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WO2025247196A1 - Polypeptide de fusion multifonctionnel - Google Patents

Polypeptide de fusion multifonctionnel

Info

Publication number
WO2025247196A1
WO2025247196A1 PCT/CN2025/097370 CN2025097370W WO2025247196A1 WO 2025247196 A1 WO2025247196 A1 WO 2025247196A1 CN 2025097370 W CN2025097370 W CN 2025097370W WO 2025247196 A1 WO2025247196 A1 WO 2025247196A1
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WO
WIPO (PCT)
Prior art keywords
domain
polypeptide
terminus
seq
heavy chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/097370
Other languages
English (en)
Chinese (zh)
Inventor
杨佳琪
张贺琳
王友平
邱庆崇
曹泽亚
任艳琴
刘志红
周晶晶
张宛麟
王晓丽
倪楠
朱坤
许国龙
何南海
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Adlai Nortye Biopharma Co Ltd
Adlai Nortye Pte Ltd
Original Assignee
Adlai Nortye Biopharma Co Ltd
Adlai Nortye Pte Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Adlai Nortye Biopharma Co Ltd, Adlai Nortye Pte Ltd filed Critical Adlai Nortye Biopharma Co Ltd
Publication of WO2025247196A1 publication Critical patent/WO2025247196A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Definitions

  • This invention relates to the field of recombinant antibodies, and more specifically, to a multifunctional specific antibody having a conditionally activated recombinant anti-CD3, anti-tumor-associated antigen (TAA) binding site and a CD86/CD80 fusion protein.
  • TAA anti-tumor-associated antigen
  • T lymphocyte activation in vivo requires two signals: the first signal is provided by the interaction between the MHC/antigen peptide complex on APCs (antigen-presenting cells) and the TCR/CD3 complex on T lymphocytes; the second signal, the co-stimulatory signal, is provided by the interaction between the co-stimulatory receptors on APCs and the co-stimulatory molecules on T lymphocytes.
  • the first signal is provided by the interaction between the MHC/antigen peptide complex on APCs (antigen-presenting cells) and the TCR/CD3 complex on T lymphocytes
  • the second signal the co-stimulatory signal
  • T lymphocytes are only fully activated upon the presence of the first signal (Baxter & Hodgkin, 2002; Bernard et al., 2002).
  • CTLs cytotoxic T lymphocytes
  • THs T helper cells
  • CTLs are the main effector cells in cell-mediated immune responses, while THs indirectly participate in cell-mediated immune responses by secreting cytokines (such as interleukin-2 (IL-2)). Since tumor immunity is mainly cell-mediated, designing anti-tumor drugs that specifically activate CTLs is of great significance in tumor immunotherapy (Foss, 2002).
  • IL-2 interleukin-2
  • CD3 and TAA bispecific antibodies are also known as Bispecific T-cell connectors (BiTEs).
  • the two arms of the CD3 and TAA bispecific antibody target the T cell surface antigen CD3 and the tumor cell surface antigen TAA, respectively, thereby bridging the gap between tumor cells and effector T cells. This bypasses the classic activation pathway where T cells must form a complex with the MHC antigen peptide and the TCR, and bind to co-stimulatory molecules before activation.
  • Activated T cells release granzymes and perforin, effectively killing tumor cells.
  • BsAbs recombinant CD3 and TAA bispecific antibodies
  • CD3xEpCAM Catumaxomab
  • CD3xCD19 Blinatumomab
  • CD3xIMCgp100 Tebentafusp
  • TAAs include, but are not limited to, CD19, CD20, CD38, DLL3, EGFR, FLT3, STEAP1, MUC16, MUC17, PD1, PDL1, GPRC5D, BCMA, PSCA, CTLA-4, VEGF, PRAME, 5T4, and GCC (Wei et al., 2022).
  • CD3 and TAA bispecific antibodies have been shown to specifically activate T lymphocytes via CD3 and significantly induce tumor-specific cell lysis, but there are still some gaps compared to CAR-T cell therapy.
  • T lymphocytes Since they do not provide co-stimulatory signals, most of them cannot fully activate T lymphocytes and may lead to activation-induced cell death (AICD) of T lymphocytes (Daniel et al., 1998) and reduce their tumor-specific cell lysis (Daniel et al., 1998).
  • AICD activation-induced cell death
  • TCEs bispecific T-cell connectors
  • PMAxCD3-TRACTr Janux's protease-activated TCE
  • PSMAxCD3-TRACTr uses a special peptide to mask the binding of CD3 antibodies and has demonstrated good efficacy and safety in clinical trials.
  • many other TCEs with CD3 antibody-masking capabilities have limited efficacy. This may be related to inappropriate CD3 antibody-masking activity, relatively simple enzymatic activation conditions, and weaker TCE function.
  • this invention proposes the development of a multifunctional fusion peptide that provides co-stimulatory signals and conditional activation. Based on a CD3 and TAA bispecific antibody, it provides dual activation signals for CTLs, enabling simultaneous interaction with multiple sites on both target and effector cells, and inducing more effective tumor-specific cell lysis.
  • CD28 is one such co-stimulatory signal.
  • This multifunctional fusion peptide possessing three binding specificities (targeting TAA, CD3, and CD28), provides dual activation signals within a single molecule. By binding to the CD3 and CD28 effector sites on T lymphocytes, it recruits and stimulates effector T cells through dual signaling pathway activation, enhancing their ability to recognize and eliminate tumor cells. Simultaneously, the safety of the multifunctional fusion peptide is improved by masking the CD3 binding site.
  • the present invention provides a fusion polypeptide comprising a first domain, a second domain and a third domain, wherein the first domain is capable of binding CD3, the second domain is capable of binding tumor-associated antigen (TAA), and the third domain is capable of binding CD28 and/or CTLA4.
  • TAA tumor-associated antigen
  • the first domain comprises an antibody or an antigen-binding fragment thereof.
  • the first domain antibody is selected from the group consisting of recombinant antibodies, single-domain antibodies, heavy chain antibodies, chimeric antibodies, and bispecific antibodies.
  • the first domain antigen-binding fragment is selected from one or more fragments from the group consisting of: Fab, Fab’, Fv fragment, F(ab’)2, F(ab)2, scFv, di-scFv, VHH, and dAb.
  • the first domain comprises an antibody, its antigen-binding fragment, or a variant thereof selected from the group consisting of: antibody OKT3, antibody UCHT1, antibody SP34, Blinatumamab, Tebentafusp, Mosunetuzumab, and Teclistamab.
  • the first domain comprises an antibody SP34 mutant or its antigen-binding site.
  • the first domain may include an antibody SP34 mutant or its antigen-binding site, wherein the mutation occurs in the CDR region.
  • the mutation occurs in the HCDR region, and the mutation comprises one or more amino acid site mutations selected from the group shown in SEQ ID NO:22 of the antibody SP34 heavy chain variable region: T31D, R50K, N100D, N97S, F100fH, S100aA, V100cT.
  • the mutation occurs in the LCDR region and comprises one or more amino acid site mutations selected from the group shown in SEQ ID NO:12 of antibody SP34 light chain variable region: N94Q.
  • the first domain comprises a single-chain immunoglobulin domain.
  • the first domain comprises a single-chain immunoglobulin IgG antibody.
  • the single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the first domain comprises a single-chain immunoglobulin Fc domain.
  • the first domain comprises the Fc domain of a single-chain immunoglobulin IgG antibody.
  • the Fc domain of the single-chain immunoglobulin IgG antibody includes human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, mouse IgG1 Fc domain, mouse IgG2a Fc domain, mouse IgG2b Fc domain, or mouse IgG3 Fc domain.
  • the first domain single-chain immunoglobulin Fc fragment contains CH2 and/or CH3 sequences, wherein the first domain CH2 is located between the variable region and the CH3 domain; preferably, the Fc fragment contains any one of the amino acid sequences selected from the group shown: SEQ ID NO: 3, SEQ ID NO: 4.
  • the first domain includes a heavy chain variable region, wherein the HCDR1, HCDR2, and/or HCDR3 amino acid sequences or variant sequences thereof of the heavy chain variable region are selected from any one of the following amino acid sequences: SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35. SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 30 7.
  • the first domain optionally further includes a light chain variable region, and the LCDR1, LCDR2 and/or LCDR3 amino acid sequences or variant sequences thereof of the light chain variable region of the first domain contain any one of the amino acid sequences selected from the following group: SEQ ID NO O: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ
  • the first domain comprises a heavy chain variable region VH of the antibody heavy chain, said VH comprising an amino acid sequence selected from any of the following groups: SEQ ID NO: 22, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 301, SEQ ID NO: 306, SEQ ID NO: 311, SEQ ID NO: 316, SEQ ID NO: 326, SEQ ID NO: 336.
  • the first domain comprises an antibody heavy chain comprising any amino acid sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 300, SEQ ID NO: 305, SEQ ID NO: 310, SEQ ID NO: 315, SEQ ID NO: 320, SEQ ID NO: 330.
  • the first domain includes a light chain variable region VL of the antibody light chain, said VL comprising any amino acid sequence selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 38, SEQ ID NO: 322, SEQ ID NO: 332.
  • the first domain comprises an antibody light chain, the antibody light chain comprising any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 16.
  • the first domain may include HCDR1, HCDR2, and HCDR3 of the antibody heavy chain, wherein:
  • the HCDR1, HCDR2, and HCDR3 may each contain the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25; or may each contain the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; or may each contain the amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35; or may each contain the amino acid sequences shown in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45; or may each contain the amino acid sequences shown in SEQ ID NO: 302, SEQ ID NO: 303, and SEQ ID NO: 304.
  • the sequence may contain, or may contain, the amino acid sequences shown in SEQ ID NO: 307, SEQ ID NO: 308, and SEQ ID NO: 309 respectively; or may contain, the amino acid sequences shown in SEQ ID NO: 312, SEQ ID NO: 313, and SEQ ID NO: 314 respectively; or may contain, the amino acid sequences shown in SEQ ID NO: 317, SEQ ID NO: 318, and SEQ ID NO: 319 respectively; or may contain, the amino acid sequences shown in SEQ ID NO: 327, SEQ ID NO: 328, and SEQ ID NO: 329 respectively; or may contain, the amino acid sequences shown in SEQ ID NO: 337, SEQ ID NO: 338, and SEQ ID NO: 339 respectively.
  • the first domain may comprise LCDR1, LCDR2, and LCDR3 of the antibody light chain, wherein:
  • the LCDR1, LCDR2, and LCDR3 may each contain the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; or may each contain the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20; or may each contain the amino acid sequences shown in SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41; or may each contain the amino acid sequences shown in SEQ ID NO: 323, SEQ ID NO: 324, and SEQ ID NO: 325; or may each contain the amino acid sequences shown in SEQ ID NO: 333, SEQ ID NO: 334, and SEQ ID NO: 335.
  • the first domain does not contain light chains.
  • the first domain does not include the CH1 domain.
  • the second domain is capable of binding tumor-associated antigens (TAAs).
  • TAAs tumor-associated antigens
  • the second domain is capable of binding to proteins or tumor antigens that are overexpressed on tumor cells relative to the corresponding non-tumor cells.
  • the second domain comprises an antibody or an antigen-binding fragment thereof.
  • the second domain antibody is selected from the group consisting of recombinant antibodies, single-domain antibodies, heavy chain antibodies, chimeric antibodies, and bispecific antibodies.
  • the second domain antigen-binding fragment is selected from one or more fragments from the group consisting of: Fab, Fab’, Fv fragment, F(ab’)2, F(ab)2, scFv, di-scFv, VHH, and dAb.
  • the second domain can bind to one or more of the targets shown in the following group: PD-L1, PD-L2, VEGF, VEGFR, FGFR, HER2, HGFR, PIP-LAR, CD44, CD147, TfR, CDCP1, ⁇ 6 ⁇ 4, ⁇ 6 ⁇ 3, Trop2, HHLA2, GCC, 5T4, EpCAM, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, DLL3, B7H3, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, PTK7, EphA2, NKG2A, Nkp36, Tim3, CD20, Her2.
  • the second domain comprises a single-chain immunoglobulin domain.
  • the second domain comprises a single-chain immunoglobulin IgG antibody.
  • the second domain single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the second domain comprises a single-chain immunoglobulin Fc domain.
  • the second domain comprises the Fc domain of a single-chain immunoglobulin IgG antibody.
  • the Fc domain of the single-chain immunoglobulin IgG antibody includes human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, mouse IgG1 Fc domain, mouse IgG2a Fc domain, mouse IgG2b Fc domain, or mouse IgG3 Fc domain.
  • the second domain single-stranded immunoglobulin Fc fragment comprises CH2 and/or CH3 sequences, with the second domain CH2 located between the variable region and the CH3 domain; preferably, the Fc fragment comprises any amino acid sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4.
  • the HCDR1, HCDR2, and/or HCDR3 amino acid sequences or variant sequences thereof in the heavy chain variable region comprise any one of the amino acid sequences selected from the group shown below: SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69.
  • SEQ ID NO: 70 SEQ ID NO: 71, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 100 2.
  • sequence or a variant thereof comprising any amino acid sequence selected from the group shown below: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 1015, SEQ ID NO: 1016, SEQ ID NO: 1017, SEQ ID NO: 2003, SEQ ID NO: 2004, SEQ ID NO: 2005.
  • the second structure includes a heavy chain variable region VH of the antibody heavy chain, the antibody heavy chain variable region VH comprising any amino acid sequence selected from the group consisting of: SEQ ID NO: 47, SEQ ID NO: 58, SEQ ID NO: 63, SEQ ID NO: 68, SEQ ID NO: 73, SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 93, SEQ ID NO: 1001, SEQ ID NO: 1010, SEQ ID NO: 2006, SEQ ID NO: 2011.
  • the second structure comprises an antibody heavy chain comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 57, SEQ ID NO: 62, SEQ ID NO: 67, SEQ ID NO: 72, SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 92, SEQ ID NO: 1000, SEQ ID NO: 1005, SEQ ID NO: 1018, SEQ ID NO: 2000, SEQ ID NO: 2010.
  • the second structure includes a light chain variable region VL of an antibody light chain, the antibody light chain comprising any amino acid sequence selected from the group consisting of: SEQ ID NO: 53, SEQ ID NO: 88, SEQ ID NO: 1014, SEQ ID NO: 2002.
  • the second domain comprises an antibody light chain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 87.
  • the second domain may comprise HCDR1, HCDR2, HCDR3 of the antibody heavy chain and LCDR1, LCDR2, and LCDR3 of the antibody light chain, wherein HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, respectively, and LCDR1, LCDR2, and LCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively.
  • the second domain may include a heavy chain variable region VH of the antibody heavy chain, the antibody heavy chain VH may include an amino acid sequence as shown in SEQ ID NO: 58, and the second domain may include a light chain variable region VL of the antibody light chain, the antibody light chain VL may include an amino acid sequence as shown in SEQ ID NO: 53.
  • the second domain may comprise an antibody heavy chain and/or an antibody light chain, wherein the antibody heavy chain may comprise an amino acid sequence as shown in SEQ ID NO: 57, and the antibody light chain may comprise an amino acid sequence as shown in SEQ ID NO: 52.
  • the second domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively.
  • the second domain may comprise the heavy chain variable region VHH of a single-domain antibody, wherein the VHH may comprise an amino acid sequence as shown in SEQ ID NO: 47.
  • the second domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 46.
  • the second domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively.
  • the second domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 63.
  • the second domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 62.
  • the second domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively.
  • the second domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 68.
  • the second domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 67.
  • the second domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, respectively.
  • the second domain may comprise the heavy chain variable region VHH of a single-domain antibody, wherein the VHH may comprise an amino acid sequence as shown in SEQ ID NO: 73.
  • the second domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 72.
  • the second domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively.
  • the second domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 78.
  • the second domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 77.
  • the second domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86, respectively.
  • the second domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 83.
  • the second domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 82.
  • the second domain may comprise HCDR1, HCDR2, HCDR3 of the antibody heavy chain and LCDR1, LCDR2, and LCDR3 of the antibody light chain, wherein HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively, and LCDR1, LCDR2, and LCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, respectively.
  • the second domain may include a heavy chain variable region VH of the antibody heavy chain, the antibody heavy chain VH may include an amino acid sequence as shown in SEQ ID NO: 93, and the second domain may include a light chain variable region VL of the antibody light chain, the antibody light chain VL may include an amino acid sequence as shown in SEQ ID NO: 88.
  • the second domain may comprise an antibody heavy chain and/or an antibody light chain, wherein the antibody heavy chain may comprise an amino acid sequence as shown in SEQ ID NO: 92, and the antibody light chain may comprise an amino acid sequence as shown in SEQ ID NO: 87.
  • the second domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may each comprise an amino acid sequence as shown in SEQ ID NO: 1002, SEQ ID NO: 1003, and SEQ ID NO: 1004.
  • the second domain may comprise the heavy chain variable region VHH of a single-domain antibody, wherein the VHH may comprise an amino acid sequence as shown in SEQ ID NO: 1001.
  • the second domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 1000 or SEQ ID NO: 1005.
  • the second domain may comprise the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 of the scFv antibody, wherein HCDR1, HCDR2, and HCDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 1011, SEQ ID NO: 1012, and SEQ ID NO: 1013, and LDR1, LCDR2, and LCDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 1015, SEQ ID NO: 1016, and SEQ ID NO: 1017.
  • the second domain may comprise a heavy chain variable region VH and a light chain variable region VL of the scFv antibody, wherein the VH may comprise an amino acid sequence as shown in SEQ ID NO: 1010, and the VL may comprise an amino acid sequence as shown in SEQ ID NO: 1014.
  • the second domain may contain an scFv antibody, which may contain an amino acid sequence as shown in SEQ ID NO: 1009 or SEQ ID NO: 1018.
  • the second domain may comprise the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 of the scFv antibody, wherein HCDR1, HCDR2, and HCDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 2007, SEQ ID NO: 2008, and SEQ ID NO: 2009, and LDR1, LCDR2, and LCDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 2003, SEQ ID NO: 2004, and SEQ ID NO: 2005.
  • the second domain may comprise a heavy chain variable region VH and a light chain variable region VL of the scFv antibody, wherein the VH may comprise an amino acid sequence as shown in SEQ ID NO: 2006, and the VL may comprise an amino acid sequence as shown in SEQ ID NO: 2002.
  • the second domain may contain an scFv antibody, which may contain an amino acid sequence as shown in SEQ ID NO: 2001 or SEQ ID NO: 2000.
  • the second domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may each comprise an amino acid sequence as shown in SEQ ID NO: 2012, SEQ ID NO: 2013, and SEQ ID NO: 2014.
  • the second domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 2011.
  • the second domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 2010.
  • the second structural domain does not contain light chains.
  • the second domain fusion peptide described above does not contain a CH1 domain.
  • the third domain can be combined with CD28 and/or CTLA4.
  • the third domain comprises CD86 or CD80 or their functionally active fragments, or variations thereof.
  • the third domain is selected from the group consisting of CD86 or CD80 derived from humans or mice, or functionally active fragments thereof or variants thereof.
  • the third domain comprises the extracellular domain of CD86 or CD80, or a functionally active fragment thereof, or a variant thereof.
  • the third domain comprises the IgV of CD86 or CD80, or a functionally active fragment thereof, or a variant thereof.
  • the third domain comprises a CD86 variant polypeptide, wherein the mutant comprises an amino acid substitution mutation of human CD86.
  • the third domain comprises a CD86 variant polypeptide comprising an IgV domain amino acid substitution mutant of CD86 and/or an extracellular domain amino acid substitution mutant of human CD86.
  • the third domain comprises a CD86 variant polypeptide, the CD86 variant polypeptide comprising an IgV domain amino acid substitution mutant of CD86, the mutation site of the CD86 IgV domain amino acid substitution mutant comprising one, two, three, four, five or more amino acid site mutations selected from the group consisting of: A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F.
  • the third domain comprises a CD86 variant polypeptide comprising an IgV domain amino acid substitution mutant of CD86, the amino acid substitution mutant comprising combinations shown below: Q25I/F33L/H90I, Q25V/F33L/H90I, Q25I/F33V/H90I, Q25V/F33V/H90I, Q25I/F33L/H90V, Q25 V/F33L/H90V, Q25I/F33V/H90V, Q25V/F33V/H90V, A13L/Q25I/F33L/H90I, A13I/Q25I/F33L/H90I, A13L/Q25V/F33L/H90I, Q25I/F33L/I89L/H90I, Q25I/F33L/M60R /H90I ⁇ Q25V/F33L/I89L/H90I ⁇ Q25V
  • the third domain comprises a CD86 variant polypeptide containing the IgV domain amino acid substitution mutant Q25I/F33L/H90I of CD86.
  • the third domain comprises a CD86 variant polypeptide, the CD86 variant polypeptide comprising an extracellular domain amino acid substitution mutant of CD86, the extracellular domain amino acid substitution mutant of CD86 comprising one, two, three, four, five or more amino acid site mutations selected from the group consisting of: A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F.
  • the third domain comprises an extracellular domain amino acid substitution mutant of CD86, the amino acid substitution mutant comprising combinations shown below: Q25I/F33L/H90I, Q25V/F33L/H90I, Q25I/F33V/H90I, Q25V/F33V/H90I, Q25I/F33L/H90V, Q25V/F33L/H90V, Q25 I/F33V/H90V, Q25V/F33V/H90V, A13L/Q25I/F33L/H90I, A13L/Q25 V/F33L/H90I, A13I/Q25V/F33L/H90I, Q25I/F33L/I89L/H90I, Q25I/F33L/M60R/H90I, Q25V /F33L/I89L/H90I, Q25V/F33L/M60R/H90I, Q25V /F33L/I89L/H90I, Q
  • the third domain comprises an extracellular domain amino acid substitution mutant of CD86, the amino acid substitution mutant comprising the combination shown below: Q25I/F33L/H90I.
  • the third domain comprises an amino acid sequence selected from SEQ ID NO: 97 to SEQ ID NO: 165.
  • the third domain comprises a CD80 variant polypeptide, wherein the mutant comprises an amino acid substitution mutation of human CD80.
  • the third domain comprises a CD80 variant polypeptide, which comprises an IgV domain amino acid substitution mutant of CD80 and/or an extracellular domain amino acid substitution mutant of human CD80.
  • the first domain is directly or indirectly connected to the second domain.
  • the heavy chain Fc of the first structural domain is directly or indirectly connected to the heavy chain Fc of the second structural domain.
  • the heavy chain Fc segment of the first structural domain is linked to the heavy chain Fc of the second structural domain via disulfide bonds.
  • the heavy chain of the first structural domain forms a heterodimer with the heavy chain of the second structural domain.
  • the heavy chain Fc segments of the first structural domain and the heavy chain Fc segments of the second structural domain are connected by a knobs-into-holes structure.
  • knots-into-holes pairing is achieved by including one or more substitutions in the Fc segment of the heavy chain, which form heterodimeric pairings between the two heavy chains.
  • CH3 in the first domain and CH3 in the second domain are substitution pairs in a pestle-mortar structure.
  • the T366 and/or Y407 amino acid residues on one of the CH3 domains of the first and second domains are replaced, and the L368 amino acid residue on the other CH3 domain is replaced.
  • T366 is replaced with tyrosine (Y) or tryptophan (W); and/or Y407 is replaced with threonine (T), alanine (A) or valine (V); and/or L368 is replaced with alanine (A).
  • the antigen-binding fragment of the first domain is directly or indirectly linked to the second domain.
  • the N-terminus of the first domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the second domain; or the C-terminus of the first domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the second domain.
  • the heavy chain of the first domain antigen-binding fragment is directly or indirectly linked to the heavy chain of the second domain; or the light chain of the first domain antigen-binding fragment is directly or indirectly linked to the heavy chain of the second domain; or the heavy chain of the first domain antigen-binding fragment is directly or indirectly linked to the light chain of the second domain; or the light chain of the first domain antigen-binding fragment is directly or indirectly linked to the light chain of the second domain.
  • the C-terminus of the heavy chain of the first domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the second domain; or the C-terminus of the light chain of the first domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the second domain; or the C-terminus of the heavy chain of the first domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the light chain of the second domain; or the N-terminus of the heavy chain of the first domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the second domain; or the N-terminus of the light chain of the first domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the second domain; or the N-terminus of the heavy chain of the first domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the second domain; or the N-terminus of the heavy chain of the first domain anti
  • the first domain is directly or indirectly linked to the antigen-binding fragment of the second domain.
  • the N-terminus of the first domain is directly or indirectly connected to the C-terminus of the antigen-binding fragment of the second domain; or the C-terminus of the first domain is directly or indirectly connected to the N-terminus of the antigen-binding fragment of the second domain.
  • the heavy chain of the first domain is directly or indirectly linked to the heavy chain of the antigen-binding fragment of the second domain; or the light chain of the first domain is directly or indirectly linked to the heavy chain of the antigen-binding fragment of the second domain; or the heavy chain of the first domain is directly or indirectly linked to the light chain of the antigen-binding fragment of the second domain; or the light chain of the first domain is directly or indirectly linked to the light chain of the antigen-binding fragment of the second domain.
  • the C-terminus of the heavy chain of the second domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the first domain; or the C-terminus of the light chain of the second domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the first domain; or the C-terminus of the heavy chain of the second domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the light chain of the first domain; or the N-terminus of the heavy chain of the second domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the first domain; or the N-terminus of the light chain of the second domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the first domain; or the N-terminus of the heavy chain of the second domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the first domain; or the N-terminus of the heavy chain of the second domain anti
  • the first domain and the third domain are directly or indirectly connected.
  • the first domain heavy chain is directly or indirectly connected to the third domain.
  • the C-terminus of the first domain heavy chain is directly or indirectly connected to the N-terminus of the third domain, or the N-terminus of the first domain heavy chain is directly or indirectly connected to the C-terminus of the third domain.
  • the first structural domain light chain is directly or indirectly connected to the third structural domain.
  • the C-terminus of the first structural domain light chain is directly or indirectly connected to the N-terminus of the third structural domain, or the N-terminus of the first structural domain light chain is directly or indirectly connected to the C-terminus of the third structural domain.
  • the second and third domains are directly or indirectly connected.
  • the second domain heavy chain is directly or indirectly connected to the third domain.
  • the C-end of the second domain heavy chain is directly or indirectly connected to the N-end of the third domain, or the N-end of the second domain heavy chain is directly or indirectly connected to the C-end of the third domain.
  • the second structural domain light chain is directly or indirectly connected to the third structural domain.
  • the C-terminus of the second structural domain light chain is directly or indirectly connected to the N-terminus of the third structural domain, or the N-terminus of the second structural domain light chain is directly or indirectly connected to the C-terminus of the third structural domain.
  • the N-terminus of the first structural domain is directly or indirectly connected to the C-terminus of the third structural domain, and the second structural domain is not directly or indirectly connected to the third structural domain; or the C-terminus of the first structural domain is directly or indirectly connected to the N-terminus of the third structural domain, and the second structural domain is not directly or indirectly connected to the third structural domain; or the N-terminus of the second structural domain is directly or indirectly connected to the C-terminus of the third structural domain, and the first structural domain is not directly or indirectly connected to the third structural domain; or the C-terminus of the second structural domain is directly or indirectly connected to the N-terminus of the third structural domain, and the first structural domain is not directly or indirectly connected to the third structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain, and the first structural domain is directly or indirectly connected to the third structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain.
  • the C-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the N-terminus of the heavy chain of the second structural domain, and the first structural domain is directly or indirectly connected to the third structural domain.
  • the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the third structural domain; or the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the third structural domain; or the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain.
  • the N-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the C-terminus of the heavy chain of the second structural domain, and the first structure is directly or indirectly connected to the third structural domain.
  • the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain; or the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain; or the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the third structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the light chain of the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain.
  • the C-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the N-terminus of the light chain of the second structural domain, and the first structural domain is directly or indirectly connected to the third structural domain.
  • the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the light chain of the second structural domain, and the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the third structural domain; or the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the light chain of the second structural domain, and the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the third structural domain; or the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the light chain of the second structural domain, and the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain.
  • the N-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the C-terminus of the light chain of the second structural domain, and the first structure is directly or indirectly connected to the third structural domain.
  • the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the light chain of the second structural domain, and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain; or the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the light chain of the second structural domain, and the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain; or the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the light chain of the second structural domain, and the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the third structural domain.
  • the light chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain.
  • the C-terminus of the light chain of the first structural domain is directly or indirectly connected to the N-terminus of the heavy chain of the second structural domain, and the first structural domain is directly or indirectly connected to the third structural domain.
  • the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the third structural domain; or the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the third structural domain; or the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain.
  • the N-terminus of the light chain of the first structural domain is directly or indirectly connected to the C-terminus of the heavy chain of the second structural domain, and the first structure is directly or indirectly connected to the third structural domain.
  • the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain; or the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain; or the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the third structural domain.
  • the light chain of the first structural domain is directly or indirectly connected to the light chain of the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain.
  • the C-terminus of the light chain of the first structural domain is directly or indirectly connected to the N-terminus of the light chain of the second structural domain, and the first structural domain is directly or indirectly connected to the third structural domain.
  • the C-terminus of the light chain of the first structural domain is directly or indirectly connected to the N-terminus of the light chain of the second structural domain, and the N-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the C-terminus of the third structural domain; or the C-terminus of the light chain of the first structural domain is directly or indirectly connected to the N-terminus of the light chain of the second structural domain, and the N-terminus of the light chain of the first structural domain is directly or indirectly connected to the C-terminus of the third structural domain; or the C-terminus of the light chain of the first structural domain is directly or indirectly connected to the N-terminus of the light chain of the second structural domain, and the C-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the N-terminus of the third structural domain.
  • the N-terminus of the light chain of the first structural domain is directly or indirectly connected to the C-terminus of the light chain of the second structural domain, and the first structure is directly or indirectly connected to the third structural domain.
  • the N-terminus of the light chain of the first structural domain is directly or indirectly connected to the C-terminus of the light chain of the second structural domain, and the C-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the N-terminus of the third structural domain; or the N-terminus of the light chain of the first structural domain is directly or indirectly connected to the C-terminus of the light chain of the second structural domain, and the C-terminus of the light chain of the first structural domain is directly or indirectly connected to the N-terminus of the third structural domain; or the N-terminus of the light chain of the first structural domain is directly or indirectly connected to the C-terminus of the light chain of the second structural domain, and the N-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the C-terminus of the third structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain
  • the second structural domain is directly or indirectly connected to the third structural domain
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the N-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the C-terminus of the heavy chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the N-end of the heavy chain of the second structural domain is directly or indirectly connected to the C-end of the third structural domain; or the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the N-end of the light chain of the second structural domain is directly or indirectly connected to the C-end of the third structural domain; or the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the C-end of the light chain of the second structural domain is directly or indirectly connected to the N-end of the third structural domain.
  • the C-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the N-terminus of the heavy chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the C-end of the light chain of the second structural domain is directly or indirectly connected to the N-end of the third structural domain; or the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the third structural domain; or the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the N-end of the light chain of the second structural domain is directly or indirectly connected to the C-end of the third structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the light chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the N-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the C-terminus of the light chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the light chain of the second structural domain, and the N-end of the heavy chain of the second structural domain is directly or indirectly connected to the C-end of the third structural domain; or the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the light chain of the second structural domain, and the N-end of the light chain of the second structural domain is directly or indirectly connected to the C-end of the third structural domain; or the N-end of the heavy chain of the first structural domain is directly or indirectly connected to the C-end of the light chain of the second structural domain, and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the third structural domain.
  • the C-terminus of the heavy chain of the first structural domain is directly or indirectly connected to the N-terminus of the light chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the light chain of the second structural domain, and the C-end of the light chain of the second structural domain is directly or indirectly connected to the N-end of the third structural domain; or the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the light chain of the second structural domain, and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the third structural domain; or the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the light chain of the second structural domain, and the N-end of the heavy chain of the second structural domain is directly or indirectly connected to the C-end of the third structural domain.
  • the light chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the N-terminus of the light chain of the first structural domain is directly or indirectly connected to the C-terminus of the heavy chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the N-end of the heavy chain of the second structural domain is directly or indirectly connected to the C-end of the third structural domain; or the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the N-end of the light chain of the second structural domain is directly or indirectly connected to the C-end of the third structural domain; or the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the heavy chain of the second structural domain, and the C-end of the light chain of the second structural domain is directly or indirectly connected to the N-end of the third structural domain.
  • the C-terminus of the light chain of the first structural domain is directly or indirectly connected to the N-terminus of the heavy chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the C-end of the light chain of the second structural domain is directly or indirectly connected to the N-end of the third structural domain; or the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the third structural domain; or the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the heavy chain of the second structural domain, and the N-end of the light chain of the second structural domain is directly or indirectly connected to the C-end of the third structural domain.
  • the light chain of the first structural domain is directly or indirectly connected to the light chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the N-terminus of the light chain of the first structural domain is directly or indirectly connected to the C-terminus of the light chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the N-terminus of the light chain of the first structural domain is directly or indirectly connected to the C-terminus of the light chain of the second structural domain, and the N-terminus of the light chain of the second structural domain is directly or indirectly connected to the C-terminus of the third structural domain; or the N-terminus of the light chain of the first structural domain is directly or indirectly connected to the C-terminus of the light chain of the second structural domain, and the N-terminus of the light chain of the second structural domain is directly or indirectly connected to the C-terminus of the third structural domain; or the N-terminus of the light chain of the first structural domain is directly or indirectly connected to the C-terminus of the light chain of the second structural domain, and the C-terminus of the heavy chain of the second structural domain is directly or indirectly connected to the N-terminus of the third structural domain.
  • the C-terminus of the light chain of the first structural domain is directly or indirectly connected to the N-terminus of the light chain of the second structural domain, and the second structural domain is directly or indirectly connected to the third structural domain.
  • the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the light chain of the second structural domain, and the C-end of the light chain of the second structural domain is directly or indirectly connected to the N-end of the third structural domain; or the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the light chain of the second structural domain, and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the third structural domain; or the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the light chain of the second structural domain, and the N-end of the heavy chain of the second structural domain is directly or indirectly connected to the C-end of the third structural domain.
  • the fusion protein further includes a fourth domain capable of binding tumor-associated antigens (TAAs).
  • TAAs tumor-associated antigens
  • the fourth domain is capable of binding to proteins or tumor antigens that are overexpressed on tumor cells relative to the corresponding non-tumor cells.
  • the fourth domain comprises an antibody or an antigen-binding fragment thereof.
  • the fourth domain antibody is selected from the group consisting of recombinant antibodies, single-domain antibodies, heavy chain antibodies, chimeric antibodies, and bispecific antibodies.
  • the fourth domain antigen-binding fragment is selected from one or more fragments from the group consisting of: Fab, Fab’, Fv fragment, F(ab’)2, F(ab)2, scFv, di-scFv, VHH, and dAb.
  • the fourth domain can bind to one or more of the following targets: PD-L1, PD-L2, VEGF, VEGFR, FGFR, HER2, HGFR, PIP-LAR, CD44, CD147, TfR, CDCP1, ⁇ 6 ⁇ 4, ⁇ 6 ⁇ 3, Trop2, HHLA2, GCC, 5T4, EpCAM, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, DLL3, B 7H3, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, PTK7, EphA2, NKG2A, Nkp36, Tim3, CD20, Her2, HHLA2, PD-L1, GCC, EGFR, DLL3 and/or B7H3, 5T4 and/or CEA.
  • targets PD-L1, PD-L2, VEGF
  • the fourth domain comprises a single-chain immunoglobulin domain.
  • the fourth domain comprises a single-chain immunoglobulin IgG antibody.
  • the fourth domain single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the fourth domain comprises a single-chain immunoglobulin Fc domain.
  • the fourth domain comprises the Fc domain of a single-chain immunoglobulin IgG antibody.
  • the Fc domain of the single-chain immunoglobulin IgG antibody includes human IgG1 Fc domain, human IgG2 Fc domain, human IgG3 Fc domain, human IgG4 Fc domain, mouse IgG1 Fc domain, mouse IgG2a Fc domain, mouse IgG2b Fc domain, or mouse IgG3 Fc domain.
  • the fourth domain single-stranded immunoglobulin Fc fragment comprises CH2 and/or CH3 sequences, wherein the first domain CH2 is located between the variable region and the CH3 domain; preferably, the Fc fragment contains any amino acid sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4.
  • the HCDR1, HCDR2, and/or HCDR3 amino acid sequences or variant sequences thereof in the heavy chain variable region contain any amino acid sequence selected from the group consisting of: SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69, S EQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 100 2.
  • the fourth structure includes a heavy chain variable region VH of the antibody heavy chain, the antibody heavy chain variable region VH comprising an amino acid sequence selected from any of the following groups: SEQ ID NO: 47, SEQ ID NO: 58, SEQ ID NO: 63, SEQ ID NO: 68, SEQ ID NO: 73, SEQ ID NO: 78, SEQ ID NO: 83, SEQ ID NO: 93, SEQ ID NO: 1001, SEQ ID NO: 1010, SEQ ID NO: 2006, SEQ ID NO: 2011.
  • the fourth structure comprises an antibody heavy chain containing an amino acid sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 57, SEQ ID NO: 62, SEQ ID NO: 67, SEQ ID NO: 72, SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 92, SEQ ID NO: 1000, SEQ ID NO: 1005, SEQ ID NO: 1018, SEQ ID NO: 2000, SEQ ID NO: 2010.
  • the fourth structure includes a light chain variable region VL of an antibody light chain, the antibody light chain comprising an amino acid sequence selected from any of the following groups: SEQ ID NO: 53, SEQ ID NO: 88, SEQ ID NO: 1014, SEQ ID NO: 2002.
  • the fourth domain comprises an antibody light chain containing an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 87.
  • the fourth domain may comprise HCDR1, HCDR2, HCDR3 of the antibody heavy chain and LCDR1, LCDR2, and LCDR3 of the antibody light chain, wherein HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, respectively, and LCDR1, LCDR2, and LCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively.
  • the fourth structural domain may include a heavy chain variable region VH of the antibody heavy chain, the antibody heavy chain VH may include an amino acid sequence as shown in SEQ ID NO: 58, and the fourth structural domain may include a light chain variable region VL of the antibody light chain, the antibody light chain VL may include an amino acid sequence as shown in SEQ ID NO: 53.
  • the fourth domain may comprise an antibody heavy chain and/or an antibody light chain, the antibody heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 57, and the antibody light chain comprising an amino acid sequence as shown in SEQ ID NO: 52.
  • the fourth domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively.
  • the fourth domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 47.
  • the fourth domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 46.
  • the fourth domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively.
  • the fourth domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 63.
  • the fourth domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 62.
  • the fourth domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively.
  • the fourth domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 68.
  • the fourth domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 67.
  • the fourth domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, respectively.
  • the fourth domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 73.
  • the fourth domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 72.
  • the fourth domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively.
  • the fourth domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 78.
  • the fourth domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 77.
  • the fourth domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86, respectively.
  • the fourth domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 83.
  • the fourth domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 82.
  • the fourth domain may comprise HCDR1, HCDR2, HCDR3 of the antibody heavy chain and LCDR1, LCDR2, and LCDR3 of the antibody light chain, wherein HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively, and LCDR1, LCDR2, and LCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, respectively.
  • the fourth structural domain may include a heavy chain variable region VH of the antibody heavy chain, the antibody heavy chain VH may include an amino acid sequence as shown in SEQ ID NO: 93, and the fourth structural domain may include a light chain variable region VL of the antibody light chain, the antibody light chain VL may include an amino acid sequence as shown in SEQ ID NO: 88.
  • the fourth structural domain may comprise an antibody heavy chain and/or an antibody light chain, wherein the antibody heavy chain may comprise an amino acid sequence as shown in SEQ ID NO: 92, and the antibody light chain may comprise an amino acid sequence as shown in SEQ ID NO: 87.
  • the fourth domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 1002, SEQ ID NO: 1003, and SEQ ID NO: 1004, respectively.
  • the fourth domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 1001.
  • the fourth domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 1000 or SEQ ID NO: 1005.
  • the fourth domain may comprise the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 of the scFv antibody, wherein HCDR1, HCDR2, and HCDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 1011, SEQ ID NO: 1012, and SEQ ID NO: 1013, and LDR1, LCDR2, and LCDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 1015, SEQ ID NO: 1016, and SEQ ID NO: 1017.
  • the fourth domain may comprise the heavy chain variable region VH and the light chain variable region VL of the scFv antibody, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO: 1010 and the VL may comprise the amino acid sequence shown in SEQ ID NO: 1014.
  • the fourth domain may comprise an scFv antibody, which may comprise an amino acid sequence as shown in SEQ ID NO: 1009 or SEQ ID NO: 1018.
  • the fourth domain may comprise the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 of the scFv antibody, wherein HCDR1, HCDR2, and HCDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 2007, SEQ ID NO: 2008, and SEQ ID NO: 2009, and LDR1, LCDR2, and LCDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 2003, SEQ ID NO: 2004, and SEQ ID NO: 2005.
  • the fourth domain may comprise the heavy chain variable region VH and the light chain variable region VL of the scFv antibody, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO: 2006 and the VL may comprise the amino acid sequence shown in SEQ ID NO: 2002.
  • the fourth domain may comprise an scFv antibody, which may comprise an amino acid sequence as shown in SEQ ID NO: 2001 or SEQ ID NO: 2000.
  • the fourth domain may comprise CDR1, CDR2, and CDR3 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 2012, SEQ ID NO: 2013, and SEQ ID NO: 2014, respectively.
  • the fourth domain may comprise the heavy chain variable region VHH of a single-domain antibody, the VHH comprising an amino acid sequence as shown in SEQ ID NO: 2011.
  • the fourth domain may comprise a heavy chain of a single-domain antibody, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 2010.
  • the fourth structural domain does not contain a light chain.
  • the fourth domain fusion peptide described above does not contain a CH1 domain.
  • the first domain and the fourth domain are directly or indirectly connected.
  • the heavy chain Fc of the first structural domain is directly or indirectly connected to the heavy chain Fc of the fourth structural domain.
  • the heavy chain Fc segment of the first structural domain is connected to the heavy chain Fc of the fourth structural domain via disulfide bonds.
  • the heavy chain of the first structural domain forms a heterodimer with the heavy chain of the fourth structural domain.
  • the heavy chain Fc segments of the first structural domain and the heavy chain Fc segments of the fourth structural domain are connected by a knobs-into-holes structure.
  • the knots-into-holes pairing is achieved by including one or more substitutions in the aforementioned heavy chain Fc fragments, which form heterodimeric pairings between heavy chains.
  • CH3 in the first structural domain and CH3 in the fourth structural domain are substitution pairs in a pestle-mortar structure.
  • the T366 and/or Y407 amino acid residues on one of the CH3 domains of the first and fourth domains are replaced, and the L368 amino acid residue on the other CH3 domain is replaced.
  • the antigen-binding fragment of the first domain is directly or indirectly linked to the fourth domain.
  • the N-terminus of the first domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the fourth domain; or the C-terminus of the first domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the fourth domain.
  • the heavy chain of the first domain antigen-binding fragment is directly or indirectly connected to the heavy chain of the fourth domain; or the light chain of the first domain antigen-binding fragment is directly or indirectly connected to the heavy chain of the fourth domain; or the heavy chain of the first domain antigen-binding fragment is directly or indirectly connected to the light chain of the fourth domain; or the light chain of the first domain antigen-binding fragment is directly or indirectly connected to the light chain of the fourth domain.
  • the C-terminus of the heavy chain of the first domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the fourth domain; or the C-terminus of the light chain of the first domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the fourth domain; or the C-terminus of the heavy chain of the first domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the light chain of the fourth domain; or the N-terminus of the heavy chain of the first domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the fourth domain; or the N-terminus of the light chain of the first domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the fourth domain; or the N-terminus of the heavy chain of the first domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the fourth domain; or the N-terminus of the heavy chain of the first domain anti
  • the first domain is directly or indirectly linked to the antigen-binding fragment of the fourth domain.
  • the N-terminus of the first domain is directly or indirectly connected to the C-terminus of the antigen-binding fragment of the fourth domain; or the C-terminus of the first domain is directly or indirectly connected to the N-terminus of the antigen-binding fragment of the fourth domain.
  • the heavy chain of the first domain is directly or indirectly connected to the heavy chain of the antigen-binding fragment of the fourth domain; or the light chain of the first domain is directly or indirectly connected to the heavy chain of the antigen-binding fragment of the fourth domain; or the heavy chain of the first domain is directly or indirectly connected to the light chain of the antigen-binding fragment of the fourth domain; or the light chain of the first domain is directly or indirectly connected to the light chain of the antigen-binding fragment of the fourth domain.
  • the C-terminus of the heavy chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the first domain; or the C-terminus of the light chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the first domain; or the C-terminus of the heavy chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the light chain of the first domain; or the N-terminus of the heavy chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the first domain; or the N-terminus of the light chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the first domain; or the N-terminus of the heavy chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the first domain; or the N-terminus of the heavy chain of the fourth domain anti
  • the fourth structural domain and the third structural domain are directly or indirectly connected.
  • the fourth structural domain heavy chain is directly or indirectly connected to the third structural domain.
  • the C-terminus of the fourth structural domain heavy chain is directly or indirectly connected to the N-terminus of the third structural domain, or the N-terminus of the fourth structural domain heavy chain is directly or indirectly connected to the C-terminus of the third structural domain.
  • the fourth structural domain light chain is directly or indirectly connected to the third structural domain.
  • the C-terminus of the fourth structural domain light chain is directly or indirectly connected to the N-terminus of the third structural domain, or the N-terminus of the fourth structural domain light chain is directly or indirectly connected to the C-terminus of the third structural domain.
  • the second and fourth structural domains are directly or indirectly connected.
  • the heavy chain Fc of the second structural domain is directly or indirectly connected to the heavy chain Fc of the fourth structural domain.
  • the heavy chain Fc segment of the second structural domain is connected to the heavy chain Fc of the fourth structural domain via a disulfide bond.
  • the heavy chain of the second structural domain forms a heterodimer with the heavy chain of the fourth structural domain.
  • the heavy chain Fc segments of the second structural domain and the heavy chain Fc segments of the fourth structural domain are connected by a knobs-into-holes structure.
  • knots-into-holes pairing is achieved by including one or more substitutions in the aforementioned heavy chain Fc fragments, which form heterodimeric pairings between heavy chains.
  • CH3 in the second structural domain and CH3 in the fourth structural domain are a pestle-mortar substitution pair.
  • the T366 and/or Y407 amino acid residues on one of the CH3 domains of the second and fourth domains are replaced, and the L368 amino acid residue on the other CH3 domain is replaced.
  • the second domain antigen-binding fragment is directly or indirectly linked to the fourth domain.
  • the N-terminus of the second domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the fourth domain; or the C-terminus of the second domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the fourth domain.
  • the heavy chain of the second domain antigen-binding fragment is directly or indirectly linked to the heavy chain of the fourth domain; or the light chain of the second domain antigen-binding fragment is directly or indirectly linked to the heavy chain of the fourth domain; or the heavy chain of the second domain antigen-binding fragment is directly or indirectly linked to the light chain of the fourth domain; or the light chain of the second domain antigen-binding fragment is directly or indirectly linked to the light chain of the fourth domain.
  • the C-terminus of the heavy chain of the second domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the fourth domain; or the C-terminus of the light chain of the second domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the fourth domain; or the C-terminus of the heavy chain of the second domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the light chain of the fourth domain; or the N-terminus of the heavy chain of the second domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the fourth domain; or the N-terminus of the light chain of the second domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the fourth domain; or the N-terminus of the heavy chain of the second domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the fourth domain; or the N-terminus of the heavy chain of the second domain anti
  • the second domain is directly or indirectly linked to the antigen-binding fragment of the fourth domain.
  • the N-terminus of the second domain is directly or indirectly connected to the C-terminus of the antigen-binding fragment of the fourth domain; or the C-terminus of the second domain is directly or indirectly connected to the N-terminus of the antigen-binding fragment of the fourth domain.
  • the heavy chain of the second domain is directly or indirectly linked to the heavy chain of the antigen-binding fragment of the fourth domain; or the light chain of the second domain is directly or indirectly linked to the heavy chain of the antigen-binding fragment of the fourth domain; or the heavy chain of the second domain is directly or indirectly linked to the light chain of the antigen-binding fragment of the fourth domain; or the light chain of the second domain is directly or indirectly linked to the light chain of the antigen-binding fragment of the fourth domain.
  • the C-terminus of the heavy chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the second domain; or the C-terminus of the light chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the heavy chain of the second domain; or the C-terminus of the heavy chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the N-terminus of the light chain of the second domain; or the N-terminus of the heavy chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the second domain; or the N-terminus of the light chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the second domain; or the N-terminus of the heavy chain of the fourth domain antigen-binding fragment is directly or indirectly connected to the C-terminus of the heavy chain of the second domain; or the N-terminus of the heavy chain of the fourth domain anti
  • the fourth structural domain may not exist.
  • the fusion protein further includes a fifth domain that conditionally masks the CD3-binding domain in the first domain.
  • the fifth domain includes a masking peptide fragment that can alter the affinity of the first domain for binding to CD3.
  • the fifth domain masking peptide fragment comprises a polypeptide sequence selected from the following: SEQ ID No:404, SEQ ID No:406, SEQ ID No:407, SEQ ID No:408, SEQ ID No:409, SEQ ID No:410, SEQ ID No:412, SEQ ID No:414, SEQ ID No:415, SEQ ID No:424, SEQ ID No:425, SEQ ID No:426, SEQ ID No:427, SEQ ID No:428, SEQ ID No:403, SEQ ID No:404. 05.
  • the fifth domain further includes an enzyme cleavage linker, through which the masking peptide fragment of the fifth domain is linked to the first domain, the second domain, or the fourth domain.
  • the enzyme-ligand contains a protease cleavage site fragment.
  • the enzyme ligand comprises one, two, three, four, five or more protease cleavage site fragments selected from SEQ ID NO:3000-3036.
  • the enzyme-ligand further includes 0, 1, 2, 3, 4, 5, or more linker amino acid sequences selected from the following: GS, GGS, GSGS, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10.
  • the linker amino acid sequence is located between two protease cleavage sites, between a masking peptide fragment and a protease cleavage site fragment, or between a protease cleavage site fragment and a first, second, or fourth domain.
  • the masking peptide fragment of the fifth domain is directly or indirectly linked to the enzyme-linked linker; preferably, the C-terminus of the masking peptide fragment of the fifth domain is directly or indirectly linked to the N-terminus of the enzyme-linked linker, or the N-terminus of the masking peptide fragment of the fifth domain is directly or indirectly linked to the C-terminus of the enzyme-linked linker.
  • the first domain and the fifth domain are directly or indirectly connected.
  • the first domain heavy chain is directly or indirectly connected to the fifth domain; preferably, the C-end of the first domain heavy chain is directly or indirectly connected to the N-end of the fifth domain, or the N-end of the first domain heavy chain is directly or indirectly connected to the C-end of the fifth domain.
  • the first structural domain light chain is directly or indirectly connected to the fifth structural domain; preferably, the C-terminus of the first structural domain light chain is directly or indirectly connected to the N-terminus of the fifth structural domain, or the N-terminus of the first structural domain light chain is directly or indirectly connected to the C-terminus of the fifth structural domain.
  • the second and fifth structural domains are directly or indirectly connected.
  • the second domain heavy chain is directly or indirectly connected to the fifth domain; preferably, the C-end of the second domain heavy chain is directly or indirectly connected to the N-end of the fifth domain, or the N-end of the second domain heavy chain is directly or indirectly connected to the C-end of the fifth domain.
  • the second structural domain light chain is directly or indirectly connected to the fifth structural domain.
  • the C-end of the second structural domain light chain is directly or indirectly connected to the N-end of the fifth structural domain, or the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the fifth structural domain.
  • the fourth and fifth structural domains are directly or indirectly connected.
  • the fourth structural domain heavy chain is directly or indirectly connected to the fifth structural domain; preferably, the C-end of the fourth structural domain heavy chain is directly or indirectly connected to the N-end of the fifth structural domain, or the N-end of the fourth structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain.
  • the fourth structural domain light chain is directly or indirectly connected to the fifth structural domain; preferably, the C-terminus of the fourth structural domain light chain is directly or indirectly connected to the N-terminus of the fifth structural domain, or the N-terminus of the fourth structural domain light chain is directly or indirectly connected to the C-terminus of the fifth structural domain.
  • the fusion protein further includes a sixth domain: the sixth domain is capable of binding CD28 and/or CTLA4, or the sixth domain may be absent.
  • the sixth domain comprises CD86 or CD80 or functionally active fragments thereof, or variations thereof.
  • the sixth domain is selected from the group consisting of CD86 or CD80 derived from humans or mice, or functionally active fragments thereof or variants thereof.
  • the sixth domain comprises the extracellular domain of CD86 or CD80, or a functionally active fragment thereof, or a variant thereof.
  • the sixth domain comprises the IgV of CD86 or CD80, or a functionally active fragment thereof, or a variant thereof.
  • the sixth domain comprises a CD86 variant polypeptide, wherein the mutant comprises an amino acid substitution mutation of human CD86.
  • the sixth domain comprises a CD86 variant polypeptide comprising an IgV domain amino acid substitution mutant of CD86 and/or an extracellular domain amino acid substitution mutant of human CD86.
  • the sixth domain comprises a CD86 variant polypeptide, the CD86 variant polypeptide comprising an IgV domain amino acid substitution mutant of CD86, wherein the mutation site of the CD86 IgV domain amino acid substitution mutant comprises one, two, three, four, five or more amino acid site mutations selected from the group consisting of: A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F.
  • the sixth domain comprises a CD86 variant polypeptide comprising an IgV domain amino acid substitution mutant of CD86, the amino acid substitution mutant comprising combinations shown below: Q25I/F33L/H90I, Q25V/F33L/H90I, Q25I/F33V/H90I, Q25V/F33V/H90I, Q25I/F33L/H90V, Q25 V/F33L/H90V, Q25I/F33V/H90V, Q25V/F33V/H90V, A13L/Q25I/F33L/H90I, A13I/Q25I/F33L/H90I, A13L/Q25V/F33L/H90I, Q25I/F33L/I89L/H90I, Q25I/F33L/M60R /H90I ⁇ Q25V/F33L/I89L/H90I ⁇ Q25V
  • the sixth domain comprises a CD86 variant polypeptide containing the IgV domain amino acid substitution mutant Q25I/F33L/H90I of CD86.
  • the sixth domain comprises a CD86 variant polypeptide, the CD86 variant polypeptide comprising an extracellular domain amino acid substitution mutant of CD86, the extracellular domain amino acid substitution mutant of CD86 comprising one, two, three, four, five or more amino acid site mutations selected from the group consisting of: A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F.
  • the sixth domain comprises an extracellular domain amino acid substitution mutant of CD86, the amino acid substitution mutant comprising combinations shown below: Q25I/F33L/H90I, 25V/F33L/H90I, Q25I/F33V/H90I, Q25V/F33V/H90I, Q25I/F33L/H90V, Q25V/F33L/H90V, Q25I /F33V/H90V, Q25V/F33V/H90V, A13L/Q25I/F33L/H90I, A13L/Q25V /F33L/H90I, A13I/Q25V/F33L/H90I, Q25I/F33L/I89L/H90I, Q25I/F33L/M60R/H90I, Q25V /F33L/I89L/H90I, Q25V/F33L/M60R/H90I, Q25V /F33L/I89L/H90I,
  • the sixth domain comprises an extracellular domain amino acid substitution mutant of CD86, the amino acid substitution mutant comprising the combination shown below: Q25I/F33L/H90I.
  • the sixth domain comprises an amino acid sequence selected from SEQ ID NO: 97 to SEQ ID NO: 165.
  • the sixth domain comprises a CD80 variant polypeptide, wherein the mutant comprises an amino acid substitution mutation of human CD80.
  • the sixth domain comprises a CD80 variant polypeptide comprising an IgV domain amino acid substitution mutant of CD80 and/or an extracellular domain amino acid substitution mutant of human CD80.
  • the first domain and the sixth domain are directly or indirectly connected.
  • the first domain heavy chain is directly or indirectly connected to the sixth domain.
  • the C-terminus of the first domain heavy chain is directly or indirectly connected to the N-terminus of the sixth domain, or the N-terminus of the first domain heavy chain is directly or indirectly connected to the C-terminus of the sixth domain.
  • the first structural domain light chain is directly or indirectly connected to the sixth structural domain.
  • the C-terminus of the first structural domain light chain is directly or indirectly connected to the N-terminus of the sixth structural domain, or the N-terminus of the first structural domain light chain is directly or indirectly connected to the C-terminus of the sixth structural domain.
  • the second and sixth structural domains are directly or indirectly connected.
  • the second domain heavy chain is directly or indirectly connected to the sixth domain.
  • the C-end of the second domain heavy chain is directly or indirectly connected to the N-end of the sixth domain, or the N-end of the second domain heavy chain is directly or indirectly connected to the C-end of the sixth domain.
  • the second structural domain light chain is directly or indirectly connected to the sixth structural domain.
  • the C-terminus of the second structural domain light chain is directly or indirectly connected to the N-terminus of the sixth structural domain, or the N-terminus of the second structural domain light chain is directly or indirectly connected to the C-terminus of the sixth structural domain.
  • the fourth and sixth structural domains are directly or indirectly connected.
  • the fourth structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the C-end of the fourth structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain, or the N-end of the fourth structural domain heavy chain is directly or indirectly connected to the C-end of the sixth structural domain.
  • the fourth structural domain light chain is directly or indirectly connected to the sixth structural domain.
  • the C-terminus of the fourth structural domain light chain is directly or indirectly connected to the N-terminus of the sixth structural domain, or the N-terminus of the fourth structural domain light chain is directly or indirectly connected to the C-terminus of the sixth structural domain.
  • the third and sixth structural domains are directly or indirectly connected.
  • the fusion protein includes a first domain, a second domain, a third domain, and a sixth domain, wherein: the first domain is directly or indirectly connected to the second domain, the first domain is directly or indirectly connected to the third domain, and the second domain is directly or indirectly connected to the sixth domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the third structural domain, and the N-end of the second structural domain heavy chain is directly or indirectly connected to the C-end of the sixth structural domain; or, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the third structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain; or, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the second structural domain heavy chain is directly or indirectly connected to the C-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the second structural domain light chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the third structural domain, and the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the sixth structural domain; or, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the third structural domain, and the C-end of the second structural domain light chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the C-end of the second structural domain light chain is directly or indirectly connected to the N-end of the sixth structural domain; or, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain light chain is directly or indirectly connected to the third structural domain, and the second structural domain light chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the N-end of the first structural domain light chain is directly or indirectly connected to the C-end of the third structural domain, and the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the sixth structural domain; or, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the N-end of the first structural domain light chain is directly or indirectly connected to the C-end of the third structural domain, and the C-end of the second structural domain light chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the third structural domain, and the C-end of the second structural domain light chain is directly or indirectly connected to the N-end of the sixth structural domain; or, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain light chain is directly or indirectly connected to the third structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the N-end of the first structural domain light chain is directly or indirectly connected to the C-end of the third structural domain, and the N-end of the second structural domain heavy chain is directly or indirectly connected to the C-end of the sixth structural domain; or, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the N-end of the first structural domain light chain is directly or indirectly connected to the C-end of the third structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the third structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain; or, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the second structural domain heavy chain is directly or indirectly connected to the C-end of the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain
  • the first structural domain is directly or indirectly connected to the fourth structural domain
  • the fourth structural domain is directly or indirectly connected to the third structural domain
  • the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain, and the first structural domain heavy chain is directly or indirectly connected to the fourth structural domain; and the fourth structural domain heavy chain is directly or indirectly connected to the third structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain; and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the fourth structural domain; and the C-end of the heavy chain of the fourth structural domain is directly or indirectly connected to the N-end of the third structural domain; and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain, and the first structural domain is directly or indirectly connected to the fourth structural domain, and the first structural domain is directly or indirectly connected to the third structural domain, and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain
  • the first structural domain heavy chain is directly or indirectly connected to the fourth structural domain
  • the first structural domain heavy chain is directly or indirectly connected to the third structural domain
  • the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain
  • the N end of the heavy chain of the first structural domain is directly or indirectly connected to the C end of the fourth structural domain
  • the C end of the heavy chain of the first structural domain is directly or indirectly connected to the N end of the third structural domain
  • the C end of the heavy chain of the second structural domain is directly or indirectly connected to the N end of the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain
  • the light chain of the first structural domain is directly or indirectly connected to the fourth structural domain
  • the heavy chain of the first structural domain is directly or indirectly connected to the third structural domain
  • the heavy chain of the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain, and the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the fourth structural domain, and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the third structural domain, and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the sixth structural domain; or, the first structural domain is directly or indirectly connected to the second structural domain, and the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the fourth structural domain, and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the third structural domain, and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain; and the second structural domain is directly or indirectly connected to the fourth structural domain; and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain
  • the first structural domain heavy chain is directly or indirectly connected to the third structural domain
  • the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain
  • the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain
  • the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain
  • the N-end of the heavy chain of the second structural domain is directly or indirectly connected to the C-end of the fourth structural domain
  • the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain
  • the heavy chain of the first structural domain is directly or indirectly connected to the third structural domain
  • the light chain of the second structural domain is directly or indirectly connected to the fourth structural domain
  • the heavy chain of the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain, and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the light chain of the second structural domain is directly or indirectly connected to the C-end of the fourth structural domain, and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the sixth structural domain; or, the first structural domain is directly or indirectly connected to the second structural domain; and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain, and the C-end of the light chain of the second structural domain is directly or indirectly connected to the N-end of the fourth structural domain; and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain or the second structural domain is directly or indirectly connected to the fifth structural domain.
  • the first domain heavy chain is directly or indirectly connected to the second domain heavy chain, and the first domain heavy chain or the second domain heavy chain is directly or indirectly connected to the fifth domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the N end of the first structural domain heavy chain or the N end of the second structural domain heavy chain is directly or indirectly connected to the C end of the fifth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain
  • the first structural domain light chain or the second structural domain light chain is directly or indirectly connected to the fifth structural domain
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the N end of the first structural domain light chain or the N end of the second structural domain light chain is directly or indirectly connected to the C end of the fifth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain; and the first structural domain is directly or indirectly connected to the fourth structural domain, and the first structural domain is directly or indirectly connected to the fifth structural domain.
  • the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain heavy chain, and the first structural domain heavy chain is directly or indirectly connected to the fourth structural domain, and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain; preferably, the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the fourth structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain.
  • the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain heavy chain, and the first structural domain heavy chain is directly or indirectly connected to the fourth structural domain, and the first structural domain light chain is directly or indirectly connected to the fifth structural domain; preferably, the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the fourth structural domain, and the N-end of the first structural domain light chain is directly or indirectly connected to the C-end of the fifth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain, and the first structural domain is directly or indirectly connected to the fourth structural domain, and the first structural domain is directly or indirectly connected to the fifth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain light chain is directly or indirectly connected to the fourth structural domain, and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain; preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the fourth structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain; or preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the N-end of the first structural domain light chain is directly or indirectly connected to the C-end of the fourth structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain light chain is directly or indirectly connected to the fourth structural domain, and the first structural domain light chain is directly or indirectly connected to the fifth structural domain; preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the fourth structural domain, and the N-end of the first structural domain light chain is directly or indirectly connected to the C-end of the fifth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the fifth structural domain, and the second structural domain is directly or indirectly connected to the fourth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain; preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end domain of the fifth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the fourth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain light chain is directly or indirectly connected to the fifth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain; preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end domain of the fifth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the fourth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain, and the second structural domain light chain is directly or indirectly connected to the fourth structural domain; preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end domain of the fifth structural domain, and the C-end of the second structural domain light chain is directly or indirectly connected to the N-end of the fourth structural domain; preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end domain of the fifth structural domain, and the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the fourth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain light chain is directly or indirectly connected to the fifth structural domain, and the second structural light chain is directly or indirectly connected to the fourth structural domain; preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end domain of the fifth structure, and the C-end of the second structural domain light chain is directly or indirectly connected to the N-end of the fourth structural domain; or preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end domain of the fifth structure, and the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the fourth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain, and the first structural domain is directly or indirectly connected to the fifth structural domain, and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain; preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the first structural domain light chain is directly or indirectly connected to the fifth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain; preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the first structural domain light chain is directly or indirectly connected to the C-end of the fifth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain, and the first structural domain is directly or indirectly connected to the third structural domain, and the first structural domain is directly or indirectly connected to the fourth structural domain, and the first structural domain is directly or indirectly connected to the fifth structural domain, and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the first structural domain light chain is directly or indirectly connected to the fourth structural domain, and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain;
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the fourth structural domain, the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the first structural domain light chain is directly or indirectly connected to the fourth structural domain, and the first structural domain light chain is directly or indirectly connected to the fifth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain;
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the fourth structural domain, the N-end of the first structural domain light chain is directly or indirectly connected to the C-end of the fifth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain; and the first structural domain is directly or indirectly connected to the fourth structural domain, and the first structural domain is directly or indirectly connected to the fifth structural domain, and the second structural domain is directly or indirectly connected to the third structural domain, and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain heavy chain
  • the first structural domain heavy chain is directly or indirectly connected to the fourth structural domain
  • the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain
  • the second structural domain heavy chain is directly or indirectly connected to the third structural domain
  • the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain;
  • the second structural domain is directly or indirectly connected to the fourth structural domain heavy chain
  • the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the fourth structural domain
  • the N-end of the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain
  • the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain
  • the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the heavy chain of the fourth structural domain
  • the first structural domain heavy chain is directly or indirectly connected to the fourth structural domain
  • the first structural domain light chain is directly or indirectly connected to the fifth structural domain
  • the second structural domain heavy chain is directly or indirectly connected to the third structural domain
  • the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain;
  • the second structural domain is directly or indirectly connected to the heavy chain of the fourth structural domain
  • the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the fourth structural domain
  • the N-end of the first structural domain light chain is directly or indirectly connected to the fifth structural domain
  • the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain
  • the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain, and the first structural domain is directly or indirectly connected to the third structural domain, and the first structural domain is directly or indirectly connected to the fifth structural domain, and the second structural domain is directly or indirectly connected to the fourth structural domain, and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain;
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain, and the N-end of the second structural domain heavy chain is directly or indirectly connected to the C-end of the fourth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain, and the second structural domain light chain is directly or indirectly connected to the fourth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain; preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain, and the N end of the light chain of the second structural domain is directly or indirectly connected to the C end of the fourth structural domain, and the C end of the heavy chain of the second structural domain is directly or indirectly connected to the N end of the sixth structural domain; or preferably, the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain, and the C end of the first structural domain heavy chain is directly or indirectly connected to the N end of the third structural domain, and the N end of the first structural domain heavy chain is directly or indirectly connected to the C end of the fifth structural domain, and the C end of the second structural domain light chain is directly or indirectly connected to the N end of the fourth structural domain, and the C end of the second structural domain heavy chain is directly or indirectly connected to the N end of the sixth structural domain.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a first domain heavy chain from its N-terminus to its C-terminus; the third polypeptide includes a second domain heavy chain from its N-terminus to its C-terminus; and the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide and the fourth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus; the third polypeptide includes a second domain heavy chain from its N-terminus to its C-terminus; and the fourth polypeptide includes a domain heavy chain from its N-terminus to its C-terminus.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a domain heavy chain from its N-terminus to its C-terminus; the third polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus; and the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide and the fourth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus; the third polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus; and the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide and the fourth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes, from its N-terminus to its C-terminus, a first domain heavy chain CD3 antigen-binding fragment, a linker, a fourth domain heavy chain, a linker, and a third domain in sequence; the third polypeptide includes, from its N-terminus to its C-terminus, a second domain heavy chain, a linker, and a sixth domain in sequence; the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first, fourth, and fifth polypeptides may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide
  • the first polypeptide comprises, from its N-terminus to its C-terminus, a first domain light chain, a linker, and a fourth domain heavy chain
  • the second polypeptide comprises, from its N-terminus to its C-terminus, a first domain heavy chain, a linker, and a third domain
  • the third polypeptide comprises, from its N-terminus to its C-terminus, a second domain heavy chain, a linker, and a sixth domain
  • the fourth polypeptide comprises, from its N-terminus to its C-terminus, a second domain light chain
  • the fifth polypeptide comprises, from its N-terminus to its C-terminus, a fourth domain light chain; wherein the fourth polypeptide and the fifth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a fourth domain heavy chain TAA antigen-binding fragment, a linker, a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus; the third polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus; the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide, the fourth polypeptide, and the fifth polypeptide may or may not
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide
  • the first polypeptide comprises, from its N-terminus to its C-terminus, a fourth domain TAA antigen-binding fragment, a linker, and a light chain of a first domain
  • the second polypeptide comprises, from its N-terminus to its C-terminus, a first domain heavy chain, a linker, and a third domain
  • the third polypeptide comprises, from its N-terminus to its C-terminus, a second domain heavy chain, a linker, and a sixth domain
  • the fourth polypeptide comprises, from its N-terminus to its C-terminus, a second domain light chain
  • the fifth polypeptide comprises, from its N-terminus to its C-terminus, a fourth domain light chain; wherein the fourth polypeptide and the fifth polypeptide may or may
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus; the third polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus; the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide, the fourth polypeptide, and the fifth polypeptide may or may not be present
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus; the third polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus; the fourth polypeptide includes a second domain light chain, a linker, and a fourth domain TAA antigen-binding fragment from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide and the fifth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus; the third polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus; the fourth polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, and a second domain light chain linker from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide and the fifth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a fifth domain, a linker, and a first domain heavy chain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent; the third polypeptide includes a second domain heavy chain from its N-terminus to its C-terminus; and the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide and the fourth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, wherein the first polypeptide includes a fifth domain, a linker, and a light chain of the first domain sequentially from the N-terminus to the C-terminus, wherein the linker may be absent; the second polypeptide includes a heavy chain of the first domain from the N-terminus to the C-terminus; the third polypeptide includes a heavy chain of the second domain from the N-terminus to the C-terminus; and the fourth polypeptide includes a light chain of the second domain from the N-terminus to the C-terminus; wherein the fourth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a fifth domain, a linker, a first domain heavy chain CD3 antigen-binding fragment, a linker, and a fourth domain heavy chain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent; the third polypeptide includes a second domain heavy chain from its N-terminus to its C-terminus; the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide, the fourth polypeptide, and the fifth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a fifth domain, a linker, and a light chain of the first domain, wherein the linker may be absent;
  • the second polypeptide comprises, from N-terminus to C-terminus, a first domain heavy chain, a CD3 antigen-binding fragment, a linker, and a fourth domain heavy chain;
  • the third polypeptide comprises, from N-terminus to C-terminus, a second domain heavy chain;
  • the fourth polypeptide comprises, from N-terminus to C-terminus, a second domain light chain;
  • the fifth polypeptide comprises, from N-terminus to C-terminus, a fourth domain light chain; wherein the fourth and fifth polypeptides may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a first domain light chain, a linker, and a fourth domain heavy chain; the second polypeptide comprises, from N-terminus to C-terminus, a fifth domain, a linker, and a first domain heavy chain, wherein the linker may be absent; the third polypeptide comprises, from N-terminus to C-terminus, a second domain heavy chain; the fourth polypeptide comprises, from N-terminus to C-terminus, a second domain light chain; and the fifth polypeptide comprises, from N-terminus to C-terminus, a fourth domain light chain; wherein the fourth and fifth polypeptides may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a fifth domain, a linker, a light chain of the first domain, a linker, and a heavy chain of the fourth domain, wherein the linker may be absent; the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first domain, wherein the linker may be absent; the third polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the second domain; the fourth polypeptide comprises, from N-terminus to C-terminus, a light chain of the second domain; and the fifth polypeptide comprises, from N-terminus to C-terminus, a light chain of the fourth domain; wherein the fourth polypeptide and the fifth polypeptide may be present or
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a fifth domain, a linker, and a first domain heavy chain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent; the third polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, and a second domain heavy chain sequentially from its N-terminus to its C-terminus; the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide, the fourth polypeptide, and the fifth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a fifth domain, a linker, and a light chain of the first domain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent; the second polypeptide includes a heavy chain of the first domain sequentially from its N-terminus to its C-terminus; the third polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, and a heavy chain of the second domain sequentially from its N-terminus to its C-terminus; the fourth polypeptide includes a light chain of the second domain sequentially from its N-terminus to its C-terminus; and the fifth polypeptide includes a light chain of the fourth domain sequentially from its N-terminus to its C-terminus; wherein the fourth polypeptide and the fifth polypeptide, where
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a fifth domain, a linker, and a first domain heavy chain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent; the third polypeptide includes a second domain heavy chain from its N-terminus to its C-terminus; the fourth polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, and a second domain light chain linker sequentially from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide and the fifth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a fifth domain, a linker, and a light chain of the first domain, wherein the linker may be absent;
  • the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first domain;
  • the third polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the second domain;
  • the fourth polypeptide comprises, from N-terminus to C-terminus, a fourth domain TAA antigen-binding fragment, a linker, and a light chain linker of the second domain;
  • the fifth polypeptide comprises, from N-terminus to C-terminus, a light chain of the fourth domain; wherein the fifth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a fifth domain, a linker, and a first domain heavy chain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent; the third polypeptide includes a second domain heavy chain from its N-terminus to its C-terminus; the fourth polypeptide includes a second domain light chain, a linker, and a fourth domain TAA antigen-binding fragment sequentially from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide and the fifth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a fifth domain, a linker, and a light chain of the first domain, wherein the linker may be absent;
  • the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first domain;
  • the third polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the second domain;
  • the fourth polypeptide comprises, from N-terminus to C-terminus, a light chain of the second domain, a linker, and a TAA antigen-binding fragment of the fourth domain;
  • the fifth polypeptide comprises, from N-terminus to C-terminus, a light chain of the fourth domain; wherein the fifth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a fifth domain, a linker, a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent; the third polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus; and the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide and the fourth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, wherein the first polypeptide includes, from N-terminus to C-terminus, a fifth domain, a linker, and a light chain of the first domain, wherein the linker may be absent; the second polypeptide includes, from N-terminus to C-terminus, a heavy chain of the first domain, a linker, and a third domain; the third polypeptide includes, from N-terminus to C-terminus, a heavy chain of the second domain, a linker, and a sixth domain; the fourth polypeptide includes, from N-terminus to C-terminus, a light chain of the second domain; wherein the fourth polypeptide may or may not be present.
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes, from its N-terminus to its C-terminus, a fifth domain, a linker, a first domain heavy chain CD3 antigen-binding fragment, a linker, a fourth domain heavy chain, a linker, and a third domain in sequence, wherein the linker may be absent; the third polypeptide includes, from its N-terminus to its C-terminus, a second domain heavy chain, a linker, and a sixth domain in sequence; the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first poly
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a fifth domain, a linker, and a light chain of the first domain, wherein the linker may be absent;
  • the second polypeptide comprises, from N-terminus to C-terminus, a first domain heavy chain CD3 antigen-binding fragment, a linker, a fourth domain heavy chain, a linker, and a third domain;
  • the third polypeptide comprises, from N-terminus to C-terminus, a second domain heavy chain, a linker, and a sixth domain;
  • the fourth polypeptide comprises, from N-terminus to C-terminus, a second domain light chain;
  • the fifth polypeptide comprises, from N-terminus to C-terminus, a fourth domain light chain; wherein the
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a first domain light chain, a linker, and a fourth domain heavy chain; the second polypeptide comprises, from N-terminus to C-terminus, a fifth domain, a linker, a first domain heavy chain, a linker, and a third domain, wherein the linker may be absent; the third polypeptide comprises, from N-terminus to C-terminus, a second domain heavy chain, a linker, and a sixth domain; the fourth polypeptide comprises, from N-terminus to C-terminus, a second domain light chain; and the fifth polypeptide comprises, from N-terminus to C-terminus, a fourth domain light chain; wherein the fourth and fifth polypeptides may or may not be present
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide, from its N-terminus to its C-terminus, sequentially includes a fifth domain, a linker, a light chain of the first domain, a linker, and a heavy chain of the fourth domain, wherein the linker may be absent; the second polypeptide, from its N-terminus to its C-terminus, sequentially includes a heavy chain of the first domain, a linker, and a third domain; the third polypeptide, from its N-terminus to its C-terminus, sequentially includes a heavy chain of the second domain, a linker, and a sixth domain; the fourth polypeptide, from its N-terminus to its C-terminus, includes a light chain of the second domain; the fifth polypeptide, from its N-terminus to its C-terminus, includes a
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a fifth domain, a linker, a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent; the third polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus; the fourth polypeptide includes a second domain light chain from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide includes a first
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide comprises, from its N-terminus to its C-terminus, a fifth domain, a linker, and a light chain of the first domain, wherein the linker may be absent;
  • the second polypeptide comprises, from its N-terminus to its C-terminus, a heavy chain of the first domain, a linker, and a third domain, wherein the linker may be absent;
  • the third polypeptide comprises, from its N-terminus to its C-terminus, a fourth domain (TAA antigen-binding fragment), a linker, a heavy chain of the second domain, a linker, and a sixth domain;
  • the fourth polypeptide comprises, from its N-terminus to its C-terminus, a light chain of the second domain;
  • the fifth polypeptide comprises
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a fifth domain, a linker, a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus, wherein the linker may or may not be present; the third polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus; the fourth polypeptide includes a second domain light chain, a linker, and a fourth domain TAA antigen-binding fragment from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a fifth domain, a linker, and a light chain of the first domain, wherein the linker may be absent;
  • the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first domain, a linker, and a third domain;
  • the third polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the second domain, a linker, and a sixth domain;
  • the fourth polypeptide comprises, from N-terminus to C-terminus, a light chain of the second domain, a linker, and a TAA antigen-binding fragment of the fourth domain;
  • the fifth polypeptide comprises, from N-terminus to C-terminus, a light
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide includes a first domain light chain from its N-terminus to its C-terminus; the second polypeptide includes a fifth domain, a linker, a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent; the third polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus; the fourth polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, and a second domain light chain linker from its N-terminus to its C-terminus; and the fifth polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus; wherein the first polypeptide includes
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a fifth domain, a linker, and a light chain of the first domain, wherein the linker may be absent;
  • the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first domain, a linker, and a third domain, wherein the linker may be absent;
  • the third polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the second domain, a linker, and a sixth domain;
  • the fourth polypeptide comprises, from N-terminus to C-terminus, a fourth domain TAA antigen-binding fragment, a linker, and a light chain linker of the second domain;
  • the fifth polypeptide comprises, from N-terminus
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, and/or a fourth polypeptide, and/or a fifth polypeptide, wherein the first polypeptide comprises, from N-terminus to C-terminus, a first domain light chain, a linker, and a fourth domain heavy chain; the second polypeptide comprises, from N-terminus to C-terminus, a fifth domain, a linker, a first domain heavy chain, a linker, and a third domain, wherein the linker may be absent; the third polypeptide comprises, from N-terminus to C-terminus, a second domain heavy chain, a linker, and a sixth domain; the fourth polypeptide comprises, from N-terminus to C-terminus, a second domain light chain; and the fifth polypeptide comprises, from N-terminus to C-terminus, a fourth domain light chain; wherein the fourth and fifth polypeptides may or may not be present
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 21, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 46, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 167, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 177, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 21, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 77, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 167, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 190, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3227, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 46, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3232, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 177, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3230, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 46, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3231, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 46, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 21, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 62, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3225, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3222, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3226, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3223, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3225, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3216, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3226, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3217, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3227, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 77, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3228, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 77, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3229, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 77, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3230, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 77, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3231, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 77, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3233, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3216, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3234, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3217, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3235, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 177, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3236, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 177, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3237, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 177, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3238, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 177, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3232, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 190, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3232, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 184, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1007, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1005, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1020, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1008, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1006, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 21, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1005, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3232, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1006, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 21, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1018, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3232, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1019, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 21, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2000, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 167, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2015, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 342, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2016, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 168, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2017, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2021, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2000, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2024, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2000, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2027, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2000, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2022, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2000, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2023, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2000, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2030, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2015, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 21, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2010, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 167, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2018, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2030, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2018, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein comprises a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 21, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 46, wherein the second polypeptide and the third polypeptide are linked by disulfide bonds in the heavy chain Fc structure or form a heterodimer (e.g., by pestle-mortar pairing in the heavy chain Fc domain).
  • the fusion protein includes a first polypeptide, and/or a second polypeptide, and/or a third polypeptide, wherein the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 11, the second polypeptide includes the amino acid sequence selected from SEQ ID NO: 440, SEQ ID NO: 443-476, and the third polypeptide includes the amino acid sequence shown in SEQ ID NO: 46.
  • the indirect connection includes a connection via a connector sub-connection.
  • the linker comprises an amino acid sequence selected from the group consisting of: SS, GS, GGS, GSGS, SEQ ID NO: 5 to SEQ ID NO: 10.
  • the present invention also provides an immunoconjugate comprising the fusion polypeptide as described above.
  • the present invention also provides a nucleic acid molecule that encodes the fusion polypeptide as described above.
  • the present invention also provides a carrier comprising the nucleic acid molecules described above.
  • the present invention also provides a cell comprising and/or expressing the fusion polypeptide, immune conjugate, nucleic acid molecule, and/or carrier as described above.
  • the present invention also provides a composition comprising the fusion peptide, immunoconjugate, nucleic acid molecule, and/or carrier, and/or cell, as described above, and optionally a pharmaceutically acceptable carrier.
  • the present invention also provides a method for preparing the fusion polypeptide as described above, comprising culturing cells as described above under conditions that enable the fusion polypeptide to be expressed.
  • the present invention also provides a method for inhibiting the growth and/or proliferation of tumors or tumor cells, comprising administering an effective amount of the fusion polypeptide, immune conjugate, nucleic acid molecule, and/or carrier, cell, and/or composition as described above.
  • the present invention also provides the use of the fusion peptides, immunoconjugates, nucleic acid molecules, and/or carriers, cells, and/or compositions described above in the preparation of medicaments, wherein the medicaments are used for the prevention, improvement, and/or treatment of tumors.
  • the tumor includes solid tumors and/or hematomas; the tumors include, but are not limited to, colon tumors, breast tumors, lung tumors, gastric tumors, melanoma, head and neck tumors, lymphoma, nasopharyngeal tumors, cervical tumors, esophageal tumors, kidney tumors, squamous cell carcinoma of the skin, endometrial tumors, liver tumors, bladder tumors, urothelial tumors, and skin tumors.
  • the tumors include, but are not limited to, colon tumors, breast tumors, lung tumors, gastric tumors, melanoma, head and neck tumors, lymphoma, nasopharyngeal tumors, cervical tumors, esophageal tumors, kidney tumors, squamous cell carcinoma of the skin, endometrial tumors, liver tumors, bladder tumors, urothelial tumors, and skin tumors.
  • Figure 1A shows an exemplary structural diagram of the multifunctional fusion peptide of this application containing CD3, TAA and CD86/CD80 variants.
  • Figure 1B shows an exemplary structural diagram of the multifunctional fusion peptide of this application containing a first domain, a second domain, a third domain, a fourth domain, and a fifth domain.
  • Figure 1C shows an exemplary structural diagram of the multifunctional fusion peptide of this application, which may contain a first structural domain, a second structural domain, a third structural domain, and a fourth structural domain.
  • the fourth or sixth structural domain involved in the antibody may not be present.
  • Figure 1D shows an exemplary structural diagram of the multifunctional fusion peptide of this application, which may contain a first domain, a second domain, a fourth domain, and a fifth domain.
  • the fourth domain which is involved in the antibody-dependent synthesis, may not be present.
  • Figure 1E shows an exemplary format of the multifunctional fusion peptide of this application containing a first domain, a second domain, a third domain, a fourth domain, and a fifth domain.
  • Figure 2A shows the structures of CD3xHer2 fusion peptide complexes TCB 160, TCB 161, PAT-003, and PAT-004 containing different CD86 mutants, and their corresponding CD3xHer2 bispecific antibodies TCB 160 and TCB 161, which do not contain CD86.
  • Figure 2B shows the killing effect of CD3xHer2 fusion peptide complexes TCB 160, TCB 161, PAT-003, PAT-004 containing different CD86 mutants and CD3xHer2 fusion peptide complex TCB 160 without CD86 on HCC824 of SHP77.
  • Figure 3A shows the antibody structures of CD3x5T4 fusion polypeptide complexes TCB 340, PAT-026, and PAT-027 containing different CD86 mutants, and their corresponding CD3x5T4 bispecific antibody TCB 340 without CD86.
  • Figure 3B shows the binding ability of CD3x5T4 fusion peptide complexes TCB 340, PAT-026, and PAT-027 containing different CD86 mutants, and their corresponding CD3x5T4 bispecific antibody TCB 340 without CD86, to tumor target cells H1975.
  • Figure 3C shows the killing effect of CD3x5T4 fusion peptide complexes TCB 340, PAT-026, and PAT-027 containing different CD86 mutants, and their corresponding CD3x5T4 bispecific antibody TCB 340 without CD86 on H226.
  • Figure 4A shows the antibody structures of CD3xEGFR fusion peptide complexes AN_PB_Ab_111, AN_PB_Ab_123, and AN_PB_Ab_124 containing different CD86 mutants, and their corresponding CD3xEGFR bispecific antibody AN_PB_Ab_110 which does not contain CD86.
  • Figure 4B shows the SDS-PAGE electrophoresis results of CD3xEGFR fusion peptide complexes AN_PB_Ab_111, AN_PB_Ab_123, and AN_PB_Ab_124 containing different CD86 mutants and their corresponding CD3xEGFR bispecific antibody AN_PB_Ab_110 without CD86.
  • Figure 4C shows the killing effect of CD3xEGFR fusion peptide complexes AN_PB_Ab-111, AN_PB_Ab-123, and AN_PB_Ab-124 containing different CD86 mutants and CD3xEGFR fusion peptide complex AN_PB_Ab-110 without CD86 on SHP77.
  • Figure 5A shows the antibody construction format used in the screening of CD3-masked peptides and the construction format of the CD3-unmasked control antibody (AN_TCB_160).
  • Figure 5B shows the binding ability of different masking peptide antibodies to Jurkat cells.
  • Figure 5C shows the results of different masking peptide antibodies regulating the killing of NUGC-4 tumor cells by primary PBMC cells before and after MTSP1 enzyme digestion.
  • Figure 6A shows the structures of CD3x5T4 fusion polypeptide complexes TCB 340, PAT-026, and PAT-027 containing different masking peptides and the same enzyme digestion fragments, and their corresponding CD3x5T4 bispecific antibody TCB 339 without masking function.
  • Figure 6B shows the binding ability of CD3x5T4 fusion polypeptide complexes TCB 340, PAT-026, and PAT-027, containing different masking peptides and the same digested fragments, and their corresponding CD3x5T4 bispecific antibody TCB 339 without masking function to tumor target cells H1975 before and after MTSP-1 digestion.
  • Figure 6C shows the killing effect of CD3x5T4 fusion polypeptide complexes TCB 340, PAT-026, and PAT-027, which contain different masking peptides and the same digestion fragments, before and after MTSP-1 digestion, and their corresponding CD3x5T4 bispecific antibody TCB 339 without masking function on H226.
  • Figure 7A shows the structural diagrams of the CD3xEGFR bispecific antibodies AN_PB_Ab-118, AN_PB_Ab-119, and AN_PB_Ab-120 with CD3 binding domain masking function and the CD3xEGFR bispecific antibody AN_PB_Ab-110 without CD3 binding domain masking function of this application.
  • Figure 7B shows the SDS-PAGE electrophoresis results of the CD3xEGFR bispecific antibodies AN_PB_Ab-118, AN_PB_Ab-119, and AN_PB_Ab-120 with CD3 binding domain masking function and the CD3xEGFR bispecific antibody AN_PB_Ab-110 without CD3 binding domain masking function of this application.
  • Figure 7C shows the results of detecting the binding ability of AN_PB_Ab-110, AN_PB_Ab-118, AN_PB_Ab-119, and AN_PB_Ab-120 to human PBMCs before and after enzyme digestion.
  • Figure 7D shows the regulation of primary PBMC cells killing H358 tumor target cells by CD3xEGFR bispecific antibodies AN_PB_Ab-118, AN_PB_Ab-119, and AN_PB_Ab-120 with CD3 binding domain masking function before and after enzyme digestion, and by CD3xEGFR bispecific antibody AN_PB_Ab-110 without CD3 binding domain masking function.
  • Figure 8A shows the killing effect of CD3xHer2 fusion peptide complexes AN_TCB_325, AN_CLA_003, AN_CLA_004, AN_CLA_005, and AN_CLA_006, which contain the same masking peptide but different digestion fragments, before and after MTSP-1 digestion, and their corresponding CD3xHer2 bispecific antibody TCB 160 without masking function on N87.
  • Figure 9A shows the structures of CD3x5T4 fusion polypeptide complexes A PAT-021, PAT-024, and PAT-025 containing the same masking peptide but different enzyme fragments, and their corresponding non-masking CD3x5T4 bispecific antibody TCB 339.
  • Figure 9B shows the binding ability of CD3x5T4 fusion polypeptide complexes A PAT-021, PAT-024, and PAT-025, containing the same masking peptide but different digestion fragments, and their corresponding CD3x5T4 bispecific antibody TCB 339 without masking function to tumor target cells H1975 before and after MTSP-1 digestion.
  • Figure 9C shows the killing effect of CD3x5T4 fusion polypeptide complexes A PAT-021, PAT-024, and PAT-025, containing the same masking peptide but different digestion fragments, and their corresponding CD3x5T4 bispecific antibody TCB 339 without masking function on H226 before and after MTSP-1 digestion.
  • Figure 10A shows the antibody structures of the CD3xEGFR bispecific antibodies AN_PB_Ab-118, AN_PB_Ab-121, and AN_PB_Ab-122 with CD3 binding domain masking function and the CD3xEGFR bispecific antibody AN_PB_Ab-110 without CD3 binding domain masking function of this application.
  • Figure 10B shows the SDS-PAGE electrophoresis results of the CD3xEGFR bispecific antibodies AN_PB_Ab-118, AN_PB_Ab-121, and AN_PB_Ab-122 with CD3 binding domain masking function and the CD3xEGFR bispecific antibody AN_PB_Ab-110 without CD3 binding domain masking function of this application.
  • Figure 10C shows the results of detecting the binding ability of AN_PB_Ab-118, AN_PB_Ab-121, AN_PB_Ab-122 before and after enzyme digestion, and AN_PB_Ab-110 for human PBMCs.
  • Figure 10D shows the regulation of primary PBMC cells killing H358 tumor target cells by CD3xEGFR bispecific antibody AN_PB_Ab-118 with CD3 binding domain masking function before and after enzyme digestion, and by CD3xEGFR bispecific antibody AN_PB_Ab-110 without CD3 binding domain masking function.
  • Figure 11A shows the antibody structures of CD3xHer2 fusion polypeptide complexes TCB 327, PAT-012, and AT-013 containing the same masking peptide, the same enzyme digestion fragment, and different CD86 mutants, and their corresponding CD3xHer2 bispecific antibody TCB 160.
  • Figure 11B shows the N87 binding ability of CD3xHer2 fusion polypeptide complexes TCB 327, PAT-012, AT-013, and their corresponding CD3xHer bispecific antibody TCB 160 to tumor target cells before and after MTSP-1 digestion, which contain the same masking peptide, the same digestion fragment, and different CD86 mutants.
  • Figure 11C shows the killing effect of CD3xHer2 fusion polypeptide complexes TCB 327, PAT-012, AT-013, and their corresponding CD3xHer2 bispecific antibody TCB 160 on N87 before and after MTSP-1 digestion, which contain the same masking peptide, the same digestion fragment, and different CD86 mutants.
  • Figure 12A shows the antibody structures of CD3xHer2 fusion polypeptide complexes TCB 327, PAT-014, and PAT-015 containing different masking peptides, the same enzyme digestion fragments, and the same CD86 mutant, and their corresponding CD3xHer2 bispecific antibody TCB 160.
  • Figure 12B shows the binding ability of CD3xHer2 fusion polypeptide complexes TCB 327, PAT-014, PAT-015, containing different masked peptides, the same digested fragments, and the same CD86 mutants, and their corresponding CD3xHer2 bispecific antibody TCB 160 to N87 tumor target cells before and after MTSP-1 digestion.
  • Figure 12C shows the SW480 killing effects of CD3xHer2 fusion polypeptide complexes TCB 327, PAT-014, PAT-015, containing different masked peptides, the same digested fragments, and the same CD86 mutants, before and after MTSP-1 digestion, and their corresponding CD3xHer2 bispecific antibody TCB 160.
  • Figure 12D shows the tolerance test of TCB 327 and TCB 160 in human CD3xHer2 double transgenic mice.
  • Figure 12E shows the pharmacokinetic analysis of TCB327 and TCB160 in mice.
  • Figure 12F shows the comparison of the inhibitory effects of PAT-014 and the masked version on tumor growth in the PBMC-NUGC4 mouse model.
  • Figure 13A shows the antibody structures of CD3xHer2 fusion polypeptide complexes TCB 327, PAT-016, and PAT-017 containing the same masking peptide, different enzyme fragments, and the same CD86 mutant, and their corresponding CD3xHer2 bispecific antibody TCB 160.
  • Figure 13B shows the binding ability of CD3xHer2 fusion polypeptide complexes TCB 327, PAT-016, PAT-017, and their corresponding CD3xHer2 bispecific antibody TCB 160 to N87 tumor target cells before and after MTSP-1 digestion, which contain the same masking peptide, different digestion fragments, and the same CD86 mutant.
  • Figure 13C shows the SW480 killing effects of CD3xHer2 fusion polypeptide complexes TCB 327, PAT-016, PAT-017, and their corresponding CD3xHer2 bispecific antibody TCB 160, which contain the same masking peptide, different digestion fragments, and the same CD86 mutant, before and after MTSP-1 digestion.
  • Figure 14A shows the antibody structures of AN_PB_Ab-110, AN_PB_Ab-111, and AN_PB_Ab-112 of this application.
  • Figure 14B shows the SDS-PAGE electrophoresis results of AN_PB_Ab-110, AN_PB_Ab-111, and AN_PB_Ab-112 of this application.
  • Figure 14C shows the results of detecting the binding ability of AN_PB_Ab-110 and AN_PB_Ab-111 to human PBMCs before and after AN_PB_Ab-112 digestion.
  • Figure 14D shows the regulation of primary PBMC cells killing H358 tumor target cells by AN_PB_Ab-111 and AN_PB_Ab-110 before and after AN_PB_Ab-112 digestion.
  • Figure 14E shows the results of the CD3xEGFR fusion peptide complex of this application regulating the activation and expression of CD69 in primary PBMC cells in the absence of relevant tumor cells.
  • Figure 15A shows the antibody structures of AN_PB_Ab-17, AN_DL_Ab-028, and AN_DL_Ab-055 of this application.
  • Figure 15B shows the SDS-page electrophoresis diagrams of AN_PB_Ab-17, AN_DL_Ab-028, and AN_DL_Ab-055 of this application.
  • Figure 15C shows the results of detecting the binding ability of AN_DL_Ab-028 and AN_DL_Ab-055 to human PBMCs before and after AN_PB_Ab-17 digestion.
  • Figure 15D shows the regulation of primary PBMC cells killing SHP77 tumor target cells by AN_DL_Ab-028 and AN_DL_Ab-055 before and after AN_PB_Ab-17 digestion.
  • Figure 16A shows the structural diagrams of the CD3xHHLA2 trispecific antibodies AN_CHH_15 and AN_CHH_38 (containing CD3 binding domain masking function and CD86 variant peptide) and the CD3xHHLA2 bispecific antibodies AN_CHH_12 and AN_CHH_36 (not containing CD3 binding domain masking function) in this application.
  • Figure 16B shows the results of SDS-PAGE analysis of the expression of the mask-linker-CD3/HHLA2/CD86 and CD3/HHLA2 fusion multifunctional peptides in this application.
  • Figure 16C shows the results of flow cytometry analysis of the binding levels of the mask-linker-CD3/HHLA2/CD86 and CD3/HHLA2 fusion multifunctional peptides of this application to Jurkat E6-1 cells.
  • Figure 16D shows the kinetics of the binding of the mask-linker-CD3/HHLA2/CD86 and CD3/HHLA2 fusion multifunctional peptides of this application to human HHLA2 protein, as detected by OCTET.
  • Figure 16E shows the kinetics of the binding of the mask-linker-CD3/HHLA2/CD86 and CD3/HHLA2 fusion multifunctional peptides of this application to human CD3 protein using OCTET.
  • Figure 16F shows the results of regulating the killing of HCC827 (HHLA2-highly expressed) tumor cells by primary PBMC cells before and after MTSP1 restriction enzyme digestion with the mask-linker-CD3/HHLA2/CD86 fusion multifunctional peptide of this application (effect-target ratio of 1:1).
  • Figure 17A shows the structural diagrams of the CD3xCEA trispecific antibody AN_CEA_21 (containing CD3 binding domain masking function and CD86 variant peptide) and the CD3xCEA bispecific antibody AN_CEA_19 (not containing CD3 binding domain masking function) in this application.
  • Figure 17B shows the results of SDS-PAGE detection of the expression of the mask-linker-CD3/CEA/CD86 and CD3/CEA fusion multifunctional peptides in this application.
  • Figure 17C shows the results of flow cytometry analysis of the binding levels of the mask-linker-CD3/CEA/CD86 and CD3/CEA fusion multifunctional peptides of this application to MKN45 cells.
  • Figure 17D shows the kinetics of the binding of the mask-linker-CD3/CEA/CD86 and CD3/CEA fusion multifunctional peptides of this application to human CD3 protein using OCTET.
  • Figure 17E shows the results of regulating the killing of MKN45 (CEA-high expression) and HT-29 (CEA-low expression) tumor cells by the mask-linker-CD3/CEA/CD86 fusion multifunctional peptide of this application before and after MTSP1 restriction enzyme digestion (effect-target ratio of 2:1).
  • the term "regulation of T cell immune response” generally refers to the regulation of lymphocyte T cell function by the multifunctional fusion polypeptide disclosed in this application.
  • it can also be extended to the effects of any other related T cell regulators, such as influencing T cell NFAT transcription factor expression, secretion of cytokines like IL-2, IFN- ⁇ , TNF ⁇ , and Granzyme B, cell proliferation, and cell killing of target cells, including tumor cells, after T cell treatment.
  • the T cells can be extended to T cell lines and/or primary T cells, including but not limited to CD4 T cells and CD4 T cell subsets Th1, Th2, Th9, Th17, TFH, and/or Treg cells; it also includes CD8 T cells, encompassing but not limited to tumor-infiltrating CD8 T cells, effector CD8 T cells, and immune memory CD8 T cells.
  • CD3 refers to the CD3 protein on T cells, generally referring to a protein complex belonging to the immunoglobulin superfamily, comprising four subunits: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • the CD3 protein plays a crucial role in the T cell receptor (TCR) complex, promoting T cell signal transduction and activation.
  • TCR T cell receptor
  • the CD3 protein interacts with the ⁇ and ⁇ chains of the TCR-CD3 complex, forming the core structure for signal transduction.
  • CD3 refers to any naturally occurring CD3 from any human source.
  • the term also encompasses "full-length" and unprocessed proteins, as well as any form of protein or one or more CD3 chains (peptides) derived from cell-processed proteins (e.g., mature peptides).
  • the term also encompasses naturally occurring variants and isotypes of CD3, such as splice variants or allelic variants.
  • www.uniprot.org/uniprot/P04234, www.uniprot.org/uniprot/P07766, and www.uniprot.org/uniprot/P09693 provide descriptions of CD3 ⁇ chains, CD3 ⁇ chains, and CD3 ⁇ chains and sequences.
  • the antibodies described herein can be derived from any animal source, including birds and mammals, including primates.
  • the antibodies are antibodies from humans, baboons, rhesus monkeys, cynomolgus monkeys, mice, donkeys, rabbits, goats, guinea pigs, camels, llama, horses, or chickens.
  • anti-CD3 antibody includes antibodies that specifically recognize a single CD3 subunit (e.g., B, ⁇ , ⁇ , or ⁇ ) and their antigen-binding fragments, as well as antibodies that specifically recognize a dimer complex of two CD3 subunits (e.g., ⁇ / ⁇ , ⁇ / ⁇ , and ⁇ / ⁇ CD3 dimers) and their antigen-binding fragments.
  • monoclonal antibodies including human, humanized, chimeric, or mouse antibodies, target the CD3 receptor in the T cell antigen receptor of mature T cells.
  • an anti-CD3 antibody could be a CD3SP34 antibody or its antigen-binding fragment.
  • tumor-associated antigen refers to antigen molecules that are not specific to a particular tumor and are also present on other tumor cells or normal cells.
  • Tumor-associated antigens used for clinical diagnosis include embryonic proteins, glycoprotein antigens, squamous cell antigens, etc. Tumor-associated antigens are not specific to tumor cells, but differ only in quantity during proliferation. Normal cells also synthesize trace amounts, hence the term "associated antigen.” Tumors of the same tissue type induced by the same carcinogen may possess the same tumor-associated antigens in different individuals.
  • the bispecific antibody is an artificial antibody prepared through cell fusion, recombinant DNA, protein engineering, and other techniques. It can simultaneously or sequentially bind specifically to two antigens or two different epitopes of the same antigen. BsAb can recognize and bind to two different antigens or antigenic epitopes, thus enabling it to connect effector cells such as immune cells or cytokines to tumor cells, thereby enhancing the killing effect on target cells.
  • this bifunctional recombinant antibody as a drug for treating tumors, has higher efficacy than monoclonal antibody drugs, achieving a synergistic effect ("1+1>2").
  • a "multispecific antibody” is a class of antibodies possessing two different antigen-binding properties, capable of simultaneously binding to two different targets.
  • This antibody structure typically consists of two separate antibody-binding fragments that can bind two different antigens within the same molecule, or two identical antigens at different sites.
  • the advantage of multispecific antibodies lies in their ability to act on two different targets simultaneously or enhance the same target, strengthening their potential in treating various diseases.
  • multispecific antibodies can effectively enhance therapeutic effects, overcome the limitations of single-target therapy, and possess stronger targeting specificity.
  • multispecific antibodies by introducing two different antigen-binding sites into their structure, achieve specific recognition of tumor cells and activation of T cells, thereby effectively enhancing the anti-tumor immune response.
  • CD86 generally refers to a class of molecules that activate cells.
  • CD86 includes its full length, variants, and/or functionally active fragments.
  • CD86 can represent a polypeptide or fragment thereof that has at least about 85% amino acid identity with the protein encoded by the NCBI Registry Gene ID: 942 gene and has CD28 (the protein encoded by the NCBI Registry Gene ID: 940 gene) and/or CTLA4 (the protein encoded by the NCBI Registry Gene ID: 1493 gene) binding activity.
  • An exemplary human CD86 amino acid sequence (SEQ ID NO: 1) is provided below.
  • CD86 extracellular region refers to the amino acid sequence of the domain in the extracellular region of the CD86 protein, which has at least about 85% amino acid identity and has CD28 and/or CTLA4 binding activity.
  • An active polypeptide or fragment thereof an exemplary CD86 extracellular domain amino acid sequence (SEQ ID NO: 2) is provided below.
  • CD86 extracellular domain IgV domain and “CD86 IgV domain” refer to a polypeptide or fragment thereof that has an amino acid sequence of at least about 85% amino acid identity in the IgV-like domain of the extracellular domain of the CD86 protein and has CD28 and/or CTLA4 binding activity.
  • CD86 extracellular domain IgV domain amino acid sequence SEQ ID NO: 3
  • the number of CD86 extracellular domains or CD86 extracellular domain IgV domains tandemly disclosed in this invention is not limited to the number shown in the examples. Any tandem combination containing this functional domain is considered to be within the scope of this invention.
  • CD80 refers to a polypeptide or fragment thereof that has at least about 85% amino acid identity with the protein encoded by the NCBI accession number Gene ID: 941 gene and has binding activity for CD28 (the protein encoded by the NCBI accession number Gene ID: 940 gene) and/or CTLA4 (the protein encoded by the NCBI accession number Gene ID: 1493 gene).
  • An exemplary human CD80 amino acid sequence (SEQ ID NO: 1) is provided below.
  • CD80 extracellular region refers to a polypeptide or fragment thereof that has at least about 85% amino acid identity with the domain in the extracellular region of the CD80 protein and has binding activity for CD28 and/or CTLA4.
  • CD80 extracellular region amino acid sequence SEQ ID NO: 294.
  • the number of tandem sequences disclosed in this invention is not limited to the number shown in the examples, and any tandem combination containing this functional domain is considered to be within the scope of this invention.
  • the term "masking” refers to the process of using a specific amino acid sequence (i.e., a masking peptide) to spatially or conformally block a target domain, particularly the CD3-binding domain, thereby affecting the binding of that domain to its natural ligand or target under normal physiological conditions and inhibiting its activity or function. This masking effect is generally achieved through steric hindrance, conformational modulation, and conditional unmasking.
  • a specific amino acid sequence i.e., a masking peptide
  • the term “masking peptide” refers to a molecule capable of "shielding” or inhibiting the function of antibodies or other therapeutic proteins through a specific amino acid sequence or peptide segment.
  • the role of a masking peptide is to control and regulate protein function by binding to a target protein or antibody under specific conditions, thereby preventing its activity.
  • the primary function of the masking peptide is to inhibit the non-specific activation of the CD3 domain in normal tissues. In the absence of tumor signals, the masking peptide, by binding to the CD3 domain, prevents premature activation of T cells, reducing potential overreaction of the immune system or cytokine storm.
  • a “specific linker” is a molecular structure used to connect two molecules (such as proteins, peptides, or drugs).
  • specific linkers are often used to connect multiple functional units or domains, enabling them to exert synergistic effects under specific conditions.
  • the specific linker connects a masking peptide to a CD3 domain and, through a sequence containing specific cleavage sites, ensures that CD3 function is activated only in the tumor microenvironment; therefore, it is also referred to as an "enzyme-cleaved linker" in this invention.
  • specific linkers are typically designed to be cleaved by specific proteases (such as MMPs, FAP, uPA, etc.) in the tumor microenvironment, thereby achieving the regulated release of functional proteins.
  • specific proteases such as MMPs, FAP, uPA, etc.
  • the introduction of specific linkers can effectively control the spatiotemporal specificity of therapeutic proteins, enabling them to function at the target site or under the target conditions, while reducing the impact on normal tissues.
  • specific proteases in the tumor microenvironment can cleave the linker, releasing the masking peptide, thereby restoring CD3 function and achieving tumor-targeted activation of T cells.
  • fusion polypeptide generally refers to a polypeptide obtained by fusing two or more proteins or polypeptides.
  • fusion polypeptide fusion polypeptide complex
  • fusion protein fusion protein
  • a fusion polypeptide may comprise a fusion polypeptide complex.
  • Fusion polypeptides can be artificially prepared using recombinant DNA technology. For example, genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be linked together to form a fusion gene or fused nucleic acid molecule that encodes the fusion polypeptide. Translation of the fusion gene can produce a single polypeptide that may possess the properties of at least one, or even each, of the two or more proteins or polypeptides before fusion.
  • multifunctional fusion polypeptide generally refers to a polypeptide or protein formed by the fusion of polypeptides or their domains from one or more sources.
  • the anti-CD3 antibody is linked via the linker to a functional fusion polypeptide comprising at least the CD86 extracellular domain and at least the functional domain for binding tumor-associated antigen (TAA).
  • TAA tumor-associated antigen
  • antibody-associated antigen-binding fragment generally refers to an amino acid fragment in an antibody responsible for specific binding to an antigen.
  • This fragment can be a key differentiator in the antibody molecule, also referred to as an antigen-binding domain, or an "epitope" or "antigen determinant.”
  • the antigen-binding domain typically consists of a variable region (VH) of the antibody heavy chain and a variable region (VL) of the antibody light chain; however, it does not necessarily include both.
  • antigen-binding domains of the antibodies disclosed in this application are not limited to the conventional VH and VL domains, but also include antigen-binding domains contained in any other type of antibody, but not limited to recombinant antibodies, single-domain antibodies, heavy-chain antibodies, chimeric antibodies, bispecific antibodies, and other unconventional antibodies and combinations thereof.
  • anti-CD3 antibodies disclosed in this invention are not limited to traditional natural antibodies, but should include any other type of antibody with anti-CD3 antibody properties, but are not limited to, for example, recombinant antibodies, single-domain antibodies, heavy chain antibodies, chimeric antibodies, bispecific antibodies and other unconventional antibodies and combinations thereof.
  • antibody fragment or "antigen-binding fragment” in this invention refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, VHH, etc. Regardless of structure, antibody fragments that bind to the same antigen are considered complete antibodies.
  • antibody fragment includes aptamers, aptamer enantiomers, and dimers.
  • antibody fragment also includes any synthetic or genetically modified protein that, like an antibody, can bind to a specific antigen to form a complex.
  • Fab generally refers to an antibody fragment composed of VL, VH, CL and CH1 domains.
  • Fab' generally refers to an antibody fragment having several additional residues at the carboxyl terminus of the CH1 domain compared to a Fab fragment.
  • Fab' may include one or more cysteine residues from the antibody hinge region.
  • F(ab)2 generally refers to an antigen-binding fragment obtained from a pair of Fab fragments linked by cysteine.
  • dAb fragment generally refers to an antibody fragment composed of VH domains (Ward et al., Nature 341: 544 546 (1989)).
  • CDR complementary determinant region
  • VL light chain variable region
  • VH heavy chain variable region
  • Fv fragment generally refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.
  • scFv generally refers to a molecule composed of the variable regions of the antibody heavy chain and the variable regions of the light chain linked by a short peptide linker, also known as a single-chain antibody.
  • single-domain antibody generally refers to an antibody consisting only of the heavy chain variable region, also known as “VHH” or “nanobody”.
  • the term "immunoglobulin” refers to an antibody comprising an amino acid fragment that specifically binds to an antigen and a constant region fragment.
  • the antigen-specific binding region is the segment in the immunoglobulin that determines key differences; it can also be called an antigen-binding domain, or an "epitope" or "antigen determinant.”
  • the antigen-binding domain typically consists of the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL). However, it does not necessarily include both.
  • the antigen-binding domain of the antibody disclosed in this invention is not limited to the conventional VH and VL domain, but also includes the antigen-binding domains contained in any other type of antibody, but not limited to, recombinant antibodies, single-domain antibodies, heavy chain antibodies, chimeric antibodies, bispecific antibodies, and other unconventional antibodies and combinations thereof.
  • the constant region refers to the common structural region of the immunoglobulin, comprising the antibody light chain constant region and heavy chain constant region.
  • immunoglobulin Fc domain generally refers to the Fc fragment formed from one Fc fragment and two identical Fab fragments after conventional antibody IgG is hydrolyzed by papain.
  • the Fc domain may contain the CH2, CH3, and hinge regions of the antibody heavy chain.
  • Conventional Fc fragments have the function of binding to Fc fragment receptors to mediate related biological effects. Site-specific mutations can alter their binding ability to the corresponding target receptors, thus affecting their biological function.
  • the immunoglobulin Fc domain disclosed in this application should include, but is not limited to, conventional Fc fragments and any other forms of Fc mutants.
  • the biological functions that the immunoglobulin Fc domain can provide include, but are not limited to, prolonging half-life, improving molecular stability, facilitating the expression and detection of fusion proteins, mediating transplacental and mucosal barriers, mediating antibody-dependent cell-mediated cytotoxicity (ADCC), mediating inflammatory responses, mediating antibody-dependent cell-mediated phagocytosis (ADCP), mediating complement-dependent cytotoxicity (CDC), mediating dendritic cell (DC) maturation, regulating cytokine secretion, and regulating B cell proliferation and differentiation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • DC dendritic cell maturation
  • regulating cytokine secretion regulating B cell proliferation and differentiation.
  • Fab-Fc, scFv-Fc, and VHH-Fc are included in the scope of "heavy chain” or “antibody heavy chain” as referred to in this invention.
  • peptide and "protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • proteins, peptides, and/or amino acid sequences involved in this application should also be understood to include at least the following range: variants or homologs that have the same or similar functions as the said protein or peptide.
  • mutant or “variant” generally refers to a peptide substantially similar to the peptide in question, and may be a protein or polypeptide whose amino acid sequence has been substituted, deleted, or added with one or more amino acids.
  • the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and/or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids.
  • the functional variant may substantially retain the biological properties of the protein or polypeptide prior to the alteration (e.g., substitution, deletion, or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the protein or polypeptide prior to the alteration.
  • the substitution may be a conserved substitution.
  • the homolog can be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody that specifically binds to the protein or a fragment thereof).
  • the polypeptide e.g., an antibody that specifically binds to the protein or a fragment thereof.
  • homology generally refers to the similarity, resemblance, or association between two or more sequences.
  • sequence homology percentage can be calculated as follows: Two sequences to be aligned are compared in a comparison window, and the number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is determined to obtain the number of matching positions.
  • nucleic acid bases e.g., A, T, C, G, I
  • amino acid residues e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys
  • sequence homology percentage is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to produce the sequence homology percentage.
  • Alignment for determining the sequence homology percentage can be performed in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region. The homology can also be determined, but is not limited to, by FASTA and BLAST.
  • N-terminus generally refers to the end of the polypeptide chain where the amino acid residue carries a free amino group.
  • C-terminus generally refers to the end of the polypeptide chain where the amino acid residue carries a free carboxyl group.
  • nucleic acid molecule generally refers to any length of isolated nucleotide, deoxyribonucleotide, or ribonucleotide or analogue thereof, isolated from its natural environment or synthesized artificially.
  • vector refers to a nucleic acid delivery vehicle that can insert a polynucleotide encoding a protein therein and enable the protein to be expressed.
  • linker generally refers to a linker molecule that connects one or more polypeptides or their domains.
  • a linker can be conformationally flexible, a short peptide chain formed by the combination of amino acid Gly (G) and Ser (S) residues, wherein the ratio of the number of amino acid Gly to the number of amino acid Ser can be ⁇ 1.
  • the linkers disclosed in this application can be extended to any short peptide having this characteristic.
  • the following provides exemplary linker amino acid sequences including but not limited to GGGGS (SEQ ID NO: 5), GGGGSGGGGS (SEQ ID NO: 6), GGGGSGGGGSGGGGS (SEQ ID NO: 7), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 8), GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 9), and GGGGSGGGS (SEQ ID NO: 10).
  • the term "enzyme-cleavable linker” generally refers to a linker containing a protease-cleavable fragment, wherein the protease-cleavable fragment can be specifically recognized and cleaved by the corresponding protease.
  • the proteases include, but are not limited to, matrix metalloproteinases, urokinase, transmembrane serine proteases, transforming factor TGF ⁇ , plasminogen, glucosinolates, human liver collagen, human ⁇ -2-macroglobulin ( ⁇ 2M), and human PZP ⁇ -2-macroglobulin-like protein (PZP), etc.
  • amino acid mutation generally refers to amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, as long as the final construct possesses the desired properties.
  • the amino acid mutation is a substitution.
  • amino acid mutation at a position refers to the substitution or deletion of a specified residue or the insertion of at least one amino acid residue adjacent to a specified residue.
  • the amino acid substitution can be conserved or non-conserved.
  • A13I can represent the substitution of alanine (A, Ala) at position 13 with isoleucine (I, Ile).
  • active fragment generally refers to a nucleic acid or amino acid fragment or variant that has a certain biological activity or function.
  • a functionally active fragment may retain or partially retain the ability of a full-length protein to bind to another molecule.
  • active fragment “functionally active fragment,” or “active functional fragment” are generally used interchangeably.
  • CD86 active fragment refers to a polypeptide or fragment thereof that has binding activity with CD28 and/or CTLA4, including but not limited to features such as regulating T cell activity, activating the immune system, and anti-tumor activity.
  • CD86 variant peptide refers to a protein or peptide whose amino acid sequence has been modified by substitution, deletion, or addition of one or more amino acids.
  • the functional variant may comprise a protein or peptide with amino acid alterations resulting from substitution, deletion, and/or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids.
  • the functional variant may substantially retain the biological characteristics of CD86 prior to the alteration (e.g., substitution, deletion, or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the protein or peptide prior to the alteration.
  • the substitution may be a conserved substitution.
  • CD86 variant peptide means an active fragment derived from the CD86 protein and possessing the functional characteristics of the CD86 protein, including but not limited to CD28 and/or CTLA4 binding activity.
  • CD80 variant peptide refers to a protein or peptide whose amino acid sequence has been altered by substitution, deletion, or addition of one or more amino acids.
  • the functional variant may comprise a protein or peptide with amino acid modifications resulting from substitution, deletion, and/or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids.
  • the functional variant may substantially retain the biological characteristics of CD80 prior to the alteration (e.g., substitution, deletion, or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the protein or peptide prior to the alteration.
  • the substitution may be a conserved substitution.
  • CD80 variant peptide means an active fragment derived from the CD80 protein and possessing the functional characteristics of the CD80 protein, including but not limited to CD28 and/or CTLA4 binding activity.
  • the co-stimulatory receptors include, but are not limited to, ICOSL, CD40L, CD137L, OX40L, CD80, CD83, and CD86.
  • the antigen phagocytosis includes, but is not limited to, the phagocytosis of bacteria, viruses, proteins, polysaccharides, etc.
  • the cytokines include, but are not limited to, IL-1beta, TNF ⁇ , IFN ⁇ , IL-6, and IL-12.
  • the chemokines include, but are not limited to, CCL1, CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL2, CXCL3, CXCL4, and CXCL5.
  • the term "about” generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.
  • This invention provides a fusion polypeptide comprising a first domain, a second domain, and a third domain.
  • the first domain binds CD3, the second domain binds a tumor-associated antigen (TAA), and the third domain binds CD28 and/or CTLA4.
  • this invention may include a fourth domain that binds a TAA. This fourth domain may bind the same or different TAA as the second domain. The fourth domain may have the same or different sequence and/or structure as the second domain. The fourth domain may be present or absent.
  • this invention may include a fifth domain that conditionally masks the CD3-binding domain in the first domain. This fifth domain may be present or absent.
  • this invention may include a sixth domain that binds CD28 and/or CTLA4. This sixth domain may have the same or different sequence and/or structure as the third domain. The sixth domain may be present or absent.
  • Figures 1A-1E show exemplary, not limiting, structural diagrams of the first, second, third, fourth, and fifth structural domains of the functional fusion peptide of the present invention.
  • the first structural domain can combine with CD3.
  • the first domain contains an antibody or an antigen-binding fragment thereof.
  • the first domain may comprise HCDR3 of the antibody heavy chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 304, SEQ ID NO: 309, SEQ ID NO: 314, and SEQ ID NO: 319.
  • the first domain may comprise HCDR2 of the antibody heavy chain, which may comprise an amino acid sequence selected from the group shown below: SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 44.
  • the first domain may comprise HCDR1 of the antibody heavy chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 43.
  • the first structural domain shown may include a heavy chain variable region VH of the antibody heavy chain, which may contain an amino acid sequence selected from the group shown: SEQ ID NO: 22, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 301, SEQ ID NO: 306, SEQ ID NO: 311, SEQ ID NO: 316.
  • the first domain may comprise an antibody heavy chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 300, SEQ ID NO: 305, SEQ ID NO: 310, SEQ ID NO: 315.
  • the first domain may comprise LCDR3 of an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 20, and SEQ ID NO: 41.
  • the first domain may comprise LCDR2 of an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 40.
  • the first domain may comprise LCDR1 of an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 39.
  • the first domain may include a light chain variable region VL of the antibody light chain
  • the antibody light chain variable region VL may include an amino acid sequence selected from the group shown below: SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 38.
  • the first domain may comprise an antibody light chain, which may comprise an amino acid sequence selected from the group shown below: SEQ ID NO: 11, SEQ ID NO: 16.
  • the fusion polypeptide includes a first domain comprising an antibody, an antigen-binding fragment thereof, or a variant thereof selected from the group consisting of: antibody OKT3, antibody UCHT1, antibody SP34, Blinatumamab, Tebentafusp, Mosunetuzumab, and Teclistamab.
  • the first domain may comprise an antibody SP34 mutant or its antigen-binding site.
  • the first domain may comprise an antibody SP34 mutant or its antigen-binding site, the mutation occurring in the CDR region.
  • the first domain may comprise an antibody SP34 mutant or its antigen-binding site, the mutation occurring in the HCDR region, the mutation comprising one or more amino acid site mutations selected from the group shown in SEQ ID NO: 22 of the antibody SP34 heavy chain variable region: T31D, R50K, N100D, N97S, F100fH, S100aA, V100cT;
  • the first domain may comprise an antibody SP34 mutant or its antigen-binding site, the mutation occurring in the LCDR region, the mutation comprising one or more amino acid site mutations selected from the group shown in SEQ ID NO: 12 of the antibody SP34 light chain variable region: N94Q.
  • the antibody may be selected from immunoglobulin antibodies, recombinant antibodies, chimeric antibodies, heavy chain antibodies, single-domain antibodies and/or bispecific antibodies;
  • the antigen-binding fragment may be selected from Fab, Fab’, Fv, F(ab)2, F(ab’)2, scFv, di-scFv, VHH and/or dAb.
  • the first domain includes a single-chain immunoglobulin domain.
  • the single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the first domain includes the Fc domain of a single-chain immunoglobulin IgG antibody, wherein the Fc domain of the single-chain immunoglobulin IgG antibody includes a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.
  • the Fc domain of the single-chain immunoglobulin IgG antibody includes a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.
  • the first domain single-chain immunoglobulin Fc fragment contains CH2 and/or CH3 sequences, and contains any amino acid sequence selected from the following group: SEQ ID NO: 3, SEQ ID NO: 4.
  • the first structural domain CH2 is located between the scFv fragment and the CH3 structural domain.
  • the first structure does not include the CH1 structural domain.
  • the first domain does not contain light chains.
  • the second or fourth domain can bind to one or more of the targets shown in the following group: PD-L1, PD-L2, VEGF, VEGFR, FGFR, HER2, HGFR, PIP-LAR, CD44, CD147, TfR, CDCP1, ⁇ 6 ⁇ 4, ⁇ 6 ⁇ 3, Trop2, HHLA2, GCC, 5T4, EpCAM, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, DLL3, B7H3, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, PTK7, EphA2, NKG2A, Nkp36 and/or Tim3.
  • the second or fourth domain binds to proteins or tumor antigens overexpressed on tumor cells relative to the corresponding non-tumor cells, such as CD20, Her2, PD-L1, HHLA2, GCC, EGFR, DLL3 and/or B7H3, 5T4 and/or CEA.
  • proteins or tumor antigens overexpressed on tumor cells such as CD20, Her2, PD-L1, HHLA2, GCC, EGFR, DLL3 and/or B7H3, 5T4 and/or CEA.
  • the second or fourth domain may comprise HCDR3 of the antibody heavy chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 50, SEQ ID NO: 61, SEQ ID NO: 66, SEQ ID NO: 71, SEQ ID NO: 76, SEQ ID NO: 81, SEQ ID NO: 86, SEQ ID NO: 96, SEQ ID NO: 2009, SEQ ID NO: 2014.
  • the second or fourth domain may comprise HCDR2 of the antibody heavy chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 49, SEQ ID NO: 60, SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 75, SEQ ID NO: 80, SEQ ID NO: 85, SEQ ID NO: 95, SEQ ID NO: 2008, SEQ ID NO: 2013.
  • the second or fourth domain may comprise HCDR1 of the antibody heavy chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 48, SEQ ID NO: 59, SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 74, SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 94, SEQ ID NO: 2007, SEQ ID NO: 2012.
  • the second or fourth domain shown may include a heavy chain variable region VH of the antibody heavy chain, which may contain an amino acid sequence selected from the group shown: SEQ ID NO: 47, SEQ ID NO: 58, SEQ ID NO: 63, SEQ ID NO: 68, SEQ ID NO: 73, SEQ ID NO: 83, SEQ ID NO: 93, SEQ ID NO: 2006, SEQ ID NO: 2011.
  • the second or fourth domain may comprise an antibody heavy chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 57, SEQ ID NO: 62, SEQ ID NO: 67, SEQ ID NO: 72, SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 92, SEQ ID NO: 2000, SEQ ID NO: 2010.
  • the second or fourth domain may comprise the LCDR3 of the antibody light chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 56, SEQ ID NO: 91, SEQ ID NO: 2005.
  • the second or fourth domain may comprise LCDR2 of the antibody light chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 55, SEQ ID NO: 90, SEQ ID NO: 2004.
  • the second or fourth domain may comprise LCDR1 of an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 54, SEQ ID NO: 89, SEQ ID NO: 2003.
  • the second or fourth domain may include a light chain variable region VL of the antibody light chain, which may contain an amino acid sequence selected from the group consisting of: SEQ ID NO: 53, SEQ ID NO: 88, SEQ ID NO: 2002.
  • the second or fourth domain may comprise an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 52, SEQ ID NO: 87.
  • the second or fourth domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 52, SEQ ID NO: 57, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 92, SEQ ID NO: 2000, SEQ ID NO: 2001, SEQ ID NO: 2010, SEQ ID NO: 2011.
  • the fourth structural domain may be associated with the same or different TAAs as the second structural domain, may have the same or different sequences and/or structures, and may or may not exist. This is also true in the more specific descriptions below, and will not be repeated here.
  • the second or fourth domain may contain HCDR3, HCDR2 and HCDR1 of the antibody heavy chain, wherein HCDR1, HCDR2 and HCDR3 may contain amino acid sequences as shown in SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively.
  • the second or fourth domain may comprise LCDR3, LCDR2, and LCDR1 of the antibody light chain, wherein LCDR1, LCDR2, and LCDR3 may comprise the amino acid sequences shown in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively.
  • the second or fourth domain may comprise HCDR3, HCDR2, HCDR1 of the antibody heavy chain and LCDR3, LCDR2, and LCDR1 of the antibody light chain, wherein HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61, respectively, and LCDR1, LCDR2, and LCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively.
  • the second or fourth domain may include a heavy chain variable region VH of the antibody heavy chain
  • the antibody heavy chain VH may include an amino acid sequence as shown in SEQ ID NO: 58
  • the second domain may include a light chain variable region VL of the antibody light chain
  • the antibody light chain VL may include an amino acid sequence as shown in SEQ ID NO: 52.
  • the second or fourth domain may be Trastuzumab or its antigen-binding fragment.
  • the second or fourth domain can be a single-domain antibody.
  • the second or fourth domain may comprise CDR3, CDR2, and CDR1 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively.
  • the second or fourth domain may contain the heavy chain variable region VHH of a single-domain antibody, wherein the VHH may contain an amino acid sequence as shown in SEQ ID NO: 47.
  • the second or fourth domain may be Her2-VHH or its antigen-binding fragment.
  • the second or fourth domain contains a single-chain immunoglobulin domain.
  • the single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the second and/or fourth domains may contain the Fc domain of a single-chain immunoglobulin IgG antibody, wherein the Fc domain of the single-chain immunoglobulin IgG antibody may contain a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.
  • the second or fourth domain single-chain immunoglobulin Fc fragment contains CH2 and/or CH3 sequences and contains any amino acid sequence selected from the following group: SEQ ID NO: 3, SEQ ID NO: 4.
  • the second or fourth domain may comprise CDR3, CDR2, and CDR1 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively.
  • the second or fourth domain may contain the heavy chain variable region VHH of a single-domain antibody, wherein the VHH may contain an amino acid sequence as shown in SEQ ID NO: 68.
  • the second or fourth domain may be PDL1-VHH-Nb02 NM-01 or its antigen-binding fragment, wherein PDL1-VHH-Nb02 NM-01 is derived from WO 2019/052508 A1.
  • the second and/or fourth domains contain single-chain immunoglobulin domains.
  • the single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the second and/or fourth domains may contain the Fc domain of a single-chain immunoglobulin IgG antibody, wherein the Fc domain of the single-chain immunoglobulin IgG antibody may contain a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.
  • the second and/or fourth domain single-chain immunoglobulin Fc fragment contains CH2 and/or CH3 sequences, and contains any amino acid sequence selected from the following group: SEQ ID NO: 3, SEQ ID NO: 4.
  • the second or fourth domain may comprise CDR3, CDR2, and CDR1 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively.
  • the second or fourth domain may contain the heavy chain variable region VHH of a single-domain antibody, wherein the VHH may contain an amino acid sequence as shown in SEQ ID NO: 78.
  • the second or fourth domain may be h5T4 VHH-HuNb1 40 or its antigen-binding fragment, wherein h5T4 VHH-HuNb1 40 is derived from CN117624366A.
  • the second or fourth domain contains a single-chain immunoglobulin domain.
  • the single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the second or fourth domain may contain the Fc domain of a single-chain immunoglobulin IgG antibody, wherein the Fc domain of the single-chain immunoglobulin IgG antibody may contain a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.
  • the second and/or fourth domain single-chain immunoglobulin Fc fragment contains CH2 and/or CH3 sequences, and contains any amino acid sequence selected from the following group: SEQ ID NO: 3, SEQ ID NO: 4.
  • the second or fourth domain can bind to HHLA2 and contains an antibody or its antigen-binding fragment.
  • the second or fourth domain may comprise CDR3, CDR2 and CDR1 of a single-domain antibody, wherein CDR1, CDR2 and CDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 1002, SEQ ID NO: 1003 and SEQ ID NO: 1004.
  • the second and/or fourth domains may contain the heavy chain variable region VHH of a single-domain antibody, wherein the VHH may contain the amino acid sequence shown in SEQ ID NO: 1001.
  • the second or fourth domain contains a single-chain immunoglobulin domain.
  • the single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the second or fourth domain may contain the Fc domain of a single-chain immunoglobulin IgG antibody, wherein the Fc domain of the single-chain immunoglobulin IgG antibody may contain a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.
  • the second or fourth domain contains the Fc domain of single-chain human immunoglobulin IgG1 and has an L234A/L235A mutation.
  • the second or fourth domain contains the Fc domain of single-stranded human immunoglobulin IgG1, has an L234A/L235A mutation, and has the necessary mutations in the knobs-into-holes (KiH) structure pair, such as: T366 on the CH3 domain of the Knob structure is replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W); or, T366 on the CH3 domain of the Hole structure is replaced by serine (S), L368 is replaced by a relatively small amino acid residue, such as alanine (A), and Y407 is replaced by a relatively small amino acid residue, such as threonine (T), alanine (A), or valine (V).
  • KiH knobs-into-holes
  • the second or fourth domain single-chain immunoglobulin Fc fragment contains CH2 and/or CH3 sequences and contains any amino acid sequence selected from the following group: SEQ ID NO: 3, SEQ ID NO: 4.
  • the second or fourth domain can be a single-domain antibody.
  • the second or fourth domain may comprise CDR3, CDR2, and CDR1 of a single-domain antibody, wherein CDR1, CDR2, and CDR3 may comprise amino acid sequences as shown in SEQ ID NO: 2012, SEQ ID NO: 2013, and SEQ ID NO: 2014, respectively.
  • the second or fourth domain may contain the heavy chain variable region VHH of a single-domain antibody, wherein the VHH may contain an amino acid sequence as shown in SEQ ID NO: 2011.
  • the second or fourth domain may be DLL3-VHH 52D04 or its antigen-binding fragment, wherein DLL3-VHH 52D04 is derived from patent CN113286817A.
  • the second or fourth domain contains a single-chain immunoglobulin domain.
  • the single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the second or fourth domain may contain the Fc domain of a single-chain immunoglobulin IgG antibody, wherein the Fc domain of the single-chain immunoglobulin IgG antibody may contain a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.
  • the second or fourth domain single-chain immunoglobulin Fc fragment contains CH2 and/or CH3 sequences and contains any amino acid sequence selected from the following group: SEQ ID NO: 3, SEQ ID NO: 4.
  • the second and/or fourth domains may comprise HCDR3, HCDR2, and HCDR1 of the antibody heavy chain, wherein HCDR1, HCDR2, and HCDR3 may comprise the amino acid sequences shown in SEQ ID NO: 1011, SEQ ID NO: 1012, and SEQ ID NO: 1013, respectively.
  • the second and/or fourth domains may comprise LCDR3, LCDR2, and LCDR1 of the antibody light chain, wherein LCDR1, LCDR2, and LCDR3 may comprise the amino acid sequences shown in SEQ ID NO: 1015, SEQ ID NO: 1016, and SEQ ID NO: 1017, respectively.
  • the second and/or fourth domains may comprise HCDR3, HCDR2, HCDR1 of the antibody heavy chain and LCDR3, LCDR2, and LCDR1 of the antibody light chain, wherein HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 1011, SEQ ID NO: 1012, and SEQ ID NO: 1013, respectively, and LCDR1, LCDR2, and LCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 1015, SEQ ID NO: 1016, and SEQ ID NO: 1017, respectively.
  • the second or fourth domain may include a heavy chain variable region VH of the antibody heavy chain
  • the antibody heavy chain VH may include an amino acid sequence as shown in SEQ ID NO: 1010
  • the second and/or fourth domain may include a light chain variable region VL of the antibody light chain
  • the antibody light chain VL may include an amino acid sequence as shown in SEQ ID NO: 1014.
  • the second domain and/or the fourth domain may be CEA (Cergutuzumab) or its antigen-binding fragment, wherein CEA (Cergutuzumab) is derived from patent number US8642742B2.
  • the second or fourth domain contains a single-chain immunoglobulin domain.
  • the single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the second or fourth domain may contain the Fc domain of a single-chain immunoglobulin IgG antibody, wherein the Fc domain of the single-chain immunoglobulin IgG antibody may contain a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.
  • the second or fourth domain single-chain immunoglobulin Fc fragment contains CH2 and/or CH3 sequences and contains any amino acid sequence selected from the following group: SEQ ID NO: 3, SEQ ID NO: 4.
  • the second or fourth domain can be an SCFV single-chain antibody.
  • the second or fourth domain may contain HCDR3, HCDR2 and HCDR1 of the antibody heavy chain, wherein HCDR1, HCDR2 and HCDR3 may contain amino acid sequences as shown in SEQ ID NO: 2007, SEQ ID NO: 2008 and SEQ ID NO: 2009, respectively.
  • the second or fourth domain may comprise LCDR3, LCDR2, and LCDR1 of the antibody light chain, wherein LCDR1, LCDR2, and LCDR3 may each comprise the amino acid sequences shown in SEQ ID NO: 2003, SEQ ID NO: 2004, and SEQ ID NO: 2005, respectively.
  • the second or fourth domain may comprise HCDR3, HCDR2, HCDR1 of the antibody heavy chain and LCDR3, LCDR2, and LCDR1 of the antibody light chain, wherein HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 2007, SEQ ID NO: 2008, and SEQ ID NO: 2009, respectively, and LCDR1, LCDR2, and LCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 2003, SEQ ID NO: 2004, and SEQ ID NO: 2005, respectively.
  • the second or fourth domain may include a heavy chain variable region VH of the antibody heavy chain
  • the antibody heavy chain VH may include an amino acid sequence as shown in SEQ ID NO: 2006
  • the second and/or fourth domain may include a light chain variable region VL of the antibody light chain
  • the antibody light chain VL may include an amino acid sequence as shown in SEQ ID NO: 2002-.
  • the second or fourth domain may be necituzumab or its antigen-binding fragment.
  • the second or fourth domain contains a single-chain immunoglobulin domain.
  • the single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the second and/or fourth domains may contain the Fc domain of a single-chain immunoglobulin IgG antibody, wherein the Fc domain of the single-chain immunoglobulin IgG antibody may contain a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.
  • the second or fourth domain single-chain immunoglobulin Fc fragment contains CH2 and/or CH3 sequences and contains any amino acid sequence selected from the following group: SEQ ID NO: 3, SEQ ID NO: 4.
  • the second or fourth domain may contain HCDR3, HCDR2 and HCDR1 of the antibody heavy chain, wherein HCDR1, HCDR2 and HCDR3 may contain amino acid sequences as shown in SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96, respectively.
  • the second or fourth domain may comprise LCDR3, LCDR2, and LCDR1 of the antibody light chain, wherein LCDR1, LCDR2, and LCDR3 may comprise the amino acid sequences shown in SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, respectively.
  • the second or fourth domain may comprise HCDR3, HCDR2, HCDR1 of the antibody heavy chain and LCDR3, LCDR2, and LCDR1 of the antibody light chain, wherein HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively, and LCDR1, LCDR2, and LCDR3 may comprise amino acid sequences as shown in SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, respectively.
  • the second or fourth domain may include a heavy chain variable region VH of the antibody heavy chain
  • the antibody heavy chain VH may include an amino acid sequence as shown in SEQ ID NO: 93
  • the second domain may include a light chain variable region VL of the antibody light chain
  • the antibody light chain VL may include an amino acid sequence as shown in SEQ ID NO: 88.
  • the second or fourth domain may be Mosunetuzumab or its antigen-binding fragment.
  • the antibody with the second or fourth domain may be selected from immunoglobulin antibodies, recombinant antibodies, chimeric antibodies, heavy chain antibodies, single-domain antibodies and/or bispecific antibodies;
  • the antigen-binding fragment may be selected from Fab, Fab’, Fv, F(ab)2, F(ab’)2, scFv, di-scFv, VHH and/or dAb.
  • the second and/or fourth domains contain single-chain immunoglobulin domains.
  • the single-chain immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.
  • the second and/or fourth domains may contain the Fc domain of a single-chain immunoglobulin IgG antibody, wherein the Fc domain of the single-chain immunoglobulin IgG antibody may contain a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.
  • the second and/or fourth domain single-chain immunoglobulin Fc fragment contains CH2 and/or CH3 sequences, and contains any amino acid sequence selected from the following group: SEQ ID NO: 3, SEQ ID NO: 4.
  • the third or sixth domain can be combined with CD28 and/or CTLA4.
  • the third or sixth domain contains CD86 or CD80 or a functionally active fragment thereof, or/and a variant of CD86 or CD80.
  • the third or sixth domain is selected from the group consisting of CD86 or CD80 or their functionally active fragments derived from humans or mice.
  • the third or sixth domain contains the IgV domain of CD86 or CD80 or a functionally active fragment thereof.
  • the third or sixth domain contains the extracellular domain of CD86 or CD80 or a functionally active fragment thereof.
  • the third or sixth domain contains a CD86 variant polypeptide, and the mutant contains an amino acid substitution mutation of humanized CD86.
  • CD86 mutant disclosed in PCT/CN2023/134527 is incorporated herein by reference.
  • the third or sixth domain comprises a CD86 variant peptide, which comprises an IgV domain amino acid substitution mutant of CD86 and/or an extracellular domain amino acid substitution mutant of humanized CD86.
  • the third or sixth domain comprises a CD86 variant polypeptide, the CD86 variant polypeptide comprising an IgV domain amino acid substitution mutant of CD86, the mutation site of the CD86 IgV domain amino acid substitution mutant comprising one, two, three, four, five or more amino acid site mutations selected from the group consisting of: A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F.
  • the third or sixth domain comprises a CD86 variant polypeptide, the CD86 variant polypeptide comprising an IgV domain amino acid substitution mutant of CD86, the amino acid substitution mutant comprising the combination shown below: Q25I/F33L/H90I, Q25V/F33L/H90I, Q25I/F33V/H90I, Q25V/F33L/H90V, Q25 V/F33L/H90V, Q25I/F33V/H90V, Q25V/F33V/H90V, A13L/Q25I/F33L/H90I, A13I/Q25I/F33L/H90I, A13L/Q25V/F33L/H90I, A13I/Q25V/F33L/H90I, Q25I/F33L/I89L/H90I, Q25I/F33L/M60R /H90I ⁇ Q25V/F33L/I89
  • the third or sixth domain comprises a CD86 variant polypeptide, which comprises the CD86 IgV domain amino acid substitution mutant Q25I/F33L/H90I.
  • the third or sixth domain comprises a CD86 variant polypeptide, the CD86 variant polypeptide comprising an extracellular domain amino acid substitution mutant of CD86, the extracellular domain amino acid substitution mutant of CD86 comprising one, two, three, four, five or more amino acid site mutations selected from the group consisting of: A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F.
  • the third or sixth domain comprises an extracellular domain amino acid substitution mutant of CD86, wherein the amino acid substitution mutant comprises the combination shown below: Q25I/F33L/H90I, Q25V/F33L/H90I, Q25I/F33V/H90I, Q25V/F33V/H90I, Q25I/F33L/H90V, Q25V/F33L/H90V, Q25 I/F33V/H90V, Q25V/F33V/H90V, A13L/Q25I/F33L/H90I, A13L/Q25 V/F33L/H90I, A13I/Q25V/F33L/H90I, Q25I/F33L/I89L/H90I, Q25I/F33L/M60R/H90I, Q25V /F33L/I89L/H90I, Q25V/F33L/M60R/H90I, Q25V /F33L/I89L/H
  • the third or sixth domain comprises an extracellular domain amino acid substitution mutant of CD86, the amino acid substitution mutant comprising the combination shown below: Q25I/F33L/H90I.
  • the third or sixth domain comprises an amino acid sequence as shown in SEQ ID NO: 97 to SEQ ID NO: 165.
  • the third or sixth domain contains a CD80 variant polypeptide, and the mutant contains an amino acid substitution mutation of humanized CD80.
  • the third or sixth domain comprises a CD80 variant peptide, which comprises an IgV domain amino acid substitution mutant of CD80 and/or an extracellular domain amino acid substitution mutant of humanized CD80.
  • CD80 mutant disclosed in PCT/CN2023/102271 is incorporated herein by reference.
  • the third structural domain and the sixth structural domain may have the same or different sequences and/or structures, and the sixth structural domain may or may not exist.
  • the fifth domain contains a peptide with CD3 binding domain masking function.
  • the peptide with CD3 binding domain masking function of the fifth domain includes peptides selected from the following that have strong masking effects: QDGAEEMGGGSQDGAEEMGGI (SEQ ID No: 404), QDGNEAMGGGSQDGNEAMGGI (SEQ ID No: 406), QDGNEEAGGGSQDGNEEAGGI (SEQ ID No: 407), QDGNEEMGGGSQDGNEEMAGI (SEQ ID No: 408), QDGNEEMGGGSQDGNEEMGAI (SEQ ID No: 409), QDGNEEMGGGSQDGNEEMGGA (SEQ ID No: 410), QDGNEDMGGGSQDGNEDMGG I(SEQ ID No:412),QDGNEDMGGGSQDGNEDMGSI(SEQ ID No:414),QDGNDEMGGGSQDGNDEMGSI(SEQ ID No:415),QDGNEEMGSITQTPYQVSISGT(SEQ ID No:424),QDGNEEMGSIGGGGS Q
  • the peptide with CD3 binding domain masking function in the fifth domain comprises peptides selected from the following with moderate masking effect: QDANEEMGGGSQDANEEMGGI (SEQ ID No: 403), QDGNAEMGGGSQDGNAEMGGI (SEQ ID No: 405), QDGNEEMGGGSQDGNEEMGSI (SEQ ID No: 411), QDGNDEMGGGSQDGNDEMGGI (SEQ ID No: 413), QEGNEEMGGGSQEGNEEMGSI (SEQ ID No: 416), QEGNEEMGGGSQEGNEEMGGI (SEQ ID No: 417), QDGNEEMGGITQTPYKVSISGT (SEQ ID No: 41).
  • the fifth domain having a CD3 binding domain masking peptide includes peptides selected from the following with weaker masking effects: ADGNEEMGGGSADGNEEMGGI (SEQ ID No: 401), QAGNEEMGGGSQAGNEEMGGI (SEQ ID No: 402), ADGNAEMGGGSADGNAEMGGI (SEQ ID No: 429), QAGNAEMGGGSQAGNAEMGGI (SEQ ID No: 430), QDANAEMGGGSQDANAEMGGI (SEQ ID No: 431).
  • the AN_PB series of antibodies were constructed by introducing different masking peptides into the AN_TCB_160 antibody for the identification of masking function.
  • the masking effect was evaluated by the strength of binding ability with human PBMCs or Jurkat cells.
  • MFI AN_PB refers to the MFI value of the corresponding AN_PB series antibody at 100 nM binding to PBMCs or Jurkat cells
  • MFI igG refers to the MFI value of human igG at 100 nM binding to PBMCs or Jurkat cells
  • MFI AN_TCB_160 refers to the MFI value of AN_TCB_160 binding to PBMCs or Jurkat cells. If the masking effect is ⁇ 99%, it is considered strong masking; if 90% ⁇ masking effect ⁇ 99%, it is considered medium masking; and if the masking effect ⁇ 90%, it is considered weak masking.
  • the fifth domain also contains an enzyme-liganded linker.
  • the enzyme cleavage linker of the ground five domain contains a protease cleavage site fragment.
  • the enzyme cleavage linker of the fifth domain contains one, two, three, four, five or more protease cleavage sites selected from the amino acid sequence of SEQ ID NO:3000-3036.
  • the protease cleavage site fragment of the fifth domain corresponds to protease types including, but not limited to, matrix metalloproteinases (MMPs): PLGLWA (SEQ ID NO: 3000), PLGLAGS (SEQ ID NO: 3001), GPLGLWA (SEQ ID NO: 3002), GPLGLWAQ (SEQ ID NO: 3003), GVPDLGRFQTFE (SEQ ID NO: 3004), GVPDVGHFSLFP (SEQ ID NO: 3005), GVPDVGHFSLFP (SEQ ID NO: 3006), GVPDVGHFSLFP (SEQ ID NO: 3007), GVPDVGHFSLFP (SEQ ID NO: 3008), GVPDVGHFSLFP (SEQ ID NO: 3009), GVPDVGHFSLFP (SEQ ID NO: 30 ...
  • MMPs matrix metalloproteinases
  • the protease cleavage site fragment of the fifth domain includes, but is not limited to, matrix metalloproteinases (MMPs) selected from the group shown below: PLGLWA (SEQ ID NO: 3000), PLGLAGS (SEQ ID NO: 3001), GPLGLWA (SEQ ID NO: 3002), GPLGLWAQ (SEQ ID NO: 3003), GVPDLGRFQTFE (SEQ ID NO: 3004), GVPDVGHFSLFP (SEQ ID NO: 3005), GVPDVGEFSLFP (SEQ ID NO: 3006), GVPDVGEFSLFP (SEQ ID NO: 3007), GVPDVGEFSLFP (SEQ ID NO: 3008), GVPDVGHFSLFP (SEQ ID NO: 3009), GVPDVGEFSLFP (SEQ ID NO: 3000), GVPDVGHFSLFP (SEQ ID NO: 30 ...
  • MMPs matrix metalloproteinases
  • the protease cleavage site fragment of the fifth domain includes, but is not limited to, the following pre-combined cleavage site fragments: LSGRSDNHGSPLGLAGS (SEQ ID NO:3025), LSGRSDNHGGSPLGLAGS (SEQ ID NO:3026), PLGLAGSGGSLSGRSDNH (SEQ ID NO:3027), PLGLGSLSGRSDNH (SEQ ID NO:3028), LSGRSDNHSPAGLAGS (SEQ ID NO:3029), LSGRSDNHSPGALGA S(SEQ ID NO:3030), LSGRSDNHSPAGLGGS(SEQ ID NO:3031), LSGRSDNHSPAGLRGS(SEQ ID NO:3032), PAGLAGSGGSLSGRSDNH(SEQ ID N O:3033), PGALGASGGSLSGRSDNH (SEQ ID NO:3034), PAGLGGSGGSLSGRSDNH (SEQ ID NO:3035), PAGLRGSGGSLSGRSDNH (SEQ ID NO:
  • the protease cleavage site fragments of the fifth domain are linked by linkers, the amino acid sequences of which include, but are not limited to, GS, GGS, GSGS, GGGGS (SEQ ID NO: 5), GGGGSGGGGS (SEQ ID NO: 6), GGGGSGGGGSGGGGS (SEQ ID NO: 7), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 8), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9), and GGGGSGGGS (SEQ ID NO: 10).
  • the linker amino acid sequence is located between two protease cleavage site fragments, between a masking peptide fragment and a protease cleavage site fragment, or between a protease cleavage site fragment and a first, second, or fourth domain.
  • the masking peptide fragment of the fifth domain is directly or indirectly linked to the protease cleavage site fragment of the fifth domain.
  • the C-terminus of the masking peptide fragment of the fifth domain is directly or indirectly linked to the N-terminus of the protease cleavage site fragment of the fifth domain.
  • the C-terminus of the masking peptide fragment of the fifth domain is directly linked to the N-terminus of the protease cleavage site fragment of the fifth domain via a linker, and the amino acid sequence includes, but is not limited to, GS, GGS, GSGS, GGGGS (SEQ ID NO: 5), GGGGSGGGGS (SEQ ID NO: 6), GGGGSGGGGSGGGGS (SEQ ID NO: 7), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 8), GGGGSGGGGSGGGGSGGGSG (SEQ ID NO: 9), and GGGGSGGGS (SEQ ID NO: 10).
  • the fifth structural domain may or may not exist.
  • the heavy chain of the first structural domain is directly connected to the heavy chain of the second structural domain.
  • the heavy chain Fc segments of the first and second structural domains are connected by a knobs-into-holes structure.
  • knots-into-holes pairing occurs by including one or more substitutions in the Fc segment of the heavy chain, which form heterodimeric pairings between the heavy chains.
  • CH3 in the first domain and CH3 in the second domain form a pestle-and-mortar substitution pair.
  • the CH3 of the first domain and the CH3 of the second domain form a club-and-mortar substitution pair, such as T366 on one CH3 domain being replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W); L368 on another CH3 domain being replaced by a relatively small amino acid residue, such as alanine (A); and Y407 on another CH3 domain being replaced by a relatively small amino acid residue, such as threonine (T), alanine (A), or valine (V).
  • T366 on one CH3 domain being replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W)
  • L368 on another CH3 domain being replaced by a relatively small amino acid residue, such as alanine (A)
  • Y407 on another CH3 domain being replaced by a relatively small amino acid residue, such as threonine (T), alanine (A), or valine (V).
  • the antigen-binding fragment of the first domain is directly or indirectly linked to the antigen-binding fragment of the second domain.
  • the indirect connection includes a connection via a linker.
  • the linker comprises an amino acid sequence selected from the group shown below: SEQ ID NO: 5 to SEQ ID NO: 10.
  • the first domain and the third domain are directly or indirectly connected.
  • the first domain heavy chain is directly or indirectly connected to the third domain.
  • the C-terminus of the first structural domain heavy chain is directly or indirectly connected to the N-terminus of the third structural domain.
  • the N-terminus of the first structural domain heavy chain is directly or indirectly connected to the C-terminus of the third structural domain.
  • the first domain light chain is directly or indirectly connected to the third domain.
  • the C-terminus of the first structural domain light chain is directly or indirectly connected to the N-terminus of the third structural domain.
  • the N-terminus of the first structural domain light chain is directly or indirectly connected to the C-terminus of the third structural domain.
  • first structural domain and the fourth structural domain are directly or indirectly connected.
  • the first domain heavy chain is directly or indirectly connected to the fourth domain.
  • the C-terminus of the first structural domain heavy chain is directly or indirectly connected to the N-terminus of the fourth structural domain.
  • the N-terminus of the first structural domain heavy chain is directly or indirectly connected to the C-terminus of the fourth structural domain.
  • the first domain light chain is directly or indirectly connected to the fourth domain.
  • the C-terminus of the first structural domain light chain is directly or indirectly connected to the N-terminus of the fourth structural domain.
  • the N-terminus of the first structural domain light chain is directly or indirectly connected to the C-terminus of the fourth structural domain.
  • the first domain and the fifth domain are directly or indirectly connected.
  • the first domain heavy chain is directly or indirectly connected to the fifth domain.
  • the C-terminus of the first structural domain heavy chain is directly or indirectly connected to the N-terminus of the fifth structural domain.
  • the N-terminus of the first structural domain heavy chain is directly or indirectly connected to the C-terminus of the fifth structural domain.
  • the first domain light chain is directly or indirectly connected to the fifth domain.
  • the C-terminus of the first structural domain light chain is directly or indirectly connected to the N-terminus of the fifth structural domain.
  • the N-terminus of the first structural domain light chain is directly or indirectly connected to the C-terminus of the fifth structural domain.
  • first structural domain and the sixth structural domain are directly or indirectly connected.
  • the first domain heavy chain is directly or indirectly connected to the sixth domain.
  • the C-terminus of the first structural domain heavy chain is directly or indirectly connected to the N-terminus of the sixth structural domain.
  • the N-terminus of the first structural domain heavy chain is directly or indirectly connected to the C-terminus of the sixth structural domain.
  • the first domain light chain is directly or indirectly connected to the sixth domain.
  • the C-terminus of the first structural domain light chain is directly or indirectly connected to the N-terminus of the sixth structural domain.
  • the N-terminus of the first structural domain light chain is directly or indirectly connected to the C-terminus of the sixth structural domain.
  • the second and third structural domains are directly or indirectly connected.
  • the second domain heavy chain is directly or indirectly connected to the third domain.
  • the C-terminus of the second structural domain heavy chain is directly or indirectly connected to the N-terminus of the third structural domain.
  • the N-terminus of the second structural domain heavy chain is directly or indirectly connected to the C-terminus of the third structural domain.
  • the second structural domain light chain is directly or indirectly connected to the third structural domain.
  • the C-terminus of the light chain in the second structural domain is directly or indirectly connected to the N-terminus of the third structural domain.
  • the N-terminus of the light chain in the second structural domain is directly or indirectly connected to the C-terminus of the third structural domain.
  • the second and fourth structural domains are directly or indirectly connected.
  • the second domain heavy chain is directly or indirectly connected to the fourth domain.
  • the C-terminus of the second structural domain heavy chain is directly or indirectly connected to the N-terminus of the fourth structural domain.
  • the N-terminus of the second structural domain heavy chain is directly or indirectly connected to the C-terminus of the fourth structural domain.
  • the second structural domain light chain is directly or indirectly connected to the fourth structural domain.
  • the C-terminus of the light chain of the second structural domain is directly or indirectly connected to the N-terminus of the fourth structural domain.
  • the N-terminus of the light chain of the second structural domain is directly or indirectly connected to the C-terminus of the fourth structural domain.
  • the heavy chain of the second structural domain is connected to the heavy chain Fc segment of the fourth structural domain through a knobs-into-holes structure.
  • knots-into-holes pairing involves one or more substitutions in the Fc segment of the heavy chain, which form heterodimer pairings between heavy chains.
  • CH3 in the second domain and CH3 in the fourth domain form a pestle-mortar substitution pair.
  • the CH3 of the second domain and the CH3 of the fourth domain form a club-and-mortar substitution pair, such as T366 on one CH3 domain being replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W); L368 on another CH3 domain being replaced by a relatively small amino acid residue, such as alanine (A); and Y407 on another CH3 domain being replaced by a relatively small amino acid residue, such as threonine (T), alanine (A), or valine (V).
  • T366 on one CH3 domain being replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W)
  • L368 on another CH3 domain being replaced by a relatively small amino acid residue, such as alanine (A)
  • Y407 on another CH3 domain being replaced by a relatively small amino acid residue, such as threonine (T), alanine (A), or valine (V).
  • the antigen-binding fragment of the second domain is directly or indirectly linked to the antigen-binding fragment of the fourth domain.
  • the indirect connection includes a sub-connection.
  • the linker comprises an amino acid sequence selected from the group shown below: SEQ ID NO: 5 to SEQ ID NO: 10.
  • the second and fifth structural domains are directly or indirectly connected.
  • the second domain heavy chain is directly or indirectly connected to the fifth domain.
  • the C-terminus of the second structural domain heavy chain is directly or indirectly connected to the N-terminus of the fifth structural domain.
  • the N-terminus of the second structural domain heavy chain is directly or indirectly connected to the C-terminus of the fifth structural domain.
  • the second structural domain light chain is directly or indirectly connected to the fifth structural domain.
  • the C-terminus of the light chain of the second structural domain is directly or indirectly connected to the N-terminus of the fifth structural domain.
  • the N-terminus of the light chain of the second structural domain is directly or indirectly connected to the C-terminus of the fifth structural domain.
  • the second and sixth structural domains are directly or indirectly connected.
  • the second domain heavy chain is directly or indirectly connected to the sixth domain.
  • the C-terminus of the second structural domain heavy chain is directly or indirectly connected to the N-terminus of the sixth structural domain.
  • the N-terminus of the second structural domain heavy chain is directly or indirectly connected to the C-terminus of the sixth structural domain.
  • the second structural domain light chain is directly or indirectly connected to the sixth structural domain.
  • the C-terminus of the second structural domain light chain is directly or indirectly connected to the N-terminus of the sixth structural domain.
  • the N-terminus of the light chain of the second structural domain is directly or indirectly connected to the C-terminus of the sixth structural domain.
  • the third and fourth structural domains are directly or indirectly connected.
  • the C-terminus of the third structural domain is directly or indirectly connected to the N-terminus of the fourth structural domain.
  • the N-terminus of the third structural domain is directly or indirectly connected to the C-terminus of the fourth structural domain.
  • the third and fifth structural domains are directly or indirectly connected.
  • the C-terminus of the third structural domain is directly or indirectly connected to the N-terminus of the fifth structural domain.
  • the N-terminus of the third structural domain is directly or indirectly connected to the C-terminus of the fifth structural domain.
  • the third and sixth structural domains are directly or indirectly connected.
  • the C-terminus of the third structural domain is directly or indirectly connected to the N-terminus of the sixth structural domain.
  • the N-terminus of the third structural domain is directly or indirectly connected to the C-terminus of the sixth structural domain.
  • the fourth and fifth structural domains are directly or indirectly connected.
  • the fourth structural domain heavy chain is directly or indirectly connected to the fifth structural domain.
  • the C-terminus of the heavy chain of the fourth structural domain is directly or indirectly connected to the N-terminus of the fifth structural domain.
  • the N-terminus of the heavy chain of the fourth structural domain is directly or indirectly connected to the C-terminus of the fifth structural domain.
  • the light chain of the fourth structural domain is directly or indirectly connected to the fifth structural domain.
  • the C-terminus of the light chain in the fourth structural domain is directly or indirectly connected to the N-terminus of the fifth structural domain.
  • the N-terminus of the light chain in the fourth structural domain is directly or indirectly connected to the C-terminus of the fifth structural domain.
  • the fourth and sixth structural domains are directly or indirectly connected.
  • the fourth structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the C-terminus of the heavy chain of the fourth structural domain is directly or indirectly connected to the N-terminus of the sixth structural domain.
  • the N-terminus of the heavy chain of the fourth structural domain is directly or indirectly connected to the C-terminus of the sixth structural domain.
  • the light chain of the fourth structural domain is directly or indirectly connected to the sixth structural domain.
  • the C-terminus of the light chain in the fourth structural domain is directly or indirectly connected to the N-terminus of the fifth structural domain.
  • the N-terminus of the light chain in the fourth structural domain is directly or indirectly connected to the C-terminus of the sixth structural domain.
  • the fifth and sixth structural domains are directly or indirectly connected.
  • the fifth structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the N-terminus of the fifth structural domain is directly or indirectly connected to the C-terminus of the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the N end of the first structural domain heavy chain is directly or indirectly connected to the C end of the third structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain light chain is directly or indirectly connected to the third structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the N end of the light chain of the first structural domain is directly or indirectly connected to the C end of the third structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the N end of the second structural domain heavy chain is directly or indirectly connected to the C end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C end of the second structural domain heavy chain is directly or indirectly connected to the N end of the sixth structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the light chain of the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the N end of the light chain of the second structural domain is directly or indirectly connected to the C end of the sixth structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the C-end of the light chain of the second structural domain is directly or indirectly connected to the N-end of the sixth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain, and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the N end of the first structural domain heavy chain is directly or indirectly connected to the C end of the third structural domain, and the N end of the second structural domain heavy chain is directly or indirectly connected to the C end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the N end of the first structural domain heavy chain is directly or indirectly connected to the C end of the third structural domain, and the C end of the second structural domain heavy chain is directly or indirectly connected to the N end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C end of the first structural domain heavy chain is directly or indirectly connected to the N end of the third structural domain, and the C end of the second structural domain heavy chain is directly or indirectly connected to the N end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the second structural domain heavy chain is directly or indirectly connected to the C-end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the second structural domain light and heavy chain is directly or indirectly connected to the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the N end of the first structural domain heavy chain is directly or indirectly connected to the C end of the third structural domain, and the N end of the second structural domain light chain is directly or indirectly connected to the C end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the N end of the first structural domain heavy chain is directly or indirectly connected to the C end of the third structural domain, and the C end of the second structural domain light chain is directly or indirectly connected to the N end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C end of the first structural domain heavy chain is directly or indirectly connected to the N end of the third structural domain, and the C end of the second structural domain light chain is directly or indirectly connected to the N end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain light chain is directly or indirectly connected to the third structural domain, and the second structural domain light and heavy chain is directly or indirectly connected to the sixth structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the N end of the light chain of the first structural domain is directly or indirectly connected to the C end of the third structural domain, and the N end of the light chain of the second structural domain is directly or indirectly connected to the C end of the sixth structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the N end of the light chain of the first structural domain is directly or indirectly connected to the C end of the third structural domain, and the C end of the light chain of the second structural domain is directly or indirectly connected to the N end of the sixth structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain, and the C-end of the light chain of the second structural domain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain light chain is directly or indirectly connected to the third structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the N end of the first structural domain light chain is directly or indirectly connected to the C end of the third structural domain, and the N end of the second structural domain heavy chain is directly or indirectly connected to the C end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the N end of the first structural domain light chain is directly or indirectly connected to the C end of the third structural domain, and the C end of the second structural domain heavy chain is directly or indirectly connected to the N end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C end of the first structural domain light chain is directly or indirectly connected to the N end of the third structural domain, and the C end of the second structural domain heavy chain is directly or indirectly connected to the N end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the second structural domain heavy chain is directly or indirectly connected to the C-end of the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain; and the first structural domain is directly or indirectly connected to the fourth structural domain; and the fourth structural domain is directly or indirectly connected to the third structural domain; and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain; and the first structural domain heavy chain is directly or indirectly connected to the fourth structural domain; and the fourth structural domain heavy chain is directly or indirectly connected to the third structural domain; and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain; and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the fourth structural domain; and the C-end of the heavy chain of the fourth structural domain is directly or indirectly connected to the N-end of the third structural domain; and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the sixth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the fourth structural domain; and the first structural domain is directly or indirectly connected to the third structural domain; and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain heavy chain is directly or indirectly connected to the fourth structural domain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain; and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the N end of the heavy chain of the first structural domain is directly or indirectly connected to the C end of the fourth structural domain; and the C end of the heavy chain of the first structural domain is directly or indirectly connected to the N end of the third structural domain, and the C end of the heavy chain of the second structural domain is directly or indirectly connected to the N end of the sixth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the light chain of the first structural domain is directly or indirectly connected to the fourth structural domain; and the heavy chain of the first structural domain is directly or indirectly connected to the third structural domain, and the heavy chain of the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the CN end of the light chain of the first structural domain is directly or indirectly connected to the C end of the fourth structural domain; and the C end of the heavy chain of the first structural domain is directly or indirectly connected to the third structural domain, and the C end of the heavy chain of the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the fourth structural domain; and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the third structural domain, and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the sixth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain; and the second structural domain is directly or indirectly connected to the fourth structural domain; and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain; and the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain; and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain; and the N-end of the heavy chain of the second structural domain is directly or indirectly connected to the C-end of the fourth structural domain; and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the sixth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the heavy chain of the first structural domain is directly or indirectly connected to the third structural domain; and the light chain of the second structural domain is directly or indirectly connected to the fourth structural domain; and the heavy chain of the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the light chain of the second structural domain is directly or indirectly connected to the C-end of the fourth structural domain; and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the C-end of the heavy chain of the first structural domain is directly or indirectly connected to the N-end of the third structural domain; and the C-end of the light chain of the second structural domain is directly or indirectly connected to the N-end of the fourth structural domain; and the C-end of the heavy chain of the second structural domain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the fifth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the N end of the first structural domain heavy chain is directly or indirectly connected to the C end of the fifth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain light chain is directly or indirectly connected to the fifth structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the N end of the light chain of the first structural domain is directly or indirectly connected to the C end of the fifth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the second structural domain is directly or indirectly connected to the fifth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the second structural domain heavy chain is directly or indirectly connected to the fifth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the N end of the second structural domain heavy chain is directly or indirectly connected to the C end of the fifth structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the light chain of the second structural domain is directly or indirectly connected to the fifth structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the N end of the light chain of the second structural domain is directly or indirectly connected to the C end of the fifth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain; and the first structural domain is directly or indirectly connected to the fourth structural domain, and the first structural domain is directly or indirectly connected to the fifth structural domain.
  • the second domain heavy chain is directly or indirectly connected to the fourth domain heavy chain; and the first domain heavy chain is directly or indirectly connected to the fourth domain, and the first domain heavy chain is directly or indirectly connected to the fifth domain.
  • the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain heavy chain; and the C end of the first structural domain heavy chain is directly or indirectly connected to the N end of the fourth structural domain, and the N end of the first structural domain heavy chain is directly or indirectly connected to the C end of the fifth structural domain.
  • the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the fourth structural domain, and the first structural domain light chain is directly or indirectly connected to the fifth structural domain.
  • the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain heavy chain; and the C end of the first structural domain heavy chain is directly or indirectly connected to the N end of the fourth structural domain, and the N end of the first structural domain light chain is directly or indirectly connected to the C end of the fifth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the fourth structural domain; and the first structural domain is directly or indirectly connected to the fifth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain light chain is directly or indirectly connected to the fourth structural domain; and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the fourth structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the N end of the light chain of the first structural domain is directly or indirectly connected to the C end of the fourth structural domain, and the N end of the heavy chain of the first structural domain is directly or indirectly connected to the C end of the fifth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain light chain is directly or indirectly connected to the fourth structural domain, and the first structural domain light chain is directly or indirectly connected to the fifth structural domain.
  • the heavy chain of the first structural domain is directly or indirectly connected to the heavy chain of the second structural domain; and the C-end of the light chain of the first structural domain is directly or indirectly connected to the N-end of the fourth structural domain, and the N-end of the light chain of the first structural domain is directly or indirectly connected to the C-end of the fifth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the fifth structural domain, and the second structural domain is directly or indirectly connected to the fourth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end domain of the fifth structure, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the fourth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain light chain is directly or indirectly connected to the fifth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end domain of the fifth structure, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the fourth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain, and the second structural domain light chain is directly or indirectly connected to the fourth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C end of the first structural domain heavy chain is directly or indirectly connected to the N end domain of the fifth structure, and the C end of the second structural domain light chain is directly or indirectly connected to the N end of the fourth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end domain of the fifth structure, and the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the fourth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain light chain is directly or indirectly connected to the fifth structural domain, and the second structural light chain is directly or indirectly connected to the fourth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end domain of the fifth structure, and the C-end of the second structural domain light chain is directly or indirectly connected to the N-end of the fourth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain light chain is directly or indirectly connected to the N-end domain of the fifth structure, and the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the fourth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain; and the first structural domain is directly or indirectly connected to the fifth structural domain; and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain; the first structural domain light chain is directly or indirectly connected to the fifth structural domain; and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the first structural domain light chain is directly or indirectly connected to the C-end of the fifth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain; and the first structural domain is directly or indirectly connected to the fourth structural domain; and the first structural domain is directly or indirectly connected to the fifth structural domain; and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain; the first structural domain light chain is directly or indirectly connected to the fourth structural domain; the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain; and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the fourth structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain; the first structural domain light chain is directly or indirectly connected to the fourth structural domain; the first structural domain light chain is directly or indirectly connected to the fifth structural domain; and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, the C-end of the first structural domain light chain is directly or indirectly connected to the N-end of the fourth structural domain, the N-end of the first structural domain light chain is directly or indirectly connected to the C-end of the fifth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain; and the first structural domain is directly or indirectly connected to the fourth structural domain, and the first structural domain is directly or indirectly connected to the fifth structural domain, and the second structural domain is directly or indirectly connected to the third structural domain, and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the fourth structural domain; and the first structural domain is directly or indirectly connected to the fourth structural domain, and the first structural domain is directly or indirectly connected to the fifth structural domain, the second structural domain is directly or indirectly connected to the third structural domain, and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the heavy chain of the fourth structural domain; and the C end of the heavy chain of the first structural domain is directly or indirectly connected to the N end of the fourth structural domain, and the N end of the heavy chain of the first structural domain is directly or indirectly connected to the fifth structural domain, the C end of the heavy chain of the second structural domain is directly or indirectly connected to the N end of the third structural domain, and the C end of the heavy chain of the second structural domain is directly or indirectly connected to the N end of the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the heavy chain of the fourth structural domain; and the heavy chain of the first structural domain is directly or indirectly connected to the fourth structural domain, and the light chain of the first structural domain is directly or indirectly connected to the fifth structural domain, the heavy chain of the second structural domain is directly or indirectly connected to the third structural domain, and the heavy chain of the second structural domain is directly or indirectly connected to the sixth structural domain.
  • the second structural domain is directly or indirectly connected to the heavy chain of the fourth structural domain; and the C end of the heavy chain of the first structural domain is directly or indirectly connected to the N end of the fourth structural domain, and the N end of the light chain of the first structural domain is directly or indirectly connected to the fifth structural domain, the C end of the heavy chain of the second structural domain is directly or indirectly connected to the N end of the third structural domain, and the C end of the heavy chain of the second structural domain is directly or indirectly connected to the N end of the sixth structural domain.
  • first structural domain is directly or indirectly connected to the second structural domain; and the first structural domain is directly or indirectly connected to the third structural domain; and the first structural domain is directly or indirectly connected to the fifth structural domain; and the second structural domain is directly or indirectly connected to the fourth structural domain; and the second structural domain is directly or indirectly connected to the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain; the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain; the second structural domain heavy chain is directly or indirectly connected to the fourth structural domain; and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain, and the N-end of the second structural domain heavy chain is directly or indirectly connected to the C-end of the fourth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the first structural domain heavy chain is directly or indirectly connected to the third structural domain, and the first structural domain heavy chain is directly or indirectly connected to the fifth structural domain, and the second structural domain light chain is directly or indirectly connected to the fourth structural domain, and the second structural domain heavy chain is directly or indirectly connected to the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain, and the N-end of the second structural domain light chain is directly or indirectly connected to the C-end of the fourth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the first structural domain heavy chain is directly or indirectly connected to the second structural domain heavy chain; and the C-end of the first structural domain heavy chain is directly or indirectly connected to the N-end of the third structural domain, and the N-end of the first structural domain heavy chain is directly or indirectly connected to the C-end of the fifth structural domain, and the C-end of the second structural domain light chain is directly or indirectly connected to the N-end of the fourth structural domain, and the C-end of the second structural domain heavy chain is directly or indirectly connected to the N-end of the sixth structural domain.
  • the heavy chain Fc fragment of the first structural domain is connected to the heavy chain Fc fragment of the second structural domain through a pestle-mortar structure pairing or disulfide bond; wherein the first structure and the fourth structural domain can be directly or indirectly linked; wherein the first structure and the fifth structural domain can be directly or indirectly linked; wherein the second structure and the sixth structural domain can be directly or indirectly linked.
  • the heavy chain Fc fragment of the first structural domain is connected to the heavy chain Fc fragment of the second structural domain through a pestle-mortar structure pairing or disulfide bond; wherein the first structure and the fourth structural domain can be directly or indirectly linked; wherein the first structure and the fifth structural domain can be directly or indirectly linked; wherein the second structure and the sixth structural domain can be directly or indirectly linked.
  • the heavy chain Fc fragment of the first structural domain is connected to the heavy chain Fc fragment of the second structural domain through a pestle-mortar structure pairing or disulfide bond; wherein the first structure and the fifth structural domain can be directly or indirectly linked; wherein the second structure and the fourth structural domain can be directly or indirectly linked; wherein the second structure and the sixth structural domain can be directly or indirectly linked.
  • the heavy chain Fc fragment of the second structural domain is connected to the heavy chain Fc fragment of the fourth structural domain through a pestle-mortar structure pairing or disulfide bond; wherein the first structure and the fourth structural domain can be directly or indirectly linked; wherein the first structure and the fifth structural domain can be directly or indirectly linked; wherein the second structure and the sixth structural domain can be directly or indirectly linked.
  • the first domain disclosed in this invention can be extended to certain anti-CD3 antibodies without Fc domains, including but not limited to single-domain antibodies, recombinant antibodies, and single-chain antibodies; and the second or fourth domain disclosed in this invention can be extended to antibody sequences that bind to certain tumor antigens without Fc domains, such as CD20, Her2, PD-L1, GCC, DLL3, EGFR, 5T4, and/or CEA, including but not limited to single-domain antibodies, recombinant antibodies, and single-chain antibodies.
  • certain tumor antigens without Fc domains such as CD20, Her2, PD-L1, GCC, DLL3, EGFR, 5T4, and/or CEA, including but not limited to single-domain antibodies, recombinant antibodies, and single-chain antibodies.
  • the multifunctional fusion polypeptide disclosed in this invention may include a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide. It should be understood that in some embodiments, the first polypeptide, the fourth polypeptide, or the fifth polypeptide may be absent.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a light chain with a first structural domain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a heavy chain with a structural domain from its N-terminus to its C-terminus;
  • the third polypeptide of the multifunctional fusion polypeptide includes a heavy chain with a second structural domain from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a light chain with a second structural domain from its N-terminus to its C-terminus.
  • the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide constitute the multifunctional fusion polypeptide; wherein the first polypeptide and the fourth polypeptide may or may not be present.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a light chain with a first structural domain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a heavy chain with a structural domain, a linker, and a third structural domain sequentially from its N-terminus to its C-terminus;
  • the third polypeptide of the multifunctional fusion polypeptide includes a heavy chain with a second structural domain from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a light chain with a second structural domain from its N-terminus to its C-terminus.
  • the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide constitute the multifunctional fusion polypeptide; wherein the first polypeptide and the fourth polypeptide may or may not be present.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a light chain with a first structural domain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a heavy chain with a structural domain from its N-terminus to its C-terminus;
  • the third polypeptide of the multifunctional fusion polypeptide includes, from its N-terminus to its C-terminus, a heavy chain with a second structural domain, a linker, and a sixth structural domain in sequence;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a light chain with a second structural domain from its N-terminus to its C-terminus.
  • the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide constitute the multifunctional fusion polypeptide; wherein the first polypeptide and the fourth polypeptide may or may not be present.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a light chain with a first structural domain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a heavy chain with a first structural domain, a linker, and a third structural domain sequentially from its N-terminus to its C-terminus;
  • the third polypeptide of the multifunctional fusion polypeptide includes a heavy chain with a second structural domain, a linker, and a sixth structural domain sequentially from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a light chain with a second structural domain from its N-terminus to its C-terminus.
  • the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide constitute the multifunctional fusion polypeptide; wherein the first polypeptide and the fourth polypeptide may or may not be present.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes, from its N-terminus to its C-terminus, a first domain heavy chain CD3 antigen-binding fragment, a linker, a fourth domain heavy chain, a linker, and a third domain;
  • the third polypeptide of the multifunctional fusion polypeptide includes, from its N-terminus to its C-terminus, a second domain heavy chain, a linker, and a sixth domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a second domain light chain from its N-terminus to its C-terminus;
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a first structural domain light chain, a linker, and a fourth structural domain heavy chain;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a first structural domain heavy chain, a linker, and a third structural domain;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a second structural domain heavy chain, a linker, and a sixth structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a second structural domain light chain;
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fourth structural domain light chain.
  • the first polypeptide, second polypeptide, third polypeptide, fourth polypeptide, and fifth polypeptide constitute
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes, from its N-terminus to its C-terminus, a fourth domain heavy chain TAA antigen-binding fragment, a linker, a first domain heavy chain, a linker, and a third domain;
  • the third polypeptide of the multifunctional fusion polypeptide includes, from its N-terminus to its C-terminus, a second domain heavy chain, a linker, and a sixth domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a second domain light chain from its N-terminus to its C-terminus;
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a fourth structural domain TAA antigen-binding fragment, a linker, and a light chain of the first structural domain;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a first structural domain heavy chain, a linker, and a third structural domain;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a second structural domain heavy chain, a linker, and a sixth structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a second structural domain light chain;
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fourth structural domain light chain.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus;
  • the third polypeptide of the multifunctional fusion polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a second domain light chain from its N-terminus to its C-terminus;
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide, second polypeptide, third polypeptide, fourth polypeptide, and fifth polypeptide
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus;
  • the third polypeptide of the multifunctional fusion polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a second domain light chain, a linker, and a fourth domain TAA antigen-binding fragment from its N-terminus to its C-terminus;
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus;
  • the third polypeptide of the multifunctional fusion polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, and a second domain light chain linker from its N-terminus to its C-terminus;
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a light chain with a first structural domain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a fifth structural domain, a linker, and a heavy chain with the first structural domain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide includes a heavy chain with a second structural domain from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a light chain with a second structural domain from its N-terminus to its C-terminus.
  • the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide constitute the multifunctional fusion polypeptide; wherein the first polypeptide and the fourth polypeptide may or may not be present.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a fifth structural domain, a linker, and a light chain of the first structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first structural domain;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the second structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a light chain of the second structural domain.
  • the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide constitute the multifunctional fusion polypeptide; wherein the fourth polypeptide may or may not be present.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes, from its N-terminus to its C-terminus, a fifth domain, a linker, a first domain heavy chain CD3 antigen-binding fragment, a linker, and a fourth domain heavy chain, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide includes a second domain heavy chain from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a second domain light chain from its N-terminus to its C-terminus; and
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, and the fifth polypeptide constitute the multifunctional fusion polypeptide; wherein the first polypeptide, the fourth polypeptide, and the fifth polypeptide may or may not be present.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a fifth structural domain, a linker, and a light chain of the first structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first structural domain, a CD3 antigen-binding fragment, a linker, and a heavy chain of the fourth structural domain;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the second structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a light chain of the second structural domain;
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a light chain of the fourth structural domain.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a first structural domain light chain, a linker, and a fourth structural domain heavy chain;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fifth structural domain, a linker, and a first structural domain heavy chain, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a second structural domain heavy chain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a second structural domain light chain;
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fourth structural domain light chain.
  • the first polypeptide, second polypeptide, third polypeptide, fourth polypeptide, and fifth polypeptide constitute the multifunctional
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention sequentially includes a fifth structural domain, a linker, a light chain of the first structural domain, a linker, and a heavy chain of the fourth structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus sequentially includes a light chain of the first structural domain, wherein the linker may be absent;
  • the fourth polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus includes a light chain of the second structural domain;
  • the fifth polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus includes a light chain of the fourth structural domain.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a fifth domain, a linker, and a first domain heavy chain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, and a second domain heavy chain sequentially from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a second domain light chain from its N-terminus to its C-terminus;
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide, second polypeptide, third polypeptide, fourth polypeptide, and fifth polypeptide constitute
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a fifth structural domain, a linker, and a light chain of the first structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first structural domain;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fourth structural domain (TAA antigen-binding fragment), a linker, and a heavy chain of the second structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a light chain of the second structural domain;
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a light chain of the fourth structural domain.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a fifth domain, a linker, and a first domain heavy chain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide includes a second domain heavy chain from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, and a second domain light chain linker sequentially from its N-terminus to its C-terminus;
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a fifth structural domain, a linker, and a light chain of the first structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first structural domain;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the second structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fourth structural domain TAA antigen-binding fragment, a linker, and a light chain linker of the second structural domain;
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a light chain of the fourth structural domain.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a light chain with a first structural domain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a fifth structural domain, a linker, and a heavy chain with the first structural domain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide includes a heavy chain with a second structural domain from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a light chain with a second structural domain, a linker, and a TAA antigen-binding fragment with a fourth structural domain sequentially from its N-terminus to its C-terminus;
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a light chain with a fourth structural domain from its N-terminus to its C-terminus.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a fifth structural domain, a linker, and a light chain of the first structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first structural domain;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the second structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a light chain of the second structural domain, a linker, and a TAA antigen-binding fragment of the fourth structural domain;
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a light chain of the fourth structural domain.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first structural domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a fifth structural domain, a linker, a first structural domain heavy chain, a linker, and a third structural domain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide includes a second structural domain heavy chain, a linker, and a sixth structural domain sequentially from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a second structural domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide constitute the multifunctional fusion polypeptide; wherein the first polypeptide and the fourth polypeptide may or may not be present.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention from N-terminus to C-terminus, sequentially includes a fifth structural domain, a linker, and a light chain of the first structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus sequentially includes a heavy chain of the first structural domain, a linker, and a third structural domain;
  • the third polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus sequentially includes a heavy chain of the second structural domain, a linker, and a sixth structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus includes a light chain of the second structural domain.
  • the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide constitute the multifunctional fusion polypeptide; wherein the fourth polypeptide may or may not be present
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes, from its N-terminus to its C-terminus, a fifth domain, a linker, a first domain heavy chain CD3 antigen-binding fragment, a linker, a fourth domain heavy chain, a linker, and a third domain, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide includes, from its N-terminus to its C-terminus, a second domain heavy chain, a linker, and a sixth domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a second domain light chain from its N-terminus to its C-terminus; and
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention sequentially includes a fifth structural domain, a linker, and a light chain of the first structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus sequentially includes a heavy chain of the first structural domain, a CD3 antigen-binding fragment, a linker, a heavy chain of the fourth structural domain, a linker, and a third structural domain;
  • the third polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus sequentially includes a heavy chain of the second structural domain, a linker, and a sixth structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus includes a light chain of the second structural domain;
  • the fifth polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus to C-terminus includes
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a first structural domain light chain, a linker, and a fourth structural domain heavy chain;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fifth structural domain, a linker, a first structural domain heavy chain, a linker, and a third structural domain, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a second structural domain heavy chain, a linker, and a sixth structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a second structural domain light chain; and
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fourth structural domain light chain.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention sequentially includes a fifth structural domain, a linker, a light chain of the first structural domain, a linker, and a heavy chain of the fourth structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus sequentially includes a heavy chain of the first structural domain, a linker, and a third structural domain;
  • the third polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus sequentially includes a heavy chain of the second structural domain, a linker, and a sixth structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus includes a light chain of the second structural domain;
  • the fifth polypeptide of the multifunctional fusion polypeptide, from N-terminus to C-terminus includes a light chain of the fourth
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a fifth domain, a linker, a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a second domain light chain from its N-terminus to its C-terminus;
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a fifth structural domain, a linker, and a light chain of the first structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first structural domain, a linker, and a third structural domain, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fourth structural domain (TAA antigen-binding fragment), a linker, a heavy chain of the second structural domain, a linker, and a sixth structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a light chain of the second structural domain;
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N-termin
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a fifth domain, a linker, a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus, wherein the linker may or may not be present;
  • the third polypeptide of the multifunctional fusion polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a second domain light chain, a linker, and a fourth domain TAA antigen-binding fragment from its N-terminus to its C-terminus;
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a fifth structural domain, a linker, and a light chain of the first structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first structural domain, a linker, and a third structural domain;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the second structural domain, a linker, and a sixth structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a light chain of the second structural domain, a linker, and a TAA antigen-binding fragment of the fourth structural domain;
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus,
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention includes a first domain light chain from its N-terminus to its C-terminus;
  • the second polypeptide of the multifunctional fusion polypeptide includes a fifth domain, a linker, a first domain heavy chain, a linker, and a third domain sequentially from its N-terminus to its C-terminus, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide includes a second domain heavy chain, a linker, and a sixth domain sequentially from its N-terminus to its C-terminus;
  • the fourth polypeptide of the multifunctional fusion polypeptide includes a fourth domain TAA antigen-binding fragment, a linker, and a second domain light chain linker from its N-terminus to its C-terminus;
  • the fifth polypeptide of the multifunctional fusion polypeptide includes a fourth domain light chain from its N-terminus to its C-terminus.
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a fifth structural domain, a linker, and a light chain of the first structural domain, wherein the linker may be absent;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first structural domain, a linker, and a third structural domain, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the second structural domain, a linker, and a sixth structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fourth structural domain TAA antigen-binding fragment, a linker, and a light chain linker of the second structural domain;
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N
  • the first polypeptide of the multifunctional fusion polypeptide of the present invention comprises, from N-terminus to C-terminus, a first structural domain light chain, a linker, and a fourth structural domain heavy chain;
  • the second polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fifth structural domain, a linker, a first structural domain heavy chain, a linker, and a third structural domain, wherein the linker may be absent;
  • the third polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a second structural domain heavy chain, a linker, and a sixth structural domain;
  • the fourth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a second structural domain light chain; and
  • the fifth polypeptide of the multifunctional fusion polypeptide comprises, from N-terminus to C-terminus, a fourth structural domain light chain.
  • the first domain CD3 antigen-binding fragment described in this invention can be used to derive any anti-CD3 antibody, including any antibody that binds to, blocks, and/or reduces the binding of CD3 to its natural receptor; including existing and future anti-CD3 antibodies.
  • the second domain TAA antigen-binding fragment and/or the fourth domain of the present invention can be used to derive any anti-tumor-associated antigen (TAA) antibody, including any antibody that binds to, blocks, or reduces tumor-associated antigen (TAA); on the other hand, the fourth domain TAA antigen-binding fragment can be used to derive any anti-tumor-associated antigen (TAA) antibody, including any antibody that binds to, blocks, or reduces tumor-associated antigen (TAA).
  • TAA anti-tumor-associated antigen
  • TAA tumor-associated antigen
  • the second and/or fourth domains can bind to one or more of the targets shown in the following group: PD-L1, PD-L2, VEGF, VEGFR, FGFR, HER2, HGFR, PIP-LAR, CD44, CD147, TfR, CDCP1, ⁇ 6 ⁇ 4, ⁇ 6 ⁇ 3, Trop2, EpCAM, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, DLL3, B7H3, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, PTK7, EphA2, NKG2A, Nkp36, and Tim3, including existing and future antitumor-associated antigen (TAA) antibodies.
  • TAA antitumor-associated antigen
  • CD3 and TAA bispecific or polyspecific antibodies act as T cell connectors, targeting T cell surface antigen CD3 and tumor cell surface antigen TAA respectively, thereby bridging the distance between tumor cells and effector T cells.
  • Activated T cells release granzymes and perforin, thus effectively killing tumor cells.
  • the third and/or sixth domains of this invention comprise extracellular regions of one or more ligands, including at least a polypeptide composed of the CD86 extracellular domain or the CD86 extracellular domain IgV region.
  • CD86 is a member protein of the B7 molecular family that co-stimulates T cell activation.
  • Mature CD86 molecules consist of an extracellular domain (ECD), a transmembrane domain, and an intracellular domain.
  • ECD extracellular domain
  • the extracellular domain (ECD) is a key region for these molecules to bind to their corresponding receptors on T cells.
  • the CD86 extracellular domains all contain immunoglobulin-like V (IgV) and immunoglobulin-like C2 (IgC2) regions, with the IgV domain being a key domain directly involved in receptor binding.
  • the CD86 extracellular domain IgV can bind to CD28 and participate in inducing T cell activation, proliferation, and effector functions.
  • CTLA4 can also bind to the CD86 IgV region and participate in immunosuppressive regulation.
  • the third and/or sixth domains comprise extracellular regions of one or more ligands, including at least a polypeptide comprising the CD80 extracellular domain or the CD80 extracellular domain IgV region.
  • CD80 is a member protein of the B7 molecular family that co-stimulates T cell activation.
  • Mature CD80 molecules consist of an extracellular domain (ECD), a transmembrane domain, and an intracellular domain.
  • the extracellular domain (ECD) is a key region for these molecules to bind to their corresponding receptors on T cells.
  • the CD80 extracellular domains all contain immunoglobulin-like V (IgV) and immunoglobulin-like C2 (IgC2) regions, with the IgV domain being a key domain directly involved in receptor binding.
  • the CD80 extracellular domain IgV can bind to CD28 and participate in inducing T cell activation, proliferation, and effector functions.
  • CTLA4 can also bind to the IgV region of CD80 to participate in immunosuppressive regulation.
  • the fifth domain of this invention comprises a masking peptide and a protease cleavage site fragment, including at least one or more masking peptide ends and one or more protease cleavage site fragments.
  • the one or more masking peptides included in the fifth domain are amino acid sequences capable of interacting with the CD3 antigen-binding portion of the first domain. Their function is to inhibit CD3 activity when specific tumor environmental conditions are not met, thereby reducing the risk of non-specific T cell activation.
  • the masking peptide interacts with the CD3 binding site through stereotactic shielding or low-affinity binding, preventing the binding of CD3 to the T cell receptor (TCR) complex.
  • the one or more protease cleavage fragments included in the fifth domain are amino acid sequences recognized by specific proteolytic enzymes. These sequences can be cleaved by proteases specifically highly expressed in the tumor microenvironment (such as MMPs, FAP, uPA, etc.), thereby releasing the CD3 binding site and enabling T cell activation.
  • proteases specifically highly expressed in the tumor microenvironment such as MMPs, FAP, uPA, etc.
  • the present invention proposes a combination of the first, second, third, and/or fourth, and/or fifth, and/or sixth structural domains in the multifunctional fusion peptide, providing a co-stimulatory signal.
  • a CD3 and TAA bispecific antibody Based on a CD3 and TAA bispecific antibody, it provides a dual activation signal for CTLA4 and induces more effective tumor-specific cell lysis.
  • Multifunctional fusion peptides with three or four binding specificities provide dual activation signals in a single molecule, enhancing T cell activation as novel agents for clinical oncology treatment.
  • the masking peptide and protein-cleaving enzyme recognition sequence fragments can inhibit non-specific CD3 activation and specifically release CD3 activity in the tumor microenvironment, significantly improving the targeting, safety, and therapeutic window of multispecific antibodies, showing broad application prospects in the field of cancer immunotherapy.
  • the following provides an exemplary fusion peptide complex comprising: a first peptide amino acid sequence of the light chain of an anti-human CD3 antibody CD3SP34 (SEQ ID NO: 11), a second peptide amino acid sequence of the heavy chain of the anti-CD3 antibody CD3SP34 (SEQ ID NO: 21), and a third peptide amino acid sequence (SEQ ID NO: 46) of an anti-tumor-associated antigen (TAA) anti-human Her2 single-domain antibody (SEQ ID NO: 47) with a C-terminus linked to a heavy chain fc fragment (SEQ ID NO: 2).
  • TAA anti-tumor-associated antigen
  • SEQ ID NO: 47 anti-human Her2 single-domain antibody
  • the second and third peptides are linked by a knockbs-in-holes structure in the heavy chain Fc domain to form the multifunctional fusion peptide complex with bispecific antibody properties.
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of the anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 167) of the heavy chain of the anti-CD3 antibody CD3SP34 (SEQ ID NO: 21) covalently linked to the CD86 extracellular domain IgV mutant Q25I/F33L/H90I (SEQ ID NO: 100) via a C-terminal linker; and a third polypeptide (SEQ ID NO: 177) of the anti-tumor-associated antigen (TAA) anti-human Her2 single-domain antibody (SEQ ID NO: 47) covalently linked to the heavy chain fc fragment (SEQ ID NO: 4) via a C-terminal linker to the CD86 extracellular domain IgV mutant Q25I/F33L/H90I (SEQ ID NO: 100).
  • the second and third polypeptides are linked by a knock-in-hole structure in
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of an anti-CD3 antibody CD3SP34, a second polypeptide (SEQ ID NO: 21) of the heavy chain of an anti-CD3 antibody CD3SP34, and a third polypeptide (SEQ ID NO: 77) of an anti-tumor-associated antigen (TAA) anti-human 5T4 single-domain antibody (SEQ ID NO: 78) with a C-terminus linked to a heavy chain fc fragment (SEQ ID NO: 4).
  • TAA anti-tumor-associated antigen
  • SEQ ID NO: 78 anti-human 5T4 single-domain antibody
  • the second and third polypeptides are linked by a knockbs-in-holes structure in the heavy chain Fc domain to form the multifunctional fusion polypeptide complex with bispecific antibody properties.
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of the anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 167) of the heavy chain of the anti-CD3 antibody CD3SP34 (SEQ ID NO: 21) covalently linked to the CD86 extracellular domain IgV mutant Q25I/F33L/H90I (SEQ ID NO: 100) via a C-terminal linker; and a third polypeptide (SEQ ID NO: 190) of the anti-tumor-associated antigen (TAA) anti-human 5T4 single-domain antibody (SEQ ID NO: 78) covalently linked to the heavy chain fc fragment (SEQ ID NO: 4) via a C-terminal linker to the CD86 extracellular domain IgV mutant Q25I/F33L/H90I (SEQ ID NO: 100).
  • the second and third polypeptides are linked by a knock-in-
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of an anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 3227) of an anti-human CD3SP34 antibody heavy chain (SEQ ID NO: 21) linked to a masking peptide (SEQ ID NO: 435) via a linker to a protease cleavage site fragment (SEQ ID NO: 3016); and a third polypeptide (SEQ ID NO: 46) of an anti-tumor-associated antigen (TAA) anti-human Her2 single-domain antibody (SEQ ID NO: 47) with a C-terminus of a heavy chain fc fragment (SEQ ID NO: 4).
  • the second and third polypeptides are linked by a knocks-in-holes structure in the Fc domain of the heavy chain to form the multifunctional fusion polypeptide complex with bispecific antibody properties.
  • the following provides an exemplary fusion peptide complex comprising: a first peptide (SEQ ID NO: 11) of the light chain of an anti-CD3 antibody CD3SP34; a masking peptide (SEQ ID NO: 435) linked to a protease cleavage site fragment (SEQ ID NO: 3016) via a linker; a heavy chain of an anti-human CD3SP34 antibody (SEQ ID NO: 21) linked to the heavy chain via a linker; and a CD86 extracellular domain IgV mutant Q25I/F3 covalently linked via a C-terminal linker.
  • the second polypeptide (SEQ ID NO: 3232) of 3L/H90I (SEQ ID NO: 100) and the third polypeptide (SEQ ID NO: 177) of the CD86 extracellular domain IgV mutant Q25I/F33L/H90I (SEQ ID NO: 100) are covalently linked via a C-terminal linker after the C-terminal linker is connected to the heavy chain fc fragment (SEQ ID NO: 4) of the anti-tumor-associated antigen (TAA) anti-human Her2 single-domain antibody (SEQ ID NO: 47).
  • the second and third polypeptides are linked by a knockbs-in-holes structure in the heavy chain Fc domain to form the multifunctional fusion polypeptide complex with bispecific antibody properties.
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of an anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 3230) of an anti-human CD3SP34 antibody heavy chain (SEQ ID NO: 21) linked to a masking peptide (SEQ ID NO: 435) via a linker and then linked to a protease cleavage site fragment (SEQ ID NO: 3026) via a linker; and a third polypeptide (SEQ ID NO: 46) of an anti-tumor-associated antigen (TAA) anti-human Her2 single-domain antibody (SEQ ID NO: 47) with a heavy chain fc fragment (SEQ ID NO: 4) linked to its C-terminus.
  • the second and third polypeptides are linked by a knocks-in-holes structure in the Fc domain of the heavy chain to form the multifunctional fusion polypeptide complex with bispecific antibody properties.
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of an anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 3231) of an anti-human CD3SP34 antibody heavy chain (SEQ ID NO: 21) linked to a masking peptide (SEQ ID NO: 435) via a linker and then linked to a protease cleavage site fragment (SEQ ID NO: 3028) via a linker; and a third polypeptide (SEQ ID NO: 46) of an anti-tumor-associated antigen (TAA) anti-human Her2 single-domain antibody (SEQ ID NO: 47) with a heavy chain fc fragment (SEQ ID NO: 4) linked to its C-terminus.
  • the second and third polypeptides are linked by a knocks-in-holes structure in the Fc domain of the heavy chain to form the multifunctional fusion polypeptide complex with bispecific antibody properties.
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of an anti-CD3 antibody CD3SP34, a second polypeptide (SEQ ID NO: 21) of the heavy chain of an anti-CD3 antibody CD3SP34, and a third polypeptide (SEQ ID NO: 62) of an anti-tumor-associated antigen (TAA) anti-human PDL-1 single-domain antibody (SEQ ID NO: 63) with a C-terminus linked to a heavy chain fc fragment (SEQ ID NO: 4).
  • TAA anti-tumor-associated antigen
  • SEQ ID NO: 63 anti-human PDL-1 single-domain antibody
  • the second and third polypeptides are linked by a knockbs-in-holes structure in the heavy chain Fc domain to form the multifunctional fusion polypeptide complex with bispecific antibody properties.
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of the anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 3225) of the heavy chain of the anti-CD3 antibody CD3SP34 (SEQ ID NO: 21) covalently linked to the CD86 extracellular domain IgV mutant Q25I/F33L/H90V (SEQ ID NO: 104) via a C-terminal linker; and a third polypeptide (SEQ ID NO: 3222) of the anti-tumor-associated antigen (TAA) anti-human 5T4 single-domain antibody (SEQ ID NO: 78) covalently linked to the heavy chain fc fragment (SEQ ID NO: 4) via a C-terminal linker to the CD86 extracellular domain IgV mutant Q25I/F33L/H90V (SEQ ID NO: 104).
  • the second and third polypeptides are linked by a knock-
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of the anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 3226) of the heavy chain of the anti-CD3 antibody CD3SP34 (SEQ ID NO: 21) covalently linked to the CD86 extracellular domain IgV mutant Q25V/F33L/H90I (SEQ ID NO: 101) via a C-terminal linker; and a third polypeptide (SEQ ID NO: 3223) of the anti-tumor-associated antigen (TAA) anti-human 5T4 single-domain antibody (SEQ ID NO: 78) covalently linked to the heavy chain fc fragment (SEQ ID NO: 4) via a C-terminal linker to the CD86 extracellular domain IgV mutant Q25V/F33L/H90I (SEQ ID NO: 101).
  • the second and third polypeptides are linked by a knock-in
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of the anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 3225) of the heavy chain of the anti-CD3 antibody CD3SP34 (SEQ ID NO: 21) covalently linked to the CD86 extracellular domain IgV mutant Q25I/F33L/H90V (SEQ ID NO: 104) via a C-terminal linker; and a third polypeptide (SEQ ID NO: 3216) of the anti-tumor-associated antigen (TAA) anti-human HER2 single-domain antibody (SEQ ID NO: 47) covalently linked to the heavy chain fc fragment (SEQ ID NO: 4) via a C-terminal linker to the CD86 extracellular domain IgV mutant Q25I/F33L/H90V (SEQ ID NO: 104).
  • the second and third polypeptides are linked by a knock-in
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of the anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 3226) of the heavy chain of the anti-CD3 antibody CD3SP34 (SEQ ID NO: 21) covalently linked to the CD86 extracellular domain IgV mutant Q25V/F33L/H90I (SEQ ID NO: 101) via a C-terminal linker; and a third polypeptide (SEQ ID NO: 3217) of the anti-tumor-associated antigen (TAA) anti-human HER2 single-domain antibody (SEQ ID NO: 47) covalently linked to the heavy chain fc fragment (SEQ ID NO: 4) via a C-terminal linker to the CD86 extracellular domain IgV mutant Q25V/F33L/H90I (SEQ ID NO: 101).
  • the second and third polypeptides are linked by a knock-in-
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of an anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 3227) of an anti-human CD3SP34 antibody heavy chain (SEQ ID NO: 21) linked to a masking peptide (SEQ ID NO: 435) via a linker and then linked to a protease cleavage site fragment (SEQ ID NO: 3016) via a linker; and a third polypeptide (SEQ ID NO: 77) of an anti-tumor-associated antigen (TAA) anti-human 5T4 single-domain antibody (SEQ ID NO: 78) with a heavy chain fc fragment (SEQ ID NO: 4) linked to its C-terminus.
  • the second and third polypeptides are linked by a knocks-in-holes structure in the Fc domain of the heavy chain to form the multifunctional fusion polypeptide complex with bispecific
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of an anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 3228) of an anti-human CD3SP34 antibody heavy chain (SEQ ID NO: 21) linked to a masking peptide (SEQ ID NO: 405) via a linker and then linked to a protease cleavage site fragment (SEQ ID NO: 3016) via a linker; and a third polypeptide (SEQ ID NO: 77) of an anti-tumor-associated antigen (TAA) anti-human 5T4 single-domain antibody (SEQ ID NO: 78) with a heavy chain fc fragment (SEQ ID NO: 4) linked to its C-terminus.
  • the second and third polypeptides are linked by a knocks-in-holes structure in the Fc domain of the heavy chain to form the multifunctional fusion polypeptide complex with bispecific
  • the following provides an exemplary fusion polypeptide complex comprising: a first polypeptide (SEQ ID NO: 11) of the light chain of an anti-CD3 antibody CD3SP34; a second polypeptide (SEQ ID NO: 3229) of an anti-human CD3SP34 antibody heavy chain (SEQ ID NO: 21) linked to a masking peptide (SEQ ID NO: 410) via a linker and then linked to a protease cleavage site fragment (SEQ ID NO: 3016) via a linker; and a third polypeptide (SEQ ID NO: 77) of an anti-tumor-associated antigen (TAA) anti-human 5T4 single-domain antibody (SEQ ID NO: 78) with a heavy chain fc fragment (SEQ ID NO: 4) linked to its C-terminus.
  • the second and third polypeptides are linked by a knocks-in-holes structure in the Fc domain of the heavy chain to form the multifunctional fusion polypeptide complex with bispecific

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Abstract

L'invention concerne un polypeptide de fusion. Le polypeptide contient un premier domaine, un deuxième domaine et un troisième domaine, le premier domaine étant capable de se lier à CD3, le deuxième domaine étant capable de se lier à un antigène associé à une tumeur (TAA), et le troisième domaine étant capable de se lier à CD28 et/ou CTLA4.
PCT/CN2025/097370 2024-05-27 2025-05-27 Polypeptide de fusion multifonctionnel Pending WO2025247196A1 (fr)

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CN202410666787 2024-05-27
CN202410666787.9 2024-05-27

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WO2025247196A1 true WO2025247196A1 (fr) 2025-12-04

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