Anti-CD3 antibodies and methods 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 TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to International Application No. PCT / CN2021 / 076626, filed February 11, 2021, and International Application No. PCT / CN2021 / 109057, filed July 28, 2021, the entire contents of which are incorporated herein by reference. Submission of sequence listing as an ASCII text file

[0002] The entire contents of the following ASCII text file submission, Sequence Listing in Computer Readable Format (CRF) (Filename: 695402000942SEQLIST.TXT, Date of Registration: February 9, 2022, Size: 432,841B), are incorporated herein by reference. [Technical Field]

[0003] This application relates to antibodies that target CD3, including multispecific antibodies that target CD3, masked and activatable anti-CD3 antibodies, and methods for making and using the same. [Background technology]

[0004] Bispecific T cell engager antibodies (BiTEs or TCEs) have been investigated as a means of recruiting cytolytic T cells to kill tumor cells. This is based on simultaneous recognition of antigens on tumor cells and binding to the CD3ε chain (CD3) in the T cell receptor complex on T cells, directly crosslinking malignant tumor cells with CD3+ T cells. Blintumomab (BLINCYTO®), the first bispecific T cell engager reactive with the B cell antigen CD19, was approved by the FDA in 2014 for the treatment of neoplasms. Although early studies have shown promising clinical efficacy, bispecific T cell engagers are hampered by severe dose-limiting toxicities, primarily manifested as cytokine release syndrome, resulting in a severely narrow therapeutic window. Activatable BiTE or TCE molecules with enhanced specificity and reduced side effects are needed.

[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 allow binding in the tumor microenvironment ("TME"), where specific activation conditions exist compared to healthy tissue, such as upregulated proteases or favorable competition due to highly localized antigen concentrations, allowing the antibody to bind to its target for tumor killing. See, e.g., WO 2019 / 149282. In this way, activatable antibodies provide antigen-specific binding proteins that remain largely inactive in healthy tissue but are primarily activated in the TME.

[0006] [Contents of the invention] The present application provides multispecific antibodies targeting CD3 and another target antigen (e.g., HER2, CD20, TROP2, BCMA, or CD19), masked antibodies (including activatable antibodies such as activatable multispecific antibodies), isolated anti-CD3 antibodies, masked antibodies (e.g., activatable) targeting HER2, and methods of treatment thereof.

[0007] One aspect of the present application provides a multispecific antibody ("multispecific T cell engager") comprising: a first antigen-binding fragment that specifically binds CD3 and is fused to a first masking moiety (MM1); and a second antigen-binding fragment that specifically binds a target antigen; wherein MM1 specifically binds to the CD3-binding moiety in competition with CD3; and the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM 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 without MM1. In some embodiments, the EC50 is determined using an ELISA assay such as that described in Example 5.

[0008] In some embodiments of any one of the multispecific antibodies described 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 MMI is fused to the N-terminus of VL1 via a first non-cleavable linker (NCL1).

[0009] One aspect of the present application provides an activatable multispecific antibody ("activatable multispecific T cell engager") comprising: a) a first antigen-binding fragment that specifically binds CD3 and is fused to a first masking moiety (MM1) via a first cleavable moiety (CM1); and b) a second antigen-binding fragment that specifically binds a target antigen, wherein CM1 comprises a first cleavage site; MM1 specifically binds to the CD3-binding moiety in competition with CD3; and the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM as determined by enzyme-linked immunosorbent assay (ELISA). In some embodiments, when CM1 is uncleaved, MM1 inhibits binding of the activatable antibody to CD3; and when 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 moiety (CM1), where CM1 comprises a first cleavage site, and when CM1 is uncleaved, MM1 inhibits binding of the multispecific antibody to CD3, and when CM1 is cleaved, the multispecific antibody binds to CD3 via the first antigen-binding fragment with higher affinity, e.g., compared to the affinity of the multispecific antibody that binds to CD3 via the first antigen-binding fragment when CM1 is uncleaved. 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 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 cleaved or has effective binding due to highly localized antigen concentrations in the TME versus normal tissue). In some embodiments, EC50 is determined using an ELISA assay as described in Example 5.

[0010] In some embodiments of 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., where the CM1 of the multispecific antibody is cleaved or has effective binding due to highly localized antigen concentrations in the TME versus normal tissue). In some embodiments, binding of the antigen-binding fragment to CD3 is measured when the antigen-binding fragment is unmasked.

[0011] In some embodiments of any one of the multispecific or activatable multispecific antibodies described above, the 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, such as the ELISA assay of Example 3. In some embodiments, the 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, such as the Jurkat NFAT assay for the antigen concentrations used in Example 3.

[0012] In some embodiments of any one of the multispecific or activatable multispecific antibodies described above, 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 an scFv. In some embodiments, the scFv comprises, from N-terminus to C-terminus, VL1, a linker, and VH1. In some embodiments, the scFv comprises, from N-terminus to C-terminus, VH1, a linker, and VL1. In some embodiments, the antibody is an activatable multispecific antibody, MMI is fused to the N-terminus of VL1 via CMI. In some embodiments, the antibody is not an activatable multispecific antibody, MMI 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 comprise a cleavable linker. In some embodiments, the masking moiety is not fused to a sequence containing a cleavage site.

[0013] In some embodiments of any one 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 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 an Fv. In some embodiments, the second antigen-binding fragment is a 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, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-1st CH3(1a); (ii) the second polypeptide comprises a structure represented by the formula: MM1-NCL1-VL1-VH1-hinge-CH2-2nd CH3(1b); and (iii) the third polypeptide comprises a structure represented by the formula: VL2-CL(1c); where: CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin heavy chain constant domain 1; CH2 is an immunoglobulin heavy chain constant domain 2; the first CH3 is a first immunoglobulin heavy chain constant domain 3; the second CH3 is a second immunoglobulin heavy chain constant domain 3; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; wherein VL1 and VH1 associate to form an scFv that specifically binds to CD3; and VL2 and VH2 associate to form an Fv that specifically binds to a 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, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-1st CH3(1a); (ii) the second polypeptide comprises a structure represented by the formula: MM1-CM1-VL1-VH1-hinge-CH2-2nd CH3(1b); and (iii) the third polypeptide comprises a structure represented by the formula: VL2-CL(1c); where: CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin heavy chain constant domain 1; CH2 is an immunoglobulin heavy chain constant domain 2; the first CH3 is a first immunoglobulin heavy chain constant domain 3; the second CH3 is a second immunoglobulin heavy chain constant domain 3; The hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; wherein VL1 and VH1 associate to form an scFv that specifically binds to CD3; and VL2 and VH2 associate to form an Fv that specifically binds to a target antigen. In some embodiments, the multispecific antibody (e.g., its second polypeptide) comprises an amino acid linker between VL1 and VH1.

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

[0015] In some embodiments of any one of the above multispecific or activatable multispecific antibodies, the CD3 is human CD3. In some embodiments, the first antigen-binding fragment is cross-reactive with CD3 polypeptide from at least one non-human species selected from the group consisting of cynomolgus monkey, mouse, rat, and dog.

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

[0017] In some embodiments of any one of the multispecific or activatable multispecific antibodies described above, the first antigen-binding fragment comprises a VH1 and a VL1 of an anti-CD3 antibody, wherein VH1 comprises 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 comprising up to about three amino acid substitutions; a 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 comprising up to about three amino acid substitutions; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 378, 395, 604, and 605. and VL1 comprises a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, and 606-609, or a variant thereof comprising up to about three amino acid substitutions, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, or a variant thereof comprising up to about three amino acid substitutions, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 381, 400-401, and 610, or a variant thereof comprising up to about three amino acid substitutions.

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

[0019] In some embodiments of 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, ​​a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 383, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 384; and VL1 comprises a light chain complementarity-determining region (CDR-L)1 comprising the amino acid sequence of SEQ ID NO: 385, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 386, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 387. In some embodiments, the VH1 comprises a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395; and the VL1 comprises a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401. In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 391, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 378; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 392, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 400.In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 392, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 401. In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 401. In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 400. In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 398, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 399, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 400.In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 391, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 391, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 378; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 400. In some embodiments, VH1 comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and VL1 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, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 378; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 391, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 378; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, the VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 391, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 378; and the VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, the VH1 comprises a polypeptide of formula (VII): EVQLVESGGGLVX1PGGSLRLSCAASGFTFX2X3YAIX4WVRQAPGKGLEWVX5RIRSKYNNYATYYAX6SVKX7RFTISRDX8SKNTLYLQX9NSLRAEDTAVYYCX. 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, and X 10 is A or V, and X 11 is F or Y and X 12is N or T); VL1 comprises an amino acid sequence of formula (VIII): XIAVVTQEPSLTVSPGGTVTLTCX2SSTGAVTTSNYX3NWX4QQKPGQAPRGLIGGTX5X6RAPGX7PARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSX8X9WVFGGGTKLTVL (SEQ ID NO: 389) (wherein XI 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 comprises 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 to 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 comprises 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 to 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 the amino acid sequence of SEQ ID NO: 67, 402, 405, 407, 409, 410, 412, 414, 415, and 416; and VL1 comprises the amino acid sequence of SEQ ID NO: 68, 403, 404, 406, 408, 411, and 413. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 388, and VL1 comprises the amino acid sequence of SEQ ID NO: 389. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 402, 405, 407, 409, 410, 412, 414, 415, and 416. and VL1 SEQ ID NOs: 403, 404, 406, 408, 411, and 413 comprise an amino acid sequence selected from the group consisting of: In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 402, and VL1 comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 402, and VL1 comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 405, and VL1 comprises the amino acid sequence of SEQ ID NO: 406. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 407, and VL1 comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 407, and VL1 comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 407, and VL1 comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 409, and VL1 comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 410, and VL1 comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 412, and VL1 comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 410, and VL1 comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 414, and VL1 comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 415, and VL1 comprises 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: 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 above-described activatable multispecific antibodies, MM1 comprises, at the N-terminus, the amino acid sequence of EVGSY (SEQ ID NO: 667). In some embodiments, MM1 comprises the amino acid sequence of formula (IX): PYDDPDCPSHX1SDCDX2 (SEQ ID NO: 668), where X1 is D or E and X2 is N or Q. In some embodiments, MM1 comprises the amino acid sequence of formula (X). In some embodiments, MM1 comprises the amino acid sequence of SEQ ID NO: 417. In some embodiments, MM1 comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, MM1 comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 597-599.

[0021] In some embodiments of any one of the above-described activatable multispecific antibodies, 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. In some embodiments, CM1 comprises the amino acid sequence of SEQ ID NO: 77 or 418.

[0022] In some embodiments of 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 alpha 5, NCAM1, PTPRC, CD138, NaPi2b, MSLN, DLL3, GPRC5D, GPNMB, ICAM1, SSTR2, or a cancer-associated antigen. In some embodiments, 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 above-described multispecific or activatable multispecific antibodies, the target antigen is HER2. In some embodiments, the second antigen-binding fragment comprises a VH2 and a VL2 of an anti-HER2 antibody, wherein the VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 423, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 424, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71; and the VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the VH2 comprises the amino acid sequence of SEQ ID NO: 75, and the VL2 comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the second antigen-binding fragment is fused to a second masking moiety (MM2) via a second cleavable moiety (CM2), wherein: a) MM2 comprises an amino acid sequence of formula (XI): ESX1X2CX3X4DPFX5CQX6 (SEQ ID NO: 670), where 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; or b) MM2 comprises an amino acid sequence of formula (XII): X1X2X3X4X5X6CX7X8DPYECX9X 10 (SEQ ID NO: 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, and X 10is A, H, or V); or c) MM2 comprises the amino acid sequence of formula (XIII): YNSDDDCX1SX2YDPYTCYY (SEQ ID NO: 672), where 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 moiety (MM2) via a second cleavable moiety (CM2), wherein 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 to 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 to 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 to SEQ ID NO: 112.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 to 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 to 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 to SEQ ID NO: 112. 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 to 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 to 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 to SEQ ID NO: 115. 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 to 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 to 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 to SEQ ID NO: 85.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 to 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 to 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 to SEQ ID NO: 685. 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 to SEQ ID NO: 425 (optionally without the 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 to SEQ ID NO: 426 (optionally without the 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 to SEQ ID NO: 112. 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 to SEQ ID NO: 427 (optionally without the 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 to SEQ ID NO: 428 (optionally without the C-terminal lysine), 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 to SEQ ID NO: 112.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 to SEQ ID NO: 429 (optionally without the 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 to SEQ ID NO: 430 (optionally without the C-terminal lysine); 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 to SEQ ID NO: 115. 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 to 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 to SEQ ID NO: 84 (optionally without the 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 to SEQ ID NO: 85 (optionally without the C-terminal lysine). 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 to 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 to SEQ ID NO: 684 (optionally without the 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 to SEQ ID NO: 685 (optionally without the C-terminal lysine).In some embodiments of 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 species comprising a C-terminal lysine and some species lacking a C-terminal lysine.

[0024] In some embodiments of any one of the above-described multispecific or activatable multispecific antibodies, the target antigen is CD20. In some embodiments, the second antigen-binding fragment comprises a VH2 and a VL2 of an anti-CD20 antibody, wherein the VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 556, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 557, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 558; and the VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 559, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 560, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 561. In some embodiments, the second antigen-binding fragment comprises a VH2 and a VL2 of an anti-CD20 antibody, wherein VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 557, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 558; and VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 559, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 560, and a CDR-L3 comprising 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, 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 to 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 to 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 to SEQ ID NO: 567.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 to 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 to 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 to SEQ ID NO: 569. 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 to 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 to SEQ ID NO: 565 (optionally without the 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 to SEQ ID NO: 567 (optionally without the C-terminal lysine). 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 to 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 to SEQ ID NO: 565 (optionally without the 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 to SEQ ID NO: 569 (optionally without the C-terminal lysine).In some embodiments of 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 species comprising a C-terminal lysine and some species lacking a 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 comprises 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 cross-linking efficiency. In some embodiments, the Fc region has reduced or no antibody-dependent cellular cytotoxicity (ADCC) effect and / or reduced or no cross-linking effect. In some embodiments, the Fc region is of the human IgG1 subclass and has an N297A amino acid substitution.

[0026] In some embodiments of 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 i) the first CH3 domain comprises a cysteine ​​(C) residue at position 390 and the second CH3 domain comprises a cysteine ​​residue at position 400, or the first CH3 domain comprises a cysteine ​​residue at position 400 and the second CH3 domain comprises a cysteine ​​residue at position 390; or ii) the first CH3 domain comprises a cysteine ​​residue at position 392. or iii) the first CH3 domain comprises a cysteine ​​residue at position 392 and the second CH3 domain comprises a cysteine ​​residue at position 397, or the first CH3 domain comprises a cysteine ​​residue at position 397 and the second CH3 domain comprises a cysteine ​​residue at position 392; or iv) the first CH3 domain comprises a cysteine ​​residue at position 392 and the second CH3 domain comprises a cysteine ​​residue at position 400, or the first CH3 domain comprises a cysteine ​​residue at position 400 and the second CH3 domain comprises a cysteine ​​residue at position 392; amino acid residue numbering is based on EU numbering. In some embodiments, i) the first CH3 domain comprises an N390C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises an N390C substitution; or ii) the first CH3 domain comprises a K392C substitution and the second CH3 domain comprises a V397C substitution, or the first CH3 domain comprises a V397C substitution and the second CH3 domain comprises a K392C substitution; or iii) the first CH3 domain comprises a K392C substitution and the second CH3 domain comprises an S400C substitution, or the first CH3 domain comprises an S400C substitution and the second CH3 domain comprises a K392C substitution. In some embodiments, i) the first CH3 domain further comprises a positively charged residue at position 357 and the second CH3 domain further comprises a negatively charged residue at position 351, or the first CH3 domain further comprises a negatively charged residue at position 351 and the second CH3 domain further comprises a positively charged residue at position 357; or ii) the first CH3 domain further comprises a positively charged residue at position 411 and the second CH3 domain further comprises a negatively charged residue at position 370, or the first CH3 domain further comprises a negatively charged residue at position 370. and the second CH3 domain further comprises a positively charged residue at position 411; or iii) the first CH3 domain further comprises a positively charged residue at position 364 and the second CH3 domain further comprises a negatively charged residue at position 370, or the first CH3 domain further comprises a negatively charged residue at position 370 and the second CH3 domain further comprises a positively charged residue at position 364; or a combination of i) and ii), or a combination of i) and iii), wherein the amino acid residue numbering is based on EU numbering. In some embodiments, the first CH3 domain further comprises a positively charged residue at position 356 and the second CH3 domain further comprises a negatively charged residue at position 439, or the first CH3 domain further comprises a negatively charged residue at position 439 and the second CH3 domain further comprises 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 comprises E357K and T411K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the first CH3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and T411K substitutions; or ii) the first CH3 domain comprises E357K and S364K substitutions and the second CH3 domain comprises L351D and K370D substitutions, or the H3 domain comprises L351D and K370D substitutions and the second CH3 domain comprises E357K and S364K substitutions; or iii) the first CH3 domain comprises D356K, E357K, and S364K substitutions and the second CH3 domain comprises L351D, K370D, and K439D substitutions, or the first CH3 domain comprises L351D, K370D, and K439D substitutions and the second CH3 domain comprises D356K, E357K, and S364K substitutions.In some embodiments, i) the first CH3 domain further comprises K392D and K409D substitutions and the second CH3 domain further comprises D356K and D399K substitutions, or the first CH3 domain further comprises D356K and D399K substitutions and the second CH3 domain further comprises K392D and K409D substitutions; or ii) the first CH3 domain further comprises L368D and K370S substitutions. or iii) the first CH3 domain further comprises L351K and T366K substitutions and the second CH3 domain further comprises L351D and L368E substitutions, or the first CH3 domain further comprises E357Q and S364K substitutions and the second CH3 domain further comprises L368D and K370S substitutions; or iv) the first CH3 domain further comprises L351K and T366K substitutions and the second CH3 domain further comprises L351D and L368E substitutions, or (iv) the first CH3 domain further comprises L351D and L368E substitutions and the second CH3 domain further comprises L351K and T366K substitutions; or (iv) the first CH3 domain further comprises P395K, P396K, and V397K substitutions and the second CH3 domain further comprises T394D, P395D, and P396D substitutions, or the first CH3 domain further comprises T394D, P395D, and P396D substitutions and the second or (v) the first CH3 domain further comprises F405E, Y407E, and K409E substitutions and the second CH3 domain further comprises F405K and Y407K substitutions; or the first CH3 domain further comprises F405K and Y407K substitutions and the second CH3 domain further comprises F405E, Y407E, and K409E substitutions.

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

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

[0029] One aspect of the present application provides an isolated antibody or antigen-binding fragment thereof ("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), where 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), where X1 is D or E, X2 is S or T, and X3 is D, G, or S; and a CDR-H2 comprising the amino acid sequence of formula (III): HGNX1GX2SYVSX3X4AY (SEQ ID NO: 384), where X1 is F or Y and X2 is N or T. or T, X3 is W or Y, and X4 is F or W; and b) a VH comprising a CDR-H3 comprising an 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), a CDR-L2 comprising an amino acid sequence of formula (V): GTX1X2RAP (SEQ ID NO: 386), (wherein X1 is K or N, and X2 is F or K), and a VL comprising a 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).In some embodiments, the VH comprises 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 comprising up to about three amino acid substitutions; a 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 comprising up to about three amino acid substitutions; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 378, 395, 604, and 605, or a variant thereof comprising up to about three amino acid substitutions. and the VL comprises a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398 and 606-609, or a variant thereof comprising up to about three amino acid substitutions, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, or a variant thereof comprising up to about three amino acid substitutions, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 381, 400-401 and 610, or a variant thereof comprising up to about three amino acid substitutions. In some embodiments, the VH comprises a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, or a variant thereof comprising up to about three amino acid substitutions, a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, or a variant thereof comprising up to about three amino acid substitutions, and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395, or a variant thereof comprising up to about three amino acid substitutions; and the VL comprises a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, or a variant thereof comprising up to about three amino acid substitutions, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, or a variant thereof comprising up to about three amino acid substitutions, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401, or a variant thereof comprising up to about three amino acid substitutions.In some embodiments, the VH comprises CDR-H1 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; the VL comprises CDR-L1 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, the VH comprises CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, and CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395; and the VL comprises CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, and CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401. In some embodiments, the VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, 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 the 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, the 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: 392, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the 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: 400. In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 392, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 401.In some embodiments, the 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: 393, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the 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, the VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the 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: 401. In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 400. In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 398, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 399, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 400. In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381.In some embodiments, the 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: 395; and the 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, the 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: 394, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the 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, the VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, 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 the 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: 400. In some embodiments, the 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: 394, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the 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, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 378; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381.In some embodiments, the 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 the 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, the 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 the 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 is of formula (VII): EVQLVESGGGLVX1PGGSLRLSCAASGFTFX2X3YAIX4WVRQAPGKGLEWVX5RIRSKYNNYATYYAX6SVKX7RFTISRDX8SKNTLYLQX9NSLRAEDTAVYYCX. 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, and X 10 is A or V, and X 11 is F or Y and X 12 is N or T); and VL comprises an amino acid sequence of formula (VIII): X1AVVTQEPSLTVSPGGTVTLTCX2SSTGAVTTSNYX3NWX4QQKPGQAPRGLIGGTX5X6RAPGX7PARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSX8X9WVFGGGTKLTVL (SEQ ID NO: 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, the VH comprises 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 to 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 the VL comprises 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 to 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, the 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 the 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 of any one of the isolated anti-CD3 antibodies or antigen-binding fragments thereof, the 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 the 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, the VH comprises the amino acid sequence of SEQ ID NO: 388, and the VL comprises the amino acid sequence of SEQ ID NO: 389.In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 402, and the VL comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 402, and the VL comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 405, and the VL comprises the amino acid sequence of SEQ ID NO: 406. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 407, and the VL comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 407, and the VL comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 407, and the VL comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 409, and the VL comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 410, and the VL comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 412, and the VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 410, and the VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 414, and the VL comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 415, and the VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 416, and the VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 416, and the VL comprises the amino acid sequence of SEQ ID NO: 411.

[0030] In some embodiments of any one of the isolated anti-CD3 antibodies or antigen-binding fragments thereof, 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, 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 alpha 5, NCAM1, PTPRC, CD138, NaPi2b, MSLN, DLL3, GPRC5D, GPNMB, ICAM1, SSTR2, a cancer associated antigen In some embodiments, 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 the application provides an activatable antibody ("activatable anti-CD3 antibody") comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a CD3-binding moiety, wherein: a) the CD3-binding portion comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; b) the CD3-binding portion comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; c) the CD3-binding portion comprises, from N-terminus to C-terminus, a VL and a VH; or d) the CD3-binding portion comprises, from N-terminus to C-terminus, a VL and a VH; the CM comprises a cleavage site; when the CM is uncleaved, the MM inhibits binding of the activatable antibody to CD3; when the CM is cleaved, the activatable antibody binds to CD3 via the VH and VL; and the activatable antibody binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM 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 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 its activated form (i.e., CM1 cleaved). In some embodiments, EC50 is determined using an ELISA assay such as that described in Example 5.

[0032] In some embodiments of any one of the above-described activatable anti-CD3 antibodies, 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 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 activating form (i.e., CM1 cleaved) of an activatable multispecific antibody.

[0033] In some embodiments of any one of the above-described activatable anti-CD3 antibodies, the MM comprises an amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of the MM. In some embodiments, the MM comprises an amino acid sequence of formula (IX): PYDDPDCPSHX1SDCDX2 (SEQ ID NO: 668), where X1 is D or E and X2 is N or Q. In some embodiments, the MM comprises an amino acid sequence of formula (X): X1X2X3DX4X5CX6X7DX8X9X 10 CX 11 X 12 (SEQ ID NO: 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, and X 10 is D, H or S, and X 11 is H, P or Y, and X 12 is N, P, or Y). In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 417. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 585-588 and 597-591. In some embodiments, the CD3 is human CD3.

[0034] One aspect of the application provides an activatable antibody ("activatable anti-CD3 antibody") comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a CD3-binding moiety, wherein: a) the CD3-binding moiety comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; b) the CD3-binding moiety comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; c) the CD3-binding moiety comprises, from N-terminus to C-terminus, a VL and a VH; or d) the CD3-binding moiety comprises, from N-terminus to C-terminus, a VL and a VH. the CM comprises a cleavage site; when the CM is not cleaved, the MM inhibits binding of the activatable antibody to CD3; when the CM is cleaved, the activatable antibody binds to CD3 via the VH and VL; a) the MM comprises the amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of the MM; b) the MM 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); or c) the MM is 10 CX 11 X 12 (SEQ ID NO: 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, and X 10 is D, H or S, and X 11 is H, P or Y, and X 12 is N, P, or Y. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NOs: 35, 417, 585-588, and 597-599. In some embodiments, the CD3 is human CD3.

[0035] In some embodiments of any one of the above-described activatable anti-CD3 antibodies, 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 an scFv. In some embodiments, the scFv comprises, from N-terminus to C-terminus, a VL, a linker, and a VH.

[0036] In some embodiments of any one of the aforementioned activatable anti-CD3 antibodies, the VH comprises a heavy chain complementarity determining region (CDR-H) 1 comprising the amino acid sequence of formula (I): X1YAX2X3 (SEQ ID NO: 382), where 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), where X1 is F or Y, X2 is N or T, and X3 is D, G, or S; and a CDR-H3 comprising the amino acid sequence of formula (III): HGNX1GX2SYVSX3X4AY (SEQ ID NO: 384), where X1 is F or Y, X2 is N or T, and X3 is W or G. or Y, and X4 is F or W; and b) the VL comprises a CDR-L1 comprising an 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), a CDR-L2 comprising an amino acid sequence of formula (V): GTX1X2RAP (SEQ ID NO: 386), (wherein X1 is K or N, and X2 is F or K), and a 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).In some embodiments, the VH comprises 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 comprising up to about three amino acid substitutions; a 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 comprising up to about three amino acid substitutions; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 378, 395, 604, and 605, or a variant thereof comprising up to about three amino acid substitutions. and the VL comprises a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398 and 606-609, or a variant thereof comprising up to about three amino acid substitutions, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, or a variant thereof comprising up to about three amino acid substitutions, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 381, 400-401 and 610, or a variant thereof comprising up to about three amino acid substitutions. In some embodiments, the VH comprises a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, or a variant thereof comprising up to about three amino acid substitutions, a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, or a variant thereof comprising up to about three amino acid substitutions, and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395, or a variant thereof comprising up to about three amino acid substitutions; and the VL comprises a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, or a variant thereof comprising up to about three amino acid substitutions, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, or a variant thereof comprising up to about three amino acid substitutions, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401, or a variant thereof comprising up to about three amino acid substitutions.In some embodiments, the VH comprises CDR-H1 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; the VL comprises CDR-L1 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, the VH comprises CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 376 and 390, CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 391-394, and CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 378 and 395; and the VL comprises CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 396-398, CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 380 and 399, and CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 381 and 400-401. In some embodiments, the VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, 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 the 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, the 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: 392, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the 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: 400. In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 392, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 401.In some embodiments, the 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: 393, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the 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, the VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the 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: 401. In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 400. In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 398, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 399, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 400. In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381.In some embodiments, the 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: 395; and the 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, the 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: 394, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the 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, the VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, 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 the 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: 400. In some embodiments, the 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: 394, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395; and the 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, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 378; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381.In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 391, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 378; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 396, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, the VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 391, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 378; and the VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 388, and the VL comprises the amino acid sequence of SEQ ID NO: 389.

[0037] In some embodiments of any one of the aforementioned activatable anti-CD3 antibodies, the VH is represented by formula (VII): EVQLVESGGGLVX1PGGSLRLSCAASGFTFX2X3YAIX4WVRQAPGKGLEWVX5RIRSKYNNYATYYAX6SVKX7RFTISRDX8SKNTLYLQX9NSLRAEDTAVYYCX 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, and X 10 is A or V, and X 11 is F or Y and X 12is N or T); and VL comprises an amino acid sequence of formula (VIII): XIAVVTQEPSLTVSPGGTVTLTCX2SSTGAVTTSNYX3NWX4QQKPGQAPRGLIGGTX5X6RAPGX7PARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSX8X9WVFGGGTKLTVL (SEQ ID NO: 389) (wherein XI 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, the VH comprises 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 to 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 the VL comprises 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 to 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, the 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 the 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 of any one of the above-described activatable anti-CD3 antibodies, the 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 the 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, the VH comprises the amino acid sequence of SEQ ID NO: 402, and the VL comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 402, and the VL comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 405, and the VL comprises the amino acid sequence of SEQ ID NO: 406. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 407, and the VL comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 407, and the VL comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 407, and the VL comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 409, and the VL comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 410, and the VL comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 412, and the VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 410, and the VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 414, and the VL comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 415, and the VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 416 and the VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 416 and the 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.

[0039] In some embodiments of any one of the above-described activatable anti-CD3 antibodies, the 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, the CM comprises the amino acid sequence of SEQ ID NO: 77 or 418.

[0040] One aspect of the present application provides a masked antibody ("masked anti-CD3 antibody") comprising, from N- to C-terminus, a masking moiety (MM), and a CD3-binding moiety, wherein: a) the CD3-binding moiety comprises a VL, and the activatable antibody comprises a second polypeptide further comprising a VH; b) the CD3-binding moiety comprises a VH, and the activatable antibody comprises a second polypeptide further comprising a VL; c) the CD3-binding moiety comprises, from N- to C-terminus, a VL and a VH; or d) the CD3-binding moiety comprises, from N- to C-terminus, a VL and a VH; the MM specifically binds to the CD3-binding moiety in competition with CD3; the activatable antibody binds to CD3 via the VH and VL; and the masked antibody binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM 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 comprises, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a CD3 binding moiety. In some embodiments, the masked anti-CD3 antibody is not an activatable antibody. In some embodiments, the masked anti-CD3 antibody comprises, from N-terminus to C-terminus, a masking moiety (MM), a non-cleavable linker (NCL), and a CD3 binding moiety.One aspect of the present application provides a masked antibody ("masked anti-CD3 antibody") comprising a masking moiety (MM) and an antibody or antigen-binding fragment thereof that binds CD3, wherein the antibody or antigen-binding fragment thereof comprises a VH and a VL; the masked antibody comprises a single-chain polypeptide, and the VH and VL of the antibody or antigen-binding fragment are part of the single-chain polypeptide, or the masked antibody comprises two polypeptide chains, and the VH and VL of the antibody or antigen-binding fragment are part of different polypeptide chains of the masked antibody; wherein the C-terminus of the MM is fused to the N-terminus of the VH or VL of the antibody or antigen-binding fragment; wherein the MM specifically binds to the antibody or antigen-binding fragment in competition with CD3; and wherein the antibody or antigen-binding fragment thereof binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM 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 comprises 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 further comprises a cleavable linker, for example, 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 comprise a cleavable linker (e.g., 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 the application provides a masked antibody ("masked anti-CD3 antibody") comprising, from N-terminus to C-terminus, a masking moiety (MM), a non-cleavable linker (NCL), and a CD3-binding moiety, wherein: a) the CD3-binding portion comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; b) the CD3-binding portion comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; c) the CD3-binding portion comprises, from N-terminus to C-terminus, a VL and a VH; or d) the CD3-binding portion comprises, from N-terminus to C-terminus, a VL and a VH; the MM1 specifically binds to the CD3-binding portion in competition with CD3; the activatable antibody binds to CD3 via the VH and VL; and the masked antibody binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 50 nM, or at least 100 nM, or about 110 nM) 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 unmasked form (e.g., without MM). In some embodiments, EC50 is determined using an ELISA assay such as described in Example 5.

[0042] In some embodiments of any one of the masked 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, the first antigen-binding fragment, when used to determine Kd, 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 a multispecific antibody in unmasked form (e.g., without MM).

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

[0044] One aspect of the present application provides a masked antibody ("masked anti-CD3 antibody") comprising, from N-terminus to C-terminus, a masking moiety (MM), and a CD3-binding moiety, wherein: a) the CD3-binding moiety comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; b) the CD3-binding moiety comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; c) the CD3-binding moiety comprises, from N-terminus to C-terminus, a VL and a VH; or d) the CD3-binding moiety comprises, from N-terminus to C-terminus, a VL and a VH. the MM comprises a VL and a VH extending therethrough; the MM1 specifically binds to the CD3 binding portion in competition with CD3; the activatable antibody binds to CD3 via the VH and VL; wherein a) the MM comprises an amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of the MM; b) the MM comprises an 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) the MM comprises an amino acid sequence of formula (X): X1X2X3DX4X5CX6X7DX8X9X 10 CX 11 X 12 (SEQ ID NO: 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, and X 10 is D, H or S, and X 11 is H, P or Y, and X 12 is N, P, or Y). In some embodiments, the masked anti-CD3 antibody is an activatable antibody. In some embodiments, the masked anti-CD3 antibody comprises, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a CD3-binding moiety. In some embodiments, the masked anti-CD3 antibody is not an activatable antibody. In some embodiments, the masked anti-CD3 antibody comprises, from N-terminus to C-terminus, a masking moiety (MM), a non-cleavable linker (NCL), and a CD3-binding moiety.

[0045] One aspect of the present application provides a masked antibody ("masked anti-CD3 antibody") comprising, from N-terminus to C-terminus, a masking moiety (MM), a non-cleavable linker (NCL), and a CD3 binding moiety, wherein: a) the CD3 binding moiety comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; b) the CD3 binding moiety comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; c) the CD3 binding moiety comprises, from N-terminus to C-terminus, a VL and a VH; or d) the CD3 binding moiety the MM comprises, from the N-terminus to the C-terminus, a VL and a VH; the MM1 specifically binds to the CD3 binding portion in competition with CD3; the activatable antibody binds to CD3 via the VH and VL; a) the MM comprises an amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of the MM; b) the MM comprises an 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) the MM comprises an amino acid sequence of formula (X): X1X2X3DX4X5CX6X7DX8X9X 10 CX 11 X 12 (SEQ ID NO: 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, and X 10 is D, H or S, and X 11 is H, P or Y, and X 12 is N, P, or Y. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NOs: 35, 417, 585-588, and 597-599. In some embodiments, the CD3 is human CD3.

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

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

[0048] In some embodiments of any one of the above-described masked anti-CD3 antibodies, the VH has formula (VII): EVQLVESGGGLVX1PGGSLRLSCAASGFTFX2X3YAIX4WVRQAPGKGLEWVX5RIRSKYNNYATYYAX6SVKX7RFTISRDX8SKNTLYLQX9NSLRAEDTAVYYCX 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, and X 10 is A or V, and X 11 is F or Y and X 12is N or T); and VL comprises an amino acid sequence of formula (VIII): X1AVVTQEPSLTVSPGGTVTLTCX2SSTGAVTTSNYX3NWX4QQKPGQAPRGLIGGTX5X6RAPGX7PARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSX8X9WVFGGGTKLTVL (SEQ ID NO: 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, the VH comprises 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 to 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 the VL comprises 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 to 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, the 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 the 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 of any one of the above-described masked anti-CD3 antibodies, the 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 the 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, the VH comprises the amino acid sequence of SEQ ID NO: 402, and the VL comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 402, and the VL comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 405, and the VL comprises the amino acid sequence of SEQ ID NO: 406. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 407, and the VL comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 407, and the VL comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 407, and the VL comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 409, and the VL comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 410, and the VL comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 412, and the VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 410, and the VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 414, and the VL comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 415, and the VL comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:416 and the VL comprises the amino acid sequence of SEQ ID NO:413.In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 416, and the 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 the application provides an activatable antibody ("activatable anti-HER2 antibody") comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a HER2-binding moiety, wherein: a) the HER2-binding moiety comprises a VL, and the activatable antibody comprises a second polypeptide comprising a VH; b) the HER2-binding moiety comprises a VH, and the activatable antibody comprises a second polypeptide comprising a VL; c) the HER2-binding moiety comprises, from N-terminus to C-terminus, a VL and a VH; or d) the HER2-binding moiety comprises, from N-terminus to C-terminus, a VH and a VL; and M comprises a cleavage site; MM inhibits binding of the activatable antibody to HER2 when the CM is not cleaved; when the CM is cleaved, the activatable antibody binds to HER2 via VH and VL, wherein MM is: a) an amino acid sequence of formula (XI): ESX1X2CX3X4DPFX5CQX6 (SEQ ID NO: 670), where 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) an amino acid sequence of formula (XII): X1X2X3X4X5X6CX7X8DPYECX9X 10 (SEQ ID NO: 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, and X 10is A, H, or V); or c) an amino acid sequence of formula (XIII): YNSDDDCX1SX2YDPYTCYY (SEQ ID NO: 672), where X1 is A, I, or V and X2 is H or R. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515. In some embodiments, the 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.

[0051] In some embodiments of any one of the above-described activatable anti-HER2 antibodies, the VH comprises 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 the VL comprises 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, the VH comprises the amino acid sequence of SEQ ID NO: 75, and the VL comprises the amino acid sequence of SEQ ID NO: 76.

[0052] One aspect of the application provides a masked antibody ("masked anti-HER2 antibody") comprising, from N-terminus to C-terminus, a masking moiety (MM), and a HER2-binding moiety, wherein: a) the HER2-binding moiety comprises a VL, and the activatable antibody comprises a second polypeptide comprising a VH; b) the HER2-binding moiety comprises a VH, and the activatable antibody comprises a second polypeptide comprising a VL; c) the HER2-binding moiety comprises, from N-terminus to C-terminus, a VL and a VH; or d) the HER2-binding moiety comprises, from N-terminus to C-terminus, a VH and and VL; the MM specifically binds to the HER2-binding portion in competition with HER2; the activatable antibody binds to HER2 via the VH and VL, wherein the MM is: a) an amino acid sequence of formula (XI): ESX1X2CX3X4DPFX5CQX6 (SEQ ID NO: 670), where 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) an amino acid sequence of formula (XII): X1X2X3X4X5X6CX7X8DPYECX9X 10 (SEQ ID NO: 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, and X 10 is A, H, or V); or c) an amino acid sequence of formula (XIII) YNSDDDCX1SX2YDPYTCYY (SEQ ID NO: 672), where 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, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a HER2-binding moiety. In some embodiments, the masked anti-HER2 antibody is not an activatable antibody. In some embodiments, the masked anti-HER2 antibody comprises, from N-terminus to C-terminus, a masking moiety (MM), a non-cleavable linker (NCL), and a HER2-binding moiety. One aspect of the 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 a VH and a VL; the masked antibody comprises a single-chain polypeptide, and the VH and VL of the antibody or antigen-binding fragment are part of the single-chain polypeptide, or the masked antibody comprises two polypeptide chains, and the VH and VL of the antibody or antigen-binding fragment are part of different polypeptide chains of the masked antibody; wherein the C-terminus of the MM is fused to the N-terminus of the VH or VL of the antibody or antigen-binding fragment; wherein the MM specifically binds to the antibody or antigen-binding fragment in competition with HER2; the MM is: a) an amino acid sequence of formula (XI): ESX1X2CX3X4DPFX5CQX6 (SEQ ID NO: 670), where 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) an amino acid sequence of formula (XII): X1X2X3X4X5X6CX7X8DPYECX9X 10 (SEQ ID NO: 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, and X 10is A, H, or V); or c) an amino acid sequence of formula (XIII)YNSDDDCX1SX2YDPYTCYY (SEQ ID NO: 672), where X1 is A, I, or V and X2 is H or R. In some embodiments, the masked antibody comprises 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 further comprises a cleavable linker, for example, 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 comprise a cleavable linker (e.g., fused to the MM or between the C-terminus of the MM and the N-terminus of the antibody or antigen-binding fragment).

[0053] One aspect of the present application provides a masked antibody ("masked anti-HER2 antibody") comprising, from N-terminus to C-terminus, a masking moiety (MM), a non-cleavable linker (NCL), and a HER2-binding moiety, wherein: a) the HER2-binding moiety comprises a VL, and the activatable antibody comprises a second polypeptide comprising a VH; b) the HER2-binding moiety comprises a VH, and the activatable antibody comprises a second polypeptide comprising a VL; c) the HER2-binding moiety comprises, from N-terminus to C-terminus, a VL and a VH; or d) the HER2-binding moiety comprises, from N-terminus to C-terminus, a VL and a VH. and a VL sequence comprising, towards the terminus, a VH and a VL; the MM competes with HER2 to specifically bind to the HER2 binding portion; the activatable antibody binds to HER2 via the VH and VL, wherein the MM is: a) an 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) an amino acid sequence of formula (XII): X1X2X3X4X5X6CX7X8DPYECX9X 10(SEQ ID NO: 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, and X 10 is A, H, or V); or c) an amino acid sequence of formula (XIII) YNSDDDCX1SX2YDPYTCYY (SEQ ID NO: 672), where X1 is A, I, or V and X2 is H or R. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515.

[0054] In some embodiments of any one of the above-described masked anti-HER2 antibodies, the VH comprises 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 the VL comprises 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, the VH comprises the amino acid sequence of SEQ ID NO: 75, and the VL comprises the amino acid sequence of SEQ ID NO: 76.

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

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

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

[0058] Also provided is a pharmaceutical composition comprising any one of the above-mentioned antibodies, multispecific antibodies, masked antibodies, activatable multispecific antibodies, isolated anti-CD3 antibodies or antigen-binding fragments thereof, 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 the present application provides a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of any one of the pharmaceutical compositions described above. In some embodiments, the pharmaceutical composition comprises an activatable multispecific antibody, wherein CM1 and CM2 are cleaved at the disease site, thereby unblocking 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 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 to provide the multispecific antibody, isolated antibody or antigen-binding fragment thereof, or masked antibody to the subject at a dose 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, the multispecific antibody, isolated antibody or antigen-binding fragment thereof, or masked antibody comprises: a first polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:427, a second polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:428, and a third polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:112; a first polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:83, a second polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:84, and a third polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:85; a first polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:683, a second polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:684, and a third polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:685;a first polypeptide comprising the amino acid sequence of SEQ ID NO:427, a second polypeptide comprising the amino acid sequence of SEQ ID NO:428, and a third polypeptide comprising the amino acid sequence of SEQ ID NO:112; a first polypeptide comprising the amino acid sequence of SEQ ID NO:83, a second polypeptide comprising the amino acid sequence of SEQ ID NO:84, and a third polypeptide comprising the amino acid sequence of SEQ ID NO:85; 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, and a third polypeptide comprising the amino acid sequence of SEQ ID NO:685; a first polypeptide comprising the amino acid sequence of SEQ ID NO:427 without the C-terminal lysine a first polypeptide comprising the amino acid sequence of SEQ ID NO: 428 without the C-terminal lysine, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 428 without the C-terminal lysine, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 112; a first 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 the C-terminal lysine, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 85 without the 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 the C-terminal lysine, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 685 without the C-terminal lysine. In some embodiments, the method further comprises administering to the subject an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the method further comprises administering to the subject a CD137 agonist or antibody. In some embodiments, the CD137 agonist or antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 comprising the amino acid sequence of TGGVGVG (SEQ ID NO: 700), CDR-H2 comprising the amino acid sequence of LIDWADDKYYSPSLKS (SEQ ID NO: 701), and CDR-H3 comprising the amino acid sequence of GGSDTVIGDWFAY (SEQ ID NO: 702);and / or the light chain variable region comprises a CDR-L1 comprising the amino acid sequence of RASQSIGSYLA (SEQ ID NO: 703), a CDR-L2 comprising the amino acid sequence of DASNLET (SEQ ID NO: 704), and a CDR-L3 comprising the amino acid sequence of QQGYYLWT (SEQ ID NO: 705). In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 706, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 7076. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 710, and / or the light chain comprises the amino acid sequence of SEQ ID NO: 711.;

[0060] Compositions, kits and articles of manufacture comprising any one of the above-described multispecific antibodies, masked antibodies, activatable multispecific antibodies, isolated anti-CD3 antibodies or antigen-binding fragments thereof, masked anti-CD3 antibodies, activatable anti-CD3 antibodies, masked anti-HER2 antibodies or activatable anti-HER2 antibodies are also provided. [Brief explanation of the drawings]

[0061] [Figure 1] 1 provides a schematic diagram of an exemplary antibody design of the present application. Antibodies can be converted into activatable antibodies by fusing one or more antigen-binding sites to a masking peptide. A schematic diagram of a Fab-Fc / Fc one-arm scaffold is shown. [Figure 2] A schematic diagram of an exemplary antibody design of the present application is provided. An 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 general 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 antigen-binding site and the second antigen-binding site can bind to different targets. [Figure 3]A schematic diagram of an exemplary antibody design of the present application is provided. An 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 antigen-binding site and the second antigen-binding site can bind to the same target or different targets. The first scFv and the second scFv can bind to the same target or different targets. [Figure 4] This application provides a schematic diagram of an exemplary antibody design.Antibodies can be converted into activatable antibodies 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 left Fab arm of a HER2xCD3 bispecific antibody binds to HER2, and the right scFv arm binds to CD3. [Figure 5] Schematics of exemplary antibody designs of the present application are provided. Antibodies can be converted into activatable antibodies by fusing one or more antigen-binding sites to a masking peptide. Figures 5A-B show schematics of activatable scaffolds. For example, the activatable antibody may be an activatable antibody targeting HER2 and CD3 (HER2xCD3 activatable antibody or SAFEbody) or an activatable antibody targeting CD20 and CD3 (CD20xCD3 activatable antibody or SAFEbody). The masking peptide (represented as a ball) may be fused to the antigen-binding fragment via a cleavable linker. [Figure 6]Characterization of bispecific antibodies by SDS-PAGE electrophoresis is shown. 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 indicate molecular weight markers, which are labeled in kilodaltons on the left side of each gel. In both gels, lane 1 indicates antibody TY24051, lane 2 indicates antibody TY24052, and lane 3 indicates antibody TY24053. [Figure 7] Figure 1 shows size-exclusion high-performance liquid chromatography 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 the heterodimeric protein (main peak), homodimeric protein, and aggregates are shown. [Figure 8] Figure 1 provides enzyme-linked immunosorbent assay (ELISA) analysis of antibodies TY24051 and TY24052. A shows HER2 binding by TY24051 (squares), TY24052 (triangles pointing up), and TY24052 after activation (triangles pointing down). B shows CD3 binding by TY24051 (squares), TY24052 (triangles pointing up), and TY24052 after activation (triangles pointing down). A and B show antibody concentration in M ​​on the x-axis and absorbance at 450 nm on the y-axis. [Figure 9] Assay of T cell-mediated cytotoxic killing upon treatment with bispecific antibodies. Antibody concentration (ng / ml) is shown on the x-axis, and percentage of cell lysis is shown on the y-axis. Target cells were incubated with T cells for 24 hours with TY24051 (circles), TY24052 (squares), isotype control (upward triangles), or no antibody (downward triangles). [Figure 10]Activation of the nuclear factor of activated T cells (NFAT) response element reporter in Jurkat cells in response to treatment with bispecific antibodies TY24051 (filled circles), TY24111 (squares), TY24052 (open circles), and TY24110 (triangles) is shown. Log-transformed antibody concentration in μg / ml is shown on the x-axis, and reporter relative light units (RLU) are shown on the y-axis. 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] Secreted IFNγ levels were measured in humanized peripheral blood mononuclear cell (PBMC) mice (huPBMC-NSG) after administration of parental antibody (TAC2245) or activatable anti-CD3 antibody (TY23104). The X-axis indicates antibody identity and sampling time. From left to right, the graphs include blank, TAC2245 sampled at 0 hours after treatment, TAC2245 sampled at 3 hours after treatment, TAC2245 sampled at 24 hours after treatment, TY23104 sampled at 0 hours after treatment, TY23104 sampled at 3 hours after treatment, and TY23104 sampled at 24 hours after treatment. The Y-axis indicates IFNγ concentration in picograms / ml. [Figure 12] Secreted IFNγ levels after administration of parental antibody (TAC2245) or activatable cross-reactive anti-CD3 antibodies (TY23115 and TY23118) in the huPBMC-NSG mouse model are shown. The X-axis indicates antibody identity and sampling time. From left to right: blank; TAC2245 sampled at 0 hours post-treatment; TAC2245 sampled at 3 hours post-treatment; TAC2245 sampled at 24 hours post-treatment; TY23115 sampled at 0 hours post-treatment; TY23115 sampled at 3 hours post-treatment; TY23115 sampled at 24 hours post-treatment; TY23118 sampled at 0 hours post-treatment; TY23118 sampled at 3 hours post-treatment; and TY23118 sampled at 24 hours post-treatment. The Y-axis indicates IFNγ concentration in picograms / ml. [Figure 13]Binding levels of the parental anti-CD3 antibody TAC2245 (circles) and the activatable anti-CD3 antibody TY23104 (squares) to Jurkat cells are shown. Log-transformed anti-CD3 antibody concentrations in nM are shown on the x-axis, and mean fluorescence intensity (MFI) of binding with a secondary anti-human IgG antibody is shown on the y-axis. [Figure 14] Activation of the NFAT response element reporter in Jurkat cells in response to treatment with parental antibody (TAC2225, circles) or activatable cross-reactive anti-CD3 antibodies (TY23115, squares; and TY23118, triangles). Log-transformed antibody concentrations in nM are shown on the x-axis, and reporter relative luminescence units (RLU) are shown on the y-axis. [Figure 15] Activation of the NFAT response element reporter in Jurkat cells in response to treatment with parental antibody (TAC2245, circles) or activatable anti-CD3 antibodies (TY23100, filled squares, TY23101, triangles, TY23102, triangles, and TY23104, open squares) is shown. Log-transformed antibody concentrations in μg / mL are shown on the x-axis, and reporter relative light units (RLU) are shown on the y-axis. Assays were performed without FcRIIb cross-linking. [Figure 16] Analysis of the masking efficiency of parental CD3 antibodies and activatable anti-CD3 antibodies is shown. (A) Binding of parental CD3 antibody (TAC2225, filled circles) and activatable anti-CD3 antibodies (TY23110, squares; TY23115, upward triangles; and TY23118, downward triangles) to recombinant human CD3δε as determined by ELISA. (B) Activation of the NFAT response element reporter in Jurkat cells in response to treatment with parental CD3 antibody (TAC2225, filled circles) and activatable anti-CD3 antibodies (TY23105, open circles; TY23110, squares; TY23115, upward triangles; and TY23118, downward triangles). Log-transformed antibody concentrations in μg / mL are shown on the x-axis, and reporter relative luminescence units (RLU) are shown on the y-axis. [Figure 17]Figure 17 shows activation of the NFAT response element reporter in Jurkat cells in response to treatment with parental antibody (TAC2225, open circles) or activatable anti-CD3 antibody. In each graph in Figure 17, the log-transformed antibody concentration in μg / mL is shown on the x-axis, the reporter relative light units (RLU) are shown on the y-axis, and the identity of the activatable anti-CD3 antibody is indicated by the shape of the data point, as indicated in each legend. Assays performed without FcRIIb cross-linking are shown. [Figure 18] The levels of Jurkat cell binding by the parental antibody TAC2245 (TAC2225, circles) and activatable anti-CD3 antibodies are shown. In each graph in Figure 18, the log-transformed concentration of anti-CD3 antibody in nM is shown on the x-axis, the mean fluorescence intensity (MFI) of binding of the secondary anti-human IgG antibody is shown on the y-axis, and the identity of the activatable anti-CD3 antibody is indicated by the shape of the data point, as shown in each legend. [Figure 19] Binding of parental and activatable anti-CD3 antibodies to recombinant human CD3δε as determined by ELISA. Log-transformed antibody concentration in M ​​is shown on the x-axis, absorbance at a wavelength of 450 nm is shown on the y-axis, and the identity of the anti-CD3 antibody is indicated by the shape of the data points, as indicated in the respective legends. [Figure 20A] Figure 1 shows the masking efficiency analysis of parental and activatable SP34 variant anti-CD3 / HER2 bispecific antibodies. Binding to recombinant human CD3δε of a low-anti-CD3 affinity parental antibody (TY25023, filled circles) and activatable antibody (TY25026, open circles), as well as a comparative parental antibody (TY24051, filled squares) and activatable antibody (TY24052, open squares), as determined by ELISA, is shown. Log-transformed antibody concentration in M ​​is shown on the x-axis, and absorbance at 450 nm is shown on the y-axis. [Figure 20B] Figure 1 shows the masking efficiency analysis of parental and activatable SP34 variant anti-CD3 / HER2 bispecific antibodies. The binding levels of anti-CD3 antibodies TY24051 (filled circles), TY24052 (open circles), and TY25023 (filled squares) to Jurkat cells are shown. Log-transformed antibody concentration in nM is shown on the x-axis, and mean fluorescence intensity (MFI) of binding with the secondary anti-human IgG antibody is shown on the y-axis. [Figure 21A] Figure 21A shows the masking efficiency and function of parental and activatable SP34 variant anti-CD3 / HER2 bispecific antibodies. Activation of the NFAT response element reporter in Jurkat cells in response to treatment with bispecific antibodies TY24051 (filled circles), TY24052 (open circles), TY25023 (filled squares), and TY25026 (open squares) is shown. Log-transformed antibody concentrations in μg / ml are shown on the x-axis, and reporter relative light units (RLU) are shown on the y-axis. In Figure 21A, NFAT reporter activity was measured in the presence of target cells (SK-OV-3). [Figure 21B] Analyses of masking efficiency and functionality of parental and activatable SP34 variant anti-CD3 / HER2 bispecific antibodies are shown. SK-OV3 tumor cell lysis levels are shown in response to treatment with bispecific antibodies TY24051 (dark gray circles), TY24052 (dark gray squares), TY25023 (light gray triangles), and TY25026 (light gray squares), as well as a reference CD3x isotype control (dark gray triangles). Log-transformed antibody concentrations in ng / mL are shown on the x-axis, and % cytotoxicity is shown on the y-axis. The table below the plots shows the EC50 cytotoxicity for each antibody in ng / mL. [Figure 21C] Figure 1 shows the masking efficiency and function analysis of parental and activatable SP34 variant anti-CD3 / HER2 bispecific antibodies. Secreted IFNγ levels in an activated CD8+ T cell assay are shown in response to treatment with bispecific antibodies TY24051 (dark gray squares), TY24052 (dark gray circles), TY25023 (light gray squares), and TY25026 (light gray circles). Log-transformed antibody concentrations in nM are shown on the x-axis, and IFNγ concentrations in picograms / mL are shown on the y-axis. [Figure 22A]Figure 24F shows cytokine release in cynomolgus monkeys treated with parental 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 indicates time in hours after administration, and the Y-axis indicates cytokine concentration in picograms / mL. The time points at which 0.2, 0.5, and 0.9 mg / kg ("mpk") antibody were administered are indicated by arrows above each plot. IL-6 release levels are also shown in Figure 24F on the log-transformed y-axis. [Figure 22B] Figure 24F shows cytokine release in cynomolgus monkeys treated with parental or activatable bispecific antibodies. Figure 24F shows the levels of cytokines IFNγ, IL-2, IL-6, TNFα, IL-5, and IL-4 released in response to treatment with bispecific antibodies TY24051, TY24052, TY25023, and TY25026. The x-axis shows time in hours after administration, and the y-axis shows the log-transformed concentration of cytokine in picograms / mL. The time points at which antibody doses of 0.2, 0.5, and 0.9 mg / kg were administered are indicated by arrows above each plot. IL-6 release levels are also shown in Figure 24F on the log-transformed y-axis. [Figure 23] Figure 1 shows the activation levels of CD4+ (left plot) and CD8+ (right plot) T cells in response to treatment with bispecific antibodies TY24051 (dark gray squares), TY24052 (dark gray circles), TY25023 (light gray squares), and TY25026 (light gray circles). The x-axis indicates time in hours after administration, and the y-axis indicates cells per μL. The time points at which antibody doses of 0.2, 0.5, and 0.9 mg / kg ("mpk") were administered are indicated by arrows above each plot. [Figure 24A]Figure 1 shows the results of a study of cynomolgus monkeys treated with parental or activatable bispecific antibodies. The T cell levels per μL of total T cells (top), CD4+ T cells (bottom, left), and CD8+ T cells (bottom, right) in monkeys are shown in response to treatment with bispecific antibodies TY24051 (dark gray squares), TY24052 (dark gray circles), TY25023 (light gray squares), and TY25026 (light gray circles). The x-axis indicates time in hours after administration, and the y-axis indicates the number of cells per μL. The time points at which antibody doses of 0.2, 0.5, and 0.9 mg / kg ("mpk") were administered are indicated by arrows above each plot. [Figure 24B] Figure 1 shows the results of a study of cynomolgus monkeys treated with parental or activatable bispecific antibodies. The levels of B cells (left) and NK cells (right) per μL in monkeys are shown in response to treatment with bispecific antibodies TY24051 (circles), TY24052 (squares), TY25023 (upward triangles), and TY25026 (downward triangles). The x-axis indicates time in hours after administration, and the y-axis indicates the number of cells per μL. The time points at which antibody doses of 0.2, 0.5, and 0.9 mg / kg ("mpk") were administered are indicated by arrows above each plot. [Figure 24C]

[0023] Figure 1 shows the results of a study of cynomolgus monkeys treated with parental or activatable bispecific antibodies. The concentrations of bispecific antibodies TY24051 (circles), TY24052 (squares), TY25023 (upward triangles), and TY25026 (downward triangles) are shown in cynomolgus monkeys. The X-axis shows time in hours after administration, and the Y-axis shows log-transformed antibody concentration in μg / mL. The time points at which antibody doses of 0.2, 0.5, and 0.9 mg / kg ("mpk") were administered are indicated by arrows above each plot. [Figure 24D] 1 shows the results of a study of cynomolgus monkeys treated with the parent antibody or the activatable bispecific antibody. Plasma concentrations and pharmacokinetic parameters are shown for monkeys treated with the bispecific antibody. [Figure 24E] 1 shows the results of a study of cynomolgus monkeys treated with parental or activatable bispecific antibodies. IL-6 release in monkeys treated with bispecific antibodies is shown. Parental bispecific antibodies are shown as squares, and activatable bispecific antibodies are shown as circles. [Figure 24F] 1 shows the results of a study of cynomolgus monkeys treated with parental or activatable bispecific antibodies. Absolute lymphocyte counts are shown for monkeys administered bispecific antibodies. Parental bispecific antibodies are shown as squares, and activatable bispecific antibodies are shown as circles. [Figure 25] Figures 25A-25B show flow cytometry analysis of yeast cell surface display of anti-HER2 antibodies. In each scatter plot, the x-axis shows the level of Fab or scFv displayed on yeast cells (detected by antibody binding to an affinity tag fused to the anti-HER2 antibody), and the y-axis shows the level of HER2 binding (detected by binding of PE-conjugated streptavidin to biotinylated human HER2-Fc). A shows binding of Fabs to HER2. B shows binding of scFv to HER2. [Figure 26] Figure 26 shows the results of four rounds (R1, R2, R3, R4) of FACS to screen the CPL yeast library for masking peptides that mask binding to 10 nM biotinylated HER2-Fc. 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] Figures 27A-27B show FACS analysis of binding to selected trastuzumab-derived activatable anti-HER2 antibodies. In each scatterplot, samples were treated with buffer PBSA (left) or TEV protease (right), and the x-axis shows the level of Fab or scFv displayed on yeast cells (detected by antibody binding to an affinity tag fused to the anti-HER2 antibody), while 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] Binding of parental antibody (trastuzumab) and activatable anti-HER2 antibodies (TY22841, TY22842, TY22839, TY22838, and TY22837) to His-tagged HER2 is shown as a measure of the masking efficiency of the activatable antibodies. The X-axis represents time (seconds) and the Y-axis represents binding levels. [Figure 29] Figure 1 shows binding of parental antibody (trastuzumab, filled circles) and activatable anti-HER2 antibodies to recombinant HER2-Fc as determined by ELISA. The x-axis shows the log-transformed antibody concentration in M, and the y-axis shows absorbance at 450 nm. In A, results are shown for TY22836, TY2237, TY2238, TY2239, TY2240, TY2241, TY2242, TY2243, and trastuzumab. In B, results are shown for TY22846, TY2247, TY2250, TY2251, TY2252, TY2253, TY2254, and trastuzumab. In C, results are shown for TY23523, TY23525, TY23526, TY23533, TY23536, TY23537, and trastuzumab. [Figure 30] A reduced SDS-PAGE showing TY22837 alone (lane 1) or in the presence of the protease MMP-9 (lane 2) is provided. [Figure 31] Figure 1 shows the binding of the parent anti-HER2 antibody (trastuzumab, filled circles) and TY22837 to recombinant HER2-Fc as determined by ELISA. TY22837 binding is shown for TY22837 alone (downward triangles) or in the presence of the protease MMP-9 (upward triangles). Log-transformed antibody concentration in M ​​is shown on the x-axis, and absorbance at a wavelength of 450 nm is shown on the y-axis. [Figure 32] Figure 1 shows the binding levels of SK-OV-3 cells by parental anti-HER2 antibody (trastuzumab, filled circles) and activating anti-HER2 antibodies (TY22837, open circles; TY23536, squares). Log-transformed antibody concentrations in nM are shown on the x-axis, and mean fluorescence intensity (MFI) of secondary anti-human IgG antibody binding is shown on the y-axis. [Figure 33A] Shown are the results of three stress tests of the activatable anti-HER2 antibodies TY22837 (left column) and TY22838 (right column). Results after three to six freeze-thaw cycles of the activatable antibodies. The x-axis shows time in minutes, and the y-axis shows the level of antibody aggregation, expressed as absorbance units at 214 nm. [Figure 33B]Shown are the results of three stress tests of the activatable anti-HER2 antibodies TY22837 (left column) and TY22838 (right column). Results are shown after incubating the activatable antibodies at 50°C for 7 days. The x-axis shows time in minutes, and the y-axis shows the level of antibody aggregation in absorbance units at 214 nm. [Figure 33C] Shown are the results of three stress tests of the activatable anti-HER2 antibodies TY22837 (left column) and TY22838 (right column). Results are shown after incubating the activatable antibodies at 40°C for 28 days. The x-axis shows time per minute, and the y-axis shows the level of antibody aggregation, expressed in absorbance units at 214 nm. [Figure 34A] Figure 1 shows the binding of parental antibody (trastuzumab) and activatable anti-HER2 antibodies to recombinant HER2-Fc as determined by ELISA. The length of the masking peptide of the activatable antibodies was varied as shown in Table 19. Results are shown for trastuzumab (circles), TY23171 (upward triangles), TY23172 (downward triangles), and TY22836 (squares). The antibody concentration, log-transformed in M, is shown on the x-axis, and absorbance at a wavelength of 450 nm is shown on the y-axis. [Figure 34B] Figure 1 shows the binding of parental antibody (trastuzumab) and activatable anti-HER2 antibodies to recombinant HER2-Fc as determined by ELISA. The length of the masking peptide of the activatable antibodies was varied as shown in Table 19. Results are shown for trastuzumab (circles), TY23173 (squares), TY23174 (downward triangles), and TY22837 (downward triangles). The antibody concentration, log-transformed in M, is shown on the x-axis, and absorbance at a wavelength of 450 nm is shown on the y-axis. [Figure 35A]Lymphocyte counts, T cell activation, and pharmacokinetic parameters are shown in cynomolgus monkeys treated with CD3-masked bispecific antibody TY25362 alone. Cell levels per μL are shown 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 in response to treatment with CD3-masked bispecific antibody TY25362. The x-axis indicates time in hours after administration, and the y-axis indicates cells per μL. The time points at which antibody doses of 1, 10, and 30 mg / kg ("mpk") were administered are indicated by arrows above each plot. [Figure 35B]

[0023] Figure 1 shows lymphocyte counts, T cell activation, and pharmacokinetic parameters in cynomolgus monkeys treated with the CD3-masked bispecific antibody TY25362 alone. The levels of CD4+ (left plot) and CD8+ (right plot) T cell activation in response to treatment with the bispecific antibody TY25362 are shown. The X-axis indicates time in hours after administration, and the Y-axis indicates the percentage of CD69+ T cells. The time points at which antibody doses of 1, 10, and 30 mg / kg ("mpk") were administered are indicated by arrows above each plot. [Figure 35C]

[0023] Figure 1 shows lymphocyte counts, T cell activation, and pharmacokinetic parameters in cynomolgus monkeys treated with only the CD3-masked bispecific antibody TY25362. TY25362 levels in cynomolgus monkeys are shown. The x-axis shows time in hours after administration, and the y-axis shows log-transformed concentration in μg / mL. The time points at which antibody doses of 1, 10, and 30 mg / kg ("mpk") were administered are indicated by arrows above each plot. [Figure 36A] Figure 1 shows the binding affinity of TY25023 and TY24051 to CD3. The EC50 and Kd for binding of TY25023 and TY24051 to human or monkey CD3δε, as determined by ELISA or Biacore interferometry, respectively, are shown. [Figure 36B]Figure 1 shows the binding affinity of TY25023 and TY24051 to CD3. Figure 2 shows the binding of TY25023 and TY24051 to human CD3δε as determined by ELISA. The EC50 of each antibody for binding to human CD3δε is plotted in nM and shown in the table to the right. [Figure 36C] Figure 1 shows the binding affinity of TY25023 and TY24051 to CD3. Figure 2 shows the binding of TY25023 and TY24051 to monkey CD3δε as determined by ELISA. The binding EC50 of each antibody to monkey CD3δε is plotted in nM and shown in the table to the right. [Figure 36D] Figure 1 shows the binding affinity of TY25023 and TY24051 to CD3. Figure 1 shows the binding of TY25023 and TY24051 to human CD3δε as determined using Biacore interferometry. [Figure 36E] Figure 1 shows the binding affinity of TY25023 and TY24051 to CD3. Figure 2 shows the binding of TY25023 and TY24051 to monkey CD3δε as determined using Biacore interferometry. [Figure 37A]

[0023] Figure 1 shows the results of a cytokine release assay in cynomolgus monkeys treated with parental antibody or activatable anti-CD3 and anti-CD20 bispecific antibody, as measured by ELISA. IL-2 levels in cynomolgus monkey serum over time are shown. The X-axis shows time in hours after administration, and the Y-axis shows IL-2 levels in pg / mL. The time point at which the 0.3 mg / kg dose of antibody was administered is indicated by an arrow. TY25455 is indicated by a circle, TY25606 by a square, TY25715 by an up-pointing triangle, and TY25816 by a down-pointing triangle. [Figure 37B]

[0023] Figure 1 shows the results of a cytokine release assay in cynomolgus monkeys treated with parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies, as measured by ELISA. Peak levels of IL-2 in cynomolgus monkey serum are shown. The X-axis indicates the identity of the antibody, and the Y-axis indicates the peak IL-2 level in pg / mL. [Figure 37C]Figure 1 shows the results of a cytokine release assay in cynomolgus monkeys treated with parental or activatable anti-CD3 and anti-CD20 bispecific antibodies, as measured by ELISA. The levels of IFN-γ in cynomolgus monkey serum over time are shown. The X-axis shows time in hours after administration, and the Y-axis shows IFN-γ levels in pg / mL. The time point at which the 0.3 mg / kg dose of antibody was administered is indicated by an arrow. TY25455 is indicated by a circle, TY25606 by a square, TY25715 by an up-pointing triangle, and TY25816 by a down-pointing triangle. [Figure 37D]

[0023] Figure 1 shows the results of a cytokine release assay in cynomolgus monkeys treated with parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies, as measured by ELISA. Peak IFN-γ levels are shown. The X-axis indicates the identity of the antibody, and the Y-axis indicates the peak IFN-γ levels in pg / mL. [Figure 38A] Pharmacodynamic markers measured by FACS in cynomolgus monkeys treated with parental or activatable anti-CD3 and anti-CD20 bispecific antibodies are shown. Lymphocyte counts (top left), CD3+ T cell counts (top right), and CD19+ B cell counts (bottom left) are shown 24 hours after antibody administration. In each plot, the x-axis represents time after administration in hours, and the y-axis represents cell counts in x109 cells / L. The time point at which the 0.3 mg / kg dose of antibody was administered is indicated by an arrow. TY25455 is represented by a circle, TY25606 by a square, TY25715 by an up-pointing triangle, and TY25816 by a down-pointing triangle. [Figure 38B] Pharmacodynamic markers measured by FACS in cynomolgus monkeys treated with parental or activatable anti-CD3 and anti-CD20 bispecific antibodies are shown. Lymphocyte counts (top left), CD3+ T cell counts (top right), and CD19+ B cell counts (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 counts in x109 cells / L. The time point at which the 0.3 mg / kg dose of antibody was administered is indicated by an arrow. TY25455 is represented by a circle, TY25606 by a square, TY25715 by an up-pointing triangle, and TY25816 by a down-pointing triangle. [Figure 38C]Pharmacodynamic markers measured using FACS in cynomolgus monkeys treated with parental or activatable anti-CD3 and anti-CD20 bispecific antibodies are shown. The numbers of CD3+CD8+ T cells (upper left), CD3+CD4+ T cells (upper right), CD8+CD69+ T cells (lower left), and CD4+CD69+ T cells (lower right) are shown 14 days after antibody administration. For each plot, the X-axis represents time in hours after administration, and the Y-axis represents the percentage of cells relative to lymphocyte levels. The time point at which the 0.3 mg / kg dose of antibody was administered is indicated by an arrow. TY25455 is represented by a circle, TY25606 by a square, TY25715 by an up-pointing triangle, and TY25816 by a down-pointing triangle. [Figure 39A] Pharmacodynamic markers measured by FACS in cynomolgus monkeys treated with the activatable anti-CD3 and anti-CD20 bispecific antibody TY25606 are shown. Lymphocyte counts (top left), CD3+ T cell counts (top right), and CD19+ B cell counts (bottom left) are shown 50 days after antibody administration. In each plot, the x-axis represents time in hours after administration, and the y-axis represents cell counts in x109 cells / L. The arrows indicate the time points after antibody administration at 0.3 mg / kg and 1 mg / kg. [Figure 39B] Pharmacodynamic markers measured using FACS in cynomolgus monkeys treated with the activatable anti-CD3 and anti-CD20 bispecific antibody TY25606 are shown. The numbers of CD3+CD8+ T cells (upper left), CD3+CD4+ T cells (upper right), CD8+CD69+ T cells (lower left), and CD4+CD69+ T cells (lower right) are shown 50 days after antibody administration. In each plot, the X-axis indicates time in hours after administration, and the Y-axis indicates the percentage of cells relative to lymphocyte levels. The time points at which the 0.3 and 1 mg / kg doses of antibody were administered are indicated by arrows. [Figure 40] Total human IgG levels in cynomolgus monkeys treated with the activatable anti-CD3 and anti-CD20 bispecific antibody TY25606, as measured using FACS. The X-axis shows time in hours after administration, and the Y-axis shows log-transformed total human IgG in μg / mL. The arrows indicate the time points at which the 0.3 and 1 mg / kg doses of antibody were administered. [Figure 41]Figure 41A shows the effect of parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies in reporter assays with or without Raji tumor cells. Figure 41A shows a reporter assay with Raji tumor cells. Log-transformed antibody concentrations in nM are shown on the x-axis, and reporter relative light units (RLU) are shown on the y-axis. The shaded area represents the calculated peak concentration in cynomolgus monkey serum at a 0.3 mg / kg dose. TAC2392 is shown as a closed circle, TAC2415 as an open circle, TY25455 as a closed square, TY25606 as an open square, TY25715 as an up-pointing triangle, TY25816 as a down-pointing triangle, and the isotype control as a diamond. Figure 41B shows a reporter assay without Raji tumor cells. Log-transformed antibody concentrations in nM are shown on the x-axis, and reporter relative light units (RLU) are shown on the y-axis. TAC2392 is shown as a closed circle, TAC2415 as an open circle, TY25455 as a closed square, TY25606 as an open square, TY25715 as an up-pointing triangle, TY25816 as a down-pointing triangle, and the isotype control as a diamond. [Figure 42] Figure 42A shows the effect of parental antibodies or activatable anti-CD3 and anti-CD20 bispecific antibodies in reporter assays with or without SU-DHL-4 tumor cells. Figure 42A shows a reporter assay with SU-DHL-4 tumor cells. Log-transformed antibody concentrations in nM are shown on the x-axis, and reporter relative light units (RLU) are shown on the y-axis. TAC2392 (filled circles), TAC2415 (open circles), TY25455 (filled squares), TY25606 (open squares), TY25715 (up-pointing triangles), TY25816 (down-pointing triangles), and isotype control (diamonds). The shaded area indicates the calculated peak concentration in cynomolgus monkey serum at a 0.3 mg / kg dose. Figure 42B shows a reporter assay without SU-DHL-4 tumor cells. Log-transformed antibody concentrations in nM are shown on the x-axis, and reporter relative light units (RLU) are shown on the y-axis. TAC2392 is shown as a closed circle, TAC2415 as an open circle, TY25455 as a closed square, TY25606 as an open square, TY25715 as an up-pointing triangle, TY25816 as a down-pointing triangle, and the isotype control as a diamond. The gray area indicates the calculated peak concentration in cynomolgus monkey serum at a dose of 0.3 mg / kg. [Figure 43]Figure 43A shows the effect 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 level of endogenous B cell killing. Log-transformed antibody concentration in nM is shown on the x-axis, and the percentage of human endogenous B cells is shown on the y-axis. AC1281 is shown as a closed circle, TAC2415 as an open circle, TY25455 as a closed square, TY25606 as an open square, TY25715 as an up-pointing triangle, TY25816 as a down-pointing triangle, and the isotype control as a diamond. The EC50 for B cell killing of each antibody in nM is shown below the plot. Figure 43B shows the level of CD8+ T cell activation. Log-transformed antibody concentration in nM is shown on the x-axis, and the percentage of CD69+ cells in the CD8+ T cell population is shown on the y-axis. TAC2392 is shown as a closed circle, TAC2415 as an open circle, TY25455 as a closed square, TY25606 as an open square, TY25715 as an up-pointing triangle, TY25816 as a down-pointing triangle, and the isotype control as a diamond. The EC50 for T cell activation for each antibody in nM is shown below the plot. [Figure 44] Binding levels of T and B cells to antibodies TAC2392 (filled circles), TY2455 (filled downward triangles), and an isotype control (open circles) are shown, as measured by FACS using PBMCs. Each plot shows the log-transformed antibody concentration in nM on the x-axis and binding levels as mean fluorescence intensity ("MFI") on the y-axis. Binding to human CD4+ T cells is shown in the upper left, human CD8+ T cells in the upper center, human B cells in the upper right, monkey CD4+ T cells in the lower left, monkey CD8+ T cells in the lower center, and monkey B cells in the lower right. The binding EC50 values ​​for TAC2392 and TY2455 on each cell type are shown below the plots, in nM. [Figure 45] Tumor volume over time in female M-NSG immunodeficient mice using human PBMCs and EMT6 mouse breast cancer cells stably transfected with HER2. Mice were dosed at 5 mg / kg with antibodies TY24051 (closed circles), TY25023 (upward triangles), TY25026 (squares), TY25362 (downward triangles), and an isotype control (open circles). The X-axis indicates days post-inoculation, with arrows indicating antibody administration times. The Y-axis indicates tumor volume in mm3. [Figure 46]A schematic of the proposed mechanism of action of SAFE bodies is shown. As shown on the left, when a SAFE body is in proximity to normal tissue (e.g., tissue lacking the epitope bound by the SAFE body), the SAFE body remains masked. Without wishing to be bound by theory, two pathways are postulated for the mechanism by which a SAFE body binds to a target site. In pathway 1, the cleavable moiety is cleaved by a protease in proximity to tumor tissue, thereby removing the masking moiety and unmasking the SAFE body so that it can bind to the target. In pathway 2, the cleavable moiety is not necessarily cleaved, and binding of the SAFE body to the target competes with binding of the SAFE body to the masking moiety. Once the SAFE body binds to the target site, the cleavable moiety can be cleaved by a protease, thereby unmasking the SAFE body. [Figure 47] Figure 1 shows induction of luciferase expression in a Jurkat / NFAT-Luc reporter line by CD20xCD3 bispecific antibodies, with target Raji cells used to screen additional CD20xCD3 bispecific antibodies. [Figure 48] Tumor growth curves of different treatment groups (N=6) of female M-NSG mice bearing established Raji tumors are shown. [Figure 49] 1 shows a PK study 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]Figure 1 shows a toxicity and pharmacology study in cynomolgus monkeys of a single-dose injection of CD20xCD3 bispecific antibody or SAFE body / bispecific antibody. A shows a plot of the normalized CD19+ B cell percentage over time in blood samples from cynomolgus monkeys administered a single dose of drug. B shows a plot of the normalized CCD3+ T cell percentage over time in blood samples from cynomolgus monkeys administered a single dose of drug. C shows pre- and post-dose levels of IFN-γ (pg / mL) in cynomolgus monkeys administered a single dose of drug. D shows pre- and post-dose levels of IL-2 (pg / mL) in cynomolgus monkeys administered a single dose of drug. [Figure 51] Figure 1 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, and activatable antibodies TY27151 and TY27008. [Figure 52] Shown are the results of an assay of killing of SKOV3 cells (FIG. 52A), MCF7 cells (FIG. 52B), and A549 cells (FIG. 52C) by CD8+ T cells in the presence of bispecific antibodies TY25023, TY24051, and TY25238. [Figure 53] The cleavage efficiency of the masking site of anti-CD3 antibody (FIG. 53A) and anti-HER2 antibody (FIG. 53B) in various HER2xCD3 bispecific antibodies is shown. [Figure 54] Figure 1 shows the in vivo antitumor efficacy of HER2xCD3 antibody and negative control against HER2-expressing tumors (SK-OV3) in a xenogeneic in vivo tumor model. Data points represent group means, and error bars represent SEM. [Figure 55]Figure 1 shows PK data in cynomolgus monkeys treated with HER2xCD3 bispecific antibody.C shows systemic cytokine release (IL-6, IFN-γ, IL-2, TNF-α) in cynomolgus monkeys. [Figure 56] Figure 56 shows in vitro cytokine release, including IFN-γ (Figure 56A) or IL-2 (Figure 56B), by human PBMC in the presence of MCF7. [Figure 57]

[0023] Figure 1 shows the in vivo antitumor effects of HER2xCD3 antibody and negative control on HER2-expressing tumors (SK-OV3) in a xenogeneic in vivo tumor model. Data points represent group means, and error bars represent SEM. [Figure 58] Lymphocyte margination induced by TY25023, TY25026, and TY25362. [Figure 59] 1 shows cytokine release levels in cynomolgus monkeys administered TY25023, TY25026, and TY25362 as determined by ELISA. [Figure 60] PK curves for cynomolgus monkeys administered TY25023, TY25026, and TY25362 are shown. [Figure 61] 1 shows the results of a luciferase-based CD3 gene reporter assay characterizing the effect of anti-HER2xCD3 activatable / bispecific antibodies on activation of CD3 signaling. [Figure 62]

[0023] Figure 1 shows the in vivo anti-tumor efficacy of the anti-HER2xCD3 bispecific parental antibody TY25238 and the activatable antibodies TY27008 and TY27151 in a PBMC-engrafted HT55 xenograft model. Data points represent group means, and error bars represent SEM. Antibody administration is indicated by arrows. [Figure 63] Figure 1 shows the in vivo anti-tumor efficacy of the anti-HER2xCD3 bispecific activatable antibody TY27151 compared to trastuzumab, DS-8201 ADC, or vehicle in a HT55 xenograft model engrafted with PBMCs. Data points represent group means, and error bars represent SEM. Antibody administration is indicated by arrows. [Figure 64]Figure 64A shows the synergistic anti-tumor effect of the anti-HER2xCD3 bispecific activatable antibody TY27151 in combination with anti-CD137 mAb in the MC38-hHER2 mouse colon cancer syngenic model. Figure 64A shows the in vivo anti-tumor effect of the anti-HER2xCD3 bispecific activatable antibody TY27151 in the MC38-hHER2 mouse colon cancer syngenic model. Antibody administration is indicated by the arrow. Figure 64B shows the results of re-challenging the MC38-hHER2 tumor without antibody administration. The arrow indicates tumor re-challenge. In both figures, data points represent group means, and error bars represent SEM. [Figure 65] Figure 1 shows the in vivo antitumor efficacy of the anti-HER2xCD3 bispecific activatable antibody TY27151 as monotherapy or in combination with anti-PD-1 mAb 2E5 in a PBMC-engrafted SK-OV3 xenograft model. Data points represent group means, and error bars represent SEM. Antibody administration is indicated by arrows. DETAILED DESCRIPTION OF THE INVENTION

[0062] The present 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, the first antigen-binding fragment being 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. Without wishing to be bound by theory, it is believed 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 affinities of the masking moiety and the antigen-binding fragment for CD3 determine 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 provides a wide therapeutic window 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 associated with the target antigen.

[0063] Thus, one aspect of the present application provides a multispecific antibody comprising: a) a first antigen-binding fragment that specifically binds CD3 and is fused to a first masking moiety (MM1); and b) a second antigen-binding fragment that specifically binds to a target antigen; wherein MM1 specifically binds to the CD3-binding moiety in competition with CD3; and the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by 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 present application provides an activatable multispecific antibody (also referred to as 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 moiety. In some embodiments, the second antigen-binding fragment is fused to a second masking site via a second cleavable moiety. An exemplary type of activatable multispecific antibody is the TAAxCD3 SAFE body bispecific T cell engager ("SAFE-bsAb"). The TAAxCD3 SAFE-bsAb molecule consists of an antigen-binding fragment of an antibody that specifically binds to a tumor-associated antigen ("TAA"), which may be 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 the circulation or in healthy tissues, activatable antibodies are inactive because the masking moiety can block antigen binding. However, upon cleavage of the cleavable moiety at the target site (e.g., disease site), the activatable antibody becomes activated and binds to both CD3 and the target antigen (e.g., TAA). Due to the weak affinity of the first antigen-binding fragment and the high masking efficiency of the first masking moiety, the activatable multispecific antibodies described herein provide a broad therapeutic window and reduce side effects associated with nonspecific binding. For example, activating forms of exemplary TAAxCD3 SAFE-bsAbs have been observed to potently stimulate T cell activation and killing of TAA+ tumor cells. Furthermore, no visible cytokine release syndrome or other adverse events were observed in exploratory toxicity studies of TAAxCD3 SAFE-bsAbs in cynomolgus monkeys, even at high dose levels (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 the parent antibodies. The activatable 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 cancers associated with the target antigen.

[0065]

[0010] Accordingly, one aspect of the present application provides an activatable multispecific antibody comprising: a) a first antigen-binding fragment that specifically binds CD3, the first antigen-binding fragment being fused to a first masking moiety (MM1) via a first cleavable moiety (CM1); and b) a second antigen-binding fragment that specifically binds a target antigen, wherein CM1 comprises a first cleavage site; when CM1 is not cleaved, MM1 inhibits binding of the activatable antibody to CD3; and when CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment; and the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by 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] Also provided are isolated anti-CD3 antibodies, masked anti-CD3 antibodies (including activatable anti-CD3 antibodies), masked anti-HER2 antibodies (including activatable anti-HER2 antibodies), compositions, methods of making and methods of use. I. Definition

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

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

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

[0070] The term "antibody" includes, but is not limited to, fragments capable of binding to antigen, such as Fv, Fab, Fab', and (Fab')2. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name reflecting their ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of 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 comprising the population are identical and / or bind to the same epitope. This excludes possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation, although such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring 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 techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0072] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence. HVRs may form structurally defined loops ("hypervariable loops"). Generally, naturally occurring four-chain antibodies contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). HVRs generally contain amino acid residues from the hypervariable loops and / or "complementarity-determining regions" (CDRs), which are those with the highest sequence variability and / or are 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)). With the exception of CDR1 of VH, CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity-determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained within regions of CDRs called truncated CDRs or a-CDRs. 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 indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0073] Table I below provides exemplary CDR definitions according to various algorithms known in the art. Table I: CDR definitions TIFF2024508671000001.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 the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain containing four framework regions (FR) and three hypervariable regions (HVR), arranged from the amino terminus to the carboxy terminus in the following order: FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4. (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. 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 specific antigen may be isolated by using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., 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 variations thereof refer to the numbering system used for the heavy chain constant domains of antibodies 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 numbered sequence with the homologous regions of the antibody's sequence.

[0076] The Kabat numbering system is commonly used to refer to residues within the variable domain (roughly corresponding to residues 1-107 in the light chain and residues 1-113 in the heavy chain) (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 shortening or insertion of the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat), inserted residues after residue 82 of the heavy chain FR (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by aligning the "standard" Kabat numbered sequence with the homologous regions of the antibody's sequence.

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

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

[0079] As used herein, the term "heavy chain constant region" refers to a region comprising at least three heavy chain constant domains, CH1, CH2, and CH3, as well as the hinge region between CH1 and CH2. Non-limiting examples of heavy chain constant regions include gamma, delta, and alpha. Non-limiting examples of heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody comprising a gamma constant region is an IgG antibody, an antibody comprising a delta constant region is an IgD antibody, and an antibody comprising an alpha constant region is an IgA antibody. Furthermore, an antibody comprising a mu constant region is an IgM antibody, and an antibody comprising an epsilon constant region is an IgE antibody. A particular isotype can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a γ1 constant region), IgG2 (containing a γ2 constant region), IgG3 (containing a γ3 constant region), and IgG4 (containing a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an α1 constant region) and IgA2 (containing an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0080] The term "CH2 domain" of the human IgG Fc region typically spans approximately residues 231 to 340 of IgG in the EU numbering system. The CH2 domain is unique in that it is not tightly paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact native IgG molecules. It has been speculated that carbohydrates may provide a surrogate for domain-domain pairing and help stabilize the CH2 domain. Burton, Molec. Immunol. 22:161-206 (1985).

[0081] The term "CH3 domain" includes the stretch of residues from the C-terminus of the Fc region to the CH2 domain (ie, from about amino acid residue 341 to about amino acid residue 447 of IgG according to the EU numbering system).

[0082] As used herein, the term "heavy chain" refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain also comprises at least a portion of a heavy chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide comprising 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 constant region" refers to a region comprising a light chain constant domain, CL. Non-limiting exemplary light chain constant regions include λ and κ.

[0084] As used herein, the term "light chain" refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain also comprises at least a portion of a light chain constant region. As used herein, the term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0085] "Affinity" refers to the strength of the sum of non-covalent interactions between the binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of molecule X for partner Y is generally determined by the dissociation constant (K d ) 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 trispecific antibodies), the affinity of the antibody to each binding specificity (i.e., target) can be measured.

[0086] The terms "bind," "specifically bind," or "specific for" refer to a measurable, reproducible interaction, such as binding between a target and an antibody, which determines the presence of a 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, avidity, more readily, and / or longer duration than it binds to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target (e.g., as 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, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but does not require, exclusive binding.

[0087] The term "multispecific" when used in connection with an antibody refers to an antibody that has polyepitopic specificity (i.e., capable of specifically binding to two, three, or more different epitopes on one biomolecule, or capable of specifically binding to epitopes on two, three, or more different biomolecules).

[0088] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs), compared to a parent antibody (not possessing such modifications), which modifications result in an improvement in the affinity of the antibody for antigen. In some examples, an affinity matured antibody refers to an antibody with one or more modifications in one or more complementarity determining regions (CDRs), compared to a parent antibody (not possessing such modifications), which modifications result in an improvement in the affinity of the antibody for antigen.

[0089] As used herein, a "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody that comprises 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 a chimeric antibody are derived from a first species, and all of the constant regions of a chimeric antibody are derived from a 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 has been replaced with the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, a humanized antibody is Fab, (Fab'), etc.

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

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

[0093] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on p. 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta), can be performed. Useful effector cells for such assays include PBMCs and NK cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo in an animal model, such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Additional polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc regions) and increased or decreased ADCC activity are described, for example, in U.S. Patent Nos. 7,923,538 and 7,994,290.

[0094] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass), which in turn binds to its cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc regions) and increased or decreased C1q binding ability are described, e.g., in U.S. Pat. Nos. 6,194,551 B1, 7,923,538, 7,994,290, and WO 1999 / 51642. See also, e.g., Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0095] Polypeptide variants with "altered" FcR binding affinity or ADCC activity are those that have either enhanced or decreased FcR binding activity and / or ADCC activity compared to the parent polypeptide or a polypeptide comprising a native-sequence Fc region. Polypeptide variants that "exhibit increased binding" to FcR bind to at least one FcR with better affinity than the parent polypeptide. Polypeptide variants that "exhibit decreased binding" to FcR bind to at least one FcR with lower affinity than the parent polypeptide. Such variants that exhibit decreased binding to FcR may have little or no appreciable binding to FcR, e.g., 0-20% of the binding to FcR compared to a native-sequence IgG Fc region.

[0096] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" may be used interchangeably and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides that comprises a nucleic acid molecule or polynucleotide.

[0097] The terms "polypeptide" and "peptide" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such a polymer of amino acid residues may contain natural or non-natural amino acid residues. Both full-length proteins and fragments thereof are included in the definition. The terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Further, "polypeptide" includes modifications to the native sequence, such as deletions, additions, and substitutions (generally conservative in nature), as long as the polypeptide maintains the desired activity. These modifications may be intentional (e.g., via site-directed mutagenesis) or accidental (e.g., via host mutations), which may result from protein or errors by PCR amplification.

[0098] A polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity with the native sequence polypeptide, after aligning the sequences to achieve the maximum percent sequence identity (not considering any conservative substitutions as part of the sequence identity) and introducing gaps if 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 sequence polypeptide.

[0099] As used herein, "percent (%) amino acid sequence identity" with respect to a peptide, polypeptide, or antibody sequence 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, using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or MEGALIGN (trademark) (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms necessary to achieve the maximum alignment over the full 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 amino acid. 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 activities, such as retention / improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC. Table A. Exemplary Amino Acid Substitutions TIFF2024508671000002.tif116170

[0101] Amino acids may be grouped according to common side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[0102] The term "vector" is used to describe a polynucleotide that can be engineered to contain cloned polynucleotide(s) that can be propagated in a host cell. A vector may contain one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters and / or enhancers) that control the expression of a polypeptide of interest, 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 a polypeptide of interest in a host cell.

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

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

[0105] The terms "individual" or "subject" are used interchangeably herein to refer to a mammal. In some embodiments, methods are provided for treating mammals, including but not limited to humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. In some examples, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder.

[0106] As used herein, "treatment" or "treating" is an approach to obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms attributable to the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, ameliorating the condition of the disease, providing remission (partial or total) of the disease, reducing the dosage of one or more other therapeutic agents required to treat the disease, slowing the progression of the disease, improving quality of life, and / or prolonging survival. Reduction of the pathological consequences of cancer is also encompassed by "treatment." The methods of the present invention contemplate any one or more of these aspects of treatment.

[0107] The term "prevent" and similar terms such as "prevented," "preventing," etc. refer to an approach for preventing, inhibiting, or reducing the likelihood of recurrence of a disease or condition, e.g., cancer. It also refers to delaying the recurrence of the disease or condition, or delaying the recurrence of symptoms of the disease or condition. As used herein, "prevention" and similar terms also include reducing the extent, impact, symptoms, and / or burden of a disease or condition prior to the recurrence of the disease or condition.

[0108] As used herein, "delaying" the onset of cancer means extending, preventing, slowing, inhibiting, stabilizing, and / or postponing the onset of the disease. This delay can be of varying duration, depending on the history of the disease and / or the individual being treated. A method of "delaying" the onset of cancer is one that reduces the likelihood of disease onset in a given timeframe and / or reduces the extent of 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 onset can be detected using standard methods, including, but not limited to, computed tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Onset also refers to the progression of cancer that was initially undetectable, including onset, recurrence, and development.

[0109] As used herein, the term "effective amount" refers to an amount of an agent or combination of agents sufficient to treat a particular disorder, condition, or disease, for example, to ameliorate, alleviate, relieve, and / or delay one or more of the symptoms. With respect to cancer, an effective amount 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 amount is an amount sufficient to delay the onset of the disease. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, suppress, slow to some extent, and preferably stop cancer cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor onset and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0110] It is understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0111] Reference herein to "about" a value or parameter includes (and accounts for) variations on that value or parameter itself. For example, a statement referring to "about X" includes the statement "X."

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

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

[0114] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0115] As used herein, the term "and / or," phrases 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 term "and / or," phrases such as "A, B, and / or C," are intended to encompass 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 Certain aspects of the present 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 the present application provides multispecific antibodies capable of binding to both T cells and target cells, such as tumor cells. In some embodiments, the multispecific antibodies are bispecific. In some embodiments, the multispecific antibodies are trispecific. In some embodiments, the multispecific antibodies bind to CD3 on the surface of T cells. Due to on-target and off-tumor effects, BiTE molecules are associated with high cytotoxicity, including central nervous system (CNS) toxicity and cytokine storm. Activatable BiTE molecules with improved specificity and reduced side effects are needed.

[0118] In some embodiments, a multispecific antibody is provided that includes: a) a first antigen-binding fragment that specifically binds CD3, the first antigen-binding fragment fused to a first masking moiety (MM1); 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 MM1 competes with CD3 to specifically bind the CD3-binding moiety; the multispecific antibody binds to CD3 via the first antigen-binding fragment; and the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5). In some embodiments, the first antigen-binding fragment binds to CD3 with 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 CD3, wherein the first antigen-binding fragment is fused to a first masking moiety (MM1); 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 MM1 competes with CD3 to specifically bind the CD3-binding moiety; the multispecific antibody binds to CD3 via the first antigen-binding fragment; the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5), and wherein 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 the present application provides activatable multispecific antibodies capable of binding to both T cells and target cells, such as tumor cells. In some embodiments, the activatable antibodies are bispecific. In some embodiments, the activatable antibodies are trispecific. For example, in some embodiments, the activatable multispecific antibodies are activatable bispecific T cell engagers ("BiTEs"). In some embodiments, the activatable multispecific antibodies bind to CD3 on the surface of T cells. Due to their on-target and off-tumor effects, BiTE molecules are associated with high cytotoxicity, including central nervous system (CNS) toxicity and cytokine storm. Activatable BiTE molecules with improved specificity and reduced side effects are needed.

[0121] The present disclosure is based, in part, 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) and masking moieties that highly efficiently reduce the binding of anti-CD3 antibodies (see, e.g., Tables 4-5). Figure 46 illustrates a possible mechanism of action of activatable BiTE molecules. Without wishing to be bound by theory, activatable BiTE molecules with relatively weak CD3 binding and / or high masking efficiency for blocking CD3 binding are believed to have fewer severe side effects than conventional BiTE molecules. This reduced severity of side effects is believed to enable the activatable BiTE molecules described herein to have a larger therapeutic window. That is, the activatable BiTE molecules described herein can be administered to effectively treat disease without producing toxic effects, such as cytokine storm, that are commonly associated with conventional BiTE molecules, e.g., BiTE molecules with stronger CD3 binding affinity. Thus, the present 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 CD3, the first antigen-binding fragment being fused to a first masking moiety (MM1) via a first cleavable moiety (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 CM1 comprises a first cleavage site; when CM1 is not cleaved, MM1 inhibits binding of the activatable antibody to CD3; and when CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment; the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5). In some embodiments, the first antigen-binding fragment binds to CD3 with 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 CD3, the first antigen-binding fragment being fused to a first masking moiety (MM1) via a first cleavable moiety (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 CM1 comprises a first cleavage site; when CM1 is not cleaved, MM1 inhibits binding of the activatable antibody to CD3; and when CM1 is cleaved, the activatable multispecific antibody binds CD3 via the first antigen-binding fragment; the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5), and wherein 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, an activatable multispecific antibody is provided, comprising: a) a first antigen-binding fragment comprising VH1 and VL1 of an anti-CD3 antibody that specifically binds 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 an antibody that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19). wherein MM1 is fused to the N-terminus of VL1 via CM1, wherein CM1 comprises a first cleavage site; when CM1 is not cleaved, MM1 inhibits binding of the activatable antibody to CD3; and when CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment; and the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) 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 an scFv comprising, from N- to C-terminus, VL1, an optional linker, and VH1.

[0125] In some embodiments, an activatable multispecific antibody is provided comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-1st CH3(1a); (ii) the second polypeptide comprises a structure represented by the formula: MM1-CM1-VL1-VH1-hinge-CH2-second CH3(1b); (iii) the third polypeptide comprises a structure represented by the formula: VL2-CL(1c); where: 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 immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; the first CH3 is the first immunoglobulin heavy chain constant domain 3; the second CH3 is a second immunoglobulin heavy chain constant domain 3; Hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the masking moiety; CM1 is a cleavable moiety containing the cleavage site; wherein VH1 and VL1 associate to form an scFv that specifically binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5), and wherein 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), wherein when CM1 is not cleaved, MM1 inhibits binding of the activatable antibody to CD3; when 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). In some embodiments, the multispecific antibody (e.g., its second polypeptide) comprises 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, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH1-CH1-hinge-CH2-first CH3(3a); (ii) the second polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-second CH3(3b); (iii) the third polypeptide comprises a structure represented by the formula: MM1-CL1-VL1-CL(3c); (iv) the fourth polypeptide comprises a structure represented by the formula: VL2-CL(3d); where: 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 immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; Hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the masking moiety; CM1 is a cleavable moiety containing the cleavage site; wherein VH1 and VL1 associate to form a first Fv that specifically binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5); and wherein 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, when CM1 is uncleaved, MM1 inhibits binding of the activatable antibody to CD3; and when 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, an activatable multispecific antibody is provided, comprising: a) a first antigen-binding fragment that specifically binds 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 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 the second masking moiety (MM2) via a second cleavable moiety (CM2); wherein CM1 comprises a first cleavage site; and wherein MM1 is cleaved when CM1 is not cleaved. inhibits binding of the activatable antibody to CD3; wherein when CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment; and wherein CM2 comprises the second cleavage site; when CM2 is not cleaved, MM2 inhibits binding of the activatable antibody to the target antigen; and wherein when CM2 is cleaved, the activatable multispecific antibody binds to the target antigen via the second antigen-binding fragment; and the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) 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).

[0128] In some embodiments, an activatable multispecific antibody is provided comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-first CH3(2a); (ii) the second polypeptide comprises a structure represented by the formula: MM1-CM1-VL1-VH1-hinge-CH2-second CH3(2b); (iii) the third polypeptide comprises a structure represented by the formula: MM2-CM2-VL2-CL(2c); where: 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 immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; the first CH3 is the first immunoglobulin heavy chain constant domain 3; the second CH3 is a second immunoglobulin heavy chain constant domain 3; Hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the first masking portion; CM1 is a first cleavable moiety comprising a first cleavage site; MM2 is the second masking portion; CM2 is a second cleavable moiety comprising a second cleavage site; wherein VH1 and VL1 associate to form an scFv that specifically binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5), and wherein 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), wherein, when CM1 is uncleaved, MM1 inhibits binding of the activatable antibody to CD3; when CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment; when CM2 is uncleaved, MM2 inhibits binding of the activatable antibody to the target antigen; and when 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). In some embodiments, the multispecific antibody (e.g., its second polypeptide) comprises an amino acid linker between VL1 and VH1.

[0129] In some embodiments, an activatable multispecific antibody is provided comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH1-CH1-hinge-CH2-1st CH3(4a); (ii) the second polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-second CH3(4b); (iii) the third polypeptide comprises a structure represented by the formula: MM1-CL1-VL1-CL(4c); (iv) the fourth polypeptide comprises a structure represented by the formula: MM2-CL2-VL2-CL(4d); where: 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 immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; Hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the first masking portion; CM1 is a first cleavable moiety comprising a first cleavage site; MM2 is the second masking portion; CM2 is a second cleavable moiety containing a second cleavage site; wherein VH1 and VL1 associate to form a first Fv that specifically binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5); and wherein 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, when CM1 is uncleaved, MM1 inhibits binding of the activatable antibody to CD3; where, when CM1 is cleaved, the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment; when CM2 is uncleaved, MM2 inhibits binding of the activatable antibody to the target antigen; and where, when 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 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 a reference antibody to CD3. In some embodiments, the first antigen-binding fragment binds to CD3 with a higher dissociation constant than a reference antibody to CD3. In some embodiments, the first antigen-binding fragment binds to CD3 with a lower dissociation constant than a reference antibody to 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 is present 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 is present in a multispecific antibody or in the activated form of an activatable multispecific antibody, i.e., when CM1 is cleaved and MM1 is not bound to the first antigen-binding fragment.

[0131] In some embodiments, the first antigen-binding fragment exhibits a half-maximal binding concentration (EC) of any one of the antibodies of 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 more (including any value or range therebetween) as determined by enzyme-linked immunosorbent assay (ELISA). 50In some embodiments, the first antigen-binding domain binds to human CD3 with an EC50 of any one of about 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 as determined by enzyme-linked immunosorbent assay (ELISA). In some embodiments, the EC50 is determined by ELISA measuring binding of the unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the first antigen-binding fragment is an scFv, and the EC50 is determined by ELISA measuring binding of the unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the EC50 is determined by ELISA measuring 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 an scFv, and the EC50 is determined by ELISA measuring binding of a parent multispecific antibody lacking CM and MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the EC50 measures binding of an antigen-binding fragment that binds to CD3 (e.g., an isolated anti-CD3 scFv or scFv-Fc fusion protein) to CD3 (e.g., human CD3 or human CD3δε) as determined by ELISA.

[0132] In some embodiments, the first antigen-binding fragment has an EC2 of the reference antibody (e.g., SP34). 50or 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 or more times higher EC 50 In some embodiments, the EC of the first antigen-binding fragment reference antibody (e.g., SP34) binds to CD3 (e.g., human CD3) at 100 MHz, including any value or range therebetween. 50 EC values ​​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 higher than 50In some embodiments, the EC50 of the first antigen-binding fragment and the reference antibody is measured under the same test conditions. In some embodiments, the EC50 of the first antigen-binding fragment and the reference antibody is 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 comprises a CD3-binding portion corresponding to SP34 (e.g., comprising the six CDRs of SP34). In some embodiments, the EC50 is determined by measuring the binding of a parent multispecific antibody lacking a CM and MM and a reference parent multispecific antibody lacking a CM and MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the reference parent multispecific antibody lacking a CM and MM comprises a CD3-binding portion corresponding to SP34 (e.g., comprising the six CDRs of SP34). In some embodiments, the Kd of the first antigen-binding fragment for binding to CD3 and the EC50 of the reference antibody are determined by ELISA, for example, the ELISA described in Example 3. In some embodiments, the Kd of the first antigen-binding fragment for binding to CD3 and the EC50 of the reference antibody are determined by a 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, at least about any one of 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 or more times weaker than the Kd of the reference antibody (including any value or range therebetween). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a dissociation constant (Kd) that 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 times weaker than the Kd of the reference antibody (e.g., SP34). In some embodiments, the Kds 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 the unmasked multispecific antibody and the unmasked multispecific reference antibody to CD3. In some embodiments, the unmasked multispecific reference antibody comprises a CD3-binding portion corresponding to SP34 (e.g., comprising the six CDRs of SP34). In some embodiments, the Kd is determined by measuring the binding of a parent multispecific antibody lacking a CM and MM and a reference parent multispecific antibody lacking a CM and MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the reference parent multispecific antibody lacking a CM and MM comprises a CD3-binding portion corresponding to SP34 (e.g., comprising the 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 a dissociation constant (Kd) of at least about any one of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 nM or more (including any value or range therebetween). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a dissociation constant (Kd) of at least about any one of 1, 10, or 100 μM (including any value or range therebetween) (when in an activated form). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a dissociation constant (Kd) of about any one of 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 a reference antibody (e.g., SP34), e.g., a koff of the reference antibody. off at least about 2, 5, 10, 20, 50, 100, 200 or more times faster than (including any value or range therebetween).

[0136] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a relatively slow on-rate (k) compared to a reference antibody (e.g., SP34), e.g., a k of the reference antibody. on at least about 2, 5, 10, 20, 50, 100, 200 or more times slower than (including any value or range therebetween).

[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), e.g., at least about any one of 2, 5, 10, 20, 50, 100, 200 or more times smaller than the Kd of the reference antibody (including any value or range therebetween).

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

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

[0141] The first antigen-binding fragment and / or the second antigen-binding fragment may be in any suitable format, including, but not limited to, Fab, Fv, scFab, and scFv. The antigen-binding fragment may have a single polypeptide chain or may have 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 an antigen-binding fragment having multiple polypeptide chains. In some embodiments, a 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, a 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 can be derived from any one of the anti-CD3 antibodies described herein, which has an EC50 of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5). The anti-CD3 antibodies and antigen-binding fragments can 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 the multispecific antibody comprises one, two, three, four, five, or six CDRs of anti-CD3 antibody TY24051, 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 a 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 antibody 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 a VH and / or VL of antibody TY25023 as shown in Table 8. In some embodiments, the first antigen-binding fragment comprises an scFv of antibody TY25023 as shown in Table 9. In some embodiments, the first antigen-binding fragment comprises a 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 to 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 ability to bind to CD3 as an antibody comprising 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 regions outside of the CDRs (i.e., in the FRs). In certain embodiments, 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 comprises a VL1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to 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 ability to bind to CD3 as an antibody comprising 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 regions outside of the CDRs (i.e., in the FRs). In certain embodiments, the VL1 comprises one, two, or three CDRs selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 400.

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

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

[0149] In some embodiments, the first antigen-binding fragment comprises a VH1 comprising CDR-H1, CDR-H2, and CDR-H3 of the VH having the sequence set forth in SEQ ID NO: 402; and a VL1 comprising CDR-L1, CDR-L2, and CDR-L3 of the VL having the sequence set forth 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 an scFv of antibody TY25238 as shown in Table 9. In some embodiments, the first antigen-binding fragment comprises a 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 to 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 an antibody comprising 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 regions outside of the CDRs (i.e., in the FRs). In certain embodiments, 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 comprises a VL1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to 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 an antibody comprising 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 regions outside of the CDRs (i.e., in the FRs). In certain embodiments, the VL1 comprises one, two, or three CDRs selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 397; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 380; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381.

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

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

[0155] In some embodiments, the first antigen-binding fragment comprises a VH1 comprising CDR-H1, CDR-H2, and CDR-H3 of the VH having the sequence set forth in SEQ ID NO: 410; and a VL1 comprising CDR-L1, CDR-L2, and CDR-L3 of the VL having the sequence set forth 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 moieties for the anti-CD3 antibodies described herein can be used, including, for example, the masking moieties in Section F, "Masking Moiety (MM)," and Tables B, 18-22, 13A, and 40. In some embodiments, the first masking moiety (MM1) comprises an amino acid sequence of formula (IX): PYDDPDCPSHX1SDCDX2 (SEQ ID NO: 668), where X1 is D or E and X2 is N or Q. In some embodiments, the first masking moiety (MM1) comprises an amino acid sequence of formula (X): X1X2X3DX4X5CX6X7DX8X9X 10 CX 11 X 12(SEQ ID NO: 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, and X 10 is D, H, or S, and X 11 is H, P, or Y, and X 12 is N, P, or Y). In some embodiments, the first masking portion (MM1) comprises the amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of MM1. In some embodiments, the first masking portion (MM1) comprises the amino acid sequence of SEQ ID NO: 417 (EVGSYPYDDPDCPSHESDCDQ). In some embodiments, the first masking portion (MM1) comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first masking portion (MM1) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 585-588. In some embodiments, the first masking portion (MM1) comprises 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 about any one of 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 any one of about 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 about any one of 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 a target (e.g., human CD3) of an activatable antibody before activation that includes a first masking moiety compared to the affinity for binding to a target (e.g., human CD3) of a corresponding unmasked antibody lacking the first masking moiety (the "parent antibody") or of the activatable antibody after activation. In some embodiments, masking efficiency is measured as the difference in activity (e.g., activation of the NFAT promoter) for binding to a target (e.g., human CD3) of an activatable antibody before activation comprising a first masking moiety compared to the activity of the parent antibody or the activatable antibody after activation.In some embodiments, masking efficiency is measured as the difference in binding level of an activatable antibody comprising a first masking moiety before activation to cells expressing its target (e.g., cells expressing human CD3) compared to the activity of the parent antibody or the activatable antibody after activation. In some embodiments, masking efficiency is measured by dividing the EC50 of the activatable antibody comprising a first masking moiety before activation by the EC50 of the parent antibody. EC50 values ​​can be measured by ELISA assay or Jurkat NFAT reporter assay, see, e.g., methods in Example 3. In some embodiments, masking efficiency is measured by dividing the kd of an activatable antibody comprising a first masking moiety before activation by the kd of the parent antibody.

[0159] Any one of the cleavage moieties described herein can be used, including, for example, the cleavage moieties in Section G, "Cleavable Moieties (CM)" and Tables 13A, 18-22, and 40. In some embodiments, the first cleavable moiety (CM1) comprises 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 moiety (CM1) comprises the amino acid sequence of SEQ ID NO: 418 (GGGPLGLAGGS). In some embodiments, the first cleavable moiety (CM1) comprises the amino acid sequence of SEQ ID NO: 77 (GGGPLGLAGSGGS).

[0160] The second antigen-binding fragment can specifically bind to a target antigen, for example, 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 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 alpha 5, NCAM1, PTPRC, CD138, NaPi2b, MSLN, DLL3, GPRC5D, GPNMB, ICAM1, SSTR2, or a cancer-associated antigen. In some embodiments, 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 Moieties (TBMs)" (e.g., anti-HER2 antibodies and anti-CD20 antibodies).

[0161] In some embodiments, the second antigen-binding fragment is fused to a second masking moiety (MM2) via a second cleavable moiety (CM2). In some embodiments, the second antigen-binding fragment is unmasked. In some embodiments, the second antigen-binding fragment is not fused to a second masking moiety. Any suitable masking moiety can be used, for example, the anti-HER2 masking moieties described in Section F, "Masking Moiety (MM)." ​​Any suitable cleavable moiety can be used, for example, the cleavable moieties described in Section G, "Cleavable Moiety (CM)."

[0162] 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 an antibody that specifically binds to HER2. In some embodiments, the second antigen-binding fragment comprises one, two, three, four, five, or six CDRs of trastuzumab. In some embodiments, the second antigen-binding fragment comprises one, two, three, four, five, or six CDRs as set forth in Table 10. In some embodiments, the VH2 comprises 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 the VL2 comprises 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, 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 moiety (MM2) via a second cleavable moiety (CM2). In some embodiments, MM2 comprises the amino acid sequence of formula (XI): ESX1X2CX3X4DPFX5CQX6 (SEQ ID NO: 670), where 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 (SEQ ID NO: 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, and X 10is A, H, or V. In some embodiments, MM2 comprises an amino acid sequence of formula (XIII): YNSDDDCX1SX2YDPYTCYY (SEQ ID NO: 672) where X1 is A, I, or V and X2 is H or R. In some embodiments, MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 419, 432-476, and 491-515. In some embodiments, MM2 comprises the amino acid sequence of SEQ ID NO: 419 (ESDACDADPFDCQA). In some embodiments, MM2 comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, CM2 comprises 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 comprises the amino acid sequence of SEQ ID NO: 420. In some embodiments, CM2 comprises 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 an antibody that specifically binds to CD20. In some embodiments, the second antigen-binding fragment comprises one, two, three, four, five, or six CDRs as set forth in Table C. In some embodiments, the VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 556, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 557, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 558, and the VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 559, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 560, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 561. In some embodiments, the VH2 comprises the amino acid sequence of SEQ ID NO: 562, and the VL2 comprises the amino acid sequence of SEQ ID NO: 563.

[0164] In some embodiments, the activatable multispecific antibody comprises 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 comprises any one of the Fc regions described in Section J, "Fc Regions and CH3 Domains." In some embodiments, the activatable multispecific antibody comprises any CH3 domain mutation described in Section J, "Fc Regions and CH3 Domains," including mutations described in Table DF.

[0165] In some embodiments, the activatable multispecific antibody comprises a first CH3 domain and a second CH3 domain, wherein the first CH3 domain comprises D356K, E357K, S364K and S400C substitutions and the second CH3 domain comprises L351D, K370D, N390C and K439D substitutions, or wherein 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 that targets human CD3 and HER2.

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

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

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

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

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

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

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

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

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

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

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

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

[0180] In some embodiments, the activatable multispecific antibody is cross-reactive with CD3 polypeptides from at least one non-human species selected from the group consisting of cynomolgus monkey, mouse, rat, and dog.

[0181] B. Masked Multispecific Anti-CD3 Antibody As described above, the present disclosure is based in part 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) and masking moieties that block binding of anti-CD3 antibodies (see, e.g., Tables 4-5). Generally, masking antibodies are comprised of a masking moiety that binds to the target-binding portion of an antibody, such that when the masking moiety is bound to the target-binding portion, it reduces binding of the antibody to its target. Masked antibodies can include a cleavable or non-cleavable linker between the masking moiety and the antigen-binding fragment. Without wishing to be bound by theory, it is believed that when a masking antibody includes a non-cleavable linker, the masking antibody can 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 affinities of the masking moiety for the target-binding moiety and the target-binding moiety for the target-binding moiety, as well as the local concentrations of the target and masking antibody, determine the extent to which the antibody actually binds to the target. Without wishing to be bound by theory, masked multispecific antibodies with relatively weak CD3 and / or high masking efficiency for blocking CD3 binding are believed to have fewer severe side effects than conventional BiTE molecules. This reduced severity of side effects is believed to allow the masked multispecific antibodies described herein to have a larger therapeutic window. That is, the masked multispecific antibodies described herein can be administered to effectively treat disease without producing toxic effects, such as cytokine storm, that are commonly associated with conventional BiTE molecules, e.g., BiTE molecules with stronger CD3 binding affinity.

[0182] In some embodiments, a multispecific antibody is provided that includes: a) a first antigen-binding fragment fused to a first masking moiety (MM1) and comprising the VH1 and VL1 of an anti-CD3 antibody that specifically binds to CD3; and b) a second antigen-binding fragment comprising the VH2 and VL2 of an antibody that specifically binds to a target antigen (e.g., a tumor antigen such as HER2, CD20, TROP2, BCMA, or CD19), wherein MM1 is fused to the N-terminus of VL1; wherein MM1 competes with CD3 to specifically bind the CD3-binding moiety; the multispecific antibody binds to CD3 via the first antigen-binding fragment; and the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) 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, hi some embodiments, the first antigen-binding fragment is an scFv comprising, from N- to C-terminus, a VL1, an optional linker, and a VH1.

[0183] In some embodiments, the masked multispecific antibody is an activatable antibody. In some embodiments, the multispecific antibody comprises a cleavable moiety. See, e.g., 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 comprise 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 M M1 is fused to the N-terminus of V L1 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 is provided comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-1st CH3(1a); (ii) the second polypeptide comprises a structure represented by the formula: MM1-NCL1-VL1-VH1-hinge-CH2-second CH3(1b); (iii) the third polypeptide comprises a structure represented by the formula: VL2-CL(1c); where: 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 immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; the first CH3 is the first immunoglobulin heavy chain constant domain 3; the second CH3 is a second immunoglobulin heavy chain constant domain 3; Hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the masking moiety; NCL1 is a non-cleavable linker; wherein VH1 and VL1 associate to form an scFv that specifically binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5), and wherein 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), wherein MM specifically binds to the CD3-binding portion in competition with CD3; 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). In some embodiments, the multispecific antibody (e.g., its second polypeptide) comprises an amino acid linker between VL1 and VH1.

[0186] In some embodiments, a multispecific antibody is provided comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH1-CH1-hinge-CH2-first CH3(3a); (ii) the second polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-second CH3(3b); (iii) the third polypeptide comprises a structure represented by the formula: MM1-NCL1-VL1-CL(3c); (iv) the fourth polypeptide comprises a structure represented by the formula: VL2-CL(3d); where: 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 immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; Hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the masking moiety; NCL1 is a non-cleavable linker; wherein VH1 and VL1 associate to form a first Fv that specifically binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5); and wherein 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), wherein MM specifically binds to the CD3-binding portion in competition with CD3; 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 CD3, the first antigen-binding fragment fused to a first masking moiety (MM1); 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 the second antigen-binding fragment is fused to the second masking moiety (MM2) via a cleavable moiety (CM); where MM1 competes with CD3 to specifically bind the CD3-binding moiety; where the CM comprises a cleavage site; where MM2 inhibits binding of the multispecific antibody to the target antigen when the CM is not cleaved; and where the multispecific antibody binds to the target antigen via the second antigen-binding fragment when the CM is cleaved; and where the first antigen-binding fragment binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) 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 (eg, the assay in Example 3).

[0188] In some embodiments, a multispecific antibody is provided comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-first CH3(2a); (ii) the second polypeptide comprises a structure represented by the formula: MM1-NCL1-VL1-VH1-hinge-CH2-second CH3(2b); (iii) the third polypeptide comprises a structure represented by the formula: MM2-NCL2-VL2-CL(2c); where: 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 immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; the first CH3 is the first immunoglobulin heavy chain constant domain 3; the second CH3 is a second immunoglobulin heavy chain constant domain 3; Hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the first masking portion; NCL1 is the first non-cleavable linker; MM2 is the second masking portion; NCL2 is a second non-cleavable linker; wherein VH1 and VL1 associate to form an scFv that specifically binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5), and wherein 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), wherein MM1 competes with CD3 to specifically bind the CD3-binding portion; the multispecific antibody binds to CD3 via the first antigen-binding fragment; MM2 inhibits binding of the multispecific antibody to the target antigen; and the multispecific antibody binds to the target antigen via the second antigen-binding fragment.

[0189] In some embodiments, a multispecific antibody is provided comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-first CH3(2a); (ii) the second polypeptide comprises a structure represented by the formula: MM1-NCL1-VL1-VH1-hinge-CH2-second CH3(2b); (iii) the third polypeptide comprises a structure represented by the formula: MM2-CM-VL2-CL(2c); where: 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 immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; the first CH3 is the first immunoglobulin heavy chain constant domain 3; the second CH3 is a second immunoglobulin heavy chain constant domain 3; Hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the first masking portion; NCL1 is a non-cleavable linker; MM2 is the second masking portion; CM is a cleavable moiety containing the cleavage site; wherein VH1 and VL1 associate to form an scFv that specifically binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5), and wherein 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), wherein MM1 competes with CD3 to specifically bind the CD3-binding portion; the multispecific antibody binds to CD3 via the first antigen-binding fragment; and wherein MM2 inhibits binding of the multispecific antibody to the target antigen when the CM is not cleaved; and when 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). In some embodiments, the multispecific antibody (e.g., its second polypeptide) comprises an amino acid linker between VL1 and VH1.

[0190] In some embodiments, a multispecific antibody is provided comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH1-CH1-hinge-CH2-1st CH3(4a); (ii) the second polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-second CH3(4b); (iii) the third polypeptide comprises a structure represented by the formula: MM1-NCL1-VL1-CL(4c); (iv) the fourth polypeptide comprises a structure represented by the formula: MM2-NCL2-VL2-CL(4d); where: 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 immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; Hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the first masking portion; NCL1 is the first non-cleavable linker; MM2 is the second masking portion; NCL2 is the first non-cleavable linker; wherein VH1 and VL1 associate to form a first Fv that specifically binds CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5); and wherein 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), wherein MM1 competes with CD3 to specifically bind the CD3-binding portion; the multispecific antibody binds to CD3 via the first antigen-binding fragment; and wherein MM2 inhibits binding of the multispecific antibody to the target antigen when the CM is not cleaved; and the multispecific antibody binds to the target antigen via the second antigen-binding fragment when the CM is cleaved. In some embodiments, a multispecific antibody is provided comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein: (i) the first polypeptide comprises a structure represented by the formula: VH1-CH1-hinge-CH2-1st CH3(4a); (ii) the second polypeptide comprises a structure represented by the formula: VH2-CH1-hinge-CH2-second CH3(4b); (iii) the third polypeptide comprises a structure represented by the formula: MM1-NCL1-VL1-CL(4c); (iv) the fourth polypeptide comprises a structure represented by the formula: MM2-CM-VL2-CL(4d); where: 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 immunoglobulin heavy chain constant domain 1; CH2 is immunoglobulin heavy chain constant domain 2; Hinge is the immunoglobulin hinge region that connects the CH1 and CH2 domains; MM1 is the first masking portion; NCL1 is a non-cleavable linker; MM2 is the second masking portion; CM is a cleavable moiety containing the cleavage site; wherein VH1 and VL1 associate to form a first Fv that specifically binds to CD3 with a half-maximal binding concentration (EC50) of the antibody of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5); and wherein 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), wherein MM1 competes with CD3 to specifically bind the CD3-binding portion; the multispecific antibody binds to CD3 via the first antigen-binding fragment; and wherein MM2 inhibits binding of the multispecific antibody to the target antigen when the CM is not cleaved; and when 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 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 a reference antibody to CD3. In some embodiments, the first antigen-binding fragment binds to CD3 with a higher dissociation constant than a reference antibody to CD3. In some embodiments, the first antigen-binding fragment binds to CD3 with a lower dissociation constant than a reference antibody to 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 is present 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 is present in a multispecific antibody.

[0192] In some embodiments, the first antigen-binding fragment has a half-maximal binding concentration (EC) of the antibody of at least about any one of 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 more (including any value or range therebetween) as determined by enzyme-linked immunosorbent assay (ELISA). 50In some embodiments, the first antigen-binding domain binds to human CD3 with an EC50 of any one of about 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 as determined by enzyme-linked immunosorbent assay (ELISA). In some embodiments, the EC50 is determined by an ELISA that measures binding of the unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the first antigen-binding fragment is an scFv, and the EC50 is determined by an ELISA that measures binding of the unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the EC50 is determined by an ELISA that measures binding of a parent multispecific antibody that lacks MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the first antigen-binding fragment is an scFv, and the EC50 is determined by an ELISA that measures binding of a parent multispecific antibody that lacks MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the EC50 measures binding of an antigen-binding fragment that binds to CD3 (e.g., an isolated anti-CD3 scFv or scFv-Fc fusion protein) to CD3 (e.g., human CD3 or human CD3δε) determined by ELISA.

[0193] In some embodiments, the first antigen-binding fragment has an EC2 of the reference antibody (e.g., SP34). 50or 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 or more times higher EC 50 In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) at an EC of 100 or less than the EC of the reference antibody (e.g., SP34), including any value or range therebetween. 50 EC values ​​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 higher than 50 In some embodiments, the EC50 of the first antigen-binding fragment and the reference antibody is measured under the same test conditions. In some embodiments, the EC50 of the first antigen-binding fragment and the reference antibody is 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 comprises a CD3-binding portion corresponding to SP34 (e.g., comprises the six CDRs of SP34). In some embodiments, the EC50 of the first antigen-binding fragment and the reference antibody is measured in the same antibody format. In some embodiments, the EC50 of the first antigen-binding fragment and the reference antibody is measured 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 comprises a CD3-binding portion corresponding to SP34 (e.g., comprises the six CDRs of SP34). In some embodiments, the EC50 of the first antigen-binding fragment and the reference antibody is measured in the same antibody format. 50is determined by measuring the binding of a parent multispecific antibody lacking MM and a reference parent multispecific antibody lacking MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the reference parent multispecific antibody lacking MM comprises a CD3-binding portion corresponding to SP34 (e.g., comprising the six CDRs of SP34). In some embodiments, the Kd of the first antigen-binding fragment for CD3 binding and the EC50 of the reference antibody are determined by ELISA, for example, the ELISA described in Example 3. In some embodiments, the Kd of the first antigen-binding fragment for CD3 binding and the EC50 of the reference antibody are determined by a cell-based assay, for example, 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, at least about any one of 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 or more times weaker than the Kd of the reference antibody (including any value or range therebetween). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a dissociation constant (Kd) that 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 times weaker than the Kd of the reference antibody (e.g., SP34). In some embodiments, the Kds 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 the unmasked multispecific antibody and the unmasked multispecific reference antibody to CD3. In some embodiments, the unmasked multispecific reference antibody comprises a CD3-binding portion corresponding to SP34 (e.g., comprising the six CDRs of SP34). In some embodiments, the Kd is determined by measuring the binding of a parent multispecific antibody lacking MM and a reference parent multispecific antibody lacking MM to CD3 (e.g., human CD3 or human CD3δε). In some embodiments, the reference parent multispecific antibody lacking MM comprises a CD3-binding portion corresponding to SP34 (e.g., comprising the six CDRs of SP34). In some embodiments, the Kd for binding to CD3 of the first antigen-binding fragment 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 any one of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 nM or more (including any value or range therebetween). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a dissociation constant (Kd) (when in an activated form) of at least about any one of 1, 10, or 100 μM (including any value or range therebetween). In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a dissociation constant (Kd) of about any one of 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 a reference antibody (e.g., SP34), e.g., a koff of the reference antibody. off at least about 2, 5, 10, 20, 50, 100, 200 or more times faster than (including any value or range therebetween).

[0197] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a relatively slow on-rate (k) compared to a reference antibody (e.g., SP34), e.g., a k of the reference antibody. on at least about 2, 5, 10, 20, 50, 100, 200 or more times slower than (including any value or range therebetween).

[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), e.g., at least about any one of 2, 5, 10, 20, 50, 100, 200 or more times smaller than the Kd of the reference antibody (including any value or range therebetween).

[0199] In some embodiments, the first antigen-binding fragment binds to CD3 (e.g., human CD3) with a relatively larger binding constant (ka) compared to a reference antibody (e.g., SP34), e.g., a binding constant greater than the k of the reference antibody. a at least about any one of 2, 5, 10, 20, 50, 100, 200 or more times gr...

Claims

1. A multispecific antibody, comprising: a) a first antigen-binding fragment that specifically binds to CD3, the first antigen-binding fragment being fused to a first masking moiety (MM1) via a first cleavable moiety (CM1) comprising a first cleavage site, the first antigen-binding fragment comprising a first immunoglobulin light chain variable domain (VL1) and a first immunoglobulin heavy chain variable domain (VH1) of an anti-CD3 antibody, the MM1 being fused to the N-terminus of the VL1 via the CM1 when the CM1 is not cleaved, the MM1 inhibiting binding of a multispecific antibody to CD3, the MM1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417, and 597-599, and the CM1 comprising 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) a second antigen-binding fragment that specifically binds the target antigen; the MM1 specifically binds to the CD3 binding portion in competition with CD3; and i) the VH1 is CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 391, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:378; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:381; ii) the VH1 is CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 392, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:400; iii) the VH1 is CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 392, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:401; iv) the VH1 CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:381; v) the VH1 is CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:401; vi) the VH1 CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:400; vii) said VH1 CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; The VL1 is CDR-L1 comprising the amino acid sequence of SEQ ID NO: 398, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 399, and comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:400; viii) said VH1 CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:381; ix) the VH1 is 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 comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:381; x) the VH1 is CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:381; xi) said VH1 is CDR-H1 comprising the amino acid sequence of SEQ ID NO: 376, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 391, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:378; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:400; xii) the VH1 is CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 393, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:378; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:381; xiii) the VH1 is 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 comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:378; The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381; or xiv) the VH1 is 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 comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:378; The VL1 is 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 A multispecific antibody comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

381.

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

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

4. 2. The multispecific antibody of 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 the target antigen.

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

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

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

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

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

10. The VH1 CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 392, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; The VL1 is 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 2. The multispecific antibody of claim 1, comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

400.

11. The VH1 CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; The VL1 is 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 2. The multispecific antibody of claim 1, comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

381.

12. The first antigen-binding fragment comprises a VH1 and a VL1, wherein: a) the VH1 is represented by the formula (VII): EVQLVESGGGLVX １ PGGSLRLSCAASGFTFX ２ X ３ YAIX ４ WVRQAPGKGLEWVX ５ RIRSKYNNYATYYAX ６ S.V.K.X. ７ RFTISRDX ８ SKNTLYLQX ９ NSLRAED TAVYYCX １０ RHGNX １１ GX １２ SYVSWFAYWGQGTLVTVSS (SEQ ID NO: 388) １ is K or Q, and X ２ is N or S, and X ３ is S or T, and X ４ is H or N, and X ５ is G or S, and X ６ is D or E, and X ７ is D or G, and X ８ is D or N, and X ９ is I or L; X １０ is A or V, and X １１ is F or Y, and X １２ is N or T; and b) The VL1 is represented by formula (VIII): X １ AVVTQEPSLTVSPGGTVTLTCX ２ SSTGAVTTSNYX ３ N.W.X. ４ QQKPGQAPRGLIGGTX ５ X ６ RAPGX ７ PARFSGSLLGGGKAALTLSGAQPEDEAEYYCALWYSX ８ X ９ WVFGGGTKLTVL (SEQ ID NO: 389) １ is E or Q, and X ２ is A, G, P, or R; X ３ is A or P, and X ４ is F or V, and X ５ is K or N, and X ６ is F or K, and X ７ is A, I, T, or V; X ８ is A, D, N, or T; X ９ The multispecific antibody of claim 1 , comprising the amino acid sequence of:

13. a) the VH1 comprises the amino acid sequence of SEQ ID NO:402 and the VL1 comprises the amino acid sequence of SEQ ID NO:403; b) the VH1 comprises the amino acid sequence of SEQ ID NO:402 and the VL1 comprises the amino acid sequence of SEQ ID NO:404; c) the VH1 comprises the amino acid sequence of SEQ ID NO:405 and the VL1 comprises the amino acid sequence of SEQ ID NO:406; d) the VH1 comprises the amino acid sequence of SEQ ID NO:407 and the VL1 comprises the amino acid sequence of SEQ ID NO:404; e) the VH1 comprises the amino acid sequence of SEQ ID NO:407 and the VL1 comprises the amino acid sequence of SEQ ID NO:403; f) the VH1 comprises the amino acid sequence of SEQ ID NO:407, and the VL1 comprises the amino acid sequence of SEQ ID NO:408; g) the VH1 comprises the amino acid sequence of SEQ ID NO:409 and the VL1 comprises the amino acid sequence of SEQ ID NO:408; h) the VH1 comprises the amino acid sequence of SEQ ID NO:410, and the VL1 comprises the amino acid sequence of SEQ ID NO:411; i) the VH1 comprises the amino acid sequence of SEQ ID NO:412 and the VL1 comprises the amino acid sequence of SEQ ID NO:413; j) the VH1 comprises the amino acid sequence of SEQ ID NO:410, and the VL1 comprises the amino acid sequence of SEQ ID NO:413; k) the VH1 comprises the amino acid sequence of SEQ ID NO:414 and the VL1 comprises the amino acid sequence of SEQ ID NO:403; l) the VH1 comprises the amino acid sequence of SEQ ID NO:415, and the VL1 comprises the amino acid sequence of SEQ ID NO:413; m) the VH1 comprises the amino acid sequence of SEQ ID NO: 416, and the VL1 comprises the amino acid sequence of SEQ ID NO: 413; or n) the VH1 comprises the amino acid sequence of SEQ ID NO: 416, and the VL1 comprises the amino acid sequence of SEQ ID NO: 411; A multispecific antibody according to claim 12.

14. The multispecific antibody of claim 13, wherein the VH1 comprises the amino acid sequence of SEQ ID NO: 402 and the VL1 comprises the amino acid sequence of SEQ ID NO:

403.

15. The multispecific antibody of claim 13, wherein the VH1 comprises the amino acid sequence of SEQ ID NO: 410 and the VL1 comprises the amino acid sequence of SEQ ID NO:

411.

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

417.

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

77.

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

418.

19. The multispecific antibody of claim 1 , wherein the target antigen is HER2.

20. the second antigen-binding fragment comprises a VH2 and a VL2 of an anti-HER2 antibody, wherein: The VH2 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 comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:71; and The VL2 is 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 20. The multispecific antibody of claim 19, comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

74.

21. 21. The multispecific antibody of claim 20, wherein the VH2 comprises the amino acid sequence of SEQ ID NO: 75 and the VL2 comprises the amino acid sequence of SEQ ID NO:

76.

22. a) MM2 is a compound represented by the formula (XI): ESX １ X ２ C.X. ３ X ４ DPFX ５ CQX ６ (SEQ ID NO: 670) (wherein, X １ is D or E, and X ２ is A, F, V, or Y; X is ３ is D or E, and X ４ is A or L, and X ５ is D or E, and X ６ is A, F, or Y; b) MM2 is a compound represented by formula (XII): １ X ２ X ３ X ４ X ５ X ６ C.X. ７ X ８ D.P.Y.E.X. ９ X １０ (SEQ ID NO: 671) (wherein, X １ is A, H, or S, and X ２ is A, D, or S, and X ３ is A, T, or V; X is ４ is P, S, or T, and X ５ is D or E, and X ６ is A or V, and X ７ is D or E, and X ８ is A or L, and X ９ is Q, S or T, and X １０ is A, H, or V; or c) MM2 has the formula (XIII) YNSDDDCCX １ S.X. ２ YDPYTCYY (SEQ ID NO: 672) １ is A, I, or V; X ２ is H or R.

23. The multispecific antibody of claim 20, wherein MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476 and 491-515.

24. 24. The multispecific antibody of claim 23, wherein the MM2 comprises the amino acid sequence of SEQ ID NO:

419.

25. The multispecific antibody of claim 24, 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.

26. 26. The multispecific antibody of claim 25, wherein the CM2 comprises the amino acid sequence of SEQ ID NO: 77 or 420.

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

28. The multispecific antibody of claim 1, wherein the target antigen is CD20.

29. The second antigen-binding fragment comprises a VH2 and a VL2 of an anti-CD20 antibody, wherein: a) the VH2 is 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 comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:558; The VL2 is 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 or comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:561; or b) the VH2 is CDR-H1 comprising the amino acid sequence of SEQ ID NO: 86, CDR-H2 comprising the amino acid sequence of SEQ ID NO:557, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:558; The VL2 is 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 29. The multispecific antibody of claim 28, comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

561.

30. 30. The multispecific antibody of claim 29, wherein the VH2 comprises the amino acid sequence of SEQ ID NO: 562 and the VL2 comprises the amino acid sequence of SEQ ID NO:

563.

31. a) a first polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:564; a second polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:565; and a third polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:567; b) a first polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:564; a second polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:565; and a third polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:569; c) a first polypeptide comprising the amino acid sequence of SEQ ID NO:564; A second polypeptide comprising the amino acid sequence of SEQ ID NO:565; and a third polypeptide comprising the amino acid sequence of SEQ ID NO:567; d) a first polypeptide comprising the amino acid sequence of SEQ ID NO:564; A second polypeptide comprising the amino acid sequence of SEQ ID NO:565; and a third polypeptide comprising the amino acid sequence of SEQ ID NO:569; e) a first polypeptide comprising the amino acid sequence of SEQ ID NO:564; a second polypeptide comprising the amino acid sequence of SEQ ID NO:565 without the C-terminal lysine; and a third polypeptide comprising the amino acid sequence of SEQ ID NO:567 without the C-terminal lysine; f) a first polypeptide comprising the amino acid sequence of SEQ ID NO:564; a second polypeptide comprising the amino acid sequence of SEQ ID NO:565 without the C-terminal lysine; and A third polypeptide comprising the amino acid sequence of SEQ ID NO:569 without the C-terminal lysine; 30. The multispecific antibody of claim 29, comprising:

32. The VH1 CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO:395; and The VL1 is 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 comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 381; The multispecific antibody of claim 2.

33. The multispecific antibody of claim 32, wherein the anti-ScFv comprises the amino acid sequence of SEQ ID NO:

422.

34. The multispecific antibody of claim 32, wherein the MM1 comprises the amino acid sequence of SEQ ID NO:

417.

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

417.

36. The multispecific antibody described in Claim 34, wherein CM1 comprises the amino acid sequence of SEQ ID NO:

77.

37. The multispecific antibody described in Claim 35, wherein CM1 comprises the amino acid sequence of SEQ ID NO:

77.

38. The multispecific antibody described in Claim 34, wherein CM1 comprises the amino acid sequence of SEQ ID NO:

418.

39. The multispecific antibody described in Claim 35, wherein CM1 comprises the amino acid sequence of SEQ ID NO:

418.

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

567.

41. The multispecific antibody described in claim 40, wherein the first polypeptide has the amino acid sequence of SEQ ID NO:

565.

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

564.

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

85.

44. The multispecific antibody of claim 43, wherein the first polypeptide has the amino acid sequence of SEQ ID NO:

84.

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

83.

46. ​​A first polypeptide comprising the amino acid sequence of SEQ ID NO:

427. A second polypeptide comprising the amino acid sequence of SEQ ID NO:428, and A third polypeptide comprising the amino acid sequence of SEQ ID NO:

112.

28. The multispecific antibody of claim 27, comprising:

47. A first polypeptide comprising the amino acid sequence of SEQ ID NO:

564. A second polypeptide comprising the amino acid sequence of SEQ ID NO:565; and A third polypeptide comprising the amino acid sequence of SEQ ID NO:

567.

32. The multispecific antibody of claim 31 , comprising:

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