 to Jurkat-CD3KO cells.

[0029] Figure 11 shows the binding of the PTM-removed variant of 136G6.3 to Jurkat cells.

[0030] Figure 12 shows the binding of the PTM-removed variant of 136G6.3 to Jurkat-CD3KO cells.

[0031] Figure 13 shows the binding of the deimmunogenic variant of 136G6.3-zom2 to Jurkat cells.

[0032] Figure 14 shows the binding of the deimmunogenic variant of 136G6.3-zom2 to Jurkat-CD3KO cells.

[0033] Figure 15 shows the binding of the humanized deimmunogenic PTM-removed variant 136G6.3M of 136G6.3 to Jurkat cells.

[0034] Figure 16 shows the binding of 136G6.3M to Jurkat-CD3KO cells.

[0035] Figure 17 shows the affinity of 136G6.3M for human CD3 (hCD3) and cynomolgus monkey CD3 (cynoCD3) antigens.136G6.3M is represented as "136G6.3-pdzom2m21m11" in the figure.

[0036] Figure 18A shows the binding activity of anti-CD20-VHH antibody against human CD20-positive tumor cells Raji.

[0037] Figure 18B shows the binding activity of anti-CD20-VHH antibody against human CD20-positive tumor cells Daudi.

[0038] Figure 19A shows the binding activity of anti-CD20-VHH antibody against HEK293-cynoCD20 cells expressing monkey CD20.

[0039] Figure 19B shows the binding activity of anti-CD20-VHH antibody against HEK293 cells that do not express CD20.

[0040] Figure 20 shows the binding activity of humanized anti-CD20-VHH antibody against human CD20-positive tumor cells Daudi.

[0041] Figure 21A shows the binding activity of the humanized anti-CD20-VHH antibody to HEK293-cynoCD20 cells expressing monkey CD20.

[0042] Figure 21B shows the binding activity of the humanized anti-CD20-VHH antibody to HEK293 cells that do not express CD20.

[0043] Figure 22A shows the endocytic activity of the humanized anti-CD20-VHH antibody V-zn6D11.m2 on Ramos cells.

[0044] Figure 22B shows the endocytic activity of the humanized anti-CD20-VHH antibody V-zn6D11.m2 on Daudi cells.

[0045] Figures 23A and 23B show schematic diagrams of the structure of the CD3xTAA (CD20 or MSLN) bispecific antibody (bsAb). In the figures, “LALA” represents the L234AL235A mutation located in the CH2 domain; k represents the Knob mutation located in the CH3 domain; and h represents the Hole mutation located in the CH3 domain corresponding to the knob mutation.

[0046] Figure 24 shows the binding of the CD3xCD20 bispecific antibody to Ramos cells.

[0047] Figure 25 shows the binding of the CD3xCD20 bispecific antibody to Jurkat cells.

[0048] Figure 26 shows the killing of Ramos cells by Donor3 PBMCs mediated by the CD3xCD20 bispecific antibody.

[0049] Figure 27 shows the killing of Ramos cells by Donor4 PBMCs mediated by the CD3xCD20 bispecific antibody.

[0050] Figure 28 shows the release of IL6 mediated by the CD3xCD20 bispecific antibody, which accompanies the killing of Ramos cells by Donor3 PBMCs.

[0051] Figure 29 shows the release of IFN-γ mediated by CD3xCD20 dual antibody, accompanying the killing of Ramos by Donor3 PBMCs.

[0052] Figure 30 shows the release of IL-6 mediated by CD3xCD20 dual antibody, accompanying the killing of Ramos by Donor4 PBMCs.

[0053] Figure 31 shows the release of IFN-γ mediated by CD3xCD20 bispecific antibody, accompanying the killing of Ramos by Donor4 PBMCs.

[0054] Figure 32 shows the binding of CD3xMSLN bispecific antibody to AsPC1 cells.

[0055] Figure 33 shows the binding of CD3xMSLN bispecific antibody to Jurkat cells.

[0056] Figure 34 shows the killing of AsPC1 cells by Donor3 PBMCs mediated by CD3xMSLN bispecific antibody. Specification 3 / 51 pages 11 CN 122255272 A

[0057] Figure 35 shows the killing of AsPC1 cells by Donor4 PBMCs mediated by CD3xMSLN bispecific antibody.

[0058] Figure 36 shows the release of IFN-γ mediated by CD3xMSLN bispecific antibody, accompanying the killing of target cells by Donor3 PBMCs.

[0059] Figure 37 shows the release of IL-6 mediated by CD3xMSLN double antibody, accompanying the killing of target cells by Donor3 PBMCs.

[0060] Figure 38 shows the release of TNF-α mediated by CD3xMSLN double antibody, accompanying the killing of target cells by Donor3 PBMCs.

[0061] Figure 39 shows the release of IFN-γ mediated by CD3xMSLN double antibody, accompanying the killing of target cells by Donor4 PBMCs.

[0062] Figure 40 shows the release of IL-6 mediated by CD3xMSLN double antibody, accompanying the killing of target cells by Donor4 PBMCs.

[0063] Figure 41 shows the release of TNF-α mediated by CD3xMSLN double antibody, accompanying the killing of target cells by Donor4 PBMCs.

[0064] Figure 42 shows the killing of Ramos cells by Donor5 PBMCs mediated by CD3xCD20 double antibody.

[0065] Figure 43 shows the killing of Raji cells by Donor5 PBMCs mediated by CD3xCD20 bispecific antibody.

[0066] Figure 44 shows the release of IFN-γ mediated by CD3xCD20 bispecific antibody in conjunction with the killing of Ramos by Donor5 PBMCs.

[0067] Figure 45 shows the release of IL-6 mediated by CD3xCD20 bispecific antibody in conjunction with the killing of Ramos by Donor5 PBMCs.

[0068] Figure 46 shows the release of IFN-γ mediated by CD3xCD20 bispecific antibody in conjunction with the killing of Raji cells by Donor5 PBMCs.

[0069] Figure 47 shows the release of IL-6 mediated by CD3xCD20 bispecific antibody in conjunction with the killing of Raji cells by Donor5 PBMCs.

[0070] Figure 48 shows the activation of CD4+ T cells mediated by CD3xCD20 bispecific antibody in conjunction with the killing of Ramos cells by Donor5 PBMCs.

[0071] Figure 49 shows CD8+ T cell activation mediated by CD3xCD20 bispecific antibody, accompanied by Donor5 PBMC killing of Ramos.

[0072] Figure 50 shows the in vivo antitumor efficacy of CD3xCD20 bispecific antibody in mice. Detailed Description of the Invention

[0074] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods and examples described herein are illustrative only and are not intended to be limiting. Other features, objects and advantages of the invention will be apparent from this specification and the accompanying drawings and from the appended claims.

[0075] Definitions

[0076] The term “about” when used in conjunction with a numerical value means to encompass a range of numerical values ​​having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

[0077] As used herein, the term “and / or”, when used in conjunction with two or more options, means any one of the options or any two or more of the options.

[0078] In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover the situation where the elements, integers, or steps mentioned are constituted. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region composed of that specific sequence.

[0079] In this document, the term “antigen-binding molecule” refers to a molecule, such as a protein or polypeptide or a molecule derived therefrom, that contains an antigen-binding domain or antigen-binding site capable of binding to a target antigen. When the target antigen is CD3 and / or a tumor-associated antigen (TAA), an antigen-binding molecule that binds CD3 and / or TAA is also referred to as a CD3-binding molecule, a TAA-binding molecule, or a CD3 / TAA-binding molecule. Antigen-binding molecules include, for example, antibodies and their antigen-binding fragments, as well as various fusions and conjugates constructed based on antibodies or antigen-binding fragments, such as immunoconjugates, antibody-drug conjugates (ADCs), multi / bispecific antibodies, and chimeric antigen receptors (CARs). As will be apparent to those skilled in the art, the antigen-binding site of an antibody typically contains amino acid residues from a “complementarity-determining region” or “CDR.” In some aspects of the invention, antigen-binding molecules based on the anti-CD3 antibody or antigen-binding fragment of the invention, or based on the multispecific antibody of the invention, are within the scope of consideration of the invention.

[0080] In this document, the term “antibody” refers to a polypeptide that contains at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. The term covers a variety of antibody structures, including, but not limited to, monoclonal antibodies.Antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, heavy-chain antibodies, murine antibodies, chimeric or humanized antibodies, intact antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0081] In this document, "intact antibody" and "full-length antibody" are used interchangeably and refer to an immunoglobulin molecule containing at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 to 4 immunoglobulin domains, CH1, CH2, and CH3, and optionally CH4. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain CL.

[0082] In this document, the term "antibody fragment" and "antigen-binding fragment" are used interchangeably and refer to a molecule distinct from an intact antibody that contains a portion of the intact antibody and is capable of binding the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, crossFab, Fab', Fab'-SH, F(ab')2; bibody; linear antibody; single-chain antibody (e.g., scFv, scFab); single-domain antibody; camelid antibody (heavy chain antibody) or fragment thereof (e.g., VHH); and monospecific, bispecific, or multispecific antibodies formed from antibody fragments. Unless otherwise stated herein or explicitly contradicted by the context, the term “antibody” is used herein in the same way as “antibody or antibody fragment thereof.”

[0083] In this document, the terms “antigen binding site” and “antigen binding domain” are used interchangeably to refer to the region in an antibody molecule that actually binds to an antigen. In the multispecific antibody of the present invention, antigen binding specificity is preferably provided by the antigen binding domain. In some embodiments, the CD3 antigen binding domain for the multispecific antibody of the present invention is preferably provided by a heavy chain variable domain (VH) and a light chain variable domain (VL) from an anti-CD3 antibody according to the present invention. In some embodiments, the TAA antigen-binding domain for the multispecific antibody of the present invention is preferably provided by a variable domain (i.e., “VHH”) from a heavy chain antibody.

[0084] In this document, the term “multispecific” refers to an antigen-binding molecule (e.g., an antibody) comprising two or more antigen-binding domains, wherein at least two of the antigen-binding domains bind to different antigenic epitopes, for example, to different epitopes on different antigens or different epitopes on the same antigen. Accordingly, “monospecific” refers to the ability to bind only one epitope. “bispecific” refers to the ability to bind two different epitopes.

[0085] In this document, the antibody-related expression “valence” or “valence number” refers to the valence of the antigen-binding site in the antibody molecule.The total number, or the number of antigen-binding sites with the same antigen-binding specificity. For example, a trivalent antibody means that the antibody molecule contains a total of 3 antigen-binding sites; the antibody molecule can be a "2+1" type bispecific antibody, that is, the antibody has two different antigen-binding specificities, of which there are 2 antigen-binding sites for one antigen-binding specificity and only 1 antigen-binding site for the other antigen-binding specificity. Such bispecific antibodies can also be called 2:1 type bispecific antibodies according to the ratio of the number of antigen-binding domains of the two different specificities.

[0086] In this document, the terms "first", "second", "third" and "fourth", etc., when used in conjunction with elements such as Fc regions or antigen-binding sites, are intended to conveniently distinguish two elements belonging to the same category. However, it should be noted that unless explicitly stated otherwise, the use of these terms is not intended to assign a specific order, orientation or position to the elements.

[0087] In this document, the term "CD3" refers to the cluster of differentiation 3 antigen expressed on the surface of T cells and interacting with the T cell receptor (TCR). CD3 has four major subunits: γ, δ, ε, and ζ, which respectively include the N-terminal extracellular region, the transmembrane domain, and the cytoplasmic tail region containing the immunoreceptor tyrosine activation motif (ITAM). These subunits interact to form three different polypeptide dimers: γ, δ, and ζζ. The CD3 complex, composed of these dimers, binds to the α and β chains of the T cell receptor (TCR) to form the complete T cell receptor complex, participating in the regulation of T cell antigen recognition, signal transduction, and T cell development. CD3 on the cell membrane surface, as a pan-T cell marker, is expressed on the surface of all mature T cells in peripheral blood and lymphoid tissues. An example of CD3-ε is the human CD3-ε protein containing the amino acid sequence UniProtKB-P07766; an example of CD3-δ is the human CD3-δ protein containing the amino acid sequence UniProtKB-P04234; an example of CD3-ζ is the human CD3-ζ protein containing the amino acid sequence UniProtKB-P20963; and an example of CD3-γ is the human CD3-γ protein containing the amino acid sequence UniProtKB-P09693. Unless explicitly specified as originating from a non-human species, all references to CD3 proteins, peptides, and protein fragments herein are intended to refer to the human version of the corresponding protein, peptide, or protein fragment. Therefore, unless specified as originating from a non-human species, such as “mouse CD3,” “monkey CD3,” etc., the expression “CD3” refers to human CD3. Accordingly, in this document,Unless otherwise specified, the term "antigen-binding specificity against CD3," that is, "antigen-binding domain that specifically binds to CD3," refers to binding specificity against human CD3.

[0088] In this document, the term "antibody that binds to CD3" or "anti-CD3 antibody" encompasses antibodies and their antigen-binding fragments that specifically recognize a single CD3 subunit (e.g., ε, δ, γ, or ζ) and antibodies and their antigen-binding fragments that specifically recognize a dimer complex (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers) of two CD3 subunits. The antibodies and antigen-binding fragments of the present invention can bind soluble CD3 and / or CD3 expressed on the cell surface. Examples of soluble CD3 include recombinant CD3 protein variants, such as monomeric and dimer CD3 constructs.

[0089] In this document, the term "CD3 expressed on the cell surface" means a CD3 protein expressed on the surface of cells in vitro or in vivo, at least a portion of which is exposed on the extracellular side of the cell membrane and accessible by the antigen-binding portion of an antibody. "CD3 expressed on cell surface" includes CD3 proteins that interact with functional T cell receptors in the cell membrane.

[0090] In this document, the term "CD3 antigen-positive cell" refers to cells in vitro or in vivo that express CD3 on their cell surface, such as CD4+ and CD8+ T cells. In this document, the term "CD3 antigen-negative cell" refers to cells in vitro or in vivo that do not express CD3 on their cell surface, such as native B cells and recombinant T cells with CD3 knockout.

[0091] In this document, the term "tumor-associated antigen," also known as TAA, refers to any antigen that is highly expressed in tumor cells or the tumor stroma. Typically, tumor-associated antigens are expressed or overexpressed in tumor cells to be treated, but expressed at low levels or not at all in normal cells. Examples of tumor-associated antigens include, but are not limited to, CD20, MSLN, etc.

[0092] In this document, the term "MSLN" refers to mesothelin. Mesothelin is a cell surface protein expressed by the mesothelin gene (MSLN). This protein is produced by the hydrolysis of a precursor protein encoded by the MSLN gene by furin protease and is anchored to the cell membrane surface via glycosylphosphatidylinositol. MSLN is highly expressed in malignant tumors but has limited expression in normal tissues, and has therefore been proposed as a candidate for tumor-targeted therapy. In this document, unless otherwise stated, the term MSLN includes any variant of human MSLN, including sequence variants, especially naturally occurring variants, allelic variants, as well as post-translational modification variants and conformational variants, and encompasses its species homologs. In some cases, the term specifically refers to the MSLN antigen expressed on the surface of tumor cells. An example of MSLN is the human MSLN protein containing the amino acid sequence UniProtKB-Q13421.White. Accordingly, in this document, unless otherwise specified, the term "antigen binding specificity against MSLN," that is, "antigen-binding domain specifically binding to MSLN," refers to the binding specificity against human MSLN.

[0093] In this document, the term "MSLN-positive" cell refers to a cell that is positive for MSLN expression on its cell surface. The MSLN expression level on the cell surface can be determined by any conventional method known in the art for determining the level of cell surface antigen expression, such as FACS detection or immunofluorescence staining. MSLN has a significantly higher expression level on various tumor cells than on normal tissues / cells. For example, MSLN has been found to be overexpressed in 90% of mesotheliomas, 80%–85% of pancreatic cancers, 60%–65% of ovarian cancers, and 60%–65% of lung cancers. Preferably, in some embodiments, MSLN-positive cells are MSLN-positive tumor cells.

[0094] In this document, the term “CD20” refers to the B lymphocyte surface antigen CD20. This antigen has been proposed as a candidate for targeted therapy of B lymphocyte tumors. In this document, unless otherwise stated, the term CD20 includes any variant of human CD20, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification variants and conformational variants, and covers its species homologs. In some cases, the term specifically refers to the CD20 antigen expressed on the surface of tumor cells. An example of CD20 is the human CD20 protein containing the amino acid sequence under UniProtKB-P11836. Accordingly, in this document, unless specifically indicated, the term “antigen-binding specificity against CD20,” that is, “antigen-binding domain that specifically binds to CD20,” refers to binding specificity against human CD20. In this document, the term “CD20-positive” cell refers to a cell that is positive for CD20 expression on its cell surface, especially CD20-positive tumor cells.

[0095] In this document, the term "T-cell connector" (TCE) refers to a multi-(bi)specific antibody comprising an antigen-binding domain specifically targeting T-cell surface antigens (such as CD3 or CD3 subunit complexes) and an antigen-binding domain specifically targeting target antigens (such as tumor-associated antigens). Through these antigen-binding domains with different specificities, the TCE can connect T cells and target cells (such as target tumor cells), forming an immune synapse between them, thereby inducing the killing effect of activated T cells on the target cells. It should be understood that in some cases, the TCE may also contain other antigen-binding domains in addition to the CD3 and TAA binding domains to, for example, further enhance or assist the tumor-suppressive efficacy of the TCE. Other aspects may be mentioned...Examples of antigen-binding domains are binding domains targeting co-stimulatory molecules.

[0096] In this document, the term “T cell activation” or “T cell activation” refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the following: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In some embodiments, the anti-CD3 antibody and multispecific antibody according to the invention are T cell activating antibodies capable of inducing T cell activation. Suitable assays for measuring T cell activation include those described herein and those known in the art.

[0097] In this document, the term “binding” or “specific binding” means that the binding of an antigen-binding site to an antigen epitope is selective and can be distinguished from unwanted or nonspecific interactions. The binding ability or binding specificity of an antigen binding site to a specific antigenic epitope can be determined by conventional binding assays known in the art, including but not limited to, detecting the binding of an antibody to an antigen by ELISA assays, detecting the binding of an antibody to cells expressing an antigen by FACS assays, or characterizing the binding affinity constant KD by surface plasmon resonance (SPR) or thin-layer interferometry (BLI) techniques.

[0098] In this document, the term “affinity” or “binding affinity” refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigenic epitope). “Binding affinity” reflects the intrinsic binding affinity of a 1:1 interaction between members of a binding pair. Binding affinity is typically expressed as a binding dissociation equilibrium constant (KD) and can be measured by commonly used methods known in the art, such as surface plasmon resonance (SPR) techniques.

[0099] In this document, the term "avidity" or "binding affinity" refers to the combined strength of the interaction between multiple binding sites of a molecule (e.g., an antibody) and the same target. Therefore, a necessary condition for affinity is the multivalent nature of the molecule (e.g., an antibody) to a target.

[0100] In this document, the term "immunoglobulin" refers to a protein having the structure of naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric sugars of approximately 150,000 Daltons, consisting of two light chains and two heavy chains linked by disulfide bonds. Each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, from the N-terminus to the C-terminus, followed by a heavy chain constant region consisting of three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable structural domain, followed by a...The light chain constant region is composed of a light chain constant domain (CL). The heavy chain of an immunoglobulin can be classified into one of five categories based on the type of its constant region, referred to as α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further subdivided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an immunoglobulin can also be classified into one of two types based on the amino acid sequence of its constant domain, referred to as κ and λ. In some embodiments of the antibody according to the invention having a constant region, the constant region refers to the constant region of an immunoglobulin or a sequence variant thereof.

[0101] In this document, the term "isotype" in relation to antibody type refers to the antibody type determined by the antibody heavy chain constant region. The antibodies according to the invention can be antibodies of IgA (e.g., IgA1 or IgA2), IgG (e.g., IgG1, IgG2 (e.g., IgG2a or IgG2b), IgG3 or IgG4), IgE, IgM, and IgD isotypes, and have a heavy chain constant region of the immunoglobulin type. It should be understood herein that when referring to an antibody having a certain isotype, it covers not only antibodies having the native sequence constant region of that isotype, but also antibodies with a few mutations introduced into the native constant region sequence of that isotype.

[0102] In this document, the term "variable region" or "variable domain" refers to a domain of the antibody's heavy or light chain involved in antibody-antigen binding. Typically, the pairing of a heavy chain variable domain (VH) with a light chain variable domain (VL) confers antigen-binding specificity; however, in some cases, a single VH domain (e.g., a single VHH domain from a heavy chain antibody) is sufficient to confer antigen-binding specificity. The heavy chain variable region is identical to the light chain variable region, containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs), arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. According to some aspects of the invention, one or more residues in the variable region of the antibody can be modified, for example, by modifying one or more CDR regions and / or one or more framework regions, particularly by substituting conserved residues, to obtain antibody variants that still substantially retain at least one biological property (e.g., antigen-binding ability) of the parent antibody. In other aspects, the antibody variable region can be modified by CDR transplantation. Since the CDR sequence is responsible for most antibody-antigen interactions, antibody variants that mimic the properties of known antibodies can be constructed. In such antibody variants, a CDR sequence from a known antibody is transplanted into the framework region of a different antibody with different properties, and one or more residue mutations, such as reversion mutations, can be made as needed to refine the antibody.The desired properties. In some cases, for the therapeutic application of antibodies or their derivatives, it is desirable to reduce their immunogenicity and improve their drugability. For this purpose, the variable domains of antibodies can be engineered to construct humanized, deimmunogenic, and / or PTM (post-translational modification) de-mutated variants. The properties of the mutated and / or modified antibodies, such as target antigen binding properties or other desired functional properties, such as T cell activation activity and / or tumor cell killing activity, can be determined and screened in vitro or in vivo using methods known in the art and described herein. It should be understood that such functional variants of any variable regions (e.g., VH and / or VL regions, VHH regions) given herein are all within the scope of this invention.

[0103] In this document, the terms “complementarity-determining region” and “CDR region”, “CDR”, and “hypervariant region” are used interchangeably and refer to regions in the variable domains of an antibody that are highly variable in sequence and form structurally defined loops (“hypervariant loops”) and / or contain antigen contact residues (“antigen contact sites”). The CDR is primarily responsible for binding to antigen epitopes. In the VH and VL domains, CDRs are sequentially numbered starting from the N-terminus, typically referred to as HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively. In the VHH domain, CDRs are sequentially numbered starting from the N-terminus, typically referred to as CDR1, CDR2, and CDR3, respectively. CDR sequences within a specific variable region can be determined using schemes known in the art, such as the Kabat, AbM, Chothia, Contact, and IMGT schemes, or any combination thereof. The Kabat complementarity-determining region (CDR) is determined based on sequence variability and is the most commonly used scheme (Kabat et al., Sequences of Proteins of..., 8 / 51, 16 CN 122255272 A Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The Chothia scheme refers to the location of the structural ring (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). AbM HVR is a compromise between Kabat HVR and the Chothia structural ring, used by the AbM antibody modeling software from Oxford Molecular. “Contact” HVR is based on the analysis of the available crystal structure of the complex.

[0104] The following are examples of CDR region ranges defined using the Kabat, AbM, IMGT, and Contact schemes.

[0105]

[0106] Unless otherwise stated, in this disclosure, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above-described methods and combinations thereof. Furthermore, a CDR may also be determined based on having the same Kabat number position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention). Moreover, it is known in the art that although CDRs differ between antibodies, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, a minimal overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. Such a minimum binding unit may be a sub-part of a CDR. The residues of the remaining portion of the CDR sequence, as will be apparent to those skilled in the art, can be determined by the antibody's structure and protein folding. Therefore, the invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues may be substituted.

[0107] In this document, unless otherwise stated, references to residue positions in the antibody variable region and CDR refer to the positions numbered according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Therefore, in this document, when referring to an amino acid substitution at a certain position of the antibody variable region sequence, the amino acid substitution is described as follows: [original amino acid residue / position / substituted amino acid residue]. For example, according to the Kabat numbering system, serine at position 27E in the light chain variable region, if substituted with threonine, can be represented as S27eT; if substituted with valine, it can be represented as S27eV; and if substituted with isoleucine, it can be represented as S27eI. Similarly, according to the Kabat numbering system, amino acid substitution V51I on the light chain variable region means that valine at position 51 is replaced with isoleucine, amino acid substitution S52Q means that serine at position 52 is replaced with glutamine, amino acid substitution V55R means that valine at position 55 is replaced with arginine, and amino acid substitution S56I means that serine at position 56 is replaced with isoleucine.

[0108] In this document, the terms “Fab” and “Fab domain” are used interchangeably to refer to antibodies similar to conventional four-chain IgG antibodies.The term refers to a structure formed by the pairing of a heavy chain variable region VH and a heavy chain constant region CH1 (VH-CH1) with a complementary light chain variable region VL and a light chain constant region CL (VL-CL). This term also encompasses structures in which CH1 and CL are exchanged, i.e., structures formed by the pairing of VH-CL and VL-CH1. In some embodiments, the Fab domain may be fused to the Fc region of an immunoglobulin, including but not limited to, by fusing a fragment containing VH to the N-terminus of the Fc region of an immunoglobulin; or by fusing a fragment containing VL to the N-terminus of the Fc region of an immunoglobulin.

[0109] In this document, the terms “scFv” and “scFv domain” are used interchangeably to refer to a single-chain polypeptide comprising a VH domain and a VL domain linked together by a flexible linker, wherein the VH domain and the VL domain located on the polypeptide chain pair to form an antigen-binding domain responsible for antigen binding. In some embodiments, the scFv domain may be fused to the Fc region of an immunoglobulin, for example, fused to the N-terminus or C-terminus of the Fc region of an immunoglobulin.

[0110] In this document, the terms “VHH” and “VHH domain” are used interchangeably to refer to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain, sometimes also called a single variable domain fragment (sVD). Thus, a VHH differs from the conventional VH of a four-chain immunoglobulin, which does not require pairing with a light chain variable domain to form an antigen-binding site. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camelids may also produce naturally occurring heavy chain antibodies lacking a light chain, and such VHHs are also within the scope of this invention.

[0111] In this document, the term “half-life extension domain” refers to a chemical structure capable of conferring an increased circulating half-life to the molecule (e.g., an antibody) bound to it after administration to an animal. Such chemical structures include, for example, flexible hydrophilic molecules (e.g., carbohydrates or PEG (polyethylene glycol)), immunoglobulin Fc regions, serum albumin, serum albumin-binding domains (e.g., small organic molecules, fatty acids, peptides, and proteins capable of binding to serum albumin), or serum albumin-binding peptides. Half-life extension domains can be linked to the antibodies of the present invention by chemical conjugation or fusion, depending on their specific properties. In some embodiments, preferably, the half-life extension domain used in the anti-CD3 antibody or multispecific antibody such as TCE of the present invention is an immunoglobulin Fc region, but the present invention also considers alternative half-life extension domains such as serum albumin-binding domains.

[0112] In this document, the term "immunoglobulin Fc region" is used interchangeably with "Fc region" and "Fc domain" to define...The C-terminal region of the immunoglobulin heavy chain comprises at least a portion of the heavy chain constant region. The term "Fc region" or "Fc domain" according to this disclosure does not include the heavy chain variable region VH and light chain variable region VL, or the heavy chain constant region CH1 and light chain constant region CL of the immunoglobulin; however, it may include all or part of the immunoglobulin hinge region. The sequence constituting the Fc region can be a native sequence or a variant sequence. Therefore, the term "Fc region" encompasses both native sequence Fc regions and variant Fc regions. In this document, unless otherwise stated, the amino acid residues in the Fc region and the heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., SEQuences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0113] In this document, the term “natural sequence Fc region” encompasses the Fc region sequences of various naturally occurring immunoglobulins, such as the Fc region sequences of various Ig subclasses and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520.). In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the C-terminus of the heavy chain. In other embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from E216 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446Lys447) of the Fc region may or may not be present.

[0114] In this document, the term "variant sequence Fc region" refers to a polypeptide containing a modified Fc region relative to a native sequence Fc region polypeptide. The modification may be the addition, deletion, and / or substitution of amino acid residues. Substitution may include naturally occurring amino acid substitutions and non-naturally occurring amino acid substitutions. The purpose of modification includes, but is not limited to, altering the binding of the Fc region to its receptor and the resulting effector function, preventing undesirable heavy chain mismatches, or site-directed introduction of amino acid mutations that can be used to alter interchain disulfide bond formation.

[0115] In this document, the term "effector function" refers to the variation in immunoglobulin Fc region attributable to immunoglobulin isotype.The biological activities of the region. Examples of immunoglobulin effector functions include: Fc receptor binding, C1q binding and complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). Depending on the intended use of the antibody molecule, the Fc region of the antibody can be selected and / or modified to give it effector functions appropriate to said use, such as attenuated, reduced, or eliminated Fcγ receptor binding, ADCC activity, and / or CDC activity compared to the wild-type IgG1 Fc region.

[0116] In this document, the term “co-stimulatory molecule” refers to the related partner of a co-stimulatory ligand, which specifically binds to the co-stimulatory ligand on T cells and thereby mediates a co-stimulatory response of T cells (e.g., but not limited to, T cell proliferation). Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, CD8, OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, and CD28.

[0117] In this document, the terms “flexible linker peptide” or “linker peptide” or “linker peptide” are used interchangeably and refer to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or from the hinge region of an immunoglobulin.

[0118] In this document, the “identity percentage (%)” of an amino acid sequence refers to the percentage of positions in the candidate sequence that have the same amino acid residues at the corresponding positions in the alignment with the specific amino acid sequence shown in this disclosure, without taking into account any conserved substitutions as part of sequence identity, after comparing the candidate sequence with the specific amino acid sequence shown in this disclosure. In some embodiments, the present invention contemplates variants of the antibody sequence of the present invention that have a considerable degree of identity with respect to the antibody sequence specifically disclosed herein within a comparison window, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. In this document, if no comparison window is specified (i.e., the antibody region of interest to be compared), the alignment is performed over the full length of the reference antibody sequence. In some embodiments, the variants may contain conserved modifications.

[0119] In this document, for a polypeptide sequence, a “conserved modification” includes substitution, deletion, or addition to the polypeptide sequence that does not significantly affect or alter the desired properties of the polypeptide containing the modification (e.g., binding characteristics and / or T cell activation characteristics).Conserved substitution tables for functionally similar amino acids are well known in the art. The following eight groups contain amino acids that are conserved substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)).

[0120] In this document, the term "chimeric antibody" refers to an antibody in which a portion (e.g., the variable region sequence) is derived from one species and another portion (e.g., the constant region sequence) is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0121] In this document, a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) of a humanized antibody correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of human antibodies. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. The term “humanized form” for an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized. In some embodiments, the humanized antibody of the present invention has a framework region sequence “derived” from a specific human lineage sequence. Here, “derived” means that the amino acid sequence of the antibody framework region has at least 85% or 90% identity with the corresponding framework region amino acid sequence encoded by the human lineage immunoglobulin gene, and that the antibody retains antigen-binding activity.

[0122] In this document, an antibody is said to be "cross-reactive" to two different antigens or antigenic determinants if its amino acid sequence is specific to two different antigens or antigenic determinants (e.g., CD3 from different mammalian species, such as human CD3 and cynomolgus monkey CD3). Antibodies exhibiting human-monkey species cross-reactivity, particularly having similar human-monkey antigen-binding affinity, is advantageous, as this property can facilitate preclinical drug development of the antibody, such as toxicological assays of antigen-binding molecules composed of antibodies. In some embodiments, the antibodies of the present invention preferably exhibit human-monkey species cross-reactivity.

[0123] In this document, "isolated" antibodies refer to artificial antibodies, recombinant antibodies, and antibodies that have been at least partially isolated from components in the natural environment in which they are produced. In some embodiments, the antibodies of the present invention are "isolated" antibodies.In some embodiments, the isolated antibody is purified to a purity of more than 90%, 95%, or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0124] In this document, the term “endocytosis” refers to the process by which a ligand / receptor complex is internalized and delivered into the cytosol or translocated to a suitable intracellular compartment, triggered by the binding of a ligand to a corresponding receptor on the cell surface. In some embodiments, the anti-CD20 antibody of the present invention initiates only weak endocytosis mediated by the CD20 receptor upon binding to CD20 expressed on the cell surface. In this document, endocytosis and endocytosis rate can be determined, for example, by the methods described in the examples, to characterize the endocytic activity of the antibody.

[0125] In this document, the term "heavy-chain antibody (hcAb)" refers to an antibody that does not have a light chain and may contain VHH-CH2-CH3 or VHH-CH1-CH2-CH3 from the N-terminus to the C-terminus; it may form a homodimer, such as a heavy-chain dimer antibody that does not have a light chain.

[0126] In this document, the term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0127] In this document, the term "expression vector" refers to a vector capable of directing the expression of a nucleotide sequence operatively linked thereto. Expression vectors typically contain a cis-acting element for the expression of said nucleotide sequence; while other elements for expression may be provided by a host cell or in an in vitro expression system. Expression vectors include, for example, but not limited to, entrapments, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0128] In this document, the term "immunoconjugate" or "immunofusion" generally refers to a molecule formed by conjugating or fusing one or more immunoglobulin-associated molecules or fragments thereof (e.g., antibodies or fragments thereof) with one or more other molecules. These other molecules may be protein-like molecules, such as peptides, polypeptides, or proteins; or non-protein-like molecules, such as chemical toxins. In cases involving multiple other molecules, these other molecules may be the same or different from each other.

[0129] In this document, the terms “individual” or “subject” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, an individual specifically refers to a human individual. Specification 12 / 51 pages 20 CN 122255272 A

[0130] In this document, the term “treatment” refers to a clinical intervention intended to alter the natural course of a disease in an individual receiving treatment. Desired therapeutic effects include, but are not limited to, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In cases involving tumor or cancer treatment, “treatment” encompasses antitumor biological effects that can be induced by human intervention (e.g., through the administration of a drug), including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0131] In this document, the term “prevention” refers to a medical intervention performed before the onset of at least one symptom of a disease to suppress, delay, or prevent the occurrence or development of the disease or a particular disease symptom. Thus, in some embodiments, prevention includes the administration of a drug to a subject before the onset of the disease or symptom.

[0132] In this document, the terms “cancer” and “tumor” are used interchangeably to refer to or describe a physiological disorder in mammals characterized by generally unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, solid tumors, and liquid tumors. In some embodiments, cancers suitable for treatment by the antibodies or immune conjugates or immune fusions of the present invention include CD20-positive and / or MSLN-positive tumors / cancers, including their metastatic forms.

[0133] The present invention is described in detail below. It will be understood by those skilled in the art that, unless the context clearly indicates otherwise, any technical feature described in any of the following sections, subsections, or embodiments may be combined with any technical feature described in any other section, subsection, or embodiment, and such combinations are all within the scope of consideration of the present invention.

[0134] I. The present invention provides an anti-CD3 antibody

[0135] The first aspect of the present invention provides an antibody that specifically binds to CD3, preferably human CD3 protein, or an antigen-binding fragment thereof. The anti-CD3 antibody of the present invention is a T-cell activating antibody. In some embodiments, the antigen-binding fragment of the antibody of the present invention is an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibodies such as scFv and scFab, (Fab')2 fragments, and linear antibodies.

[0136] Antibody CDR Region

[0137] The CDR region is the amino acid region in the variable region of the antibody that is primarily responsible for binding to the antigen epitope. Some exemplary anti-CD3 antibodies of the present inventionThe VH and VL sequence combinations are given in Table A below: Table A

[0138] In some embodiments, the antibodies of the present invention comprise the HCDR1, HCDR2, and HCDR3 sequences contained in the VH of any antibody shown in Table A and the LCDR1, LCDR2, and LCDR3 sequences contained in the VL. In some embodiments, the present invention also contemplates including humanized, deimmunized, and / or PTM-removed antibodies in the said CDR sequences.

[0139] In some embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: three heavy chain complementarity-determining regions (HCDR1, HCDR2 and HCDR3) contained in a heavy chain variable region (VH) sequence selected from SEQ ID NO: 31 and 67-69, and three light chain complementarity-determining regions (LCDR1, LCDR2 and LCDR3) contained in a light chain variable region (VL) sequence selected from SEQ ID NO: 32 and 70-71. In some embodiments, the antibody or its antigen-binding fragment optionally comprises the following amino acid substitutions: (a) amino acid substitutions selected from S27eT, S27eV, and S27eI in LCDR1, (b) amino acid substitutions selected from V51I, S52Q, V55R, and S56I in LCDR2, or a combination of (a) and (b), wherein the variable region amino acid residues are numbered according to the Kabat numbering system.

[0140] In some embodiments, the present invention provides an anti-CD3 antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained in the heavy chain variable region (VH) sequence of SEQ ID NO: 31, and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained in the light chain variable region (VL) sequences selected from SEQ ID NO: 32 and 72-74. In some embodiments, the antibody or its antigen-binding fragment optionally comprises the following amino acid substitutions: (a) amino acid substitutions selected from S27eT, S27eV, and S27eI in LCDR1; (b) amino acid substitutions selected from V51I, S52Q, V55R, and S56I in LCDR2; or a combination of (a) and (b), wherein the variable region amino acid residues are numbered according to the Kabat numbering system.

[0141] In some embodiments, the present invention provides an anti-CD3 antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises: three heavy chain complementarities contained in the heavy chain variable region (VH) sequence selected from SEQ ID NO: 68.The antibody comprises the antibody-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementary determinant regions (LCDR1, LCDR2, and LCDR3) contained in the light chain variable region (VL) sequences selected from SEQ ID NO: 70-74, 78-82, and 87. In some embodiments, the antibody or its antigen-binding fragment optionally comprises the following amino acid substitutions: (a) amino acid substitutions selected from S27eT, S27eV, and S27eI in LCDR1, (b) amino acid substitutions selected from V51I, S52Q, V55R, and S56I in LCDR2, or a combination of (a) and (b), wherein the variable region amino acid residues are numbered according to the Kabat numbering system.

[0142] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3 contained in one of the following VH and VL sequence pairs: (a) the VH sequence of SEQ ID NO: 31 and the VL sequence of one of SEQ ID NOs: 32 and 72-74; (b) the VH sequence of one of SEQ ID NOs: 67-69 and the VL sequence of one of SEQ ID NOs: 70-71; (c) the VH sequence of SEQ ID NO: 68 and the VL sequence of one of SEQ ID NOs: 78-82; or (d) the VH sequence of SEQ ID NO: 68 and the VL sequence of SEQ ID NO: 87.

[0143] In other embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 contained in the VH sequence of SEQ ID NO: 47 and LCDR1-3 contained in the VL sequence of SEQ ID NO: 48.

[0144] In other embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 contained in the VH sequence of SEQ ID NO: 7 and LCDR1-3 contained in the VL sequence of SEQ ID NO: 8. In other embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 contained in the VH sequence of SEQ ID NO: 15 and LCDR1-3 contained in the VL sequence of SEQ ID NO: 16. In other embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 contained in the VH sequence of SEQ ID NO: 23, and in SEQ ID NO:LCDR1-3 contained in the VL sequence of SEQ ID NO: 39. In other embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises HCDR1-3 contained in the VH sequence of SEQ ID NO: 39 and LCDR1-3 contained in the VL sequence of SEQ ID NO: 40. Specification 14 / 51 pages 22 CN 122255272 A

[0145] The CDR according to the present invention can be determined using any CDR definition scheme known in the art. In some embodiments, it is defined according to AbM, Chothia, Kabat, IMGT or any combination thereof. In other embodiments, preferably, the CDR according to the present invention is defined according to Kabat or AbM or a combination thereof, more preferably, according to AbM.

[0146] Table B below provides some exemplary CDR sequence combinations of the present invention:

[0147] Table C below provides some other exemplary CDR sequence combinations of the present invention:

[0148] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the antibody comprises: (i) six CDR sequences contained in the VH and VL sequences of any antibody listed in Table A; or (ii) six CDR sequences contained in any combination listed in Table B; or (iii) six CDR sequences contained in any combination listed in Table C.

[0149] In some preferred embodiments, the antibody or antigen-binding fragment of the present invention comprises: (i) HCDR1-3 and LCDR1-3 comprising or composed of the amino acid sequences of SEQ ID NOs:25-27 and SEQ ID NOs:28-30, respectively; (ii) HCDR1-3 comprising or composed of the amino acid sequences of SEQ ID NOs:25-27, respectively, and LCDR1 comprising or composed of the amino acid sequences of SEQ ID NOs:75, 76 or 77, respectively, and LCDR2-3 comprising or composed of the amino acid sequences of SEQ ID NOs:29-30, respectively; (iii) HCDR1-3 comprising or composed of the amino acid sequences of SEQ ID NOs:25-27, respectively, and LCDR1 and LCDR3 comprising or composed of the amino acid sequences of SEQ ID NOs:28 and 30, respectively, and LCDR1 comprising or composed of the amino acid sequences of SEQ ID NOs:25-27, respectively, and LCDR2-3 comprising or composed of the amino acid sequences of SEQ ID NOs:28 and 30, respectively. The amino acid sequence of 83, 84, 85 or 86 or LCDR2 composed therefrom; or (iv) the amino acid sequences comprising SEQ ID NOs:25-27 and SEQ ID NOs:75, 86 and 30 respectively or composed therefrom.The HCDR1-3 and LCDR1-3 are composed of

[0150] . In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein - HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 25, - HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 26, - HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 27, - LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 28, - LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 29, and - LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 30.

[0151] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein: - HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 25; - HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 26; - HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 27; - LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 75; - LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 29; and - LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 30.

[0152] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein: - HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 25; - HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 26; - HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 27; - LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 28; - LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 86; and - LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 30.

[0153] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein: - HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 25; -HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 26, - HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 27, - LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 75, - LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 86, and - LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 30. Specification 16 / 51 pages 24 CN 122255272 A

[0154] Antibody Variable Region

[0155] The variable region of the antibody is composed of CDR and frame region. Since the antigen-binding properties of the antibody are mainly responsible for the CDR sequence, different frame regions can be selected based on the CDR region according to the present invention to construct a variety of anti-CD3 antibodies of the present invention that bind CD3 equally effectively. Therefore, in one embodiment, the present invention relates to an anti-CD3 antibody or its antigen-binding fragment, which comprises the CDR sequence of the heavy chain and light chain variable regions from one of the antibodies shown in Table A, but with different frame region sequences. The frame region sequence for such replacement can be obtained from public DNA databases, such as germline DNA databases of human heavy and light chain variable region genes. Sequence similarity search tools (e.g., Gapped BLAST) can be used to compare the antibody protein sequence with protein sequences in the database to find suitable frame candidates. Preferably, the frame sequence for replacement has sequence identity with the original antibody's frame sequence, for example, at least 80%, 85%, or 90% sequence identity, or more preferably more than 95%, 96%, 97%, 98%, or 99% sequence identity.

[0156] In some embodiments, therefore, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region, which comprises or is composed of the VH sequence of any antibody listed in Table A or a variant thereof. In some embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain variable region, which comprises or is composed of the VL sequence of any antibody listed in Table A or a variant thereof. In one embodiment, the variant VH / VL sequence has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity in the amino acid sequence compared to the reference VH / VL sequence. In one embodiment, the variant VH / VL sequence contains at least one and no more than 30, 10 or 5, 4, 3, 2 amino acid modifications (preferably amino acid substitutions, preferably conservative substitutions) in the amino acid sequence compared to the reference VH / VL sequence. Preferably, the sequence modification does not occur in the CDR region.

[0157] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable regionThe VH region comprises or consists of a sequence selected from the amino acid sequence shown in SEQ ID NO: 7, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or consists of a sequence selected from the amino acid sequence shown in SEQ ID NO: 8, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises or consists of the amino acid sequence shown in SEQ ID NO: 7; and the light chain variable region (VL) comprises or consists of the amino acid sequence shown in SEQ ID NO: 8.

[0158] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or consists of a sequence selected from the amino acid sequence shown in SEQ ID NO: 15, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or consists of a sequence selected from the amino acid sequence shown in SEQ ID NO: 16, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO: 15; and the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO: 16.

[0159] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the following sequence: the amino acid sequence shown in SEQ ID NO: 23, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, as described on page 17 / 51 of CN 122255272 A, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or is composed of the following sequence: the amino acid sequence shown in SEQ ID NO: 24, or having at least 80%, 85%, or 90%, or preferably at leastSequences with 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO: 23; and the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO: 24.

[0160] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the following sequence: the amino acid sequence shown in SEQ ID NO: 39, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO: 40, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO: 39; and the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO: 40.

[0161] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or consists of the amino acid sequence shown in SEQ ID NO: 47, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or consists of the amino acid sequence shown in SEQ ID NO: 48, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the amino acid sequence shown in SEQ ID NO: 47; and the light chain variable region (VL) comprises or is composed of the amino acid sequence shown in SEQ ID NO: 48.

[0162] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the following sequences: SEQ ID NO: 31 and 67-69The amino acid sequence shown in one of SEQ ID NO: 32, 70-74, 78-82, and 87, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain variable region, wherein the light chain variable region (VL) comprises or is composed of the following sequences: the amino acid sequence shown in one of SEQ ID NO: 32, 70-74, 78-82, and 87, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some preferred embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises or is composed of the following sequence: the amino acid sequence shown in SEQ ID NO: 31, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and a light chain variable region (VL), wherein the light chain variable region (VL) comprises or is composed of the following sequence: the amino acid sequence shown in one of SEQ ID NO: 32 and 72-74, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some other preferred embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the following sequence: the amino acid sequence shown in SEQ ID NO: 67 or 69, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and a light chain variable region, wherein the light chain variable region (VL) comprises or is composed of the following sequence: the amino acid sequence shown in SEQ ID NO: 70 or 71, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some other preferred embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region (VH) comprises or is composed of the following sequence: the amino acid sequence shown in SEQ ID NO: 68, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with it; and a light chain variable region, wherein the light chain variable region (VL) comprises or is composed of the following sequence: the amino acid sequence shown in one of SEQ ID NO: 70-71, 78-82, and 87, or a sequence having at least 80%, 85%, or 90%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0163] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 31, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 32.

[0164] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 31, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 72, 73, or 74.

[0165] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 67 or 69, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 70 or 71.

[0166] In some more preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 68, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 70 or 71.

[0167] In some more preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 68, and a VL containing or consisting of the amino acid sequence shown in one of SEQ ID NO: 78-82.

[0168] In some more preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 68, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 82.

[0169] In some preferred embodiments, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof comprising: a VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 68, and a VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 87.

[0170] Antibody Heavy Chain and Light Chain

[0171] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain constant region, such as an Fc region of an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody of the present invention comprises a κ or λ light chain constant region, such as a human κ light chain constant region.

[0172] In some preferred embodiments, the anti-CD3 antibody of the present invention comprises an Fc region containing the amino acid sequence of SEQ ID NO: 111, or relative to SEQ ID NO:The amino acid sequence of SEQ ID NO: 111 contains at least one, two, or three, but no more than 20, 10, or 5 amino acid sequences that are modified with respect to the amino acid sequence of SEQ ID NO: 111, or a sequence that has at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 111. In some embodiments, the Fc region contains a mutation that reduces or eliminates the binding of the Fc region to FcγR, for example, the L234AL235A mutation.

[0173] In some preferred embodiments, the anti-CD3 antibody of the present invention contains a heavy chain constant region that contains the amino acid sequence of SEQ ID NO: 61, or contains at least one, two, or three, but no more than 20, 10, or 5 amino acid sequences that are modified with respect to the amino acid sequence of SEQ ID NO: 61, or a sequence that has at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 61.

[0174] In some preferred embodiments, the anti-CD3 antibody of the present invention comprises a light chain constant region. In a preferred embodiment, page 19 / 51 of CN 122255272 A, the light chain constant region is a human κ light chain constant region. In a further preferred embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence comprising at least 1, 2, or 3, but no more than 20, 10, or 5 amino acid modifications relative to the amino acid sequence of SEQ ID NO:62, or a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:62.

[0175] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence selected from SEQ ID NOs: 49, 51, 53, 55, 57, and 59. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain, wherein the light chain comprises an amino acid sequence selected from SEQ ID NOs: 50, 52, 54, 56, 58, and 60. In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain / light chain pair selected from the following: heavy chain / light chain pairs of the amino acid sequences shown in SEQ ID NOs: 49 / 50, 51 / 52, 53 / 54, 55 / 56, 57 / 58, and 59 / 60. In some embodiments, the anti-CD3 antibody according to the present invention is a full-length antibody of type IgG1.

[0176] In some embodiments, the anti-CD3 antibody of the present invention comprises a heavy chain, wherein the heavy chain comprises a heavy chain variable region selected from SEQ ID NOs: 31 and 67-69 and an immunoglobulin IgG heavy chain constant region. In some embodiments, the anti-CD3 antibody of the present invention comprises a light chain, wherein the light chain comprises a heavy chain variable region selected from SEQ ID NOs: 31 and 67-69 and an immunoglobulin IgG heavy chain constant region.NOs: 32, 70-74, 78-82 and 87 light chain variable regions and immunoglobulin light chain constant regions. In some embodiments, the anti-CD3 antibody according to the invention is a full-length antibody comprising the heavy chain and the light chain. In some embodiments, the anti-CD3 antibody according to the invention is a full-length IgG1 antibody. In some embodiments, the full-length anti-CD3 antibody according to the invention comprises the heavy chain constant region of SEQ ID NO: 61 and the light chain constant region of SEQ ID NO: 62.

[0177] Properties of the anti-CD3 antibody of the invention

[0178] In some embodiments, the anti-CD3 antibody or antigen-binding fragment of the invention has one or more or all of the following characteristics: (a) specific binding to human CD3 antigen; (b) immune cross-reactivity with human and monkey CD3 antigen; (c) substantially no nonspecific binding to surface CD3 antigen-negative cells; and (d) activation of CD4+ and CD8+ T cells.

[0179] In some embodiments, the anti-CD3 antibody or antigen-binding fragment of the invention specifically binds to human CD3 antigen. In some embodiments, as measured by in vitro surface plasmon resonance (SPR) binding analysis, the binding affinity KD value of the anti-CD3 antibody of the present invention is approximately 10 x 10⁻⁷ M to approximately 1 x 10⁻⁸ M, preferably approximately 5 x 10⁻⁷ M to approximately 5 x 10⁻⁸ M. Preferably, the binding affinity KD value is determined according to Example 3.7.

[0180] In some embodiments, the anti-CD3 antibody or antigen-binding fragment of the present invention has immune cross-reactivity with human and monkey CD3 antigens. In some embodiments, as measured by in vitro surface plasmon resonance (SPR) binding analysis, the binding affinity KD values ​​for human and monkey CD3 antigens differ by no more than approximately 5 times, preferably no more than approximately 3 times. Preferably, the binding affinity KD value is determined according to Example 3.7.

[0181] In some embodiments, the anti-CD3 antibody of the present invention specifically binds to surface human CD3 antigen-positive T cells. In some embodiments, the EC50 value of the anti-CD3 antibody of the present invention, when bound to the positive T cells in a bivalent form, is determined by flow cytometry on Jurkat cells to be approximately 0.01-10 nM, preferably 0.1-5 nM. In some embodiments, the anti-CD3 antibody of the present invention has lower cell-binding activity compared to the BMK8 reference positive antibody, preferably with a bivalent cell-binding EC50 value approximately 5-50 times or 10-30 times that of the reference antibody. Preferably, the assay is performed according to Example 2.2. In this document, "BMK8 reference positive antibody" refers to the anti-CD3 antibody of the present invention compared to the one being compared, except for differences in VH and VL.A reference antibody with the same antibody structure, wherein the VH and VL of the reference antibody respectively contain the amino acid sequences of SEQ ID NO: 63 and 64, page 20 / 51, CN 122255272 A.

[0182] In some embodiments, the anti-CD3 antibody of the present invention binds substantially nonspecifically to surface CD3 antigen-negative cells. Preferably, the binding on CD3 knockout Jurkat cells is determined by flow cytometry, for example, according to Example 2.2.

[0183] In some embodiments, the anti-CD3 antibody of the present invention specifically binds to surface monkey CD3 antigen-positive T cells. In some embodiments, by flow cytometry determination on cynomolgus monkey CD4+ or CD8+ T cells, when the anti-CD3 antibody of the present invention binds to the T cells in a bivalent form, the EC50 value is approximately 0.1-20 nM, preferably 1-10 nM. In some embodiments, in the assay, the antibody according to the invention has lower cynomolgus monkey T cell binding activity compared to the BMK8 reference positive antibody, preferably with an EC50 value of about 5-50 times or 10-30 times that of the reference antibody. Preferably, the assay is performed according to Example 2.3.

[0184] In some embodiments, the anti-CD3 antibody of the invention activates CD4+ and CD8+ T cells. In some embodiments, by a T cell activation assay, the bivalent form of the anti-CD3 antibody of the invention has intermediate T cell activation function compared to the BMK8 and BMK9 reference positive antibodies. In some embodiments, the T cell activation assay is performed by detecting an increase in the proportion of antibody-induced CD25-positive CD4+ and CD8+ T cells. Preferably, the assay is performed according to Example 2.4.

[0185] II. Anti-CD20 antibody of the invention

[0186] In a second aspect, the present invention provides an anti-CD20 antibody that binds to CD20. In some embodiments, the anti-CD20 antibody according to the invention comprises a VHH domain. In some embodiments, the anti-CD20 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences in a variable region having amino acid sequences selected from those shown in SEQ ID NOs: 102 and 115-124. Preferably, the CDRs are defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. More preferably, the CDRs are defined according to Kabat or AbM, or combinations thereof, and more preferably, the CDRs are defined according to AbM. However, it should be understood that the CDRs may also be defined in any other manner known in the art. In some embodiments, the CD20 VHH domain according to the present invention comprises complementarity-determining regions CDR1, CDR2, and CDR3, wherein: (i) the CDR1 comprises SEQ ID NOs: 102, 115-124 ...(ii) The CDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 103 or 137; (iii) The CDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 104.

[0187] Since the antigen-binding properties of the antibody are mainly responsible for the CDR sequence, different framework regions can be selected based on the CDR region of the present invention to construct a variety of anti-CD20 VHH domains of the present invention and anti-CD20 antibodies containing them that bind CD20 equally effectively. These anti-CD20 VHH domains and anti-CD20 antibodies are all within the scope of the present invention.

[0188] Accordingly, in one embodiment, the present invention provides an anti-CD20 VHH domain and an anti-CD20 antibody or antigen-binding fragment containing it, wherein the VHH domain contains three CDR sequences from one of the VHH domains of SEQ ID NOs: 102 and 115-124, but has a different framework region sequence. The frame sequence used for replacement preferably has a certain sequence identity in the frame region with one of SEQ ID NOs: 102 and 115-124, for example, at least 80%, 85%, or 90% sequence identity, or more preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the VHH domain is humanized.

[0189] In some specific embodiments, the present invention provides an anti-CD20 VHH domain and an anti-CD20 antibody or antigen-binding fragment comprising therein, wherein the VHH domain comprises a variable region sequence having an amino acid sequence selected from SEQ ID NOs: 102 and 115-124. In some further embodiments, the anti-CD20 VHH domain according to the invention comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with an amino acid sequence selected from SEQ ID Nos: 102 and 115-124 and retaining the ability to specifically bind CD20. In some further embodiments, the anti-CD20 VHH domain according to the invention comprises an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions (e.g., conservative substitutions) compared to an amino acid sequence selected from SEQ ID Nos: 102 and 115-124 and retaining the ability to specifically bind CD20. Preferably, the addition, deletion, and / or substitution of said amino acids does not occur in the CDR region.

[0190] In some preferred embodiments, the present invention provides an anti-CD20 VHH domain and an anti-CD20 antibody containing therefrom.A body or antigen-binding fragment, wherein the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of the amino acid sequence of SEQ ID NO: 102. In some embodiments, the CDRs are defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. In other embodiments, preferably, the CDRs are defined according to AbM. In some embodiments, CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 103; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 104; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 105. In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the VHH domain is composed of the amino acid sequence of SEQ ID NO: 102.

[0191] In some embodiments, the anti-CD20 VHH domain according to the invention has one or more of the following properties: (i) strong binding specificity and affinity for human CD20; (ii) smaller size, higher stability and deeper tissue penetration compared to conventional four-chain full-length antibodies; (iii) the ability to recognize and precisely target CD20 antigens on the surface of tumor cells; and (iv) weak endocytic activity.

[0192] In some aspects, the strong antigen-binding and weak endocytic properties of the anti-CD20 VHH domain according to the invention make it suitable as a component for constructing full-length antibodies, bispecific antibodies or multispecific antibodies, especially TCEs. In other aspects, the good tumor targeting properties of the anti-CD20 antibody according to the invention make it suitable for use in tumor therapy in the form of nanobodies (VHHs), for example, conjugates designed to deliver payloads (e.g., drugs or radioisotopes), thereby facilitating the delivery of the carried payload to tumor tissue to provide better applications such as tumor killing, immune modulation or disease detection. Therefore, in some embodiments, the present invention provides an anti-CD20 antibody comprising the anti-CD20 VHH domain of the present invention, which includes an immunoglobulin Fc region linked to the VHH domain (i.e., having a VHH-Fc form). In other embodiments, the present invention provides an anti-CD20 antibody comprising the anti-CD20 VHH domain of the present invention, wherein the anti-CD20 antibody is a heavy chain antibody. In other embodiments, the present invention provides an anti-CD20 antibody comprising the anti-CD20 VHH domain of the present invention, wherein the anti-CD20 antibody is a nanobody. In other embodiments, the present invention provides an anti-CD20 antibody comprising the anti-CD20 VHH domain of the present invention, wherein the anti-CD20 antibody is a bispecific antibody or a multispecific antibody. In other embodiments, the present invention...An anti-CD20 antibody comprising the anti-CD20 VHH domain of the present invention is provided, wherein the anti-CD20 antibody is a TCE.

[0193] In addition to the antibody forms described above, it should be understood that the anti-CD20 antibody of the present invention comprising the anti-CD20 VHH domain of the present invention may also have any other suitable antibody structure, such as, but not limited to, single-chain or multi-chain antibodies, monovalent or multivalent antibodies, linear antibodies; single-domain antibodies or multi-domain antibodies.

[0194] III. Multispecific antibody of the present invention

[0195] In a third aspect, the present invention provides a multispecific antibody comprising a CD3 antigen-binding domain. The multispecific antibody of the present invention comprises at least two different antigen-binding specificities. In some embodiments, in addition to CD3 binding specificity, the multispecific antibody of the present invention also comprises at least one different antigen-binding specificity. In some embodiments, in addition to CD3 binding specificity, the multispecific antibody of the present invention also comprises at least two, three, or four different antigen-binding specificities. The binding specificity that can be incorporated into the anti-CD3 multispecific antibody of the present invention can be selected from, for example, but not limited to, tumor-associated antigens (TAAs), other immune-associated molecules, and co-stimulatory molecules. Depending on the included binding specificity, the anti-CD3 multispecific antibody of the present invention can be a bispecific antibody, a trispecific antibody, a tetraspecific antibody, or an antibody with more specificities. In some embodiments, the present invention provides a multispecific antibody having binding specificity to CD3 and at least one (e.g., 1-3 different) TAAs. In other embodiments, the present invention provides a multispecific antibody having binding specificity to CD3 and at least one (e.g., 1-3 different) TAAs and binding specificity to at least one (e.g., 1) co-stimulatory molecule. In some cases, the TAA can be selected from solid tumor surface antigens or blood tumor surface antibodies. In other cases, the co-stimulatory molecule can be selected from CD28, OX40, CD137, CD8, ICOS, CD27, GITR, CD2, IL-2RP, CD58, CD80, CD7, and MyD88 / CD40. In some particular aspects, the present invention particularly provides T-cell connectors comprising a CD3 antigen-binding domain and a tumor-associated antigen (TAA)-binding domain. Preferably, in some embodiments, at least one CD3 antigen-binding domain of the multispecific antibody according to the present invention comprises or is derived from, or is composed of, an anti-CD3 antibody or an antigen-binding fragment thereof according to the first aspect of the present invention.

[0196] Structure of the multispecific antibody of the present invention

[0197] Therefore, in some embodiments, the present invention provides a multispecific antibody comprising one or more specific antigen-binding domains.A first antigen-binding domain that heterologously binds to CD3 and a second antigen-binding domain with different binding specificities (and optionally, a third, fourth, and / or a fifth antigen-binding domain with different binding specificities), wherein the one or more CD3 antigen-binding domains comprise, or are composed of, an anti-CD3 antibody or an antigen-binding fragment thereof according to the first aspect of the invention.

[0198] The second antigen-binding domain (and optionally, a third, fourth, and / or a fifth antigen-binding domain) included in the multispecific antibody of the invention is not particularly limited. In some embodiments, the second antigen-binding domain (and optionally, a third, fourth, and / or a fifth antigen-binding domain) specifically binds to a target cell antigen. In some embodiments, the target cell antigen is an antigenic determinant presented on the surface of a target cell (e.g., a tumor cell or a cell in the tumor stroma, or a T cell).

[0199] In some preferred aspects, the multispecific antibody according to the invention comprises a first and a second antigen-binding domain, wherein the first antigen-binding domain is a CD3 antigen-binding domain according to the invention, and the second antigen-binding domain is an antigen-binding domain that specifically binds to a tumor-associated antigen (TAA). The multispecific antibody of this invention, comprising a combination of a TAA antigen-binding domain and a CD3-binding domain, facilitates the formation of an immune synapse between target tumor cells expressing the TAA and T cells, thereby achieving the killing of the target tumor. The TAA used in this invention can be a solid tumor cell surface antigen or a hematologic tumor cell surface antigen.

[0200] In some embodiments, the multispecific antibody of the present invention specifically binds to one or more TAAs, wherein each of the one or more TAAs is independently selected from CD19, BCMA, TSHR, CD171, CS-1, CLL-1, GD3, TnAg, FLT3, CD38, CD123, CD44v6, B7H3, B7H4, KIT, IL-13Ra2, IL-11Ra, PSCA, PSMA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, sLe, GM3, TGS5, HMWMAA, GD2, FOLR1, FOLR2, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY‑BR‑1, UPK2, HAVCR1, ADRB3, PANX3, GPR20,LY6K, OR51E2, TAARP, WT1, ETV6‑AML, SPA17, XAGE1, Tie 2, MAD‑CT‑1, MAD‑CT‑2, FOSL1, hTERT, ML‑IAP, ERG, NA17, PAX3, AR, Cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY‑TES1, LCK, AKAP‑4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, Instructions 23 / 51 Page 31 CN 122255272 A LY75, GPC3, FCRL5, IGLL1, CD20, CD30, HER2, ROR1, FLT3, TAAG72, CD22, CD33, GD2, gp100Tn, FAP, TYR, EPCAM, CEA, IGF‑1R, EphB2, MSLN, Claudin18.2, CDH17, CD32b, EGFRvIII, GPNMB, GPR64, HER3, LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C and TACSTD2.

[0201] In some embodiments, the multispecific antibody of the present invention specifically binds to surface antigens of solid tumor cells, selected from, for example, mesothelin (MSLN), carcinoembryonic antigen (CEA), epithelial cell adhesion factor (EpCAM), human epidermal growth factor receptor-2 (HER2), and prostate-specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Claudin18.2, and CDH17. In other embodiments, the multispecific antibody of the present invention specifically binds to surface antigens of blood tumor cells, selected from, for example, CD19, CD20, CD79b, CD33, BCMA, and GPRC5D.

[0202] Therefore, in some embodiments, the present invention provides a multispecific antibody comprising a CD3 antigen-binding domain and a TAA antigen-binding domain. In some embodiments, the CD3 antigen-binding domain of the antibody according to the present invention has a weak monovalent binding affinity for CD3 (preferably human CD3), for example, a binding affinity KD value of 10 x 10⁻⁷ M to 1 x 10⁻⁸ M, or 5 x 10⁻⁷ M to 5 x 10⁻⁸ M.

[0203] In some cases, to balance the effects of the affinity of the CD3 antigen-binding domain and the TAA antigen-binding domain, it is advantageous to adjust the ratio of the CD3 and TAA antigen-binding domains in the multispecific antibody of the present invention. In some embodiments, the present invention provides a multispecific antibody wherein the ratio of the number or valence of the CD3 antigen-binding domain to the TAA antigen-binding domain is 1:1 or 1:2. In some embodiments, the valence (i.e., the total number of antigen-binding domains) of the multispecific antibody according to the present invention is 2-4 valences, preferably 3 valences. In some embodiments, the multispecific antibody according to the present invention is a trivalent antibody comprising one CD3 binding domain with weak binding affinity and two TAA binding domains with medium to high affinity.

[0204] In some embodiments, the CD3 antigen-binding domain contained in the multispecific antibody of the present invention comprises or is composed of Fab, scFab, or scFv domains. In other embodiments, the TAA antigen-binding domain contained in the multispecific antibody of the present invention comprises or is composed of Fab, scFab, scFv, or VHH domains.

[0205] In some embodiments, the multispecific antibody according to the present invention may further comprise an immunoglobulin Fc region in addition to the aforementioned antigen-binding domain. In other embodiments, alternatively, it may also comprise a half-life extension domain, such as serum albumin or a serum albumin-binding peptide, to adjust the circulating half-life of the antibody after administration to an animal. In some embodiments, preferably, the antibody of the present invention comprises an immunoglobulin Fc region.

[0206] The immunoglobulin Fc region used in the multispecific antibody of the present invention may be an Fc region from any immunoglobulin. In some embodiments, the immunoglobulin Fc region comprises at least an immunoglobulin CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin Fc region further comprises a hinge region or a partially hinge region. In some embodiments, the immunoglobulin Fc region comprises, from the N-terminus to the C-terminus, an immunoglobulin hinge region or a partially hinge region, a CH2 domain and a CH3 domain, or is composed thereof. In some embodiments, the immunoglobulin Fc region comprises, or is composed of, a CH2 domain and a CH3 domain from the N-terminus to the C-terminus. In some embodiments, the immunoglobulin Fc region is preferably derived from IgG1, IgG2, or IgG4, or a subtype thereof. Preferably, the immunoglobulin Fc region comprises an Fc region sequence derived from humans.

[0207] The immunoglobulin Fc region can be fused to the antigen-binding domain and / or other domains according to the invention via its N- or C-terminus. The C-terminal fusion of the immunoglobulin Fc region can be a direct fusion, but in some cases it is preferred to be a linker fusion. The N-terminal fusion of the immunoglobulin Fc region can be a direct fusion, but in some cases it is preferred to be a linker fusion.The fusion is performed on the protein hinge region sequence.

[0208] The immunoglobulin Fc region used in the multispecific antibody of the present invention can be the native Fc region sequence. Alternatively, the Fc region can contain a mutation relative to the native Fc sequence. Mutations include substitutions, insertions, and / or deletions. Such mutations can be made for the purpose of introducing desired therapeutic properties. A Knob-into-Hole (KiH) mutation can be introduced into the CH3 domain to promote heterodimerization in order to facilitate proper antibody assembly. In the case of introducing the KIH mutation, one Fc chain will be designed to contain a large protruding residue (i.e., Knob), while the other Fc chain is designed to contain a complementary pocket (i.e., Hole). Suitable locations for the KiH mutation are known in the art. Exemplary KiH mutations include, but are not limited to, combinations of Knob mutation T366W and Hole mutations T366S, L368A, Y407V; and combinations of Knob mutation T366Y and Hole mutation Y407T. When the multispecific antibody of the present invention comprises an asymmetric double-stranded structure, the Fc region preferably contains a KiH mutation that promotes proper heterodimerization of the antibody polypeptide chain. Alternatively or additionally, a cysteine ​​mutation may be introduced into the Fc region to increase disulfide bond linkages in the dimerized Fc region; for example, the mutation S354C may be introduced into one Fc chain, and the mutation Y349C into another Fc chain.

[0209] Furthermore, depending on the specific application of the antibody or antibody-based molecule according to the present invention, the Fc region may also contain mutations that alter effector function. For example, in the case of the multispecific antibody of the present invention being a TCE, the Fc region preferably contains mutations that reduce or eliminate effector function, such as the LALA mutation where leucine (L) at positions 234 and 235 of the Fc region is replaced with alanine (A).

[0210] According to the specific application of the antibody or antibody-based molecule of the present invention, the Fc region may also contain other mutations, such as mutations for increasing binding to FcRn and / or removing protease sites, and / or introducing amino acid modifications that can be used to couple active molecules. Additionally or alternatively, the Fc region may be mutated to remove or replace amino acids that may undergo post-translational modifications (e.g., glycosylation) to provide improved drugability and developability of the therapeutic antibody.

[0211] In some embodiments, the multispecific antibody of the present invention comprises a first Fc and a second Fc that form a dimer. In some embodiments, the first Fc region and the second Fc region are IgG isotype Fc regions, for example, IgG1, IgG2, or IgG4 isotype Fc regions, preferably derived from human IgG1 or human IgG4 Fc regions. In some embodiments, the first Fc region and the second Fc region contain amino acid mutations that promote the formation of the Fc dimer. In some embodiments, the first Fc region...The first Fc region contains T336W and S354C, and the second Fc region contains T366S, L368A, Y407V, and Y349C, or vice versa. In some embodiments, the first Fc region and the second Fc region also contain mutations that reduce or eliminate the binding of the Fc region to FcγR, for example, the L234AL235A mutation. In some embodiments, the first and second Fc regions contain the amino acid sequences of SEQ ID NO: 108 and SEQ ID NO: 107, or amino acid sequences that are at least 95%, 96%, 98%, or 99% identical to them.

[0212] In the multispecific antibody according to the invention, antibody components (i.e., antigen-binding domains and optionally immunoglobulin Fc or half-life-binding domains) can be linked by linkers. There are no particular limitations on the linkers that can be used in the antibodies of the invention. Linker sequences are generally flexible. They can consist mainly of amino acids with large side chains that do not have the potential to limit flexibility, such as glycine, alanine, and serine. Alternatively, it can consist of sequences from the hinge region of immunoglobulins. Depending on the linker location and the components to be linked, those skilled in the art can readily determine the available linker sequence or optimal length. Suitable linker lengths can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, or longer. In some cases, the linker sequence length can be shorter, for example less than about 20 or 15 amino acids, such as 2–15 amino acids or 5–10 amino acids. Suitable connective sequences include, but are not limited to, G4S (SEQ ID NO: 138); (G4S)2 (SEQ ID NO: 106); (G4S)3 (SEQ ID NO: 136); GGGSG (SEQ ID NO: 139); GGSGG (SEQ ID NO: 140); GSGGG (SEQ ID NO: 141); GSGGGP (SEQ ID NO: 142); GGEPS (SEQ ID NO: 143); GGEGGGP (SEQ ID NO: 144) and GGEGGGSEGGGS (SEQ ID NO: 145); and (G4S)n (SEQ ID NO: 112), where n is an integer equal to or greater than 1; TS(G4S)n (SEQ ID NO: 146), where n is an integer equal to or greater than 1; G(G4S)n (SEQ ID NO: 112) ... 147), where n is an integer equal to or greater than 1; (G4)n (SEQ ID NO. 25 / 51, page 25 of the specification)33 CN 122255272 A NO: 148), where n is an integer equal to or greater than 1; (GRPGS)n (SEQ ID NO: 149), where n is an integer equal to or greater than 1. Suitable flexible linker peptides can be rationally designed using computer programs to simulate the three-dimensional structures of proteins and peptides, or by phage display methods. In some embodiments, the linker used in the antibody of the present invention is a flexible linker peptide of 5-50 amino acids, preferably comprising a linker peptide containing glycine (G) and / or serine (S) and / or threonine residues (T). In one embodiment, the linker has a length of 5-50 amino acids, for example, 5, 10, 15, 20, 25, or 30 amino acids, or has an amino acid length falling between any two integers. In some embodiments, the linker comprises the amino acid sequence (G4S)n (SEQ ID NO: 112), where n is an integer equal to or greater than 1, for example, n is an integer from 1 to 7, such as n = 1, 2, 3, 4, 5, 6, or 7.

[0213] The multispecific antibody according to the invention can take any suitable form, such as single-chain or multi-chain form. In some embodiments, the multispecific antibody according to the invention comprises a CD3 arm providing at least one CD3 antigen-binding domain and a TAA arm providing at least one TAA antigen-binding domain; and optionally at least one additional antigen-binding domain (e.g., at least one additional TAA antigen-binding domain) connected to said CD3 arm, TAA arm, or both.

[0214] In some embodiments, the multispecific antibody according to the present invention comprises: - a first structural portion comprising, from the N-terminus to the C-terminus, a first antigen-binding domain, optionally a linker, and a first immunoglobulin Fc region; and - a second structural portion comprising, from the N-terminus to the C-terminus, a second antigen-binding domain, optionally a linker, and a second immunoglobulin Fc region; - a third antigen-binding domain optionally connected to the N-terminus or C-terminus of the first or second structural portion via the linker; wherein the first and second immunoglobulin Fc regions dimerize to form an Fc dimer. In some embodiments, the first antigen-binding domain binds CD3 and comprises or is composed of Fab, scFab, or scFv domains. In some embodiments, the second and third antigen-binding domains bind TAA and comprise or are composed of Fab, scFab, scFv, or VHH domains. Preferably, the first antigen-binding domain is a Fab domain that binds CD3, and the second and third antigen-binding domains are VHH domains that bind TAA. In some embodiments, preferably, the third antigen-binding domain binds to the N-terminus of the first structural portion, or to the N-terminus of the second structural portion. In some embodiments, the second and third antigens...The binding domain binds to the same TAA antigen. In some embodiments, the linker is 5-25 amino acids in length, or contains an amino acid sequence of SEQ ID NO: 106, 136, or 112.

[0215] In some embodiments, the multispecific antibody according to the invention is an anti-CD3xTAA multispecific antibody comprising the following structural portions: - a first structural portion comprising, from the N-terminus to the C-terminus: a CD3 antigen-binding domain, optionally a linker, and a first immunoglobulin Fc region; and - a second structural portion comprising, from the N-terminus to the C-terminus: a first TAA antigen-binding domain, optionally a linker, and a second immunoglobulin Fc region; - a second TAA antigen-binding domain optionally linked to the first or second portion via the linker; wherein the first and second immunoglobulin Fc regions dimerize to form an Fc dimer. Preferably, the second TAA antigen-binding domain is linked to the N-terminus of the first or second structural portion; however, C-terminal linkage is also considered in this disclosure.

[0216] In some embodiments, the anti-CD3xTAA multispecific antibody comprises: - a first structural portion from the N-terminus to the C-terminus comprising: a second TAA antigen-binding domain, optionally a linker, a CD3 antigen-binding domain, optionally a linker, and a first immunoglobulin Fc region; and - a second structural portion from the N-terminus to the C-terminus comprising: a first TAA antigen-binding domain, optionally a linker, and a second immunoglobulin Fc region; wherein the first and second immunoglobulin Fc regions dimerize to form an Fc dimer.

[0217] In some embodiments, the anti-CD3xTAA multispecific antibody comprises: - a first structural portion comprising, from the N-terminus to the C-terminus, a CD3 antigen-binding domain, optionally a linker, and a first immunoglobulin Fc region; and - a second structural portion comprising, from the N-terminus to the C-terminus, a second TAA antigen-binding domain, optionally a linker, a first TAA antigen-binding domain, optionally a linker, and a second immunoglobulin Fc region; wherein the first and second immunoglobulin Fc regions dimerize to form an Fc dimer.

[0218] In some embodiments of the anti-CD3xTAA multispecific antibody according to the present invention, preferably, the CD3 antigen-binding domain is a Fab domain that binds CD3, and the first and second TAA antigen-binding domains are VHH domains that bind TAA. In some embodiments, preferably, the linker is 5-25 amino acids in length, or comprises an amino acid sequence of SEQ ID NO: 106, 136, or 112. In some embodiments, the Fab domain comprises VH-CH1Alternatively, a fragment of VH-CL may be fused to the N-terminus of the Fc region. In other embodiments, the Fab domain is fused to the N-terminus of the Fc region by a fragment comprising VH-CL or VL-CH1.

[0219] In some embodiments, the multispecific antibody according to the invention comprises first, second, and third polypeptide chains, wherein: the first polypeptide chain comprises, from the N-terminus to the C-terminus: a VHH domain, a linker, a VH domain, a CH1 domain, and a first Fc region; the second polypeptide chain comprises, from the N-terminus to the C-terminus: a VL domain and a CL domain; and the third polypeptide chain comprises, from the N-terminus to the C-terminus: a VHH domain and a second Fc region. In some embodiments, the VH domain and the VL domain pair to form a CD3 binding domain according to the invention; and the VHH domain comprises or is composed of a TAA binding domain according to the invention. Preferably, the linker is 10-25 amino acids long, for example, about 15 amino acids long, or comprises an amino acid sequence of SEQ ID NO: 136 or 112.

[0220] In some embodiments, the multispecific antibody according to the present invention comprises a first, second, and third polypeptide chain, wherein: the first polypeptide chain comprises a VH domain, a CH1 domain, and a first Fc region from the N-terminus to the C-terminus; the second polypeptide chain comprises a VL domain and a CL domain from the N-terminus to the C-terminus; and the third polypeptide chain comprises a VHH domain, a linker, a VHH domain, and a second Fc region from the N-terminus to the C-terminus. In some embodiments, the VH domain and the VL domain pair to form a CD3 binding domain according to the present invention; and the VHH domain comprises or is composed of a TAA binding domain according to the present invention. Preferably, the linker is 5-15 amino acids long, for example, about 10 amino acids long, or comprises an amino acid sequence of SEQ ID NO: 106 or 112.

[0221] Exemplary CD3xCD20 Multispecific Antibody

[0222] In some embodiments of the multispecific antibody according to the present invention described above, preferably, the multispecific antibody according to the present invention comprises an antigen-binding domain of a TAA on the surface of hematologic malignancies. In some embodiments, the TAA is CD20. In some embodiments, the TAA antigen-binding domain comprises or is composed of a VHH domain that specifically binds to CD20. In some embodiments, the anti-CD20 VHH domain comprises the CDR1, CDR2, and CDR3 sequences of one of the amino acid sequences in SEQ ID NO: 102 or 115-124; preferably, the CDR1, CDR2, and CDR3 sequences comprise or are composed of the amino acid sequences in SEQ ID NOs: 103, 104, and 105, respectively, or comprise or are composed of the amino acid sequences in SEQ ID NOs: 137, 104, and 105, respectively. In some embodiments, the anti-CD20...The VHH domain comprises an amino acid sequence of one of SEQ ID NO: 102 or 115-124, or has at least 85%, 90%, 95%, or 99% identity with said amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed of said amino acid sequence.

[0223] In some embodiments, the multispecific antibody according to the invention is an anti-CD3 multispecific antibody comprising a CD3 antigen-binding domain combined with the anti-CD20 antigen-binding domain of any of the above embodiments. In some embodiments, said CD3 antigen-binding domain may comprise any anti-CD3 antibody according to the invention or its antigen-binding fragment, or a combination of its 6 CDRs or its VH / VL combination. In some embodiments, the CD3 antigen-binding domain comprises HCDR1-3 and LCDR1-3 contained in one of the following VH and VL sequence pairs: (a) the VH sequence of SEQ ID NO: 31 and the VL sequence of one of SEQ ID NOs: 32 and 72-74; (b) the VH sequence of one of SEQ ID NOs: 67-69 and the VL sequence of one of SEQ ID NOs: 70-71; (c) the VH sequence of SEQ ID NO: 68 and the VL sequence of one of SEQ ID NOs: 78-82; or (d) the VH sequence of SEQ ID NO: 68 and the VL sequence of SEQ ID NO: 87.

[0224] In other embodiments, the anti-CD3 antigen-binding domain comprises HCDR1-3 contained in the VH sequence of SEQ ID NO: 47 and LCDR1-3 contained in the VL sequence of SEQ ID NO: 48. In some preferred embodiments, the CD3 antigen-binding domain comprises a VH / VL amino acid sequence selected from the following: SEQ ID NOs: 31 and 32; one of SEQ ID NOs: 67 and SEQ ID NOs: 70-71; one of SEQ ID NOs: 69 and SEQ ID NOs: 70-71; one of SEQ ID NOs: 68 and SEQ ID NOs: 70-71 and 78-82; SEQ ID NO: 68 and SEQ ID NO: 87. In some preferred embodiments, the CD3 antigen-binding domain comprises the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32. In some preferred embodiments, the CD3 antigen-binding domain comprises the VH sequence of SEQ ID NO: 68 and the VL sequence of SEQ ID NO: 69 and 70-71.The VL sequence of SEQ ID NO: 47. In some other preferred embodiments, the CD3 antigen-binding domain comprises the VH sequence of SEQ ID NO: 47 and the VL sequence of SEQ ID NO: 48.

[0225] In some embodiments, the multispecific antibody according to the invention is an anti-CD3 and CD20 bispecific antibody, preferably a CD3xCD20 bispecific T cell connector. In some embodiments, the multispecific antibody according to the invention comprises: (i) a first, second, and third polypeptide chain comprising, respectively, SEQ ID NOs: 88, 89, and 101, or having at least 95%, 96%, 97%, 98%, or 99% identity with them, or (ii) a first, second, and third polypeptide chain comprising, respectively, SEQ ID NOs: 90, 91, and 101, or having at least 95%, 96%, 97%, 98%, or 99% identity with them. In some preferred embodiments, the first, second, and third polypeptide chains comprise, respectively, the amino acid sequences of SEQ ID NOs: 88, 89, and 101.

[0226] In some embodiments, the multispecific antibody according to the present invention comprises: (i) a first, second, and third polypeptide chain comprising, respectively, amino acid sequences of SEQ ID NOs: 130, 131, and 132, or having at least 95%, 96%, 97%, 98%, or 99% identity with them; or (ii) a first, second, and third polypeptide chain comprising, respectively, amino acid sequences of SEQ ID NOs: 133, 134, and 135, or having at least 95%, 96%, 97%, 98%, or 99% identity with them. In some preferred embodiments, the first, second, and third polypeptide chains comprise, respectively, the amino acid sequences of SEQ ID NOs: 130, 131, and 132; or comprise, respectively, the amino acid sequences of SEQ ID NOs: 133, 134, and 135.

[0227] Exemplary CD3xMSLN Multispecific Antibody

[0228] In some embodiments of the multispecific antibody according to the present invention described above, preferably, the multispecific antibody according to the present invention comprises an antigen-binding domain of a TAA on the surface of solid tumor cells. In some embodiments, the TAA is an MSLN. In some embodiments, the TAA antigen-binding domain comprises or is composed of a VHH domain that specifically binds to MSLN. In some embodiments, the anti-MSLN VHH domain comprises the CDR1, CDR2, and CDR3 sequences in the amino acid sequence of SEQ ID NO: 97 (page 28 / 51, 36 CN 122255272 A); preferably, the CDR1, CDR2, and CDR3 sequences comprise SEQ ID NOs: 98, 99, and 99, respectively.The amino acid sequence of 100 or thereof. In some embodiments, the anti-MSLN VHH domain comprises the amino acid sequence of SEQ ID NO: 97, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence with one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or comprises thereof.

[0229] In some embodiments, the multispecific antibody according to the invention is an anti-CD3 multispecific antibody comprising a CD3 antigen-binding domain combined with the anti-MSLN antigen-binding domain of any of the above embodiments. In some embodiments, the CD3 antigen-binding domain may comprise any anti-CD3 antibody according to the invention or its antigen-binding fragment, or a combination comprising its 6 CDRs, or its VH / VL combination. In some embodiments, the CD3 antigen-binding domain comprises HCDR1-3 and LCDR1-3 contained in one of the following VH and VL sequence pairs: (a) the VH sequence of SEQ ID NO: 31 and the VL sequence of one of SEQ ID NOs: 32 and 72-74; (b) the VH sequence of one of SEQ ID NOs: 67-69 and the VL sequence of one of SEQ ID NOs: 70-71; (c) the VH sequence of SEQ ID NO: 68 and the VL sequence of one of SEQ ID NOs: 78-82; or (d) the VH sequence of SEQ ID NO: 68 and the VL sequence of SEQ ID NO: 87.

[0230] In other embodiments, the anti-CD3 antigen-binding domain comprises HCDR1-3 contained in the VH sequence of SEQ ID NO: 47 and LCDR1-3 contained in the VL sequence of SEQ ID NO: 48. In some preferred embodiments, the CD3 antigen-binding domain comprises a VH / VL amino acid sequence pair selected from the following: SEQ ID NOs: 31 and 32; one of SEQ ID NOs: 67 and SEQ ID NOs: 70-71; one of SEQ ID NOs: 69 and SEQ ID NOs: 70-71; one of SEQ ID NO: 68 and SEQ ID NOs: 70-71 and 78-82; SEQ ID NO: 68 and SEQ ID NO: 87. In some preferred embodiments, the CD3 antigen-binding domain comprises the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32. In other preferred embodiments, the CD3 antigen-binding domain comprises the VH sequence of SEQ ID NO: 47 and the VL sequence of SEQ ID NO: 48.

[0231] In some embodiments, the multispecific antibody according to the present invention is a bispecific antibody against CD3 and MSLN, preferably a CD3xMSLN bispecific T-cell connector. In some embodiments, the multispecific antibody according to the present invention comprises: (i) first, second, and third polypeptide chains comprising, respectively, amino acid sequences of SEQ ID NOs: 88, 89, and 96, or having at least 95%, 96%, 97%, 98%, or 99% identity with them; or (ii) first, second, and third polypeptide chains comprising, respectively, amino acid sequences of SEQ ID NOs: 90, 91, and 96, or having at least 95%, 96%, 97%, 98%, or 99% identity with them. In some preferred embodiments, the first, second, and third polypeptide chains comprise the amino acid sequences of SEQ ID NOs: 88, 89, and 96, respectively. In other embodiments, the first, second, and third polypeptide chains comprise the amino acid sequences of SEQ ID NOs: 90, 91, and 96, respectively.

[0232] Properties of the Multispecific Antibody of the Invention

[0233] As shown in the examples, the anti-CD3 sequence of the present invention exhibits unique behavior. When used alone, especially in the monovalent case, its binding to CD3 on the cell surface is very weak. However, when spliced ​​into a TCE, it exhibits very strong T cell activation and killing ability by means of the TAA arm binding to tumor cells. Therefore, in some embodiments, the present disclosure provides a multispecific antibody according to the invention, said multispecific antibody being a TCE. In some embodiments, the TCE according to the invention has one or more of the following properties: (i) binding to surface TAA-positive tumor cells. In some embodiments, this binding activity of the TCE according to the invention is comparable to that of a corresponding TCE composed of a BMK8 reference antibody.

[0234] (ii) exhibiting TAA-positive tumor cell-dependent T cell activation activity. According to the test results of the T cell activation assay (page 29 / 51, CN 122255272 A), in the absence of relevant tumor cells, the TCE molecule having the CD3 domain of the present invention only showed weak activation activity against T cells; while in the presence of relevant tumor cells, the CD3 domain of the present invention showed significant T cell activation by binding to TAA-containing tumor cells via the anti-TAA arm. In some embodiments, the T cell activation assay is performed by detecting the release of cytokines induced by T cells or the tumor cell killing activity of T cells.

[0235] In some further embodiments, the TCE of the present invention also exhibits one or more of the following properties: (i) in the absence of target tumor cells expressing TAA, it weakly binds to surface CD3-positive T cells, preferably by flow cytometry on Jurkat cells, showing extremely weak binding activity.T cell binding. In some embodiments, the binding activity of the TCE according to the invention is equivalent to that of a corresponding TCE composed of a BMK9 reference antibody. Preferably, the assay is performed according to Examples 5.2 and 6.2.

[0236] (ii) Inducing T cell killing of said tumor cells in the presence of surface TAA-positive tumor cells and T cells. In some embodiments, said tumor cells are hematologic malignancies. In other embodiments, said tumor cells are solid tumor cells. In some embodiments, the killing activity of the TCE according to the invention is equivalent to or even stronger than that of a corresponding TCE composed of a BMK8 reference antibody. Preferably, the assay is performed according to Examples 5.3 or 6.3.

[0237] (iii) At an antibody dose that induces half-kill of target tumor cells, non-substantially inducing the release of cytokines from T cells. In some embodiments, the release of cytokines IL-6, IFNγ, TNF-α, or any combination thereof can be measured. In some embodiments, in the same assay, for the TCE according to the invention, the ratio of the cytokine release EC50 value to the tumor killing EC50 value is greater than 1.2, 1.3, 1.5, or greater than 3 in some embodiments, or 4 to 10, for example 5 to 7, in other embodiments. In some embodiments, the TCE drug according to the invention has a lower EC50 ratio and thus better safety compared to the corresponding TCE composed of BMK8 by separating tumor cell killing activity from cytokine release activity. In some embodiments, the tumor cells are hematologic malignancies. In other embodiments, the tumor cells are solid tumor cells. In some embodiments, the assay is performed according to Example 5.3 or 6.3.

[0238] In some embodiments, the multispecific antibody according to the invention is a 2:1 type asymmetric TCE bispecific antibody structure, such as the TCE molecule with the configuration shown in FIG23. Although not bound by any theory, it is believed that in the 2:1 form, since the CD3 antigen-binding domain provided by the anti-CD3 antibody of the present invention has only a weak monovalent binding affinity to T cells, in some cases, the bivalent affinity of the antibody for TAA can play a role in balancing antibody efficacy and specificity. On the other hand, although not bound by any theory, it is believed that the weak T cell binding activity of the monovalent CD3 antigen-binding domain of the present invention will help avoid excessive activation and attenuation of T cells, and reduce the risk of adverse reactions such as cytokine release syndrome (CRS) and neurotoxicity.

[0239] IV. Production and purification of the antibody of the present invention

[0240] In a fourth aspect, the present invention provides an anti-CD3 antibody encoding the first aspect of the present invention and an anti-CD20 antibody encoding the second aspect of the present invention.The nucleic acid of the antibody or the multispecific antibody of the third aspect, the host cell containing it, and the method for producing the anti-CD3 antibody, the anti-CD20 antibody, or the multispecific antibody.

[0241] To produce the antibody of the present invention, the polypeptide chain of the antibody of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production, and assembled under suitable conditions. For recombinant production, polynucleotides encoding any one and / or multiple polypeptide chains of the antibody can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. The polynucleotides can be easily isolated and sequenced using conventional methods. In one embodiment, a polynucleotide encoding one or more polypeptide chains of the antibody of the present invention is provided. In yet another embodiment, the present invention provides a vector comprising one or more polynucleotides of the present invention, preferably expressing a vector. Therefore, in one embodiment, the present invention provides a method for producing the antibody of the present invention, the method comprising: culturing a host cell containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the antibody; and assembling the polypeptide chain to produce the antibody under conditions suitable for assembling the polypeptide chain into the antibody.

[0242] Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, granulocytes, λ phages, or yeast artificial chromosomes (YAC).

[0243] In one embodiment, the present invention also provides a host cell containing one or more of the polynucleotides of the present invention. In some embodiments, a host cell containing an expression vector of the present invention is provided. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK293 or 293F cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63, and Sp2 / O, etc. In a preferred embodiment, the host cell is CHO or HEK293 cells.

[0244] Antibodies prepared by the methods described herein can be processed using known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, etc.Purification is performed using methods such as exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. After purification, the purity of the antibody of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography. The physical / chemical properties and / or biological activity of the antibody provided herein can be identified, screened, or characterized by a variety of assays known in the art.

[0245] In a preferred embodiment, the antibody of the present invention exhibits good production properties when recombinantly produced in mammalian host cells such as CHO cells, particularly good expression yield and good byproduct profile.

[0246] V. Immunofusions and Immunoconjugates

[0247] In a fifth aspect, the present invention provides antigen-binding molecules, such as immunofusions or immunoconjugates, generated by fusing or conjugating the antibody of the present invention to a heterologous molecule.

[0248] In one embodiment, in the immunofusion, the antibody of the present invention (or its antigen-binding fragment) is linked to a heterologous peptide or polypeptide molecule directly or through an amino acid linker. Heterogeneous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that impart another functional activity to the fusion, or tagged peptides that facilitate the purification or detection of the immunofusion. For example, a chimeric antigen receptor (CAR) comprising the antibody of the present invention or its antigen-binding fragment is an example of an immunofusion according to the present invention.

[0249] In one embodiment, in the immunoconjugate, the antibody of the present invention (or its antigen-binding fragment) is conjugated to a conjugated portion (e.g., a therapeutic agent, diagnostic agent, or detectable agent). In the conjugate, a linker may be used to covalently link different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers. Advantageously, the linker is a “cleavable linker” that facilitates the release of the conjugated portion after delivery of the conjugate to the target site. For example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers may be used.

[0250] In embodiments conjugated with a therapeutic agent, suitable therapeutic agents for the conjugate include, but are not limited to, cytotoxins (e.g., cell growth inhibitors or cell killers), pharmaceuticals, or radioisotopes.

[0251] In embodiments conjugated with diagnostic or detectable agents, such conjugates can be used as part of clinical testing methods (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnostics and detections can be achieved by conjugating antibodies with detectable agents, including but not limited to enzymes such as horseradish peroxidase; cofactors such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metal and non-radioactive paramagnetic metal ions used in various positron emission tomography (PET) imaging techniques.

[0252] VI. Pharmaceutical Compositions, Pharmaceutical Conjugates, and Kits

[0253] In a sixth aspect, the present invention provides compositions, such as pharmaceutical compositions, comprising an antibody of the first, second, or third aspect of the present invention, or an antigen-binding molecule of the fifth aspect of the present invention (e.g., an immunoconjugate or immunofusion), formulated with a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. Pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, the antibody of the present invention or the immunoconjugate or immunofusion of the present invention is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise the antibody or the immunoconjugate or immunofusion of the present invention described herein, along with other therapeutic agents.

[0254] In another aspect, the present invention also provides pharmaceutical conjugates comprising the antibody or the immunoconjugate or immunofusion of the present invention described herein, along with other therapeutic agents.

[0255] The therapeutic agents suitable for use in the pharmaceutical compositions and combinations thereof of the present invention may be therapeutic agents selected from any of the following categories (i)-(iv): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression (e.g., anti-PD-L1 antibodies); (iv) drugs that have anti-tumor effects.

[0256] The pharmaceutical compositions of the present invention may contain an "effective therapeutic amount" or an "effective preventive amount" of the antibody described herein. An "effective therapeutic amount" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. The effective therapeutic amount may vary depending on various factors such as disease state, individual age, sex, and weight. An effective therapeutic amount is any amount in which any toxic or harmful effects are less than the beneficial therapeutic effects. "Therapeutic effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%, relative to untreated subjects. The ability of the antibodies of the present invention to inhibit measurable parameters (e.g., tumor volume) can be evaluated in animal model systems that predict efficacy in human tumors. "Prophylactic effective amount" refers to the amount that effectively achieves the desired preventive outcome at the required dose and for the required duration. Typically, because the prophylactic dose is used in subjects before or at an earlier stage of the disease, the prophylactic effective amount is less than the therapeutic effective amount.

[0257] Kits containing the antibodies described herein are also within the scope of the present invention. Kits may include one or more other elements, such as: instructions for use; other reagents, such as markers or reagents for conjugation; pharmaceutically acceptable carriers;And devices or other materials for administration to a subject.

[0258] VII. Uses and Methods

[0259] In a seventh aspect, the present invention provides the use and methods of antibodies according to the first, second and third aspects of the invention or antigen-binding molecules (e.g., immune conjugates or fusions) according to the fifth aspect of the invention in the treatment and prevention of CD3-related diseases and / or cancer, or CD20-related diseases and / or cancer.

[0260] Overexpression of tumor-associated antigens such as CD20 and MSLN on the surface of cancerous tissue cells makes them suitable targets for cancer immunotherapy. In one aspect, therefore, the present invention provides the use of the multispecific antibodies or TCEs of the present invention comprising CD3 and TAA antigen-binding domains for the prevention and / or treatment of said TAA-related tumors (i.e., TAA-positive tumors) in a subject. In another aspect, the present invention provides the use of the anti-CD20 antibody or multispecific antibody of the present invention, comprising the CD20 binding domain of the present invention, for the prevention and / or treatment of CD20-related tumors (i.e., CD20-positive tumors) in a subject. In said application, the antibody of the present invention may be administered to the subject as the sole active agent or may be administered to the subject in combination with other therapies or therapeutic agents. The other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells and eliminate tumors by binding to and / or blocking these molecules; drugs that activate the immune system of the subject, prompting it to spontaneously eliminate tumors. In yet another aspect, the present invention also provides a method for the prevention or treatment of cancer in a subject, comprising administering the antibody of the present invention or its antigen-binding fragment to a subject in need.

[0261] TAA-positive tumors or CD20-positive tumors suitable for the methods and applications of the present invention may be selected from various solid tumors or hematologic malignancies. In some embodiments, the TAA is an MSLN, and the tumor is selected from, for example, mesothelioma (such as malignant mesothelioma), pancreatic cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), and colorectal cancer. In some embodiments, the TAA is a CD20, and the tumor is selected from B-cell lymphomas (such as DLBCL and LBCL). The tumor suitable for the method and application of the present invention can be an early, intermediate, or late-stage or metastatic cancer. In addition, the tumor suitable for the method and application of the present invention can be a tumor that has previously received treatment and has escaped immune response.

[0262] In any of the above embodiments of the method of the present invention, the administration of the antibody or fragment thereof according to the present invention may include 1) a therapeutic measure that cures, slows down, alleviates or reduces the symptoms of a diagnosed pathological condition or disease and / or stops the progression of the diagnosed pathological condition or disease; or 2) a preventive or protective measure that prevents and / or slows down the progression of the disease.The development of a disease or condition. Therefore, in the method of the present invention, the subject may be an individual who has already contracted a disease, an individual who is susceptible to a disease, or an individual who wishes to prevent a disease. The individual will benefit from the therapeutic or preventive measures and will show a reduction or improvement in the occurrence, recurrence, or development of the disease, condition, symptom, and / or symptoms compared to an individual who has not received the treatment. In some embodiments, the present invention relates to the treatment of a disease or condition; in other embodiments, the present invention relates to the prevention of a disease or condition.

[0263] The antibody or fragment thereof according to the present invention, and other therapeutic agents optionally used in combination therewith, may be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Various dosing schedules are covered herein, including, but not limited to, single administration or multiple administration at multiple time points, bolus administration, and pulsatile infusion.

[0264] For the prevention or treatment of disease, the appropriate dosage of the antibody or its fragment according to the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the specific type of drug used, the severity and course of the disease, whether the drug is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0265] In some embodiments, the invention also provides for the use of the antibody or antibody fragment of the invention as a drug or for the preparation of a drug. In some embodiments, the drug is a drug for use in the foregoing treatment and prevention methods.

[0266] Any or all of the features described above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention; however, it should be understood that the examples are for illustrative purposes only and should not be construed as constituting any limitation.

[0267] Examples

[0268] Example 1: Immunization and Acquisition of Hybridoma Clones

[0269] 1.1 Animal Immunization

[0270] Different strains of mice (Balb / c, SJL) and different forms of immunogens (huCD3ED dimer (ACRO, CAT# CDD-H52W1), huCD3EG dimer (ACRO, CAT# CDG-H52W5), huCD3E (ACRO, CAT# CDE-H5223), etc.) were selected to carry out animal immunization in various combinations. By monitoring the antigen or cell-specific serum antibody titers, mice with titers meeting the requirements were selected for hybridoma fusion experiments. Positive antibodies were selected by ELISA and FACS screening. (Instructions 33 / 51 pages 41 CN 122255272 A)Hybridoma clones were formed; after subcloning, ELISA and FACS were used for rescreening to confirm the specific binding of the molecules to human and cynomolgus CD3.

[0271] 1.2 Screening of hybridomas

[0272] RNA was extracted from hybridoma cells and reverse transcribed into cDNA using a reverse transcription kit. The cDNA was then amplified by PCR using degenerate primers (synthesized by Azenta primers). The PCR product was cloned into the pMD18-T vector (TaKaRa, Cat# 6013), transformed, amplified, and sequenced. Six clones were selected based on the sequenced sequences: H4 (27E2.1, also known as 27E2-1D10), H45 (119D3.4), H10 (112F4.1), H36 (136G6.3), H54 (217B11.1), and H61 (250E4.1) to construct plasmids for expressing chimeric antibodies and identifying functional activity. The CDR sequences and heavy and light chain variable region sequences of the obtained antibodies are shown in the sequence listing SEQ ID NOs: 1-48.

[0273] Example 2: Expression and functional determination of hybridoma chimeric antibodies

[0274] 2.1 Construction and expression of chimeric antibodies

[0275] The hybridoma candidate clones obtained in Example 1 were used to extract the antibody light and heavy chain gene sequences and construct human-mouse chimeric antibodies. In short, the VH and VL genes of the mouse monoclonal antibody were amplified again using cloning primers containing appropriate restriction sites and cloned into the expression vector pcDNA3.4 to generate corresponding clones of chimeric antibodies with the constant region of human IgG1. The clones were transfected into Expi-CHOS cells for transient transfection expression, purified with Protein A, and the obtained proteins were identified. Chimeric antibodies V-HD-27E2.1, V-HD-119D3.4, V-HD-112F4.1, V-HD-136G6.3, V-HD-217B11.1, and V-HD-250E4.1 were obtained, and their full-length heavy chain and light chain sequences are shown in the sequence listing SEQ ID NOs: 49-60, respectively.

[0276] 2.2 Flow Cytometry Binding Assay

[0277] Freshly cultured Jurkat cells and CD3 knockout Jurkat-CD3KO cells (Aikon) culture suspensions were collected into 50 mL centrifuge tubes, centrifuged at 300 g for 5 minutes, washed once with 20 mL of pre-cooled 4℃ FACS buffer (PBS+2%FBS), and resuspended in 10 mL of FACS buffer. 1 mL of cell suspension was taken for counting, and the cell suspension was diluted to 1E6 cells / mL. Add 100 μL of cell suspension to a 96-well U-plate, centrifuge at 300g for 5 minutes and discard the supernatant. Add 100 μL of serially diluted chimeric antibody to each well and incubate at 4°C for 1 hour. Add pre-chilled FACS at 4°C.Wash three times with buffer. Add the fluorescent secondary antibody Goat anti-Human IgG Fc, PE (eBioscience™, CAT#12-4998-82, 1:200 dilution) diluted with FACS buffer and incubate at 4°C for 0.5 hours. Wash once with pre-chilled FACS buffer at 4°C, resuspend the cells in 100 μL of FACS buffer, and detect the cell surface fluorescence intensity using FACS.

[0278] The assay included anti-CD3 antibody BMK8 as a positive control. The BMK8 sequence (SEQ ID NOs: 63 and 64, sequence derived from patent US10781264B2) is the anti-CD3 antibody sequence used in the second-generation BITE with extended half-life developed by Amgen, with representative drugs including IMDELLTRATM, etc. Its characteristics include strong CD3 binding activity and T cell activating ability. It has been reported that, unlike the CD3 sequence OKT3 used in Amgen's first-generation BITE, BMK8 has good monkey CD3 cross-activity. (See: A Bispecific DLL3 / CD3 IgG-Like T-Cell Engaging Antibody Induces Antitumor Responses in Small Cell Lung Cancer. Clin Cancer Res. 2020 Oct 1;26(19):5258-5268. doi: 10.1158 / 1078-0432.CCR-20-0926.) As a control antibody for testing, BMK8 contains the human IgG1 constant region of SEQ ID NO: 61 and the human lambda light chain constant region of SEQ ID NO: 114, and is expressed according to the procedure described in Example 2.1.

[0279] As shown in Figure 1, all chimeric antibodies can bind to Jurkat cells. As shown in Figure 2, among the antibodies tested, BMK8 and chimeric antibody 250E4.1 have a certain degree of non-specific binding to CD3 knockout Jurkat cells.

[0280] 2.3 CynoCD3 Cross-binding Flow Cytometry Assay Instructions 34 / 51 pages 42 CN 122255272 A

[0281] Resuscitate frozen cynomolgus monkey PBMC (cynoPBMC) cells (from Ausen Biotech), wash once with 20 mL of pre-chilled 4℃ FACS buffer (PBS+2%FBS), and resuspend in 10 mL of FACS buffer. Take 1 mL of cell suspension for counting, and dilute the cell suspension to 1E6 cells / mL. Add 100 μL of cell suspension to a 96-well U plate, centrifuge at 300g for 5 minutes and discard the supernatant, then add 100 μL of the supernatant to each well.Cells were serially diluted with μL of chimeric antibody and incubated at 4°C for 1 hour. They were then washed three times with pre-chilled FACS buffer at 4°C. Fluorescent secondary antibodies Goat anti-Human IgG Fc, PE (eBioscience™, CAT#12-4998-82, 1:200 dilution) diluted with FACS buffer, and directly labeled flow cytometry antibodies anti-CD4, APC (BD Pharmingen™, CAT#551980, 1:200 dilution) and anti-CD8 BV421 (BioLegend, CAT# 301036, 1:200) were added. The cells were incubated at 4°C for 0.5 hours. After washing once with pre-chilled FACS buffer at 4°C, the cells were resuspended in 100 μL of FACS buffer, and the cell surface fluorescence intensity was detected using FACS. Anti-CD3 antibody BMK8 was included as a positive control in the assay.

[0282] As shown in Figures 3 and 4, all chimeric antibodies specifically bind to cyno CD4+ T and CD8+ T cells.

[0283] 2.4 T Cell Activation Assay

[0284] The selected chimeric antibodies were further subjected to PBMC functional activation assay. Specifically, frozen PBMCs (Donor1 and Donor2, from Ausen Biotech) from two healthy volunteers were revived and diluted to 1E6 cells / mL with RPMI 1640 complete medium. 100 μL of cell suspension was added to a 96-well U-plate, and 100 μL of serially diluted chimeric antibody was added to each well. The 96-well plate was then incubated at 37°C in a 5% CO2 incubator for 40 hours. Preparation of staining solutions: Human TruStain FcX™ (BioLegend, CAT# 422302, 1:100); anti-human CD4, FITC (Biolegend, CAT# 317408, 1:200); anti-human CD8, APC (eBioscience™, CAT# 17-0087-42, 1:200); anti-human CD25, BV421 (Biolegend, CAT# 302630, 1:200). Wash cells in 96-well plates once with FACS buffer, add 100 μL of cell staining solution to each well, and incubate at 4°C for 0.5 hours. Wash once with pre-chilled FACS buffer at 4°C, resuspend cells in 100 μL of FACS buffer, and detect cell surface fluorescence intensity using FACS. The assay included anti-CD3 antibodies BMK8 and BMK9 (Teneobio-F2B) as positive controls. Teneobio-F2B is a...CD3 antibodies with weak T cell binding and activation activity, when incorporated into TCE molecules in monovalent form, exhibit cytotoxic activity against hematologic malignancies, but were ineffective against solid tumors in a recent clinical trial (NCT04740034) (J Clin Oncol 42, e14587(2024). DOI: 10.1200 / JCO.2024.42.16_suppl.e14587). As a control antibody for testing, BMK9 contains the variable region sequence of Teneobio-F2B (SEQ ID NOs: 65 and 66), as well as the constant region of the human IgG1 heavy chain (SEQ ID NO: 61) and the constant region of the human kappa light chain (SEQ ID NO: 62), and is expressed according to the procedure described in Example 2.1.

[0285] As shown in Figures 5-8, 136G6.3 and 250E4.1 showed weaker activation function compared to the control antibody BMK8. However, existing reports indicate that for CD3 antibodies, the excessive T-cell activation function similar to BMK8 may actually predict potential toxicity and the risk of T-cell exhaustion and activation-induced T-cell death (AICD) (Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies. MAbs. 2019 May / Jun;11(4):639-652.). Therefore, 136G6.3 and 250E4.1 are expected to have better therapeutic windows.

[0286] Example 3: Antibody engineering of 136G6.3

[0287] 3.1 Sequence modification of 136G6.3 humanization and PTM removal

[0288] By comparing the human antibody variable region germline gene database in the Biophi (Humanize Antibody - BioPhi Antibody design platform (dichlab.org)) database, the heavy chain and light chain variable region germline genes with high homology with V-HD-136G6.3 were selected as templates using MOE (Molecular Operating Environment) software. The CDRs sequences of the mouse antibody determined based on the AbM protocol were transplanted into the corresponding human templates to form the variable region sequence "FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4". V-HD- Instructions for Use, 35 / 51 pages, 43 CN 122255272 A 136G6.3. The humanized template for the murine antibody is IGHV1-18 01, IGHJ1.01 and IGKV2-30 01, IGKJ4 01. Some amino acids in the FR region sequence of the V-HD-136G6.3 humanized antibody were reverted to the amino acids corresponding to the mouse antibody. Table 1 below shows the constructed humanized antibody and its variable region version and sequence.

[0289] Table 1. 136G6.3 humanized antibody

[0290] According to the PTM risk assessment, the V-HD-1 36G 6.3 antibody sequence was modified by post-translational modification (PTM) to form a PTM-removed variant. Table 2 below shows the constructed PTM-removed antibody and its variable region version and sequence.

[0291] Table 2. PTM-removed antibody of 136G6.3

[0292] The encoding genes of the humanized antibody and the PTM-removed antibody were synthesized by the Azenta gene. The synthesized gene was cloned into the expression vector, expressed to produce a humanized antibody with the human IgG1 constant region and purified by protein A.

[0293] 3.2 Cell Binding Detection of Humanized and PTM-Removed Variants of 136G6.3

[0294] The binding of each variant to Jurkat and Jurkat-CD3KO cells was compared with that of the parent cell using the flow cytometry binding assay procedure described in Example 2.2. Specific data are shown in Figures 9-12.

[0295] 3.3 Immunogenization Modification of 136G6.3-zom2

[0296] The immunogenicity of 136G6.3-zom2 was predicted using Wemol 3.0 and designed using MOE software to ensure the original affinity and reduce the risk of immunogenicity. Primers with corresponding amino acid site mutations were designed based on the nucleotide sequence of 136G6.3-zom2, and PCR was performed using a vector containing the 136G6.3-zom2 fragment as a template. After PCR was completed, Dpn I enzyme was added and digested at 37°C for 1 hour. The target fragment was recovered by 1% agarose gel electrophoresis and then transformed into DH5α competent cells. After colonies grew the next day, single clones were picked and sent for sequencing. After sequencing identification, nucleotide sequences that were completely consistent with the target sequence were selected for plasmid extraction, expression and purification. The generated deimmunogenic antibodies and their variable region sequences are shown in Table 3 below, where the mutation introduced in the VL sequence (m1L, SEQ ID NO: 70) of 136G6.3-zom2 is shown in parentheses after m1L.

[0297] Table 3. Deimmunogenic variants of 136G6.3 humanized antibody

[0298] 3.4 Cell binding detection of 136G6.3-zom2 deimmunogenic variants

[0299] The binding of each variant to Jurkat and Jurkat-CD3KO cells was compared with the parent cell binding assay procedure of Example 2.2. See Figures 13 and 14 for specific data. (Explanation)Book 36 / 51, page 44, CN 122255272 A

[0300] 3.5 Combination of 136G6.3-dzom2.m21 and 136G6.3-pom11 sequences

[0301] Primers with corresponding amino acid site mutations were designed based on the nucleotide sequence of 136G6.3-dzom2.m21, and PCR was performed using a vector containing the 136G6.3-dzom2.m21 fragment as a template. After PCR, Dpn I enzyme was added and digested at 37°C for 1 hour. The target fragment was recovered by 1% agarose gel electrophoresis and then transformed into DH5α competent cells. After colonies grew the next day, single clones were picked and sent for sequencing. After sequencing identification, nucleotide sequences completely consistent with the target sequence were selected for plasmid extraction, expression, and purification to obtain 136G6.3M (VH sequence: SEQ ID NO: 68; VL sequence: SEQ ID NO: 87; HCDR1-3: SEQ ID NOs: 25-27, LCDR1-3: SEQ ID NOs: 75, 86, and 30).

[0302] 3.6 Cell binding detection of 136G6.3M

[0303] The binding of 136G6.3M to Jurkat and Jurkat-CD3KO cells was detected according to the flow cytometry binding assay procedure in Example 2.2. See Figures 15-16 for specific data.

[0304] 3.7 Antigen Affinity Detection of 136G6.3M

[0305] The affinity of 136G6.3M for human CD3 and cyno CD3 antigens was detected using the SPR method on a Biocore 8K instrument. Specifically, a fixed concentration of antibody (10 μg / mL) was captured using a protein A chip (Cytiva, CAT# 10323089). Serially diluted antigens were used as the mobile phase. The binding time was set to 120 seconds, and the dissociation time was set to 200 seconds. After each dissociation, regeneration was performed with 20 mM glycine (pH 2.0). Data were analyzed using a 1:1 binding model.

[0306] As shown in Figure 17 and Table 4, the affinity of 136G6.3M for human CD3 and cyno CD3 antigens was comparable, differing by less than 3 times.

[0307] Table 4. Affinity of 136G6.3M to human CD3 and cyno CD3 antigens

[0308] Example 4: Screening and characterization of anti-CD20-VHH antibodies

[0309] 4.1 Screening, recombinant expression and identification of anti-CD20-VHH

[0310] Alpaca immunoassay and magnetic sorting techniques were used to screen candidate VHH sequences that bind to CD20 through preliminary characterization. In short, Raji (from ICON) and Daudi (from ICON) cells expressing human CD20 membrane protein were used alternately.Alpacas were immunized 14 days apart. Peripheral blood was collected seven days after each immunization, starting with the second immunization, and serum titers were monitored using FACS (Fluorescence Antibody-Cellular Synthesis) assays. Once the serum titer reached the standard for blood bank construction, peripheral blood was collected from immunized alpacas, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted from PBMCs, and cDNA was prepared using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara) as a template for reverse transcription. Using the cDNA as a template, a first-round PCR amplification produced nucleic acid fragments of conventional IgG (containing VH) and pure heavy chain IgG lacking the CH1 domain (containing VHH). These two types of nucleic acids were separated on an agarose gel. The VHH-encoding nucleic acid was extracted, purified, and then subjected to a second-round PCR amplification. The VHH fragment was then separated, purified, and recovered using gel electrophoresis. The recovered VHH gene fragment and the linearized yeast display vector pDisaplay (see page 37 / 51, CN 122255272 A) were mixed and co-transfected into competent yeast cells by electroporation to generate a yeast display library displaying VHH antibodies on the surface of yeast cells. Yeast cells bound to the target antigen were enriched from the constructed library by magnetic sorting using streptavidin magnetic beads that had been pre-incubated with the target antigen and thus bound to it.

[0311] The yeast culture obtained after magnetic bead sorting was plated on SDCAA plates, and single-clone cells were picked and cultured. After 48 hours of induction, the single-clone cell cultures were incubated sequentially with biotin-antigen and PE-Streptavidin. After incubation, flow cytometry (FACS) was performed to identify positive single-clone yeast cells bound to the target antigen. Genomic DNA was extracted from the cultures of the obtained positive yeast cell clones for PCR amplification of antibody sequences and sequencing.

[0312] Based on the sequencing results, candidate VHH sequences were selected and ligated into the expression vector pcDNA3.4 in the form of C-terminal fusion with human IgG1 Fc sequence. After the vector was verified by sequencing, it was transiently transfected into HEK-293F cells (hereinafter referred to as "293F cells"). The culture supernatant was taken to characterize the binding and endocytic properties of the expressed antibody, and finally the anti-CD20 antibody V-n6D11 with the VHH sequence of SEQ ID NO: 115 was obtained. The CDR1-3 sequence of this antibody is shown in SEQ ID NOs: 103-105 according to the AbM protocol.

[0313] 4.2 Expression and purification of candidate VHH-Fc antibody

[0314] The encoding gene of the above VHH antibody sequence was synthesized and inserted into the expression vector pcDNA3.4, so that the hIgG1 Fc sequence (SEQ ID NO: 115) was fused to the C-terminus.125). The constructed expression vector was transiently transfected into 293F cells. After continuous culture of transfected cells for 7 days, the culture supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was transferred to a sterile centrifuge tube, the antibody was purified using a Protein A column, and the purity of the antibody product was determined by SEC-HPLC.

[0315] 4.3 Flow cytometry assay of human CD20 positive tumor cells

[0316] The binding of anti-CD20 VHH-Fc candidate antibody molecule V-n6D11 to CD20 positive target cells was detected using the FACS binding assay. The specific experimental conditions were as follows: Raji / Daudi target cells (2×105 / well) + VHH-Fc or reference antibody (375 nM, 5× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ 0.5h). This experiment included the reference antibodies Ofatumumab and Obinutuzumab as controls. The Ofatumumab sequence (SEQ ID NOs: 126 and 127) was obtained from the IMGT database, and its sequence was retrieved as: IMGT / 3Dstructure-DB card, IMGT / 2Dstructure-DB card for INN: 8606. The Obinutuzumab sequence (SEQ ID NOs: 128 and 129) was obtained from the IMGT database, and its sequence was retrieved as: IMGT / 3Dstructure-DB card, IMGT / 2Dstructure-DB card for INN: 9043.

[0317] In short, according to the above experimental conditions, the target cells were seeded into 96-well plates at the cell density described above, and centrifuged at 300g for 5 minutes at 4°C. The test sample was added, and the plate was incubated at 4°C for 1 hour. After centrifugation at 4°C, the supernatant was removed, and the plate was washed twice with FACS buffer (1% BSA or 2% FBS in PBS), and then centrifuged at 4°C. Add the above-mentioned flow cytometry secondary antibody, resuspend the cells, and incubate at 4°C in the dark for 0.5 h. Wash twice with FACS buffer, resuspend the cells in 100 µl / well of FACS buffer, and perform flow cytometry analysis. Measure the MFI of the cells using a flow cytometer (Beckman Coulter).

[0318] The FACS binding results are shown in Figure 18. The candidate antibodies showed good target cell binding properties on both tested cell lines. In the FACS binding assay of Raji cells (Figure 18A), V-n6D11 showed better maximum binding (Bmax) than Ofatumumab and Obinutuzumab; in the FACS binding assay of Daudi cells (Figure 18B), V-n6D11 showed similar binding to the reference antibody.Ofatumumab has a similar EC50 value and maximum binding (Bmax), and is superior to Obinutuzumab in maximum binding (Bmax).

[0319] 4.4 Flow Cytometry Assay for Cross-Binding of Monkey CD20

[0320] The binding of the anti-CD20 VHH-Fc candidate antibody molecule V-n6D11 to cynoCD20 overexpressing cells was detected using the FACS binding assay. The FACS experimental procedure was basically as described in 4.3. The experiment was performed under the following conditions: HEK293 empty cells or HEK293-cynoCD20 target cells (2×105 / well) + VHH-Fc or reference antibody (375 nM, 5× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ 0.5h).

[0321] The FACS binding results are shown in Figure 19. The candidate antibody showed good target cell binding properties on HEK293-cynoCD20 cells, with a better maximum binding (Bmax) than the control antibodies Ofatumumab and Obinutuzumab (Figure 19A); and no obvious binding signal was observed with blank negative cells HEK293 that do not express CD20 (Figure 19B).

[0322] 4.5 Humanization of anti-CD20-VHH

[0323] The original V-n6D11 VHH sequence was humanized using the "best-matching method". The amino acid sequence of the VHH framework region was compared and analyzed using the human germline V gene database to select the best germline sequence. The best-matching human CDR sequence was replaced with the VHH CDR sequence to generate the humanized VHH sequence. The V-n6D11 sequence was analyzed and found to have no post-translational modification (PTM) that needed to be removed. The reverse-translated humanized sequence was synthesized by Genewiz (Shanghai, China). It was then constructed into the pcDNA 3.4 expression vector, with the C-terminus fused with the hIgG1 Fc sequence (SEQ ID NO: 125) to generate humanized VHHs in the form of human IgG1, thereby obtaining the VHH antibody protein. Table 5 below shows the VHH sequences of V-n6D11 and its humanized antibodies.

[0324] Table 5. Anti-CD20 VHH Sequence

[0325] 4.6 Cell Binding Detection of V-n6D11 Humanized Variants

[0326] As described in 4.3, the obtained antibodies V-zn6D11.m1-m9 were subjected to FACS detection. The results are shown in Figure 20: The binding of all humanized antibody variants to target cells Daudi was similar to that of the parent antibody.

[0327] The obtained antibodies V-zn6D11.m1 to m9 were subjected to FACS assay to verify their human-monkey cross-reactivity. As shown in Figures 21A and 21B, the binding of the humanized antibody to the target cells HEK293-cynoCD20 (Figure 21A) was similar to that of the maternal antibody, and neither bound to the negative cells HEK293 (Figure 21B).

[0328] 4.7 Internalization assay of V-n6D11 humanized variant

[0329] The internalization ability of CD20 antibody molecule V-zn6D11.m2 and control antibody Rituximab (derived from Baiying Biotechnology) on different target cells Ramos and Daudi cells was detected using an endocytosis assay.

[0330] Specifically: Target cells were plated at 2×105 cells per well, and the test sample diluted to an appropriate concentration was added. The cells were incubated at 2-8 ℃ for about 0.5 h to allow the test sample to bind to the cells. Centrifuge at 400 xg for 4 min at 4 ℃, remove the supernatant, and wash cells 2-3 times with pre-chilled 200 µl / well FACS buffer to remove excess unbound sample. Resuspend cells in pre-chilled 100 µl / well FACS buffer. Divide cells into two groups and incubate at 4 ℃ and 37 ℃ for 4 h, respectively. Immediately after incubation, add ice-cold FACS buffer to stop the endocytosis experiment. Centrifuge at 400 xg for 4 min at 4 ℃, and wash cells 2-3 times with pre-chilled 200 µl / well FACS buffer. Immediately add 100 µl / well of flow cytometry secondary antibody diluted to the target dilution, resuspend cells, and incubate at 2-8 ℃ in the dark for 30 min to approximately 1 h. After washing cells 2-3 times with 200 µL / well FACS buffer, resuspend the cells in 100 µL / well FACS buffer and perform flow cytometry. Measure the MFI of the cells using a Beckman Coulter flow cytometer. Calculate the internalization level of the antibody bound to the cell surface using the following formula: Internalization rate (%) = 100% - (MFI of the sample incubated at 37°C / MFI of the sample incubated at 4°C) × 100%. As shown in Figure 22, the internalization of V-zn6D11.m2 in both Ramos (Figure 22A) and Daudi (Figure 22B) cell lines was weaker than that of the control antibody Rituximab.

[0331] Based on the data from Example 4, V-n6D11 is a novel anti-CD20 nanobody (VHH). Nanobodies are small antibody molecules composed of a single heavy chain antibody molecule, possessing high specificity and affinity, and exhibiting smaller size, higher stability, and deeper tissue penetration compared to traditional antibodies. This gives them enormous potential in cancer treatment, enabling precise therapy by identifying and targeting specific antigens on the surface of tumor cells, and allowing them to break through the barriers that commonly penetrate tumor cells.Deep tumor tissues that conventional antibodies cannot reach. Nanobodies can be designed to deliver drugs or radioisotopes to tumor cells to kill tumor cells. In addition, nanobodies have a natural advantage in the construction of bispecific and multispecific antibodies and do not have the problem of light and heavy chain mismatch. V-n6D11 has a better binding force to human and monkey CD20 than the control antibodies Ofatumuma b and Obinutuzumab; at the same time, in the internalization experiment of tumor cells, it showed much weaker endocytic activity than the control antibody Retuximab. The above strong binding and weak endocytic properties suggest that V-n6D11 is suitable for splicing TCE bispecific and multispecific antibodies.

[0332] Example 5: CD3xCD20 bispecific antibody splicing and activity verification

[0333] 5.1 CD3xCD20 bispecific antibody splicing

[0334] Using the CD20 VHH clone screened above as CD20 arm, four CD3xCD20 bispecific antibodies were constructed. Bispecific antibodies are bispecific antibodies against human IgG1 subtypes constructed using a knock-in-hole approach. The "CD3 arm" and "CD20 arm" are each linked to a human IgG1 region sequence via hinge regions, where human IgG1 exhibits L234A and L235A mutations, i.e., IgG1 LALA. The CD3xCD20 bispecific antibodies, constructed using 136G6.3, 250E4.1, BMK8, and BMK9 (Teneobio-F2B) as the CD3 arms, are named V-F1F1S1.4, V-F1F1S2.5, V-F1F1S3.6, and V-F1F1S4.7, respectively (see Table 6 below). A schematic diagram of the bispecific antibody structure is shown in Figure 23A.

[0335] Table 6. Composition of CD3xCD20 bispecific antibody

[0336] 5.2 Cell binding assay of CD3xCD20 bispecific antibody CD3 arm and CD20 arm

[0337] Referring to the flow cytometry cell binding assay procedure in Example 2.2, the cell binding activity of the CD3 arm and CD20 arm of the CD3xCD20 bispecific antibody was measured and compared. In the CD3 arm binding assay, Jurkat cells were used as target cells. In the CD20 arm binding assay, Ramos cells (derived from ICON Biotech) were used as target cells. In the assay, the commercially available antibody CD3xCD20 bispecific antibody glofitamab analog (derived from Bio-Tech) was selected as a positive control.

[0338] As shown in Figure 24, the binding of the four bispecific antibody molecules to the CD20 arm is comparable, and CD20 can maintain good activity when matched with different CD3 molecules. However, as shown in Figure 25, the binding of the CD3 arms in monovalent forms exhibits significant differences. The CD3 arm constructed from the BMK8 antibody sequence shows the strongest CD3 binding, followed by...The CD3 arm of the glofitamab analog. The Jurkat monovalent binding of the CD3 arm composed of the 136G6.3 sequence is extremely weak, and the Jurkat monovalent binding of the CD3 arm composed of the control antibody BMK9 sequence is also extremely weak.

[0339] 5.3 PBMC killing and cytokine release of CD3xCD20 bispecific antibody

[0340] The commercially available antibody CD3xCD20 bispecific antibody glofitamab analog (from SAILYBIO) was selected as a positive control to compare the target cell killing activity of the four CD3xCD20 bispecific antibodies. PBMCs from two healthy human donors were selected as effector cell sources (strong immune response donor Donor3, weak immune response donor Donor4, from SAILYBIO). Specifically, the frozen Donor3 and Donor4 PBMCs were thawed and diluted to 4E6 cells / mL with RPMI 1640 complete medium. CD20-positive Ramos cells were selected as target cells, and the cell density was adjusted to 2E5 cells / mL. A 4-fold serial dilution of CD3xCD20 bispecific antibody was prepared. 50 μL of PBMC suspension, 100 μL of target cell suspension, and 50 μL of bispecific antibody dilution were added to a 96-well U-plate, with an effector-to-target ratio of 10:1. The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 48 hours. 50 μL of culture supernatant was collected, and the killing effect on target cells was calculated by detecting LDH release. The usage method for LDH (kit from Dojindo, CAT#CK12) is described in the instruction manual. The release of cytokines such as IFN-γ (kit from Thermo, CAT# 88-7316-88) and IL-6 (kit from Thermo, CAT# 88-7066-86) was detected in the culture supernatant using an ELISA kit.

[0341] As shown in Figures 26 and 27, regardless of whether it is the strong donor Donor3 or the weak donor Donor4, the bispecific antibody V-F1F1S1.4 composed of 136G6.3 exhibits strong killing activity comparable to the glofitamab analogue, and is superior to the bispecific antibody V-F1F1S3.6 with the BMK8 sequence and the bispecific antibody V-F1F1S4.7 with the BMK9 sequence. Figures 28 to 31 show that the bispecific antibody composed of 136G6.3 is comparable to the glofitamab analogue in IL-6 secretion, but shows a lower level in IFNγ secretion, suggesting that the bispecific antibody composed of 136G6.3 has better safety.

[0342] Example 6: CD3xMSLN bispecific antibody splicing and activity verification

[0343] 6.1 CD3xMSLN bispecific antibody splicing

[0344] Three CD3xMSLN bispecific antibodies were constructed using the publicly disclosed mesothelin-binding (MSLN) antibody sequence 2A2 (from patent applications: WO2018209298 (A1), WO2018209304). The bispecific antibodies are bispecific antibodies against human IgG1 subtypes constructed using a knock-in-hole approach. The "CD3 arm" and "MSLN arm" are each linked to a human IgG1 region sequence via a hinge region, where human IgG1 exhibits the L234A / L235A mutation, i.e., IgG1 LALA. The CD3xMSLN bispecific antibodies, with 136G6.3, BMK8, and BMK9 as the CD3 arms, were named V-T1T1S1.8, V-T1T1S3.9, and V-T1T1S4.10, respectively (see Table 7 below). A schematic diagram of the bispecific antibody structure is shown in Figure 23A.

[0345] Table 7. Composition of CD3x MSLN Bispecific Antibody 41 / 51 pages 49 CN 122255272 A

[0346] 6.2 Cell Binding Assay of CD3xMSLN Bispecific Antibody CD3 Arm and MSLN Arm

[0347] Referring to the flow cytometry cell binding assay procedure in Example 2.2, the cell binding activity of the CD3 arm and MSLN arm of the CD3xMSLN bispecific antibody was measured and compared. In the CD3 arm binding assay, Jurkat cells were used as target cells. In the MSLN arm binding assay, AsPC1 cells (derived from Nanjing Kebai) were used as target cells.

[0348] As shown in Figure 32, the binding of the three bispecific antibody molecules to the MSLN arm was comparable, and the MSLN arm maintained good activity when matched with different CD3 molecules. However, as can be seen from the binding of the CD3 arm shown in Figure 33, the binding of the monovalent form of the CD3 arm showed a large difference, and the BMK8 antibody sequence showed the strongest CD3 binding. The binding of Jurkat monovalent to 136G6.3 was extremely weak.

[0349] 6.3 PBMC killing and cytokine release of CD3xMSLN bispecific antibodies

[0350] PBMCs from two healthy human donors (strong immune response donor Donor3 and weak immune response donor Donor4, derived from SAILYBIO) were selected as effector cell sources. The target cell killing activity of the three CD3xMSLN bispecific antibodies was compared. Specifically, MLSN-positive AsPC-1 cells were selected as target cells, and the cell density was adjusted to 1E5 cells / mL. 100 μL of target cell suspension was added to a 96-well V plate and placed in a 37°C 5% CO2 incubator to allow it to adhere to the plate. Donor3 and Donor4 PBMCs were thawed and frozen, and diluted to 2E6 cells / mL with RPMI 1640 complete medium. 4-fold serial dilutions of CD3xMSLN bispecific antibodies were prepared. 50 μL of PBMC suspension and 50 μL of PBMC suspension were added to the plate.μL of double antibiotic dilution buffer was added to a 96-well U-type plate with an effector-to-target ratio of 10:1. The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 48 hours. 50 μL of culture supernatant was aspirated, and the killing effect on target cells was calculated by detecting LDH release. The release of cytokines such as IFN-γ (Thermo, CAT# 88-7316-88), TNF-α (Thermo, CAT# 88-7346-86), and IL-6 (Thermo, CAT# 88-7066-86) in the culture supernatant was detected using an ELISA kit.

[0351] As shown in Figures 34 and 35, the cytotoxic effect of the bispecific antibody V-T1T1S1.8 composed of 136G6.3 was only slightly weaker than that of the bispecific antibody V-T1T1S3.9 composed of the BMK8 sequence, regardless of whether it was the strong donor Donor3 or the weak donor Donor4; it was far superior to the bispecific antibody V-T1T1S4.10 composed of BMK9 (Teneobio-F2B). Figures 36-41 show that the bispecific antibody composed of 136G6.3 induced a lower level of cytokine secretion compared with the bispecific antibody composed of the BMK8 sequence, suggesting that 136G6.3 has better safety.

[0352] Based on the data from Examples 5 and 6, 136G6.3 is a moderately potent CD3 agonist that can be used for the assembly of TCEs against hematologic malignancies as well as against solid tumors. The results of in vitro evaluation show that 136G6.3 has the best potential among similar antibodies. For the hematologic malignancy target CD20, bispecific antibodies composed of CD20 cells showed superior cell killing compared to bispecific antibodies composed of BMK8 cells. Furthermore, compared to the approved 2+1 bispecific antibody Golifitamab, it produced relatively lower cytokine release while achieving similar cell killing activity. For the solid tumor target MSLN, bispecific antibodies composed of 136G6.3 cells were as effective as those composed of BMK8 cells in killing solid tumor cells. However, bispecific antibodies composed of Teneobio-F2B cells failed to mediate effective killing of solid tumor cells due to their low affinity for CD3.

[0353] Example 7: CD3xCD20 Bispecific Antibody Intrinsic and Extracellular Activity Validation Manual 42 / 51 pages 50 CN 122255272 A

[0354] Based on the previous description, the CD3 arm in V-F1F1S1.4 was replaced with the 136G6.3M sequence on the structure shown in Figure 23A to obtain V-F1S5.14; at the same time, a new bispecific antibody structure V-F1S5.15 was constructed. A schematic diagram of V-F1S5.15 is shown in Figure 23B.

[0355] Table 8. Composition of CD3xCD20 Bispecific Antibody

[0356] 7.1Assay of PBMC killing, cytokine release and T cell activation by CD3xCD20 bispecific antibody

[0357] The commercially available CD3xCD20 bispecific antibody glofitamab analog (from SAILYBIO) was selected as a positive control. The target cell killing activity of the two CD3xCD20 bispecific antibodies was compared. PBMCs from healthy human donors (Donor5, from SAILYBIO) were selected as the effector cell source. Specifically, Donor5 PBMCs were thawed and diluted to 4E6 cells / mL with RPMI 1640 complete medium. CD20-positive Ramos and Raji cells were selected as target cells, and the cell density was adjusted to 2E5 cells / mL. 6-fold serial dilutions of CD3xCD20 bispecific antibody were prepared. 50 μL of PBMC suspension, 100 μL of target cell suspension and 50 μL of bispecific antibody dilution were added to a 96-well U plate with an effector-to-target ratio of 10:1. Incubate 96-well plates at 37°C with 5% CO2 for 48 hours. Aspirate 50 μL of culture supernatant and calculate target cell killing by detecting LDH release. Refer to the instruction manual for LDH (kit from Dojindo, CAT#CK12) usage. Detect the release of cytokines such as IFN-γ (kit from Thermo, CAT# 88-7316-88) and IL-6 (kit from Thermo, CAT# 88-7066-86) in the culture supernatant using an ELISA kit. Co-cultured cells are then used to detect CD25 expression on CD4 and CD8 T cells. Specifically: Prepare the staining solution: Human TruStain FcX™ (BioLegend, CAT# 422302, 1:100); anti-human CD4, FITC (Biolegend, CAT# 317408, 1:200); anti-human CD8, APC (eBioscience™, CAT# 17-0087-42, 1:200); anti-human CD25, BV421 (Biolegend, CAT# 302630, 1:200). Wash the cells in the 96-well plate once with FACS buffer, add 100 μL of cell staining solution to each well, and incubate at 4°C for 0.5 hours. Wash once with pre-chilled FACS buffer at 4°C, resuspend the cells in 100 μL of FACS buffer, and detect the cell surface fluorescence intensity using FACS.

[0358] As shown in Figures 42-49, the in vitro killing and cytokine activity of the candidate CD3xCD20 bispecific antibody drug V-F1S5.14 of the present invention are demonstrated.The release and T cell activation were stronger than those of the glofitamab analogue. Although the in vitro killing effect of the candidate molecule V-F1S5.15 was weaker than that of the glofitamab analogue due to the occlusion of the CD3 arm, the weak cytokine release and T cell activation suggest that the molecule has better safety.

[0359] 7.2 Antitumor effect of CD3xCD20 bispecific antibody in subcutaneous transplantation model of hPBMC reconstructed mouse WSU-DLCL2

[0360] This experiment was used to evaluate the efficacy of the test product (including the CD3xCD20 bispecific antibody of the present invention) in subcutaneous transplantation model of hPBMC reconstructed NSG mouse WSU-DLCL2. The control antibodies used were the bispecific antibodies V-F1F1S3.6 and V-F1F1S4.7 constructed in Example 5.

[0361] Model Construction and Grouping

[0362] Model Construction: 7-8 week old female NSG mice (NSG, from Shanghai Southern Model Biotechnology Co., Ltd.) were used. After acclimatization for 1 week, WSU-DLCL2 (CBP60273, Nanjing Kebai Biomedical Technology Co., Ltd., 43 / 51 pages, CN 122255272 A) was injected into the right scapula. The cells were resuspended in 50% matrix gel mixed with PBS (1×107 cells / animal). When the tumor volume grew to 30-60 mm3, hPBMCs (8×106 cells / animal) were injected via the tail vein according to the average tumor volume for immune reconstitution. When the tumor volume grew to 100-200 mm3, the mice were randomly divided into groups (n=6-8). The day of grouping was defined as D0. The test product was administered intravenously once a week for a total of 3 times.

[0363] Dosage volume: Adjusted according to mouse body weight (mouse dosing volume = 10 μL / g × mouse body weight (g))

[0364] Data collection: After the start of administration, the mouse body weight was measured twice a week, the tumor volume was measured twice a week, and the animals were observed twice a day.

[0365] Experimental endpoint: The endpoint was determined based on the tumor volume (1500-2000 cm3) or the animal's condition. At the endpoint, all surviving animals were euthanized and the tumors were collected. The tumors were photographed, weighed, and then processed.

[0366] Endpoint analysis

[0367] At the end of the experiment, the following indicators were analyzed: tumor volume change (TGITV) and body weight change.

[0368] TGITV calculation formula: TGITV = {1-[(Vt-V0) / (Ct-C0)]} Vt: mean tumor volume of mice in the test drug administration group on day t; V0: mean tumor volume of mice in the test drug administration group on day 0; Ct: mean tumor volume of mice in the solvent group on day t; C0: mean tumor volume of mice in the solvent group on day 0.

[0369] Statistical analysis

[0370] Analysis, processing and reporting: the quantitative indicators are expressed as mean ± standard error.(Mean ± SEM / SD) description. Quantitative indicators were analyzed using one-way ANOVA or two-way ANOVA. For inter-group comparisons, the t-test was used, and p < 0.05 was considered statistically significant. Statistical and biological significance were considered in the results analysis.

[0371] As shown in Figure 50, the antitumor efficacy of the bispecific antibodies V-F1S5.14 and V-F1S5.15 composed of CD3 antibodies in this invention is far superior to that of the control bispecific antibody composed of CD3 molecules. The TGITV of each group is shown in Table 9.

[0372] Table 9. Antitumor effects of CD3xCD20 bispecific antibody in subcutaneous transplantation model of hPBMC reconstructed mouse WSU-DLCL2

[0373] Sequence listing overview Specification 44 / 51 pages 52 CN 122255272 A Specification 45 / 51 pages 53 CN 122255272 A Specification 46 / 51 pages 54 CN 122255272 A Specification 47 / 51 pages 55 CN 122255272 A Specification 48 / 51 pages 56 CN 122255272 A Specification 49 / 51 pages 57 CN 122255272 A Specification 50 / 51 pages 58 CN 122255272 A Specification 51 / 51 pages 59 CN 122255272 A Figure 1 Figure 2 Figure 3 Specification Figures 1 / 31 pages 60 CN 122255272 A Figure 4 Figure 5 Appendix 2 / 31 Page 61 CN 122255272 A Figure 6 Figure 7 Appendix 3 / 31 Page 62 CN 122255272 A Figure 8 Figure 9 Appendix 4 / 31 Page 63 CN 122255272 A Figure 10 Figure 11 Appendix 5 / 31 Page 64 CN 122255272 A Figure 12 Figure 13 Appendix 6 / 31 Page 65 CN 122255272 A Figure 14 Figure 15 Appendix 7 / 31 Page 66 CN 122255272 A Figure 16 Figure 17 Appendix 8 / 31 Page 67 CN 122255272 A Figure 18A Appendix 9 / 31 Page 68 CN 122255272 A Figure 18B Appendix 10 / 31 Page 69 CN 122255272 A Figure 19A Figure 19B Instruction Manual Drawings 11 / 31 Page 70 CN 122255272Figure 20 Figure 21A Instruction Manual Drawings, Page 12 / 31, 71 CN 122255272 A Figure 21B Instruction Manual Drawings, Page 13 / 31, 72 CN 122255272 A Figure 22A Instruction Manual Drawings, Page 14 / 31, 73 CN 122255272 A Figure 22B Instruction Manual Drawings, Page 15 / 31, 74 CN 122255272 A Figure 23A Instruction Manual Drawings, Page 16 / 31, 75 CN 122255272 A Figure 23B Figure 24 Instruction Manual Drawings, Page 17 / 31, 76 CN 122255272 A Figure 25 Figure 26 Instruction Manual Drawings, Page 18 / 31, 77 CN 122255272 A Figure 27 Figure 28 Figure 29 Instruction Manual Drawings, Page 19 / 31, 78 CN 122255272 A Figure 30 Figure 31 Figure 32 Figure 33 on page 20 / 31 of the instruction manual (CN 122255272 A); Figure 34 on page 21 / 31 of the instruction manual (CN 122255272 A); Figure 35 on page 22 / 31 of the instruction manual (CN 122255272 A); Figure 37 on page 23 / 31 of the instruction manual (CN 122255272 A); Figure 39 on page 24 / 31 of the instruction manual (CN 122255272 A); Figure 41 on page 25 / 31 of the instruction manual (CN 122255272 A); Figure 42 on page 26 / 31 of the instruction manual (CN 122255272 A); Figure 43 on page 27 / 31 of the instruction manual (CN 122255272 A); Figure 45 on page 28 / 31 of the instruction manual (CN 122255272 A); Figure 47 on page 28 / 31 of the instruction manual (CN 122255272 A). 122255272 A Figure 47 Figure 48 Appendix to the Specification Page 29 / 31 88 CN 122255272 A Figure 49 Appendix to the Specification Page 30 / 31 89 CN 122255272 A Figure 50 Appendix to the Specification Page 31 / 31 90 CN 122255272 A Abstract Provided are a T-cell activating anti-CD3 antibody and a multispecific antibody comprisingsame, in particular a T-cell engager. Also provided are a composition comprising the antibody or multispecific antibody, and a therapeutic application thereof.

Claims

1. An anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: - Three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained in the heavy chain variable region (VH) sequence selected from SEQ ID NOs: 31 and 67-69, and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained in the light chain variable region (VL) sequence selected from SEQ ID NOs: 32 and 70-71, and optionally containing the following amino acid substitutions: amino acid substitutions selected from S27eT, S27eV, and S27eI in LCDR1, and / or amino acid substitutions selected from V51I, S52Q, V55R, and S56I in LCDR2, wherein the variable region amino acid residues are numbered according to the Kabat numbering system; - HCDR1-3 contained in the VH sequence of SEQ ID NO: 7 and LCDR1-3 contained in the VL sequence of SEQ ID NO: 8; - HCDR1-3 contained in the VH sequence of SEQ ID NO: 15 and LCDR1-3 contained in the VL sequence of SEQ ID NO: 16; - HCDR1-3 contained in the VH sequence of SEQ ID NO: 23 and LCDR1-3 contained in the VL sequence of SEQ ID NO: 24; - HCDR1-3 contained in the VH sequence of SEQ ID NO: 39, and LCDR1-3 contained in the VL sequence of SEQ ID NO: 40; or - HCDR1-3 contained in the VH sequence of SEQ ID NO: 47 and LCDR1-3 contained in the VL sequence of SEQ ID NO: 48; Preferably, the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3 contained in the following VH and VL sequence pairs: (a) The VH sequence of SEQ ID NO: 31 and the VL sequence of one of SEQ ID NOs: 32 and 72-74; (b) VH sequence of one of SEQ ID NOs: 67-69 and VL sequence of one of SEQ ID NOs: 70-71; (c) The VH sequence of SEQ ID NO: 68 and the VL sequence of one of SEQ ID NOs: 78-82; or (d) The VH sequence of SEQ ID NO: 68 and the VL sequence of SEQ ID NO:

87.

2. The anti-CD3 antibody or its antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3, wherein: - HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs: 25, 26 and 27, and LCDR1 contains or consists of the amino acid sequences selected from SEQ ID NOs: 28 and 75-77, LCDR2 contains or consists of the amino acid sequences selected from SEQ ID NOs: 29 and 83-86, and LCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 30; - HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs: 1, 2 and 3, and LCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs: 4, 5 and 6; - HCDR1-3 comprises or consists of the amino acid sequences of SEQ ID NOs: 9, 10 and 11, respectively, and LCDR1-3 comprises or consists of the amino acid sequences of SEQ ID NOs: 12, 13 and 14, respectively; - HCDR1-3 comprises or consists of the amino acid sequences of SEQ ID NOs: 17, 18 and 19, respectively, and LCDR1-3 comprises or consists of the amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively; - HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs: 33, 34 and 35, and LCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs: 36, 37 and 38; or - HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs: 41, 42 and 43, and LCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs: 44, 45 and 46. Preferably, the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3, wherein: (i) HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:25, 26 and 27, and LCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:28, 29 and 30; (ii) HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:25, 26 and 27, LCDR1 contains or consists of the amino acid sequences of SEQ ID NOs:75, 76 or 77, and LCDR2-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:29 and 30. (iii) HCDR1-3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:25, 26 and 27, and LCDR1 and LCDR3 respectively contain or consist of the amino acid sequences of SEQ ID NOs:28 and 30, and LCDR2 contains or consists of the amino acid sequences of SEQ ID NOs:83, 84, 85 or 86; or (iv) HCDR1-3 contain or consist of the amino acid sequences of SEQ ID NOs:25, 26 and 27 respectively, and LCDR1-3 contain or consist of the amino acid sequences of SEQ ID NOs:75, 86 and 30 respectively.

3. The anti-CD3 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) The heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 31, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed thereof; and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 32 and 72-74, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed thereof; (b) The heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 67-69, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed thereof; and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 70-71 and SEQ ID NOs: 78-82 and 87, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed thereof; (c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the thereof; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the thereof; (d) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed thereof; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed thereof; (e) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 23, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed thereof; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed thereof; (f) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 39, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 40, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof; or (g) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 47, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed thereof; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed thereof.

4. The anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-3, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) The heavy chain variable region comprises, or is composed of, the amino acid sequence of SEQ ID NO: 31; and the light chain variable region comprises, or is composed of, the amino acid sequences selected from SEQ ID NOs: 32 and 72-74; (b) The heavy chain variable region comprises, or is composed of, an amino acid sequence selected from SEQ ID NOs: 67-69; and the light chain variable region comprises, or is composed of, an amino acid sequence selected from SEQ ID NOs: 70-71, 78-82 and 87; (c) The heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 7; and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 8; (d) The heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 15; and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO:

16. (e) The heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 23; and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 24; (f) The heavy chain variable region comprises, or is composed of, the amino acid sequence of SEQ ID NO: 39; and the light chain variable region comprises, or is composed of, the amino acid sequence of SEQ ID NO: 40; or (g) The heavy chain variable region comprises, or is composed of, the amino acid sequence of SEQ ID NO: 47; and the light chain variable region comprises, or is composed of, the amino acid sequence of SEQ ID NO:

48. Preferably, the antibody or antigen-binding fragment comprises: (i) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 or thereof, and a light chain variable region comprising one of the amino acid sequences of SEQ ID NO: 32 and 72-74; or (ii) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 68 or thereof, and a light chain variable region comprising the amino acid sequence selected from SEQ ID NOs: 70-71, 78-82 and 87 or thereof; or (iii) A heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 67 or 69 or thereof, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 70 or 71 or thereof.

5. The anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-4, wherein said antibody or antigen-binding fragment has one or more of the following characteristics: (a) Specific binding to human CD3 antigen, preferably, wherein the binding affinity K is measured by in vitro surface plasmon resonance (SPR) binding assay. D The value is approximately 10x10 -7 M to approximately 1x10 -8 M, preferably approximately 5x10 -7 M to approximately 5x10 -8 M; (b) Possesses immunoreactivity with human and monkey CD3 antigens, preferably wherein the binding affinity K for human and monkey CD3 antigens is measured, as determined by in vitro surface plasmon resonance (SPR) binding assay. D The values ​​should not differ by more than approximately 5 times, preferably not by more than approximately 3 times; (c) It exhibits essentially no nonspecific binding to CD3 antigen-negative cells; and (d) Activate CD4+ and CD8+ T cells.

6. The anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-5, wherein: - The antibody or its antigen-binding fragment is murine, chimeric, or humanized; and / or - The antibody or its antigen-binding fragment is selected from full-length antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv and scFab), crossFab, F(ab')2, or linear antibodies.

7. The anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-6, wherein the antibody comprises an immunoglobulin Fc region, and preferably the Fc region is an IgG isotype, such as the human IgG1 or IgG4 isotype Fc region.

8. A multispecific antibody comprising at least two different antigen-binding specificities, wherein the first binding specificity is provided by a first antigen-binding domain that specifically binds to CD3, and wherein the CD3 antigen-binding domain comprises or is composed of an anti-CD3 antibody according to any one of claims 1-7 or an antigen-binding fragment thereof.

9. The multispecific antibody of claim 8, wherein, in addition to CD3 binding specificity, the multispecific antibody further comprises at least one, two, three, four or five different antigen binding specificities, optionally the antigens being independently selected from tumor-associated antigens (TAAs), other immune-associated molecules and co-stimulatory molecules.

10. The multispecific antibody of claim 9, wherein the multispecific antibody has binding specificity to CD3 and at least one (e.g., 1-3 different) TAAs, and optionally also has binding specificity to at least one (e.g., 1) co-stimulatory molecule.

11. The multispecific antibody according to any one of claims 9-10, wherein the TAA is a solid tumor cell surface antigen or a hematologic tumor cell surface antigen, and is optionally selected from CD19, BCMA, TSHR, CD171, CS-1, CLL-1, GD3, TnAg, FLT3, CD38, CD123, CD44v6, B7H3, B7H4, KIT, IL-13Ra2, IL-11Ra, PSCA, PSMA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, sLe, GM3, TGS5, HMWMAA, GD2, FOLR1, FOLR2, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY-BR-1,UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TAARP, WT1, ETV6-AML, SPA17,XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, FOSL1, hTERT, ML-IAP, ERG, NA17, PAX3, AR,Cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4,SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2,LY75, GPC3, FCRL5, IGLL1, CD20, CD30, HER2, ROR1, FLT3, TAAG72, CD22, CD33,GD2, gp100Tn, FAP, TYR, EPCAM, CEA, IGF-1R, EphB2, MSLN, Claudin18.2, CDH17,CD32b, EGFRvIII, GPNMB, GPR64, HER3, LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C and TACSTD2.

12. The multispecific antibody according to any one of claims 9-11, wherein the co-stimulatory molecule is selected from CD28, OX40, CD137, CD8, ICOS, CD27, GITR, CD2, IL-2RP and MyD88 / CD40.

13. The multispecific antibody according to any one of claims 8-12, wherein the multispecific antibody comprises a second antigen-binding domain that specifically binds to a tumor-associated antigen (TAA), preferably, the TAA is a solid tumor cell surface antigen selected from, for example, MSLN, CEA, EpCAM, HER2, PSMA, EGFR, Claudin18.2 and CDH17, or the TAA is a hematologic tumor cell surface antigen selected from, for example, CD19, CD20, CD79b, CD33, BCMA and GPRC5D.

14. The multispecific antibody according to any one of claims 8-13, wherein the antibody comprises a CD3 antigen-binding domain and a TAA antigen-binding domain, and has one or more of the following features: (a) The valence ratio of the TAA antigen-binding domain to the CD3 antigen-binding domain is 1:1 or 2:1, preferably 2:1; (b) The antibody is a bivalent, trivalent, or quadrivalent bispecific antibody, preferably a trivalent bispecific antibody; and (c) The antibody is a T-cell adaptor (TCE).

15. The multispecific antibody according to any one of claims 8-14, wherein the antibody further comprises an Fc dimer having first and second immunoglobulin Fc regions, and optionally wherein: (i) The first Fc region and the second Fc region contain amino acid mutations that promote the formation of the Fc dimer; (ii) The first Fc region is a human IgG Fc region containing T336W and S354C, and the second Fc region is a human IgG Fc region containing T366S, L368A, Y407V, and Y349C. (iii) The first Fc region and the second Fc region respectively contain mutations that reduce or eliminate the binding interaction between the Fc region and FcγR, for example, L234A and L235A mutations. (iv) The first Fc region and the second Fc region are IgG1 or IgG4 isotypes; and / or (v) The first and second Fc regions respectively contain the amino acid sequences of SEQ ID NO:108 and SEQ ID NO:107, or amino acid sequences that are at least 95%, 96%, 98% or 99% identical to them.

16. The multispecific antibody according to any one of claims 8-15, wherein the antibody comprises - A first structural portion comprising the following components from the N-terminus to the C-terminus: a first antigen-binding domain, optionally a linker, and a first immunoglobulin Fc region; - A second structural portion from the N-terminus to the C-terminus comprising the following components: a second antigen-binding domain, an optional linker, and a second immunoglobulin Fc region; and -Optionally, a third antigen-binding domain is connected to the N-terminus or C-terminus of the first or second structural portion via a linker; The Fc regions of the first and second immunoglobulins dimerize to form Fc dimers. Preferably, the linker is 5-25 amino acids in length, or contains an amino acid sequence of SEQ ID NO: 106, 136 or 112.

17. The multispecific antibody according to any one of claims 8-16, wherein: - The first antigen-binding domain binds to CD3 and includes or is composed of Fab, scFab, or scFv domains; and / or - The second and third antigen-binding domains bind to the TAA and contain or consist of Fab, scFab, scFv, or VHH domains. Preferably, the first antigen-binding domain is a Fab domain that binds to CD3, and the second and third antigen-binding domains are VHH domains that bind to TAA.

18. The multispecific antibody according to any one of claims 8-17, wherein the antibody comprises a TAA antigen-binding domain, and The TAA antigen-binding domain specifically binds to CD20. Preferably, the CD20 antigen-binding domain comprises or is composed of a VHH domain. More preferably, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of one of the amino acid sequences in SEQ ID NO: 102 and 115-124; More preferably, the CDR1, CDR2 and CDR3 sequences respectively contain or are composed of the amino acid sequences of SEQ ID NOs:103, 104 and 105, or respectively contain or are composed of the amino acid sequences of SEQ ID NOs:137, 104 and 105; More preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NO: 102 and 115-124, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed of such an amino acid sequence. Alternatively, the TAA antigen-binding domain may specifically bind to MSLN; preferably, the MSLN antigen-binding domain comprises or is composed of a VHH domain. More preferably, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of the amino acid sequence of SEQ ID NO: 97; More preferably, the CDR1, CDR2 and CDR3 sequences respectively contain or consist of the amino acid sequences of SEQ ID NOs:98, 99 and 100; More preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 97, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the above.

19. The multispecific antibody according to any one of claims 8-18, wherein the antibody is a bispecific antibody against CD3 and CD20, and comprises: (i) First, second, and third polypeptide chains comprising SEQ ID NOs: 88, 89, and 101, respectively, or having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them. (ii) First, second, and third polypeptide chains comprising SEQ ID NOs: 90, 91, and 101, respectively, or having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them. (iii) First, second, and third polypeptide chains comprising SEQ ID NOs: 130, 131, and 132, respectively, or having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them, or (iv) First, second, and third polypeptide chains comprising SEQ ID NOs: 133, 134, and 135, respectively, or having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them. Or the antibody described therein is a bispecific antibody against CD3 and MSLN, and contains: (v) First, second, and third polypeptide chains comprising SEQ ID NOs: 88, 89, and 96, respectively, or having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them, or (vi) The first, second, and third polypeptide chains respectively containing SEQ ID NOs: 90, 91, and 96, or having an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with them. Preferably, the first, second, and third polypeptide chains contain amino acid sequences of SEQ ID NOs: 88, 89, and 96, respectively, or contain amino acid sequences of SEQ ID NOs: 88, 89, and 101, respectively.

20. An anti-CD20 antibody or an antigen-binding fragment thereof, comprising a VHH domain that specifically binds to CD20, and The VHH domain contains the CDR1, CDR2, and CDR3 sequences of one of the amino acid sequences in SEQ ID NO: 102, 115-124; Preferably, the CDR1, CDR2 and CDR3 sequences respectively contain or consist of the amino acid sequences of SEQ ID NOs:103, 104 and 105, or respectively contain or consist of the amino acid sequences of SEQ ID NOs:137, 104 and 105; More preferably, the VHH domain comprises an amino acid sequence of one of SEQ ID NO: 102, 115-124, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids. Most preferably, the VHH domain comprises, or is composed of, the amino acid sequence of SEQ ID NO:

102.

21. A polynucleotide encoding the anti-CD3 antibody or its antigen-binding fragment as claimed in any one of claims 1-7, the multispecific antibody as claimed in any one of claims 8-19, or the anti-CD20 antibody or its antigen-binding fragment as claimed in claim 20.

22. A vector, preferably an expression vector, comprising the polynucleotide of claim 21.

23. A host cell comprising the polynucleotide of claim 21 or the vector of claim 22, wherein the host cell is optionally a mammalian cell.

24. A method for producing the anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-7, the multispecific antibody according to any one of claims 8-19, or the anti-CD20 antibody or its antigen-binding fragment according to claim 20, the method comprising: Host cells containing polynucleotides encoding the polypeptide chains are cultured under conditions suitable for producing the antibody or its antigen-binding fragments or polypeptide chains of the multispecific antibody.

25. An antigen-binding molecule comprising, for example, an immunoconjugate or an immunofusion compound, the anti-CD3 antibody of any one of claims 1-7 or its antigen-binding fragment, the multispecific antibody of any one of claims 8-19 or the anti-CD20 antibody of claim 20 or its antigen-binding fragment.

26. A pharmaceutical composition comprising the anti-CD3 antibody of any one of claims 1-7 or an antigen-binding fragment thereof, the multispecific antibody of any one of claims 8-19, the anti-CD20 antibody of claim 20 or an antigen-binding fragment thereof, or the antigen-binding molecule of claim 25, and a pharmaceutically acceptable carrier.

27. The use of the anti-CD3 antibody or its antigen-binding fragment according to any one of claims 1-7, the multispecific antibody according to any one of claims 8-19, the anti-CD20 antibody according to claim 20 or its antigen-binding fragment, or the antigen-binding molecule according to claim 25 as a drug or for the preparation of a drug.

28. The use of claim 27, wherein the drug is used to treat and / or prevent cancer in an individual.

29. A method of treating or preventing cancer, comprising administering to an individual in need an effective amount of the anti-CD3 antibody of any one of claims 1-7 or an antigen-binding fragment thereof, the multispecific antibody of any one of claims 8-19, the anti-CD20 antibody of claim 20 or an antigen-binding fragment thereof, the antigen-binding molecule of claim 25, or the pharmaceutical composition of claim 26.

30. The use or method of claim 28 or 29, wherein the cancer is a solid tumor or hematologic malignancy, preferably wherein: The solid tumor is an MSLN-positive solid tumor, selected from, for example, mesothelioma (such as malignant mesothelioma), pancreatic cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), and colorectal cancer; The hematologic malignancy is a CD20-positive hematologic malignancy, selected from, for example, B-cell lymphomas (such as DLBCL and LBCL).

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