Anti-CD3 antibody and method of use thereof

Abstract

The present application provides antibodies, including antigen-binding fragments of anti-CD3 antibodies, with tailored affinity in the low to medium range for CD3. In some embodiments, the antibodies further comprise antigen-binding fragments that specifically bind to a target antigen, such as HER2, CD20, TROP2, BCMA, or CD19. Also provided are anti-CD3 antibodies, masked anti-CD3 antibodies (including activatable anti-CD3 antibodies), anti-CD20 antibodies, and masked anti-HER2 antibodies (including activatable anti-HER2 antibodies). The antibodies described herein are useful for the treatment of cancer.

Description

Cross-reference of related applications

[0001] This application claims the benefit of priority from International Application PCT / CN2021 / 076626, filed on 11 February 2021, and International Application PCT / CN2021 / 109057, filed on 28 July 2021, the entire contents of which are incorporated herein by reference. Submission of the sequence list for the ASCII text file.

[0002] The entire contents of the following ASCII text file submission, a computer-readable format (CRF) sequence listing (filename: 695402000942SEQLIST.TXT, registration date: February 9, 2022, size: 432,841B), are incorporated herein by reference. [Technical Field]

[0003] This application relates to CD3-targeting antibodies, including CD3-targeting multispecific antibodies and masked and activatable anti-CD3 antibodies, as well as methods for producing and using the same. [Background technology]

[0004] Bispecific T-cell engager antibodies (BiTEs or TCEs) have been studied as a means of recruiting cytolytic T cells to kill tumor cells. This is based on the simultaneous recognition of antigens on tumor cells and binding to the CD3ε chain (CD3) within the T-cell receptor complex on T cells, directly cross-linking malignant tumor cells with CD3+ T cells. Blinatumomab (BLINCYTO®) is the first bispecific T-cell engager that reacts with the B-cell antigen CD19 and was approved by the FDA in 2014 as a treatment for neoplasms. Although early studies showed promising clinical efficacy, bispecific T-cell engagers are hampered by severe dose-limiting toxicity, mainly manifesting as cytokine release syndrome, resulting in an extremely narrow therapeutic range. There is a need for activatable BiTE or TCE molecules with enhanced specificity and reduced side effects.

[0005] Activatable antibodies, also known as SAFEBODY®, are designed to mask the antigen-binding interface with a masking motif, preventing the antibody from binding to its target in healthy tissue. The masking motif is designed to activate or unmask the antibody to enable binding in the tumor microenvironment ("TME"), where specific activation conditions exist, such as favorable competition due to upregulated proteases or highly localized antigen concentrations compared to healthy tissue, allowing the antibody to bind to its target for tumor killing. See, for example, WO2019 / 149282. Thus, activatable antibodies provide antigen-specific binding proteins that remain largely inactive in healthy tissue while being primarily activated in the TME.

[0006] [Contents of the invention] This application provides multispecific antibodies targeting CD3 and other target antigens (e.g., HER2, CD20, TROP2, BCMA, or CD19), mask antibodies (including activatable antibodies such as activatable multispecific antibodies), isolated anti-CD3 antibodies, HER2-targeting mask antibodies (e.g., activatable), and methods for treating them.

[0007] One aspect of this application provides a multispecific antibody ("multispecific T cell engager") comprising: a first antigen-binding fragment that specifically binds to CD3 and is fused to a first masking moiety (MM1); and a second antigen-binding fragment that specifically binds to a target antigen; where MM1 specifically binds to the CD3-binding moiety in competition with CD3; and the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM of the antibody, as determined by enzyme-linked immunosorbent assay (ELISA). In some embodiments, the EC50 is at least 50 nM. In some embodiments, the EC50 is at least 100 nM (e.g., about 110 nM). In some embodiments, when used to determine the EC50, the first antigen-binding fragment is an scFv such as an isolated anti-CD3 scFv, an isolated anti-CD3 scFv-Fc fusion protein, or an anti-CD3 scFv fragment in a multispecific (e.g., bispecific) antibody that does not contain MM1. In some embodiments, EC50 is determined using an ELISA assay such as that described in Example 5.

[0008] In some embodiments described in any one of the multispecific antibodies above, the multispecific antibody is not an activatable multispecific antibody. In some embodiments, the first antigen-binding fragment comprises a first immunoglobulin light chain variable domain (VL1) and a first immunoglobulin heavy chain variable domain (VH1) of an anti-CD3 antibody, and MM1 is fused to the N-terminus of VL1 via a first non-cleavable linker (NCL1).

[0009] One aspect of this application provides an activatable multispecific antibody ("activatable multispecific T cell engager") comprising: a) a first antigen-binding fragment that specifically binds to CD3 and is fused to a first masking fragment (MM1) via a first cleavable portion (CM1); and b) a second antigen-binding fragment that specifically binds to a target antigen, wherein CM1 comprises the first cleavage site; MM1 specifically binds to the CD3 binding portion in competition with CD3; and the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM of antibody as determined by enzyme-linked immunosorbent assay (ELISA). In some embodiments, if CM1 is not cleaved, MM1 inhibits the binding of the activatable antibody to CD3; and if CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment. In some embodiments, the first antigen-binding fragment is fused to MM1 via a first cleavable portion (CM1), where CM1 includes a first cleavage site. When CM1 is not cleaved, MM1 inhibits the binding of a multispecific antibody to CD3; when CM1 is cleaved, the multispecific antibody binds to CD3 via the first antigen-binding fragment with higher affinity compared to, for example, the affinity of the multispecific antibody that binds to CD3 via the first antigen-binding fragment when CM1 is not cleaved. In some embodiments, EC50 is at least 50 nM. In some embodiments, EC50 is at least 100 nM (e.g., about 110 nM). In some embodiments, when used to determine EC50, the first antigen-binding fragment is an scFv such as an isolated anti-CD3 scFv, an isolated anti-CD3 scFv-Fc fusion protein, or an anti-CD3 scFv fragment in a multispecific (e.g., bispecific) antibody, or an activatable multispecific antibody in an activated form (i.e., CM1 is cleaved or has effective binding due to a highly localized antigen concentration in TME versus normal tissue). In some embodiments, EC50 is determined using an ELISA assay such as that described in Example 5.

[0010] In some embodiments described in any one of the multispecific or activatable multispecific antibodies described above, the first antigen-binding fragment binds to CD3 with a dissociation constant (Kd) of at least 50 nM or at least 100 nM. In some embodiments, when used to determine Kd, the first antigen-binding fragment is an scFv such as an isolated anti-CD3 scFv, an isolated anti-CD3 scFv-Fc fusion protein, or an anti-CD3 scFv fragment in a multispecific (e.g., bispecific) antibody, or an activatable multispecific antibody in an activated form (e.g., CM1 of the multispecific antibody is cleaved, or it has effective binding due to a highly localized antigen concentration in TME versus normal tissue). In some embodiments, the binding of the antigen-binding fragment to CD3 is measured when the antigen-binding fragment is unmasked.

[0011] In some embodiments described in any one of the multispecific or activatable multispecific antibodies described above, MM1 has a masking efficiency of at least 250 (e.g., at least 500, 1000, 2000, 3000, 5000, 10000 or more) as determined by an ELISA assay, e.g., the ELISA assay of Example 3. In some embodiments, MM1 has a masking efficiency of at least 50 (e.g., at least 100, 200, 300, 400, 500, 600, 800, 1000 or more) as determined by a Jurkat NFAT reporter assay, e.g., a Jurkat NFAT assay for the antigen concentration used in Example 3.

[0012] In some embodiments described in any one of the above-mentioned multispecific or activatable multispecific antibodies, the first antigen-binding fragment comprises a first immunoglobulin light chain variable domain (VL1) and a first immunoglobulin heavy chain variable domain (VH1) of an anti-CD3 antibody. In some embodiments, the first antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab, and scFv. In some embodiments, the first antigen-binding fragment is scFv. In some embodiments, scFv comprises VL1, a linker, and VH1 from the N-terminus to the C-terminus. In some embodiments, scFv comprises VH1, a linker, and VL1 from the N-terminus to the C-terminus. In some embodiments, the antibody is an activatable multispecific antibody, MM1 is fused to the N-terminus of VL1 via CM1. In some embodiments, the antibody is not an activatable multispecific antibody, MM1 is fused to the N-terminus of VL1 via NCL1. In some embodiments, the multispecific antibody is not an activatable multispecific antibody. In some embodiments, the multispecific antibody does not contain a cleavable linker. In some embodiments, the masking portion is not fused with the sequence containing the cutting portion.

[0013] In some embodiments of the multispecific or activatable multispecific antibodies described above, the second antigen-binding fragment comprises a second immunoglobulin light chain variable domain (VL2) and a second immunoglobulin heavy chain variable domain (VH2) of the antibody that specifically binds to the target antigen. In some embodiments, the second antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab, and scFv. In some embodiments, the second antigen-binding fragment is Fv. In some embodiments, the second antigen-binding fragment is Fab. In some embodiments where the antibody is not an activatable multispecific antibody, the multispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide, where: (i) The first polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-First CH3(1a); (ii) The second polypeptide comprises a structure represented by the following formula: MM1-NCL1-VL1-VH1-Hinge-CH2-Second CH3(1b); and (iii) The third polypeptide comprises a structure represented by the following formula: VL2-CL(1c); Here: CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The first CH3 is the first immunoglobulin heavy chain constant domain 3; the second CH3 is the second immunoglobulin heavy chain constant domain 3; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; Here, VL1 and VH1 associate to form scFv which binds specifically to CD3; and VL2 and VH2 associate to form Fv which binds specifically to the target antigen. In some embodiments where the antibody is an activatable multispecific antibody, the activatable multispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide, where: (i) The first polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-First CH3(1a); (ii) The second polypeptide comprises a structure represented by the following formula: MM1-CM1-VL1-VH1-Hinge-CH2-Second CH3(1b); and (iii) The third polypeptide comprises a structure represented by the following formula: VL2-CL(1c); Here: CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The first CH3 is the first immunoglobulin heavy chain constant domain 3; the second CH3 is the second immunoglobulin heavy chain constant domain 3; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; Here, VL1 and VH1 associate to form scFv which specifically binds to CD3; and VL2 and VH2 associate to form Fv which specifically binds to the target antigen. In some embodiments, the multispecific antibody (e.g., its second polypeptide) contains an amino acid linker between VL1 and VH1.

[0014] In some embodiments described in any one of the above-mentioned multispecific or activatable multispecific antibodies, a second antigen-binding fragment is fused to a second masking moiety (MM2), where MM2 competes with the target antigen to specifically bind the second antigen-binding fragment. In some embodiments, the second antigen-binding fragment is fused to MM2 via a second non-cleavable linker (NCL2). In some embodiments, the second antigen-binding fragment is fused to MM2 via a second cleavable moiety (CM2), where CM2 includes a second cleavage site, where MM2 inhibits the binding of the multispecific antibody to the target antigen if CM2 is not cleaved, and where CM2 is cleaved, the multispecific antibody binds to the target antigen via the second antigen-binding fragment. In some embodiments, the second antigen-binding fragment includes VH2 and VL2 of an antibody that specifically binds to the target antigen, where MM2 is fused to the N-terminus of VL2 via CM2. In some embodiments, a multispecific or activatable multispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide, where: (i) The first polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-First CH3(2a); (ii) The second polypeptide comprises a structure represented by the following formula: MM1-CM1-VL1-VH1-Hinge-CH2-Second CH3(2b); and (iii) The third polypeptide comprises a structure represented by the following formula: MM2-CM2-VL2-CL(2c); Here: CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The first CH3 is the first immunoglobulin heavy chain constant domain 3; the second CH3 is the second immunoglobulin heavy chain constant domain 3; The hinge is an immunoglobulin hinge region that links the CH1 and CH2 domains; where VL1 and VH1 associate to form scFv which specifically binds to CD3; and VL2 and VH2 associate to form Fv which specifically binds to the target antigen. In some embodiments, the multispecific antibody (e.g., its second polypeptide) includes an amino acid linker between VL1 and VH1.

[0015] In some embodiments described in any one of the above-mentioned multispecific or activatable multispecific antibodies, CD3 is human CD3. In some embodiments, the first antigen-binding fragment is cross-reactive with CD3 polypeptides derived from at least one non-human species selected from the group consisting of cynomolgus monkeys, mice, rats, and dogs.

[0016] In some embodiments described above for any one of the multispecific or activatable multispecific antibodies, the first antigen-binding fragment comprises VH1 and VL1 of an anti-CD3 antibody, wherein VH1 comprises a heavy chain complementarity-determining region (CDR-H)1 containing the amino acid sequence of formula (I):X1YAX2X3(SEQ ID NO: 382) (wherein X1 is D, S, or T, X2 is I, L, or M, and X3 is N or T), and formula (II):RI The CDR-H2 contains the amino acid sequence RSKYNNYATYYAX1X2VKX3 (SEQ ID NO: 383) (wherein X1 is F or Y, X2 is N or T, and X3 is D, G, or S), and the CDR-H3 contains the amino acid sequence (III):HGNX1GX2SYVSX3X4AY (SEQ ID NO: 384) (wherein X1 is F or Y, X2 is N or T, X3 is W or Y, and X4 is F or W). In some embodiments, VL1 includes CDR-L1 comprising the amino acid sequence of formula (IV):X1SSTGAVTX2X3NYX4N (SEQ ID NO: 385), (wherein X1 is A, G, or R, X2 is S, or T, X3 is G, or S, and X4 is A, P, or V); CDR-L2 comprising the amino acid sequence of formula (V):GTX1X2RAP (SEQ ID NO: 386), (wherein X1 is K, or N, and X2 is F, or K); and CDR-L3 comprising the amino acid sequence of formula (VI):ALWYSX1X2WV (SEQ ID NO: 387), (wherein X1 is D, N, or T, and X2 is L, or R).

[0017] In some embodiments described in any one of the multispecific or activatable multispecific antibodies described above, the first antigen-binding fragment comprises VH1 and VL1 of an anti-CD3 antibody, wherein VH1 comprises a heavy chain complementarity-determining region (CDR-H) 1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 376, 390, 601, and 602, or a variant thereof containing up to about three amino acid substitutions, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 377, 391-394, and 603, or a variant thereof containing up to about three amino acid substitutions, and SEQ ID NOs. 378, 395, 604, and 605. CDR-H3 includes an amino acid sequence selected from the group, or a variant thereof containing up to approximately three amino acid substitutions; and VL1 includes CDR-L1, or a variant thereof containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 396-398 and 606-609, or a variant thereof containing up to approximately three amino acid substitutions; CDR-L2, or a variant thereof containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 380 and 399, or a variant thereof containing up to approximately three amino acid substitutions; and CDR-L3, or a variant thereof containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 381, 400-401 and 610, or a variant thereof containing up to approximately three amino acid substitutions.

[0018] In some embodiments described in any one of the multispecific or activatable multispecific antibodies described above, the first antigen-binding fragment comprises VH1 and VL1 of an anti-CD3 antibody, wherein VH1 comprises CDR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 376 and 390, or a variant thereof containing up to about three amino acid substitutions, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 391-394, or a variant thereof containing up to about three amino acid substitutions, and an amino acid sequence selected from the group consisting of SEQ ID NOs. 378 and 395. Includes CDR-H3, or a variant thereof containing up to approximately three amino acid substitutions; and VL1 includes CDR-L1, or a variant thereof containing up to approximately three amino acid substitutions, containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 396-398; CDR-L2, or a variant thereof containing up to approximately three amino acid substitutions, containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 380 and 399; and CDR-L3, or a variant thereof containing up to approximately three amino acid substitutions, containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 381 and 400-401.

[0019] In some embodiments described in any one of the multispecific or activatable multispecific antibodies described above, the first antigen-binding fragment comprises VH1 and VL1 of an anti-CD3 antibody, wherein VH1 comprises a heavy chain complementarity-determining region (CDR-H)1 comprising the amino acid sequence of SEQ ID NO: 382, ​​CDR-H2 comprising the amino acid sequence of SEQ ID NO: 383, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 384; VL1 comprises a light chain complementarity-determining region (CDR-L)1 comprising the amino acid sequence of SEQ ID NO: 385, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 386, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 387. In some embodiments, VH1 includes CDR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, and CDR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395; and VL1 includes CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, CDR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, and CDR-L3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401. In some embodiments, VH1 includes CDR-H1 containing the amino acid sequence of SEQ ID NOs: 376, CDR-H2 containing the amino acid sequence of SEQ ID NOs: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 378; and VL1 includes CDR-L1 containing the amino acid sequence of SEQ ID NOs: 396, CDR-L2 containing the amino acid sequence of SEQ ID NOs: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 381. In some embodiments, VH1 includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL1 includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400.In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 401. In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 401. In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 398, CDR-L2 containing the amino acid sequence of SEQ ID NO: 399, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400.In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381.In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH1 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL1 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH1 is formula (VII):EVQLVESGGGLVX1PGGSLRLSCAASGFTFX2X3YAIX4WVRQAPGKGLEWVX5RIRSKYNNYATYYAX6SVKX7RFTISRDX8SKNTLYLQX9NSLRAEDTAVYYCX. 10 RHGNX 11 GX 12 SYVSWFAYWGQGTLVTVSS(Sequence ID 388)(wherein X1 is K or Q, X2 is N or S, X3 is S or T, X4 is H or N, X5 is G or S, X6 is D or E, X7 is D or G, X8 is D or N, X9 is I or L, X 10 is A or V, and X 11 is F or Y, and X 12VL1 contains the amino acid sequence of formula (VIII): X1AVVTQEPSLTVSPGGTVTLTCX2SSTGAVTTSNYX3NWX4QQKPGQAPRGLIGGTX5X6RAPGX7PARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSX8X9WVFGGGTKLTVL (Sequence ID 389) (wherein X1 is E or Q, X2 is A, G, P, or R, X3 is A or P, X4 is F or V, X5 is K or N, X6 is F or K, X7 is A, I, T, or V, X8 is A, D, N, or T, and X9 is H or L). In some embodiments, VH1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414-416, and 611-640, or a variant having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414-416, and 611-640; and VL1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, 413, and 641-666, or a variant having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, 413, and 641-666. In some embodiments, VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414, 415, and 416; and VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, and 413. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NOs: 388, and VL1 comprises the amino acid sequence of SEQ ID NOs: 389. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NOs: 402, 405, 407, 409, 410, 412, 414, 415, and 416 The amino acid sequence is selected from the group consisting of; and VL1 SEQ ID NOs. 403, 404, 406, 408, 411, and 413 are selected from the group consisting of. In some embodiments, VH1 contains the amino acid sequence of SEQ ID NO 402, and VL1 contains the amino acid sequence of SEQ ID NO 403. In some embodiments, the amino acid sequence of SEQ ID NO 402 is included, and VL1 contains the amino acid sequence of SEQ ID NO 404. In some embodiments, VH1 contains the amino acid sequence of SEQ ID NO 405, and VL1 contains the amino acid sequence of SEQ ID NO 406. In some embodiments, VH1 contains the amino acid sequence of SEQ ID NO 407, and VL1 contains the amino acid sequence of SEQ ID NO 404. In some embodiments, VH1 contains the amino acid sequence of SEQ ID NO 407, and VL1 contains the amino acid sequence of SEQ ID NO 403. In some embodiments, VH1 contains the amino acid sequence of SEQ ID NO 407, and VL1 contains the amino acid sequence of SEQ ID NO 408. In some embodiments, VH1 includes the amino acid sequence of SEQ ID NO: 409, and VL1 includes the amino acid sequence of SEQ ID NO: 408. In some embodiments, VH1 includes the amino acid sequence of SEQ ID NO: 410, and VL1 includes the amino acid sequence of SEQ ID NO: 411. In some embodiments, VH1 includes the amino acid sequence of SEQ ID NO: 412, and VL1 includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH1 includes the amino acid sequence of SEQ ID NO: 410, and VL1 includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH1 includes the amino acid sequence of SEQ ID NO: 414, and VL1 includes the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH1 includes the amino acid sequence of SEQ ID NO: 415, and VL1 includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH1 includes the amino acid sequence of SEQ ID NO: 416, and VL1 includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 416, and VL1 comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, the first antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 421.In some embodiments, the first antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 422.

[0020] In some embodiments of any one of the activatable multispecific antibodies described above, MM1 contains the amino acid sequence EVGSY (SEQ ID NO: 667) at its N-terminus. In some embodiments, MM1 contains the amino acid sequence of formula (IX):PYDDPDCPSHX1SDCDX2 (SEQ ID NO: 668) (wherein X1 is D or E and X2 is N or Q). In some embodiments, MM1 contains the amino acid sequence of formula (X). In some embodiments, MM1 contains the amino acid sequence of SEQ ID NO: 417. In some embodiments, MM1 contains the amino acid sequence of SEQ ID NO: 35. In some embodiments, MM1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 597-599.

[0021] In some embodiments described in any one of the above-mentioned activatable multispecific antibodies, CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 127-129, 418, 420, 431, 477-490, and 516-555. In some embodiments, CM1 comprises the amino acid sequence of SEQ ID NO: 77 or 418.

[0022] In some embodiments described in any one of the multispecific or activatable multispecific antibodies described above, the target antigen is a tumor antigen. In some embodiments, the tumor antigen is CD19, CD20, EpCAM, CEA, PSMA, CD33, EGFR, HER2, EphA2, MCSP, ADAM17, PSCA, 17-A1, NKG2D, TROP2, CD79B, Nectin-4, BCMA, CD22, CD38, EGFR, GD2, SLAMF7, CD30, EpCAM, MUC1, MUC16, CD123, CD37, FOLR1, MET, FLT3, GPC3, CEACAM5, CLDN18, CSF1, integrin α5, NCAM1, PTPRC, CD138, NaPi2b, MSLN, DLL3, GPRC5D, GPNMB, ICAM1, SSTR2, or cancer-related antigen. The tumor antigen is selected from the group consisting of CTAA16, CA9, ENG, ACVRL1, CD80, CSPG4, EGFL7, FLT1, HAVCR1, HGF, HLA-DRB, IGF1R, TPBG, ERBB3, and STEAP2. In some embodiments, the tumor antigen is HER2. In some embodiments, the tumor antigen is CD20. In some embodiments, the tumor antigen is TROP2. In some embodiments, the tumor antigen is BCMA. In some embodiments, the tumor antigen is CD19.

[0023] In some embodiments of any one of the multispecific or activatable multispecific antibodies described above, the target antigen is HER2. In some embodiments, the second antigen-binding fragment comprises VH2 and VL2 of the anti-HER2 antibody, where VH2 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 423, CDR-H2 containing the amino acid sequence of SEQ ID NO: 424, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 71; and VL2 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 75, and VL2 comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, a second antigen-binding fragment is fused to a second masking moiety (MM2) via a second cleavable moiety (CM2), where: a) MM2 comprises the amino acid sequence of formula (XI):ESX1X2CX3X4DPFX5CQX6 (SEQ ID NO: 670) (wherein X1 is D or E, X2 is A, F, V or Y, X3 is D or E, X4 is A or L, X5 is D or E, and X6 is A, F or Y); b) MM2 comprises the amino acid sequence of formula (XII):X1X2X3X4X5X6CX7X8DPYECX9X 10 (Sequence number 671) (wherein X1 is A, H, or S, X2 is A, D, or S, X3 is A, T, or V, X4 is P, S, or T, X5 is D or E, X6 is A or V, X7 is D or E, X8 is A or L, X9 is Q, S, or T, X 10c) MM2 comprises an amino acid sequence of formula (XIII):YNSDDDCX1SX2YDPYTCYY(SEQ ID NO: 672) (wherein X1 is A, I, or V, and X2 is H or R). In some embodiments, the second antigen-binding fragment is fused to the second masking fragment (MM2) via a second cleavable portion (CM2), where MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515. In some embodiments, MM2 comprises the amino acid sequence of SEQ ID NO: 419. In some embodiments, CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 127-129, 418, 420, 431, and 477-490, and 516-555. In some embodiments, CM2 comprises the amino acid sequence of SEQ ID NO: 420. In some embodiments, CM2 comprises the amino acid sequence of SEQ ID NO: 77. In some embodiments, the multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 425; a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 426; and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 112.In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 427; a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 428; and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 112. In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 429; a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 430; and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 115. In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 83; a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 84; and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 85.In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 683; a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 684; and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 685. In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 425 (optionally, without C-terminal lysine); a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 426 (optionally, without C-terminal lysine); and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 112. In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 427 (optionally, without C-terminal lysine); a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 428 (optionally, without C-terminal lysine); and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 112.In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 429 (optionally, without C-terminal lysine); a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 430 (optionally, without C-terminal lysine); and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 115. In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 83; a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 84 (optionally, without C-terminal lysine); and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 85 (optionally, without C-terminal lysine). In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 683; a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 684 (optionally, without C-terminal lysine); and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 685 (optionally, without C-terminal lysine).In some embodiments described in any one of the multispecific or activatable multispecific antibodies described above, the multispecific or activatable multispecific antibody comprises a mixture of heavy chain species, some of which contain C-terminal lysine, and some of which lack C-terminal lysine.

[0024] In some embodiments described in any one of the above-mentioned multispecific or activatable multispecific antibodies, the target antigen is CD20. In some embodiments, the second antigen-binding fragment comprises VH2 and VL2 of an anti-CD20 antibody, where VH2 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 556, CDR-H2 containing the amino acid sequence of SEQ ID NO: 557, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 558; and VL2 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 559, CDR-L2 containing the amino acid sequence of SEQ ID NO: 560, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 561. In some embodiments, the second antigen-binding fragment comprises VH2 and VL2 of an anti-CD20 antibody, where VH2 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 86, CDR-H2 containing the amino acid sequence of SEQ ID NO: 557, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 558; and VL2 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 559, CDR-L2 containing the amino acid sequence of SEQ ID NO: 560, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 561. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 562, and VL2 comprises the amino acid sequence of SEQ ID NO: 563. In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 564; a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 565; and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 567.In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 564; a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 565; and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 569. In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 564; a second polypeptide (optionally without C-terminal lysine) comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 565; and a third polypeptide (optionally without C-terminal lysine) comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 567. In some embodiments, a multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 564; a second polypeptide (optionally without C-terminal lysine) comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 565; and a third polypeptide (optionally without C-terminal lysine) comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 569.In some embodiments described in any one of the multispecific or activatable multispecific antibodies described above, the multispecific or activatable multispecific antibody comprises a mixture of heavy chain species, some of which contain C-terminal lysine, and some of which lack C-terminal lysine.

[0025] In some embodiments of any one of the multispecific or activatable multispecific antibodies described above, the multispecific or activatable multispecific antibody includes an Fc region. In some embodiments, the Fc region is of the human IgG1 subclass. In some embodiments, the Fc region is of the human IgG2 subclass. In some embodiments, the Fc region is of the human IgG4 subclass. In some embodiments, the Fc region has enhanced ADCC and / or crosslinking efficiency. In some embodiments, the Fc region has reduced antibody-dependent cell-mediated cytotoxicity (ADCC) effect or no antibody-dependent cell-mediated cytotoxicity (ADCC) effect and / or reduced crosslinking effect or no crosslinking effect. In some embodiments, the Fc region is of the human IgG1 subclass and has the N297A amino acid substitution.

[0026] In some embodiments described in any one of the multispecific or activatable multispecific antibodies described above, the multispecific or activatable multispecific antibody comprises a first CH3 domain and a second CH3 domain, i) the first CH3 domain contains a cysteine ​​(C) residue at position 390 and the second CH3 domain contains a cysteine ​​residue at position 400, or the first CH3 domain contains a cysteine ​​residue at position 400 and the second CH3 domain contains a cysteine ​​residue at position 390; or ii) the first CH3 domain contains a cysteine ​​residue at position 392 The first CH3 domain contains a cysteine ​​residue, and the second CH3 domain contains a cysteine ​​residue at position 397, or the first CH3 domain contains a cysteine ​​residue at position 397 and the second CH3 domain contains a cysteine ​​residue at position 392; or iii) the first CH3 domain contains a cysteine ​​residue at position 392 and the second CH3 domain contains a cysteine ​​residue at position 400, or the first CH3 domain contains a cysteine ​​residue at position 400 and the second CH3 domain contains a cysteine ​​residue at position 392; amino acid residue numbering is based on EU numbering. In some embodiments, i) the first CH3 domain includes an N390C substitution and the second CH3 domain includes an S400C substitution, or the first CH3 domain includes an S400C substitution and the second CH3 domain includes an N390C substitution; or ii) the first CH3 domain includes a K392C substitution and the second CH3 domain includes a V397C substitution, or the first CH3 domain includes a V397C substitution and the second CH3 domain includes a K392C substitution; or iii) the first CH3 domain includes a K392C substitution and the second CH3 domain includes an S400C substitution, or the first CH3 domain includes an S400C substitution and the second CH3 domain includes a K392C substitution. In some embodiments, i) the first CH3 domain further contains a positively charged residue at position 357 and the second CH3 domain further contains a load-charged residue at position 351, or the first CH3 domain further contains a load-charged residue at position 351 and the second CH3 domain further contains a positively charged residue at position 357; or ii) the first CH3 domain further contains a positively charged residue at position 411 and the second CH3 domain further contains a load-charged residue at position 370, or the first CH3 domain further contains a load-charged residue at position 370 The first CH3 domain contains a residue, and the second CH3 domain further contains a positively charged residue at position 411; or iii) the first CH3 domain further contains a positively charged residue at position 364 and the second CH3 domain further contains a negatively charged residue at position 370, or the first CH3 domain further contains a negatively charged residue at position 370 and the second CH3 domain further contains a positively charged residue at position 364; or a combination of i) and ii), or a combination of i) and iii), and the amino acid residue numbering is based on EU numbering. In some embodiments, the first CH3 domain further includes a positively charged residue at position 356 and the second CH3 domain further includes a negatively charged residue at position 439, or the first CH3 domain further includes a negatively charged residue at position 439 and the second CH3 domain further includes a positively charged residue at position 356; amino acid residue numbering is based on EU numbering. In some embodiments, i) the positively charged residue is a lysine (K) residue and the negatively charged residue is an aspartic acid (D) residue; or ii) the positively charged residue is a lysine (K) residue and the negatively charged residue is a glutamic acid (E) residue; or iii) the positively charged residue is an arginine (R) residue and the negatively charged residue is an aspartic acid (D) residue; or iv) the positively charged residue is an arginine (R) residue and the negatively charged residue is a glutamic acid (E) residue.In some embodiments, i) the first CH3 domain includes E357K and T411K substitutions and the second CH3 domain includes L351D and K370D substitutions, or the first CH3 domain includes L351D and K370D substitutions and the second CH3 domain includes E357K and T411K substitutions; or ii) the first CH3 domain includes E357K and S364K substitutions and the second CH3 domain includes L351D and K370D substitutions, or the first C The H3 domain includes L351D and K370D substitutions, and the second CH3 domain includes E357K and S364K substitutions; or iii) the first CH3 domain includes D356K, E357K, and S364K substitutions, and the second CH3 domain includes L351D, K370D, and K439D substitutions, or the first CH3 domain includes L351D, K370D, and K439D substitutions, and the second CH3 domain includes D356K, E357K, and S364K substitutions.In some embodiments, i) the first CH3 domain further includes K392D and K409D substitutions, and the second CH3 domain further includes D356K and D399K substitutions; or the first CH3 domain further includes D356K and D399K substitutions, and the second CH3 domain further includes K392D and K409D substitutions; or ii) the first CH3 domain further includes L368D and K370S substitutions. (i) The second CH3 domain further includes E357Q and S364K substitutions, or the first CH3 domain further includes E357Q and S364K substitutions, and the second CH3 domain further includes L368D and K370S substitutions; or iii) The first CH3 domain further includes L351K and T366K substitutions, and the second CH3 domain further includes L351D and L368E substitutions, or the first CH3 The first CH3 domain further includes L351D and L368E substitutions, and the second CH3 domain further includes L351K and T366K substitutions; or (iv) the first CH3 domain further includes P395K, P396K, and V397K substitutions, and the second CH3 domain further includes T394D, P395D, and P396D substitutions, or the first CH3 domain further includes T394D, P395D, and P396D substitutions, and the second The CH3 domain of (v) further includes P395K, P396K, and V397K substitutions, or (v) the first CH3 domain further includes F405E, Y407E, and K409E substitutions, and the second CH3 domain further includes F405K and Y407K substitutions, or the first CH3 domain further includes F405K and Y407K substitutions, and the second CH3 domain further includes F405E, Y407E, and K409E substitutions.

[0027] In some embodiments described in any one of the multispecific or activatable multispecific antibodies described above, the multispecific or activatable multispecific antibody comprises a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises E357K, S364K, and N390C substitutions and the second CH3 domain comprises L351D, K370D, and S400C substitutions, or the first CH3 domain comprises L351D, K370D, and S400C substitutions and the second CH3 domain comprises E357K, S364K, and N390C substitutions. In some embodiments, the first CH3 domain includes the substitutions E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, and N390C, or the first CH3 domain includes the substitutions L351D, K370D, and N390C, and the second CH3 domain includes the substitutions E357K, S364K, and S400C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and S400C, and the second CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, or the first CH3 domain includes the substitutions L351D, K370D, N390C, and K439D, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and S400C. In some embodiments, the first CH3 domain includes the substitutions D356K, E357K, S364K, and N390C, and the second CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, or the first CH3 domain includes the substitutions L351D, K370D, K439D, and S400C, and the second CH3 domain includes the substitutions D356K, E357K, S364K, and N390C.

[0028] In some embodiments described in any one of the above-mentioned multispecific or activatable multispecific antibodies, the multispecific or activatable multispecific antibody is a bispecific antibody.

[0029] One aspect of this application provides an isolated antibody or its antigen-binding fragment ("anti-CD3 antibody") that specifically binds to CD3, comprising: a heavy chain complementarity-determining region (CDR-H) 1 comprising the amino acid sequence of formula (I): X1YAX2X3 (SEQ ID NO: 382), (wherein X1 is D, S, or T, X2 is I, L, or M, and X3 is N or T); a CDR-H2 comprising the amino acid sequence of formula (II): RIRSKYNNYATYYAX1X2VKX3 (SEQ ID NO: 383), (wherein X1 is D or E, X2 is S, or T, and X3 is D, G, or S); and a formula (III): HGNX1GX2SYVSX3X4AY (SEQ ID NO: 384), (wherein X1 is F, or Y, X2 is N, VH containing CDR-H3 containing the amino acid sequence (wherein X1 is T, X3 is W or Y, and X4 is F or W); and b) CDR-L1 containing the amino acid sequence of formula (IV): X1SSTGAVTX2X3NYX4N (SEQ ID NO: 385), (wherein X1 is A, G or R, X2 is S or T, X3 is G or S, and X4 is A, P or V); CDR-L2 containing the amino acid sequence of formula (V): GTX1X2RAP (SEQ ID NO: 386), (wherein X1 is K or N, and X2 is F or K); and VL containing CDR-L3 containing the amino acid sequence of formula (VI: ALWYSX1X2WV (SEQ ID NO: 387), (wherein X1 is D, N or T, and X2 is L or R)).In some embodiments, VH includes an amino acid sequence heavy chain complementarity determining region (CDR-H) 1 selected from the group consisting of SEQ ID NOs: 376, 390, 601, and 602, or a variant thereof containing up to about three amino acid substitutions; CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 377, 391-394, and 603, or a variant thereof containing up to about three amino acid substitutions; and CDR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 378, 395, 604, and 605, or up to about three amino acid substitutions. VL includes variants of the same, which include substitutions; and VL includes CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398 and 606-609, or a variant thereof containing up to about three amino acid substitutions; CDR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, or a variant thereof containing up to about three amino acid substitutions; and CDR-L3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 381, 400-401 and 610, or a variant thereof containing up to about three amino acid substitutions. In some embodiments, VH includes CDR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, or a variant thereof containing up to about three amino acid substitutions; CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, or a variant thereof containing up to about three amino acid substitutions; and CDR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395, or a variant thereof containing up to about three amino acid substitutions; and VL includes CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, or a variant thereof containing up to about three amino acid substitutions; CDR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, or a variant thereof containing up to about three amino acid substitutions; and CDR-L3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401, or a variant thereof containing up to about three amino acid substitutions.In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 382, ​​CDR-H2 containing the amino acid sequence of SEQ ID NO: 383, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 384; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 385, CDR-L2 containing the amino acid sequence of SEQ ID NO: 386, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 387. In some embodiments, VH includes CDR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, and CDR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395; and VL includes CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, CDR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, and CDR-L3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 401.In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 401. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 398, CDR-L2 containing the amino acid sequence of SEQ ID NO: 399, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381.In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381.In some embodiments, VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 391, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 378; and VL comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 391, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 378; and VL comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH has the formula (VII): EVQLVESGGGLVX1PGGSLRLSCAASGFTFX2X3YAIX4WVRQAPGKGLEWVX5RIRSKYNNYATYYAX6SVKX7RFTISRDX 10 RHGNX 11 GX 12 SYVSWFAYWGQGTLVTVSS (SEQ ID NO 388) (wherein X1 is K or Q, X2 is N or S, X3 is S or T, X4 is H or N, X5 is G or S, X6 is D or E, X7 is D or G, X8 is D or N, X9 is I or L, X 10 is A or V, X 11 is F or Y, X 12 is N or T) and VL has the formula (VIII): The amino acid sequence X1AVVTQEPSLTVSPGGTVTLTCX2SSTGAVTTSNYX3NWX4QQKPGQAPRGLIGGTX5X6RAPGX7PARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSX8X9WVFGGGTKLTVL (Sequence ID 389) (wherein X1 is E or Q, X2 is A, G, P, or R, X3 is A or P, X4 is F or V, X5 is K or N, X6 is F or K, X7 is A, I, T, or V, X8 is A, D, N, or T, and X9 is H or L) is included. In some embodiments, VH includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414-416, and 611-640, or a variant having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414-416, and 611-640; and VL includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, 413, and 641-666, or a variant having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, 413, and 641-666. In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414, 415, and 416; and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, and 413. In some embodiments described in any one of the isolated anti-CD3 antibodies or its antigen-binding fragments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 402, 405, 407, 409, 410, 412, 414, 415, and 416; and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 403, 404, 406, 408, 411, and 413. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 388, and VL comprises the amino acid sequence of SEQ ID NO: 389.In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 402, and VL includes the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 402, and VL includes the amino acid sequence of SEQ ID NO: 404. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 405, and VL includes the amino acid sequence of SEQ ID NO: 406. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 407, and VL includes the amino acid sequence of SEQ ID NO: 404. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 407, and VL includes the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 407, and VL includes the amino acid sequence of SEQ ID NO: 408. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 409, and VL includes the amino acid sequence of SEQ ID NO: 408. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 410, and VL includes the amino acid sequence of SEQ ID NO: 411. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 412, and VL includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 410, and VL includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 414, and VL includes the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 415, and VL includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 416, and VL includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 416, and VL includes the amino acid sequence of SEQ ID NO: 411.

[0030] In some embodiments described for any one of the isolated anti-CD3 antibodies or their antigen-binding fragments, the anti-CD3 antibody further comprises a second antigen-binding fragment that specifically binds to a target antigen. In some embodiments, the target antigen is a tumor antigen. In some embodiments, tumor antigens include CD19, CD20, EpCAM, CEA, PSMA, CD33, EGFR, HER2, EphA2, MCSP, ADAM17, PSCA, 17-A1, NKG2D, TROP2, CD79B, Nectin-4, BCMA, CD22, CD38, EGFR, GD2, SLAMF7, CD30, EpCAM, MUC1, MUC16, CD123, CD37, FOLR1, MET, FLT3, GPC3, CEACAM5, CLDN18, CSF1, integrin α5, NCAM1, PTPRC, CD138, NaPi2b, MSLN, DLL3, GPRC5D, GPNMB, ICAM1, SSTR2, and cancer-related antigens. The tumor antigen is selected from the group consisting of CTAA16, CA9, ENG, ACVRL1, CD80, CSPG4, EGFL7, FLT1, HAVCR1, HGF, HLA-DRB, IGF1R, TPBG, ERBB3, and STEAP2. In some embodiments, the tumor antigen is HER2. In some embodiments, the tumor antigen is CD20. In some embodiments, the tumor antigen is TROP2. In some embodiments, the tumor antigen is BCMA. In some embodiments, the tumor antigen is CD19.

[0031] One aspect of this application provides an activatable antibody ("activatable anti-CD3 antibody") comprising a masking moiety (MM), a cleavable moiety (CM), and a CD3-binding moiety from the N-terminus to the C-terminus, wherein: a) the CD3-binding moiety comprises VL, and the activatable antibody further comprises a second polypeptide comprising VH; b) the CD3-binding moiety comprises VH, and the activatable antibody further comprises a second polypeptide comprising VL; c) the CD3-binding moiety comprises VL and VH from the N-terminus to the C-terminus; or d) the CD3-binding moiety comprises VL and VH from the N-terminus to the C-terminus; the CM comprises a cleavage site; if the CM is not cleaved, the MM inhibits the binding of the activatable antibody to CD3; if the CM is cleaved, the activatable antibody binds to CD3 via VH and VL; and the activatable antibody binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM of the antibody as determined by enzyme-linked immunosorbent assay (ELISA) (e.g., at least 50 nM, or at least 100 nM, or about 110 nM). In some embodiments, when used to determine EC50, the first antigen-binding fragment is an isolated anti-CD3 scFv, an isolated anti-CD3 scFv-Fc fusion protein, or an scFv such as an anti-CD3 scFv fragment in a multispecific (e.g., bispecific) antibody, or an activatable multispecific antibody in an activated form (i.e., with CM1 cleaved). In some embodiments, EC50 is determined using an ELISA assay as described in Example 5.

[0032] In some embodiments described for any one of the activatable anti-CD3 antibodies described above, the first antigen-binding fragment binds to CD3 with a dissociation constant (Kd) of at least 50 nM. In some embodiments, when used to determine Kd, the first antigen-binding fragment is an isolated anti-CD3 scFv, an isolated anti-CD3 scFv-Fc fusion protein, or an scFv such as an anti-CD3 scFv fragment in a multispecific (e.g., bispecific) antibody, or an activatable multispecific antibody in an activated form (i.e., with CM1 cleaved).

[0033] In some embodiments described in any one of the above-mentioned activatable anti-CD3 antibodies, MM contains the amino acid sequence EVGSY (SEQ ID NO: 667) at the N-terminus of MM. In some embodiments, MM contains the amino acid sequence of formula (IX):PYDDPDCPSHX1SDCDX2 (SEQ ID NO: 668) (wherein X1 is D or E and X2 is N or Q). In some embodiments, MM contains the amino acid sequence of formula (X):X1X2X3DX4X5CX6X7DX8X9X 10 CX 11 X 12 (Sequence ID 669) (wherein X1 is A or D, X2 is A, D, or P, X3 is D, H, or P, X4 is F or P, X5 is D or P, X6 is D or P, X7 is A or P, X8 is D, N, or P, X9 is A, N, or P, X 10 is D, H or S, X 11 is H, P, or Y, and X 12 The amino acid sequence includes (where is N, P, or Y). In some embodiments, MM includes the amino acid sequence of SEQ ID NO: 35. In some embodiments, MM includes the amino acid sequence of SEQ ID NO: 417. In some embodiments, MM includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 585-588 and 597-591. In some embodiments, CD3 is human CD3.

[0034] One aspect of this application provides an activatable antibody ("activatable anti-CD3 antibody") comprising a masking moiety (MM), a cleavable moiety (CM), and a CD3-binding moiety from the N-terminus to the C-terminus, wherein: a) the CD3-binding moiety comprises VL, and the activatable antibody further comprises a second polypeptide comprising VH; b) the CD3-binding moiety comprises VH, and the activatable antibody further comprises a second polypeptide comprising VL; c) the CD3-binding moiety comprises VL and VH from the N-terminus to the C-terminus; or d) the CD3-binding moiety comprises VL and VH from the N-terminus to the C-terminus Contains;CM contains a cleavage site;If CM is not cleaved, MM inhibits the binding of the activatable antibody to CD3;If CM is cleaved, the activatable antibody binds to CD3 via VH and VL;a)MM contains the amino acid sequence EVGSY (SEQ ID NO: 667) at the N-terminus of MM;b)MM contains the amino acid sequence of formula (IX):PYDDPDCPSHX1SDCDX2 (SEQ ID NO: 668) (wherein X1 is D or E and X2 is N or Q);or c)MM contains the amino acid sequence of formula (X):X1X2X3DX4X5CX6X7DX8X9X 10 CX 11 X 12 (Sequence ID 669) (wherein X1 is A or D, X2 is A, D, or P, X3 is D, H, or P, X4 is F or P, X5 is D or P, X6 is D or P, X7 is A or P, X8 is D, N, or P, X9 is A, N, or P, X 10 is D, H or S, X 11 is H, P, or Y, and X 12 The amino acid sequence (where is N, P, or Y) is included. In some embodiments, MM includes the amino acid sequences of SEQ ID NOs. 35, 417, 585-588, and 597-599. In some embodiments, CD3 is human CD3.

[0035] In some embodiments of any one of the activatable anti-CD3 antibodies described above, the activatable anti-CD3 antibody comprises an anti-CD3 antigen-binding fragment selected from the group consisting of Fab, Fv, scFab, and scFv. In some embodiments, the anti-CD3 antigen-binding fragment is scFv. In some embodiments, scFv comprises a VL, a linker, and a VH from the N-terminus to the C-terminus.

[0036] In some embodiments described in any one of the above-mentioned activatable anti-CD3 antibodies, VH comprises a heavy chain complementation determining region (CDR-H)1 containing the amino acid sequence of formula (I):X1YAX2X3 (SEQ ID NO: 382), (wherein X1 is D, S, or T, X2 is I, L, or M, and X3 is N or T), a CDR-H2 containing the amino acid sequence of formula (II):RIRSKYNNYATYYAX1X2VKX3 (SEQ ID NO: 383), (wherein X1 is F or Y, X2 is N or T, and X3 is D, G, or S), and formula (III):HGNX1GX2SYVSX3X4AY (SEQ ID NO: 384), (wherein X1 is F or Y, X2 is N or T, and X3 is W b) VL comprises CDR-H3 containing the amino acid sequence of formula (IV):X1SSTGAVTX2X3NYX4N(SEQ ID NO: 385)(wherein X1 is A, G, or R, X2 is S or T, X3 is G or S, and X4 is A, P, or V), CDR-L2 containing the amino acid sequence of formula (V):GTX1X2RAP(SEQ ID NO: 386)(wherein X1 is K or N, and X2 is F or K), and CDR-L3 containing the amino acid sequence of formula (VI):ALWYSX1X2WV(SEQ ID NO: 387)(wherein X1 is D, N, or T, and X2 is L or R).In some embodiments, VH is a heavy chain complementarity determining region (CDR-H) 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 376, 390, 601, and 602, or a variant thereof containing up to about three amino acid substitutions; CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 377, 391-394, and 603, or a variant thereof containing up to about three amino acid substitutions; and CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 378, 395, 604, and 605, or up to about three amino acid substitutions. VL includes variants of the same, which include substitutions; and VL includes CDR-L1, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398 and 606-609, or a variant thereof containing up to approximately three amino acid substitutions; CDR-L2, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, or a variant thereof containing up to approximately three amino acid substitutions; and CDR-L3, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 381, 400-401 and 610, or a variant thereof containing up to approximately three amino acid substitutions. In some embodiments, VH includes CDR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, or a variant thereof containing up to about three amino acid substitutions; CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, or a variant thereof containing up to about three amino acid substitutions; and CDR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395, or a variant thereof containing up to about three amino acid substitutions; and VL includes CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, or a variant thereof containing up to about three amino acid substitutions; CDR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, or a variant thereof containing up to about three amino acid substitutions; and CDR-L3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401, or a variant thereof containing up to about three amino acid substitutions.In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 382, ​​CDR-H2 containing the amino acid sequence of SEQ ID NO: 383, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 384; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 385, CDR-L2 containing the amino acid sequence of SEQ ID NO: 386, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 387. In some embodiments, VH includes CDR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, and CDR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395; and VL includes CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, CDR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, and CDR-L3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 401.In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 401. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 398, CDR-L2 containing the amino acid sequence of SEQ ID NO: 399, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381.In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381.In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 388, and VL includes the amino acid sequence of SEQ ID NO: 389.

[0037] In some embodiments described for any one of the activatable anti-CD3 antibodies described above, VH is formula (VII):EVQLVESGGGLVX1PGGSLRLSCAASGFTFX2X3YAIX4WVRQAPGKGLEWVX5RIRSKYNNYATYYAX6SVKX7RFTISRDX8SKNTLYLQX9NSLRAEDTAVYYCX 10 RHGNX 11 GX 12 SYVSWFAYWGQGTLVTVSS(Sequence ID 388)(wherein X1 is K or Q, X2 is N or S, X3 is S or T, X4 is H or N, X5 is G or S, X6 is D or E, X7 is D or G, X8 is D or N, X9 is I or L, X 10 is A or V, and X 11 is F or Y, and X 12X1 contains an amino acid sequence of formula (VIII): X1AVVTQEPSLTVSPGGTVTLTCX2SSTGAVTTSNYX3NWX4QQKPGQAPRGLIGGTX5X6RAPGX7PARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSX8X9WVFGGGTKLTVL (Sequence ID 389) (wherein X1 is E or Q, X2 is A, G, P, or R, X3 is A or P, X4 is F or V, X5 is K or N, X6 is F or K, X7 is A, I, T, or V, X8 is A, D, N, or T, and X9 is H or L). In some embodiments, VH includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414-416, and 611-640, or a variant having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414-416, and 611-640; and VL includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, 413, and 641-666, or a variant having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, 413, and 641-666. In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414, 415, and 416; and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, and 413.

[0038] In some embodiments described in any one of the above-described activatable anti-CD3 antibodies, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 402, 405, 407, 409, 410, 412, 414, 415, and 416; and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 403, 404, 406, 408, 411, and 413. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 402, and VL comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 402, and VL comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 405, and VL comprises the amino acid sequence of SEQ ID NO: 406. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 407, and VL comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 407, and VL includes the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 407, and VL includes the amino acid sequence of SEQ ID NO: 408. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 409, and VL includes the amino acid sequence of SEQ ID NO: 408. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 410, and VL includes the amino acid sequence of SEQ ID NO: 411. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 412, and VL includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 410, and VL includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 414, and VL includes the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 415, and VL includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 416, and VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 416, and VL comprises the amino acid sequence of SEQ ID NO: 411.In some embodiments, the CD3 binding portion includes the amino acid sequence of SEQ ID NO: 421 or SEQ ID NO: 422.

[0039] In some embodiments described in any one of the above-mentioned activatable anti-CD3 antibodies, CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 127-129, 418, 420, 431, and 477-490, and 516-555. In some embodiments, CM comprises the amino acid sequence of SEQ ID NO: 77 or 418.

[0040] One aspect of this application provides a masked antibody ("masked anti-CD3 antibody") comprising a masking moiety (MM) and a CD3-binding moiety from the N-terminus to the C-terminus, wherein: a) the CD3-binding moiety comprises VL, and the activatable antibody further comprises a second polypeptide comprising VH; b) the CD3-binding moiety comprises VH, and the activatable antibody further comprises a second polypeptide comprising VL; c) the CD3-binding moiety comprises VL and VH from the N-terminus to the C-terminus; or d) the CD3-binding moiety comprises VL and VH from the N-terminus to the C-terminus; MM1 specifically binds to the CD3-binding moiety in competition with CD3; the activatable antibody binds to CD3 via VH and VL; and the masked antibody binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM of the antibody as determined by enzyme-linked immunosorbent assay (ELISA) (e.g., at least 50 nM, or at least 100 nM, or about 110 nM). In some embodiments, the masked anti-CD3 antibody is an activatable antibody. In some embodiments, the masked anti-CD3 antibody includes a masking moiety (MM), a cleavable moiety (CM), and a CD3-binding moiety from the N-terminus to the C-terminus. In some embodiments, the masked anti-CD3 antibody is not an activatable antibody. In some embodiments, the masked anti-CD3 antibody includes a masking moiety (MM), a non-cleavable linker (NCL), and a CD3-binding moiety from the N-terminus to the C-terminus.One aspect of the present application provides a masked antibody ("masked anti-CD3 antibody") comprising a masking portion (MM) and an antibody or antigen-binding fragment that binds to CD3, wherein the antibody or antigen-binding fragment comprises VH and VL; the masked antibody comprises a single-chain polypeptide, where VH and VL of the antibody or antigen-binding fragment are parts of the single-chain polypeptide; or the masked antibody comprises two polypeptide chains, where VH and VL of the antibody or antigen-binding fragment are parts of different polypeptide chains of the masked antibody; wherein the C-terminus of MM is fused to the N-terminus of VH or VL of the antibody or antigen-binding fragment; wherein MM specifically binds to the antibody or antigen-binding fragment in competition with CD3; wherein the antibody or antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM of the antibody as determined by enzyme-linked immunosorbent assay (ELISA) (e.g., at least 50 nM, or at least 100 nM, or about 110 nM). In some embodiments, the masked antibody includes an amino acid linker between the C-terminus of the MM and the N-terminus of the VH or VL of the antibody or antigen-binding fragment. In some embodiments, the masked antibody includes, for example, a further cleavable linker between the C-terminus of the MM and the N-terminus of the VH or VL of the antibody or antigen-binding fragment. In some embodiments, the masked antibody does not include a cleavable linker (for example, one fused to the MM or between the C-terminus of the MM and the N-terminus of the antibody or antigen-binding fragment).

[0041] One aspect of this application provides a masked antibody ("masked anti-CD3 antibody") comprising a masking moiety (MM), a non-cleavable linker (NCL), and a CD3-binding moiety from the N-terminus to the C-terminus, wherein: a) the CD3-binding moiety comprises VL, and the activatable antibody further comprises a second polypeptide comprising VH; b) the CD3-binding moiety comprises VH, and the activatable antibody further comprises a second polypeptide comprising VL; c) the CD3-binding moiety comprises VL and VH from the N-terminus to the C-terminus; or d) the CD3-binding moiety comprises VL and VH from the N-terminus to the C-terminus; MM1 specifically binds to the CD3-binding moiety in competition with CD3; the activatable antibody binds to CD3 via VH and VL; and the masked antibody binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 50 nM, or at least 100 nM, or about 110 nM) of the antibody as determined by enzyme-linked immunosorbent assay (ELISA). In some embodiments, when used to determine EC50, the first antigen-binding fragment is an scFv such as an isolated anti-CD3 scFv, an isolated anti-CD3 scFv-Fc fusion protein, or an anti-CD3 antigen-binding fragment in a multispecific (e.g., bispecific) antibody, or a multispecific antibody in an unmasked form (e.g., without MM). In some embodiments, EC50 is determined using an ELISA assay such as that described in Example 5.

[0042] In some embodiments described in any one of the masked anti-CD3 antibodies above, the first antigen-binding fragment binds to CD3 with a dissociation constant (Kd) of at least 50 nM. In some embodiments, the first antigen-binding fragment is an isolated anti-CD3 scFv, an isolated anti-CD3 scFv-Fc fusion protein, or an scFv such as an anti-CD3 scFv fragment in a multispecific (e.g., bispecific) antibody, or a multispecific antibody in an unmasked form (e.g., without MM).

[0043] In some embodiments described in any one of the masked anti-CD3 antibodies above, MM contains the amino acid sequence EVGSY (SEQ ID NO: 667) at the N-terminus of MM. In some embodiments, MM contains the amino acid sequence of formula (IX):PYDDPDCPSHX1SDCDX2 (SEQ ID NO: 668) (wherein X1 is D or E and X2 is N or Q). In some embodiments, MM contains the amino acid sequence of formula (X):X1X2X3DX4X5CX6X7DX8X9X 10 CX 11 X 12 (Sequence ID 669) (wherein X1 is A or D, X2 is A, D, or P, X3 is D, H, or P, X4 is F or P, X5 is D or P, X6 is D or P, X7 is A or P, X8 is D, N, or P, X9 is A, N, or P, X 10 is D, H or S, X 11 is H, P, or Y, and X 12 The amino acid sequence includes (where is N, P, or Y). In some embodiments, MM includes the amino acid sequence of SEQ ID NO: 35. In some embodiments, MM includes the amino acid sequence of SEQ ID NO: 417. In some embodiments, MM includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 585-588 and 597-591. In some embodiments, CD3 is human CD3.

[0044] One aspect of this application provides a masked antibody ("masked anti-CD3 antibody") comprising a masking moiety (MM) and a CD3-binding moiety from the N-terminus to the C-terminus, wherein: a) the CD3-binding moiety comprises VL and the activatable antibody further comprises a second polypeptide comprising VH; b) the CD3-binding moiety comprises VH and the activatable antibody further comprises a second polypeptide comprising VL; c) the CD3-binding moiety comprises VL and VH from the N-terminus to the C-terminus; or d) the CD3-binding moiety comprises VL and VH from the N-terminus to the C-terminus The molecule contains VL and VH at its ends; MM1 specifically binds to the CD3 binding site in competition with CD3; the activatable antibody binds to CD3 via VH and VL; where a) MM contains the amino acid sequence EVGSY (SEQ ID NO: 667) at its N-terminus; b) MM contains the amino acid sequence of formula (IX): PYDDPDCPSHX1SDCDX2 (SEQ ID NO: 668) (wherein X1 is D or E and X2 is N or Q); or c) MM contains the amino acid sequence of formula (X): X1X2X3DX4X5CX6X7DX8X9X 10 CX 11 X 12 (Sequence ID 669) (wherein X1 is A or D, X2 is A, D, or P, X3 is D, H, or P, X4 is F or P, X5 is D or P, X6 is D or P, X7 is A or P, X8 is D, N, or P, X9 is A, N, or P, X 10 is D, H or S, X 11 is H, P, or Y, and X 12 The amino acid sequence (where is N, P, or Y) is included. In some embodiments, the masked anti-CD3 antibody is an activatable antibody. In some embodiments, the masked anti-CD3 antibody has a masking moiety (MM), a cleavable moiety (CM), and a CD3 binding moiety from the N-terminus to the C-terminus. In some embodiments, the masked anti-CD3 antibody is not an activatable antibody. In some embodiments, the masked anti-CD3 antibody has a masking moiety (MM), a non-cleavable linker (NCL), and a CD3 binding moiety from the N-terminus to the C-terminus.

[0045] One aspect of this application provides a masked antibody ("masked anti-CD3 antibody") comprising a masking moiety (MM), a non-cleavable linker (NCL), and a CD3 binding moiety from the N-terminus to the C-terminus, wherein: a) the CD3 binding moiety comprises VL, and the activatable antibody further comprises a second polypeptide comprising VH; b) the CD3 binding moiety comprises VH, and the activatable antibody further comprises a second polypeptide comprising VL; c) the CD3 binding moiety comprises VL and VH from the N-terminus to the C-terminus; or d) the CD3 binding moiety The fraction contains VL and VH from the N-terminus to the C-terminus; MM1 specifically binds to the CD3 binding site in competition with CD3; the activatable antibody binds to CD3 via VH and VL; a) MM contains the amino acid sequence EVGSY (SEQ ID NO: 667) at the N-terminus of MM; b) MM contains the amino acid sequence of formula (IX): PYDDPDCPSHX1SDCDX2 (SEQ ID NO: 668) (wherein X1 is D or E and X2 is N or Q); or c) MM contains the amino acid sequence of formula (X): X1X2X3DX4X5CX6X7DX8X9X 10 CX 11 X 12 (Sequence ID 669) (wherein X1 is A or D, X2 is A, D, or P, X3 is D, H, or P, X4 is F or P, X5 is D or P, X6 is D or P, X7 is A or P, X8 is D, N, or P, X9 is A, N, or P, X 10 is D, H or S, X 11 is H, P, or Y, and X 12 The amino acid sequence (where is N, P, or Y) is included. In some embodiments, MM includes the amino acid sequences of SEQ ID NOs. 35, 417, 585-588, and 597-599. In some embodiments, CD3 is human CD3.

[0046] In some embodiments described in any one of the masked anti-CD3 antibodies above, the activatable anti-CD3 antibody comprises an anti-CD3 antigen-binding fragment selected from the group consisting of Fab, Fv, scFab, and scFv. In some embodiments, the anti-CD3 antigen-binding fragment is scFv. In some embodiments, scFv comprises a VL, a linker, and a VH from the N-terminus to the C-terminus.

[0047] In some embodiments described in any one of the masked anti-CD3 antibodies described above, VH is a heavy chain complementarity determining region (CDR-H)1 comprising the amino acid sequence of formula (I):X1YAX2X3 (SEQ ID NO: 382), (wherein X1 is D, S, or T, X2 is I, L, or M, and X3 is N or T), CDR-H2 comprising the amino acid sequence of formula (II):RIRSKYNNYATYYAX1X2VKX3 (SEQ ID NO: 383), (wherein X1 is F or Y, X2 is N or T, and X3 is D, G, or S), and formula (III):HGNX1GX2SYVSX3X4AY (SEQ ID NO: 384), (wherein X1 is F or Y, X2 is N or T, and X3 is W b) VL comprises CDR-H3 containing the amino acid sequence of formula (IV):X1SSTGAVTX2X3NYX4N(SEQ ID NO: 385)(wherein X1 is A, G, or R, X2 is S or T, X3 is G or S, and X4 is A, P, or V), CDR-L2 containing the amino acid sequence of formula (V):GTX1X2RAP(SEQ ID NO: 386)(wherein X1 is K or N, and X2 is F or K), and CDR-L3 containing the amino acid sequence of formula (VI):ALWYSX1X2WV(SEQ ID NO: 387)(wherein X1 is D, N, or T, and X2 is L or R).In some embodiments, VH is a heavy chain complementarity determining region (CDR-H) 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 376, 390, 601, and 602, or a variant thereof containing up to about three amino acid substitutions; CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 377, 391-394, and 603, or a variant thereof containing up to about three amino acid substitutions; and CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 378, 395, 604, and 605, or up to about three amino The variants include acid substitutions; and VL includes CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398 and 606-609, or a variant thereof containing up to approximately three amino acid substitutions; CDR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, or a variant thereof containing up to approximately three amino acid substitutions; and CDR-L3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 381, 400-401 and 610, or a variant thereof containing up to approximately three amino acid substitutions. In some embodiments, VH includes CDR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, or a variant thereof containing up to about three amino acid substitutions; CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, or a variant thereof containing up to about three amino acid substitutions; and CDR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395, or a variant thereof containing up to about three amino acid substitutions; and VL includes CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, or a variant thereof containing up to about three amino acid substitutions; CDR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, or a variant thereof containing up to about three amino acid substitutions; and CDR-L3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401, or a variant thereof containing up to about three amino acid substitutions.In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 382, ​​CDR-H2 containing the amino acid sequence of SEQ ID NO: 383, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 384; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 385, CDR-L2 containing the amino acid sequence of SEQ ID NO: 386, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 387. In some embodiments, VH includes CDR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, and CDR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395; and VL includes CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, CDR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, and CDR-L3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 401.In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 401. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 398, CDR-L2 containing the amino acid sequence of SEQ ID NO: 399, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381.In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381.In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; and VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 388, and VL includes the amino acid sequence of SEQ ID NO: 389.

[0048] In some embodiments described in any one of the masked anti-CD3 antibodies above, VH is formula (VII): EVQLVESGGGLVX1PGGSLRLSCAASGFTFX2X3YAIX4WVRQAPGKGLEWVX5RIRSKYNNYATYYAX6SVKX7RFTISRDX8SKNTLYLQX9NSLRAEDTAVYYCX 10 RHGNX 11 GX 12 SYVSWFAYWGQGTLVTVSS(Sequence ID 388)(wherein X1 is K or Q, X2 is N or S, X3 is S or T, X4 is H or N, X5 is G or S, X6 is D or E, X7 is D or G, X8 is D or N, X9 is I or L, X 10 is A or V, and X 11 is F or Y, and X 12The amino acid sequence (where is N or T) is included; and VL is formula (VIII): X1AVVTQEPSLTVSPGGTVTLTCX2SSTGAVTTSNYX3NWX4QQKPGQAPRGLIGGTX5X6RAPGX7PARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSX8X9WVFGGGTKLTVL (Sequence ID 389) (wherein X1 is E or Q, X2 is A, G, P, or R, X3 is A or P, X4 is F or V, X5 is K or N, X6 is F or K, X7 is A, I, T, or V, X8 is A, D, N, or T, and X9 is H or L) contains the amino acid sequence. In some embodiments, VH includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414-416, and 611-640, or a variant having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414-416, and 611-640; and VL includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, 413, and 641-666, or a variant having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, 413, and 641-666. In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414, 415, and 416; and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, and 413.

[0049] In some embodiments described in any one of the masked anti-CD3 antibodies described above, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 402, 405, 407, 409, 410, 412, 414, 415, and 416; and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 403, 404, 406, 408, 411, and 413. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 402, and VL comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 402, and VL comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 405, and VL comprises the amino acid sequence of SEQ ID NO: 406. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 407, and VL comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 407, and VL includes the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 407, and VL includes the amino acid sequence of SEQ ID NO: 408. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 409, and VL includes the amino acid sequence of SEQ ID NO: 408. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 410, and VL includes the amino acid sequence of SEQ ID NO: 411. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 412, and VL includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 410, and VL includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 414, and VL includes the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH includes the amino acid sequence of SEQ ID NO: 415, and VL includes the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 416, and VL comprises the amino acid sequence of SEQ ID NO: 413.In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 416, and VL comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, the CD3 binding portion comprises the amino acid sequence of SEQ ID NO: 421 or SEQ ID NO: 422.

[0050] One aspect of this application provides an activatable antibody ("activatable anti-HER2 antibody") comprising a masking moiety (MM), a cleavable moiety (CM), and a HER2-binding moiety from the N-terminus to the C-terminus, wherein: a) the HER2-binding moiety comprises VL and the activatable antibody comprises a second polypeptide comprising VH; b) the HER2-binding moiety comprises VH and the activatable antibody comprises a second polypeptide comprising VL; c) the HER2-binding moiety comprises VL and VH from the N-terminus to the C-terminus; or d) the HER2-binding moiety comprises VH and VL from the N-terminus to the C-terminus; and wherein C M contains the cleavage site; MM inhibits the binding of the activatable antibody to HER2 if CM is not cleaved; if CM is cleaved, the activatable antibody binds to HER2 via VH and VL, where MM is: a) amino acid sequence of formula (XI): ESX1X2CX3X4DPFX5CQX6 (SEQ ID NO: 670) (wherein X1 is D or E, X2 is A, F, V, or Y, X3 is D or E, X4 is A or L, X5 is D or E, and X6 is A, F, or Y); b) formula (XII): X1X2X3X4X5X6CX7X8DPYECX9X 10 (Sequence number 671) (wherein X1 is A, H, or S, X2 is A, D, or S, X3 is A, T, or V, X4 is P, S, or T, X5 is D or E, X6 is A or V, X7 is D or E, X8 is A or L, X9 is Q, S, or T, X 10The amino acid sequence is (wherein X1 is A, H, or V); or c) the amino acid sequence of formula (XIII):YNSDDDCX1SX2YDPYTCYY(SEQ ID NO: 672) (wherein X1 is A, I, or V, and X2 is H or R). In some embodiments, MM includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515. In some embodiments, CM includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 127-129, 418, 420, 431, and 477-490, and 516-555.

[0051] In some embodiments of any one of the activatable anti-HER2 antibodies described above, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 423, CDR-H2 containing the amino acid sequence of SEQ ID NO: 424, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 71, and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 75, and VL comprises the amino acid sequence of SEQ ID NO: 76.

[0052] One aspect of this application provides a masked antibody ("masked anti-HER2 antibody") comprising a masking moiety (MM) and a HER2-binding moiety from the N-terminus to the C-terminus, wherein: a) the HER2-binding moiety comprises VL and the activatable antibody comprises a second polypeptide comprising VH; b) the HER2-binding moiety comprises VH and the activatable antibody comprises a second polypeptide comprising VL; c) the HER2-binding moiety comprises VL and VH from the N-terminus to the C-terminus; or d) the HER2-binding moiety comprises VH and The antibody contains VH and VL; MM specifically binds to the HER2 binding site in competition with HER2; the activatable antibody binds to HER2 via VH and VL, where MM is: a) amino acid sequence of formula (XI): ESX1X2CX3X4DPFX5CQX6 (SEQ ID NO: 670) (wherein X1 is D or E, X2 is A, F, V, or Y, X3 is D or E, X4 is A or L, X5 is D or E, and X6 is A, F, or Y); b) formula (XII): X1X2X3X4X5X6CX7X8DPYECX9X 10 (Sequence code 671) (wherein X1 is A, H, or S, X2 is A, D, or S, X3 is A, T, or V, X4 is P, S, or T, X5 is D or E, X6 is A or V, X7 is D or E, X8 is A or L, X9 is Q, S, or T, X 10 (where is A, H, or V); or c) an amino acid sequence of formula (XIII)YNSDDDCX1SX2YDPYTCYY(SEQ ID NO: 672) (wherein X1 is A, I, or V, and X2 is H or R). In some embodiments, the masked anti-HER2 antibody is an activatable antibody. In some embodiments, the masked anti-HER2 antibody comprises a masking moiety (MM), a cleavable moiety (CM), and a HER2-binding moiety from the N-terminus to the C-terminus. In some embodiments, the masked anti-HER2 antibody is not an activatable antibody. In some embodiments, the masked anti-HER2 antibody comprises a masking moiety (MM), a non-cleavable linker (NCL), and a HER2-binding moiety from the N-terminus to the C-terminus. One aspect of this application provides a masked antibody ("masked anti-HER2 antibody") comprising a masking moiety (MM) and an antibody or antigen-binding fragment that binds to HER2, wherein the antibody or antigen-binding fragment comprises VH and VL; the masked antibody comprises a single-chain polypeptide, where VH and VL of the antibody or antigen-binding fragment are parts of the single-chain polypeptide; or the masked antibody comprises two polypeptide chains, where VH and VL of the antibody or antigen-binding fragment are parts of different polypeptide chains of the masked antibody; here The C-terminus of MM is fused to the N-terminus of the VH or VL of the antibody or antigen-binding fragment; here MM specifically binds to the antibody or antigen-binding fragment in competition with HER2; MM is: a) amino acid sequence of formula (XI): ESX1X2CX3X4DPFX5CQX6 (SEQ ID NO: 670) (wherein X1 is D or E, X2 is A, F, V, or Y, X3 is D or E, X4 is A or L, X5 is D or E, and X6 is A, F, or Y); b) formula (XII): X1X2X3X4X5X6CX7X8DPYECX9X 10 (Sequence number 671) (wherein X1 is A, H, or S, X2 is A, D, or S, X3 is A, T, or V, X4 is P, S, or T, X5 is D or E, X6 is A or V, X7 is D or E, X8 is A or L, X9 is Q, S, or T, X 10The amino acid sequence is (where is A, H, or V); or c) the amino acid sequence of formula (XIII)YNSDDDCX1SX2YDPYTCYY(SEQ ID NO: 672) (wherein X1 is A, I, or V, and X2 is H or R). In some embodiments, the masked antibody includes an amino acid linker between the C-terminus of MM and the N-terminus of VH or VL of the antibody or antigen-binding fragment. In some embodiments, the masked antibody includes a further cleavable linker, for example, between the C-terminus of MM and the N-terminus of VH or VL of the antibody or antigen-binding fragment. In some embodiments, the masked antibody does not include a cleavable linker (for example, fused to MM, or between the C-terminus of MM and the N-terminus of the antibody or antigen-binding fragment).

[0053] One aspect of this application provides a masked antibody ("masked anti-HER2 antibody") comprising a masking moiety (MM), a non-cleavable linker (NCL), and a HER2-binding moiety from the N-terminus to the C-terminus, wherein: a) the HER2-binding moiety comprises VL and the activatable antibody comprises a second polypeptide comprising VH; b) the HER2-binding moiety comprises VH and the activatable antibody comprises a second polypeptide comprising VL; c) the HER2-binding moiety comprises VL and VH from the N-terminus to the C-terminus; or d) the HER2-binding moiety comprises C from the N-terminus to the C-terminus Towards the end, it contains VH and VL; MM specifically binds to the HER2 binding site in competition with HER2; the activatable antibody binds to HER2 via VH and VL, where MM is: a) amino acid sequence of formula (XI): ESX1X2CX3X4DPFX5CQX6 (SEQ ID NO: 670) (wherein X1 is D or E, X2 is A, F, V or Y, X3 is D or E, X4 is A or L, X5 is D or E, and X6 is A, F or Y); b) formula (XII): X1X2X3X4X5X6CX7X8DPYECX9X 10(Sequence number 671) (wherein X1 is A, H, or S, X2 is A, D, or S, X3 is A, T, or V, X4 is P, S, or T, X5 is D or E, X6 is A or V, X7 is D or E, X8 is A or L, X9 is Q, S, or T, X 10 (where is A, H, or V); or c) an amino acid sequence of formula (XIII)YNSDDDCX1SX2YDPYTCYY(SEQ ID NO: 672) (wherein X1 is A, I, or V, and X2 is H or R). In some embodiments, MM includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515.

[0054] In some embodiments described in any one of the masked anti-HER2 antibodies described above, VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 423, CDR-H2 containing the amino acid sequence of SEQ ID NO: 424, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 71, and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 75, and VL comprises the amino acid sequence of SEQ ID NO: 76.

[0055] In another embodiment, the Disclosure provides an anti-HER2 antibody comprising six CDR and / or VH and VL sequences of any anti-HER2 binding domain provided herein. In some embodiments, the anti-HER2 antibody comprises VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 423, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 424, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71, and VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 75, and VL comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the anti-HER2 antibody comprises VH, which includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 69, CDR-H2 containing the amino acid sequence of SEQ ID NO: 70, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 71, and VL, which includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 74.

[0056] In another embodiment, the Disclosure provides an anti-CD20 antibody comprising six CDR and / or VH and VL sequences of any anti-CD20 binding domain provided herein. In some embodiments, the anti-CD20 antibody comprises VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 556, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 557, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 558, and VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 559, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 560, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 561. In some embodiments, the anti-CD20 antibody comprises VH, which includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 86, CDR-H2 containing the amino acid sequence of SEQ ID NO: 557, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 558; and VL, which includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 559, CDR-L2 containing the amino acid sequence of SEQ ID NO: 560, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 561. In some embodiments, VH contains the amino acid sequence of SEQ ID NO: 562, and VL contains the amino acid sequence of SEQ ID NO: 563.

[0057] One aspect of this application provides one or more isolated nucleic acids encoding any one of the above-mentioned antibodies, multispecific antibodies, masked antibodies, activatable multispecific antibodies, isolated anti-CD3 antibodies or their antigen-binding fragments, masked anti-CD3 antibodies, activatable anti-CD3 antibodies, masked anti-HER2 antibodies, or activatable anti-HER2 antibodies. In some embodiments, a vector comprising one or more nucleic acids comprising any one of the above-mentioned nucleic acids is provided. In some embodiments, a host cell comprising one or more nucleic acids comprising any one of the above-mentioned nucleic acids or any one of the above-mentioned vectors is provided. In some embodiments, methods are provided for preparing masked antibodies, multispecific antibodies, activatable multispecific antibodies, isolated anti-CD3 antibodies or their antigen-binding fragments, masked anti-CD3 antibodies, activatable anti-CD3 antibodies, masked anti-HER2 antibodies, or activatable anti-HER2 antibodies, the methods comprising: a) culturing any host cells under conditions that allow expression of one or more nucleic acids or vectors; and b) recovering multispecific antibodies, activatable multispecific antibodies, anti-CD3 antibodies or their antigen-binding fragments, masked anti-CD3 antibodies, activatable anti-CD3 antibodies, masked anti-HER2 antibodies, or activatable antibodies from the host cell culture.

[0058] Also provided are pharmaceutical compositions comprising the aforementioned antibodies, multispecific antibodies, masked antibodies, activatable multispecific antibodies, isolated anti-CD3 antibodies or their antigen-binding fragments, masked anti-CD3 antibodies, activatable anti-CD3 antibodies, masked anti-HER2 antibodies, or activatable anti-HER2 antibodies, and a pharmaceutically acceptable carrier.

[0059] Another aspect of this application provides a method for treating a disease or condition in a subject requiring treatment of the disease or condition, the method comprising administering an effective amount of one of the above-described pharmaceutical compositions to the subject. In some embodiments, the pharmaceutical composition comprises an activatable multispecific antibody, where CM1 and CM2 are cleaved at the disease site, thereby preventing the binding of the multispecific activatable antibody to CD3 and the target antigen at the disease site. In some embodiments, the disease or condition is a cancer, such as a liquid cancer or a solid cancer. In some embodiments, the target antigen is HER2, and the cancer is selected from the group consisting of breast cancer, ovarian cancer, and lung cancer. In some embodiments, the target antigen is CD20, and the cancer is lymphoma or leukemia. In some embodiments, the target antigen is TROP2, and the cancer is breast cancer or lymphoma. In some embodiments, the pharmaceutical composition is administered so that a multispecific antibody, an isolated antibody or its antigen-binding fragment, or a masked antibody is provided to the target in doses of 0.02 mg / kg, 0.2 mg / kg, 2 mg / kg, 10 mg / kg, 30 mg / kg, or 60 mg / kg. In some embodiments, a multispecific antibody, an isolated antibody or its antigen-binding fragment, or a masked antibody includes: a first polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 427, a second polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 428, and a third polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 112; a first polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 83, a second polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84, and a third polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 85; a first polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 683, a second polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 684, and a third polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 685;A first polypeptide containing the amino acid sequence of SEQ ID NO: 427, a second polypeptide containing the amino acid sequence of SEQ ID NO: 428, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 112; a first polypeptide containing the amino acid sequence of SEQ ID NO: 83, a second polypeptide containing the amino acid sequence of SEQ ID NO: 84, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 85; a first polypeptide containing the amino acid sequence of SEQ ID NO: 683, a second polypeptide containing the amino acid sequence of SEQ ID NO: 684, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 685; a first polypeptide containing the amino acid sequence of SEQ ID NO: 427 without C-terminal lysine A first polypeptide comprising the amino acid sequence of SEQ ID NO: 428 without C-terminal lysine, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 112; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 83, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 84 without C-terminal lysine, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 85 without C-terminal lysine; or a first polypeptide comprising the amino acid sequence of SEQ ID NO: 683, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 684 without C-terminal lysine, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 685 without C-terminal lysine. In some embodiments, the method further comprises administering an anti-PD-1 or anti-PD-L1 antibody to the subject. In some embodiments, the method further comprises administering a CD137 agonist or antibody to the subject. In some embodiments, the CD137 agonist or antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR-H1 containing the amino acid sequence TGGVGVG (SEQ ID NO: 700), CDR-H2 containing the amino acid sequence LIDWADDKYYSPSLKS (SEQ ID NO: 701), and CDR-H3 containing the amino acid sequence GGSDTVIGDWFAY (SEQ ID NO: 702);The light chain variable region includes CDR-L1 containing the amino acid sequence of RASQSIGSYLA (SEQ ID NO: 703), CDR-L2 containing the amino acid sequence of DASNLET (SEQ ID NO: 704), and CDR-L3 containing the amino acid sequence of QQGYYLWT (SEQ ID NO: 705). In some embodiments, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 706, and / or the light chain variable region includes the amino acid sequence of SEQ ID NO: 7076. In some embodiments, the heavy chain includes the amino acid sequence of SEQ ID NO: 710, and / or the light chain includes the amino acid sequence of SEQ ID NO: 711.

[0060] Compositions, kits, and products are also provided that include one of the above-mentioned multispecific antibodies, masked antibodies, activatable multispecific antibodies, isolated anti-CD3 antibodies or their antigen-binding fragments, masked anti-CD3 antibodies, activatable anti-CD3 antibodies, masked anti-HER2 antibodies, or activatable anti-HER2 antibodies. [Brief explanation of the drawing]

[0061] [Figure 1] This invention provides a schematic diagram of an exemplary antibody design. The antibody can be converted into an activatable antibody by fusing one or more antigen-binding sites to a masking peptide. A schematic diagram of a Fab-Fc / Fc 1-arm scaffold is shown. [Figure 2] This invention provides a schematic diagram of an exemplary antibody design. The antibody can be converted into an activatable antibody by fusing one or more antigen-binding sites to a masking peptide. A schematic diagram of a typical light chain scaffold is shown. A bispecific antibody has a first antibody heavy chain, a second antibody heavy chain, and two copies of a common light chain. The first antibody heavy chain and the first common light chain form a first antigen-binding site, and the second antibody heavy chain and the second common light chain form a second antigen-binding site. The first and second antigen-binding sites can bind to different targets. [Figure 3]This invention provides a schematic diagram of an exemplary antibody design. The antibody can be converted into an activatable antibody by fusing one or more antigen-binding sites to a masking peptide. A schematic diagram of a Morrison-format bispecific scaffold is shown. The antibody has a first heavy chain fused to a first scFv, a second heavy chain fused to a second scFv, and two copies of a common light chain. The first antibody heavy chain and the first common light chain form a first antigen-binding site, and the second antibody heavy chain and the second common light chain form a second antigen-binding site. The first and second antigen-binding sites can bind to the same target or to different targets. The first and second scFvs may bind to the same target or to different targets. [Figure 4] This invention provides a schematic diagram of an exemplary antibody design. The antibody can be converted into an activatable antibody by fusing one or more antigen-binding sites to a masking peptide. A schematic diagram of an ScFv bispecific scaffold is shown. For example, in this format, the HER2xCD3 bispecific antibody has the Fab arm on the left bound to HER2 and the scFv arm on the right bound to CD3. [Figure 5] This application provides schematic diagrams of exemplary antibody designs. Antibodies can be converted into activatable antibodies by fusing one or more antigen-binding sites to masking peptides. Figures 5A-B show schematic diagrams of activatable scaffolds. For example, the activatable antibody may be an activatable antibody targeting HER2 and CD3 (HER2xCD3 activatable antibody or SAFE body) or an activatable antibody targeting CD20 and CD3 (CD20xCD3 activatable antibody or SAFE body). Masking peptides (represented as balls) can be fused to antigen-binding fragments via cleavable linkers. [Figure 6]This provides characterization of bispecific antibodies by SDS-PAGE electrophoresis. The gel on the left is a 12% SDS-PAGE gel under reducing conditions, and the gel on the right is a 4-15% SDS-PAGE gel under non-reducing conditions. The MW lanes show molecular weight markers, which are labeled in kilodaltons on the left side of each gel. In both gels, lane 1 shows antibody TY24051, lane 2 shows antibody TY24052, and lane 3 shows antibody TY24053. [Figure 7] This provides size-exclusion high-performance liquid chromatography (HCM) analysis of bispecific antibodies. The top plot shows the TY24051 antibody, the middle plot shows the TY24105 antibody, and the bottom plot shows the TY24106 antibody. In each plot, time is shown on the x-axis and relative protein abundance on the y-axis. Peaks corresponding to heterodimer proteins (main peak), homodimer proteins, and aggregates are shown. [Figure 8] This document provides enzyme-linked immunosorbent assay (ELISA) analysis of antibodies TY24051 and TY24052. A shows the binding of HER2 by TY24051 (square), TY24052 (upward-pointing triangle), and activated TY24052 (downward-pointing triangle). B shows the binding of CD3 by TY24051 (square), TY24052 (upward-pointing triangle), and activated TY24052 (downward-pointing triangle). In A and B, the antibody concentration is shown on the x-axis in units of M, and the absorbance at 450 nm is shown on the y-axis. [Figure 9] This shows an assay for T cell-mediated cytotoxic killing when treated with a bispecific antibody. Antibody concentration (ng / ml) is shown on the x-axis, and the percentage of cell lysis is shown on the y-axis. Target cells were incubated with T cells for 24 hours under the conditions of TY24051 (circle), TY24052 (square), isotype control (upward triangle), or no antibody (downward triangle). [Figure 10]This graph shows the activation of activated T cell nuclear factor (NFAT) response element reporters in Jurkat cells in response to treatment with the bispecific antibodies TY24051 (black circles), TY24111 (squares), TY24052 (white circles), and TY24110 (triangles). The x-axis shows the antibody concentration, logarithmically transformed in μg / ml, and the y-axis shows the relative luminescence units (RLU) of the reporter. In A, NFAT reporter activity was measured in the absence of target cells (SK-OV-3). In B, NFAT reporter activity was measured in the presence of target cells. [Figure 11] This graph shows the secretion levels of IFNγ in humanized peripheral blood mononuclear cell (PBMC) model mice (huPBMC-NSG) after administration of either the parent antibody (TAC2245) or the activatable anti-CD3 antibody (TY23104). The X-axis indicates the antibody identity and sampling time. From left to right, the graph includes a blank, TAC2245 sampled 0 hours after treatment, TAC2245 sampled 3 hours after treatment, TAC2245 sampled 24 hours after treatment, TY23104 sampled 0 hours after treatment, TY23104 sampled 3 hours after treatment, and TY23104 sampled 24 hours after treatment. The Y-axis shows the IFNγ concentration in picograms / ml. [Figure 12] This graph shows the secretion of IFNγ after administration of the parental antibody (TAC2245) or activatable cross-reactive anti-CD3 antibodies (TY23115 and TY23118) in the huPBMC-NSG mouse model. The X-axis shows the antibody attributes and sampling time. From left to right: blank, TAC2245 sampled 0 hours after treatment, TAC2245 sampled 3 hours after treatment, TAC2245 sampled 24 hours after treatment, TY23115 sampled 0 hours after treatment, TY23115 sampled 3 hours after treatment, TY23115 sampled 24 hours after treatment, TY23118 sampled 0 hours after treatment, TY23118 sampled 3 hours after treatment, and TY23118 sampled 24 hours after treatment. The Y-axis shows the IFNγ concentration in picograms / ml. [Figure 13]This graph shows the binding levels of the parental anti-CD3 antibody TAC2245 (circles) and the activatable anti-CD3 antibody TY23104 (squares) to Jurkat cells. The x-axis shows the concentration of the anti-CD3 antibody, logarithmically transformed in nM, and the y-axis shows the mean fluorescence intensity (MFI) of binding to the secondary anti-human IgG antibody. [Figure 14] This shows the activation of NFAT response element reporters in Jurkat cells in response to treatment with parental antibody (TAC2225, circle) or activatable cross-reactive anti-CD3 antibodies (TY23115, square; and TY23118, triangle). The x-axis shows the logarithmically transformed antibody concentration in nM, and the y-axis shows the relative luminescence units (RLU) of the reporter. [Figure 15] This shows the activation of NFAT response element reporters in Jurkat cells in response to treatment with the parent antibody (TAC2245, circle) or activatable anti-CD3 antibodies (TY23100, black square, TY23101, triangle, TY23102, triangle, and TY23104, white square). The x-axis shows the antibody concentration, logarithmically transformed in μg / mL, and the y-axis shows the relative luminescence units (RLU) of the reporter. The assay was performed without FcRIIb crosslinking. [Figure 16] The results of the analysis of the masking efficiency of parental CD3 antibodies and activatable anti-CD3 antibodies are shown. A shows the binding of parental CD3 antibody (TAC2225, black circle) and activatable anti-CD3 antibodies (TY23110, square; TY23115, upward triangle; and TY23118, downward triangle), determined by ELISA, to recombinant human CD3δε. B shows the activation of NFAT response element reporters in Jurkat cells in response to treatment with parental CD3 antibody (TAC2225, black circle) and activatable anti-CD3 antibodies (TY23105, white circle; TY23110, square; TY23115, upward triangle; and TY23118, downward triangle). The x-axis shows the antibody concentration, logarithmically transformed in μg / mL, and the y-axis shows the relative luminescence units (RLU) of the reporter. [Figure 17]Figure 17 shows the activation of the NFAT response element reporter in Jurkat cells in response to treatment with the parent antibody (TAC2225, white circle) or an activatable anti-CD3 antibody. In each graph in Figure 17, the x-axis shows the antibody concentration, logarithmically transformed in μg / mL, and the y-axis shows the relative light units (RLU) of the reporter. The identity of the activatable anti-CD3 antibody is indicated by the shape of the data point, as shown in each legend. Assays performed without FcRIIb crosslinking are shown. [Figure 18] Figure 18 shows the levels of Jurkat cell binding by the parent antibody TAC2245 (TAC2225, circled) and the activatable anti-CD3 antibody. In each graph in Figure 18, the x-axis shows the concentration of the anti-CD3 antibody, logarithmically transformed in nM, the y-axis shows the mean fluorescence intensity (MFI) of the binding of the secondary anti-human IgG antibody, and the identity of the activatable anti-CD3 antibody is indicated by the shape of the data points, as shown in each legend. [Figure 19] This graph shows the binding of parental and activatable anti-CD3 antibodies to recombinant human CD3δε, as determined by ELISA. The x-axis shows the antibody concentration, logarithmically transformed by M, and the y-axis shows the absorbance at a wavelength of 450 nm. The identity of the anti-CD3 antibody is indicated by the shape of the data points, as shown in each legend. [Figure 20A] This section shows the results of the analysis of the masking efficiency of the parent antibody and the activatable SP34 variant anti-CD3 / HER2 bispecific antibody. The binding of the parent antibody (TY25023, black circle) and activatable antibody (TY25026, white circle) with low anti-CD3 affinity, as determined by ELISA, to recombinant human CD3δε, as well as the comparative parent antibody (TY24051, black square) and activatable antibody (TY24052, white square). The x-axis shows the antibody concentration, logarithmically transformed by M, and the y-axis shows the absorbance at a wavelength of 450 nm. [Figure 20B] This shows the results of the analysis of the masking efficiency of the parent antibody and the activatable SP34 variant anti-CD3 / HER2 bispecific antibody. The binding levels of the anti-CD3 antibodies TY24051 (black circles), TY24052 (white circles), and TY25023 (black squares) to Jurkat cells are shown. The x-axis represents the logarithmically transformed antibody concentration in nM, and the y-axis represents the mean fluorescence intensity (MFI) of binding to the secondary anti-human IgG antibody. [Figure 21A] This figure shows the results of the analysis of the masking efficiency and function of the parent antibody and the activatable SP34 variant anti-CD3 / HER2 bispecific antibody. It shows the activation of the NFAT response element reporter in Jurkat cells in response to treatment with the bispecific antibodies TY24051 (black circles), TY24052 (white circles), TY25023 (black squares), and TY25026 (white squares). The x-axis shows the antibody concentration, logarithmically transformed in μg / ml, and the y-axis shows the relative luminescence units (RLU) of the reporter. In Figure 21A, NFAT reporter activity was measured in the presence of target cells (SK-OV-3). [Figure 21B] This section presents the results of analyses of the masking efficiency and function of the parent antibody and the activatable SP34 variant anti-CD3 / HER2 bispecific antibody. It shows the lysis levels of SK-OV3 tumor cells in response to treatment with the bispecific antibodies TY24051 (dark gray circles), TY24052 (dark gray squares), TY25023 (light gray triangles), TY25026 (light gray squares), and the reference CD3x isotype control (dark gray triangles). The x-axis shows the antibody concentration, logarithmically transformed in ng / mL, and the y-axis shows the cytotoxicity %. The table below the plot shows the EC50 cytotoxicity of each antibody in ng / mL. [Figure 21C] This section presents the results of analyses of the masking efficiency and function of the parent antibody and the activatable SP34 variant anti-CD3 / HER2 bispecific antibody. The levels of secreted IFNγ in the activated CD8+ T cell assay are shown in response to treatment with the bispecific antibodies TY24051 (dark gray square), TY24052 (dark gray circle), TY25023 (light gray square), and TY25026 (light gray circle). The x-axis shows the antibody concentration, logarithmically transformed in nM, and the y-axis shows the IFNγ concentration in picograms / mL. [Figure 22A]This figure shows cytokine release in cynomolgus monkeys treated with parental antibodies or activatable bispecific antibodies. The levels of cytokines IFNγ, IL-2, IL-6, TNFα, IL-5, and IL-4 released in response to treatment with bispecific antibodies TY24051 (dark gray squares), TY24052 (dark gray circles), TY25023 (light gray squares), and TY25026 (light gray circles) are shown. The X-axis represents time after administration in hours, and the Y-axis represents cytokine concentration in picograms / mL. Arrows above each plot indicate the time points at which 0.2, 0.5, and 0.9 mg / kg ("mpk") of antibodies were administered. IL-6 release levels are also shown in Figure 24F on a logarithmically transformed y-axis. [Figure 22B] This figure shows cytokine release in cynomolgus monkeys treated with parental antibodies or activatable bispecific antibodies. The levels of cytokines released in response to treatment with bispecific antibodies TY24051, TY24052, TY25023, and TY25026 are shown. The X-axis represents time after administration in hours, and the Y-axis represents the logarithmically converted cytokine concentration in picograms / mL. Arrows above each plot indicate the time points at which antibodies at doses of 0.2, 0.5, and 0.9 mg / kg were administered. IL-6 release levels are also shown in Figure 24F, on the logarithmically converted y-axis. [Figure 23] The graphs show the activation levels of CD4+ (left plot) and CD8+ (right plot) T cells in response to treatment with the bispecific antibodies TY24051 (dark gray square), TY24052 (dark gray circle), TY25023 (light gray square), and TY25026 (light gray circle). The x-axis represents time after administration in hours, and the y-axis represents the number of cells per μL. Arrows above each plot indicate the time points at which antibodies were administered at doses of 0.2, 0.5, and 0.9 mg / kg ("mpk"). [Figure 24A]The results of studies on cynomolgus monkeys treated with parental antibodies or activatable bispecific antibodies are shown. The T cell levels per μL for total T cells (top), CD4+ T cells (bottom left), and CD8+ T cells (bottom right) of monkeys in response to treatment with the bispecific antibodies TY24051 (dark gray square), TY24052 (dark gray circle), TY25023 (light gray square), and TY25026 (light gray circle) are shown. The x-axis represents time after administration in hours, and the y-axis represents cell count per μL. Arrows above each plot indicate the time points at which antibodies were administered at doses of 0.2, 0.5, and 0.9 mg / kg ("mpk"). [Figure 24B] The results of studies on cynomolgus monkeys treated with parental antibodies or activatable bispecific antibodies are shown. The levels of B cells (left) and NK cells (right) per μL in monkeys in response to treatment with bispecific antibodies TY24051 (circles), TY24052 (squares), TY25023 (upward triangles), and TY25026 (downward triangles) are shown. The x-axis represents time after administration in hours, and the y-axis represents cell count per μL. Arrows above each plot indicate the time points at which antibodies were administered at doses of 0.2, 0.5, and 0.9 mg / kg ("mpk"). [Figure 24C] The results of studies on cynomolgus monkeys treated with parental antibodies or activatable bispecific antibodies are shown. The concentrations of the bispecific antibodies TY24051 (circles), TY24052 (squares), TY25023 (upward triangles), and TY25026 (downward triangles) in cynomolgus monkeys are shown. The X-axis represents time after administration in hours, and the Y-axis represents logarithmically transformed antibody concentration in μg / mL. Arrows above each plot indicate the time points at which antibodies were administered at doses of 0.2, 0.5, and 0.9 mg / kg ("mpk"). [Figure 24D] The results of studies on cynomolgus monkeys treated with parental antibodies or activatable bispecific antibodies are presented. Plasma concentrations and pharmacokinetic parameters of monkeys treated with bispecific antibodies are shown. [Figure 24E] The results of studies on cynomolgus monkeys treated with parental antibodies or activatable bispecific antibodies are shown. IL-6 release levels in monkeys treated with bispecific antibodies are indicated. Parental bispecific antibodies are shown as squares, and activatable bispecific antibodies are shown as circles. [Figure 24F] The results of a study on cynomolgus monkeys treated with parental antibodies or activatable bispecific antibodies are shown. The absolute lymphocyte count of monkeys administered with bispecific antibodies is shown. Parental bispecific antibodies are indicated by squares, and activatable bispecific antibodies are indicated by circles. [Figure 25] This paper provides flow cytometry analysis of the surface display of anti-HER2 antibodies on yeast cells. In the scatter plots of Figures 25A and 25B, the x-axis represents the level of Fab or scFv displayed on yeast cells (detected by antibody binding to affinity tags fused to anti-HER2 antibodies), and the y-axis represents the level of HER2 binding (detected by binding of PE-conjugated streptavidin to biotinylated human HER2-Fc). A shows the binding of Fabs to HER2. B shows the binding of scFv to HER2. [Figure 26] The results of four rounds (R1, R2, R3, R4) of FACS screening a CPL yeast library for masking peptides that mask binding to 10 nM biotinylated HER2-Fc are shown. In each scatter plot in Figure 26, the x-axis represents the level of myc-tagged anti-HER2 antibody, and the y-axis represents the level of HER2 binding. [Figure 27] FACS analysis of binding with selected trastuzumab-derived activatable anti-HER2 antibodies is shown. In each scatter plot of Figures 27A and 27B, samples were treated with buffered PBSA (left) or TEV protease (right). The x-axis shows the level of Fab or scFv displayed on yeast cells (detected by antibody binding to affinity tags fused to anti-HER2 antibodies), and the y-axis shows the level of HER2 binding (detected by binding of PE-conjugated streptavidin to biotinylated human HER2-Fc). In A, the anti-HER2 antibody (B14126) is in scFv format. In B, the anti-HER2 antibody (B14132) is in Fab format. [Figure 28] The binding of the parent antibody (trastuzumab) to His-tagged HER2 with activatable anti-HER2 antibodies (TY22841, TY22842, TY22839, TY22838, and TY22837) is shown as a measure of the masking efficiency of the activatable antibodies. The X axis represents time (seconds), and the Y axis represents the binding level. [Figure 29] The binding of parental antibodies (trastuzumab, black circles) and activatable anti-HER2 antibodies to recombinant HER2-Fc, as determined by ELISA, is shown. The x-axis shows the antibody concentration, logarithmically transformed by M, and the y-axis shows the absorbance at a wavelength of 450 nm. A shows the results for TY22836, TY2237, TY2238, TY2239, TY2240, TY2241, TY2242, TY2243, and trastuzumab. B shows the results for TY22846, TY2247, TY2250, TY2251, TY2252, TY2253, TY2254, and trastuzumab. C shows the results for TY23523, TY23525, TY23526, TY23533, TY23536, TY23537, and trastuzumab. [Figure 30] This provides a reduced SDS-PAGE showing TY22837 alone (lane 1) or in the presence of the protease MMP-9 (lane 2). [Figure 31] This graph shows the binding of the parental anti-HER2 antibody (trastuzumab, black circles) and TY22837 to recombinant HER2-Fc, as determined by ELISA. TY22837 binding is shown for TY22837 alone (downward triangle) or in the presence of the protease MMP-9 (upward triangle). The x-axis shows the antibody concentration, logarithmically transformed by M, and the y-axis shows the absorbance at a wavelength of 450 nm. [Figure 32] This chart shows the binding levels of parental anti-HER2 antibodies (trastuzumab, black circles) and activated anti-HER2 antibodies (TY22837, white circles; TY23536, squares) to SK-OV-3 cells. The x-axis shows the antibody concentration, logarithmically transformed in nM, and the y-axis shows the mean fluorescence intensity (MFI) of the binding of the secondary anti-human IgG antibody. [Figure 33A] The results of three stress tests for the activatable anti-HER2 antibodies TY22837 (left column) and TY22838 (right column) are shown. The results are after 3 to 6 freeze-thaw cycles of the activatable antibodies. The X-axis represents time per minute, and the Y-axis represents the antibody aggregation level in absorbance units at 214 nm. [Figure 33B]The results of three stress tests for the activatable anti-HER2 antibodies TY22837 (left column) and TY22838 (right column) are shown. The results after incubation of the activatable antibodies at 50°C for 7 days are also shown. The X axis represents time per minute, and the Y axis represents the antibody aggregation level in absorbance units at 214 nm. [Figure 33C] The results of three stress tests for the activatable anti-HER2 antibodies TY22837 (left column) and TY22838 (right column) are shown. The results after incubation of the activatable antibodies at 40°C for 28 days are also shown. The X axis represents time per minute, and the Y axis represents the antibody aggregation level in absorbance units at 214 nm. [Figure 34A] The binding of the parental antibody (trastuzumab) and the activatable anti-HER2 antibody to recombinant HER2-Fc, as determined by ELISA, is shown. As shown in Table 19, the length of the masking peptide of the activatable antibody was changed. Results for trastuzumab (circles), TY23171 (upward triangles), TY23172 (downward triangles), and TY22836 (squares) are shown. The x-axis shows the antibody concentration, logarithmically transformed by M, and the y-axis shows the absorbance at a wavelength of 450 nm. [Figure 34B] The binding of the parental antibody (trastuzumab) and the activatable anti-HER2 antibody to recombinant HER2-Fc, as determined by ELISA, is shown. As shown in Table 19, the length of the masking peptide of the activatable antibody was changed. Results for trastuzumab (circles), TY23173 (squares), TY23174 (downward triangles), and TY22837 (downward triangles) are shown. The x-axis shows the antibody concentration, logarithmically transformed by M, and the y-axis shows the absorbance at a wavelength of 450 nm. [Figure 35A]This graph shows lymphocyte counts, T cell activation, and pharmacokinetic parameters in cynomolgus monkeys treated with CD3-masked bispecific antibody TY25362 alone. The x-axis shows the cell levels per μL for total T cells (top left), CD4+ T cells (top center), CD8+ T cells (top right), B cells (bottom left), and NK cells (bottom right) in monkeys responding to treatment with CD3-masked bispecific antibody TY25362. The x-axis represents time after administration in hours, and the y-axis represents the cell count per μL. Arrows above each plot indicate the time points at which 1, 10, and 30 mg / kg ("mpk") of the antibody were administered. [Figure 35B] This graph shows lymphocyte counts, T cell activation, and pharmacokinetic parameters in cynomolgus monkeys treated with the CD3-masked bispecific antibody TY25362 alone. The graphs show the activation levels of CD4+ (left plot) and CD8+ (right plot) T cells in response to treatment with the bispecific antibody TY25362. The X-axis represents time after administration in hours, and the Y-axis represents the percentage of CD69+ T cells. Arrows above each plot indicate the time points at which 1, 10, and 30 mg / kg ("mpk") of the antibody were administered. [Figure 35C] This graph shows lymphocyte count, T cell activation, and pharmacokinetic parameters in cynomolgus monkeys treated with CD3-masked bispecific antibody TY25362 alone. It also shows TY25362 levels in cynomolgus monkeys. The x-axis represents time after administration in hours, and the y-axis represents logarithmically transformed concentration in μg / mL. Arrows above each plot indicate the time points at which 1, 10, and 30 mg / kg ("mpk") doses of the antibody were administered. [Figure 36A] This shows the binding affinity of TY25023 and TY24051 to CD3. The EC50 and Kd values ​​for binding of TY25023 and TY24051 to human or monkey CD3δε, determined by ELISA or Biacore interferometry, are also shown. [Figure 36B]This shows the binding affinity of TY25023 and TY24051 to CD3. The binding of TY25023 and TY24051 to human CD3δε, as determined by ELISA, is shown. The EC50 of each antibody's binding to human CD3δε is shown in nM in the table to the right of the plot. [Figure 36C] This shows the binding affinity of TY25023 and TY24051 to CD3. The binding of TY25023 and TY24051 to monkey CD3δε, as determined by ELISA, is shown. The binding EC50 of each antibody to monkey CD3δε, in nM, is shown in the table to the right of the plot. [Figure 36D] This shows the binding affinity of TY25023 and TY24051 to CD3. It also shows the binding of TY25023 and TY24051 to human CD3δε, as determined using Biacore interferometry. [Figure 36E] This shows the binding affinity of TY25023 and TY24051 to CD3. It also shows the binding of TY25023 and TY24051 to monkey CD3δε, as determined by Biacore interferometry. [Figure 37A] This shows the results of cytokine release assays in cynomolgus monkeys treated with parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies, measured by ELISA. The levels of IL-2 in cynomolgus monkey serum over time are shown. The X-axis represents time after administration in hours, and the Y-axis represents the IL-2 level in pg / mL. Arrows indicate the time at which a 0.3 mg / kg antibody dose was administered. TY25455 is represented by a circle, TY25606 by a square, TY25715 by an upward-pointing triangle, and TY25816 by a downward-pointing triangle. [Figure 37B] The results of cytokine release assays in cynomolgus monkeys treated with parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies, measured by ELISA, are shown. The peak levels of IL-2 in cynomolgus monkey serum are indicated. The X-axis represents antibody identity, and the Y-axis represents the peak level of IL-2 in pg / mL. [Figure 37C]This graph shows the results of cytokine release assays in cynomolgus monkeys treated with parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies, measured by ELISA. It also shows the levels of IFN-γ in cynomolgus monkey serum over time. The X-axis represents time after administration in hours, and the Y-axis represents the IFN-γ level in pg / mL. Arrows indicate the time at which a 0.3 mg / kg antibody dose was administered. TY25455 is represented by a circle, TY25606 by a square, TY25715 by an upward-pointing triangle, and TY25816 by a downward-pointing triangle. [Figure 37D] The results of cytokine release assays in cynomolgus monkeys treated with parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies, measured by ELISA, are shown. The peak levels of IFN-γ are indicated. The X-axis represents antibody identity, and the Y-axis represents the peak level of IFN-γ in pg / mL. [Figure 38A] This shows pharmacodynamic marker measurements in cynomolgus monkeys treated with parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies, as measured using FACS. Lymphocyte count (top left), CD3+ T cell count (top right), and CD19+ B cell count (bottom left) 24 hours after antibody administration are shown. In each plot, the X-axis represents time after administration in hours, and the Y-axis represents cell count in x10⁹ cells / L. An arrow indicates the time at which a 0.3 mg / kg antibody dose was administered. TY25455 is represented by a circle, TY25606 by a square, TY25715 by an upward-pointing triangle, and TY25816 by a downward-pointing triangle. [Figure 38B] This graph shows pharmacodynamic marker measurements in cynomolgus monkeys treated with parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies, as measured using FACS. Lymphocyte count (top left), CD3+ T cell count (top right), and CD19+ B cell count (bottom left) are shown 14 days after antibody administration. In each plot, the X-axis represents time after administration in hours, and the Y-axis represents cell count in x10⁹ cells / L. An arrow indicates the time point at which a 0.3 mg / kg antibody dose was administered. TY25455 is represented by a circle, TY25606 by a square, TY25715 by an upward-pointing triangle, and TY25816 by a downward-pointing triangle. [Figure 38C]This shows pharmacodynamic marker measurements in cynomolgus monkeys treated with parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies, as measured using FACS. The numbers of CD3+CD8+ T cells (top left), CD3+CD4+ T cells (top right), CD8+CD69+ T cells (bottom left), and CD4+CD69+ T cells (bottom right) are shown 14 days after antibody administration. In each plot, the X-axis represents time after administration in hours, and the Y-axis represents the percentage of cells relative to lymphocyte levels. An arrow indicates the time at which a 0.3 mg / kg antibody dose was administered. TY25455 is represented by a circle, TY25606 by a square, TY25715 by an upward-pointing triangle, and TY25816 by a downward-pointing triangle. [Figure 39A] This graph shows pharmacodynamic marker measurements in cynomolgus monkeys treated with the activatable anti-CD3 and anti-CD20 bispecific antibody TY25606, as measured using FACS. Lymphocyte count (top left), CD3+ T cell count (top right), and CD19+ B cell count (bottom left) are shown 50 days after antibody administration. In each plot, the X-axis represents time after administration in hours, and the Y-axis represents cell count in x10⁹ cells / L. Arrows indicate the time points at which 0.3 mg / kg and 1 mg / kg of antibody were administered. [Figure 39B] This graph shows pharmacodynamic marker measurements in cynomolgus monkeys treated with the activatable anti-CD3 and anti-CD20 bispecific antibody TY25606, as measured using FACS. The graphs show the number of CD3+CD8+ T cells (top left), CD3+CD4+ T cells (top right), CD8+CD69+ T cells (bottom left), and CD4+CD69+ T cells (bottom right) 50 days after antibody administration. In each plot, the X-axis represents time after administration in hours, and the Y-axis represents the percentage of cells relative to lymphocyte levels. Arrows indicate the time points at which 0.3 and 1 mg / kg doses of the antibody were administered. [Figure 40] Total human IgG levels in cynomolgus monkeys treated with the activatable anti-CD3 and anti-CD20 bispecific antibody TY25606, as measured by FACS. The X-axis shows time after administration in hours, and the Y-axis shows the logarithmically transformed total human IgG amount in μg / mL. Arrows indicate the time points at which 0.3 and 1 mg / kg doses of the antibody were administered. [Figure 41]This figure shows the effects of parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies in reporter assays with and without Raji tumor cells. Figure 41A shows the reporter assay with Raji tumor cells. The x-axis shows the antibody concentration, logarithmically transformed in nM, and the y-axis shows the reporter's relative luminescence units (RLU). The gray area shows the calculated peak concentration in cynomolgus monkey serum at a dose of 0.3 mg / kg. TAC2392 is represented by a black circle, TAC2415 by a white circle, TY25455 by a black square, TY25606 by a white square, TY25715 by an upward-pointing triangle, TY25816 by a downward-pointing triangle, and the isotype control by a diamond. Figure 41B shows the reporter assay without Raji tumor cells. The x-axis shows the antibody concentration, logarithmically transformed in nM, and the y-axis shows the reporter's relative luminescence units (RLU). TAC2392 is represented by a black circle, TAC2415 by a white circle, TY25455 by a black square, TY25606 by a white square, TY25715 by an upward-pointing triangle, TY25816 by a downward-pointing triangle, and isotype counterparts by a diamond. [Figure 42] This figure shows the effects of the parent antibody or an activatable anti-CD3 and anti-CD20 bispecific antibody in reporter assays with and without SU-DHL-4 tumor cells. Figure 42A shows the reporter assay with SU-DHL-4 tumor cells. The x-axis shows the antibody concentration, logarithmically transformed in nM, and the y-axis shows the reporter's relative luminescence units (RLU). TAC2392 is represented by a black circle, TAC2415 by a white circle, TY25455 by a black square, TY25606 by a white square, TY25715 by an upward-pointing triangle, TY25816 by a downward-pointing triangle, and the isotype control by a diamond. The gray area shows the calculated peak concentration in cynomolgus monkey serum at a dose of 0.3 mg / kg. Figure 42B shows the reporter assay without SU-DHL-4 tumor cells. The x-axis shows the antibody concentration, logarithmically transformed in nM, and the y-axis shows the reporter's relative luminescence units (RLU). TAC2392 is represented by a black circle, TAC2415 by a white circle, TY25455 by a black square, TY25606 by a white square, TY25715 by an upward-pointing triangle, TY25816 by a downward-pointing triangle, and the isotype control by a diamond. The gray area shows the calculated peak concentration in cynomolgus monkey serum at a dose of 0.3 mg / kg. [Figure 43]This figure shows the effects of parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies in an in vitro B cell killing assay using PBMCs. Figure 43A shows the killing levels of endogenous B cells. The x-axis shows the antibody concentration, logarithmically transformed in nM, and the y-axis shows the killing rate of human endogenous B cells. AC1281 is represented by black circles, TAC2415 by white circles, TY25455 by black squares, TY25606 by white squares, TY25715 by upward-pointing triangles, TY25816 by downward-pointing triangles, and the isotype control by diamonds. Below the plot, the EC50 of B cell killing for each antibody is shown in nM. Figure 43B shows the activation levels of CD8+ T cells. The x-axis shows the antibody concentration, logarithmically transformed in nM, and the y-axis shows the proportion of CD69+ cells in the CD8+ T cell population. TAC2392 is represented by a black circle, TAC2415 by a white circle, TY25455 by a black square, TY25606 by a white square, TY25715 by an upward-pointing triangle, TY25816 by a downward-pointing triangle, and the isotype control by a diamond. Below the plot, the EC50 of T cell activation for each antibody is shown in nM. [Figure 44] This plot shows the binding levels of T cells and B cells to the antibodies TAC2392 (black circles), TY2455 (black downward-pointing triangles), and isotype control (white circles), measured by FACS using PBMCs. Each plot shows the antibody concentration, logarithmically transformed in nM, on the x-axis, and the binding level, mean fluorescence intensity ("MFI"), on the y-axis. Binding to human CD4+ T cells is shown in the upper left, binding to human CD8+ T cells in the upper center, binding to human B cells in the upper right, binding to monkey CD4+ T cells in the lower left, binding to monkey CD8+ T cells in the lower center, and binding to monkey B cells in the lower right. Below the plot, the binding EC50 for TAC2392 and TY2455 for each cell type is shown in nM. [Figure 45] This graph shows the time course of tumor volume in female M-NSG immunodeficient mice using human PBMCs and EMT6 mouse mammary cancer cells stably transfected with HER2. Mice were administered 5 mg / kg of antibodies TY24051 (black circles), TY25023 (upward triangles), TY25026 (squares), TY25362 (downward triangles), and isotype control (white circles). The X-axis shows the number of days after inoculation, with arrows indicating the time of antibody administration, and the Y-axis shows the tumor volume in mm³. [Figure 46]A schematic diagram of the proposed mechanism of action of SAFE bodies is shown. As shown on the left, when a SAFE body is close to normal tissue (e.g., tissue lacking the epitope to which the SAFE body binds), the SAFE body remains masked. While we do not wish to be bound by theory, two pathways are hypothesized for the mechanism by which SAFE bodies bind to the target site. In pathway 1, the cleavable portion is cleaved by a protease close to the tumor tissue, thereby removing the masking portion and demasking the SAFE body so that it can bind to the target. In pathway 2, the cleavable portion is not necessarily cleaved, and the binding of the SAFE body to the target competes with the binding of the SAFE body to the masking portion. Once the SAFE body binds to the target site, the cleavable portion may be cleaved by a protease, thereby demasking the SAFE body. [Figure 47] This demonstrates the induction of luciferase expression in the Jurkat / NFAT-Luc reporter line by a CD20xCD3 bispecific antibody, accompanied by target Raji cells used for screening additional CD20xCD3 bispecific antibodies. [Figure 48] The tumor growth curves of female M-NSG mice carrying established Raji tumors, treated in different ways (N=6), are shown. [Figure 49] This shows PK studies of TY25455 and TY25606 in tumor-bearing mice. A shows the concentration of TY25455 in tumor-bearing mice at different time points using different dose-setting strategies. B shows the concentration of TY25606 in tumor-bearing mice at different time points using different dose-setting strategies. [Figure 50]This shows toxicity and pharmacological studies of single-dose injections of CD20xCD3 bispecific antibodies or SAFE body / bispecific antibodies in cynomolgus monkeys. A shows a normalized time-course plot of CD19+ B cell percentage in blood samples from cynomolgus monkeys administered a single dose of the drug. B shows a normalized time-course plot of CCD3+ T cell percentage in blood samples from cynomolgus monkeys administered a single dose of the drug. C shows the pre- and post-administration levels (pg / mL) of IFN-γ in cynomolgus monkeys administered a single dose of the drug. D shows the pre- and post-administration levels (pg / mL) of IL-2 in cynomolgus monkeys administered a single dose of the drug. [Figure 51] This shows the binding affinity of HER2xCD3 bispecific antibodies to CD3 and HER2, as determined by enzyme-linked immunosorbent assay (ELISA). A shows the CD3δε ELISA binding curves for bispecific antibodies TY24051, TY25238, and TY25023. B shows the CD3δε ELISA binding curves for bispecific antibody TY25238, activatable antibodies TY27151, and TY27008. C shows the HER2 ELISA binding curves for trastuzumab, bispecific antibody TY25238, activatable antibodies TY27151, and TY27008. [Figure 52] The results of CD8+ T cell-mediated killing assays of SKOV3 cells (Figure 52A), MCF7 cells (Figure 52B), and A549 cells (Figure 52C) in the presence of bispecific antibodies TY25023, TY24051, and TY25238 are shown. [Figure 53] This shows the cleavage efficiency of the masking sites of anti-CD3 antibodies (Figure 53A) and anti-HER2 antibodies (Figure 53B) in various HER2xCD3 bispecific antibodies. [Figure 54] This shows the in vivo antitumor effects of HER2xCD3 antibodies and negative controls against HER2-expressing tumors (SK-OV3) in a heterogeneous in vivo tumor model. Data points represent group means, and error bars represent SEM. [Figure 55]The PK data for cynomolgus monkeys treated with a HER2xCD3 bispecific antibody is shown. C indicates systemic cytokine release (IL-6, IFN-γ, IL-2, TNF-α) in cynomolgus monkeys. [Figure 56] This shows the in vitro cytokine release by human PBMCs in the presence of MCF7, including IFN-γ (Figure 56A) or IL-2 (Figure 56B). [Figure 57] This shows the in vivo antitumor effects of HER2xCD3 antibodies and negative controls against HER2-expressing tumors (SK-OV3) in a heterogeneous in vivo tumor model. Data points represent group means, and error bars represent SEM. [Figure 58] Peripheral trend of lymphocytes induced by TY25023, TY25026, and TY25362. [Figure 59] This shows the cytokine release levels in cynomolgus monkeys administered TY25023, TY25026, and TY25362, as determined by ELISA. [Figure 60] The PK curves for cynomolgus monkeys administered TY25023, TY25026, and TY25362 are shown. [Figure 61] This paper presents the results of a luciferase-based CD3 gene reporter assay characterizing the effect of an anti-HER2xCD3 activatable / bispecific antibody on the activation of CD3 signaling. [Figure 62] This study shows the in vivo antitumor effects of the anti-HER2xCD3 bispecific parental antibody TY25238, as well as the activatable antibodies TY27008 and TY27151, in an HT55 xenograft model transplanted with PBMCs. Data points represent group mean values, and error bars represent SEM values. Antibody administration is indicated by arrows. [Figure 63] This report compares the in vivo antitumor effect of the anti-HER2xCD3 bispecific activatable antibody TY27151 with trastuzumab, DS-8201 ADC, or vehicle in an HT55 xenograft model using PBMCs. Data points represent group means, and error bars represent SEM. Antibody administration is indicated by arrows. [Figure 64]This study demonstrates the synergistic antitumor effect of the anti-HER2xCD3 bispecific activatable antibody TY27151 in combination with an anti-CD137 mAb in a syngenic model of MC38-hHER2 mouse colon cancer. Figure 64A shows the in vivo antitumor effect of the anti-HER2xCD3 bispecific activatable antibody TY27151 in a syngenic model of MC38-hHER2 mouse colon cancer. Antibody administration is indicated by arrows. Figure 64B shows the results of re-challenging the MC38-hHER2 tumor without antibody administration. Arrows indicate tumor re-challenge. In both figures, data points represent the group mean, and error bars represent SEM. [Figure 65] This study demonstrates the in vivo antitumor effects of the anti-HER2xCD3 bispecific activatable antibody TY27151 as monotherapy or in combination with the anti-PD-1 mAb 2E5 in an SK-OV3 xenograft model using transplanted PBMCs. Data points represent group mean values, and error bars represent SEM values. Antibody administration is indicated by arrows. [Modes for carrying out the invention]

[0062] This application provides a masked multispecific antibody comprising a first antigen-binding fragment that specifically binds to CD3 with weak affinity and a second antigen-binding fragment that specifically binds to a target antigen, wherein the first antigen-binding fragment is fused to a first masking moiety. The masking moiety may be fused to the first antigen-binding fragment via a cleavable or non-cleavable linker. While we do not wish to be bound by theory, it is conceivable that a multispecific antibody comprising a first masking moiety may be in a dynamic equilibrium between a masked state in which the antigen-binding fragment that specifically binds to CD3 is bound to the masking moiety and a CD3-bound state in which the antigen-binding fragment that specifically binds to CD3 is bound to CD3. Therefore, the relative binding affinity of the masking moiety and the antigen-binding fragment to CD3 determines the extent to which the antibody actually binds to CD3. Due to the weak affinity of the first antigen-binding fragment and the high masking efficiency of the first masking moiety, the multispecific antibody described herein offers a broad therapeutic range and reduces side effects associated with nonspecific binding. The multispecific antibodies described herein provide a safe and effective therapeutic approach for the treatment of a variety of diseases and conditions, including liquid and solid tumors, related to target antigens.

[0063] Accordingly, one aspect of the present application provides a multispecific antibody comprising: a) a first antigen-binding fragment that specifically binds to CD3 and is fused to a first masking moiety (MM1); and b) a second antigen-binding fragment that specifically binds to a target antigen; where MM1 specifically binds to the CD3-binding moiety in competition with CD3; and the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an enzyme-linked immunosorbent assay (ELISA, e.g., the ELISA assay of Example 3). In some embodiments, MM1 comprises the amino acid sequence of SEQ ID NO: 35 or 417. In some embodiments, the target antigen is HER2. In some embodiments, the target antigen is CD20.

[0064] In some embodiments, the application provides an activatable multispecific antibody (also called an "activatable multispecific T cell engager" or "SAFE body multispecific T cell engager") comprising a first antigen-binding fragment that specifically binds to CD3 with weak affinity and a second antigen-binding fragment that specifically binds to a target antigen, wherein the first antigen-binding fragment is fused to a first masking site via a first cleavable portion. In some embodiments, the second antigen-binding fragment is fused to a second masking site via a second cleavable portion. An exemplary type of the activatable multispecific antibody is the TAAxCD3 SAFE body bispecific T cell engager ("SAFE-bsAb"). The TAAxCD3 SAFE-bsAb molecule comprises an antigen-binding fragment of an antibody that specifically binds to tumor-associated antigens ("TAAs") whether masked or unmasked, and includes a masked anti-CD3 antigen-binding fragment. Exemplary SAFE-bsAbs described herein include HER2xCD3 SAFE bodies (see, e.g., Examples 1-2, 5-8, and 13) and CD20xCD3 SAFE bodies (see, e.g., Examples 9-12). In circulation or in healthy tissue, activatable antibodies are inactive because the masking portion can block antigen binding. However, when the cleavable portion is cleaved at a target site (e.g., disease site), the activatable antibody is activated and binds to both CD3 and the target antigen (e.g., TAA). Due to the low affinity of the first antigen-binding fragment and the high masking efficiency of the first masking portion, the activatable multispecific antibodies described herein offer a broad therapeutic range and reduce side effects associated with nonspecific binding. For example, the exemplary activated form of TAAxCD3 SAFE-bsAbs has been observed to potently stimulate T cell activation and TAA+ tumor cell killing. Furthermore, even at high dose levels, no visible cytokine release syndrome or other adverse events were observed in exploratory toxicity studies of TAAxCD3 SAFE-bsAbs in cynomolgus monkeys (see, e.g., Figures 50C-50D and 59).Furthermore, the activatable multispecific antibodies described herein exhibit improved stability and more robust expression levels compared to parental antibodies. These activatable multispecific antibodies provide a safe and effective therapeutic approach for the treatment of a variety of diseases and conditions, including liquid and solid tumors related to target antigens.

[0065] Accordingly, one aspect of the present application provides an activatable multispecific antibody comprising: a) a first antigen-binding fragment that specifically binds to CD3, wherein the first antigen-binding fragment is fused to a first masking fragment (MM1) via a first cleavable portion (CM1); and b) a second antigen-binding fragment that specifically binds to a target antigen, wherein CM1 comprises a first cleavage site; if CM1 is not cleaved, MM1 inhibits the binding of the activatable antibody to CD3; wherein CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment; the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an enzyme-linked immunosorbent assay (ELISA, e.g., the ELISA assay of Example 3). In some embodiments, MM1 comprises the amino acid sequence of SEQ ID NO: 35 or 417. In some embodiments, CM1 comprises the amino acid sequence of SEQ ID NO: 77 or 418. In some embodiments, the target antigen is HER2. In some embodiments, the target antigen is CD20.

[0066] Isolated anti-CD3 antibodies, masked anti-CD3 antibodies (including activatable anti-CD3 antibodies), masked anti-HER2 antibodies (including activatable anti-HER2 antibodies), compositions, manufacturing methods, and methods of use are also provided. I. Definition

[0067] Unless otherwise defined below, terms are used herein in the sense that they are commonly used in the art.

[0068] The term "antibody" as used herein is used in its broadest sense and is not limited to a wide range of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments provided they exhibit desired antigen-binding activity.

[0069] The term "antigen-binding fragment" refers to one or more parts of an antibody that retain the ability to bind to an antigen. Examples of antibody "antigen-binding fragments" include, but are not limited to, (i) Fab fragments (monovalent fragments consisting of VL, VH, CL, and CH1 domains); (ii) F(ab')2 fragments (bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region); (iii) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (v) single-stranded Fv fragments containing the VH and VL domains of an antibody (where the VH and VL domains are fused to each other); and (vi) single-stranded Fab fragments containing a single polypeptide containing the VL, VH, CL, and CH1 domains.

[0070] The term "antibody" includes, but is not limited to, fragments capable of binding to an antigen, such as Fv, Fab, Fab', and (Fab')2. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site and the remaining "Fc" fragment; its name reflects its ability to readily crystallize. Pepsin treatment produces an F(ab')2 fragment having two antigen-binding sites and still capable of crosslinking antigens. The term "antibody" also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as mouse, human, and cynomolgus monkey.

[0071] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope. However, this excludes possible variant antibodies, such as those containing naturally occurring mutations or arising during the production of monoclonal antibody preparations, although such variants are generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of the antibody such that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of methods, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0072] As used herein, the terms “hypervariable region” or “HVR” refer to each region of the antibody variable domain whose sequence is hypervariable. HVRs may form structurally defined loops (“hypervariable loops”). Generally, natural quadruple-chain antibodies have six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs generally contain amino acid residues derived from hypervariable loops and / or “complementarity-determining regions” (CDRs), the CDRs being the most sequence-variable and / or involved in antigen recognition. Exemplary hypervariable loops are located at amino acid residues 26–32 (L1), 50–52 (L2), 91–96 (L3), 26–32 (H1), 53–55 (H2), and 96–101 (H3). (Chothia and Lesk, J.Mol.Biol.196:901–917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are located at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Except for VH CDR1, CDRs generally contain amino acid residues that form a hypervariable loop. CDRs also contain "specificity-determining residues" or "SDRs," which are residues that come into contact with the antigen. SDRs are contained within a region of the CDR called a shortened CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are located at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)).Unless otherwise specified, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered herein in accordance with Kabat et al., cited above.

[0073] Table I below provides exemplary CDR definitions according to various algorithms known in the relevant field. Table I: CDR definitions TIFF0007902187000001.tif39170 1 Residue numbering follows the nomenclature of Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991). 2 Residue numbering follows the nomenclature of Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997). 3 Residue numbering follows the nomenclature of MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol.,45: 3832-3839 (2008). 4 Residue numbering follows the nomenclature of Lefranc MP et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Plueckthun, J. Mol. Biol., 309:657-670 (2001). 5 Residue numbering follows the nomenclature of Honegger and Plueckthun, J. Mol. Biol., 309:657-670 (2001).

[0074] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four framework regions (FRs) and three hypervariable regions (HVRs), arranged from the amino terminus to the carboxyl terminus in the following order: FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated using VH or VL domains derived from the antigen-binding antibody, respectively, to screen for a library of complementary VL or VH domains. For example, see Portolano et al., J.Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0075] The terms "EU numbering" or "amino acid position numbering based on EU numbering" and their variations refer to the numbering system used for the heavy chain constant domain of antibody editing in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). The EU numbering of residues can be determined for a given antibody by aligning the "standard" EU numbering sequence with homologous regions of the antibody sequence.

[0076] The Kabat numbering system is generally used to refer to residues within the variable domain (approximately corresponding to light chain residues 1-107 and heavy chain residues 1-113) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of FR or HVR in the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat), and a residue inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by aligning the "standard" Kabat numbering sequence with homologous regions of the antibody sequence.

[0077] For heterodimer proteins having two CH3 domains (e.g., activatable multispecific antibodies), a given amino acid position in the first CH3 domain is denoted as X, and the corresponding amino acid position in the second CH3 domain is denoted as X'. For example, N390C-S400'C refers to a heterodimer protein (e.g., activatable multispecific antibodies) having a first CH3 domain with the N390C mutation and a second CH3 domain with the S400C mutation. All mutations or substitutions in heterodimer proteins (e.g., activatable multispecific antibodies) described herein refer to in relation to the wild-type naturally occurring CH3 domain.

[0078] Unless otherwise indicated, all polypeptide chain formulas described herein list the polypeptide components in order from the N-terminus to the C-terminus. For example, the formula VH2-CH1-hinge-CH2-first CH3 indicates that the polypeptide contains the following structural components from the N-terminus to the C-terminus: VH2, CH1, hinge, CH2, and first CH3.

[0079] As used herein, the term “heavy chain constant region” refers to a region containing at least three heavy chain constant domains, CH1, CH2, and CH3, as well as a hinge region between CH1 and CH2. Unrestricted heavy chain constant regions include γ, δ, and α. Unrestricted heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. Furthermore, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (including the γ1 constant region), IgG2 (including the γ2 constant region), IgG3 (including the γ3 constant region), and IgG4 (including the γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (including the α1 constant region) and IgA2 (including the α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0080] The term "CH2 domain" in the human IgG Fc region typically refers to approximately 231–340 residues of IgG in the EU numbering system. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched glycans are inserted between the two CH2 domains of an intact, native IgG molecule. It is hypothesized that carbohydrates may provide a substitute for domain-domain pairing and help stabilize the CH2 domain. (Burton, Molec. Immunol. 22:161-206 (1985)).

[0081] The term "CH3 domain" refers to the sequence of residues from the C-terminus of the Fc region to the CH2 domain (i.e., approximately amino acid residues 341 to 447 of IgG in the EU numbering system).

[0082] As used herein, the term “heavy chain” refers to a polypeptide that includes at least a heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain includes at least a portion of the heavy chain constant region. As used herein, the term “full-length heavy chain” refers to a polypeptide that includes both a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0083] As used herein, the term “light chain steady region” refers to the region containing the light chain steady domain CL. A non-limiting exemplary light chain steady region includes λ and κ.

[0084] As used herein, the term “light chain” refers to a polypeptide that includes at least a light chain variable region, with or without a leader sequence. In some embodiments, the light chain includes at least a portion of the light chain constant region. As used herein, the term “full-length light chain” refers to a polypeptide that includes both a light chain variable region and a light chain constant region, with or without a leader sequence.

[0085] "Affinity" refers to the total strength of non-covalent interactions between a molecule's (e.g., antibody) binding site and its binding partner (e.g., antigen). The affinity of molecule X to partner Y is generally expressed by the dissociation constant (K). d Affinity can be expressed as ). Affinity can be measured by common methods known in the art, including those described herein. In the context of multispecific antibodies (e.g., bispecific or triplicate antibodies), affinity can be measured for each binding specificity (i.e., target) of the antibody.

[0086] The terms “binding,” “specifically binding,” or “specific to” refer to measurable and reproducible interactions, such as binding between a target and an antibody, which determine the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, binding strength, more readily, and / or for a longer duration than an antibody that binds to other targets. In some embodiments, the degree of antibody binding to unrelated targets is less than about 10% of the degree of antibody binding to the target (e.g., measured by radioimmunoassay (RIA)). In some embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved between proteins of different species. In some embodiments, specific binding may include, but does not require, exclusive binding.

[0087] The term "multispecificity," used in conjunction with antibodies, refers to antibodies that possess polyepitope specificity (i.e., they are capable of specifically binding to two, three, or more different epitopes on a given biomolecule, or to two, three, or more different epitopes on a given biomolecule).

[0088] A "affinity-mature" antibody is an antibody that has one or more modifications in one or more hypervariable regions (HVRs) compared to a parent antibody (without such modifications), where such modifications result in an improvement in the antibody's affinity for the antigen. In some examples, affinity-mature antibodies are antibodies that have one or more modifications in one or more complementarity-determining regions (CDRs) compared to a parent antibody (without such modifications), where such modifications result in an improvement in the antibody's affinity for the antigen.

[0089] As used herein, “chimeric antibody” refers to an antibody in which a portion of the heavy chain and / or light chain originates from a particular source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species. In some embodiments, a chimeric antibody refers to an antibody comprising at least one variable region derived from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region derived from a second species (e.g., human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, all of the variable regions of the chimeric antibody originate from the first species, and all of the constant regions of the chimeric antibody originate from the second species.

[0090] As used herein, “humanized antibody” refers to an antibody in which at least one amino acid in the framework region of a non-human variable region is replaced with a corresponding amino acid derived from a human variable region. In some embodiments, the humanized antibody comprises at least one human constant region or a fragment thereof. In some embodiments, the humanized antibody is Fab, (Fab')2, etc.

[0091] As used herein, “HVR-transplanted antibody” refers to a humanized antibody in which one or more hypervariable regions (HVRs) of a first (non-human) species are transplanted into framework regions (FRs) of a second (human) species. In some examples, as used herein, “CDR-transplanted antibody” refers to a humanized antibody in which one or more complementarity-determining regions (CDRs) of a first (non-human) species are transplanted into framework regions (FRs) of a second (human) species.

[0092] As used herein, "human antibodies" refers to antibodies produced in humans, antibodies produced in non-human animals containing human immunoglobulin genes, such as XENOMOUSE®, and antibodies selected using in vitro methods such as phage display, and the antibody repertoire is based on human immunoglobulin sequences.

[0093] Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a form of cytotoxicity in which secreted immunoglobulin (Ig) bound to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) specifically binds these cytotoxic effector cells to antigen-carrying target cells, which are then killed by cytotoxins. Primary cells that mediate ADCC, such as NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, for example, as described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta). Effector cells useful for such assays include PBMC cells and NK cells. Alternatively, the ADCC activity of a target molecule may be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al. Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Modified Fc region amino acid sequences (polypeptides having variant Fc regions) and additional polypeptide variants having increased or decreased ADCC activity are described, for example, in U.S. Patent No. 7,923,538 and U.S. Patent No. 7,994,290.

[0094] Complement-dependent cell injury, or CDC, refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding the first component of the complement system (C1q) to an antibody (of a preferred subclass), which then binds to a congener antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J.Immunol.Methods202:163 (1996). Polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc region) and increased or decreased C1q binding ability are described, for example, in U.S. Patent Nos. 6,194,551B1, 7,923,538, 7,994,290, and WO1999 / 51642. See also, for example, Idusogie et al., J.Immunol.164:4178-4184 (2000).

[0095] Polypeptide variants with "modified" FcR binding affinity or ADCC activity are those that exhibit either enhanced or decreased FcR binding activity and / or ADCC activity compared to the parent polypeptide or the polypeptide containing the natural sequence Fc region. Polypeptide variants that "show increased binding" to FcR bind to at least one FcR with better affinity than the parent polypeptide. Polypeptide variants that "show decreased binding" to FcR bind to at least one FcR with lower affinity than the parent polypeptide. These variants that show decreased binding to FcR may have little to no binding to recognizable FcRs, for example, 0-20% of the FcR binding compared to the natural sequence IgGFc region.

[0096] The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” may be used interchangeably and refer to polymers of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, including, but not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to a linear sequence of nucleotides, including a nucleic acid molecule or polynucleotide.

[0097] The terms “polypeptide” and “peptide” are used interchangeably to refer to polymers of amino acid residues and are not limited to the minimum length. Such polymers of amino acid residues may contain native or non-native amino acid residues. Both full-length proteins and their fragments are included in the definition. The term also includes post-expression modifications of polypeptides, e.g., glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, “polypeptide” includes modifications to the native sequence, e.g., deletions, additions, and substitutions (generally inherently conserved), as long as the polypeptide maintains the desired activity. These modifications may be planned (e.g., via site-directed mutagenesis) or accidental (e.g., via host mutations), resulting in protein or error upon PCR amplification.

[0098] A polypeptide "variant" means a bioactive polypeptide having at least about 80% amino acid sequence identity with the native polypeptide after aligning the sequence to achieve the maximum percentage of sequence identity (without considering any conservative substitutions as part of sequence identity) and introducing gaps as necessary. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, the variant has at least about 80% amino acid sequence identity. In some embodiments, the variant has at least about 90% amino acid sequence identity. In some embodiments, the variant has at least about 95% amino acid sequence identity with the native polypeptide.

[0099] As used herein, “percent (%) amino acid sequence identity” for peptide, polypeptide, or antibody sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular peptide or polypeptide sequence after aligning the sequences to achieve maximum percent sequence identity (without considering any conservative substitutions as part of the sequence identity) and introducing gaps as necessary. Alignment for determining percent amino acid sequence identity can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN® (DNASTAR) software. Those skilled in the art can determine preferred parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0100] Amino acid substitutions may include, but are not limited to, replacing one amino acid in a polypeptide with another. Exemplary substitutions are shown in Table A. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, such as retention / improvement of antigen binding, decreased immunogenicity, or improvement of ADCC or CDC. Table A. Exemplary amino acid substitutions TIFF0007902187000002.tif116170

[0101] Amino acids may be grouped according to their general side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basicity: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions would require replacing one member of these classes with one of another.

[0102] The term “vector” is used to describe a polynucleotide that can be manipulated to contain cloned polynucleotides that can be replicated within a host cell. A vector may contain one or more of the following elements: an origin of replication, one or more regulatory sequences that regulate the expression of the polypeptide of interest (e.g., promoters and / or enhancers), and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). The term “expression vector” refers to a vector used to express the polypeptide of interest in a host cell.

[0103] "Host cell" refers to a cell that can or is a recipient of a vector or isolated polynucleotide. The host cell may be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, e.g., primate or non-primate animal cells; fungal cells, e.g., yeast; plant cells; and insect cells. Non-exclusive mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, as well as their derivatives, e.g., 293-6E and DG44 cells, respectively. The term "cell" includes primary target cells and their offspring.

[0104] As used herein, the term “isolated” refers to a molecule that has been separated from at least some of the components with which it is normally found or produced in nature. For example, a polypeptide is called “isolated” if it has been separated from at least some of the components of the cell in which it was produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing it from the cell that produced the polypeptide is considered “isolated” the polypeptide. Similarly, a polynucleotide is called “isolated” if it is not part of a larger polynucleotide that is normally found in nature (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide), or, for example, an RNA polynucleotide, if it has been separated from at least some of the components of the cell in which it was produced. Thus, a DNA polynucleotide contained in a vector within a host cell may be called “isolated.”

[0105] The terms “individual” or “subject” are used interchangeably herein to refer to mammals. In some embodiments, methods for treating mammals are provided, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sports animals, and mammalian pets. In some examples, “individual” or “subject” refers to an individual or subject requiring treatment for a disease or disorder.

[0106] As used herein, “treatment” or “to treat” is an approach to obtain beneficial or desirable outcomes, including clinical outcomes. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, one or more of the following: reducing one or more symptoms caused by the disease; reducing the severity of the disease; stabilizing the disease (e.g., preventing or delaying disease exacerbation); preventing or delaying the spread of the disease (e.g., metastasis); preventing or delaying disease recurrence; delaying or slowing disease progression; improving the condition; providing (partial or total) remission of the disease; reducing the dosage of one or more other therapeutic agents required to treat the disease; delaying disease progression; improving quality of life; and / or extending survival. Reduction of the pathological outcomes of cancer is also encompassed by “treatment.” The methods of the present invention aim to achieve any one or more of these aspects of treatment.

[0107] The terms “prevent,” and similar terms such as “preventable,” and “preventing,” refer to approaches to prevent, inhibit, or reduce the likelihood of recurrence of a disease or condition, such as cancer. It also refers to delaying the recurrence of a disease or condition, or delaying the recurrence of symptoms of a disease or condition. As used herein, “prevention” and similar terms also include reducing the severity, impact, symptoms, and / or burden of a disease or condition before it recurs.

[0108] As used herein, “delaying” cancer development means extending, interfering with, slowing, suppressing, stabilizing, and / or postponing the onset of the disease. This delay may be of varying durations depending on the disease history and / or the individual being treated. A method of “delaying” cancer development is a method that reduces the likelihood of disease development and / or the severity of the disease in a given timeframe compared to not using the method. Such comparisons are usually based on clinical studies using a statistically significant number of individuals. Cancer development can be detected using standard methods, including but not limited to computed tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasonography, coagulation tests, angiography, or biopsy. Development also refers to the progression of cancer that was initially undetectable and includes development, recurrence, and onset.

[0109] As used herein, the term “effective dose” refers to an amount of an agent or combination of agents sufficient to treat a particular disorder, condition, or disease, for example, to improve, alleviate, reduce, and / or delay one or more of its symptoms. With respect to cancer, an effective dose includes an amount sufficient to shrink a tumor and / or reduce the rate of tumor growth (e.g., inhibit tumor growth) or prevent or delay other undesirable cell proliferation. In some embodiments, an effective dose is an amount sufficient to delay the onset of the disease. In some embodiments, an effective dose is an amount sufficient to prevent or delay recurrence. An effective dose may be administered in one or more doses. An effective dose of an agent or composition may (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, suppress, to some extent slow, preferably stop, the invasion of cancer cells into peripheral organs; (iv) inhibit tumor metastasis (i.e., to some extent slow, preferably stop); (v) inhibit tumor growth; (vi) prevent or delay tumor development and / or recurrence; and / or (vii) alleviate to some extent one or more of the symptoms associated with cancer.

[0110] Embodiments of the present invention described herein are understood to include "consisting of" and / or "essentially consisting of" embodiments.

[0111] In this specification, references to values ​​or parameters "about" include (and describe) variations in the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X".

[0112] Where used herein, the reference "not" to a value or parameter generally means and describes "other than" a value or parameter. For example, the statement that a method is not used to treat type X cancer means that the method is used to treat cancers other than type X.

[0113] As used herein, the term "approximately XY" has the same meaning as "approximately X to approximately Y".

[0114] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise explicitly stated in the context.

[0115] As used herein, the terms “and / or,” expressions such as “A and / or B,” are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the terms “and / or,” expressions such as “A, B, and / or C,” are intended to include each of the following embodiments: 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).

[0116] II. Antibodies Some aspects of this application relate to masked antibodies such as multispecific antibodies, activatable antibodies (activatable multispecific antibodies such as activatable bispecific T cell engager molecules), antigen-binding fragments thereof, or derivatives of such antibodies.

[0117] One aspect of this application provides a multispecific antibody capable of binding to both T cells and target cells such as tumor cells. In some embodiments, the multispecific antibody is bispecific. In some embodiments, the multispecific antibody is tripspecific. In some embodiments, the multispecific antibody binds to CD3 on the surface of T cells. Due to on-target and off-tumor effects, BiTE molecules are associated with high cytotoxicity, including toxicity to the central nervous system (CNS) and cytokine storms. There is a need for activatable BiTE molecules with improved specificity and reduced side effects.

[0118] In some embodiments, a multispecific antibody is provided comprising: a) a first antigen-binding fragment that specifically binds to CD3 and is fused to a first masking moiety (MM1); and b) a second antigen-binding fragment that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19); where MM1 specifically binds to the CD3 binding moiety in competition with CD3; the multispecific antibody binds to CD3 via the first antigen-binding fragment; the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of the antibody, as determined by an ELISA assay (e.g., as described in Example 5). In some embodiments, the first antigen-binding fragment binds to CD3 at a dissociation constant (kd) of at least 50 nM.

[0119] In some embodiments, a multispecific antibody is provided comprising: a) a first antigen-binding fragment that specifically binds to CD3 and is fused to a first masking moiety (MM1); and b) a second antigen-binding fragment that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19); where MM1 specifically binds to the CD3 binding moiety in competition with CD3; the multispecific antibody binds to CD3 via the first antigen-binding fragment; the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of the antibody as determined by an ELISA assay (e.g., described in Example 5), and where MM1 has a masking efficiency of at least 50 as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3).

[0120] A. Activatable multispecific T cell engagers One aspect of this application provides an activatable multispecific antibody capable of binding to both T cells and target cells such as tumor cells. In some embodiments, the activatable antibody is bispecific. In some embodiments, the activatable antibody is tripspecific. For example, in some embodiments, the activatable multispecific antibody is an activatable bispecific T cell engager ("BiTE"). In some embodiments, the activatable multispecific antibody binds to CD3 on the surface of T cells. Due to on-target, off-tumor effects, BiTE molecules are associated with high cytotoxicity, including toxicity to the central nervous system (CNS) and cytokine storms. There is a need for activatable BiTE molecules with improved specificity and reduced side effects.

[0121] This disclosure is partly based on the discovery of anti-CD3 BiTE molecules that bind to CD3 with relatively weak binding affinity (see, e.g., Figures 21A–21C and Table 6), as well as masking moieties that efficiently reduce the binding of anti-CD3 antibodies (see, e.g., Tables 4–5). Figure 46 shows a possible mechanism of action of the activatable BiTE molecule. While we do not wish to be constrained by theory, activatable BiTE molecules with relatively weak CD3 binding and / or high masking efficiency are thought to have fewer serious side effects than conventional BiTE molecules. This reduction in the severity of side effects is thought to enable a wider therapeutic range for the activatable BiTE molecules described herein. That is, the activatable BiTE molecules described herein can be administered to effectively treat diseases without producing toxic effects such as cytokine storms commonly associated with conventional BiTE molecules, e.g., BiTE molecules with stronger CD3 binding affinity. Accordingly, this application provides antibodies or antigen-binding fragments thereof, activatable antibodies, activatable multispecific antibodies, activatable antibody fragments, and polypeptides that specifically bind to human CD3 with relatively weak binding affinity.

[0122] In some embodiments, an activatable multispecific antibody is provided, comprising: a) a first antigen-binding fragment that specifically binds to CD3, fused to a first masking fragment (MM1) via a first cleavable portion (CM1); and b) a second antigen-binding fragment that specifically binds a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19); where CM1 comprises a first cleavage site; if CM1 is not cleaved, MM1 inhibits the binding of the activatable antibody to CD3; where CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment; the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of the antibody, as determined by an ELISA assay (e.g., as described in Example 5). In some embodiments, the first antigen-binding fragment binds to CD3 at a dissociation constant (kd) of at least 50 nM.

[0123] In some embodiments, an activatable multispecific antibody is provided, comprising: a) a first antigen-binding fragment that specifically binds to CD3, and is fused to a first masking fragment (MM1) via a first cleavable portion (CM1); and b) a second antigen-binding fragment that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19); where CM1 comprises a first cleavage site; if CM1 is not cleaved, MM1 inhibits the binding of the activatable antibody to CD3; where CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment; the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of the antibody as determined by an ELISA assay (e.g., described in Example 5), and where MM1 has a masking efficiency of at least 50 as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3).

[0124] In some embodiments, activatable multispecific antibodies are provided, comprising: a) a first antigen-binding fragment comprising VH1 and VL1 of anti-CD3 antibodies that specifically bind to CD3, wherein the first antigen-binding fragment is fused to a first masking moiety (MM1) via a first cleavable moiety (CM1); and b) a second antigen-binding fragment comprising VH2 and VL2 of antibodies that specifically bind to a target antigen (e.g., tumor antigens such as HER2, CD20, TROP2, BCMA, or CD19). Here, MM1 is fused to the N-terminus of VL1 via CM1, where CM1 includes a first cleavage site; if CM1 is not cleaved, MM1 inhibits the binding of the activatable antibody to CD3; if CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment; the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an ELISA assay (e.g., described in Example 5). In some embodiments, MM1 has a masking efficiency of at least 50, as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3). In some embodiments, the first antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab, and scFv. In some embodiments, the first antigen-binding fragment is scFv containing VL1, an optional linker, and VH1 from the N-terminus to the C-terminus.

[0125] In some embodiments, an activatable multispecific antibody comprising a first polypeptide, a second polypeptide, and a third polypeptide is provided, where: (i) The first polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-First CH3(1a); (ii) The second polypeptide comprises a structure represented by the following formula: MM1-CM1-VL1-VH1-Hinge-CH2-Second CH3(1b); (iii) The third polypeptide comprises a structure represented by the following formula: VL2-CL(1c); Here: VL1 is the first immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The first CH3 is the first immunoglobulin heavy chain constant domain 3; The second CH3 is the second constant domain 3 of the immunoglobulin heavy chain; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the masking area; CM1 is the cleavable portion, including the cutting site; Here, VH1 and VL1 associate to form an scFv that specifically binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an ELISA assay (e.g., described in Example 5), where VH2 and VL2 associate to form an Fv that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), where MM1 inhibits the binding of the activatable antibody to CD3 if CM1 is not cleaved; if CM1 is cleaved, the activatable multispecific antibody binds to CD3 via a first antigen-binding fragment. In some embodiments, MM1 has a masking efficiency of at least 50, as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3). In some embodiments, the multispecific antibody (e.g., its second polypeptide) includes an amino acid linker between VL1 and VH1.

[0126] In some embodiments, an activatable multispecific antibody is provided comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, where: (i) The first polypeptide comprises a structure represented by the following formula: VH1-CH1-Hinge-CH2-First CH3(3a); (ii) The second polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-Second CH3(3b); (iii) The third polypeptide comprises a structure represented by the following formula: MM1-CL1-VL1-CL(3c); (iv) The fourth polypeptide comprises a structure represented by the following formula: VL2-CL(3d); Here: VL1 is the first immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the masking area; CM1 is the cleavable portion, including the cutting site; Here, VH1 and VL1 associate to form a first Fv that specifically binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an ELISA assay (e.g., described in Example 5); here, VL2 and VH2 associate to form a second Fv that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), where MM1 inhibits the binding of the activatable antibody to CD3 if CM1 is not cleaved; and if CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment. In some embodiments, MM1 has a masking efficiency of at least 50, as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3).

[0127] In some embodiments, activatable multispecific antibodies are provided, comprising: a) a first antigen-binding fragment that specifically binds to CD3, and is fused to a first masking region (MM1) via a first cleavable region (CM1); and b) a second antigen-binding fragment that specifically binds a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), wherein the second antigen-binding fragment is fused to a second masking region (MM2) via a second cleavable region (CM2); where CM1 comprises the first cleavage site; and if CM1 is not cleaved, MM1 CM1 inhibits the binding of the activatable antibody to CD3; if CM1 is cleaved, the activatable multispecific antibody binds to CD3 via a first antigen-binding fragment; here CM2 contains a second cleavage site; if CM2 is not cleaved, MM2 inhibits the binding of the activatable antibody to the target antigen; if CM2 is cleaved, the activatable multispecific antibody binds to the target antigen via a second antigen-binding fragment; the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an ELISA assay (e.g., described in Example 5). In some embodiments, MM1 has a masking efficiency of at least 50, as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3).

[0128] In some embodiments, an activatable multispecific antibody comprising a first polypeptide, a second polypeptide, and a third polypeptide is provided, where: (i) The first polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-First CH3(2a); (ii) The second polypeptide comprises a structure represented by the following formula: MM1-CM1-VL1-VH1-Hinge-CH2-Second CH3(2b); (iii) The third polypeptide comprises a structure represented by the following formula: MM2-CM2-VL2-CL(2c); Here: VL1 is the first immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the immunoglobulin light chain constant domain; CH1 is the immunoglobulin heavy chain constant domain 1; CH2 is the immunoglobulin heavy chain constant domain 2; The first CH3 is the first immunoglobulin heavy chain constant domain 3; The second CH3 is the second immunoglobulin heavy chain constant domain 3; The hinge is the immunoglobulin hinge region connecting the CH1 and CH2 domains; MM1 is the first masking portion; CM1 is the first cleavable portion containing the first cleavage site; MM2 is the second masking portion; CM2 is the second cleavable portion containing the second cleavage site; Here, VH1 and VL1 associate to form scFv that specifically bind to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an ELISA assay (e.g., described in Example 5), where VH2 and VL2 associate to form Fv that specifically bind to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), where MM1 inhibits the binding of the activatable antibody to CD3 if CM1 is not cleaved; where CM1 is cleaved, the activatable multispecific antibody binds to CD3 via a first antigen-binding fragment; where CM2 is not cleaved, MM2 inhibits the binding of the activatable antibody to the target antigen; and where CM2 is cleaved, the activatable multispecific antibody binds to the target antigen via a second antigen-binding fragment. In some embodiments, MM1 has a masking efficiency of at least 50 as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3). In some embodiments, the multispecific antibody (e.g., its second polypeptide) contains an amino acid linker between VL1 and VH1.

[0129] In some embodiments, an activatable multispecific antibody comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide is provided, where: (i) The first polypeptide comprises a structure represented by the following formula: VH1-CH1-Hinge-CH2-First CH3(4a); (ii) The second polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-Second CH3(4b); (iii) The third polypeptide comprises a structure represented by the following formula: MM1-CL1-VL1-CL(4c); (iv) The fourth polypeptide comprises a structure represented by the following formula: MM2-CL2-VL2-CL(4d); Here: VL1 is the first immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the first masking section; CM1 is a first cleavable portion including the first cutting site; MM2 is the second masking section; CM2 is the second cleavable portion, which includes the second cutting site; Here, VH1 and VL1 associate to form a first Fv that specifically binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an ELISA assay (e.g., described in Example 5); here, VL2 and VH2 associate to form a second Fv that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), where, if CM1 is not cleaved, MM1 inhibits the binding of the activatable antibody to CD3; here, if CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment; here, if CM2 is not cleaved, MM2 inhibits the binding of the activatable antibody to the target antigen; and here, if CM2 is cleaved, the activatable multispecific antibody binds to the target antigen via the second antigen-binding fragment. In some embodiments, MM1 has a masking efficiency of at least 50, as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3).

[0130] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a weak binding affinity. In some embodiments, the first antigen-binding fragment binds to CD3 with a relatively weak binding affinity compared to the KD of the reference antibody against CD3. In some embodiments, the first antigen-binding fragment binds to CD3 with a higher dissociation constant than the reference antibody against CD3. In some embodiments, the first antigen-binding fragment binds to CD3 with a lower dissociation constant than the reference antibody against CD3. In some embodiments, the reference antibody is SP34. In some embodiments, the binding affinity of the first antigen-binding fragment to CD3 is measured when the first antigen-binding fragment exists as an isolated antigen-binding fragment or as part of a monospecific antibody. In some embodiments, the binding affinity of the first antigen-binding fragment to CD3 is measured when the first antigen-binding fragment exists in a multispecific antibody or in an activated form of an activatable multispecific antibody, i.e., CM1 is cleaved and MM1 is not bound to the first antigen-binding fragment.

[0131] In some embodiments, the first antigen-binding fragment is at least about 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, 145, 150, 160, 175, 200, 250 nM or higher (including any value or range between these values) of the semi-maximal binding concentration (EC) of any one antibody determined by enzyme-linked immunosorbent assay (ELISA). 50) binds to CD3 (e.g., human CD3). In some embodiments, the first antigen-binding domain binds to human CD3 with one of the following EC50 values, determined by enzyme-linked immunosorbent assay (ELISA): 10-50, 50-100, 100-150, 150-200, 10-100, 10-110, 9-111, 10-115, 75-150, 100-150, 10-150, 10-200, 50-125, 10-20, 20-50, 50-75, 75-125, 90-120, 100-120, 100-110, 110-120, 50-150, 50-200, or 10-250 nM. In some embodiments, EC50 is determined by an ELISA measuring the binding of an unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the first antigen-binding fragment is scFv, and EC50 is determined by an ELISA measuring the binding of an unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, EC50 is determined by an ELISA measuring the binding of a parent multispecific antibody lacking CM and MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the first antigen-binding fragment is scFv, and EC50 is determined by an ELISA measuring the binding of a parent multispecific antibody lacking CM and MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, EC50 measures the binding of an antigen-binding fragment (e.g., isolated anti-CD3 scFv or scFv-Fc fusion protein) to CD3 (e.g., human CD3 or human CD3δε), as determined by an ELISA.

[0132] In some embodiments, the first antigen-binding fragment is the EC of a reference antibody (e.g., SP34). 50Therefore, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500 times or more, whichever is one higher EC. 50 It binds to CD3 (e.g., human CD3) (including any value or range between these values). In some embodiments, the EC of the first antigen-binding fragment reference antibody (e.g., SP34) 50 More specifically, one of the following EC values ​​is higher: approximately 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-200, 200-500, 2-5, 5-10, 5-20, 5-30, 5-40, 5-50, 5-55, 5-60, 10-20, 10-30, 10-40, 10-50, 10-60, 20-40, 20-55, 30-60, 10-30, or 5-100 times. 50It binds to CD3 (e.g., human CD3). In some embodiments, the EC50 of the first antigen-binding fragment and the reference antibody are measured under the same test conditions. In some embodiments, the EC50 of the first antigen-binding fragment and the reference antibody are measured in the same antibody format. In some embodiments, the EC50 is determined by measuring the binding of an unmasked multispecific antibody and an unmasked multispecific reference antibody to CD3. In some embodiments, the unmasked multispecific reference antibody includes a CD3 binding moiety corresponding to SP34 (e.g., including six CDRs of SP34). In some embodiments, the EC50 is determined by measuring the binding of a parent multispecific antibody lacking CM and MM and a reference parent multispecific antibody lacking CM and MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the reference parent multispecific antibody lacking CM and MM includes a CD3 binding moiety corresponding to SP34 (e.g., including six CDRs of SP34). In some embodiments, the Kd binding to CD3 of the first antigen-binding fragment and the EC50 of the reference antibody are determined by ELISA, for example, the ELISA described in Example 3. In some embodiments, the Kd binding to CD3 of the first antigen-binding fragment and the EC50 of the reference antibody are determined by cell-based assay, for example, the Jurkat NFAT reporter assay described in Example 3.

[0133] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a relatively weak dissociation constant (Kd) compared to a reference antibody (e.g., SP34), for example, one weaker than the Kd of the reference antibody by at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500 times or more (including any value or range between these values). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a dissociation constant (Kd) that is one less than approximately 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-200, 200-500, 2-5, 5-10, 5-20, 5-30, 5-40, 5-50, 5-55, 5-60, 10-20, 10-30, 10-40, 10-50, 10-60, 20-40, 20-55, 30-60, 10-30, or 5-100 times weaker than the Kd of the reference antibody (e.g., SP34). In some embodiments, the Kd of the first antigen-binding fragment and the reference antibody are measured under the same test conditions. In some embodiments, the kd of the first antigen-binding fragment and the reference antibody are measured in the same antibody format. In some embodiments, the kd is determined by measuring the binding of an unmasked multispecific antibody and an unmasked multispecific reference antibody to CD3. In some embodiments, the unmasked multispecific reference antibody includes a CD3 binding moiety corresponding to SP34 (e.g., including six CDRs of SP34). In some embodiments, the kd is determined by measuring the binding of a parent multispecific antibody lacking CM and MM and a reference parent multispecific antibody lacking CM and MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the reference parent multispecific antibody lacking CM and MM includes a CD3 binding moiety corresponding to SP34 (e.g., including six CDRs of SP34). In some embodiments, the kd of the first antigen-binding fragment binding to CD3 and the kd of the reference antibody are determined by ELISA.

[0134] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with at least one dissociation constant (Kd) of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 nM or higher (including any value or range between these values). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with at least one dissociation constant (Kd) of about 1, 10, or 100 μM (including any value or range between these values) (when in the activated form). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a dissociation constant (Kd) of approximately one of the following: 10-50, 50-100, 100-200, 200-500, 500-1000, 10-100, 100-500, 100-1000, 50-200, 50-250, 50-500, or 10-1000 nM.

[0135] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a relatively fast off-rate (koff) compared to the reference antibody (e.g., SP34), e.g., the k of the reference antibody. off It is at least one of the following speeds: approximately 2, 5, 10, 20, 50, 100, 200 times, or more (including any value or range between these values).

[0136] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) at a relatively slow on-rate (kon) compared to the reference antibody (e.g., SP34), e.g., the k of the reference antibody. on It is at least one of the following values ​​slower: approximately 2, 5, 10, 20, 50, 100, 200 times, or more (including any value or range between these values).

[0137] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a relatively small dissociation constant (Kd) compared to a reference antibody (e.g., SP34), for example, at least about 2, 5, 10, 20, 50, 100, 200 times or more than the Kd of the reference antibody (including any value or range between these values).

[0138] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a relatively large binding constant (ka) compared to the reference antibody (e.g., SP34), e.g., k of the reference antibody. a It is at least one of the following: approximately 2, 5, 10, 20, 50, 100, 200 times, or more.

[0139] Methods for measuring the ability of an antibody (e.g., an activatable multispecific antibody) to bind to an antigen are known in the art and include, but are not limited to, via BIAcore analysis, surface plasmon resonance, ELISA, flow cytometry, and cell-based assays (e.g., measuring binding to Jurkat cells) (see, e.g., Example 5 and Table 6). The EC50, dissociation constant (Kd), association constant (ka), off-rate (koff), and / or on-rate (kon) of binding to CD3 (e.g., human CD3) can be measured in various contexts. In some embodiments, binding to CD3 (e.g., human CD3) is measured using an antigen-binding fragment that binds to CD3 (e.g., scFv or scFv-Fc fusion protein). In some embodiments, binding to CD3 (e.g., human CD3) is measured using an unmasked multispecific antibody. In some embodiments, binding to CD3 (human CD3) is measured using an activatable antibody (e.g., an activatable multispecific antibody), and the cleavable portion associated with the anti-CD3 antigen-binding fragment is cleaved. In some embodiments, binding to human CD3δε is measured. In some embodiments, binding to human CD3δε fused to an Fc fragment is measured. In some embodiments, binding to Jurkat cells is measured.

[0140] In some embodiments, ELISA is performed using human CD3 (a heterodimer of the ε and δ chains) fused to a human Fc fragment as a binding substrate. An exemplary ELISA method is as follows: 1. Prepare 2 μg / mL of human CD3 (a heterodimer of the ε and δ chains) fused to a human Fc fragment and coat an ELISA plate overnight at 2-8°C. 2. After washing and blocking, add 50 μL of serially diluted IgG (e.g., the first antigen-binding fragment, unmasked multispecific antibody, or activatable antibody (e.g., an activatable multispecific antibody)) and incubate at 37°C for 1 hour. 3. Wash the plate three times and incubate it with 50 μL / well of TMB substrate at room temperature for approximately 20 minutes. 4. Stop the reaction. 5. Measure the absorbance at 450 nm. 6. The concentration of each antibody that showed semi-maximal binding to CD3εδ is determined as EC50 (nM).

[0141] The first antigen-binding fragment and / or the second antigen-binding fragment may be in any preferred format, including but not limited to Fab, Fv, scFab, and scFv. The antigen-binding fragment may have a single-stranded polypeptide or two or more polypeptide chains. A masking moiety (e.g., MM1 or MM2) may be fused to the N-terminus of any one polypeptide chain of the antigen-binding fragment having multiple polypeptide chains. In some embodiments, the masking moiety (e.g., MM1 or MM2) is fused to the N-terminus of the VL (e.g., VL1 or VL2) of the antigen-binding fragment. In some embodiments, the masking moiety (e.g., MM1 or MM2) is fused to the N-terminus of the VH (e.g., VH1 or VH2) of the antigen-binding fragment.

[0142] The first antigen-binding fragment may be derived from any one of the anti-CD3 antibodies described herein, which have an EC50 of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., described in Example 5). The anti-CD3 antibody and antigen-binding fragment may be any one of those described in Section i) "Anti-CD3 Antibodies" and Tables 5B-5H.

[0143] In some embodiments, the first antigen-binding fragment comprises one, two, three, four, five, or six CDRs of an antibody as shown in Table 7. In some embodiments, the first antigen-binding fragment of a multispecific antibody comprises one, two, three, four, five, or six CDRs of an anti-CD3 antibody TY25051, TY25236, TY25023, TY25024, TY25237, TY25228, TY25227, TY25230, TY25229, TY25238, TY25239, TY25243, TY25231, TY25244, TY25241, or TY25240 as shown in Table 7. In some embodiments, the first antigen-binding fragment comprises VH1 and / or VL1 as shown in Table 8. In some embodiments, the first antigen-binding fragment comprises VH1 and / or VL1 of anti-CD3 antibodies TY24051, TY25236, TY25023, TY25024, TY25237, TY25228, TY25227, TY25230, TY25229, TY25238, TY25239, TY25243, TY25231, TY25244, TY25241, or TY25240, as shown in Table 8.

[0144] In some embodiments, the first antigen-binding fragment comprises one, two, three, four, five, or six CDRs of antibody TY25023 as shown in Table 7. In some embodiments, the first antigen-binding fragment comprises the VH and / or VL of antibody TY25023 as shown in Table 8. In some embodiments, the first antigen-binding fragment comprises the scFv of antibody TY25023 as shown in Table 9. In some embodiments, the first antigen-binding fragment comprises the heavy chain of antibody TY25023 as shown in Table 12.

[0145] In some embodiments, the first antigen-binding fragment comprises a VH1 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 402. In certain embodiments, the VH1 sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 402, but retains the same CD3-binding ability as the antibody containing SEQ ID NO: 402. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 402. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In a particular embodiment, VH1 comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 392, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395.

[0146] In some embodiments, the first antigen-binding fragment includes a VL1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 403. In certain embodiments, the VL1 sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 403, but retains the same CD3-binding ability as the antibody containing SEQ ID NO: 403. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 403. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In a particular embodiment, VL1 comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 400.

[0147] In some embodiments, the first antigen-binding fragment includes VH1, which includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395, and VL1, which includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 400.

[0148] In some embodiments, the first antigen-binding fragment includes VH1, which contains the amino acid sequence of SEQ ID NO: 402, and VL1, which contains the amino acid sequence of SEQ ID NO: 403.

[0149] In some embodiments, the first antigen-binding fragment includes VH1, comprising VH CDR-H1, CDR-H2, and CDR-H3 having the sequence described in SEQ ID NO: 402; and VL1, comprising VL CDR-L1, CDR-L2, and CDR-L3 having the sequence described in SEQ ID NO: 403.

[0150] In some embodiments, the first antigen-binding fragment comprises one, two, three, four, five, or six CDRs of antibody TY25238 as shown in Table 7. In some embodiments, the first antigen-binding fragment comprises VH1 and / or VL1 of antibody TY25238 as shown in Table 8. In some embodiments, the first antigen-binding fragment comprises the scFv of antibody TY25238 as shown in Table 9. In some embodiments, the first antigen-binding fragment comprises the heavy chain of antibody TY25238 as shown in Table 12.

[0151] In some embodiments, the first antigen-binding fragment comprises a VH1 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 410. In certain embodiments, the VH1 sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 410, but retains the same ability to bind to human CD3 as the antibody containing SEQ ID NO: 410. In certain embodiments, a total of 1 to 13 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 410. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In a particular embodiment, VH1 comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395.

[0152] In some embodiments, the first antigen-binding fragment includes a VL1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 411. In certain embodiments, the VL1 sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 411, but retains the same ability to bind to human CD3 as the antibody containing SEQ ID NO: 411. In certain embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 411. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In a particular embodiment, VL1 comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381.

[0153] In some embodiments, the first antigen-binding fragment includes VH1, which includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 395, and VL1, which includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381.

[0154] In some embodiments, the first antigen-binding fragment includes VH1, which contains the amino acid sequence of SEQ ID NO: 410, and VL1, which contains the amino acid sequence of SEQ ID NO: 411.

[0155] In some embodiments, the first antigen-binding fragment includes VH1 comprising VH CDR-H1, CDR-H2, and CDR-H3 having the sequence described in SEQ ID NO: 410; and VL1 comprising VL CDR-L1, CDR-L2, and CDR-L3 having the sequence described in SEQ ID NO: 411.

[0156] In some embodiments, the first antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 421 or SEQ ID NO: 422.

[0157] Any one of the masking sites for the anti-CD3 antibody described herein can be used, for example, including the masking sites in Section F, “Masking Sites (MM),” and Tables B, 18-22, 13A, and 40. In some embodiments, the first masking site (MM1) comprises the amino acid sequence of formula (IX):PYDDPDCPSHX1SDCDX2 (SEQ ID NO: 668) (wherein X1 is D or E, and X2 is N or Q). In some embodiments, the first masking site (MM1) comprises the amino acid sequence of formula (X):X1X2X3DX4X5CX6X7DX8X9X 10 CX 11 X 12(Sequence ID 669) (wherein X1 is A or D, X2 is A, D or P, X3 is D, H or P, X4 is F or P, X5 is D or P, X6 is D or P, X7 is A or P, X8 is D, N or P, X9 is A, N or P, X 10 is D, H, or S, X 11 is H, P, or Y, and X 12 The first masking portion (MM1) includes an amino acid sequence of (where is N, P, or Y). In some embodiments, the first masking portion (MM1) includes the amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of MM1. In some embodiments, the first masking portion (MM1) includes the amino acid sequence of SEQ ID NO: 417 (EVGSYPYDDPDCPSHESDCDQ). In some embodiments, the first masking portion (MM1) includes the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first masking portion (MM1) includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 585-588. In some embodiments, the first masking portion (MM1) includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 597-599.

[0158] In some embodiments, the masking efficiency of MM1 is at least one of approximately 2, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 490, 500, 510, 550, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 10000, or more. In some embodiments, the masking efficiency of MM1 is one of approximately 2-10, 10-20, 20-50, 50-100, 40-510, 50-500, 100-200, 100-500, 200-500, 300-500, 400-500, 400-600, 500-1000, 1000-5000, 5000-10000, 10-100, 100-500, 100-1000, 1000-10000, 10-1000, or 100-10000. In some embodiments, the masking efficiency of MM1 is at least 50. In some embodiments, the masking efficiency of MM1 is at least one of approximately 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In some embodiments, the masking efficiency of MM1 is 50–500. In some embodiments, the masking efficiency of MM1 is 500. In some embodiments, the masking efficiency is measured as the difference in affinity for binding to the target (e.g., human CD3) of a pre-activatable antibody containing the first masking moiety compared to the affinity for binding to the target (e.g., human CD3) of a corresponding unmasked antibody ("parent antibody") or a post-activatable antibody lacking the first masking moiety. In some embodiments, masking efficiency is measured as the difference in activity (e.g., activation of the NFAT promoter) of the pre-activation activatable antibody containing the first masking moiety to bind to a target (e.g., human CD3) compared to the activity of the parent antibody or the activated activatable antibody.In some embodiments, masking efficiency is measured as the difference in binding levels of the pre-activation activatable antibody containing the first masking moiety to cells expressing its target (e.g., cells expressing human CD3), compared to the activity of the parent antibody or the activated activatable antibody. In some embodiments, masking efficiency is measured by dividing the EC50 of the pre-activation activatable antibody containing the first masking moiety by the EC50 of the parent antibody. The EC50 value can be measured by an ELISA assay or a Jurkat NFAT reporter assay; see, for example, the method in Example 3. In some embodiments, masking efficiency is measured by dividing the kd of the pre-activation activatable antibody containing the first masking moiety by the kd of the parent antibody.

[0159] Any one of the cleavable segments described herein can be used, for example, including the cleavable segments in Section G, “Cleavable Segments (CM),” and Tables 13A, 18-22, and 40. In some embodiments, the first cleavable segment (CM1) includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 418, 420, 431, and 477-490, and 516-555. In some embodiments, the first cleavable segment (CM1) includes the amino acid sequence of SEQ ID NO: 418 (GGGPLGLAGGS). In some embodiments, the first cleavable segment (CM1) includes the amino acid sequence of SEQ ID NO: 77 (GGGPLGLAGSGGS).

[0160] The second antigen-binding fragment can specifically bind to a target antigen, such as a tumor antigen. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the target antigen is a tumor-associated antigen (TAA). In some embodiments, the target antigens are CD19, CD20, EpCAM, CEA, PSMA, CD33, EGFR, HER2, EphA2, MCSP, ADAM17, PSCA, 17-A1, NKG2D, TROP2, CD79B, Nectin-4, BCMA, CD22, CD38, EGFR, GD2, SLAMF7, CD30, EpCAM, MUC1, MUC16, CD123, CD37, FOLR1, MET, FLT3, GPC3, CEACAM5, CLDN18, CSF1, Integrin α5, NCAM1, PTPRC, CD138, NaPi2b, MSLN, DLL3, GPRC5D, GPNMB, ICAM1, SSTR2, and cancer-related antigens. The target antigen is selected from the group consisting of CTAA16, CA9, ENG, ACVRL1, CD80, CSPG4, EGFL7, FLT1, HAVCR1, HGF, HLA-DRB, IGF1R, TPBG, ERBB3, and STEAP2. In some embodiments, the target antigen is HER2. In some embodiments, the target antigen is CD20. In some embodiments, the target antigen is TROP2. The second antigen-binding fragment may be derived from any one of the non-CD3 antibodies described in Section H, “Target-Binding Molecules (TBMs)” (e.g., an anti-HER2 antibody and an anti-CD20 antibody).

[0161] In some embodiments, the second antigen-binding fragment is fused to the second masking portion (MM2) via the second cleavable portion (CM2). In some embodiments, the second antigen-binding fragment is not masked. In some embodiments, the second antigen-binding fragment is not fused to the second masking portion. Any suitable masking portion can be used, for example, the anti-HER2 masking portion described in Section F, “Masking Portion (MM)”. Any suitable cleavable portion can be used, for example, the cleavable portion described in Section G, “Cleavable Portion (CM)”.

[0162] In some embodiments, the activatable multispecific antibody comprises a second antigen-binding fragment containing a second immunoglobulin light chain variable domain (VL2) and a second immunoglobulin heavy chain variable domain (VH2) of the antibody that specifically binds to HER2. In some embodiments, the second antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs of trastuzumab. In some embodiments, the second antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs as shown in Table 10. In some embodiments, VH2 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 423, CDR-H2 containing the amino acid sequence of SEQ ID NO: 424, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 71, and VL2 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 75, and VL2 comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the second antigen-binding fragment is fused to the second masking portion (MM2) via a second cleavable portion (CM2). In some embodiments, MM2 comprises the amino acid sequence of formula (XI):ESX1X2CX3X4DPFX5CQX6 (SEQ ID NO: 670) (wherein X1 is D or E, X2 is A, F, V or Y, X3 is D or E, X4 is A or L, X5 is D or E, and X6 is A, F or Y). In some embodiments, MM2 comprises the amino acid sequence of formula (XII):X1X2X3X4X5X6CX7X8DPYECX9X 10 (Sequence ID 671) (wherein X1 is A, H or S, X2 is A, D or S, X3 is A, T or V, X4 is P, S or T, X5 is D or E, X6 is A or V, X7 is D or E, X8 is A or L, X9 is Q, S or T, X 10The amino acid sequence includes (where X1 is A, H, or V). In some embodiments, MM2 includes the amino acid sequence of formula (XIII):YNSDDDCX1SX2YDPYTCYY(SEQ ID NO: 672) (wherein X1 is A, I, or V, and X2 is H or R). In some embodiments, MM2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 419, 432-476, and 491-515. In some embodiments, MM2 includes the amino acid sequence of SEQ ID NO: 419 (ESDACDADPFDCQA). In some embodiments, MM2 includes the amino acid sequence of SEQ ID NO: 36. In some embodiments, CM2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 418, 420, 431, and 477-490, and 516-555. In some embodiments, CM2 includes the amino acid sequence of SEQ ID NO: 420. In some embodiments, CM2 includes the amino acid sequence of SEQ ID NO: 77.

[0163] In some embodiments, the activatable multispecific antibody comprises a second antigen-binding fragment comprising a second immunoglobulin light chain variable domain (VL2) and a second immunoglobulin heavy chain variable domain (VH2) of the antibody that specifically binds to CD20. In some embodiments, the second antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs as shown in Table C. In some embodiments, VH2 comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 556, CDR-H2 containing the amino acid sequence of SEQ ID NO: 557, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 558, and VL2 comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 559, CDR-L2 containing the amino acid sequence of SEQ ID NO: 560, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 561. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 562, and VL2 comprises the amino acid sequence of SEQ ID NO: 563.

[0164] In some embodiments, the activatable multispecific antibody includes an Fc region. In some embodiments, the Fc region is of the human IgG1 subclass. In some embodiments, the Fc region is of the human IgG4 subclass. In some embodiments, the activatable multispecific antibody includes one of the Fc regions described in Section J, "Fc Region and CH3 Domain". In some embodiments, the activatable multispecific antibody includes one of the CH3 domain mutations described in Section J, "Fc Region and CH3 Domain," including the mutations described in Table DF.

[0165] In some embodiments, the activatable multispecific antibody comprises a first CH3 domain and a second CH3 domain, where the first CH3 domain comprises D356K, E357K, S364K and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C and K439D substitutions, or the first CH3 domain comprises L351D, K370D, N390C and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K and S400C substitutions.

[0166] In some embodiments, the activatable multispecific antibody is a bispecific antibody. In some embodiments, the activatable multispecific antibody is an activatable BiTE molecule. Exemplary activatable BiTE molecules are shown, for example, in Tables 2 and 3A.

[0167] In some embodiments, the activatable multispecific antibody is an activatable BiTE targeting human CD3 and HER2.

[0168] In some embodiments, an activatable HER2xCD3 BiTE is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 115, a second polypeptide containing the amino acid sequence of SEQ ID NO: 116, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 117.

[0169] In some embodiments, an activatable HER2xCD3 BiTE is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 425, a second polypeptide containing the amino acid sequence of SEQ ID NO: 426, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 112.

[0170] In some embodiments, an activatable HER2xCD3 BiTE is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 427, a second polypeptide containing the amino acid sequence of SEQ ID NO: 428, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 112.

[0171] In some embodiments, an activatable HER2xCD3 BiTE is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 429, a second polypeptide containing the amino acid sequence of SEQ ID NO: 430, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 115.

[0172] In some embodiments, an activatable HER2xCD3 BiTE is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 83, a second polypeptide containing the amino acid sequence of SEQ ID NO: 84, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 85.

[0173] In some embodiments, an activatable HER2xCD3 BiTE is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 683, a second polypeptide containing the amino acid sequence of SEQ ID NO: 684, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 685.

[0174] In some embodiments, the activatable multispecific antibody is an activatable BiTE targeting human CD3 and CD20.

[0175] In some embodiments, an activatable CD20xCD3 BiTE is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 564, a second polypeptide containing the amino acid sequence of SEQ ID NO: 565, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 567.

[0176] In some embodiments, an activatable CD20xCD3 BiTE is provided, comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 564, a second polypeptide containing the amino acid sequence of SEQ ID NO: 565, and a third polypeptide containing the amino acid sequence of SEQ ID NO: 569.

[0177] In some embodiments, the activatable multispecific antibody is an activatable BiTE targeting human CD3 and TROP2.

[0178] In some embodiments, the activatable multispecific antibody is an activatable BiTE targeting human CD3 and BCMA.

[0179] In some embodiments, the activatable multispecific antibody is an activatable BiTE targeting human CD3 and CD19.

[0180] In some embodiments, the activatable multispecific antibody is cross-reactive with CD3 polypeptides derived from at least one non-human species selected from the group consisting of cynomolgus monkeys, mice, rats, and dogs.

[0181] B. Masked multispecific anti-CD3 antibody As stated above, this disclosure is partly based on the discovery of anti-CD3 antibodies that bind to CD3 with relatively weak binding affinity (see, e.g., Figures 21A-21C and Table 6), as well as masking moieties that block the binding of anti-CD3 antibodies (see, e.g., Tables 4-5). Generally, masking antibodies consist of a masking moiety that binds to the target binding moiety of the antibody, and when the masking moiety is bound to the target binding, it reduces the binding of the antibody to the target. Masked antibodies may contain a cleavable or non-cleavable linker between the masking moiety and the antigen-binding fragment. While we do not wish to be bound by theory, if a masking antibody contains a non-cleavable linker, it is thought that the masking antibody may be in a dynamic equilibrium between a masked state in which the target binding moiety is bound to the masking moiety and a target-bound state in which the target binding moiety is bound to the target. Therefore, the relative binding affinity of the masking moiety to the target binding moiety and the target binding moiety to the target binding moiety, as well as the local concentrations of the target and masking antibodies, determine the extent to which the antibody actually binds to the target. While we do not wish to be bound by theory, masked multispecific antibodies with relatively weak CD3 binding and / or high masking efficiency are thought to have fewer serious side effects than conventional BiTE molecules. This reduction in the severity of side effects is thought to enable a wider therapeutic range for the masked multispecific antibodies described herein. That is, the masked multispecific antibodies described herein can be administered to effectively treat diseases without causing toxic effects such as cytokine storms commonly associated with conventional BiTE molecules, for example, BiTE molecules with stronger CD3 binding affinity.

[0182] In some embodiments, a multispecific antibody is provided comprising: a) a first antigen-binding fragment comprising VH1 and VL1 of an anti-CD3 antibody fused to a first masking moiety (MM1) and specifically binding to CD3; and b) a second antigen-binding fragment comprising VH2 and VL2 of an antibody specifically binding to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19); where MM1 is fused to the N-terminus of VL1; where MM1 specifically binds to the CD3 binding moiety in competition with CD3; the multispecific antibody binds to CD3 via the first antigen-binding fragment; and the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of the antibody, as determined by an ELISA assay (e.g., as described in Example 5). In some embodiments, MM1 has a masking efficiency of at least 50, as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3). In some embodiments, the first antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab, and scFv. In some embodiments, the first antigen-binding fragment is scFv containing VL1, an optional linker, and VH1 from the N-terminus to the C-terminus.

[0183] In some embodiments, the masked multispecific antibody is an activatable antibody. In some embodiments, the multispecific antibody contains a cleavable moiety. See, for example, activatable multispecific T cell engagers.

[0184] In some embodiments, the multispecific antibody is not an activatable multispecific antibody. In some embodiments, the multispecific antibody does not contain a cleavable moiety. In some embodiments, the first antigen-binding fragment comprises a first immunoglobulin light chain variable domain (VL1) and a first immunoglobulin heavy chain variable domain (VH1) of an anti-CD3 antibody, wherein MM1 is fused to the N-terminus of VL1 via a first non-cleavable linker (NCL1). In some embodiments, NCL1 is any one of the non-cleavable linkers known in the art. In some embodiments, NCL1 is any one of the non-cleavable linkers described in Section I. Linkers.

[0185] In some embodiments, a multispecific antibody comprising a first polypeptide, a second polypeptide, and a third polypeptide is provided, where: (i) The first polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-First CH3(1a); (ii) The second polypeptide comprises a structure represented by the following formula: MM1-NCL1-VL1-VH1-Hinge-CH2-Second CH3(1b); (iii) The third polypeptide comprises a structure represented by the following formula: VL2-CL(1c); Here: VL1 is the first immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The first CH3 is the first immunoglobulin heavy chain constant domain 3; The second CH3 is the second constant domain 3 of the immunoglobulin heavy chain; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the masking area; NCL1 is a non-cuttable linker; Here, VH1 and VL1 associate to form an scFv that specifically binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an ELISA assay (e.g., described in Example 5), where VH2 and VL2 associate to form an Fv that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), where MM competes with CD3 to specifically bind to the CD3 binding site; the multispecific antibody binds to CD3 via a first antigen-binding fragment. In some embodiments, MM1 has a masking efficiency of at least 50, as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3). In some embodiments, the multispecific antibody (e.g., its second polypeptide) includes an amino acid linker between VL1 and VH1.

[0186] In some embodiments, a multispecific antibody comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide is provided, where: (i) The first polypeptide comprises a structure represented by the following formula: VH1-CH1-Hinge-CH2-First CH3(3a); (ii) The second polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-Second CH3(3b); (iii) The third polypeptide comprises a structure represented by the following formula: MM1-NCL1-VL1-CL(3c); (iv) The fourth polypeptide comprises a structure represented by the following formula: VL2-CL(3d); Here: VL1 is the first immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the masking area; NCL1 is a non-cuttable linker; Here, VH1 and VL1 associate to form a first Fv that specifically binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an ELISA assay (e.g., described in Example 5); here, VL2 and VH2 associate to form a second Fv that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), where MM competes with CD3 to specifically bind to the CD3 binding site; and the multispecific antibody binds to CD3 via the first antigen-binding fragment. In some embodiments, MM1 has a masking efficiency of at least 50, as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3).

[0187] In some embodiments, a multispecific antibody is provided comprising: a) a first antigen-binding fragment that specifically binds to CD3 and is fused to a first masking portion (MM1); and b) a second antigen-binding fragment that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), wherein the second antigen-binding fragment is fused to a second masking portion (MM2) via a cleavable portion (CM); where MM1 specifically binds to the CD3 binding portion in competition with CD3; where CM includes a cleavage site; where MM2 inhibits the binding of the multispecific antibody to the target antigen if CM is not cleaved; where CM is cleaved, the multispecific antibody binds to the target antigen via the second antigen-binding fragment; and the first antigen-binding fragment binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of the antibody, as determined by an ELISA assay (e.g., as described in Example 5). In some embodiments, MM1 has a masking efficiency of at least 50 as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3).

[0188] In some embodiments, a multispecific antibody comprising a first polypeptide, a second polypeptide, and a third polypeptide is provided, where: (i) The first polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-First CH3(2a); (ii) The second polypeptide comprises a structure represented by the following formula: MM1-NCL1-VL1-VH1-Hinge-CH2-Second CH3(2b); (iii) The third polypeptide comprises a structure represented by the following formula: MM2-NCL2-VL2-CL(2c); Here: VL1 is the first immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The first CH3 is the first immunoglobulin heavy chain constant domain 3; The second CH3 is the second constant domain 3 of the immunoglobulin heavy chain; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the first masking section; NCL1 is the first non-cuttable linker; MM2 is the second masking section; NCL2 is the second non-cuttable linker; Here, VH1 and VL1 associate to form scFv that specifically bind to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody determined by an ELISA assay (e.g., described in Example 5), where VH2 and VL2 associate to form Fv that specifically bind to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), where MM1 specifically binds to the CD3 binding site in competition with CD3; the multispecific antibody binds to CD3 via a first antigen-binding fragment; MM2 inhibits the binding of the multispecific antibody to the target antigen; and the multispecific antibody binds to the target antigen via a second antigen-binding fragment.

[0189] In some embodiments, a multispecific antibody comprising a first polypeptide, a second polypeptide, and a third polypeptide is provided, where: (i) The first polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-First CH3(2a); (ii) The second polypeptide comprises a structure represented by the following formula: MM1-NCL1-VL1-VH1-Hinge-CH2-Second CH3(2b); (iii) The third polypeptide comprises a structure represented by the following formula: MM2-CM-VL2-CL(2c); Here: VL1 is the first immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The first CH3 is the first immunoglobulin heavy chain constant domain 3; The second CH3 is the second constant domain 3 of the immunoglobulin heavy chain; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the first masking section; NCL1 is a non-cuttable linker; MM2 is the second masking section; CM is the dicable portion, including the cutting site; Here, VH1 and VL1 associate to form an scFv that specifically binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an ELISA assay (e.g., described in Example 5), where VH2 and VL2 associate to form an Fv that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), where MM1 specifically binds to the CD3 binding moiety in competition with CD3; the multispecific antibody binds to CD3 via a first antigen-binding fragment; where MM2 inhibits the binding of the multispecific antibody to the target antigen if the CM is not cleaved; if the CM is cleaved, the multispecific antibody binds to the target antigen via a second antigen-binding fragment. In some embodiments, MM1 has a masking efficiency of at least 50 as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3). In some embodiments, the multispecific antibody (e.g., its second polypeptide) contains an amino acid linker between VL1 and VH1.

[0190] In some embodiments, a multispecific antibody comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide is provided, where: (i) The first polypeptide comprises a structure represented by the following formula: VH1-CH1-Hinge-CH2-First CH3(4a); (ii) The second polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-Second CH3(4b); (iii) The third polypeptide comprises a structure represented by the following formula: MM1-NCL1-VL1-CL(4c); (iv) The fourth polypeptide comprises a structure represented by the following formula: MM2-NCL2-VL2-CL(4d); Here: VL1 is the first immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the first masking section; NCL1 is the first non-cuttable linker; MM2 is the second masking section; NCL2 is the first non-cuttable linker; Here, VH1 and VL1 associate to form a first Fv that specifically binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody determined by an ELISA assay (e.g., described in Example 5); here, VL2 and VH2 associate to form a second Fv that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), where MM1 competes with CD3 to specifically bind to the CD3 binding portion; the multispecific antibody binds to CD3 via the first antigen-binding fragment; here, MM2 inhibits the binding of the multispecific antibody to the target antigen if the CM is not cleaved; if the CM is cleaved, the multispecific antibody binds to the target antigen via the second antigen-binding fragment. In some embodiments, a multispecific antibody comprising a first polypeptide, a second polypeptide, a third polypeptide and a fourth polypeptide is provided, where: (i) The first polypeptide comprises a structure represented by the following formula: VH1-CH1-Hinge-CH2-First CH3(4a); (ii) The second polypeptide comprises a structure represented by the following formula: VH2-CH1-Hinge-CH2-Second CH3(4b); (iii) The third polypeptide comprises a structure represented by the following formula: MM1-NCL1-VL1-CL(4c); (iv) The fourth polypeptide comprises a structure represented by the following formula: MM2-CM-VL2-CL(4d); Here: VL1 is the first immunoglobulin light chain variable domain; VH1 is the first immunoglobulin heavy chain variable domain; VL2 is the second immunoglobulin light chain variable domain; VH2 is the second immunoglobulin heavy chain variable domain; CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the first masking section; NCL1 is a non-cuttable linker; MM2 is the second masking section; CM is the dicable portion, including the cutting site; Here, VH1 and VL1 associate to form a first Fv that specifically binds to CD3 at a semi-maximal binding concentration (EC50) of at least 10 nM (e.g., at least 100 nM) of antibody, as determined by an ELISA assay (e.g., described in Example 5); here, VL2 and VH2 associate to form a second Fv that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), where MM1 competes with CD3 to specifically bind to the CD3 binding portion; the multispecific antibody binds to CD3 via the first antigen-binding fragment; here, MM2 inhibits the binding of the multispecific antibody to the target antigen if the CM is not cleaved; if the CM is cleaved, the multispecific antibody binds to the target antigen via the second antigen-binding fragment. In some embodiments, MM1 has a masking efficiency of at least 50, as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3).

[0191] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a weak binding affinity. In some embodiments, the first antigen-binding fragment binds to CD3 with a relatively weak binding affinity compared to the KD of the reference antibody against CD3. In some embodiments, the first antigen-binding fragment binds to CD3 with a higher dissociation constant than the reference antibody against CD3. In some embodiments, the first antigen-binding fragment binds to CD3 with a lower dissociation constant than the reference antibody against CD3. In some embodiments, the reference antibody is SP34. In some embodiments, the binding affinity of the first antigen-binding fragment to CD3 is measured when the first antigen-binding fragment exists as an isolated antigen-binding fragment or as part of a monospecific antibody. In some embodiments, the binding affinity of the first antigen-binding fragment to CD3 is measured when the first antigen-binding fragment exists within a multispecific antibody.

[0192] In some embodiments, the first antigen-binding fragment is used to bind at least about 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, 145, 150, 160, 175, 200, 250 nM or higher to the semi-maximal binding concentration (EC) of an antibody at any value or range between these values, as determined by an enzyme-linked immunosorbent assay (ELISA). 50) binds to CD3 (e.g., human CD3). In some embodiments, the first antigen-binding domain binds to human CD3 with one of the following EC50 values, determined by enzyme-linked immunosorbent assay (ELISA): 10-50, 50-100, 100-150, 150-200, 10-100, 10-110, 9-111, 10-115, 75-150, 100-150, 10-150, 10-200, 50-125, 10-20, 20-50, 50-75, 75-125, 90-120, 100-120, 100-110, 110-120, 50-150, 50-200, or 10-250 nM. In some embodiments, EC50 is determined by an ELISA measuring the binding of an unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the first antigen-binding fragment is scFv, and EC50 is determined by an ELISA measuring the binding of an unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, EC50 is determined by an ELISA measuring the binding of a parent multispecific antibody lacking MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the first antigen-binding fragment is scFv, and EC50 is determined by an ELISA measuring the binding of a parent multispecific antibody lacking MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, EC50 measures the binding of an antigen-binding fragment (e.g., isolated anti-CD3 scFv or scFv-Fc fusion protein) that binds to CD3 (e.g., human CD3 or human CD3δε), as determined by an ELISA.

[0193] In some embodiments, the first antigen-binding fragment is the EC of a reference antibody (e.g., SP34). 50is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500 fold or higher than any one of them in EC 50 binds to CD3 (e.g., human CD3) (including any value or range between these values). In some embodiments, the EC of the first antigen-binding fragment is higher than that of a reference antibody (e.g., SP34) 50 is about 2 - 10, 10 - 20, 20 - 30, 30 - 40, 40 - 50, 50 - 60, 60 - 75, 75 - 100, 100 - 200, 200 - 500, 2 - 5, 5 - 10, 5 - 20, 5 - 30, 5 - 40, 5 - 50, 5 - 55, 5 - 60, 10 - 20, 10 - 30, 10 - 40, 10 - 50, 10 - 60, 20 - 40, 20 - 55, 30 - 60, 10 - 30, or 5 - 100 fold higher than any one of them in EC 50 binds to CD3 (e.g., human CD3). In some embodiments, the EC50 of the first antigen-binding fragment and the reference antibody are measured under the same test conditions. In some embodiments, the EC50 of the first antigen-binding fragment and the reference antibody are measured in the same antibody format. In some embodiments, the EC50 is determined by measuring the binding of the unmasked multispecific antibody and the unmasked multispecific reference antibody to CD3. In some embodiments, the unmasked multispecific reference antibody includes a CD3-binding portion corresponding to SP34 (e.g., including 6 CDRs of SP34). In some embodiments, the EC 50This is determined by measuring the binding of the MM-deficient parental multispecific antibody and the MM-deficient reference parental multispecific antibody to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the MM-deficient reference parental multispecific antibody contains a CD3 binding moiety corresponding to SP34 (e.g., containing six CDRs of SP34). In some embodiments, the Kd binding to CD3 of the first antigen-binding fragment and the EC50 of the reference antibody are determined by ELISA, e.g., the ELISA described in Example 3. In some embodiments, the Kd binding to CD3 of the first antigen-binding fragment and the EC50 of the reference antibody are determined by cell-based assay, e.g., the Jurkat NFAT reporter assay described in Example 3.

[0194] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a relatively weak dissociation constant (Kd) compared to a reference antibody (e.g., SP34), for example, one weaker than the Kd of the reference antibody by at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500 times or more (including any value or range between these values). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a dissociation constant (Kd) that is one less than approximately 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-200, 200-500, 2-5, 5-10, 5-20, 5-30, 5-40, 5-50, 5-55, 5-60, 10-20, 10-30, 10-40, 10-50, 10-60, 20-40, 20-55, 30-60, 10-30, or 5-100 times weaker than the Kd of the reference antibody (e.g., SP34). In some embodiments, the Kd of the first antigen-binding fragment and the reference antibody are measured under the same test conditions. In some embodiments, the kd of the first antigen-binding fragment and the reference antibody are measured in the same antibody format. In some embodiments, the Kd is determined by measuring the binding of an unmasked multispecific antibody and an unmasked multispecific reference antibody to CD3. In some embodiments, the unmasked multispecific reference antibody includes a CD3 binding moiety corresponding to SP34 (e.g., including six CDRs of SP34). In some embodiments, the Kd is determined by measuring the binding of a MM-less parental multispecific antibody and a MM-less reference parental multispecific antibody to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the MM-less reference parental multispecific antibody includes a CD3 binding moiety corresponding to SP34 (e.g., including six CDRs of SP34). In some embodiments, the Kd of the first antigen-binding fragment binding to CD3 and the Kd of the reference antibody are determined by ELISA.

[0195] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a dissociation constant (Kd) of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 nM or higher (including any value or range between these values). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a dissociation constant (Kd) of at least about 1, 10, or 100 μM (including any value or range between these values) (when in the activated form). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with one of the following dissociation constants (Kd): approximately 10-50, 50-100, 100-200, 200-500, 500-1000, 10-100, 100-500, 100-1000, 50-200, 50-250, 50-500, or 10-1000 nM.

[0196] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a relatively fast off-rate (koff) compared to the reference antibody (e.g., SP34), e.g., the k of the reference antibody. off It is at least one of the following speeds: approximately 2, 5, 10, 20, 50, 100, 200 times, or more (including any value or range between these values).

[0197] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) at a relatively slow on-rate (kon) compared to the reference antibody (e.g., SP34), e.g., the k of the reference antibody. on It is at least one of the following values ​​slower: approximately 2, 5, 10, 20, 50, 100, 200 times, or more (including any value or range between these values).

[0198] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a relatively small dissociation constant (Kd) compared to a reference antibody (e.g., SP34), for example, at least about 2, 5, 10, 20, 50, 100, 200 times or more than the Kd of the reference antibody (including any value or range between these values).

[0199] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a relatively large binding constant (ka) compared to the reference antibody (e.g., SP34), e.g., k of the reference antibody. a It is at least one of the following: approximately 2, 5, 10, 20, 50, 100, 200 times, or more.

[0200] Methods for measuring the ability of an antibody (e.g., an activatable multispecific antibody) to bind to an antigen are known in the art and include, but are not limited to, BIAcore analysis, surface plasmon resonance, ELISA, flow cytometry, and cell-based assays (e.g., measuring binding to Jurkat cells) (see, for example, Examples 5 and Table 6). The EC50, dissociation constant (Kd), binding constant (ka), off-rate (koff), and / or on-rate (kon) of binding to CD3 (e.g., human CD3) can be measured in various contexts. In some embodiments, binding to CD3 (e.g., human CD3) is measured using an antigen-binding fragment that binds to CD3 ...

Claims

1. It is a multispecific antibody, a) A first antigen-binding fragment that specifically binds to CD3, wherein a first masking portion (MM1) is fused to a first cleavable portion (CM1) containing a first cleavable site, and comprises a first immunoglobulin light chain variable domain (VL1) and a first immunoglobulin heavy chain variable domain (VH1) of an anti-CD3 antibody, and if the CM1 is not cleaved, the MM1 is fused to the N-terminus of the VL1 via the CM1, the MM1 inhibits the binding of the multispecific antibody to CD3, the MM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417, and 597-599, and the CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 127-129, 418, 420, 431, and 477-490, and 516-555; and b) comprising a second antigen-binding fragment that specifically binds to a target antigen; i) The VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 381; ii) The above VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 381; iii) The above VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 400; iv) The VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 401; v) The above VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 381; vi) The above VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 401; vii) The above VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 400; viiii) The above VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 398, CDR-L2 containing the amino acid sequence of SEQ ID NO: 399, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 400; ix) The VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 381; x) The above VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 381; xi) The VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 376, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 400; xi) The VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 393, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 381; xiiii) The above VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 396, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381; or xiv) The above VH1 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 391, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 378; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and A multispecific antibody containing CDR-L3, which includes the amino acid sequence of SEQ ID NO:

381.

2. The multispecific antibody according to claim 1, wherein the first antigen-binding fragment is an scFv comprising VL1, a linker, and VH1 from the N-terminus to the C-terminus.

3. The multispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide, where: (i) The first polypeptide comprises a structure represented by the following formula: VH2-CH1-hinge-CH2-first CH3(1a); (ii) The second polypeptide comprises the structure represented by the following formula: MM1-CM1-VL1-VH1-Hinge-CH2-Second CH3(1b); and (iii) The third polypeptide comprises a structure represented by the following formula: VL2-CL (1c); Here: CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The first CH3 is the first immunoglobulin heavy chain constant domain 3; The second CH3 is the second immunoglobulin heavy chain constant domain 3; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; VL1 and VH1 associate to form scFv, which specifically binds to CD3; The multispecific antibody according to claim 2, wherein VL2 and VH2 associate to form Fv, which specifically binds to the target antigen.

4. The multispecific antibody according to claim 1, wherein the second antigen-binding fragment comprises a second immunoglobulin light chain variable domain (VL2) and a second immunoglobulin heavy chain variable domain (VH2) of an antibody that specifically binds to a target antigen.

5. The multispecific antibody according to claim 4, wherein the second antigen-binding fragment is selected from the group consisting of Fab, Fv, scFab, and scFv.

6. The multispecific antibody according to claim 5, wherein the second antigen-binding fragment is fused to a second masking portion (MM2) via a second cleavable portion (CM2), and the CM2 includes a second cleavage site. If the CM2 is not cleaved, the MM2 inhibits the binding of the multispecific antibody to the target antigen, and if the CM2 is cleaved, the multispecific antibody binds to the target antigen via the second antigen-binding fragment.

7. The multispecific antibody according to claim 6, wherein the MM2 is fused to the N-terminus of the VL2 via the CM2.

8. The multispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide, where: (i) The first polypeptide comprises a structure represented by the following formula: VH2-CH1-hinge-CH2-first CH3(2a); (ii) The second polypeptide comprises the structure represented by the following formula: MM1-CM1-VL1-VH1-Hinge-CH2-Second CH3(2b); and (iii) The third polypeptide comprises a structure represented by the following formula: MM2-CM2-VL2-CL (2c); Here: CL is the constant domain of the immunoglobulin light chain; CH1 is the constant domain 1 of the immunoglobulin heavy chain; CH2 is the constant domain 2 of the immunoglobulin heavy chain; The first CH3 is the first immunoglobulin heavy chain constant domain 3; The second CH3 is the second immunoglobulin heavy chain constant domain 3; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; VL1 and VH1 associate to form scFv, which specifically binds to CD3; The multispecific antibody according to claim 7, wherein VL2 and VH2 associate to form Fv, which specifically binds to the target antigen.

9. The multispecific antibody according to claim 1, wherein the CD3 is human CD3.

10. The aforementioned VH1 is CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 392, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and The multispecific antibody according to claim 1, comprising CDR-L3 containing the amino acid sequence of SEQ ID NO:

400.

11. The aforementioned VH1 is CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and The multispecific antibody according to claim 1, comprising CDR-L3 containing the amino acid sequence of SEQ ID NO:

381.

12. The multispecific antibody according to claim 1, wherein the first antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 421 or SEQ ID NO:

422.

13. The first antigen-binding fragment comprises VH1 and VL1, where: a) said VH1 has the amino acid sequence of formula (VII): EVQLVESGGGLVX １ PGGSRLSCAAGSFTFX ２ X ３ YAIX ４ WVRQAPGKGLVWX ５ RIRSKYNNYYATYYAX ６ SVKX ７ RFTSRDX ８ SKNTLYLQX ９ NSLAEDTAVYYCX １０ RHGNX １１ GX １２ SYVSWFAYWGQGTLVTVSS (SEQ ID NO: 388) (where X １ is K or Q, X ２ is N or S, X ３ is S or T, X ４ is H or N, X ５ is G or S, X ６ is D or E, X ７ is D or G, X ８ is D or N, X ９ is I or L, X １０ is A or V, X １１ is F or Y, X １２ is N or T); and b) The VL1 is, formula (VIII): X １ AVVTQEPSLTVSPGGTVTLTCX ２ SSTGAVTTSNYX ３ NWX ４ QQKPGQAPRGLIGGTX ５ X ６ RAPGX ７ PARFSGSLLGGGKAALTLSGAQPEDEAEYYCALWYSX ８ X ９ WVFGGGTKLTVL (Sequence No. 389) (wherein X １ is E or Q, X ２ is A, G, P, or R, and X ３ is A or P, X ４ is F or V, X ５ is K or N, X ６ is F or K, and X ７ is A, I, T, or V, and X ８ is A, D, N, or T, X ９ A multispecific antibody according to claim 1, comprising an amino acid sequence (where is H or L).

14. a) Whether VH1 contains the amino acid sequence of SEQ ID NO: 402 and VL1 contains the amino acid sequence of SEQ ID NO: 403; b) The VH1 contains the amino acid sequence of SEQ ID NO: 402, and the VL1 contains the amino acid sequence of SEQ ID NO: 404; c) The VH1 contains the amino acid sequence of SEQ ID NO: 405, and the VL1 contains the amino acid sequence of SEQ ID NO: 406; d) Whether VH1 contains the amino acid sequence of SEQ ID NO: 407 and VL1 contains the amino acid sequence of SEQ ID NO: 404; e) The VH1 contains the amino acid sequence of SEQ ID NO: 407, and the VL1 contains the amino acid sequence of SEQ ID NO: 403; f) Whether VH1 contains the amino acid sequence of SEQ ID NO: 407 and VL1 contains the amino acid sequence of SEQ ID NO: 408; g) The VH1 contains the amino acid sequence of SEQ ID NO: 409, and the VL1 contains the amino acid sequence of SEQ ID NO: 408; h) Whether VH1 contains the amino acid sequence of SEQ ID NO: 410 and VL1 contains the amino acid sequence of SEQ ID NO: 411; i) VH1 contains the amino acid sequence of SEQ ID NO: 412, and VL1 contains the amino acid sequence of SEQ ID NO: 413; j) Whether VH1 contains the amino acid sequence of SEQ ID NO: 410 and VL1 contains the amino acid sequence of SEQ ID NO: 413; k) The VH1 contains the amino acid sequence of SEQ ID NO: 414, and the VL1 contains the amino acid sequence of SEQ ID NO: 403; l) The VH1 contains the amino acid sequence of SEQ ID NO: 415, and the VL1 contains the amino acid sequence of SEQ ID NO: 413; m) The VH1 contains the amino acid sequence of SEQ ID NO: 416, and the VL1 contains the amino acid sequence of SEQ ID NO: 413; or n) The VH1 contains the amino acid sequence of SEQ ID NO: 416, and the VL1 contains the amino acid sequence of SEQ ID NO: 411, The multispecific antibody according to claim 13.

15. The multispecific antibody according to claim 14, wherein VH1 comprises the amino acid sequence of SEQ ID NO: 402, and VL1 comprises the amino acid sequence of SEQ ID NO:

403.

16. The multispecific antibody according to claim 14, wherein VH1 comprises the amino acid sequence of SEQ ID NO: 410, and VL1 comprises the amino acid sequence of SEQ ID NO:

411.

17. The multispecific antibody according to claim 1, wherein MM1 comprises the amino acid sequence of SEQ ID NO:

417.

18. The multispecific antibody according to claim 1, wherein CM1 comprises the amino acid sequence of SEQ ID NO:

77.

19. The multispecific antibody according to claim 1, wherein CM1 contains the amino acid sequence of SEQ ID NO:

418.

20. The multispecific antibody according to claim 1 or claim 11, wherein the target antigen is HER2.

21. The second antigen-binding fragment comprises VH2 and VL2 of the anti-HER2 antibody, where: The aforementioned VH2 is CDR-H1 containing the amino acid sequence of SEQ ID NO: 423, CDR-H2 containing the amino acid sequence of SEQ ID NO: 424, and It contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 71; and The aforementioned VL2 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 72, CDR-L2 containing the amino acid sequence of SEQ ID NO: 73, and The multispecific antibody according to claim 20, comprising CDR-L3 containing the amino acid sequence of SEQ ID NO:

74.

22. The multispecific antibody according to claim 21, wherein VH2 contains the amino acid sequence of SEQ ID NO: 75, and VL2 contains the amino acid sequence of SEQ ID NO:

76.

23. The second antigen-binding fragment is fused to a second masking portion (MM2) via a second cleavable portion (CM2), wherein the CM2 includes a second cleavage site, and if the CM2 is not cleaved, the MM2 inhibits the binding of the multispecific antibody to the target antigen; if the CM2 is cleaved, the multispecific antibody binds to the target antigen via the second antigen-binding fragment. a) MM2 is given by equation (XI): ESX １ X ２ CX ３ X ４ DPFX ５ CQX ６ (Sequence No. 670) (wherein X １ is D or E, X ２ is A, F, V, or Y, and X ３ is D or E, X ４ is A or L, X ５ is D or E, X ６ It contains an amino acid sequence of A, F, or Y; b) MM2 is given by equation (XII): X １ X ２ X ３ X ４ X ５ X ６ CX ７ X ８ DPYECX ９ X １０ (Sequence No. 671) (wherein X １ is A, H, or S, and X ２ is A, D, or S, X ３ is A, T, or V, and X ４ is P, S, or T, X ５ is D or E, X ６ is A or V, X ７ is D or E, X ８ is A or L, X ９ is Q, S or T, and X １０ It contains an amino acid sequence of (where is A, H, or V); or c) MM2 is given by formula (XIII) YNSDDDCX １ SX ２ YDPYTCYY (Sequence No. 672) (where X １ is A, I, or V, and X ２ The multispecific antibody according to claim 21, comprising an amino acid sequence (where is H or R).

24. The multispecific antibody according to claim 21, wherein MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476 and 491-515.

25. The multispecific antibody according to claim 24, wherein MM2 comprises the amino acid sequence of SEQ ID NO:

419.

26. The multispecific antibody according to claim 25, wherein CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 127-129, 418, 420, 431, 477-490, and 516-555.

27. The multispecific antibody according to claim 26, wherein the CM2 comprises the amino acid sequence of SEQ ID NO: 77 or 420.

28. a) A first polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 425, A second polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 426, and A third polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 112; b) A first polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 427, A second polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 428, and A third polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 112; c) A first polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 429, A second polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 430, and A third polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115; d) A first polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 83, A second polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 84, and A third polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 85; e) A first polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 683, A second polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 684, and A third polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 685; f) A first polypeptide containing the amino acid sequence of SEQ ID NO: 425, A second polypeptide containing the amino acid sequence of SEQ ID NO: 426, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 112; g) A first polypeptide containing the amino acid sequence of SEQ ID NO: 427, A second polypeptide containing the amino acid sequence of SEQ ID NO: 428, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 112; h) A first polypeptide containing the amino acid sequence of SEQ ID NO: 429, A second polypeptide containing the amino acid sequence of SEQ ID NO: 430, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 115; i) A first polypeptide containing the amino acid sequence of SEQ ID NO: 83, A second polypeptide containing the amino acid sequence of SEQ ID NO: 84, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 85; j) A first polypeptide containing the amino acid sequence of SEQ ID NO: 683, A second polypeptide containing the amino acid sequence of SEQ ID NO: 684, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 685; k) A first polypeptide containing the amino acid sequence of SEQ ID NO: 425 without a C-terminal lysine, A second polypeptide containing the amino acid sequence of SEQ ID NO: 426 without a C-terminal lysine, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 112; l) A first polypeptide containing the amino acid sequence of SEQ ID NO: 427 without a C-terminal lysine, A second polypeptide containing the amino acid sequence of SEQ ID NO: 428 without a C-terminal lysine, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 112; m) A first polypeptide containing the amino acid sequence of SEQ ID NO: 429 without a C-terminal lysine, A second polypeptide containing the amino acid sequence of SEQ ID NO: 430 without C-terminal lysine, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 115; n) A first polypeptide containing the amino acid sequence of SEQ ID NO: 83, A second polypeptide containing the amino acid sequence of SEQ ID NO: 84 without C-terminal lysine, and A third polypeptide comprising the amino acid sequence of SEQ ID NO: 85 without a C-terminal lysine; or o) A first polypeptide containing the amino acid sequence of SEQ ID NO: 683, A second polypeptide containing the amino acid sequence of SEQ ID NO: 684 without C-terminal lysine, and The multispecific antibody according to claim 27, comprising a third polypeptide having the amino acid sequence of SEQ ID NO: 685 without a C-terminal lysine.

29. The multispecific antibody according to claim 1 or claim 11, wherein the target antigen is CD20.

30. The second antigen-binding fragment comprises VH2 and VL2 of the anti-CD20 antibody, where: a) The VH2 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 556, CDR-H2 containing the amino acid sequence of SEQ ID NO: 557, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 558; The aforementioned VL2 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 559, CDR-L2 containing the amino acid sequence of SEQ ID NO: 560, and Contains CDR-L3 containing the amino acid sequence of SEQ ID NO: 561; or b) The VH2 is, CDR-H1 containing the amino acid sequence of SEQ ID NO: 86, CDR-H2 containing the amino acid sequence of SEQ ID NO: 557, and Contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 558; The aforementioned VL2 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 559, CDR-L2 containing the amino acid sequence of SEQ ID NO: 560, and The multispecific antibody according to claim 29, comprising CDR-L3 containing the amino acid sequence of SEQ ID NO:

561.

31. The multispecific antibody according to claim 30, wherein VH2 comprises the amino acid sequence of SEQ ID NO: 562 and VL2 comprises the amino acid sequence of SEQ ID NO:

563.

32. a) A first polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 564, A second polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 565, and A third polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 567; b) A first polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 564, A second polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 565, and A third polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 569; c) A first polypeptide containing the amino acid sequence of SEQ ID NO: 564, A second polypeptide containing the amino acid sequence of SEQ ID NO: 565, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 567; d) A first polypeptide containing the amino acid sequence of SEQ ID NO: 564, A second polypeptide containing the amino acid sequence of SEQ ID NO: 565, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 569; e) A first polypeptide containing the amino acid sequence of SEQ ID NO: 564, A second polypeptide containing the amino acid sequence of SEQ ID NO: 565 without C-terminal lysine, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 567 without a C-terminal lysine; f) A first polypeptide containing the amino acid sequence of SEQ ID NO: 564, A second polypeptide containing the amino acid sequence of SEQ ID NO: 565 without C-terminal lysine, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 569 without C-terminal lysine, A multispecific antibody according to claim 30, comprising:

33. The aforementioned VH1 is CDR-H1 containing the amino acid sequence of SEQ ID NO: 390, CDR-H2 containing the amino acid sequence of SEQ ID NO: 394, and It contains CDR-H3 containing the amino acid sequence of SEQ ID NO: 395; and The aforementioned VL1 is CDR-L1 containing the amino acid sequence of SEQ ID NO: 397, CDR-L2 containing the amino acid sequence of SEQ ID NO: 380, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 381, The multispecific antibody according to claim 2.

34. The multispecific antibody according to claim 33, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

422.

35. The multispecific antibody according to claim 33, wherein MM1 comprises the amino acid sequence of SEQ ID NO:

417.

36. The multispecific antibody according to claim 34, wherein MM1 comprises the amino acid sequence of SEQ ID NO:

417.

37. The multispecific antibody according to claim 35, wherein CM1 comprises the amino acid sequence of SEQ ID NO:

77.

38. The multispecific antibody according to claim 36, wherein CM1 comprises the amino acid sequence of SEQ ID NO:

77.

39. The multispecific antibody according to claim 35, wherein CM1 comprises the amino acid sequence of SEQ ID NO:

418.

40. The multispecific antibody according to claim 36, wherein CM1 comprises the amino acid sequence of SEQ ID NO:

418.

41. The multispecific antibody according to claim 3, wherein the second polypeptide has the amino acid sequence of SEQ ID NO:

567.

42. The multispecific antibody according to claim 41, wherein the first polypeptide has the amino acid sequence of SEQ ID NO:

565.

43. The multispecific antibody according to claim 42, wherein the third polypeptide has the amino acid sequence of SEQ ID NO:

564.

44. The multispecific antibody according to claim 8, wherein the second polypeptide has the amino acid sequence of SEQ ID NO:

85.

45. The multispecific antibody according to claim 44, wherein the first polypeptide has the amino acid sequence of SEQ ID NO:

84.

46. The multispecific antibody according to claim 45, wherein the third polypeptide has the amino acid sequence of SEQ ID NO:

83.

47. The first polypeptide containing the amino acid sequence of SEQ ID NO: 427, A second polypeptide containing the amino acid sequence of SEQ ID NO: 428, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 112 A multispecific antibody according to claim 28, comprising:

48. The first polypeptide containing the amino acid sequence of SEQ ID NO: 564, A second polypeptide containing the amino acid sequence of SEQ ID NO: 565, and A third polypeptide containing the amino acid sequence of SEQ ID NO: 567 A multispecific antibody according to claim 32, comprising:

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