Antigen-binding molecule and pharmaceutical use thereof

Abstract

An antigen-binding molecule and the pharmaceutical use thereof. Also disclosed are a multispecific activatable antibody that specifically binds to CD3, a pharmaceutical composition containing same, a preparation method therefor, and the pharmaceutical use thereof.

Description

Antigen-binding molecules and their medicinal uses

[0001] This application claims priority to Chinese patent application CN202411987611.X filed on December 31, 2024 and Chinese patent application 202511857448.X filed on December 10, 2025. Technical Field

[0002] This disclosure pertains to the field of biotechnology, and more specifically, to antigen-binding molecules and their pharmaceutical uses. Background Technology

[0003] The statements herein are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0004] T-cell engagers (TCEs) act as connectors between T cells and tumor cells. By simultaneously binding to tumor-associated antigens (TAAs) and CD3 on T cells, they activate T cells, thereby killing tumor cells. Currently, eight bispecific TCE antibodies targeting hematological malignancies have been approved for marketing. Among them, the approval of Tarlatamab from Amgen, which targets DLL3 / CD3, in 2024 marked the first breakthrough for CD3-based bispecific antibodies in the field of solid tumors. However, CD3 bispecific antibodies have a low killing threshold, making it impossible to distinguish between tumor tissues with high expression of TAs and normal tissues with low expression. On-target toxicity (off-tumor toxicity) leading to cytokine storms and a narrow safety window resulting in insufficient efficacy have become bottlenecks limiting the efficacy of TCEs in solid tumors.

[0005] To enhance the safety window of T-cell adjuvants in solid tumors and reduce the killing effect of T-cell adjuvants on normal tissues and the release of corresponding cytokines, this disclosure provides TCE-activatable antibodies that are conditionally activated in tumor tissues. These antibodies are designed by utilizing the expression differences of proteases in tumor tissues and non-tumor environments to conditionally activate TCEs in the tumor microenvironment. Summary of the Invention

[0006] This disclosure provides antigen-binding molecules, activatable antibodies (e.g., trispecific or pentaspecific activatable antibodies), their encoded nucleic acids, vectors, host cells, pharmaceutical compositions, methods of preparation, and methods for treating, preventing, or improving diseases or conditions, and related pharmaceutical uses.

[0007] antigen-binding molecules

[0008] In one aspect, this disclosure provides an antigen-binding molecule comprising:

[0009] An antigen-binding domain that specifically binds to CD3, wherein the antigen-binding domain specifically binding to CD3 is Fab, and the Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1); and

[0010] The extended half-life domain is a single variable domain of an immunoglobulin that specifically binds to human serum albumin (HSA).

[0011] The antigen-binding molecule further includes a linker, and the half-life extension domain is linked to the CD3-specific antigen-binding domain via the linker.

[0012] In some embodiments, as described above, the linker is linker 1 in the antigen-binding molecule.

[0013] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the antigen-binding domain that specifically binds to CD3 is a first antigen-binding domain.

[0014] In another aspect, this disclosure provides an antigen-binding molecule comprising:

[0015] The first antigen-binding domain that specifically binds to CD3 is Fab, wherein the Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1); and

[0016] The extended half-life domain is a single variable domain of an immunoglobulin that specifically binds to HSA.

[0017] The antigen-binding molecule further includes a linker 1, and the half-life extension domain is connected to the first antigen-binding domain through the linker 1.

[0018] In some embodiments, as described above, the antigen-binding molecule has a single variable domain (VHH) of the immunoglobulin that specifically binds to HSA.

[0019] In some embodiments, as described in any of the preceding embodiments, the extended half-life domain is connected to the N-terminus or C-terminus of the first antigen-binding domain via linker 1. In some embodiments, as described in any of the preceding embodiments, the extended half-life domain is connected to the N-terminus of the VL or VH of the first antigen-binding domain via linker 1. In some embodiments, as described in any of the preceding embodiments, the extended half-life domain is connected to the N-terminus of the VL of the first antigen-binding domain via linker 1. In some embodiments, as described in any of the preceding embodiments, the extended half-life domain is connected to the N-terminus of the VH of the first antigen-binding domain via linker 1.

[0020] In some embodiments, as described in any of the preceding antigen-binding molecules, wherein the linker 1 is a cleavable linker or a non-cleavable linker. In some embodiments, as described in any of the preceding antigen-binding molecules, the linker 1 is a cleavable linker. In some embodiments, as described in any of the preceding antigen-binding molecules, the linker 1 comprises a masking peptide and / or a protease-cleavable sequence. In some embodiments, as described in any of the preceding antigen-binding molecules, the linker 1 comprises a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus.

[0021] In another aspect, this disclosure provides an antigen-binding molecule comprising:

[0022] An antigen-binding domain that specifically binds to CD3, wherein the antigen-binding domain that specifically binds to CD3 is Fab; and

[0023] Half-life extended structural domains; and

[0024] A linker comprising, from N-terminus to C-terminus, a masking peptide and a protease-cleavable sequence;

[0025] The extended half-life domain is connected to the N-terminus of the antigen-binding domain that specifically binds to CD3 via a linker.

[0026] In some embodiments, as described above, the linker is linker 1 in the antigen-binding molecule.

[0027] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the antigen-binding domain that specifically binds to CD3 is a first antigen-binding domain.

[0028] In another aspect, this disclosure provides an antigen-binding molecule comprising:

[0029] An antigen-binding domain that specifically binds to CD3, wherein the antigen-binding domain specifically binding to CD3 is Fab, and the Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1); and

[0030] Half-life extended structural domains; and

[0031] A linker comprising a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus;

[0032] The extended half-life domain is connected to the N-terminus of the VL or VH of the antigen-binding domain that specifically binds to CD3 via a linker.

[0033] In some embodiments, as described above, the linker is linker 1 in the antigen-binding molecule.

[0034] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the antigen-binding domain that specifically binds to CD3 is a first antigen-binding domain.

[0035] In another aspect, this disclosure provides an antigen-binding molecule comprising:

[0036] The first antigen-binding domain that specifically binds to CD3 is Fab, wherein the Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1); and

[0037] Half-life extended structural domains; and

[0038] Linker 1, wherein linker 1 comprises, from N-terminus to C-terminus, a masking peptide and a protease-cleavable sequence;

[0039] The half-life extension domain is connected to the N-terminus of the VL or VH of the first antigen-binding domain via linker 1.

[0040] In some embodiments, as described in any of the preceding embodiments, the masking peptide inhibits or reduces the binding of the first antigen-binding domain to CD3. In some embodiments, as described in any of the preceding embodiments, the masking peptide inhibits or reduces the binding of the first antigen-binding domain to the N-terminus of CD3ε.

[0041] In some embodiments, as described in any of the preceding antigen-binding molecules, the masking peptide binds to the first antigen-binding domain via ionic interactions, electrostatic interactions, hydrophobic interactions, π-stacking interactions, and hydrogen bonding interactions, or combinations thereof. In some embodiments, as described in any of the preceding antigen-binding molecules, the masking peptide comprises a peptide sequence of at least 1 to no more than 40 amino acids. In some embodiments, as described in any of the preceding antigen-binding molecules, the masking peptide comprises a peptide sequence of at least 1 to no more than 20 amino acids. In some embodiments, as described in any of the preceding antigen-binding molecules, the masking peptide comprises a peptide sequence of at least 1 to no more than 20 amino acids. In some embodiments, as described in any of the preceding antigen-binding molecules, the masking peptide comprises a peptide sequence of at least 5 to no more than 10 amino acids. In some embodiments, as described in any of the preceding antigen-binding molecules, the masking peptide comprises a peptide sequence of 8 amino acids.

[0042] In some embodiments, as described in any of the preceding embodiments, the masking peptide comprises a cyclic peptide or a linear peptide. In some embodiments, as described in any of the preceding embodiments, the masking peptide comprises a cyclic peptide. In some embodiments, as described in any of the preceding embodiments, the masking peptide comprises a linear peptide.

[0043] In some embodiments, the masking peptide, as described in any of the preceding embodiments, comprises SEQ ID NO: 18, or an amino acid sequence having at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with it.

[0044] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the protease is a protease expressed or overexpressed in the tumor microenvironment. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the protease is selected from one or more of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamate proteases, gelatinases, and asparagine peptide lysins. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the protease is selected from one or two of metalloproteinases and serine proteases. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the metalloproteinase is a matrix metalloproteinase (MMP), and the serine protease is urokinase (uPA), protein lyase (MTSP1), or hepsin, or combinations thereof. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the matrix metalloproteinase is MMP2, MMP7, MMP9, MMP13, or MMP14, or combinations thereof.

[0045] In some embodiments, as described in any of the preceding antigen-binding molecules, the protease-cleavable sequence is a peptide sequence comprising at least 1 to no more than 50 amino acids. In some embodiments, as described in any of the preceding antigen-binding molecules, the protease-cleavable sequence is a peptide sequence comprising at least 1 to no more than 30 amino acids. In some embodiments, as described in any of the preceding antigen-binding molecules, the protease-cleavable sequence is a peptide sequence comprising at least 1 to no more than 20 amino acids. In some embodiments, as described in any of the preceding antigen-binding molecules, the protease-cleavable sequence is a peptide sequence comprising at least 1 to no more than 10 amino acids. In some embodiments, as described in any of the preceding antigen-binding molecules, the protease-cleavable sequence is a peptide sequence comprising at least 6 amino acids. In some embodiments, as described in any of the preceding antigen-binding molecules, the protease-cleavable sequence is a peptide sequence comprising 6 amino acids.

[0046] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the protease-cleavable sequence comprises SEQ ID NO: 10, or an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with it.

[0047] In other embodiments, as described in any of the preceding embodiments, the protease-cleavable sequence of the antigen-binding molecule is recognized by MMP-2, MMP-9, or a combination thereof. In yet another embodiment, as described in any of the preceding embodiments, the protease-cleavable sequence comprises the sequences in GPLGMLSQ (SEQ ID NO: 64), GPLGLWAQ (SEQ ID NO: 65), GPLGLAG (SEQ ID NO: 66), KKNPAELIGPVD (SEQ ID NO: 67), KKQPAANLVAPED (SEQ ID NO: 68), GPLGIAGQ (SEQ ID NO: 69), or PVGLIG (SEQ ID NO: 70). In some embodiments, as described in any of the preceding embodiments, the protease-cleavable sequence of the antigen-binding molecule includes any protease cleavage site (protease-cleavable sequence) known in the art that is sensitive to proteases present in the tumor environment, such as, but not limited to, the protease cleavage sites disclosed in Eckhard, U et al., Matrix Biol. Jan; 49:37-60.

[0048] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments includes a protease-cleavable sequence comprising a uPA-cleavable sequence comprising sequences such as NSGRAV (SEQ ID NO: 71), SGRSA (SEQ ID NO: 72), LGGSGRSANAILE (SEQ ID NO: 73), SGRS (SEQ ID NO: 74), GGSGRSANK (SEQ ID NO: 75), LGGSGRSANAILEC (SEQ ID NO: 76), GGGRR (SEQ ID NO: 77), TGRGPS (SEQ ID NO: 78), LSGRSDNH (SEQ ID NO: 79), or PLTGRSGG (SEQ ID NO: 80).

[0049] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, the protease-cleavable sequence includes sequences such as QRRVVGG (SEQ ID NO: 81), QAR, AANL (SEQ ID NO: 82), PTNL (SEQ ID NO: 83), PTN, or SAN.

[0050] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, used as a substrate for the protease-cleavable sequence of MMP2 / 9, uPA, protein lyase, and asparagine endopeptidase, comprises the amino acid sequence of PLGLAGSGRSDNH (SEQ ID NO: 84).

[0051] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, when the protease-cleavable sequence is cleaved by a protease, the masking peptide changes from binding to non-binding with the first antigen-binding domain, thereby exposing the first antigen-binding domain to CD3.

[0052] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the linker 1 further comprises an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 85.

[0053] In some embodiments, as described in any of the preceding antigen-binding molecules, the linker 1 comprises a structure of L1-masking peptide-L2-protease-cleavable sequence-L3. In some embodiments, as described in any of the preceding antigen-binding molecules, the linker 1 has a structure as shown in the L1-masking peptide-L2-protease-cleavable sequence-L3, and the shown structure is arranged from the N-terminus to the C-terminus. In some embodiments, the antigen-binding molecules as described in any of the preceding claims, wherein L1, L2, L3 are each independently selected from (GS)n, (GGS)n, (GGGS)n (SEQ ID NO: 107), (GGSG)n (SEQ ID NO: 108), (GGSGG)n (SEQ ID NO: 109), (GGGGS)n (SEQ ID NO: 110), (GGGGG)n (SEQ ID NO: 111), or (GGG)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 85, LPETG (SEQ ID NO: 87), (GGGGSGGGS) (SEQ ID NO: 88), or SGGG (SEQ ID NO: 89). In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein L1, L2, and L3 have an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 85. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the amino acid sequence of L1 is as shown in SEQ ID NO: 3. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the amino acid sequence of L2 is as shown in SEQ ID NO: 85. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the amino acid sequence of L3 is as shown in SEQ ID NO: 6.

[0054] In some specific embodiments, as described in any of the preceding antigen-binding molecules, linker 1 is a peptide sequence containing at least 1 to no more than 50 amino acids. In some specific embodiments, as described in any of the preceding antigen-binding molecules, linker 1 is a peptide sequence containing at least 5 to no more than 50 amino acids. In some specific embodiments, as described in any of the preceding antigen-binding molecules, linker 1 is a peptide sequence containing at least 10 to no more than 50 amino acids. In some specific embodiments, as described in any of the preceding antigen-binding molecules, linker 1 is a peptide sequence containing at least 20 to no more than 40 amino acids. In some specific embodiments, as described in any of the preceding antigen-binding molecules, linker 1 is a peptide sequence containing at least 30 to no more than 40 amino acids. In some specific embodiments, as described in any of the preceding antigen-binding molecules, linker 1 is a peptide sequence containing 36 amino acids. In some specific embodiments, such as the antigen-binding molecule described in any of the preceding claims, the amino acid sequence of the linker 1 is shown as GGGGSGGGGSQDGNEEMGGGSGGSPLGLAGGGSGGS (SEQ ID NO: 86).

[0055] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the extended half-life domain binds to a serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35 / CR1; or the extended half-life domain is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered large-volume serum protein or a fragment thereof.

[0056] In some embodiments, the circulating immunoglobulin or fragment thereof, as described in any of the preceding embodiments, comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD, or fragment thereof.

[0057] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the extended half-life domain binds to a serum protein or a fragment thereof, or CD35 / CR1; or the extended half-life domain is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered large-volume serum protein.

[0058] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the engineered scaffold comprises sdAb, scFv, Fab, VHH, type III fibronectin domain, immunoglobulin-like scaffold, DARPin, cysteine ​​knot peptide, lipid carrier protein, triple helix bundle scaffold, protein G-associated albumin-binding module, or DNA or RNA aptamer scaffold. In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the bulk serum protein is albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, or pentamer IgM.

[0059] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the serum protein comprises albumin, thyroxine-binding protein, thyroxine carrier protein, 1-acid glycoprotein, transferrin, transferrin receptor or its transferrin-binding moiety, or fibrinogen. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the serum protein is albumin. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the serum protein is human serum albumin (HSA). In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the half-life extension domain is an antibody that specifically binds to HSA. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the half-life extension domain is an immunoglobulin single variable domain, scFv, Fd, Fv, dsFv, Fab, Fab′, or F(ab′)2. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the immunoglobulin single variable domain is VHH.

[0060] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the VHH is camel-derived, humanized, reverse-mutated, affinity-matured, T-cell epitope-removed, antibody deamidated, and / or antibody isomerized.

[0061] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the half-life extension domain comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 of the half-life extension domain comprise CDR1, CDR2 and CDR3 of SEQ ID NO: 11, respectively.

[0062] In some embodiments, as described in any of the preceding antigen-binding molecules, the CDR1, CDR2, and CDR3 of the half-life extension domain are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, as described in any of the preceding antigen-binding molecules, the CDR1, CDR2, and CDR3 of the half-life extension domain are defined according to the Kabat numbering rule. In some embodiments, as described in any of the preceding antigen-binding molecules, the CDR1, CDR2, and CDR3 of the half-life extension domain are defined according to the IMGT numbering rule. In some embodiments, as described in any of the preceding antigen-binding molecules, the CDR1, CDR2, and CDR3 of the half-life extension domain are defined according to the Chothia numbering rule. In some embodiments, as described in any of the preceding antigen-binding molecules, the CDR1, CDR2, and CDR3 of the half-life extension domain are defined according to the AbM numbering rule. In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the CDR1, CDR2, and CDR3 of the half-life extension domains are defined according to the Contact numbering rules.

[0063] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 51, CDR2 comprises the amino acid sequence of SEQ ID NO: 52, and CDR3 comprises the amino acid sequence of SEQ ID NO: 53.

[0064] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the CDR1, CDR2, and CDR3 of the half-life extension domain are defined according to the Kabat numbering rules.

[0065] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the half-life extension domain comprises SEQ ID NO: 11, or an amino acid sequence having at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with it.

[0066] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the VH of the first antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, and the VL of the first antigen-binding domain comprises LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, and HCDR3 of the VH comprise HCDR1, HCDR2, and HCDR3 as described in SEQ ID NO: 12, and the LCDR1, LCDR2, and LCDR3 of the VL comprise LCDR1, LCDR2, and LCDR3 as described in SEQ ID NO: 13 or 14, respectively.

[0067] In some embodiments, as described in any of the preceding embodiments, the HCDR1, HCDR2, and HCDR3 of the VH and the LCDR1, LCDR2, and LCDR3 of the VL are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, as described in any of the preceding embodiments, the HCDR1, HCDR2, and HCDR3 of the VH and the LCDR1, LCDR2, and LCDR3 of the VL are defined according to the Kabat numbering rule. In some embodiments, as described in any of the preceding embodiments, the HCDR1, HCDR2, and HCDR3 of the VH and the LCDR1, LCDR2, and LCDR3 of the VL are defined according to the IMGT numbering rule. In some embodiments, as described in any of the preceding embodiments, the HCDR1, HCDR2, and HCDR3 of the VH and the LCDR1, LCDR2, and LCDR3 of the VL are defined according to the Chothia numbering rule. In some embodiments, as described in any of the preceding embodiments, the HCDR1, HCDR2, and HCDR3 of the VH and the LCDR1, LCDR2, and LCDR3 of the VL are defined according to the AbM numbering rules. In some embodiments, as described in any of the preceding embodiments, the HCDR1, HCDR2, and HCDR3 of the VH and the LCDR1, LCDR2, and LCDR3 of the VL are defined according to the Contact numbering rules.

[0068] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the HCDR1 of the VH comprises the amino acid sequence of SEQ ID NO: 54, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 55, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 56, and the LCDR1 of the VL comprises the amino acid sequence of SEQ ID NO: 57, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 58, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 59; or

[0069] The VH's HCDR1 contains the amino acid sequence of SEQ ID NO: 54, HCDR2 contains the amino acid sequence of SEQ ID NO: 55, and HCDR3 contains the amino acid sequence of SEQ ID NO: 56; the VL's LCDR1 contains the amino acid sequence of SEQ ID NO: 60, LCDR2 contains the amino acid sequence of SEQ ID NO: 58, and LCDR3 contains the amino acid sequence of SEQ ID NO: 61.

[0070] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, wherein the HCDR1, HCDR2, and HCDR3 of the VH and the LCDR1, LCDR2, and LCDR3 of the VL are defined according to the Kabat numbering rules.

[0071] In some embodiments, as described in any of the preceding embodiments, the VH comprises SEQ ID NO: 12, or an amino acid sequence having at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with it, and the VL comprises SEQ ID NO: 12. NO: 13 or 14, or an amino acid sequence that is at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to it.

[0072] In some embodiments, as described in any of the preceding embodiments, the first antigen-binding domain is an antibody derived from 82# or an antigen-binding fragment thereof. In some embodiments, as described in any of the preceding embodiments, the 82# antibody is derived from PCT / CN2024 / 141016 (included herein by reference in its entirety).

[0073] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments further comprises an antigen-binding domain that specifically binds to a tumor-associated antigen (TAA). In some embodiments, the antigen-binding domain that specifically binds to the TAA is a second antigen-binding domain.

[0074] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the second antigen-binding domain is an immunoglobulin single variable domain, scFv, Fd, Fv, dsFv, Fab, Fab′, or F(ab′)2. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the second antigen-binding domain is an immunoglobulin single variable domain. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the immunoglobulin single variable domain is VHH.

[0075] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the VHH is camel-derived, humanized, reverse-mutated, affinity-matured, T-cell epitope-removed, antibody deamidated, and / or antibody isomerized.

[0076] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises:

[0077] (a) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is Fab;

[0078] (b) A second antigen-binding domain that specifically binds to a tumor-associated antigen (TAA), wherein the second antigen-binding domain is VHH; and

[0079] (c) A half-life extension domain that specifically binds to HSA, wherein the half-life extension domain is VHH, and wherein the half-life extension domain is connected to the N-terminus of the VL or VH of the first antigen-binding domain via a linker 1.

[0080] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises:

[0081] (a) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is Fab;

[0082] (b) A second antigen-binding domain that specifically binds to a tumor-associated antigen (TAA), wherein the second antigen-binding domain is VHH; and

[0083] (c) A half-life extension domain that specifically binds to HSA, wherein the half-life extension domain is VHH, wherein the half-life extension domain is operably connected to the N-terminus of the VL or VH of the first antigen-binding domain via a linker 1, the second antigen-binding domain is operably connected to the first antigen-binding domain, and the second antigen-binding domain and the linker 1 are operably connected to different ends of the first antigen-binding domain.

[0084] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises:

[0085] (a) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is Fab;

[0086] (b) Specifically binds to the second antigen-binding domain of TAA, wherein the second antigen-binding domain is VHH;

[0087] (c) A half-life extension domain that specifically binds to HSA, wherein the half-life extension domain is VHH; and

[0088] (d) Linker 1, which comprises a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus;

[0089] The half-life extension domain is connected to the N-terminus of the VL or VH of the first antigen-binding domain via linker 1.

[0090] In some embodiments, the antigen-binding molecule as described in the preceding one, wherein:

[0091] The extended half-life domain is operably connected to the N-terminus of VL of the first antigen-binding domain via linker 1, and the second antigen-binding domain is operably connected to the C-terminus of CH1, the C-terminus of CL, or the N-terminus of VH of the first antigen-binding domain; or

[0092] The extended half-life domain is operably connected to the N-terminus of the VH of the first antigen-binding domain via linker 1, and the second antigen-binding domain is operably connected to the C-terminus of CH1, the C-terminus of CL, or the N-terminus of VL of the first antigen-binding domain.

[0093] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises a first chain and a second chain, wherein:

[0094] (i) The first chain contains a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, VL and CL that specifically bind to CD3, and

[0095] The second chain contains a VHH that specifically binds to TAA, a VH that specifically binds to CD3, and CH1; or

[0096] (ii) The first chain contains a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, VL and CL that specifically bind to CD3, and

[0097] The second chain comprises a VH that specifically binds to CD3, a CH1 that specifically binds to TAA, and a VHH that specifically binds to TAA; or

[0098] (iii) The first chain comprises a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, a VL and CL that specifically bind to CD3, and a VHH that specifically binds to TAA.

[0099] The second chain contains VH and CH1 that specifically bind CD3; or

[0100] (iv) The first chain contains VHH, which specifically binds to TAA, VL and CL, which specifically bind to CD3, and

[0101] The second chain contains a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, and VH and CH1 that specifically bind to CD3; or

[0102] (v) The first chain comprises VL and CL, which specifically bind to CD3, and VHH, which specifically binds to TAA.

[0103] The second chain contains a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, and VH and CH1 that specifically bind to CD3; or

[0104] (vi) The first chain contains VL and CL that specifically bind to CD3, and

[0105] The second chain contains a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, a VH that specifically binds to CD3, CH1, and a VHH that specifically binds to TAA.

[0106] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises a first chain and a second chain, wherein:

[0107] (i) The first chain, from the N-terminus to the C-terminus, includes a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, VL and CL that specifically bind to CD3, and

[0108] The second chain, from the N-terminus to the C-terminus, includes a VHH that specifically binds to TAA, a VH that specifically binds to CD3, and CH1; or

[0109] (ii) The first chain, from the N-terminus to the C-terminus, includes a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, VL and CL that specifically bind to CD3, and

[0110] The second chain, from the N-terminus to the C-terminus, includes a VH and CH1 that specifically bind CD3 and a VHH that specifically binds TAA; or

[0111] (iii) The first chain, from the N-terminus to the C-terminus, includes a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, VL and CL that specifically bind to CD3, and a VHH that specifically binds to TAA.

[0112] The second chain contains VH and CH1 that specifically bind CD3 from the N-terminus to the C-terminus; or

[0113] (iv) The first chain, from the N-terminus to the C-terminus, includes VHH, which specifically binds to TAA, and VL and CL, which specifically bind to CD3, and

[0114] The second chain, from the N-terminus to the C-terminus, includes a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, and VH and CH1 that specifically bind to CD3; or

[0115] (v) The first chain, from the N-terminus to the C-terminus, includes VL and CL, which specifically bind CD3, and VHH, which specifically binds TAA.

[0116] The second chain, from the N-terminus to the C-terminus, includes a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, and VH and CH1 that specifically bind to CD3; or

[0117] (vi) The first chain contains VL and CL, which specifically bind CD3, from the N-terminus to the C-terminus.

[0118] The second chain contains, from the N-terminus to the C-terminus, a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, a VH that specifically binds to CD3, CH1, and a VHH that specifically binds to TAA.

[0119] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises a first chain and a second chain, specifically as follows from the N-terminus to the C-terminus:

[0120] (i) The first chain sequentially comprises α-HSA-VHH, a masking peptide, a protease-cleavable sequence, α-CD3-VL, and CL, wherein the C-terminus of α-HSA-VHH is operatively linked to the N-terminus of the masking peptide, the C-terminus of the masking peptide is operatively linked to the N-terminus of the protease-cleavable sequence, the C-terminus of the protease-cleavable sequence is operatively linked to the N-terminus of α-CD3-VL, and the C-terminus of α-CD3-VL is operatively linked to the N-terminus of CL; and

[0121] The second chain sequentially comprises α-TAA-VHH, α-CD3-VH, and CH1, wherein the C-terminus of α-TAA-VHH is operably connected to the N-terminus of α-CD3-VH, and the C-terminus of α-CD3-VH is operably connected to the N-terminus of CH1; or

[0122] (ii) The first chain sequentially comprises α-HSA-VHH, a masking peptide, a protease-cleavable sequence, α-CD3-VL, and CL, wherein the C-terminus of α-HSA-VHH is operatively linked to the N-terminus of the masking peptide, the C-terminus of the masking peptide is operatively linked to the N-terminus of the protease-cleavable sequence, the C-terminus of the protease-cleavable sequence is operatively linked to the N-terminus of α-CD3-VL, and the C-terminus of α-CD3-VL is operatively linked to the N-terminus of CL; and

[0123] The second chain sequentially comprises α-CD3-VH, CH1, and α-TAA-VHH, wherein the C-terminus of α-CD3-VH is operatively connected to the N-terminus of CH1, and the C-terminus of CH1 is operatively connected to the N-terminus of α-TAA-VHH; or

[0124] (iii) The first chain sequentially comprises α-HSA-VHH, a masking peptide, a protease-cleavable sequence, α-CD3-VL, CL, and α-TAA-VHH, wherein the C-terminus of α-HSA-VHH is operatively linked to the N-terminus of the masking peptide, the C-terminus of the masking peptide is operatively linked to the N-terminus of the protease-cleavable sequence, the C-terminus of the protease-cleavable sequence is operatively linked to the N-terminus of α-CD3-VL, the C-terminus of α-CD3-VL is operatively linked to the N-terminus of CL, and the C-terminus of CL is operatively linked to the N-terminus of α-TAA-VHH;

[0125] The second chain sequentially comprises α-CD3-VH and CH1, wherein the C-terminus of α-CD3-VH is operatively connected to the N-terminus of CH1; or

[0126] (iv) The first chain sequentially comprises α-TAA-VHH, α-CD3-VL, and CL, wherein the C-terminus of α-TAA-VHH is operably connected to the N-terminus of α-CD3-VL, and the C-terminus of α-CD3-VL is operably connected to the N-terminus of CL; and

[0127] The second chain sequentially comprises α-HSA-VHH, a masking peptide, a protease-cleavable sequence, α-CD3-VH, and CH1, wherein the C-terminus of α-HSA-VHH is operatively linked to the N-terminus of the masking peptide, the C-terminus of the masking peptide is operatively linked to the N-terminus of the protease-cleavable sequence, the C-terminus of the protease-cleavable sequence is operatively linked to the N-terminus of α-CD3-VH, and the C-terminus of α-CD3-VH is operatively linked to the N-terminus of CH1; or

[0128] (v) The first chain sequentially comprises α-CD3-VL, CL, and α-TAA-VHH, wherein the C-terminus of α-CD3-VL is operably connected to the N-terminus of CL, and the C-terminus of CL is operably connected to the N-terminus of α-TAA-VHH; and

[0129] The second chain sequentially comprises α-HSA-VHH, a masking peptide, a protease-cleavable sequence, α-CD3-VH, and CH1, wherein the C-terminus of α-HSA-VHH is operatively linked to the N-terminus of the masking peptide, the C-terminus of the masking peptide is operatively linked to the N-terminus of the protease-cleavable sequence, the C-terminus of the protease-cleavable sequence is operatively linked to the N-terminus of α-CD3-VH, and the C-terminus of α-CD3-VH is operatively linked to the N-terminus of CH1; or

[0130] (vi) The first chain sequentially comprises α-CD3-VL and CL, wherein the C-terminus of α-CD3-VL is operatively connected to the N-terminus of CL, and

[0131] The second chain sequentially comprises α-HSA-VHH, a masking peptide, a protease-cleavable sequence, α-CD3-VH, CH1, and α-TAA-VHH, wherein the C-terminus of α-HSA-VHH is operatively linked to the N-terminus of the masking peptide, the C-terminus of the masking peptide is operatively linked to the N-terminus of the protease-cleavable sequence, the C-terminus of the protease-cleavable sequence is operatively linked to the N-terminus of α-CD3-VH, the C-terminus of α-CD3-VH is operatively linked to the N-terminus of CH1, and the C-terminus of CH1 is operatively linked to the N-terminus of α-TAA-VHH.

[0132] In some embodiments, the antigen-binding molecule as described in any of the preceding claims comprises:

[0133] (i) A first chain having the structure shown in equation (a), and a second chain having the structure shown in equation (b), or

[0134] (ii) A first chain having the structure shown in equation (a), and a second chain having the structure shown in equation (c), or

[0135] (iii) A first chain having the structure shown in equation (d), and a second chain having the structure shown in equation (e), or

[0136] (iv) A first chain having the structure shown in equation (f), and a second chain having the structure shown in equation (g), or

[0137] (v) A first chain having the structure shown in formula (h), and a second chain having the structure shown in formula (g), or

[0138] (vi) A first chain having the structure shown in equation (i), and a second chain having the structure shown in equation (j);

[0139] in,

[0140] (a)[α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0141] (b)[α-TAA-VHH]-[connector 2a]-[α-CD3-VH]-[CH1];

[0142] (c)[α-CD3-VH]-[CH1]-[linker 2b]-[α-TAA-VHH];

[0143] (d)[α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2]c-[α-TAA-VHH];

[0144] (e)[α-CD3-VH]-[CH1];

[0145] (f)[α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0146] (g)[α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1];

[0147] (h)[α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0148] (i)[α-CD3-VL]-[CL];

[0149] (j)[α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1]-[connector 2f]-[α-TAA-VHH];

[0150] Wherein, linker 1 and linkers 2a, 2b, 2c, 2d, 2e and 2f are the same or different peptide linkers, or linkers 2a, 2b, 2c, 2d, 2e and 2f do not exist;

[0151] Optionally, the positions of CH1 and CL in the first and second chains can be interchanged; the technical solution after the interchange is still covered within the scope of this disclosure.

[0152] In some embodiments, as described in any of the preceding antigen-binding molecules, linkers 2a, 2b, 2c, 2d, 2e, and 2f are the same linker. In some embodiments, as described in any of the preceding antigen-binding molecules, linkers 2a, 2b, 2c, 2d, 2e, and 2f are linker 2.

[0153] In some embodiments, as described in any of the preceding antigen-binding molecules, linker 1 and linkers 2a, 2b, 2c, 2d, 2e, and 2f are different peptide linkers. In some embodiments, as described in any of the preceding antigen-binding molecules, linker 1 is a cleavable linker that comprises, from its N-terminus to its C-terminus, a CD3-masking peptide and a protease-cleavable sequence.

[0154] In some embodiments, the antigen-binding molecule as described in any of the preceding claims comprises:

[0155] (i) A first chain having the structure shown in equation (a), and a second chain having the structure shown in equation (b), or

[0156] (ii) A first chain having the structure shown in equation (a), and a second chain having the structure shown in equation (c), or

[0157] (iii) A first chain having the structure shown in equation (d), and a second chain having the structure shown in equation (e), or

[0158] (iv) A first chain having the structure shown in equation (f), and a second chain having the structure shown in equation (g), or

[0159] (v) A first chain having the structure shown in formula (h), and a second chain having the structure shown in formula (g), or

[0160] (vi) A first chain having the structure shown in equation (i), and a second chain having the structure shown in equation (j);

[0161] in,

[0162] (a) [α-HSA-VHH]-[masking peptide]-[protease-cleavable sequence]-[α-CD3-VL]-[CL];

[0163] (b)[α-TAA-VHH]-[connector 2a]-[α-CD3-VH]-[CH1];

[0164] (c)[α-CD3-VH]-[CH1]-[linker 2b]-[α-TAA-VHH];

[0165] (d) [α-HSA-VHH]-[masking peptide]-[protease-cleavable sequence]-[α-CD3-VL]-[CL]-[linker 2c]-[α-TAA-VHH];

[0166] (e)[α-CD3-VH]-[CH1];

[0167] (f)[α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0168] (g)[α-HSA-VHH]-[masking peptide]-[protease-cleavable sequence]-[α-CD3-VH]-[CH1];

[0169] (h)[α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0170] (i)[α-CD3-VL]-[CL];

[0171] (j)[α-HSA-VHH]-[masking peptide]-[protease-cleavable sequence]-[α-CD3-VH]-[CH1]-[linker 2f]-[α-TAA-VHH];

[0172] Optionally, the positions of CH1 and CL in the first and second chains can be interchanged.

[0173] In some embodiments, as described in any of the preceding antigen-binding molecules, linkers 2a, 2b, 2c, 2d, 2e, and 2f are the same linker. In some embodiments, as described in any of the preceding antigen-binding molecules, linkers 2a, 2b, 2c, 2d, 2e, and 2f are linker 2.

[0174] In some embodiments, the linker 2 is an uncleavable linker, as described in any of the preceding embodiments. In some embodiments, the linker 2 is selected from (GS)n, (GGS)n, (GGGS)n (SEQ ID NO: 107), (GGSG)n (SEQ ID NO: 108), (GGSGG)n (SEQ ID NO: 109), (GGGGS)n (SEQ ID NO: 110), (GGGGG)n (SEQ ID NO: 111), or (GGG)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 85, LPETG (SEQ ID NO: 87), (GGGGSGGGS) (SEQ ID NO: 88), or SGGG (SEQ ID NO: 89).

[0175] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the linker 2 has an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 85. In some specific embodiments, the amino acid sequence of the linker 2 is as shown in SEQ ID NO: 3 or 4.

[0176] In some embodiments, the antigen-binding molecules as described in any of the preceding embodiments have structures represented by formulas (a), (b), (c), (d), (e), (f), (g), (h), (i), and (j) arranged from the N-terminus to the C-terminus.

[0177] In other embodiments, the antigen-binding molecules described in any of the preceding embodiments have structures represented by formulas (a), (b), (c), (d), (e), (f), (g), (h), (i), and (j) arranged from the C-terminus to the N-terminus.

[0178] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments is shown in Figure 1A.

[0179] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0180] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0181] The second chain has a structure as shown in [α-TAA-VHH]-[connector 2a]-[α-CD3-VH]-[CH1];

[0182] Wherein linker 1 and linker 2a are the same or different peptide linkers, or linker 2a is absent;

[0183] The structures shown in the first and second chains are arranged from the N end to the C end.

[0184] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the structure of the antigen-binding molecule is shown in Figure 1B.

[0185] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0186] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0187] The second chain has a structure as shown in [α-CD3-VH]-[CH1]-[connector 2b]-[α-TAA-VHH];

[0188] The linker 1 and the linker 2b are the same or different peptide linkers, or the linker 2b is absent;

[0189] The structures shown in the first and second chains are arranged from the N end to the C end.

[0190] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the structure of the antigen-binding molecule is shown in Figure 1C.

[0191] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0192] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH];

[0193] The second chain has a structure as shown in [α-CD3-VH]-[CH1];

[0194] The linker 1 and the linker 2c are the same or different peptide linkers, or the linker 2c is absent;

[0195] The structures shown in the first and second chains are arranged from the N end to the C end.

[0196] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments has the structure shown in Figure 1D.

[0197] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0198] The first chain has a structure as shown in [α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0199] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1];

[0200] Wherein linker 1 and linker 2d are the same or different peptide linkers, or linker 2d is absent;

[0201] The structures shown in the first and second chains are arranged from the N end to the C end.

[0202] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments is shown in Figure 1E.

[0203] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0204] The first chain has a structure as shown in [α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0205] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1];

[0206] Wherein linker 1 and linker 2e are the same or different peptide linkers, or linker 2e is absent;

[0207] The structures shown in the first and second chains are arranged from the N end to the C end.

[0208] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments has the structure shown in Figure 1F.

[0209] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0210] The first chain has a structure as shown in [α-CD3-VL]-[CL];

[0211] The second chain has a structure as shown in [α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1]-[linker 2f]-[α-TAA-VHH];

[0212] Wherein linker 1 and linker 2f are the same or different peptide linkers, or linker 2f is absent;

[0213] The structures shown in the first and second chains are arranged from the N end to the C end.

[0214] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the structure of the antigen-binding molecule is shown in Figure 2A.

[0215] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0216] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0217] The second chain has a structure as shown in [α-TAA-VHH]-[connector 2a]-[α-CD3-VH]-[CH1-G1];

[0218] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2a is an uncleavable linker.

[0219] CH1-G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0220] The structures shown in the first and second chains are arranged from the N end to the C end.

[0221] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the structure of the antigen-binding molecule is shown in Figure 2B.

[0222] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0223] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0224] The second chain has a structure as shown in [α-CD3-VH]-[CH1-G1]-[connector 2b]-[α-TAA-VHH];

[0225] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2b is an uncleavable linker.

[0226] CH1-G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0227] The structures shown in the first and second chains are arranged from the N end to the C end.

[0228] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the structure of the antigen-binding molecule is shown in Figure 2C.

[0229] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0230] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH];

[0231] The second chain has a structure as shown in [α-CD3-VH]-[CH1-G1];

[0232] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2c is an uncleavable linker.

[0233] CH1-G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0234] The structures shown in the first and second chains are arranged from the N end to the C end.

[0235] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the structure of the antigen-binding molecule is shown in Figure 2D.

[0236] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0237] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0238] The second chain has a structure as shown in [α-TAA-VHH]-[connector 2a]-[α-CD3-VH]-[CH1-G4];

[0239] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2a is an uncleavable linker.

[0240] CH1-G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0241] The structures shown in the first and second chains are arranged from the N end to the C end.

[0242] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments is shown in Figure 2E.

[0243] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the antigen-binding molecule comprises a first chain and a second chain:

[0244] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0245] The second chain has a structure as shown in [α-CD3-VH]-[CH1_G4]-[connector 2b]-[α-TAA-VHH];

[0246] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2b is an uncleavable linker.

[0247] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0248] The structures shown in the first and second chains are arranged from the N end to the C end.

[0249] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments has the structure shown in Figure 2F.

[0250] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0251] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH];

[0252] The second chain has a structure as shown in [α-CD3-VH]-[CH1_G4];

[0253] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2c is an uncleavable linker.

[0254] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0255] The structures shown in the first and second chains are arranged from the N end to the C end.

[0256] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments has the structure shown in Figure 2G.

[0257] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0258] The first chain has a structure as shown in [α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0259] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G1];

[0260] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2d is an uncleavable linker.

[0261] CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0262] The structures shown in the first and second chains are arranged from the N end to the C end.

[0263] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the structure of the antigen-binding molecule is shown in Figure 2H.

[0264] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0265] The first chain has a structure as shown in [α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0266] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G1];

[0267] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2e is an uncleavable linker.

[0268] CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0269] The structures shown in the first and second chains are arranged from the N end to the C end.

[0270] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the structure of the antigen-binding molecule is shown in Figure 2I.

[0271] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0272] The first chain has a structure as shown in [α-CD3-VL]-[CL];

[0273] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G1]-[connector 2f]-[α-TAA-VHH];

[0274] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2f is an uncleavable linker.

[0275] CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0276] The structures shown in the first and second chains are arranged from the N end to the C end.

[0277] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments has the structure shown in Figure 2J.

[0278] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0279] The first chain has a structure as shown in [α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0280] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G4];

[0281] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2d is an uncleavable linker.

[0282] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0283] The structures shown in the first and second chains are arranged from the N end to the C end.

[0284] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the structure of the antigen-binding molecule is shown in Figure 2K.

[0285] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0286] The first chain has a structure as shown in [α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0287] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G4];

[0288] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2e is an uncleavable linker.

[0289] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0290] The structures shown in the first and second chains are arranged from the N end to the C end.

[0291] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments has the structure shown in Figure 2L.

[0292] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0293] The first chain has a structure as shown in [α-CD3-VL]-[CL];

[0294] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G4]-[connector 2f]-[α-TAA-VHH];

[0295] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2f is an uncleavable linker.

[0296] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0297] The structures shown in the first and second chains are arranged from the N end to the C end.

[0298] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments further comprises one or more identical or different target antigen-binding domains, said target antigen-binding domains specifically binding to tumor-associated antigens, co-stimulatory molecules, and / or effector cell antigens. In some embodiments, the target antigen-binding domain of the antigen-binding molecule as described in any of the preceding embodiments specifically binds to tumor-associated antigens and co-stimulatory molecules.

[0299] In some embodiments, as described in any of the preceding embodiments, the target antigen-binding domain is an immunoglobulin single variable domain, scFv, Fd, Fv, dsFv, Fab, Fab′, or F(ab′)2. In some embodiments, as described in any of the preceding embodiments, the target antigen-binding domain is an immunoglobulin single variable domain. In some embodiments, as described in any of the preceding embodiments, the immunoglobulin single variable domain is VHH.

[0300] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments further comprises a third antigen-binding domain that specifically binds to the TAA and a fourth domain that specifically binds to the co-stimulatory molecule.

[0301] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the tumor-associated antigen (TAA) is selected from extracellular epitopes of tumor cell surface antigens, tumor microenvironment antigens, matrix antigens in the tumor microenvironment (TME), angiogenic antigens in the TME, antigens on blood vessels in the TME, cytokine antigens in the TME, and any combination thereof.

[0302] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the co-stimulatory molecule is selected from one or more of CD28, 41-BB, CD2, OX40, ICOS, CD40, CD27, and CD244. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the co-stimulatory molecule is CD28.

[0303] In some embodiments, as described in any of the preceding embodiments, the third and fourth antigen-binding domains are immunoglobulin single variable domains, scFv, Fd, Fv, dsFv, Fab, Fab′, or F(ab′)2. In some embodiments, as described in any of the preceding embodiments, the third and fourth antigen-binding domains are immunoglobulin single variable domains. In some embodiments, as described in any of the preceding embodiments, the immunoglobulin single variable domain is VHH.

[0304] In some embodiments, the antigen-binding molecule as described in any of the preceding claims further comprises a second antigen-binding domain specifically binding to target A, a third antigen-binding domain specifically binding to target B, and a fourth antigen-binding domain specifically binding to target C, wherein target A, target B, and target C are the same or different. In some embodiments, the antigen-binding molecule as described in any of the preceding claims contains different targets A, target B, and target C. In some embodiments, the antigen-binding molecule as described in any of the preceding claims contains at least one of target A, target B, and target C as a tumor-associated antigen. In some embodiments, the antigen-binding molecule as described in any of the preceding claims contains one of target A, target B, and target C as a tumor-associated antigen, and the other two are the same or different, and are each independently selected from tumor-associated antigens and effector cell antigens.

[0305] In some embodiments, the antigen-binding molecule as described in the preceding one, wherein:

[0306] When target A is an effector cell antigen, targets B and C are tumor-associated antigens; or

[0307] When target C is an effector cell antigen, targets A and B are tumor-associated antigens.

[0308] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the tumor-associated antigen is selected from extracellular epitopes of tumor cell surface antigens, tumor microenvironment antigens, matrix antigens in the tumor microenvironment (TME), angiogenic antigens in the TME, antigens on blood vessels in the TME, cytokine antigens in the TME, and any combination thereof. In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the tumor-associated antigen is selected from one or both of EGFR and PSMA.

[0309] In some embodiments, the effector cell antigen of the antigen-binding molecule as described in any of the preceding embodiments is selected from T cell antigens, B cell antigens, and NK cell antigens. In some embodiments, the effector cell antigen of the antigen-binding molecule as described in any of the preceding embodiments is a T cell antigen. In some embodiments, the effector cell antigen of the antigen-binding molecule as described in any of the preceding embodiments is selected from one or more of CD28, 41-BB, CD2, OX40, ICOS, CD40, CD27, and CD244. In some embodiments, the effector cell antigen of the antigen-binding molecule as described in any of the preceding embodiments is CD28.

[0310] In some embodiments, as described in any of the preceding embodiments, the second, third, and fourth antigen-binding domains are immunoglobulin single variable domains, scFv, Fd, Fv, dsFv, Fab, Fab′, or F(ab′)2. In some embodiments, as described in any of the preceding embodiments, the immunoglobulin single variable domain is VHH. In some embodiments, as described in any of the preceding embodiments, the second, third, and fourth antigen-binding domains are VHH.

[0311] In some embodiments, the antigen-binding molecule as described in any of the preceding claims comprises:

[0312] (a) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is Fab;

[0313] (b) Specifically binds to the second antigen-binding domain of target A, wherein the second antigen-binding domain is VHH;

[0314] (c) Specifically binds to the third antigen-binding domain of target B, wherein the third antigen-binding domain is VHH;

[0315] (d) Specifically binds to the fourth antigen-binding domain of target C, wherein the fourth antigen-binding domain is VHH;

[0316] (e) A half-life extension domain that specifically binds to HSA, wherein the half-life extension domain is VHH;

[0317] The half-life extension domain is connected to the N-terminus of the VL or VH of the first antigen-binding domain via a linker (preferably linker 1).

[0318] In some embodiments, the antigen-binding molecule as described in any of the preceding claims comprises:

[0319] (vii) A first chain having the structure shown in equation (k), and a second chain having the structure shown in equation (l), or

[0320] (ⅷ) A first chain having the structure shown in formula (m), and a second chain having the structure shown in formula (n);

[0321] in,

[0322] (k)[α-HSA-VHH]-[linker 1]-[α-CD3-VL]-[CL]-[linker 2g]-[α-target A-VHH];

[0323] (l)[α-target B-VHH]-[linker 3a]-[α-CD3-VH]-[CH1]-[linker 4a]-[α-target C-VHH];

[0324] (m)[α-target B-VHH]-[connector 3b]-[α-CD3-VL]-[CL]-[connector 2h]-[α-target A-VHH];

[0325] (n)[α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1]-[connector 4b]-[α-target C-VHH];

[0326] Wherein, the linker 1, the linkers 2g and 2h, the linkers 3a and 3b or the linkers 4a and 4b are the same or different peptide linkers, or the linkers 2g and 2h, the linkers 3a and 3b and / or the linkers 4a and 4b are absent;

[0327] The structures shown in equations (k), (l), (m), and (n) are arranged from the N end to the C end;

[0328] Optionally, the positions of CH1 and CL in the first and second chains can be interchanged.

[0329] In some embodiments, the antigen-binding molecule as described in any of the preceding claims comprises:

[0330] (vii) A first chain having the structure shown in equation (k), and a second chain having the structure shown in equation (l), or

[0331] (ⅷ) A first chain having the structure shown in formula (m), and a second chain having the structure shown in formula (n);

[0332] in,

[0333] (k)[α-HSA-VHH]-[masking peptide]-[protease-cleavable sequence]-[α-CD3-VL]-[CL]-[linker 2g]-[α-target A-VHH];

[0334] (l)[α-target B-VHH]-[linker 3a]-[α-CD3-VH]-[CH1]-[linker 4a]-[α-target C-VHH];

[0335] (m)[α-target B-VHH]-[connector 3b]-[α-CD3-VL]-[CL]-[connector 2h]-[α-target A-VHH];

[0336] (n)[α-HSA-VHH]-[masking peptide]-[protease-cleavable sequence]-[α-CD3-VH]-[CH1]-[linker 4b]-[α-target C-VHH];

[0337] Wherein, the linkers 2g and 2h, the linkers 3a and 3b, or the linkers 4a and 4b are the same or different peptide linkers, or the linkers 2g and 2h, the linkers 3a and 3b, and / or the linkers 4a and 4b are absent;

[0338] The structures shown in equations (k), (l), (m), and (n) are arranged from the N end to the C end;

[0339] Optionally, the positions of CH1 and CL in the first and second chains can be interchanged.

[0340] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the structure of the antigen-binding molecule is shown in Figure 4A.

[0341] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0342] The first chain has a structure as shown in [α-target B-VHH]-[connector 3b]-[α-CD3-VL]-[CL]-[connector 2h]-[α-target A-VHH].

[0343] The second chain has a structure as shown in [α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1]-[linker 4b]-[α-target C-VHH];

[0344] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2h, 3b, and 4b are non-cleavable linkers.

[0345] The first and second chains are arranged from the N end to the C end.

[0346] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, the structure of the antigen-binding molecule is shown in Figure 4B.

[0347] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0348] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2g]-[α-target A-VHH];

[0349] The second chain has a structure as shown in [α-target B-VHH]-[linker 3a]-[α-CD3-VH]-[CH1]-[linker 4a]-[α-target C-VHH];

[0350] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3a and 4a are non-cleavable linkers.

[0351] Both the first and second chains are arranged from the N end to the C end.

[0352] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0353] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2g]-[α-target A-VHH];

[0354] The second chain has a structure as shown in [α-target B-VHH]-[connector 3a]-[α-CD3-VH]-[CH1_G1]-[connector 4a]-[α-target C-VHH];

[0355] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3a and 4a are non-cleavable linkers.

[0356] CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0357] Both the first and second chains are arranged from the N end to the C end.

[0358] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0359] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2g]-[α-target A-VHH];

[0360] The second chain has a structure as shown in [α-target B-VHH]-[connector 3a]-[α-CD3-VH]-[CH1_G4]-[connector 4a]-[α-target C-VHH];

[0361] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3a and 4a are non-cleavable linkers.

[0362] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0363] Both the first and second chains are arranged from the N end to the C end.

[0364] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0365] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2g]-[α-target A-VHH];

[0366] The second chain has a structure as shown in [α-target B-VHH]-[connector 3a]-[α-CD3-VH]-[CH1_G1]-[connector 4a]-[α-target C-VHH];

[0367] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3a and 4a are non-cleavable linkers.

[0368] CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0369] Both the first and second chains are arranged from the N end to the C end.

[0370] Wherein, target A is an effector cell antigen, and target B and target C are TAAs.

[0371] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0372] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2g]-[α-target A-VHH];

[0373] The second chain has a structure as shown in [α-target B-VHH]-[connector 3a]-[α-CD3-VH]-[CH1_G4]-[connector 4a]-[α-target C-VHH];

[0374] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3a and 4a are non-cleavable linkers.

[0375] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0376] Both the first and second chains are arranged from the N end to the C end.

[0377] Wherein target C is an effector cell antigen, and target A and target B are TAAs.

[0378] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0379] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2g]-[α-CD28-VHH];

[0380] The second chain has a structure as shown in [α-PSMA-VHH]-[linker 3a]-[α-CD3-VH]-[CH1_G1]-[linker 4a]-[α-EGFR-VHH];

[0381] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3a and 4a are non-cleavable linkers.

[0382] CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0383] Both the first and second chains are arranged from the N end to the C end.

[0384] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0385] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2g]-[α-EGFR-VHH];

[0386] The second chain has a structure as shown in [α-PSMA-VHH]-[linker 3a]-[α-CD3-VH]-[CH1_G1]-[linker 4a]-[α-CD28-VHH];

[0387] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3a and 4a are non-cleavable linkers.

[0388] CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0389] Both the first and second chains are arranged from the N end to the C end.

[0390] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0391] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2g]-[α-CD28-VHH];

[0392] The second chain has a structure as shown in [α-PSMA-VHH]-[linker 3a]-[α-CD3-VH]-[CH1_G4]-[linker 4a]-[α-EGFR-VHH];

[0393] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3a and 4a are non-cleavable linkers.

[0394] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0395] Both the first and second chains are arranged from the N end to the C end.

[0396] In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein:

[0397] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2g]-[α-EGFR-VHH];

[0398] The second chain has a structure as shown in [α-PSMA-VHH]-[linker 3a]-[α-CD3-VH]-[CH1_G4]-[linker 4a]-[α-CD28-VHH];

[0399] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3a and 4a are non-cleavable linkers.

[0400] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0401] Both the first and second chains are arranged from the N end to the C end.

[0402] In some embodiments, as described in the preceding claim, the antigen-binding molecule, wherein linkers 2g and 2h are the same linker. In some embodiments, as described in the preceding claim, the antigen-binding molecule, wherein linkers 2g and 2h are linker 2.

[0403] In some embodiments, as described in the preceding claim, the antigen-binding molecule, wherein linkers 3a and 3b are the same linker. In some embodiments, as described in the preceding claim, the antigen-binding molecule, wherein linkers 3a and 3b are linker 3.

[0404] In some embodiments, as described in the preceding claim, the antigen-binding molecule, wherein linkers 4a and 4b are the same linker. In some embodiments, as described in the preceding claim, the antigen-binding molecule, wherein linkers 4a and 4b are linker 4.

[0405] In some embodiments, in the antigen-binding molecule as described in any of the preceding claims, wherein the linker 2, the linker 3, or the linker 4 is an uncleavable linker or a cleavable linker. In some embodiments, in the antigen-binding molecule as described in any of the preceding claims, wherein the linker 2, the linker 3, or the linker 4 is an uncleavable linker. In some embodiments, the antigen-binding molecule as described in any of the preceding claims, wherein the linker 2, the linker 3, or the linker 4 is each independently selected from (GS)n, (GGS)n, (GGGS)n (SEQ ID NO: 107), (GGSG)n (SEQ ID NO: 108), (GGSGG)n (SEQ ID NO: 109), (GGGGS)n (SEQ ID NO: 110), (GGGGG)n (SEQ ID NO: 111), or (GGG)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 85, LPETG (SEQ ID NO: 87), (GGGGSGGGS) (SEQ ID NO: 88), or SGGG (SEQ ID NO: 89). In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the linker 2, the linker 3, or the linker 4 has an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 85.

[0406] In some embodiments, as described in any of the preceding embodiments, the first and second chains are linked by disulfide bonds in CH1 and CL to form a heterologous antigen-binding molecule. In some embodiments, as described in any of the preceding embodiments, the positions of CH1 and CL may be interchanged.

[0407] In some embodiments, such as the antigen-binding molecule described in any of the preceding embodiments, wherein the first chain and the second chain form a heterodimer.

[0408] In some embodiments, as described in any of the preceding embodiments, the CH1 is derived from IgG. In some embodiments, as described in any of the preceding embodiments, the CH1 is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, as described in any of the preceding embodiments, the CH1 is derived from IgG1 or IgG4. In some embodiments, as described in any of the preceding embodiments, the CH1 comprises SEQ ID NO: 16 or 17, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it. In some embodiments, as described in any of the preceding embodiments, the amino acid sequence of the CH1 is as shown in SEQ ID NO: 16 or 17. In some embodiments, as described in any of the preceding embodiments, the CH1 is derived from IgG1. In some embodiments, as described in any of the preceding embodiments, the amino acid sequence of the CH1 is as shown in SEQ ID NO: 16. In some embodiments, as described in any of the preceding antigen-binding molecules, the CH1 is derived from IgG4. In some embodiments, as described in any of the preceding antigen-binding molecules, the amino acid sequence of the CH1 is shown in SEQ ID NO: 17.

[0409] In some embodiments, as described in any of the preceding embodiments, the CL is derived from the light chain constant region of human kappa or lambda. In some embodiments, as described in any of the preceding embodiments, the CL is derived from the light chain constant region of human kappa. In some embodiments, as described in any of the preceding embodiments, the CL comprises SEQ ID NO: 15, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it. In some embodiments, as described in any of the preceding embodiments, the amino acid sequence of the CL is as shown in SEQ ID NO: 15.

[0410] In some embodiments, as described in any of the preceding embodiments, the sequences of the first and second chains are different. In some embodiments, as described in any of the preceding embodiments, the first and second chains are used only to distinguish amino acid sequences and do not limit the positional relationship between the polypeptide and the protein. When either one is the first chain, the other is the second chain.

[0411] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments is an antibody. In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments is an activatable antibody. In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments is a multispecific activatable antibody. In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments is a trispecific or pentaspecific activatable antibody. In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments is a trispecific activatable antibody. In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments is a pentaspecific activatable antibody.

[0412] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments does not contain an Fc domain. In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments does not contain a CH2 domain. In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments does not contain a CH3 domain.

[0413] Activated antibodies

[0414] In another aspect, this disclosure provides an activatable antibody comprising:

[0415] (a) An antigen-binding domain that specifically binds to CD3, wherein the antigen-binding domain that specifically binds to CD3 is Fab;

[0416] (b) An antigen-binding domain that specifically binds to a tumor-associated antigen (TAA), wherein the antigen-binding domain specifically binding to the TAA is VHH; and

[0417] (c) A half-life extension domain that specifically binds to human serum albumin (HSA), wherein the half-life extension domain that specifically binds to HSA is VHH;

[0418] (d) A linker comprising a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus;

[0419] The extended half-life domain is connected via a linker to the N-terminus of the light chain variable region (VL) or heavy chain variable region (VH) of the antigen-binding domain that specifically binds to CD3.

[0420] In some implementations, such as the activatable antibody described in any of the preceding embodiments, the linker is linker 1.

[0421] In some embodiments, such as the activatable antibody described in any of the preceding embodiments, the antigen-binding domain that specifically binds to CD3 is a first antigen-binding domain.

[0422] In some embodiments, such as the activatable antibody described in any of the preceding embodiments, wherein the antigen-binding domain that specifically binds to the TAA is a second antigen-binding domain.

[0423] In some implementations, such as the activatable antibody described in the preceding one, wherein:

[0424] The extended half-life domain is operably connected to the N-terminus of VL of the first antigen-binding domain via linker 1, and the second antigen-binding domain is operably connected to the C-terminus of CH1, the C-terminus of CL, or the N-terminus of VH of the first antigen-binding domain; or

[0425] The extended half-life domain is operably connected to the N-terminus of the VH of the first antigen-binding domain via linker 1, and the second antigen-binding domain is operably connected to the C-terminus of CH1, the C-terminus of CL, or the N-terminus of VL of the first antigen-binding domain.

[0426] In some embodiments, the activatable antibody as described above has the structure as described in any of the preceding embodiments.

[0427] In some embodiments, the activatable antibody as described above has a masking peptide as described in any of the preceding embodiments.

[0428] In some embodiments, the activatable antibody as described above has a protease-cleavable sequence as described in any of the preceding embodiments.

[0429] In some embodiments, the activatable antibody as described above has a first antigen-binding domain that specifically binds to CD3 as described in any of the preceding embodiments.

[0430] In some embodiments, the activatable antibody as described above has a half-life extension domain as described in any of the preceding embodiments.

[0431] Trispecific activated antibodies

[0432] In another aspect, this disclosure provides a trispecific activatable antibody, wherein the trispecific activatable antibody comprises:

[0433] (a) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is Fab;

[0434] (b) A second antigen-binding domain that specifically binds to a tumor-associated antigen (TAA), wherein the second antigen-binding domain is VHH; and

[0435] (c) Specifically binds to the half-life extension domain of human serum albumin (HSA), wherein the half-life extension domain is VHH;

[0436] The extended half-life domain is connected to the N-terminus of the light chain variable region or heavy chain variable region of the first antigen-binding domain via a linker (preferably linker 1), and the linker (preferably linker 1) sequentially comprises a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; or

[0437] The trispecific activatable antibody comprises:

[0438] (a) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is scFv;

[0439] (b) A second antigen-binding domain that specifically binds to a tumor-associated antigen (TAA), wherein the second antigen-binding domain is VHH; and

[0440] (c) Specifically binds to the half-life extension domain of human serum albumin (HSA), wherein the half-life extension domain is VHH;

[0441] The extended half-life domain is connected to the N-terminus of the light chain variable region of the first antigen-binding domain via a linker (preferably linker 1). Linker 1 contains a masking peptide and a protease-cleavable sequence sequentially from the N-terminus to the C-terminus. The second antigen-binding domain is connected to the C-terminus of the heavy chain variable region of the first antigen-binding domain via a linker (preferably linker 3c).

[0442] In some embodiments, the trispecific activatable antibody as described above comprises:

[0443] (a) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is Fab;

[0444] (b) A second antigen-binding domain that specifically binds to TAA, wherein the second antigen-binding domain is VHH; and

[0445] (c) A half-life extension domain that specifically binds to HSA, wherein the half-life extension domain is VHH;

[0446] The extended half-life domain is connected to the N-terminus of the light chain variable region or the heavy chain variable region of the first antigen-binding domain via linker 1. Linker 1 contains a masking peptide and a protease-cleavable sequence sequentially from the N-terminus to the C-terminus.

[0447] In some implementations, such as the trispecific activatable antibody described in the preceding one, wherein:

[0448] The extended half-life domain is operably connected to the N-terminus of VL of the first antigen-binding domain via linker 1, and the second antigen-binding domain is operably connected to the C-terminus of CH1, the C-terminus of CL, or the N-terminus of VH of the first antigen-binding domain; or

[0449] The extended half-life domain is operably connected to the N-terminus of the VH of the first antigen-binding domain via linker 1, and the second antigen-binding domain is operably connected to the C-terminus of CH1, the C-terminus of CL, or the N-terminus of VL of the first antigen-binding domain.

[0450] In some embodiments, the antigen-binding molecule comprises:

[0451] (i) A first chain having the structure shown in equation (a), and a second chain having the structure shown in equation (b), or

[0452] (ii) A first chain having the structure shown in equation (a), and a second chain having the structure shown in equation (c), or

[0453] (iii) A first chain having the structure shown in equation (d), and a second chain having the structure shown in equation (e), or

[0454] (iv) A first chain having the structure shown in equation (f), and a second chain having the structure shown in equation (g), or

[0455] (v) A first chain having the structure shown in formula (h), and a second chain having the structure shown in formula (g), or

[0456] (vi) A first chain having the structure shown in equation (i), and a second chain having the structure shown in equation (j);

[0457] in,

[0458] (a)[α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0459] (b)[α-TAA-VHH]-[connector 2a]-[α-CD3-VH]-[CH1];

[0460] (c)[α-CD3-VH]-[CH1]-[linker 2b]-[α-TAA-VHH];

[0461] (d)[α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH];

[0462] (e)[α-CD3-VH]-[CH1];

[0463] (f)[α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0464] (g)[α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1];

[0465] (h)[α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0466] (i)[α-CD3-VL]-[CL];

[0467] (j)[α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1]-[connector 2f]-[α-TAA-VHH];

[0468] Wherein, linker 1 and linkers 2a, 2b, 2c, 2d, 2e and 2f are the same or different peptide linkers, or linkers 2a, 2b, 2c, 2d, 2e and 2f are not present; linker 1 contains a masking peptide and a protease-cleavable sequence sequentially from the N-terminus to the C-terminus;

[0469] The structures shown in equations (a), (b), (c), (d), (e), (f), (g), (h), (i), and (j) are arranged from the N end to the C end;

[0470] Optionally, the positions of CH1 and CL in the first and second chains can be interchanged.

[0471] In some embodiments, the trispecific activatable antibody as described above comprises:

[0472] (a) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is scFv;

[0473] (b) A second antigen-binding domain that specifically binds to TAA, wherein the second antigen-binding domain is VHH; and

[0474] (c) A half-life extension domain that specifically binds to HSA, wherein the half-life extension domain is VHH;

[0475] The extended half-life domain is connected to the N-terminus of the light chain variable region of the scFv of the first antigen-binding domain via linker 1. Linker 1 contains a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus. The second antigen-binding domain is connected to the C-terminus of the heavy chain variable region of the scFv of the first antigen-binding domain via linker 3c.

[0476] In some embodiments, the trispecific activatable antibody comprises, from the N-terminus to the C-terminus, the following:

[0477] [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[connector 2i]-[α-CD3-VL]-[connector 3c]-[α-TAA-VHH];

[0478] The linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence sequentially from the N-terminus to the C-terminus.

[0479] In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the masking peptide inhibits or reduces the binding of the first antigen-binding domain to CD3. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the masking peptide inhibits or reduces the binding of the first antigen-binding domain to the N-terminus of CD3ε. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the masking peptide comprises SEQ ID NO: 18, or an amino acid sequence having at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with it.

[0480] In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the protease is a protease expressed or overexpressed in the tumor microenvironment. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the protease is selected from one or more of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamate proteases, gelatinases, and asparagine peptidases. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the protease is selected from one or two of metalloproteinases and serine proteases. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the metalloproteinase is a matrix metalloproteinase (MMP), and the serine protease is urokinase (uPA), protein lyase (MTSP1), or hepsin, or combinations thereof. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the matrix metalloproteinase is MMP2, MMP7, MMP9, MMP13, or MMP14, or combinations thereof.

[0481] In some embodiments, such as the trispecific activatable antibody described in any of the preceding embodiments, wherein the protease-cleavable sequence comprises SEQ ID NO: 10, or an amino acid sequence having at least 70% or 80% (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with it.

[0482] In some embodiments, such as the trispecific activatable antibody described in any of the preceding embodiments, wherein the linker 1 further comprises an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 85.

[0483] In some embodiments, the trispecific activatable antibody as described in any of the preceding claims, wherein the linkers 2a, 2b, 2c, 2d, 2e, 2f, 2i, and 3c are uncleavable or cleavable linkers. In some embodiments, the trispecific activatable antibody as described in any of the preceding claims, wherein the linkers 2a, 2b, 2c, 2d, 2e, 2f, 2i, and 3c are uncleavable linkers. In some embodiments, the trispecific activatable antibody as described in any of the preceding claims, wherein the linkers 2a, 2b, 2c, 2d, 2e, 2f, 2i, and 3c have an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 85. In some embodiments, the trispecific activatable antibody as described in any of the preceding claims, wherein the amino acid sequences of the linkers 2a, 2b, 2c, 2d, 2e, 2f, 2i, and 3c are as shown in SEQ ID NO: 3.

[0484] In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments does not contain a CH2 domain. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments does not contain a CH3 domain. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments does not contain both CH2 and CH3 domains. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments does not contain an Fc domain.

[0485] Five specific activated antibodies

[0486] In another aspect, this disclosure provides a five-specific activatable antibody comprising:

[0487] (a) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is Fab;

[0488] (b) Specifically binds to the second antigen-binding domain of target A, wherein the second antigen-binding domain is VHH;

[0489] (c) Specifically binds to the third antigen-binding domain of target B, wherein the third antigen-binding domain is VHH;

[0490] (d) A fourth antigen-binding domain that specifically binds to target C, wherein the fourth antigen-binding domain is VHH; and

[0491] (e) A half-life extension domain that specifically binds to HSA, wherein the half-life extension domain is VHH;

[0492] The extended half-life domain is connected to the N-terminus of the first antigen-binding domain via a linker (preferably linker 1), and the linker (preferably linker 1) contains a masking peptide and a protease-cleavable sequence sequentially from the N-terminus to the C-terminus.

[0493] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments further comprises a second antigen-binding domain specifically binding to target A, a third antigen-binding domain specifically binding to target B, and a fourth antigen-binding domain specifically binding to target C, wherein target A, target B, and target C are the same or different. In some embodiments, target A, target B, and target C are different. In some embodiments, at least one of target A, target B, and target C is a tumor-associated antigen. In some embodiments, one of target A, target B, and target C is a tumor-associated antigen, and the other two are the same or different, and are each independently selected from tumor-associated antigens and effector cell antigens.

[0494] In some implementations, the antigen-binding molecule as described above, wherein:

[0495] When target A is an effector cell antigen, targets B and C are tumor-associated antigens; or

[0496] When target C is an effector cell antigen, targets A and B are tumor-associated antigens.

[0497] In some embodiments, the five-specific activatable antibody as described above comprises:

[0498] (vii) A first chain having the structure shown in equation (k), and a second chain having the structure shown in equation (l), or

[0499] (ⅷ) A first chain having the structure shown in formula (m), and a second chain having the structure shown in formula (n);

[0500] in,

[0501] (k)[α-HSA-VHH]-[linker 1]-[α-CD3-VL]-[CL]-[linker 2g]-[α-target A-VHH];

[0502] (l)[α-target B-VHH]-[linker 3a]-[α-CD3-VH]-[CH1]-[linker 4a]-[α-target C-VHH];

[0503] (m)[α-target B-VHH]-[connector 3b]-[α-CD3-VL]-[CL]-[connector 2h]-[α-target A-VHH];

[0504] (n)[α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1]-[connector 4b]-[α-target C-VHH];

[0505] Wherein, the linker 1, the linker 2g, 2h, the linker 3a, 3b or the linker 4a, 4b are the same or different peptide linkers, or the linker 2g, 2h, the linker 3a, 3b and / or the linker 4a, 4b are not present;

[0506] The structures shown in equations (k), (l), (m), and (n) are arranged from the N end to the C end;

[0507] Optionally, the positions of CH1 and CL in the first and second chains can be interchanged.

[0508] In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments, wherein the masking peptide inhibits or reduces the binding of the first antigen-binding domain to CD3. In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments, wherein the masking peptide inhibits or reduces the binding of the first antigen-binding domain to the N-terminus of CD3ε. In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments, wherein the masking peptide comprises SEQ ID NO: 18, or an amino acid sequence having at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with it.

[0509] In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments, wherein the protease is a protease expressed or overexpressed in the tumor microenvironment. In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments, wherein the protease is selected from one or more of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamate proteases, gelatinases, and asparagine peptidases. In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments, wherein the protease is selected from one or two of metalloproteinases and serine proteases. In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments, wherein the metalloproteinase is a matrix metalloproteinase (MMP), and the serine protease is urokinase (uPA), protein lyase (MTSP1), or hepsin, or combinations thereof. In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments, wherein the matrix metalloproteinase is MMP2, MMP7, MMP9, MMP13, or MMP14, or combinations thereof.

[0510] In some embodiments, such as the five-specific activatable antibody described in any of the preceding embodiments, wherein the protease-cleavable sequence comprises SEQ ID NO: 10, or an amino acid sequence having at least 70% or 80% (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with it.

[0511] In some embodiments, such as the five-specific activatable antibody described in any of the preceding embodiments, wherein the linker 1 further comprises an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 85.

[0512] In some embodiments, as described in the preceding claim, the linkers 2g and 2h are the same linker. In some embodiments, as described in the preceding claim, the linkers 2g and 2h are linker 2.

[0513] In some embodiments, as described in the preceding claim, the antigen-binding molecule, wherein the linkers 3a and 3b are the same linker. In some embodiments, as described in the preceding claim, the linkers 3a and 3b are linker 3.

[0514] In some embodiments, as described in the preceding claim, the linkers 4a and 4b are the same linker. In some embodiments, as described in the preceding claim, the linkers 4a and 4b are linker 4.

[0515] In some embodiments, such as the five-specific activatable antibody described in any of the preceding embodiments, wherein the linker 2, the linker 3, or the linker 4 has an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 85.

[0516] In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments does not contain a CH2 domain. In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments does not contain a CH3 domain. In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments does not contain both CH2 and CH3 domains. In some embodiments, the five-specific activatable antibody as described in any of the preceding embodiments does not contain an Fc domain.

[0517] In another aspect, this disclosure provides one or more nucleic acid molecules (e.g., isolated nucleic acid molecules) that encode an antigen-binding molecule as described in any of the preceding claims, or an activated antibody as described in any of the preceding claims, or a trispecific activated antibody as described in any of the preceding claims, or a pentaspecific activated antibody as described in any of the preceding claims.

[0518] In another aspect, this disclosure provides one or more vectors that encode one or more nucleic acid molecules (e.g., isolated nucleic acid molecules) as described in any of the preceding claims.

[0519] In another aspect, this disclosure provides one or more host cells that comprise nucleic acid molecules (e.g., isolated nucleic acid molecules) as described in any of the preceding embodiments. In some embodiments, the host cell is a non-human host cell. In some embodiments, the host cell is a bacterium, yeast, or mammalian cell. In some embodiments, the host cell is a mammalian cell.

[0520] In another aspect, this disclosure provides a pharmaceutical composition comprising an antigen-binding molecule as described in any of the preceding claims, or an activated antibody as described in any of the preceding claims, or a trispecific activated antibody as described in any of the preceding claims, or a pentaspecific activated antibody as described in any of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0521] In another aspect, this disclosure provides a method for preparing an antigen-binding molecule as described in any of the preceding claims, or an activatable antibody as described in any of the preceding claims, or a trispecific activatable antibody as described in any of the preceding claims, or a pentaspecific activatable antibody as described in any of the preceding claims. In some embodiments, the method includes expressing one or more nucleic acids (e.g., isolated nucleic acids) as described above, or culturing one or more host cells as described above to generate the antigen-binding molecule, activatable antibody, trispecific activatable antibody, or pentaspecific activatable antibody.

[0522] In another aspect, this disclosure provides a molecule (e.g., an activated molecule) derived from an antigen-binding molecule as described in any of the preceding claims, or an activatable antibody as described in any of the preceding claims, or a trispecific activatable antibody as described in any of the preceding claims, or a pentaspecific activatable antibody as described in any of the preceding claims, via protease cleavage. In some embodiments, the molecule as described above, wherein the protease is a protease expressed or overexpressed in the tumor microenvironment. In some embodiments, the molecule as described in any of the preceding claims, wherein the protease is selected from one or more of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamate proteases, gelatinases, and asparagine peptide lysases. In some embodiments, the molecule as described in any of the preceding claims, wherein the protease is selected from one or two of metalloproteinases and serine proteases. In some embodiments, the metalloproteinase is a matrix metalloproteinase (MMP), and the serine protease is urokinase (uPA), protein lyase (MTSP1), or hepsin, or combinations thereof. In some embodiments, the matrix metalloproteinase, as described in any of the preceding embodiments, is MMP2, MMP7, MMP9, MMP13, or MMP14, or combinations thereof.

[0523] In some implementations, the activated molecule, as described above, is an activated antibody.

[0524] In some embodiments, activatable antibodies have reduced adverse effects (e.g., side effects) compared to activated antibodies. In some embodiments, activatable antibodies have reduced adverse effects compared to activated antibodies, wherein the activatable antibody can be enzymatically activated or converted into an activated antibody, i.e., the active form of the antibody.

[0525] In some embodiments, the activatable antibody has an extended half-life compared to the activated antibody. In other embodiments, the activatable antibody has an extended half-life compared to the activated antibody, wherein the activatable antibody can be enzymatically activated or converted into the active form of the activated antibody, and the active form has a reduced half-life compared to the activatable antibody.

[0526] In some embodiments, the ability of the activatable antibody to bind to T cells is reduced. In some embodiments, the ability of the activatable antibody to activate T cells is reduced compared to an activated antibody. In some embodiments, the ability of the activatable antibody to bind to T cells is reduced. In some embodiments, the ability of the activatable antibody to activate T cells is reduced compared to an activated antibody, wherein the activatable antibody can be enzymatically activated or converted into the active form of the antibody.

[0527] In some embodiments, compared to activated antibodies, activatable antibodies have both an extended half-life and a reduced ability to activate T cells. In some embodiments, compared to activated antibodies, activatable antibodies have both an extended half-life and a reduced ability to bind to T cells. In some embodiments, compared to activated antibodies, activatable antibodies have both an extended half-life and a reduced ability to activate T cells, wherein the activatable antibody can be enzymatically activated or converted into the active form of the antibody. In some embodiments, compared to activated antibodies, activatable antibodies have both an extended half-life and a reduced ability to bind to T cells, wherein the activatable antibody can be enzymatically activated or converted into the active form of the antibody.

[0528] In some embodiments, the activatable antibody is synthesized in vitro. In some embodiments, when the activatable antibody is present in vivo in a non-tumor microenvironment (e.g., in circulation), it does not convert to its active antibody form.

[0529] In some implementations, the activatable antibodies disclosed herein do not induce a cytokine storm or induce the release of cytokines sufficient to induce toxic side effects.

[0530] In some embodiments, the activatable antibody disclosed herein exhibits good drug-like properties. In some embodiments, the SEC purity of the activatable antibody disclosed herein after storage at 40°C for 14 days is greater than 70% (e.g., greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%). In some embodiments, the change in the SEC peak value of the activatable antibody disclosed herein after storage at 40°C for 14 days is less than 30% (e.g., less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%).

[0531] In another aspect, this disclosure provides a method for treating, preventing, or improving a disease or condition, comprising administering to a subject in need a therapeutically effective amount of an antigen-binding molecule as described in any of the preceding claims, or an activated antibody as described in any of the preceding claims, or a trispecific activated antibody as described in any of the preceding claims, or a pentaspecific activated antibody as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims.

[0532] In another aspect, this disclosure provides use in the preparation of a medicament for treating, preventing, or improving a disease or condition, comprising administering to a subject a therapeutically effective amount of an antigen-binding molecule as described in any of the preceding claims, or an activated antibody as described in any of the preceding claims, or a trispecific activated antibody as described in any of the preceding claims, or a pentaspecific activated antibody as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims.

[0533] In another aspect, this disclosure provides an antigen-binding molecule as described in any of the preceding claims, or an activated antibody as described in any of the preceding claims, or a trispecific activated antibody as described in any of the preceding claims, or a pentaspecific activated antibody as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims, for use as a medicament.

[0534] In some embodiments, the disease or condition is a tumor. In some embodiments, the tumor is a solid tumor.

[0535] In some implementations, solid tumors as described above include sarcomas or carcinomas, fibrosarcomas, myxosarcomas, liposarcomas, chondrosarcomas, osteosarcomas, chordomas, angiosarcomas, endothelial sarcomas, lymphangiosarcomas, lymphangioendothelial sarcomas, synovial tumors, mesotheliomas, Ewing sarcomas, leiomyosarcomas, rhabdomyosarcomas, colon cancer, pancreatic cancer or tumors, breast cancer or tumors, ovarian cancer or tumors, prostate cancer or tumors, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, etc. Cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer or tumor, uterine cancer or tumor, testicular cancer or tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, or retinoblastoma. In some implementations, solid tumors include adrenocortical tumors (adenomas and carcinomas), carcinoma, colorectal cancer, desmoidoma, fibroproliferative small round cell tumors, endocrine tumors, Ewing sarcoma, germ cell tumors, hepatoblastoma, hepatocellular carcinoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, soft tissue sarcoma other than rhabdomyosarcoma, and Wilms' tumor. In some embodiments, the solid tumor is a breast tumor. In another embodiment, the solid tumor is prostate cancer. In yet another embodiment, the solid tumor is colon cancer. In some embodiments, the tumor is a brain tumor. In another embodiment, the tumor is a pancreatic tumor. In yet another embodiment, the tumor is a colorectal tumor.

[0536] The multispecific activatable antibodies disclosed herein possess excellent drug-like properties (e.g., higher Tm values, higher SEC purity, and fewer SEC aggregates) and biological activity: These multispecific activatable antibodies exhibit almost no tumor cell killing function before (or before) activation, but after (or after) activation, they effectively activate T cells, enabling T cells to kill tumor cells. The structures of the multispecific activatable antibodies disclosed herein (e.g., Format F series activatable antibodies) are particularly suitable for the CD3 antibodies disclosed herein. Attached Figure Description

[0537] Figures 1A-1F show schematic diagrams of the structures of trispecific antibodies Format F (e.g., Format F_A, Format F_B, Format F_C, Format F_D, Format F_E, Format F_F), with the black solid lines representing linkers.

[0538] Figure 1A shows a schematic diagram of the structure of Format F_A;

[0539] Figure 1B shows a schematic diagram of the structure of Format F_B;

[0540] Figure 1C shows a schematic diagram of the structure of Format F_C;

[0541] Figure 1D shows a schematic diagram of the structure of Format F_D;

[0542] Figure 1E shows a schematic diagram of the structure of Format F_E;

[0543] Figure 1F shows a schematic diagram of the structure of Format F_F.

[0544] Figures 2A-2L show schematic diagrams of the structures of trispecific activatable antibodies Format F (e.g., Format F1_A, Format F1_B, Format F1_C, Format F4_A, Format F4_B, Format F4_C, Format F1_D, Format F1_E, Format F1_F, Format F4_D, Format F4_E, Format F4_F). The solid black lines represent linkers, and the light gray rectangles on the solid black lines represent CD3 masking peptides.

[0545] Figure 2A shows a schematic diagram of the structure of Format F1_A;

[0546] Figure 2B shows a schematic diagram of the structure of Format F1_B;

[0547] Figure 2C shows a schematic diagram of the structure of Format F1_C;

[0548] Figure 2D shows a schematic diagram of the structure of Format F4_A;

[0549] Figure 2E shows a schematic diagram of the structure of Format F4_B;

[0550] Figure 2F shows a schematic diagram of the structure of Format F4_C;

[0551] Figure 2G shows a schematic diagram of the structure of Format F1_D;

[0552] Figure 2H shows a schematic diagram of the structure of Format F1_E;

[0553] Figure 2I shows a schematic diagram of the structure of Format F1_F;

[0554] Figure 2J shows a schematic diagram of the structure of Format F4_D;

[0555] Figure 2K shows a schematic diagram of the structure of Format F4_E;

[0556] Figure 2L shows a schematic diagram of the structure of Format F4_F.

[0557] Figure 3 shows a schematic diagram of the structure of the trispecific activatable antibody Format A. The black solid line represents the linker, and the light gray rectangles on the black solid line represent the CD3 masking peptide.

[0558] Figures 4A and 4B show the structural diagrams of the five specific activatable antibodies Format F'_plus and Format F_plus. The black solid lines represent linkers, and the light gray rectangles on the black solid lines represent CD3 masking peptides.

[0559] Figure 4A shows a schematic diagram of the structure of Format F'_plus;

[0560] Figure 4B shows a schematic diagram of the structure of Format F_plus.

[0561] Figures 5A-5D show schematic diagrams of the structures of five specific activatable antibodies Format F plus (e.g., Format F1_plus A, Format F1_plus B, Format F4_plus A, Format F4_plus B). The black solid lines represent linkers, and the light gray rectangles on the black solid lines represent CD3 masking peptides.

[0562] Figure 5A shows a schematic diagram of the structure of Format F1_plus A;

[0563] Figure 5B shows a schematic diagram of the structure of Format F1_plus B;

[0564] Figure 5C shows a schematic diagram of the structure of Format F4_plus A;

[0565] Figure 5D shows a schematic diagram of the structure of Format F4_plus B.

[0566] Figures 6A to 6F show the CD3 masking efficiency of EGFR / CD3 / HSA trispecific activatable antibodies with Format F structure—a CD3 antigen binding capacity assay based on an ELISA method.

[0567] Figure 6A shows the CD3 masking efficiency of EGFR / CD3 / HSA trispecific activatable antibodies with Format F1_A and Format F4_A structures;

[0568] Figure 6B shows the CD3 masking efficiency of EGFR / CD3 / HSA trispecific activatable antibodies with the Format F1_A structure;

[0569] Figure 6C shows the CD3 masking efficiency of EGFR / CD3 / HSA trispecific activatable antibodies with Format F1_B and Format F1_C structures;

[0570] Figure 6D shows the CD3 masking efficiency of EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_A, Format F4_B and Format F4_C structures;

[0571] Figure 6E shows the CD3 masking efficiency of EGFR / CD3 / HSA trispecific activatable antibodies with Format F1_D, Format F1_E and Format F1_F structures;

[0572] Figure 6F shows the CD3 masking efficiency of EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_D, Format F4_E and Format F4_F structures.

[0573] Figure 7 shows the ability of EGFR / CD3 / HSA trispecific activatable antibodies with Format A structure to induce PBMC cells to kill HT29 tumor cells before and after in vitro activation.

[0574] Figures 8A to 8E illustrate the ability of EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_A and Format A structures to induce tumor cell killing in PBMC cells before and after in vitro activation.

[0575] Figure 8A shows the ability of EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_A and Format A structures to induce the killing ability of PBMC cells against H292 tumor cells before and after in vitro activation (in vitro incubation for 24 hours);

[0576] Figure 8B shows the killing ability of PBMC cells against HT-29 tumor cells induced by EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_A and Format A structures before and after in vitro activation (in vitro incubation for 24 hours);

[0577] Figure 8C shows the killing ability of PBMC cells against HCT116 tumor cells induced by EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_A and Format A structures before and after in vitro activation (in vitro incubation for 24 hours).

[0578] Figure 8D shows the killing ability of PBMC cells against HT-29 tumor cells induced by EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_A and Format A structures before and after in vitro activation (in vitro incubation for 48 hours);

[0579] Figure 8E shows the killing ability of PBMC cells against HCT116 tumor cells induced by EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_A and Format A structures before and after in vitro activation (in vitro incubation for 48 hours).

[0580] Figures 9A to 9D show the killing ability of trispecific activatable antibodies with Format F1_B and Format F1_C structures against tumor cells before and after in vitro activation.

[0581] Figure 9A shows the killing ability of PBMC cells against HCT116 tumor cells induced by the EGFR / CD3 / HSA trispecific activatable antibody with Format F1_C structure before and after in vitro activation (in vitro incubation for 48 hours).

[0582] Figure 9B shows the killing ability of PBMC cells against HT-29 tumor cells induced by the EGFR / CD3 / HSA trispecific activatable antibody with Format F1_C structure before and after in vitro activation (in vitro incubation for 48 hours).

[0583] Figure 9C shows the killing ability of PBMC cells against HCT116 tumor cells induced by the EGFR / CD3 / HSA trispecific activatable antibody with Format F1_B structure before and after in vitro activation (in vitro incubation for 48 hours).

[0584] Figure 9D shows the killing ability of PBMC cells against HT-29 tumor cells induced by the EGFR / CD3 / HSA trispecific activatable antibody with Format F1_B structure before and after in vitro activation (in vitro incubation for 48 hours).

[0585] Figures 10A to 10D illustrate the ability of trispecific activatable antibodies with Format F1_A, Format F1_B, and Format F1_C structures to induce tumor cell killing in PBMCs after in vitro activation.

[0586] Figure 10A shows the ability of activated antibodies with EGFR / CD3 / HSA trispecific activatable antibodies of Format F1_A, Format F1_B and Format F1_C to induce the killing ability of PBMC cells against HT-29 cells (in vitro incubation for 48 hours);

[0587] Figure 10B shows the ability of activated antibodies with EGFR / CD3 / HSA trispecific activatable antibodies of Format F1_A, Format F1_B and Format F1_C to induce the killing ability of PBMC cells against HCT116 cells (in vitro incubation for 48 hours);

[0588] Figure 10C shows the ability of EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_A, Format F4_B and Format F4_C structures to induce PBMC cells to kill HT-29 cells after in vitro activation (in vitro incubation for 48 hours);

[0589] Figure 10D shows the ability of EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_A, Format F4_B and Format F4_C structures to induce the killing of HCT116 cells by PBMC cells after in vitro activation (in vitro incubation for 48 hours).

[0590] Figures 11A to 11D illustrate the ability of EGFR / CD3 / HSA trispecific activatable antibodies with Format F1_D, Format F1_E, Format F1_F, Format F4_D, Format F4_E, and Format F4_F structures to induce tumor cell killing in PBMC cells after in vitro activation.

[0591] Figure 11A shows the ability of EGFR / CD3 / HSA trispecific activatable antibodies with Format F1_D, Format F1_E and Format F1_F structures to induce the killing effect of PBMC cells on HCT116 cells after in vitro activation (in vitro incubation for 48 hours).

[0592] Figure 11B shows the ability of EGFR / CD3 / HSA trispecific activatable antibodies with Format F1_D, Format F1_E and Format F1_F structures to induce PBMC cells to kill HT-29 cells after in vitro activation (in vitro incubation for 48 hours);

[0593] Figure 11C shows the ability of EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_D, Format F4_E and Format F4_F structures to induce the killing effect of PBMC cells on HCT116 cells after in vitro activation (in vitro incubation for 48 hours).

[0594] Figure 11D shows the ability of EGFR / CD3 / HSA trispecific activatable antibodies with Format F4_D, Format F4_E and Format F4_F structures to induce PBMC cells to kill HT-29 cells after in vitro activation (in vitro incubation for 48 hours).

[0595] Figure 12 shows the ability of activated antibodies with Format F1_A and Format F1_B structures to induce PBMC cells to kill MSLN-positive KURAMOCHI cells (in vitro incubation for 48 hours).

[0596] Figures 13A and 13B show the ability of PSMA / EGFR / CD3 / CD28 / HSA five-specific activatable antibodies with Format F1_plus A and Format F1_plus B structures to induce tumor cell killing in PBMC cells after in vitro activation (in vitro incubation for 48 hours).

[0597] Figure 13A shows the ability of PSMA / EGFR / CD3 / CD28 / HSA five-specific activatable antibodies with Format F1_plus A and Format F1_plus B structures to induce the killing ability of PBMC cells against LNCaP tumor cells after in vitro activation (in vitro incubation for 48 hours).

[0598] Figure 13B shows the ability of PSMA / EGFR / CD3 / CD28 / HSA five-specific activatable antibodies with Format F1_plus A and Format F1_plus B structures to induce the killing ability of PBMC cells against 22Rv1 tumor cells after in vitro activation (in vitro incubation for 48 hours). Detailed Implementation

[0599] Terminology (Definition)

[0600] To facilitate understanding of this disclosure, certain technical and scientific terms are described below. Unless otherwise expressly defined in this disclosure, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0601] The singular forms “a,” “an,” and “the” used in the specification and claims include plural references unless the context clearly indicates otherwise.

[0602] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0603] The term "and / or" implies both "and" and "or". For example, the phrase "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0604] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0605] The term "CD3" refers to an antigen expressed on T cells as a portion of the multimolecular T cell receptor (TCR), composed of homodimers or heterodimers formed from two of the following four receptor chains: CD3-ε (CD3E or CD3 epsilon), CD3-δ (CD3D), CD3-ζ, and CD3-γ. For example, human CD3-ε (hCD3ε) comprises the amino acid sequence described in UniProtKB / Swiss-Prot: P07766.2. For example, human CD3-δ (hCD3δ) comprises the amino acid sequence described in UniProtKB / Swiss-Prot: P04234.1. Therefore, unless explicitly stated to be from a non-human species, such as "mouse CD3," "monkey CD3," etc., the term "CD3" refers to human CD3, and the term also covers its naturally occurring variants.

[0606] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0607] The term "amino acid mutation" includes amino acid substitution (also known as amino acid replacement), deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced or absent binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide chain. A specific amino acid mutation can be an amino acid substitution. In some embodiments, an amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid that has a different structure and / or chemical properties. Amino acid substitution includes substitution by non-naturally occurring amino acids or by derivatives of 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering that alter amino acid side chain groups, such as chemical modification, are also expected to be available. Various names may be used in this disclosure to refer to the same amino acid mutation. In this disclosure, the amino acid residue at a specific site may be represented by the format "position + amino acid residue". For example, 102S indicates that the amino acid residue at position 102 is S. C102S indicates that the amino acid residue at position 102 has mutated from C to S. When the residue at a specific site is defined in the claim using the format "position + amino acid residue", the original residue at that site does not limit the scope of protection.

[0608] The term “antibody” is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and antigen-binding fragments (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

[0609] The term "antigen-binding molecule" is used in the broadest sense to encompass molecules that specifically bind antigens, including but not limited to antibodies, other peptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of both, as well as any molecule containing the aforementioned antibodies, peptides, or antibody fusion proteins, provided they exhibit the desired antigen-binding activity. The antigen-binding molecules disclosed herein comprise a variable region (VH) and a variable region (VL), which together constitute the antigen-binding domain. The antigen-binding molecules disclosed herein comprise a single variable domain of an immunoglobulin (e.g., a single-domain antibody, nanobody, VHH). Exemplarily, the antigen-binding molecules in this disclosure are antibodies or activatable antibodies, multispecific antibodies or multispecific activatable antibodies (e.g., trispecific antibodies or trispecific activatable antibodies, pentaspecific antibodies or pentaspecific activatable antibodies).

[0610] The terms “activatable antibody,” “protease-activated antibody,” and “intact activated antibody” are used interchangeably herein to refer to recombinant “masked” or “masked” binding compounds designed to exhibit antibody-like binding specificity to biological targets only after activation by exposure to certain proteases. Structurally, an activated antibody comprises at least: an antigen-binding domain, a masking portion (e.g., a masking peptide), and a cleavable portion (e.g., a protease-cleavable sequence).

[0611] The term "natural antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH, also known as the variable heavy domain or heavy chain variable region), followed by a heavy chain constant region. The natural IgG heavy chain constant region typically contains three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL, also known as the variable light domain or light chain variable domain), followed by a constant light domain (light chain constant region, CL).

[0612] The term "variable region" or "variable domain" in an antibody refers to the domain in the antibody heavy or light chain involved in antibody binding to the antigen. In this disclosure, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also known as the N-terminus) to the carboxyl terminus (also known as the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0613] The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In this disclosure, the term "operably linked" means linking two or more amino acid segments to produce a functional polypeptide. For example, in the context of antigen-binding molecules of this disclosure, individual antigen-binding domains may be linked directly or via peptide linkers. In the context of nucleic acids encoding fusion proteins, such as polypeptide chains of antigen-binding molecules of this disclosure, "operably linked" means linking two nucleic acids such that the amino acid sequences encoded by the two nucleic acids remain within the frame.

[0614] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. The correspondence between various numbering systems is well known to those skilled in the art and is exemplified as shown in Table 1 below.

[0615] Table 1. Relationship between CDR numbering systems

[0616] Unless otherwise stated, the variable regions and CDRs in this disclosure embodiment are governed by the "Kabat" numbering rule. Although the Kabat numbering rule is used in specific implementations to define amino acid residues, corresponding technical solutions using other numbering systems are considered equivalent.

[0617] The term "antibody fragment" refers to a molecule that is distinct from the intact antibody but contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, dsFv, Fab, Fab′, Fab′-SH, Fd, F(ab′)2, and single-domain antibodies (sdAbs, such as VH, VL, VHH, or V). HH ), single-chain Fab (scFab), biantibody, linear antibody, single-chain antibody (e.g., scFv, sc(Fv)2); and multispecific antibodies formed from antibody fragments.

[0618] The term “antigen-binding fragment” encompasses Fab, modified Fab, Fab', Fab'-SH, modified Fab', F(ab')2, Fv, dsFv, Fab-Fv, Fab-dsFv, Fd, single-domain antibody (sdAb, e.g., VH or VL or VHH), single-chain Fab (scFab), single-chain antibody (e.g., scFv, sc(Fv)2), biantibody, linear antibody, bivalent or trivalent or quadrivalent antibody, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0619] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interacting with other variable domains (e.g., without the VH / VL interaction required between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domain" include nanobodies (including VHH, humanized VHH, and / or camelified VH, such as camelified human VH), IgNAR, domains, and (single-domain) antibodies (such as dAbs) that are VH domains or derived from VH domains. TM ) and antibodies that are VL domains or derived from VL domains (such as dAbs) TMImmunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is the “VHH domain” (or simply “VHH”) as defined below.

[0620] The antigen-binding site of a "single-domain antibody" is located on and formed by a single variable domain of an immunoglobulin. This distinguishes a "single-domain antibody" from "conventional" immunoglobulins or fragments thereof (such as Fab, scFv, etc.) (where two immunoglobulin variable domains, specifically two variable domains, interact to form the antigen-binding site). Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both VH and VL contribute to the antigen-binding site; a total of six CDRs are involved in the formation of the antigen-binding site. Conversely, the binding site of a single-domain antibody is formed by a single VH, VHH, or VL domain. Therefore, the antigen-binding site of a single variable domain of an immunoglobulin is formed by no more than three CDRs.

[0621] The term "VHH domain," also known as heavy chain single-domain antibody, VHH, V H The H domain, VHH antibody fragment, VHH antibody, and nanobody are variable domains of antigen-binding immunoglobulins called "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH" is used to distinguish the variable domain from the heavy chain variable domain (referred to herein as the "VH domain" or "VH") present in conventional 4-chain antibodies and the light chain variable domain (referred herein as the "VL domain" or "VL") present in conventional 4-chain antibodies. The VHH domain can specifically bind epitopes in the absence of other antigen-binding domains (unlike the VH or VL domains in conventional 4-chain antibodies, where the epitope is recognized by both the VL and VH domains).

[0622] The VHH domain is a small, stable, and highly efficient antigen recognition unit formed by a single immunoglobulin domain. In some cases, the VHH is derived from naturally occurring antibodies lacking the light chain found in camels or cartilaginous fish, or from synthetic and non-immunogenic VHHs that can be constructed accordingly. In some cases, the VHH is a natural VHH, such as a VHH derived from camels, or a recombinant protein containing a heavy-chain variable domain. In some embodiments, the VHH is derived from species selected from camels, llamas, vicuñas, guanacos, and cartilaginous fish (e.g., but not limited to sharks). In some embodiments, the VHH is derived from alpacas (e.g., but not limited to Huacaya alpaca or Surialpaca). In this disclosure, the terms VHH domain, VHH, VHH antibody fragment, VHH antibody, and "nanobody" and "single-domain antibody" are used interchangeably and refer to an immunoglobulin single variable domain having a FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure and specifically binding to an epitope without the presence of another immunoglobulin variable domain.

[0623] VHHs include, but are not limited to, naturally occurring antibodies produced by camelids, or antibodies produced by camelids that have been humanized, or those obtained through phage display technology. The total number of amino acid residues in the VHH of natural camelids is typically in the range of 110 to 120, often between 112 and 115. It should be understood that the number of residues in humanized VHHs may vary slightly due to modifications in the frame region. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in this disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J Biol Chem., 287(2012)13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17June, 2009 and WO94 / 04678.

[0624] In this disclosure, the term "α-HSA-VHH" refers to a VHH antibody that binds to HSA. The term "α-CD3-VL" refers to the light chain variable region of an anti-CD3 antibody. The term "α-CD3-VH" refers to the heavy chain variable region of an anti-CD3 antibody. The term "α-TAA-VHH" refers to a VHH antibody that binds to TAA. The term "α-target A-VHH" refers to a VHH antibody that binds to target A. The term "α-target B-VHH" refers to a VHH antibody that binds to target B. The term "α-target C-VHH" refers to a VHH antibody that binds to target C.

[0625] As is known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or encoding rules include Chothia, IMGT, and AbM.

[0626] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and modified Fc regions. An Fc region comprises two "Fc region subunits" that bind together to form the Fc region. In some embodiments, the Fc region comprises the same or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. The Fc regions of the antibodies described in this disclosure include those of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may also vary, for example, by omitting the C-terminal lysine (residue 447 according to the EU numbering system) or omitting both the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system). Unless otherwise stated, the Fc region is numbered according to the EU numbering system, also known as the EU index.

[0627] The Fc region can be appropriately obtained by partially digesting IgG monoclonal antibodies with proteolytic enzymes such as pepsin, followed by eluting the components adsorbed on the protein A or protein G column. As the proteolytic enzyme, any enzyme capable of restrictively digesting full-length antibodies to produce Fab and F(ab')2 by appropriately setting the enzyme reaction conditions such as pH is acceptable; there is no particular limitation, and examples include pepsin and papain.

[0628] In this disclosure, the term "Fc region" or "Fc domain" refers to an antibody region that contains at least a CH2 domain and a CH3 domain. In this disclosure, the term "CH2 region" or "CH2 domain" is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering system (according to the IMGT website). However, the CH2 region can also be any other antibody isotype as described in this disclosure.

[0629] In this disclosure, the terms “CH3 region,” “CH3 domain,” or “CH3 structural domain” are intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system (according to the IMGT website). However, the CH3 region can also be any other antibody isotype as described in this disclosure.

[0630] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0631] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0632] The terms "human antibody," "fully human antibody," and "completely human antibody" are used interchangeably, referring to antibodies whose variable and constant regions are human sequences. This term encompasses antibodies derived from human genes but with sequence alterations, such as reduced potential immunogenicity, increased affinity, or the elimination of cysteine ​​residues or glycosylation sites that might cause undesirable folding. This term also covers antibodies recombined in non-human cells (which may confer glycosylations not characteristic of human cells). The term also includes antibodies generated in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The meaning of "human antibody" explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0633] The term "pre-existing antidrug antibody" or "pre-ADA" refers to an ADA already present in a subject or individual to whom a drug (e.g., a protein or peptide drug, more specifically, an antibody drug) will be administered or given. Pre-existing antidrug antibodies may be present in a subject or individual being used for the first time in an experiment (i.e., a subject or individual to whom the drug has never been previously administered). In some embodiments, the antigen-binding molecule described in this disclosure, the nanobody (VHH), has its C-terminus modified to reduce its binding to pre-ADA or ADA, such as that present in serum. Reduced binding to pre-ADA or ADA means that the molecule binds to pre-ADA (or ADA) with reduced affinity or reduced avidity.

[0634] In some embodiments, the C-terminal modification includes C-terminal modifications derived from WO2023093899A1 (included herein by reference in its entirety) and CN202111429892.3, and the patents that have priority to these patents (included herein by reference in its entirety). Other techniques used in this disclosure for C-terminal modification to reduce antibody binding to pre-ADA are also well known in the art, such as WO2012175741A3 (Ablynx), WO2013024059A3 (GSK), and WO2015173325A2 (Ablynx).

[0635] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art, including those described herein.

[0636] As used herein, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. For example, it can be measured using a biosensing system such as a system for measuring surface plasmon resonance (e.g., Biacore), or by measuring affinity in solution using solution equilibrium titration (SET).

[0637] The term “surface plasmon resonance” refers to the optical phenomenon of analyzing real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0638] The term "effector function" refers to biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region with amino acid sequence mutations) and that vary across antibody isotypes. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0639] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibody formulations typically contain multiple different antibodies with different amino acid sequences in their variable structural domains, and they generally specifically target different epitopes. "Monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0640] The term "antigen" refers to a molecule or molecular part that can be bound by antigen-binding proteins, including, for example, antibodies. An antigen may have one or more epitopes that can interact with different antigen-binding proteins, such as antibodies.

[0641] The term "epitope" refers to a region on an antigen that is capable of specifically binding to an antibody or its antigen-binding fragment. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain discontinuous amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., the tertiary folding of an antigen as a protein). The difference between conformational and linear epitopes is that in the presence of a denaturing solvent, the antibody loses binding to the conformational epitope. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0642] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than to other antigens or epitopes. In some embodiments, the KD of antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by... Surface plasmon resonance assays are used to measure this. However, antibodies that specifically bind to antigens or epitopes within antigens may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous) (such as humans or monkeys, such as the cynomolgus (cyno), chimpanzee (chimp), or common marmoset (marmoset)).

[0643] The term "non-binding" means that, in a specified detection method, the antibody cannot be detected binding to an antigen or its epitope in the manner described above specific binding.

[0644] The term "linker" refers to a connecting unit that links two polypeptide fragments. In this disclosure, linkers appearing in the same structural formula may be the same or different. A linker may be a "peptide linker" containing one or more amino acids, typically about 1-30, 2-24, or 3-15 amino acids. Linkers used in this disclosure may be the same or different. When a "-" appears in a structural formula, it indicates that the units on either side are directly connected by a covalent bond.

[0645] The term "peptide linker" can be any suitable peptide chain, as long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, a peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues.

[0646] The terms “antibody-dependent cell cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to mechanisms that induce cell death that rely on the interaction between antibody-coated target cells and lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies described herein can be assessed in vitro using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) from lysed cells.

[0647] The term "antibody-dependent phagocytosis (ADCP)" refers to the mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells).

[0648] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q. C1q then activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0649] The terms “tumor microenvironment” (TME), “cancer microenvironment”, and “tumor environment” are used interchangeably, possessing the same properties and meanings and encompassing the microenvironment in which tumors develop. While the normal cellular microenvironment can inhibit the growth of malignant cells, changes occurring in the tumor microenvironment can synergistically support cell proliferation.

[0650] Furthermore, tumor-secreted proteins alter the microenvironment by contributing growth factors and proteases that degrade the extracellular matrix and influence cell motility and adhesion. Stromal cells secrete ECM proteins, cytokines, growth factors, proteases, protease inhibitors, and endoglucosidases such as heparanase. Matrix metalloproteinases (MMPs) are important secreted proteins closely associated with cancer development. Tumor-associated epithelial cells express MMPs at higher levels compared to normal epithelial cells. In some embodiments, the tumor microenvironment contains enhanced protease activity compared to the non-tumor environment.

[0651] The term "C-terminus" of a polypeptide, such as carboxyl-terminus, C-terminus, C-tail, C-terminus, or COOH-terminus, is the end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH). When a protein is translated from messenger RNA, it is produced from the N-terminus to the C-terminus. The convention for writing peptide sequences is to place the C-terminus on the right and write the sequence from N to C. In some embodiments, the C-terminus of a polypeptide includes up to the last amino acid residue of the polypeptide, which contributes its amino group to form a peptide bond with the carboxyl group of its adjacent amino acid residue.

[0652] The term "N-terminus" of a polypeptide, such as amino-terminus, NH2-terminus, N-terminus, N-terminus, or amine-terminus, is the beginning of a protein or polypeptide and refers to the free amino group (-NH2) located at the end of the polypeptide. Normally, the amino group bonds to another carboxyl group in the protein to make it chain, but since only one of the two regions at the end of a protein is chained, the free amino group refers to the N-terminus. As mentioned above, by convention, peptide sequences in LTR language are written from N-terminus to C-terminus, from left to right. This associates the translation direction with the text direction (because when a protein is translated from messenger RNA, it is produced from N-terminus to C-terminus—an amino acid is added to the carbonyl terminus). In some embodiments, the N-terminus of the polypeptide contains the first amino acid of the polypeptide, which contributes its carboxyl group to form a peptide bond with the amino group of its adjacent amino acid residue.

[0653] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this disclosure and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-naturally occurring, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). "Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from its components in its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that typically contain such molecules but are present outside the chromosome or at a chromosomal location different from their natural chromosomal location. Isolated nucleic acids encoding polypeptides or fusion proteins refer to one or more nucleic acid molecules encoding polypeptides or fusion proteins, including one or more such nucleic acid molecules in a single or separate vector, and one or more such nucleic acid molecules present at one or more locations in the host cell. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues.

[0654] The terms “polypeptide” and “protein” are used interchangeably in this disclosure.

[0655] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions; wherein, when performing optimal alignment of two sequences, gaps are introduced where necessary to obtain the maximum percentage of sequence identity, and no conserved substitutions are considered part of the sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0656] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which an additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming host cells and containing a nucleic acid sequence that directs and / or controls (alongside the host cell) the expression of one or more heterologous coding regions operatively linked to it. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, influence RNA splicing of coding regions operatively linked to them.

[0657] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This disclosure includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stipitis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. The fungi include *C. polymorpha*, *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium* sp., *Fusarium graminearum*, *Fusarium venenatum*, *Physcomitrella patens*, and *Neurospora crassa*.

[0658] As used in this disclosure, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Therefore, the terms “transformer” and “transformed cell” include primary subject cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that, due to intentional or unintentional mutations, not all progeny will have identical DNA contents. This includes mutant progeny that have the same function or biological activity as the original transformed cells from which they were selected.

[0659] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0660] The term "pharmaceutical composition" refers to a mixture containing one or more antigen-binding molecules described herein along with other chemical components, such as physiological / pharmaceutical carriers and excipients.

[0661] The term “pharmaceutically acceptable carrier, diluent or excipient” refers to a component in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject; as an example, pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0662] The terms “subject” or “individual” include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms “patient” or “subject” are used interchangeably in this disclosure. In some embodiments, the individual or subject is a human being.

[0663] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0664] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within the subject's body. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0665] "Treatment" and "treatment" (and their grammatical variations) refer to clinical interventions that attempt to alter the natural processes of the individual being treated, and can be implemented for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, antibodies disclosed herein are used to delay disease onset or slow disease progression.

[0666] The terms "recurrence," "relapse," and "relapsed" refer to the recovery of cancer or disease after a clinical assessment of disease resolution. A diagnosis of distant metastasis or local recurrence can be considered a recurrence.

[0667] The terms "refractory" or "resistant" refer to cancers or diseases that do not respond to treatment.

[0668] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate such symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or mitigate damage caused by or associated with a disease state. In some embodiments, an effective dose is a therapeutically effective dose or a preventatively effective dose. A "therapeuticly effective dose" is an amount sufficient to treat a disease state or symptom, especially a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A "preventatively effective dose" is an amount that, when administered to a subject, will have a predetermined preventative effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. A complete therapeutic or preventative effect may not occur after a single dose is administered, but may occur after a series of doses. Therefore, a therapeutically or preventatively effective dose may be administered once or multiple times. "Therapeutic effective dose" and "preventive effective dose" can vary depending on a number of factors, such as an individual's disease state, age, sex, and weight, as well as the ability of the treatment or combination of treatments to elicit the desired response in the individual. Exemplary indicators of an effective treatment or combination of treatments include, for example, improved health status in the patient.

[0669] Exemplary antigen-binding molecules (e.g., trispecific activatable antibodies)

[0670] This disclosure provides an antigen-binding molecule with numerous advantageous properties, such as antigen-binding activity, cytotoxicity, in vitro T-cell activation ability, good therapeutic activity, safety, pharmacokinetic properties, and drugability (e.g., solubility, viscosity, purity, and stability).

[0671] The three-specific activated antibodies disclosed herein

[0672] For example, the trispecific activatable antibody disclosed herein comprises:

[0673] (a) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is Fab;

[0674] (b) A second antigen-binding domain that specifically binds to a tumor-associated antigen (TAA), wherein the second antigen-binding domain is VHH; and

[0675] (c) Specifically binds to the half-life extension domain of human serum albumin (HSA), wherein the half-life extension domain is VHH;

[0676] The extended half-life domain is connected to the N-terminus of the light chain variable region or the heavy chain variable region of the first antigen-binding domain via a linker (preferably linker 1), and the linker (preferably linker 1) contains a masking peptide and a protease-cleavable sequence sequentially from the N-terminus to the C-terminus.

[0677] For example, the trispecific activatable antibody disclosed herein comprises:

[0678] (i) A first chain having the structure shown in equation (a), and a second chain having the structure shown in equation (b), or

[0679] (ii) A first chain having the structure shown in equation (a), and a second chain having the structure shown in equation (c), or

[0680] (iii) A first chain having the structure shown in equation (d), and a second chain having the structure shown in equation (e), or

[0681] (iv) A first chain having the structure shown in equation (f), and a second chain having the structure shown in equation (g), or

[0682] (v) A first chain having the structure shown in formula (h), and a second chain having the structure shown in formula (g), or

[0683] (vi) A first chain having the structure shown in equation (i), and a second chain having the structure shown in equation (j);

[0684] in,

[0685] (a)[α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0686] (b)[α-TAA-VHH]-[connector 2a]-[α-CD3-VH]-[CH1];

[0687] (c)[α-CD3-VH]-[CH1]-[linker 2b]-[α-TAA-VHH];

[0688] (d)[α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH];

[0689] (e)[α-CD3-VH]-[CH1];

[0690] (f)[α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0691] (g)[α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1];

[0692] (h)[α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0693] (i)[α-CD3-VL]-[CL];

[0694] (j)[α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1]-[connector f]-[α-TAA-VHH];

[0695] Wherein, linker 1 and linkers 2a, 2b, 2c, 2d, 2e and 2f are the same or different peptide linkers, or linkers 2a, 2b, 2c, 2d, 2e and 2f are not present; linker 1 contains a masking peptide and a protease-cleavable sequence sequentially from the N-terminus to the C-terminus;

[0696] The structures shown in equations (a), (b), (c), (d), (e), (f), (g), (h), (i), and (j) are arranged from the N end to the C end.

[0697] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0698] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0699] The second chain has a structure as shown in [α-TAA-VHH]-[connector 2a]-[α-CD3-VH]-[CH1];

[0700] The linker 1 and the linker 2a are the same or different peptide linkers, or the linker 2a is absent; the linker 1 contains a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus.

[0701] The structures shown in the first and second chains are arranged from the N end to the C end.

[0702] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0703] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0704] The second chain has a structure as shown in [α-CD3-VH]-[CH1]-[connector 2b]-[α-TAA-VHH];

[0705] The linker 1 and the linker 2b are the same or different peptide linkers, or the linker 2b is absent; the linker 1 contains a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus.

[0706] The structures shown in the first and second chains are arranged from the N end to the C end.

[0707] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0708] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH];

[0709] The second chain has a structure as shown in [α-CD3-VH]-[CH1];

[0710] The linker 1 and the linker 2c are the same or different peptide linkers, or the linker 2c is absent; the linker 1 contains a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus.

[0711] The structures shown in the first and second chains are arranged from the N end to the C end.

[0712] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0713] The first chain has a structure as shown in [α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0714] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1];

[0715] The linker 1 and the linker 2d are the same or different peptide linkers, or the linker 2d is absent; the linker 1 contains a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus.

[0716] The structures shown in the first and second chains are arranged from the N end to the C end.

[0717] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0718] The first chain has a structure as shown in [α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0719] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1];

[0720] The linker 1 and the linker 2e are the same or different peptide linkers, or the linker 2e is absent; the linker 1 contains a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus.

[0721] The structures shown in the first and second chains are arranged from the N end to the C end.

[0722] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0723] The first chain has a structure as shown in [α-CD3-VL]-[CL];

[0724] The second chain has a structure as shown in [α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1]-[linker 2f]-[α-TAA-VHH];

[0725] The linker 1 and the linker 2f are the same or different peptide linkers, or the linker 2f is absent; the linker 1 contains a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus.

[0726] The structures shown in the first and second chains are arranged from the N end to the C end.

[0727] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2A.

[0728] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0729] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0730] The second chain has a structure as shown in [α-TAA-VHH]-[connector 2a]-[α-CD3-VH]-[CH1-G1];

[0731] The linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0732] The connector 2a is an indivisible connector, or the connector 2a does not exist;

[0733] CH1-G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0734] The structures shown in the first and second chains are arranged from the N end to the C end.

[0735] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2B.

[0736] In some embodiments, a trispecific activatable antibody as described in any of the preceding embodiments, wherein the trispecific activatable antibody comprises a first chain and a second chain, wherein:

[0737] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0738] The second chain has a structure as shown in [α-CD3-VH]-[CH1-G1]-[connector 2b]-[α-TAA-VHH];

[0739] The linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0740] The connector 2b is an indivisible connector, or the connector 2b does not exist;

[0741] CH1-G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0742] The structures shown in the first and second chains are arranged from the N end to the C end.

[0743] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2C.

[0744] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0745] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH];

[0746] The second chain has a structure as shown in [α-CD3-VH]-[CH1-G1];

[0747] The linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0748] The connector 2c is an uncuttable connector, or the connector 2c does not exist;

[0749] CH1-G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0750] The structures shown in the first and second chains are arranged from the N end to the C end.

[0751] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2D.

[0752] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0753] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0754] The second chain has a structure as shown in [α-TAA-VHH]-[connector 2a]-[α-CD3-VH]-[CH1-G4];

[0755] The linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0756] The connector 2a is an indivisible connector, or the connector 2a does not exist;

[0757] CH1-G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0758] The structures shown in the first and second chains are arranged from the N end to the C end.

[0759] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2E.

[0760] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain:

[0761] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0762] The second chain has a structure as shown in [α-CD3-VH]-[CH1_G4]-[connector 2b]-[α-TAA-VHH];

[0763] The linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0764] The connector 2b is an indivisible connector, or the connector 2b does not exist;

[0765] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0766] The structures shown in the first and second chains are arranged from the N end to the C end.

[0767] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2F.

[0768] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0769] The first chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH];

[0770] The second chain has a structure as shown in [α-CD3-VH]-[CH1_G4];

[0771] The linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0772] The connector 2c is an uncuttable connector, or the connector 2c does not exist;

[0773] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0774] The structures shown in the first and second chains are arranged from the N end to the C end.

[0775] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2G.

[0776] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0777] The first chain has a structure as shown in [α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0778] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G1];

[0779] The linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0780] The connector 2d is an uncuttable connector, or the connector 2d does not exist;

[0781] CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0782] The structures shown in the first and second chains are arranged from the N end to the C end.

[0783] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2H.

[0784] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0785] The first chain has a structure as shown in [α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0786] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G1];

[0787] Among them, linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0788] The connector 2e is an indivisible connector, or the connector 2e does not exist;

[0789] CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0790] The structures shown in the first and second chains are arranged from the N end to the C end.

[0791] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2I.

[0792] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0793] The first chain has a structure as shown in [α-CD3-VL]-[CL];

[0794] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G1]-[connector 2f]-[α-TAA-VHH];

[0795] Among them, linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0796] The connector 2f is an uncuttable connector, or the connector 2f does not exist;

[0797] CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1;

[0798] The structures shown in the first and second chains are arranged from the N end to the C end.

[0799] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2J.

[0800] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0801] The first chain has a structure as shown in [α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0802] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G4];

[0803] Among them, linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0804] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0805] The connector 2d is an uncuttable connector, or the connector 2d does not exist;

[0806] The structures shown in the first and second chains are arranged from the N end to the C end.

[0807] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2K.

[0808] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0809] The first chain has a structure as shown in [α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0810] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G4];

[0811] Among them, linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0812] The connector 2e is an indivisible connector, or the connector 2e does not exist;

[0813] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0814] The structures shown in the first and second chains are arranged from the N end to the C end.

[0815] For example, the structure of the trispecific activatable antibody disclosed herein is shown in Figure 2L.

[0816] For example, the trispecific activatable antibody disclosed herein comprises a first chain and a second chain, wherein:

[0817] The first chain has a structure as shown in [α-CD3-VL]-[CL];

[0818] The second chain has a structure as shown in [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G4]-[connector 2f]-[α-TAA-VHH];

[0819] Among them, linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus.

[0820] The connector 2f is an uncuttable connector, or the connector 2f does not exist;

[0821] CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4;

[0822] The structures shown in the first and second chains are arranged from the N end to the C end.

[0823] In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the masking peptide inhibits or reduces the binding of the first antigen-binding domain to CD3. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the masking peptide inhibits or reduces the binding of the first antigen-binding domain to the N-terminus of CD3ε. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the masking peptide comprises SEQ ID NO: 18, or an amino acid sequence having at least 80% identity with it.

[0824] In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the protease is a protease expressed or overexpressed in the tumor microenvironment. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the protease is selected from one or more of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamate proteases, gelatinases, and asparagine peptidases. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the protease is selected from one or two of metalloproteinases and serine proteases. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the metalloproteinase is a matrix metalloproteinase (MMP), and the serine protease is urokinase (uPA), protein lyase (MTSP1), or hepsin, or combinations thereof. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments, wherein the matrix metalloproteinase is MMP2, MMP7, MMP9, MMP13, or MMP14, or combinations thereof.

[0825] In some embodiments, such as the trispecific activatable antibody described in any of the preceding embodiments, wherein the protease-cleavable sequence comprises SEQ ID NO: 10, or an amino acid sequence having at least 70% or 80% identity with it.

[0826] In some embodiments, such as the trispecific activatable antibody described in any of the preceding embodiments, wherein the linker 1 further comprises an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 85.

[0827] In some embodiments, the trispecific activatable antibody as described in any of the preceding claims, wherein the linker 2 is an uncleavable linker. In some embodiments, the trispecific activatable antibody as described in any of the preceding claims, wherein the linker 2 is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 85.

[0828] In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments does not contain an Fc domain. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments does not contain a CH2 domain. In some embodiments, the trispecific activatable antibody as described in any of the preceding embodiments does not contain a CH3 domain.

[0829] antibody structure

[0830] In some implementations, the antibodies provided in this disclosure are full-length antibodies.

[0831] In some implementations, the antibodies provided in this disclosure are antibody fragments.

[0832] In some embodiments, the antibody fragment is a Fab, Fab′, Fab′-SH, or F(ab′)2 fragment, particularly a Fab fragment. “Fab” is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. A “Fab fragment” can be generated by cleavage of an antibody with papain. “Fab′” contains VL, CL, VH, and CH1, and also contains a region between the CH1 and CH2 domains, allowing interchain disulfide bonds to form between the two heavy chains of two Fab′ fragments to form an F(ab′)2 molecule. “Fab′-SH” is a Fab′ fragment in which the cysteine ​​residues in the constant region have free thiol groups. “F(ab′)2” is a divalent fragment comprising two Fab fragments linked by disulfide bonds in the hinge region.

[0833] In other embodiments, the antibody fragment is a biantibody, triantibody, or tetraantibody. A biantibody is an antibody fragment having two antigen-binding sites. In some embodiments, the antibody fragment contains linked VH and VL domains on the same polypeptide chain (VH-VL). By using a short linker that prevents pairing between two domains on the same chain, these domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites, the two antigens of which may be the same or different. In some embodiments, the antibody fragment contains a Fab domain and a VHH domain, thereby creating two or more antigen-binding sites.

[0834] In other embodiments, the antibody fragment is a single-chain Fab fragment. A “single-chain Fab fragment” or “scFab” is a polypeptide consisting of VH, CH1, VL, CL, and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In some embodiments, the linker is a polypeptide having at least 30 amino acids. In other embodiments, the linker is a polypeptide having between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between CL and CH1. Additionally, these single-chain Fab molecules can be further stabilized by inserting cysteine ​​residues (e.g., at position 44 in the heavy chain variable region and position 100 in the light chain variable region, according to Kabat numbering) to create interchain disulfide bonds.

[0835] In other embodiments, the antibody fragment is an Fv fragment composed of the VH and VL domains of a single arm of the antibody.

[0836] In other embodiments, the antibody fragment is a single-chain variable fragment (scFv). An “scFv” is a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked by a short, flexible peptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, in this disclosure, the scFv may have VL and VH variable regions in any order; for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

[0837] In other embodiments, the antibody fragment is dsFv, which is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on prediction of the antibody's three-dimensional structure using known methods (Protein Engineering. 7:697 (1994)).

[0838] In other embodiments, the antibody fragment is a single-domain antibody (dAb). A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, a single-domain antibody refers to a single-domain antibody that does not contain a light chain and can specifically bind to an epitope in the absence of other antigen-binding domains. A single-domain antibody is a small, stable, and highly efficient antigen recognition unit formed by a single immunoglobulin domain.

[0839] In some embodiments, the antibodies provided in this disclosure are chimeric antibodies. In some embodiments, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, the chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from the class or subclass of the parent antibody.

[0840] In some embodiments, the antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable regions, wherein the CDR or a portion thereof is derived from the non-human antibody, and the FR or a portion thereof is derived from the human antibody. Optionally, the humanized antibody may also contain a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody may be replaced with corresponding residues from the non-human antibody (e.g., an antibody providing the CDR sequence).

[0841] Humanized antibodies and their generation methods are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337,7,527,791,6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “resurfuacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describes the “guided selection” method for FR shuffling).

[0842] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best-fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions of the common sequence of human antibodies derived from specific subgroups of light chain variable regions or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); mature human (somatic mutant) frame regions or human germline frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and the frame regions obtained by screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0843] Variants of antigen-binding molecules

[0844] In some embodiments, amino acid sequence variants of the antigen-binding molecules provided in this disclosure are included. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antigen-binding molecule. Amino acid sequence variants of the antigen-binding molecule can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antigen-binding molecule, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion, and / or substitution of residues within the amino acid sequence of the antigen-binding molecule. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen-binding properties.

[0845] Replace, insert, and delete variants

[0846] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Substitution mutagenesis sites of interest include CDR and FR. Conserved substitutions are shown in Table 2 under the heading “Preferred Substitutions.” More substantial variations are provided in Table 2 under the heading “Exemplary Substitutions” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0847] Table 2. Substitution of amino acids

[0848] Based on common side-chain characteristics, amino acids can be grouped as follows:

[0849] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0850] (2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0851] (3) Acidic: Asp, Glu;

[0852] (4) Alkaline: His, Lys, Arg;

[0853] (5) Residues that affect chain orientation: Gly, Pro;

[0854] (6) Aromatic: Trp, Tyr, Phe.

[0855] Non-conservative replacement would require replacing a member of one of these categories with a member of another category.

[0856] One class of substitution variants involves replacing one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further research will have alterations (e.g., improvements) to certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques (such as those described herein). In short, one or more CDR residues are mutated, and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity). CDRs can be altered (e.g., substituted), for example, to improve antibody affinity. Such alterations can be made to CDR “hotspots,” residues encoded by codons that undergo mutations at a high frequency during somatic maturation, and / or residues that contact the antigen, while testing the binding affinity of the resulting variant VH or VL. In some implementations of affinity maturation, diversity is introduced into the selected variant gene for maturation using any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed approaches, where several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. In particular, HCDR3 and LCDR3 are frequently targeted.

[0857] In some embodiments, substitution, insertion, or deletion can occur within one or more CDRs, provided that such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided in this disclosure) can be made to the CDRs that do not materially reduce binding affinity. Such changes can, for example, be external to the antigen-contacting residues in the CDR. In some embodiments of the variant VH and VL sequences provided above, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0858] One method for identifying residues or regions in an antibody that can serve as mutagenic targets is called "alanine scan mutagenesis." In this method, a residue or target group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Furthermore, the contact points between the antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0859] Amino acid sequence insertions include fusion of the amino and / or carboxyl ends of peptides ranging in length from 1 residue to 100 or more residues, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme or a peptide that extends the serum half-life of the antibody.

[0860] Recombination method

[0861] Antigen-binding molecules can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the antigen-binding molecule are provided.

[0862] In some embodiments, this disclosure provides isolated nucleic acids encoding antigen-binding molecules as described in any of the preceding embodiments. Such nucleic acids may be derived from independent polypeptide chains encoding any of the foregoing (e.g., polypeptide chains comprising any CDR, VH, and / or VL, heavy and / or light chains as described in this disclosure). In another aspect, this disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In yet another aspect, this disclosure provides host cells comprising such nucleic acids. In some embodiments, a method for preparing a polypeptide or fusion protein is provided, wherein the method comprises culturing host cells comprising nucleic acids encoding said polypeptide or fusion protein, as provided above, under conditions suitable for expression, and optionally recovering said antigen-binding molecules from the host cells (or host cell culture medium).

[0863] To generate antigen-binding molecules through recombination, nucleic acids encoding proteins are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be readily isolated and sequenced using standard procedures, or generated through recombinant methods or obtained through chemical synthesis.

[0864] Suitable host cells for cloning or expressing vectors encoding antigen-binding molecules include prokaryotic or eukaryotic cells as described herein. For example, they can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, the vector can be separated from the bacterial cell paste in a soluble fraction and further purified.

[0865] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding fusion proteins, including fungal and yeast strains. Suitable host cells for expressing fusion proteins can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells; plant cell cultures can also be used as hosts, such as US5959177, US 6040498, US6420548, US 7125978, and US6417429; and vertebrate cells, such as mammalian cell lines adapted for growth in suspension, can also be used as hosts. Other examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293T cells); young hamster kidney cells (BHK); mouse seltoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0866] Measurement

[0867] The antigen-binding molecules provided herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activities using a variety of assays known in the art. In one aspect, the activity of the antigen-binding molecules disclosed herein can be tested, for example, by known methods such as ELISA, Western blotting, etc.

[0868] Treatment methods and routes of administration

[0869] Any antigen-binding molecules provided in this disclosure may be used for therapeutic purposes. In another aspect, the antigen-binding molecules provided in this disclosure are used in the manufacture or preparation of medicaments. In some embodiments, the disease is a tumor. In some embodiments, the tumor is a solid tumor.

[0870] In another aspect, pharmaceutical compositions comprising the said antigen-binding molecule are provided, for example, for any of the pharmaceutical uses or therapeutic methods described above. In one embodiment, the pharmaceutical composition comprises any antigen-binding molecule provided herein or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0871] The antigen-binding molecules disclosed herein can be used alone or in combination with other agents for treatment. For example, the antibodies disclosed herein can be administered co-administered with at least one other therapeutic agent.

[0872] The antigen-binding molecules (and any other therapeutic agents) disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0873] The antigen-binding molecules disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner. The antigen-binding molecules may be formulated with or without one or more agents currently used for the prevention or treatment of the stated condition. The effective amount of such other agents depends on the amount present in the pharmaceutical composition, the type of condition or treatment, and other factors. These are generally used at the same dosage and route of administration as described herein, or at approximately 1% to 99% of the dosage described herein, or at other dosages, and in any route determined empirically / clinically as appropriate.

[0874] For the prevention or treatment of disease, the appropriate dosage of the antigen-binding molecule disclosed herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and duration of the disease, whether it is administered for preventive or therapeutic purposes, prior treatment, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is appropriately administered to the patient either once or after a series of treatments.

[0875] Products

[0876] In another aspect of this disclosure, an article of manufacture (such as a medicine box) is provided, comprising materials that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article of manufacture comprises a container and a label or package insert on or in conjunction with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container contains a composition, alone or in combination with another composition, that is effective in treating, preventing, and / or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper puncturable by a hypodermic needle). At least one active agent in the composition is an antigen-binding molecule of this disclosure. The label or package insert indicates that the use of the composition is for the treatment of a selected condition. Furthermore, the article of manufacture may comprise: (a) a first container containing a composition, wherein the composition contains an antigen-binding molecule of this disclosure; and (b) a second container containing a composition, wherein the composition contains an additional therapeutic agent. The article of manufacture in embodiments of this disclosure may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer solution. From a commercial and user perspective, it may further include other materials as desired, including additional buffers, diluents, filters, needles, and syringes.

[0877] Example

[0878] The present disclosure is further described below with reference to examples and test cases, but these examples and test cases are not intended to limit the scope of the disclosure. Experimental methods in the examples and test cases of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

[0879] Example 1. Structure of a multispecific antibody

[0880] For example, the structures of the multispecific antibodies disclosed herein include three classes: Format F, Format F plus, and Format A. Among them, Format F further includes the structures shown in Format F_A (e.g., Format F1_A, Format F4_A), Format F_B (e.g., Format F1_B, Format F4_B), Format F_C (e.g., Format F1_C, Format F4_C), Format F_D (e.g., Format F1_D, Format F4_D), Format F_E (e.g., Format F1_E, F4_E), and Format F_F (e.g., Format F1_F, Format F4_F).

[0881] Format F plus further includes the structures shown in Format F1_plus and Format F4_plus, with the specific structure described below:

[0882] 1. Format F_A is an asymmetric structure containing a first chain and a second chain, as shown in Figure 1A. The details are as follows:

[0883] First chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0884] Second chain: [α-TAA-VHH]-[linker 2a]-[α-CD3-VH]-[CH1];

[0885] The connector 1 is either an indivisible connector or a divisible connector, and the structure of the first chain and the second chain is arranged from the N end to the C end.

[0886] 2. Format F_B is an asymmetric structure containing a first chain and a second chain, as shown in Figure 1B. The details are as follows:

[0887] First chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0888] Second chain: [α-CD3-VH]-[CH1]-[linker 2b]-[α-TAA-VHH];

[0889] The connector 1 is either an indivisible connector or a divisible connector, and the structure of the first chain and the second chain is arranged from the N end to the C end.

[0890] 3. Format F_C is an asymmetric structure containing a first chain and a second chain, as shown in Figure 1C:

[0891] First chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH];

[0892] Second chain: [α-CD3-VH]-[CH1];

[0893] The connector 1 is either an indivisible connector or a divisible connector, and the structure of the first chain and the second chain is arranged from the N end to the C end.

[0894] 4. Format F_D is an asymmetric structure containing a first chain and a second chain, as shown in Figure 1D. The details are as follows:

[0895] First chain: [α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0896] Second chain: [α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1];

[0897] The connector 1 is either an indivisible connector or a divisible connector, and the structure of the first chain and the second chain is arranged from the N end to the C end.

[0898] 5. Format F_E is an asymmetric structure containing a first chain and a second chain, as shown in Figure 1E. The details are as follows:

[0899] First chain: [α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0900] Second chain: [α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1];

[0901] The connector 1 is either an indivisible connector or a divisible connector, and the structure of the first chain and the second chain is arranged from the N end to the C end.

[0902] 6. Format F_F is an asymmetric structure containing a first chain and a second chain, as shown in Figure 1F. The details are as follows:

[0903] First chain: [α-CD3-VL]-[CL];

[0904] Second chain: [α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1]-[linker 2f]-[α-TAA-VHH];

[0905] The connector 1 is either an indivisible connector or a divisible connector, and the structure of the first chain and the second chain is arranged from the N end to the C end.

[0906] 7. Format F1_A is an asymmetric structure containing a first chain and a second chain, as shown in Figure 2A. The details are as follows:

[0907] First chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0908] Second chain: [α-TAA-VHH]-[linker 2a]-[α-CD3-VH]-[CH1-G1];

[0909] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linker 2a is an uncleavable linker; CH1-G1 represents the heavy chain constant region 1 (CH1) derived from IgG1; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0910] 8. Format F1_B is an asymmetric structure containing a first chain and a second chain, as shown in Figure 2B. The details are as follows:

[0911] First chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0912] Second chain: [α-CD3-VH]-[CH1-G1]-[linker 2b]-[α-TAA-VHH];

[0913] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linker 2b is an uncleavable linker; CH1-G1 represents the heavy chain constant region 1 (CH1) derived from IgG1; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0914] 9. Format F1_C is an asymmetric structure containing a first chain and a second chain, as shown in Figure 2C. The details are as follows:

[0915] First chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH];

[0916] Second chain: [α-CD3-VH]-[CH1-G1];

[0917] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2c is an uncleavable linker; CH1-G1 represents the heavy chain constant region 1 (CH1) derived from IgG1; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0918] 10. Format F4_A is an asymmetric structure containing a first chain and a second chain, as shown in Figure 2D. Details are as follows:

[0919] First chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0920] Second chain: [α-TAA-VHH]-[linker 2a]-[α-CD3-VH]-[CH1-G4];

[0921] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linker 2a is an uncleavable linker; CH1-G4 represents the heavy chain constant region 1 (CH1) derived from IgG4; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0922] 11. Format F4_B is an asymmetric structure containing a first chain and a second chain, as shown in Figure 2E. The details are as follows:

[0923] First chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0924] Second chain: [α-CD3-VH]-[CH1_G4]-[connector 2b]-[α-TAA-VHH];

[0925] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2b is an uncleavable linker; CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0926] 12. Format F4_C is an asymmetric structure containing two chains: the first chain and the second chain. Its schematic diagram is shown in Figure 2F, as follows:

[0927] First chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH];

[0928] Second chain: [α-CD3-VH]-[CH1_G4];

[0929] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linker 2c is an uncleavable linker; CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0930] 13. Format F1_D is an asymmetric structure containing a first chain and a second chain, as shown in Figure 2G. The details are as follows:

[0931] First chain: [α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0932] Second chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G1];

[0933] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linker 2d is an uncleavable linker; CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0934] 14. Format F1_E is an asymmetric structure containing a first chain and a second chain, as shown in Figure 2H. The details are as follows:

[0935] First chain: [α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0936] Second chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G1];

[0937] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linker 2e is an uncleavable linker; CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0938] 15. Format F1_F is an asymmetric structure containing a first chain and a second chain, as shown in Figure 2I. The details are as follows:

[0939] First chain: [α-CD3-VL]-[CL];

[0940] Second chain: [α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1_G1]-[linker 2f]-[α-TAA-VHH];

[0941] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2f is an uncleavable linker; CH1-IgG1 represents the heavy chain constant region 1 (CH1) derived from IgG1; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0942] 16. Format F4_D is an asymmetric structure containing a first chain and a second chain, as shown in Figure 2J. The details are as follows:

[0943] First chain: [α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL];

[0944] Second chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G4];

[0945] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linker 2d is an uncleavable linker; CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0946] 17. Format F4_E is an asymmetric structure containing a first chain and a second chain, as shown in Figure 2K. The details are as follows:

[0947] First chain: [α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH];

[0948] Second chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1_G4];

[0949] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linker 2e is an uncleavable linker; CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0950] 18. Format F4_F is an asymmetric structure containing a first chain and a second chain, as shown in Figure 2L. The details are as follows:

[0951] First chain: [α-CD3-VL]-[CL];

[0952] Second chain: [α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1_G4]-[linker 2f]-[α-TAA-VHH];

[0953] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linker 2f is an uncleavable linker; CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0954] 19. Format F'_plus is an asymmetric structure containing a first chain and a second chain, as shown in Figure 4A. The details are as follows:

[0955] First chain: [α-target B-VHH]-[connector 3b]-[α-CD3-VL]-[CL]-[connector 2h]-[α-target A-VHH];

[0956] Second chain: [α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1]-[linker 4b]-[α-target C-VHH];

[0957] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linkers 2h, 3b, and 4b are non-cleavable linkers; the structures of the first and second strands are both arranged from the N-terminus to the C-terminus.

[0958] 20. Format F_plus is an asymmetric structure containing a first chain and a second chain, as shown in Figure 4B. The details are as follows:

[0959] First chain: [α-HSA-VHH]-[linker 1]-[α-CD3-VL]-[CL]-[linker 2g]-[α-target A-VHH];

[0960] Second chain: [α-target B-VHH]-[linker 3a]-[α-CD3-VH]-[CH1]-[linker 4a]-[α-target C-VHH];

[0961] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3h, and 4h are non-cleavable linkers; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0962] 21. Format F1_plus is an asymmetric structure containing a first chain and a second chain, as shown in Figure 5A or Figure 5B, specifically as follows:

[0963] First chain: [α-HSA-VHH]-[linker 1]-[α-CD3-VL]-[CL]-[linker 2g]-[α-target A-VHH];

[0964] Second chain: [α-target B-VHH]-[connector 3a]-[α-CD3-VH]-[CH1_G1]-[connector 4a]-[α-target C-VHH];

[0965] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3a and 4a are non-cleavable linkers; CH1_G1 represents the heavy chain constant region 1 (CH1) derived from IgG1; the structures of the first and second chains are arranged from the N-terminus to the C-terminus.

[0966] 22. Format F4_plus is an asymmetric structure containing a first chain and a second chain, as shown in Figure 5C or Figure 5D, specifically as follows:

[0967] First chain: [α-HSA-VHH]-[linker 1]-[α-CD3-VL]-[CL]-[linker 2g]-[α-target A-VHH];

[0968] Second chain: [α-target B-VHH]-[connector 3a]-[α-CD3-VH]-[CH1_G4]-[connector 4a]-[α-target C-VHH];

[0969] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; linkers 2g, 3a and 4a are non-cleavable linkers; CH1_G4 represents the heavy chain constant region 1 (CH1) derived from IgG4; the structures of the first and second chains are arranged from the N-terminus to the C-terminus.

[0970] Technical personnel understand that although specific target points are illustrated in Figures 5A to 5D, the Format F1_plus and Format F4_plus structures are not limited to these specific target points.

[0971] 23. Format A contains a chain, as shown in Figure 3. The specific structure is as follows:

[0972] From N to C, the sequence is: [α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[connector 2i]-[α-CD3-VL-]-[connector 3c]-[α-target A-VHH];

[0973] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linkers 2i and 3c are non-cleavable linkers.

[0974] Table 3. Indivisible Connective Subsequences

[0975] Table 4. Amino acid sequences that can be cleaved by proteases

[0976] The variable region sequence of the anti-HSA single-domain antibody is derived from WHO INN PL105 (2011), and the specific sequence is as follows:

[0977] >α-HSA-VHH

[0978] Table 5. CDR sequences of anti-HSA single-domain antibodies

[0979] Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0980] The variable region sequence of the anti-CD3 antibody is derived from patent PCT / CN2024 / 141016, and the specific sequence is as follows:

[0981] >82_VH_H7

[0982] >82_VL_L8

[0983] >82_VL_L8 S27eP,L94V

[0984] The CDR sequences of anti-CD3 antibodies are shown in the table below:

[0985] Table 6. CDR of anti-CD3 antibodies

[0986] Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0987] >CL_kappa

[0988] CH1_IgG1

[0989] CH1_IgG4

[0990] CD3 antibody masking peptide

[0991] The 9G8 variable region sequence of the anti-EGFR single-domain antibody is derived from Structural Evaluation of EGFR Inhibition Mechanisms for Nanobodies / VHH Domains (Schmitz KR, et al. structure. 2013; 21:1214-1224), and the specific sequence is as follows:

[0992] >α-EGFR-VHH

[0993] The variable region sequence of the anti-MSLN single-domain antibody is derived from patent CN110913908B, and the specific sequence is as follows:

[0994] >α-MSLN-VHH

[0995] The variable region sequence of the anti-PSMA single-domain antibody is derived from US20180162949A1, and the specific sequence is as follows:

[0996] >α-PSMA-VHH

[0997] The variable region sequence of the anti-CD28 single-domain antibody is derived from US20230348600A1, and the specific sequence is as follows:

[0998] >α-CD28-VHH

[0999] The control antibody PC-4 (EGFR / CD3 / HSA) sequence is derived from patent WO2022240637A2, and the specific sequence is as follows:

[1000] PC-4LC

[1001] PC-4HC

[1002] The sequence of the control antibody JNJ (MSLN / CD3) is derived from patent WO2024089551A1, and the specific sequence is as follows:

[1003] JNJ HC1

[1004] JNJ HC2

[1005] JNJ LC

[1006] The control antibody HPN (MSLN / HSA / CD3) sequence is derived from patent CN110913908B, and the specific sequence is as follows:

[1007] The negative control antibody used in this disclosure is C25-hIgG1 (where the VH / VL sequence is derived from the C25 antibody of patent US6114143A).

[1008] >C25-HC

[1009] C25-LC

[1010] Note: The gray background area is the variable area, and the italicized area is the constant area.

[1011] 1.1 Sequence of EGFR / CD3 / HSA antibody

[1012] Table 7-1. Amino acid sequence of EGFR / CD3 / HSA antibody chain 1

[1013] Table 7-2. Amino acid sequence of EGFR / CD3 / HSA antibody chain 2

[1014] Table 7-3. Amino acid sequences of control molecules

[1015] Note: PC-4 is an activated antibody; PC-4_act is the molecule after PC-4 activation, which is actually an EGFR-CD3 bispecific antibody; PC-4_NCL is an uncleavable EGFR / CD3 / HSA antibody containing a CD3 masking peptide.

[1016] Table 7-4. Amino acid sequences of EGFR / CD3 / HSA antibodies

[1017] Among them: PRO-10, PRO-11, and PRO-12 are cleavable EGFR / CD3 / HSA antibodies containing CD3 masking peptides, and their linker 1 is an cleavable linker; PRO-13, PRO-14, and PRO-15 are EGFR / CD3 antibodies, representing activated EGFR / CD3 / HSA antibodies; PRO-17 is a control antibody without an anti-EGFR single-domain antibody structural domain.

[1018] Table 7-5. Amino acid sequences of EGFR / CD3 / HSA antibodies

[1019] 1.2 Sequence of MSLN / CD3 / HSA antibody

[1020] Table 8-1. Amino acid sequence of MSLN / CD3 / HSA antibody chain 1

[1021] Table 8-2. Amino acid sequence of MSLN / CD3 / HSA antibody chain 2

[1022] Table 8-3. Amino acid sequences of MSLN control molecules

[1023] Table 8-4. Amino acid sequences of MSLN / CD3 / HSA antibodies

[1024] Table 8-5. Amino acid sequences of MSLN / CD3 / HSA antibodies

[1025] 1.3 Sequence of PSMA / EGFR / CD3 / CD28 / HSA antibody

[1026] Table 9-1. Amino acid sequence of PSMA / EGFR / CD3 / CD28 / HSA antibody chain 1

[1027] Table 9-2. Amino acid sequence of PSMA / EGFR / CD3 / CD28 / HSA antibody chain 2

[1028] Table 9-3. Amino acid sequences of PSMA / EGFR / CD3 / CD28 / HSA antibodies

[1029] Table 9-4. Amino acid sequences of PSMA / EGFR / CD3 / CD28 / HSA antibodies

[1030] Example 2. Expression and purification of multispecific antibodies

[1031] Preparation of antibodies with Format F series structures

[1032] Antibodies with Format F structure were prepared using the Expi-293F expression system. Nucleotide sequences encoding the first and second strands of each antibody were synthesized and inserted into the vector pcDNA3.4 to obtain the corresponding recombinant plasmids. These recombinant plasmids were used for cloning and / or expression in host cells. Expi-293F cells (Thermo Fisher Scientific) were passaged in OPM-293CD05 medium (OPM, Cat. #81075). Cell density was measured one day before transfection and adjusted to 1.6 × 10⁶ cells / year using fresh cell culture medium. 6 Cells / mL, cell density before transfection was 3 × 10⁶. 6 -3.5×10 6 Cells / mL, transfection plasmid dosage is 1 μg corresponding to 3 × 10⁻⁶ cells / mL. 6 The ratio of plasmid to Polyethylenimine Max (MW 40,000*)-High Potency Linear PEI (polysciences, Cat.#24765-1) was 1:3, and the transfection system was 10% of the expression system. Taking a 100mL transfection system as an example, after filtering the plasmid, adjust the volume to 5mL using Opit-MEM (Gibco, Cat.#11058-021). Mix 300μL (1mg / mL) of PEI with 4.7mL of Opit-MEM and incubate at room temperature for 5 minutes. Add the prepared PEI to the plasmid system, mix well, and incubate at room temperature for 15 minutes. Slowly add the PEI-plasmid complex to the Expi293F cell suspension and mix well. Incubate on a shaker. 24 hours after transfection, add 5% OPM-293ProFeed (OPM, Cat.#F081918) at a volume ratio. 6 days after transfection, collect the supernatant and analyze it using KappaSelect. TMAffinity column (Cytiva, Cat.#17545801) and CaptureSelect TM Purified IgG-CH1 protein (Thermo, Cat.#1943462010).

[1033] KappaSelect TM Affinity purification: Before purification, equilibrate the chromatography column with 10-20 column volumes of 1×PBS (Sangon Biotech, Cat.#SD8117) until UV light returns to baseline; load the collected culture supernatant onto the chromatography column, and wash the column with 1×PBS until UV light returns to baseline to remove non-specifically bound proteins; rinse the chromatography column with elution buffer (0.1M glycine hydrochloride buffer, pH 3.0) and collect the eluent; then neutralize to neutral with 1M Tris-HCl (Sangon Biotech, Cat.#B548139-0500) buffer for further purification.

[1034] CH1 affinity purification: Before purification, equilibrate the chromatography column with 10 column volumes of 1×PBS (Sangon Biotech, Cat.#SD8117) until UV light returns to baseline; load the neutralized KappaSelect purification eluent onto the chromatography column, and wash the column with 10 column volumes of 1×PBS until UV light returns to baseline to remove non-specifically binding proteins; wash the chromatography column with CH1 eluent (20 mM acetic acid + 150 mM sodium chloride, pH 3.5) and collect the eluent; transfer the eluent to Amicon Ultra-15 Centrifugal Filters (Merck, Cat: UFC901024), add 50 AA (30 mM CH3COONa, pH = 5.0) to the maximum mark, centrifuge at 3500 rpm for 10 minutes at 4°C, repeat this operation three times to complete the buffer exchange and concentration, transfer the concentrated sample to a 15 mL centrifuge tube, and adjust the concentration to 1-2 mg / mL. After sterilization using a 0.22 μm syringe filter in a biosafety cabinet, the sample was transferred to a 1.5 mL centrifuge tube. 100 μL of the sample was then subjected to SDS-PAGE and SEC analysis. Further qualitative and quantitative analysis of the antibody composition and content was performed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[1035] Preparation of antibodies with Format A structure

[1036] Antibodies with Format A structures were prepared using the Expi-293F expression system. The nucleotide sequences encoding the first strand of each antibody were synthesized and inserted into the vector pcDNA3.4 to obtain the corresponding recombinant plasmids. These recombinant plasmids were used for cloning and / or expression in host cells. Expi-293F cell passage and transfection conditions were the same as described above. Six days after transfection, the supernatant was collected and processed using… Proteins were purified using AF-Protein L-650F affinity packing material (Tosoh Bioscience, Cat.#0023487) or Ni Sepharose excel affinity packing material (Cytiva, Cat.#17371201).

[1037] AF-Protein L-650F affinity purification: Before purification, equilibrate the chromatography column with 10-20 column volumes of 1×PBS until UV back to baseline; load the collected supernatant onto the chromatography column, and wash the column with 1×PBS until UV back to baseline to remove non-specifically bound proteins; wash the column with the first-step elution buffer (0.1M acetate-hydrochloride buffer with 100mM arginine hydrochloride added, pH 3.0) and collect the eluent, then neutralize to neutral with 1M Tris-HCl (Sangon Biotech, Cat.#B548139-0500) buffer; then wash the column with the second-step elution buffer (0.1M acetate buffer, pH 3.0) and collect the eluent, neutralizing to neutral with 1M Tris-HCl (Sangon Biotech, Cat.#B548139-0500) buffer. The neutralization and eluents obtained from the two steps were mixed and transferred to an Amicon Ultra-15 Centrifugal Filter. 1×PBS was added to the maximum mark, and the sample was centrifuged at 3500 rpm for 10 minutes at 4°C. This process was repeated three times to complete the concentration. The concentrated sample was then transferred to a 15 mL centrifuge tube, and the concentration was adjusted to 1-2 mg / mL. After sterilization using a 0.22 μm syringe filter in a biosafety cabinet, the sample was transferred to a 1.5 mL centrifuge tube. 100 μL of the sample was taken for SDS-PAGE and SEC analysis. Further qualitative and quantitative analysis of the antibody composition and content was performed using a combination of CE-SDS and LC-MS.

[1038] Ni Sepharose Excel affinity purification: Before purification, equilibrate the column with 10-20 column volumes of 1×PBS until UV back to baseline; load the collected culture supernatant onto the column, and wash the column with 1×PBS until UV back to baseline to remove non-specifically binding proteins; wash the column with 5-10 column volumes of first-step purification buffer (PBS solution with 20 mM imidazole, pH 7.5); then wash the column with 5-10 column volumes of second-step purification buffer (PBS solution with 30 mM imidazole, pH 7.5); finally, wash the column with 5-10 column volumes of elution buffer (PBS solution with 300 mM imidazole, pH 7.5) and collect the eluent, neutralizing to neutral with 1M Tris-HCl buffer. The neutralized eluent was transferred to an Amicon Ultra-15 Centrifugal Filter, and 1×PBS was added to the maximum mark. The sample was centrifuged at 3500 rpm for 10 minutes at 4°C. This process was repeated three times to complete the concentration. The concentrated sample was then transferred to a 15 mL centrifuge tube, and the concentration was adjusted to 1-2 mg / mL. After sterilization using a 0.22 μm syringe filter in a biosafety cabinet, the sample was transferred to a 1.5 mL centrifuge tube. 100 μL of the sample was taken for SDS-PAGE and SEC analysis. Further qualitative and quantitative analysis of the antibody composition and content was performed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[1039] The purification method described above is also applicable to other antibodies disclosed herein.

[1040] Test case

[1041] Test Example 1. ELISA detection of the masking efficiency of the masking peptide on CD3.

[1042] Dilute the antigen CD3e-His&hFc tag (Cat.#10977-H03H, Sino Biological) to 5 μg / mL with PBS, add 50 μL / well to the microplate, and incubate at 37°C for 1 hour; wash the plate once with PBST solution (PBS containing 0.1% Tween 20) (300 μL / well). TM Casein (Cat.#37528, Thermo Scientific) 100 μL / well, seal at 37°C for 0.5 hours, then spin dry. Use a solution containing 10% Blocker. TMAntibodies were prepared using Casein PBS at concentrations up to 200 nM (activated molecules) or 2000 nM (trispecific activated antibodies), serially diluted 3-fold, 50 μL / well, and incubated at 37°C for 1 hour. The plates were washed 3 times with PBST solution (300 μL / well). Working concentration of anti-Fab-HRP (Ca.#109-035-006, Jackson) antibody was added, 50 μL / well, and incubated at 37°C for 0.5 hours. The plates were washed 3 times with PBST solution (300 μL / well). 100 μL of TMB (Cat.#5120-0077, Seracare) chromogenic buffer was added, and the plates were incubated at room temperature for 5-10 minutes. 100 μL of 2M H₂SO₄ was added to stop the incubation process, and the OD450 value was read using a microplate reader (BRN 07861, Molecular Devices). Results are shown in Figures 6A to 6F.

[1043] The results showed that the masking peptides of the activatable antibodies disclosed herein could effectively mask the binding of the activatable antibodies to the CD3 antigen.

[1044] Test Example 2. Killing Function of EGFR / CD3 / HSA Trispecific Activatable Antibody After In Vitro Activation

[1045] In vitro activation method for EGFR / CD3 / HSA trispecific activatable antibodies: Recombinant human metalloproteinase 2 (rhMMP2) (Cat.#902-MP, R&D) was activated with APMA (Cat.#A9563, Sigma) according to the instructions. Then, the activated rhMMP2 was diluted to 1000 nM with 1xTCNB buffer (Cat.#FY19556, Nantong Feiyu Biotechnology) and aliquoted into 10 μL and frozen. Before in vitro activation, the EGFR / CD3 / HSA trispecific activatable antibody was diluted to a final concentration of 20 μM with PBS. Then, 2xTCNB buffer (100 mM Tris, 20 mM CaCl2, 300 mM NaCl, 0.1% Brij 35, pH 7.5) was added at a volume ratio of 1:1, resulting in a total digestion volume of 40 μL. Then, the required amount of rhMMP2 (final concentration 10 nM) was added according to a molar ratio of rhMMP2 to EGFR / CD3 / HSA trispecific activatable antibody of 1:1000. The digestion was incubated overnight, and the supernatant was collected by centrifugation at 12000 rpm for 2 minutes. 1 μg of the sample before and after digestion was subjected to SDS-PAGE to verify the digestion effect.

[1046] To test the cytotoxic activity of human peripheral blood mononuclear cells (PBMCs) against tumor cells mediated by in vitro activation of EGFR / CD3 / HSA trispecific activatable antibodies, EGFR-positive tumor cells NCI-H292 (ATCC, CRL-1848), HCT116 (ATCC, CCL247), and HT-29 (ATCC, HTB-38) were centrifuged at 1000 rpm for 5 minutes, resuspended in RPMI 1640 complete medium containing 10% FBS, and counted. The cell density was adjusted to 5 × 10⁶ cells / year. 5 Cells / mL. Frozen PBMCs (Shanghai Xuanfeng Biotechnology, Shanghai Saili Biotechnology) were rapidly thawed at 37℃. Cells were slowly added to 20 mL of preheated complete culture medium, centrifuged at 1500 rpm for 10 minutes, resuspended in complete culture medium, counted, and adjusted to a cell density of 2.5 × 10⁻⁶ cells / mL. 6 Cells / mL. The test antibody, serially diluted with complete culture medium, was added to a 96-well plate. Then, EGFR-positive tumor cells and PBMCs were mixed in a specific ratio to obtain a cell mixture with a final effector-to-target ratio of 10:1, 5:1, or 1:1. The mixture was then added at 160 μL / well to each well of the 96-well plate. The 96-well plate was incubated at 37°C with 5% CO2 for 48 hours. The supernatant was then aspirated, and the LDH (lactate dehydrogenase) content released from dead cells was measured according to the LDH detection kit instructions (G1781, Promega) to determine the percentage of antibody-induced tumor cell death. Results are shown in Figures 7, 8A-8E, 9A-9D, 10A-10D, and 11A-11D.

[1047] The results showed that the activated antibodies disclosed herein, after in vitro activation or their corresponding activated forms, could induce human PBMC cells to kill EGFR-positive tumor cells.

[1048] Test Example 3. Binding ability of EGFR / CD3 / HSA trispecific activatable antibody to human serum albumin

[1049] 1 μg / mL biotinylated human serum albumin (HSA) (Cat.#HSA-H82E3, ACRO) was coupled to an SA biosensor chip (Series S SA biosensor chip, Cat.#BR-1005-31, Cytiva) (approximately 200 RU), and excess sites on the chip were blocked with 10 μM biotin. The antibody sample to be tested was then passed through the chip surface for 180 s, followed by dissociation for 720 s. The reaction signal was detected in real time using a Biacore 8K (Cytiva) to obtain binding and dissociation curves. After each cycle, the chip was washed and regenerated with 10 mM glycine-hydrochloric acid at pH 1.5 (Cat.#BR100354, GE). After the experiment, the data were fitted using a (1:1) Langmuir model using GE Biacore T200 Evaluation version 3.0 software to calculate the affinity values.

[1050] To test whether the fusion of anti-EGFR single-domain antibodies at the N-terminus of the heavy chain in Format F1_A and Format F4_A affects the binding ability of anti-HSA single-domain antibodies fused at the N-terminus of the light chain to HSA, the binding kinetics of PRO-2, PRO-1, PRO-3, and the control antibody PRO-17 without the anti-EGFR single-domain antibody domain to HSA were compared. The experimental results showed no significant difference in the binding of each molecule to HSA, suggesting that the fusion of anti-EGFR single-domain antibodies at the N-terminus of the heavy chain did not affect the binding ability of anti-HSA single-domain antibodies fused at the N-terminus of the light chain to HSA. The specific results are shown in Table 10.

[1051] Table 10. Binding ability of anti-HSA single-domain antibodies to HSA

[1052] Test Example 4. Drug-like properties of activatable antibodies

[1053] A solution containing 20 mg / mL of the trispecific activatable antibody was prepared using a buffer solution of 20 mM acetic acid, pH 5.0, and 8% sucrose. The sample was divided into two portions and incubated at 4°C and 40°C respectively for 14 days. After incubation, the SEC purity of the antibody samples was measured. The results are shown in Tables 11-1 to 11-15. The experimental results indicate that the trispecific activatable antibody with the structure shown in Format F disclosed herein has excellent stability.

[1054] Table 11-1. Changes in SEC purity before and after PRO-2 acceleration stability.

[1055] Table 11-2. Changes in SEC purity before and after PRO-1 acceleration stability.

[1056] Table 11-3. Changes in SEC purity before and after PRO-4 acceleration stability.

[1057] Table 11-4. Changes in SEC purity before and after PRO-5 acceleration stability testing

[1058] Table 11-5 Changes in SEC purity before and after PRO-7 acceleration stability testing

[1059] Table 11-6. Changes in SEC purity before and after PRO-8 acceleration stability testing

[1060] Table 11-7 Changes in SEC purity before and after PRO-6 acceleration stability testing

[1061] Table 11-8. Changes in SEC purity before and after PRO-9 acceleration stability.

[1062] Table 11-9. Changes in SEC purity before and after PRO-18 acceleration stability.

[1063] Table 11-10. Changes in SEC purity before and after PRO-19 acceleration stability.

[1064] Table 11-11. Changes in SEC purity before and after PRO-20 acceleration stability.

[1065] Table 11-12. Changes in SEC purity before and after PRO-21 acceleration stability.

[1066] Table 11-13. Changes in SEC purity before and after PRO-22 acceleration stability.

[1067] Table 11-14. Changes in SEC purity before and after PRO-23 acceleration stability testing.

[1068] Table 11-15. Changes in SEC purity of PC-4 control antibody before and after accelerated stability enhancement

[1069] Note: HMW represents aggregate peak, Main represents main peak, Δ represents the change value of main peak, and LMW represents fragment peak.

[1070] Test Example 5. Killing Function of MSLN / CD3 / HSA Trispecific Activatable Antibody After In Vitro Activation

[1071] The in vitro activation method for the MSLN / CD3 / HSA trispecific activatable antibody is the same as that in test example 2.

[1072] To test the cytotoxic activity of human peripheral blood mononuclear cells (PBMCs) mediated by in vitro activation of MSLN / CD3 / HSA trispecific activatable antibodies against tumor cells, MSLN-positive tumor cells KURAMOCHI (ATCC, CRL-1848) were centrifuged at 1000 rpm for 5 minutes, resuspended in RPMI 1640 complete medium containing 10% FBS, and counted to adjust the cell density to 5 × 10⁶ cells / year. 5 Cells / mL. The frozen PBMCs (Shanghai Xuanfeng Biotechnology, Shanghai Saili Biotechnology) were rapidly thawed at 37℃. The cells were slowly added to 20 mL of preheated complete culture medium, centrifuged at 1500 rpm for 10 minutes, resuspended in complete culture medium, and counted. The cell density was adjusted to 2.5 × 10⁻⁶ cells / mL. 6 Cells / mL. The test antibody, serially diluted with complete culture medium, was added to a 96-well plate. MSLN-positive tumor cells and PBMCs were then mixed at a specific ratio to obtain a final effector-to-target ratio of 5:1. 160 μL of this mixture was added to each well of the 96-well plate. The 96-well plate was incubated at 37°C with 5% CO2 for 48 hours. The supernatant was then collected, and the LDH (lactate dehydrogenase) content released from dead cells was measured according to the LDH detection kit instructions (G1781, Promega) to determine the percentage of antibody-induced tumor cell death. The results are shown in Figure 12.

[1073] The results showed that the activated antibody disclosed herein, after in vitro activation, could induce human PBMC cells to kill MSLN-positive tumor cells.

[1074] Test Example 6. The killing function of PSMA / EGFR / CD3 / CD28 / HSA activated antibodies after in vitro activation.

[1075] The in vitro activation procedure for the five-specific activatable antibodies against PSMA / EGFR / CD3 / CD28 / HSA was the same as in Test Example 2. To detect the killing effect of PBMCs on tumor cells mediated by the in vitro activation of the five-specific activatable antibodies against PSMA / EGFR / CD3 / CD28 / HSA in the examples, LNCaP cells (ATCC, CRL-1740) and 22Rv1 cells (ATCC, CCL247) simultaneously expressing PSMA and EGFR were centrifuged at 1000 rpm for 5 minutes, resuspended in RPMI 1640 complete medium containing 10% FBS, and the cells were counted and the cell density was adjusted to 2.5 × 10⁻⁶. 5Cells / mL. The frozen PBMCs (Shanghai Saili Biotechnology) were rapidly thawed at 37℃. The cells were slowly added to 20 mL of preheated complete culture medium, centrifuged at 1500 rpm for 10 minutes, resuspended in complete culture medium, and counted. The cell density was adjusted to 1.25 × 10⁻⁶ cells / mL. 6 Cells / mL. The test antibody, serially diluted with complete culture medium, was added to 96-well plates. Then, cell mixtures obtained by mixing PSMA-positive LNCaP cells and 22Rv1 cells with PBMC at a final effector-to-target ratio of 5:1 or 1:1 were added to the 96-well plates. After incubating the 96-well plates at 37°C and 5% CO2 for 48 hours, the supernatant was aspirated, and the LDH content released from dead cells was measured according to the LDH detection kit instructions (G1781, Promega) to determine the percentage of antibody-induced tumor cell death. The results are shown in Figures 13A and 13B. The experimental results indicate that the five-specific activated antibody PSMA / EGFR / CD3 / CD28 / HSA disclosed herein has a stronger killing ability than the three-specific activated antibody PSMA / CD3 / HSA.

[1076] Although the invention has been described in detail with the aid of accompanying drawings and examples for clarity of understanding, these descriptions and examples should not be construed as limiting the scope of this disclosure. All patent and scientific literature disclosures cited in this disclosure are clearly and fully incorporated by reference.

Claims

1. An antigen-binding molecule comprising: An antigen-binding domain that specifically binds to CD3, wherein the antigen-binding domain specifically binding to CD3 is Fab, and the Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1); and Half-life extended structural domains; and A linker comprising a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; The extended half-life domain is connected to the N-terminus of the VL or VH of the antigen-binding domain that specifically binds to CD3 via a linker.

2. The antigen-binding molecule according to claim 1, wherein the masking peptide inhibits or reduces the binding of the antigen-binding domain that specifically binds to CD3 to CD3; Preferably, the masking peptide inhibits or reduces the binding of the antigen-binding domain that specifically binds to CD3 to the N-terminus of CD3ε.

3. The antigen-binding molecule according to claim 1 or 2, wherein the protease is a protease expressed or overexpressed in the tumor microenvironment; Preferably, the protease is selected from one or more of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamate proteases, gelatinase, and asparagine peptide lysase.

4. The antigen-binding molecule according to any one of claims 1 to 3, wherein the extended half-life domain binds to a serum protein or a fragment thereof, or the extended half-life domain is a serum protein or a fragment thereof; Preferably, the extended half-life domain specifically binds to human serum albumin (HSA).

5. The antigen-binding molecule according to claim 4, wherein the extended half-life domain is an immunoglobulin single variable domain, scFv, Fd, Fv, dsFv, Fab, Fab′ or F(ab′)2; Preferably, the half-life extension domain is a single variable domain of an immunoglobulin; More preferably, the immunoglobulin single variable domain is VHH.

6. The antigen-binding molecule according to any one of claims 1 to 5, further comprising an antigen-binding domain that specifically binds to tumor-associated antigens (TAAs).

7. The antigen-binding molecule according to claim 6, wherein the antigen-binding domain that specifically binds to TAA is an immunoglobulin single variable domain, scFv, Fd, Fv, dsFv, Fab, Fab′ or F(ab′)2. Preferably, the antigen-binding domain that specifically binds to TAA is a single variable domain of an immunoglobulin. More preferably, the immunoglobulin single variable domain is VHH.

8. The antigen-binding molecule according to any one of claims 1 to 7, comprising: (a) An antigen-binding domain that specifically binds to CD3, wherein the antigen-binding domain that specifically binds to CD3 is Fab; (b) An antigen-binding domain that specifically binds to TAA, wherein the antigen-binding domain that specifically binds to TAA is VHH; (c) A half-life extension domain that specifically binds to HSA, wherein the half-life extension domain that specifically binds to HSA is VHH; and (d) A linker comprising a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; The half-life extension domain that specifically binds to HSA is connected to the N-terminus of the VL or VH of the antigen-binding domain that specifically binds to CD3 via a linker.

9. The antigen-binding molecule according to any one of claims 1 to 8, wherein the antigen-binding molecule comprises a first chain and a second chain, wherein: (i) The first chain contains a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, VL and CL that specifically bind to CD3, and The second chain comprises a VH that specifically binds to CD3, a CH1 that specifically binds to TAA, and a VHH that specifically binds to TAA; or (ii) The first chain contains a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, VL and CL that specifically bind to CD3, and The second chain contains a VHH that specifically binds to TAA, a VH that specifically binds to CD3, and CH1; or (iii) The first chain comprises a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, a VL and CL that specifically bind to CD3, and a VHH that specifically binds to TAA. The second chain contains VH and CH1 that specifically bind CD3; or (iv) The first chain contains VHH, which specifically binds to TAA, VL and CL, which specifically bind to CD3, and The second chain contains a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, and VH and CH1 that specifically bind to CD3; or (v) The first chain comprises VL and CL, which specifically bind to CD3, and VHH, which specifically binds to TAA. The second chain contains a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, and VH and CH1 that specifically bind to CD3; or (vi) The first chain contains VL and CL that specifically bind to CD3, and The second chain contains a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, a VH that specifically binds to CD3, CH1, and a VHH that specifically binds to TAA.

10. The antigen-binding molecule according to any one of claims 1 to 9, wherein the antigen-binding molecule comprises: (i) A first chain having the structure shown in equation (a), and a second chain having the structure shown in equation (c), or (ii) A first chain having the structure shown in equation (a), and a second chain having the structure shown in equation (b), or (iii) A first chain having the structure shown in equation (d), and a second chain having the structure shown in equation (e), or (iv) A first chain having the structure shown in equation (f), and a second chain having the structure shown in equation (g), or (v) A first chain having the structure shown in formula (h), and a second chain having the structure shown in formula (g), or (vi) A first chain having the structure shown in equation (i), and a second chain having the structure shown in equation (j); in, (a)[α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]; (b)[α-TAA-VHH]-[connector 2a]-[α-CD3-VH]-[CH1]; (c)[α-CD3-VH]-[CH1]-[linker 2b]-[α-TAA-VHH]; (d)[α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL]-[connector 2c]-[α-TAA-VHH]; (e)[α-CD3-VH]-[CH1]; (f)[α-TAA-VHH]-[connector 2d]-[α-CD3-VL]-[CL]; (g)[α-HSA-VHH]-[linker 1]-[α-CD3-VH]-[CH1]; (h)[α-CD3-VL]-[CL]-[connector 2e]-[α-TAA-VHH]; (i)[α-CD3-VL]-[CL]; (j)[α-HSA-VHH]-[connector 1]-[α-CD3-VH]-[CH1]-[connector 2f]-[α-TAA-VHH]; Wherein, linker 1 and linkers 2a, 2b, 2c, 2d, 2e and 2f are the same or different peptide linkers, or linkers 2a, 2b, 2c, 2d, 2e and 2f do not exist; The linker 1 contains a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; The structures shown in equations (a), (b), (c), (d), (e), (f), (g), (h), (i), and (j) are arranged from the N end to the C end; Optionally, the positions of CH1 and CL in the first chain and the second chain can be interchanged.

11. The antigen-binding molecule according to any one of claims 1 to 10, further comprising one or more identical or different target antigen-binding domains, said target antigen-binding domains specifically binding to tumor-associated antigens, co-stimulatory molecules, and / or effector cell antigens.

12. The antigen-binding molecule according to claim 11, wherein the target antigen-binding domain is an immunoglobulin single variable domain, scFv, Fd, Fv, dsFv, Fab, Fab′ or F(ab′)2. Preferably, the target antigen-binding domain is a single variable domain of an immunoglobulin; More preferably, the immunoglobulin single variable domain is VHH.

13. The antigen-binding molecule according to any one of claims 1 to 12, wherein the VH of the antigen-binding domain specifically binding CD3 comprises HCDR1, HCDR2 and HCDR3, and the VL of the antigen-binding domain specifically binding CD3 comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2 and HCDR3 of the VH respectively comprise HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 12, and the LCDR1, LCDR2 and LCDR3 of the VL respectively comprise LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 14 or 13; Preferably, The VH's HCDR1 contains the amino acid sequence of SEQ ID NO: 54, HCDR2 contains the amino acid sequence of SEQ ID NO: 55, and HCDR3 contains the amino acid sequence of SEQ ID NO: 56; and the VL's LCDR1 contains the amino acid sequence of SEQ ID NO: 60, LCDR2 contains the amino acid sequence of SEQ ID NO: 58, and LCDR3 contains the amino acid sequence of SEQ ID NO: 61; or The VH's HCDR1 contains the amino acid sequence of SEQ ID NO: 54, HCDR2 contains the amino acid sequence of SEQ ID NO: 55, and HCDR3 contains the amino acid sequence of SEQ ID NO: 56; the VL's LCDR1 contains the amino acid sequence of SEQ ID NO: 57, LCDR2 contains the amino acid sequence of SEQ ID NO: 58, and LCDR3 contains the amino acid sequence of SEQ ID NO:

59. More preferably, The VH contains SEQ ID NO: 12, or an amino acid sequence having at least 80% identity with it, and the VL contains SEQ ID NO: 14 or 13, or an amino acid sequence having at least 80% identity with it.

14. The antigen-binding molecule according to any one of claims 1 to 13, wherein the antigen-binding molecule is an antibody; preferably an activatable antibody.

15. An activatable antibody comprising: (a) An antigen-binding domain that specifically binds to CD3, wherein the antigen-binding domain that specifically binds to CD3 is Fab; (b) An antigen-binding domain that specifically binds to TAA, wherein the antigen-binding domain that specifically binds to TAA is VHH; and (c) A half-life extension domain that specifically binds to HSA, wherein the half-life extension domain that specifically binds to HSA is VHH; (d) A linker comprising a masking peptide and a protease-cleavable sequence from the N-terminus to the C-terminus; The half-life extension domain that specifically binds to HSA is connected to the N-terminus of the light chain variable region or the heavy chain variable region of the antigen-binding domain that specifically binds to CD3 via the linker. Preferably, the activatable antibody has the structure defined as claimed in claim 9 or 10.

16. A nucleic acid molecule encoding an antigen-binding molecule as described in any one of claims 1 to 14, or an activatable antibody as described in claim 15.

17. A host cell comprising the nucleic acid molecule as described in claim 16.

18. A pharmaceutical composition comprising an antigen-binding molecule as described in any one of claims 1 to 14, or an activatable antibody as described in claim 15, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

19. A method for preparing an antigen-binding molecule as described in any one of claims 1 to 14, or an activatable antibody as described in claim 15, the method comprising expressing a nucleic acid molecule as described in claim 16, or culturing a host cell as described in claim 17, to produce the antigen-binding molecule or the activatable antibody.

20. A molecule derived from the antigen-binding molecule of any one of claims 1 to 14, or the activatable antibody of claim 15, by protease cleavage; The protease mentioned therein is a protease expressed or overexpressed in the tumor microenvironment; Preferably, the protease is selected from one or more of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamate proteases, gelatinase, and asparagine peptide lysase.

21. A method of treating, preventing or improving a disease or condition, comprising administering to a subject in need a therapeutically effective amount of an antigen-binding molecule as described in any one of claims 1 to 14, or an activatable antibody as described in claim 15, or a pharmaceutical composition as described in claim 18, or a molecule as described in claim 20; Preferably, the disease or symptom is a tumor; More preferably, the tumor is a solid tumor.

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