WO2022170619A1 - Anti-cd3 antibodies and methods of use thereof - Google Patents

Anti-cd3 antibodies and methods of use thereof Download PDF

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Publication number
WO2022170619A1
WO2022170619A1 PCT/CN2021/076626 CN2021076626W WO2022170619A1 WO 2022170619 A1 WO2022170619 A1 WO 2022170619A1 CN 2021076626 W CN2021076626 W CN 2021076626W WO 2022170619 A1 WO2022170619 A1 WO 2022170619A1
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amino acid
seq
acid sequence
cdr
antibody
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French (fr)
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Peter Peizhi Luo
Fangyong Du
Yan Li
Guizhong Liu
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Adagene Pte Ltd
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Adagene Pte Ltd
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Priority to PCT/CN2021/076626 priority Critical patent/WO2022170619A1/en
Priority to PCT/CN2021/109057 priority patent/WO2022170740A1/en
Priority to AU2022219133A priority patent/AU2022219133A1/en
Priority to JP2023547585A priority patent/JP2024508671A/ja
Priority to PCT/CN2022/076000 priority patent/WO2022171192A1/en
Priority to EP22752352.9A priority patent/EP4291578A4/en
Priority to CA3206603A priority patent/CA3206603A1/en
Priority to TW111105162A priority patent/TW202246337A/zh
Priority to KR1020237030742A priority patent/KR20230162930A/ko
Priority to CN202280025216.6A priority patent/CN117255801A/zh
Priority to US18/546,246 priority patent/US20250326860A1/en
Publication of WO2022170619A1 publication Critical patent/WO2022170619A1/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present application relates to antibodies targeting CD3, including multispecific antibodies targeting CD3, masked and activatable antibodies targeting CD3, methods of preparation, and methods of use thereof.
  • Bispecific T-cell engager antibodies have been explored as a means to recruit cytolytic T-cells to kill tumor cells. This is based on the simultaneous recognition of an antigen on tumor cells and binding to the CD3 epsilon chain, or CD3, within the T-cell receptor complex on T-cells that bridges malignant tumor cells directly to CD3+ T-cells. Blinatumomab, or 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.
  • An activatable antibody also known as a SAFEBODY TM , is designed to mask an antigen-binding interface with a masking motif, which then prevents an antibody from binding to its target in healthy tissues.
  • the masking motif is designed to activate, or unmask, the antibody to allow binding in the tumor microenvironment ( “TME” ) where certain activation conditions such as a protease is upregulated as compared to healthy tissues, allowing the antibody to bind to its target for tumor killing.
  • TME tumor microenvironment
  • Activatable antibodies thus provide antigen-specific binding proteins that are activated predominantly in the TME while remaining largely in an inactive state in healthy tissues.
  • 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 (e.g., activatable) antibodies targeting HER2, and methods of treatment thereof.
  • a 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 (e.g., activatable) antibodies targeting HER2, and methods of treatment thereof.
  • multispecific T cell engager comprising: a first antigen-binding fragment that specifically binds CD3, wherein the first antigen-binding fragment is fused to a first masking moiety (MM1) ; and a second antigen-binding fragment that specifically binds a target antigen; wherein the MM1 competes with CD3 to specifically bind the CD3-binding moiety; and wherein the first antigen-binding fragment binds CD3 with half-maximal binding at a concentration of antibody (EC50) that is at least 10 nM as determined by an enzyme-linked immunosorbent assay (ELISA) . In some embodiments, the EC 50 is at least 50 nM.
  • the EC 50 is at least 100 nM (e.g., about 110 nM) .
  • the first antigen-binding fragment is a 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 the MM1, when used to determine the EC 50 .
  • the EC 50 is determined using the ELISA assay as described in Example 5.
  • the multispecific antibody is a not an activatable multispecific antibody.
  • 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 wherein the MM1 is fused to the N-terminus of the VL1 via a first non-cleavable linker (NCL1) .
  • an activatable multispecific antibody 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; wherein the CM1 comprises a first cleavage site; wherein the MM1 competes with CD3 to specifically bind the CD3-binding moiety; and wherein the first antigen-binding fragment binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM as determined by an enzyme-linked immunosorbent assay (ELISA) .
  • a concentration of antibody EC 50
  • ELISA enzyme-linked immunosorbent assay
  • the MM1 inhibits binding of the activatable antibody to CD3 when the CM1 is not cleaved; and the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment when the CM1 is cleaved.
  • the EC 50 is at least 50 nM. In some embodiments, the EC 50 is at least 100 nM (e.g., about 110 nM) .
  • the first antigen-binding fragment is a 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., with CM1 cleaved) , when used to determine the EC 50 .
  • the EC 50 is determined using the ELISA assay as described in Example 5.
  • the first antigen-binding fragment binds CD3 with a dissociation constant (Kd) of at least 50 nM.
  • the first antigen-binding fragment is a 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., with CM1 of the multispecific antibody cleaved) , when used to determine the Kd.
  • the MM1 has a masking efficiency of at least 250 (e.g., at least 500, 1000, 2000, 3000, 5000, 10000 or higher) as determined by an ELISA assay, e.g., the ELISA assay in 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 higher) as determined by a Jurkat NFAT reporter assay, e.g., the Jurkat NFAT assay in Example 3.
  • a Jurkat NFAT reporter assay e.g., the Jurkat NFAT assay in Example 3.
  • 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.
  • the first antigen-binding fragment is selected from the group consisting of a Fab, a Fv, a scFab and a scFv.
  • the first antigen-binding fragment is a scFv.
  • the scFv comprises from N-terminus to C-terminus, VL1, a linker, and VH1.
  • the scFv comprises from N-terminus to C-terminus, VH1, a linker, and VL1.
  • the MM1 is fused to the N-terminus of the VL1 via the CM1.
  • the MM1 is fused to the N-terminus of the VL1 via the NCL1.
  • 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 the target antigen.
  • the second antigen-binding fragment is selected from the group consisting of a Fab, a Fv, a scFab and a scFv.
  • the second antigen-binding fragment is a Fv.
  • the second antigen-binding fragment is a Fab.
  • the multispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • 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;
  • first CH3 is a first immunoglobulin heavy chain constant domain 3;
  • second CH3 is a second immunoglobulin heavy chain constant domain 3;
  • the hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; wherein the VL1 and the VH1 associate to form a scFv that specifically binds CD3; and wherein the VL2 and the VH2 associate to form a Fv that specifically binds the target antigen.
  • the activatable multispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • 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;
  • first CH3 is a first immunoglobulin heavy chain constant domain 3;
  • second CH3 is a second immunoglobulin heavy chain constant domain 3;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • VL1 and the VH1 associate to form a scFv that specifically binds CD3; and wherein the VL2 and the VH2 associate to form a Fv that specifically binds the target antigen.
  • the second antigen-binding fragment is fused to a second masking moiety (MM2) , wherein the MM2 competes with the target antigen to specifically bind the second antigen-binding fragment.
  • the second antigen-binding fragment is fused to the MM2 via a second non-cleavable linker (NCL2) .
  • the second antigen-binding fragment is fused to the MM2 via a second cleavable moiety (CM2) , wherein the CM2 comprises a second cleavage site, wherein the MM2 inhibits binding of the multispecific antibody to the target antigen when the CM2 is not cleaved, and wherein the multispecific antibody binds the target antigen via the second antigen-binding fragment when the CM2 is cleaved.
  • the second antigen-binding fragment comprises a VH2 and a VL2 of an antibody that specifically binds the target antigen
  • the MM2 is fused to the N-terminus of the VL2 via the CM2.
  • the multispecific or activatable multispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • 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;
  • first CH3 is a first immunoglobulin heavy chain constant domain 3;
  • second CH3 is a second immunoglobulin heavy chain constant domain 3;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • VL1 and the VH1 associate to form a scFv that specifically binds CD3; and wherein the VL2 and the VH2 associate to form a Fv that specifically binds the target antigen.
  • the CD3 is human CD3.
  • the first antigen-binding fragment is cross-reactive with a CD3 polypeptide from at least one non-human species selected from the group consisting of cynomolgus monkey, mouse, rat and dog.
  • the VH1 comprises a heavy chain complementarity determining region (CDR-H) 1 comprising the amino acid sequence according to Formula (I) : X 1 YAX 2 X 3 (SEQ ID NO: 382) , wherein X 1 is D, S, or T, X 2 is I, L, or M, and X 3 is N or T, a CDR-H2 comprising the amino acid sequence according to Formula (II) : RIRSKYNNYATYYAX 1 X 2 VKX 3 (SEQ ID NO: 383) , wherein X 1 is D or E, X 2 is S or T, and X 3 is D, G, or S, and a CDR-H3 comprising the amino acid sequence according to Formula (III) : HGNX 1 GX 2 SYVSX 3
  • the VL1 comprises a CDR-L1 comprising the amino acid sequence according to Formula (IV) : X 1 SSTGAVTX 2 X 3 NYX 4 N (SEQ ID NO: 385) , wherein X 1 is A, G, or R, X 2 is S or T, X 3 is G or S, and X 4 is A, P, or V, a CDR-L2 comprising the amino acid sequence according to Formula (V) : GTX 1 X 2 RAP (SEQ ID NO: 386) , wherein X 1 is K or N, and X 2 is F or K, and a CDR-L3 comprising the amino acid sequence according to Formula (VI) : ALWYSX 1 X 2 WV (SEQ ID NO: 387) , wherein X 1 is D, N, or T, and X 2 is L or R.
  • Formula (IV) : X 1 SSTGAVTX 2 X 3 NYX 4 N (SEQ ID NO: 385) , wherein X
  • the 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 3 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 3 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 3 amino acid substitutions; and the VL1 comprises a CDR-L1 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 3 amino acid substitutions, a CDR-H2 comprising an amino acid sequence selected from the group consisting of S
  • the 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 3 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 3 amino acid substitutions
  • 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 3 amino acid substitutions
  • the 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 3 amino acid substitutions
  • 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 3 amino acid substitutions
  • the 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 the 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.
  • CDR-H heavy chain complementarity determining region
  • CDR-L light chain complementarity determining region
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the VH1 comprises the amino acid sequence according to Formula (VII) : EVQLVESGGGLVX 1 PGGSLRLSCAASGFTFX 2 X 3 YAIX 4 WVRQAPGKGLEWVX 5 RIRSKY NNYATYYAX 6 SVKX 7 RFTISRDX 8 SKNTLYLQX 9 NSLRAEDTAVYYCX 10 RHGNX 11 GX 12 S YVSWFAYWGQGTLVTVSS (SEQ ID NO: 388) , wherein X 1 is K or Q, X 2 is N or S, X 3 is S or T, X 4 is H or N, X 5 is G or S, X 6 is D or E, X 7 is D or G, X 8 is D or N, X 9 is I or L, X 10 is A or V, X 11 is F or Y, X 12 is N or T; and the VL1 comprises the amino acid sequence according to Formula (VIII) : X 1
  • the 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 thereof having at least about 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 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 thereof having at least about 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.
  • the VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 402, 405, 407, 409, 410, 412, 414, 415, and 416; and the VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 403, 404, 406, 408, 411, and 413.
  • the VH1 comprises the amino acid sequence of SEQ ID NO: 388
  • the VL1 comprises the amino acid sequence of SEQ ID NO: 389.
  • the VH1 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 VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 403, 404, 406, 408, 411, and 413.
  • 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.
  • 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.
  • 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
  • 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
  • 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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.
  • the MM1 comprises the amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of the MM1.
  • the MM1 comprises an amino acid sequence according to Formula (IX) : PYDDPDCPSHX 1 SDCDX 2 (SEQ ID NO: 668) , wherein X 1 is D or E, and X 2 is N or Q.
  • the MM1 comprises an amino acid sequence according to Formula (X) .
  • the MM1 comprises the amino acid sequence of SEQ ID NO: 417.
  • the MM1 comprises the amino acid sequence of SEQ ID NO: 35.
  • the MM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 597-599.
  • the CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 127-129, 418, 420, 431 and 477-490, and 516-555. In some embodiments, the CM1 comprises the amino acid sequence of SEQ ID NO: 418.
  • the target antigen is a tumor antigen.
  • the tumor antigen is selected from the group consisting of 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, carcinoma associated antigen CTAA16, CA9, ENG, ACVRL1, CD80, CSPG4, EGFL7, FLT1, HAVCR
  • 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.
  • the target antigen is HER2.
  • 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.
  • the VH2 comprises the amino acid sequence of SEQ ID NO: 75
  • the VL2 comprises the amino acid sequence of SEQ ID NO: 76.
  • the second antigen-binding fragment is fused to a second masking moiety (MM2) via a second cleavable moiety (CM2)
  • a) the MM2 comprises an amino acid sequence according to Formula (XI) : ESX1X 2 CX 3 X 4 DPFX 5 CQX 6 (SEQ ID NO: 670) , wherein X 1 is D or E, X 2 is A, F, V, or Y, X 3 is D or E, X 4 is A or L, X 5 is D or E, and X 6 is A, F, or Y; b) the MM2 comprises an amino acid sequence according to Formula (XII) : X 1 X 2 X 3 X 4 X 5 X 6 CX 7 X 8 DPYECX 9 X 10 (
  • the second antigen-binding fragment is fused to a second masking moiety (MM2) via a second cleavable moiety (CM2) , wherein the MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515.
  • the MM2 comprises the amino acid sequence of SEQ ID NO: 419.
  • the CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 127-129, 418, 420, 431 and 477-490, and 516-555.
  • the CM2 comprises the amino acid sequence of SEQ ID NO: 420.
  • the multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 425, a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 426, and a third polypeptide comprising an amino acid sequence having at least at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 112.
  • the multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 427, a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 428, and a third polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 112.
  • the multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 429, a second polypeptide comprising an amino acid sequence having at least at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 430, and a third polypeptide comprising an amino acid sequence having at least at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 115.
  • the target antigen is CD20.
  • 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.
  • 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.
  • the multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 564, a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 565, and a third polypeptide comprising an amino acid sequence having at least at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 567.
  • the multispecific or activatable multispecific antibody comprises: a first polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 564, a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 565, and a third polypeptide comprising an amino acid sequence having at least at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 569.
  • the multispecific or activatable multispecific antibody comprises an Fc region.
  • the Fc region is of the human IgG1 subclass.
  • the Fc region is of the human IgG2 subclass.
  • the Fc region is of the human IgG4 subclass.
  • the Fc region has enhanced ADCC and/or cross-linking efficiency.
  • the Fc region has reduced or no antibody-dependent cell cytotoxicity (ADCC) effect and/or reduced or no cross-linking effect.
  • the Fc region is of the human IgG1 subclass and has an N297A amino acid substitution.
  • the multispecific or activatable multispecific antibody comprises a first CH3 domain and a second CH3 domain
  • the first CH3 domain comprises a cysteine (C) residue at position 390 and the second CH3 domain comprises a cysteine residue at position 400
  • the first CH3 domain comprises a cysteine residue at position 400 and the second CH3 domain comprises a cysteine residue at position 390
  • the first CH3 domain comprises a cysteine residue at position 392 and the second CH3 domain comprises a cysteine residue at position 397
  • the first CH3 domain comprises a cysteine residue at position 397 and the second CH3 domain comprises a cysteine residue at position 392
  • the first CH3 domain comprises a cysteine residue at position 392 and the second CH3 domain comprises a cysteine residue at position 400
  • the first CH3 domain comprises a cysteine residue at position 400 and the second CH3 domain
  • the first CH3 domain comprises N390C substitution and the second CH3 domain comprises S400C substitution, or the first CH3 domain comprises S400C substitution and the second CH3 domain comprises N390C substitution; or ii) the first CH3 domain comprises K392C substitution and the second CH3 domain comprises V397C substitution, or the first CH3 domain comprises V397C substitution and the second CH3 domain comprises K392C substitution; or iii) the first CH3 domain comprises K392C substitution and the second CH3 domain comprises S400C substitution, or the first CH3 domain comprises S400C substitution and the second CH3 domain comprises K392C substitution.
  • 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)
  • 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 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; and wherein the amino acid residue numbering is based on EU numbering.
  • 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.
  • 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 first CH3 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.
  • 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 and the second CH3 domain further comprises E357Q and S364K substitutions, or the first CH3 domain further comprises E357Q and S364K substitutions and the second 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 L351D and L368E substitutions and the second CH3 domain further comprises L351K and T366K substitutions; or (iv) the
  • the multispecific or activatable multispecific antibody comprises a first CH3 domain and a second CH3 domain
  • the first CH3 domain comprises E357K, S364K and N390C substitutions and the second CH3 domain comprises L351D, K370D, and S400C substitutions
  • the first CH3 domain comprises L351D, K370D, and S400C substitutions
  • the second CH3 domain comprises E357K, S364K and N390C substitutions.
  • 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.
  • 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.
  • 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.
  • the multispecific or activatable multispecific antibody is a bispecific antibody.
  • an isolated antibody or antigen-binding fragment thereof that specifically binds CD3 ( “anti-CD3 antibody” ) , comprising: a VH comprising a heavy chain complementarity determining region (CDR-H) 1 comprising the amino acid sequence according to Formula (I) : X 1 YAX 2 X 3 (SEQ ID NO: 382) , wherein X 1 is D, S, or T, X 2 is I, L, or M, and X 3 is N or T, a CDR-H2 comprising the amino acid sequence according to Formula (II) : RIRSKYNNYATYYAX 1 X 2 VKX 3 (SEQ ID NO: 383) , wherein X 1 is D or E, X 2 is S or T, and X 3 is D, G, or S, and a CDR-H3 comprising the amino acid sequence according to Formula (III) : HGNX 1 GX 2 SYVSX 3 X 4 AY (SEQ ID NO: 38
  • 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 3 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 3 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 3 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 3 amino acid substitutions, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SDR-L
  • 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 3 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 3 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 3 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 3 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 3 amino acid substitutions, and a CDR
  • the VH comprises a CDR-H1 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 a VL comprising a CDR-L1 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.
  • the VH 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 VL 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.
  • 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: 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.
  • 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: 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.
  • 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.
  • 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: 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.
  • 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: 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.
  • 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.
  • 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.
  • 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.
  • 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: 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: 381.
  • 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: 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.
  • 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: 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: 400.
  • 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: 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.
  • 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.
  • 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.
  • 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.
  • the VH comprises the amino acid sequence according to Formula (VII) : EVQLVESGGGLVX 1 PGGSLRLSCAASGFTFX 2 X 3 YAIX 4 WVRQAPGKGLEWVX 5 RIRSKY NNYATYYAX 6 SVKX 7 RFTISRDX 8 SKNTLYLQX 9 NSLRAEDTAVYYCX 10 RHGNX 11 GX 12 S YVSWFAYWGQGTLVTVSS (SEQ ID NO: 388) , wherein X 1 is K or Q, X 2 is N or S, X 3 is S or T, X 4 is H or N, X 5 is G or S, X 6 is D or E, X 7 is D or G, X 8 is D or N, X 9 is I or L, X 10 is A or V, X 11 is F or Y, X 12 is N or T; and the VL comprises the amino acid sequence according to Formula (VIII) : X 1 AV
  • 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 thereof having at least about 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 thereof having at least about 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the anti-CD3 antibody further comprises a second antigen-binding fragment that specifically binds a target antigen.
  • the target antigen is a tumor antigen.
  • the tumor antigen is selected from the group consisting of 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, carcinoma associated antigen CTAA16, CA9, ENG, ACVRL1, CD80, CSPG4, EGFL7, FLT1, HAVCR1, HGF, HLA-DRB, IGF1R, TPBG, ERBB3, and STEAP2.
  • 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.
  • 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 the N-terminus to the C-terminus, a VL and a VH; or d) the CD3-binding moiety comprise from the N-terminus to the C-terminus, a VH and a VL; wherein the CM comprises a cleavage site; wherein the MM inhibits binding of the activatable antibody to CD
  • the first antigen-binding fragment is a 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., with CM1 cleaved) , when used to determine the EC 50 .
  • the EC 50 is determined using the ELISA assay as described in Example 5.
  • the first antigen-binding fragment binds CD3 with a dissociation constant (Kd) of at least 50 nM.
  • the first antigen-binding fragment is a 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., with CM1 cleaved) , when used to determine the Kd.
  • the MM comprises the amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of the MM.
  • the MM comprises an amino acid sequence according to Formula (IX) : PYDDPDCPSHX 1 SDCDX 2 (SEQ ID NO: 668) , wherein X 1 is D or E, and X 2 is N or Q.
  • the MM comprises an amino acid sequence according to Formula (X) : X 1 X 2 X 3 DX 4 X 5 CX 6 X 7 DX 8 X 9 X 10 CX 11 X 12 (SEQ ID NO: 669) , wherein X 1 is A or D, X 2 is A, D, or P, X 3 is D, H, or P, X 4 is F or P, X 5 is D or P, X 6 is D or P, X 7 is A or P, X 8 is D, N, or P, X 9 is A, N, or P, X 10 is D, H, or S, X 11 is H, P, or Y, and X 12 is N, P, or Y.
  • Formula (X) : X 1 X 2 X 3 DX 4 X 5 CX 6 X 7 DX 8 X 9 X 10 CX 11 X 12 (SEQ ID NO: 669) , wherein X 1 is A or D, X 2 is
  • 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.
  • 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 the N-terminus to the C-terminus, a VL and a VH; or d) the CD3-binding moiety comprise from the N-terminus to the C-terminus, a VH and a VL; wherein the CM comprises a cleavage site; wherein the MM inhibits binding of the activatable antibody to CD
  • the activatable anti-CD3 antibody comprises an anti-CD3 antigen-binding fragment selected from the group consisting of a Fab, a Fv, a scFab and a scFv.
  • the anti-CD3 antigen-binding fragment is a scFv.
  • the scFv comprises from the N-terminus to the C-terminus, the VL, a linker and the VH.
  • the VH comprising a heavy chain complementarity determining region (CDR-H) 1 comprising the amino acid sequence according to Formula (I) : X 1 YAX 2 X 3 (SEQ ID NO: 382) , wherein X 1 is D, S, or T, X 2 is I, L, or M, and X 3 is N or T, a CDR-H2 comprising the amino acid sequence according to Formula (II) : RIRSKYNNYATYYAX 1 X 2 VKX 3 (SEQ ID NO: 383) , wherein X 1 is D or E, X 2 is S or T, and X 3 is D, G, or S, and a CDR-H3 comprising the amino acid sequence according to Formula (III) : HGNX 1 GX 2 SYVSX 3 X 4 AY (SEQ ID NO: 384) , wherein X 1 is F or Y, X 2 is N or
  • 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 3 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 3 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 3 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 3 amino acid substitutions, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SDR-L
  • 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 3 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 3 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 3 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 3 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 3 amino acid substitutions, and a CDR
  • the VH comprises a CDR-H1 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 the VL comprises a CDR-L1 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.
  • the VH 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 VL 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.
  • 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: 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.
  • 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: 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.
  • 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.
  • 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: 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.
  • 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: 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.
  • 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.
  • 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.
  • 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.
  • 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: 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: 381.
  • 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: 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.
  • 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: 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: 400.
  • 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: 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the VH comprises the amino acid sequence according to Formula (VII) : EVQLVESGGGLVX 1 PGGSLRLSCAASGFTFX 2 X 3 YAIX 4 WVRQAPGKGLEWVX 5 RIRSKY NNYATYYAX 6 SVKX 7 RFTISRDX 8 SKNTLYLQX 9 NSLRAEDTAVYYCX 10 RHGNX 11 GX 12 S YVSWFAYWGQGTLVTVSS (SEQ ID NO: 388) , wherein X 1 is K or Q, X 2 is N or S, X 3 is S or T, X 4 is H or N, X 5 is G or S, X 6 is D or E, X 7 is D or G, X 8 is D or N, X 9 is I or L, X 10 is A or V, X 11 is F or Y, X 12 is N or T; and the VL comprises the amino acid sequence according to Formula (VII) : EVQLVESGGGLVX
  • 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 thereof having at least about 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 thereof having at least about 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.
  • 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.
  • 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.
  • the VH comprises the amino acid sequence of SEQ ID NO: 402
  • the VL comprises the amino acid sequence of SEQ ID NO: 403.
  • the VH comprises the amino acid sequence of SEQ ID NO: 402
  • the VL comprises the amino acid sequence of SEQ ID NO: 404.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 moiety comprises the amino acid sequence of SEQ ID NO: 421 or SEQ ID NO: 422.
  • the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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: 418.
  • a masked antibody comprising, from N-terminus to C-terminus, a masking moiety (MM) , and a CD3-binding moiety
  • the CD3-binding moiety comprises a VL and the activatable antibody further comprises a second polypeptide comprising a VH
  • the CD3-binding moiety comprises a VH and the activatable antibody further comprises a second polypeptide comprising a VL
  • the CD3-binding moiety comprises from the N-terminus to the C-terminus, a VL and a VH
  • the CD3-binding moiety comprise from the N-terminus to the C-terminus, a VH and a VL
  • the MM competes with CD3 to specifically bind the CD3-binding moiety
  • the activatable antibody binds CD3 via the VH and the VL
  • the masked anti-CD3 antibody is an activatable antibody. In some embodiments, the masked anti-CD3 antibody comprises, from N- terminus to C-terminus, the masking moiety (MM) , a cleavable moiety (CM) , and the CD3-binding moiety. In some embodiments, the masked anti-CD3 antibody is a not an activatable antibody. In some embodiments, the masked anti-CD3 antibody comprises, from N-terminus to C-terminus, the masking moiety (MM) , a non-cleavable linker (NCL) , and the CD3-binding moiety.
  • NCL non-cleavable linker
  • a masked antibody comprising, from N-terminus to C-terminus, a masking moiety (MM) , a non-cleavable linker (NCL) , and a CD3-binding moiety
  • the CD3-binding moiety comprises a VL and the activatable antibody further comprises a second polypeptide comprising a VH
  • the CD3-binding moiety comprises a VH and the activatable antibody further comprises a second polypeptide comprising a VL
  • the CD3-binding moiety comprises from the N-terminus to the C-terminus, a VL and a VH
  • the CD3-binding moiety comprise from the N-terminus to the C-terminus, a VH and a VL
  • the MM competes with CD3 to specifically bind the CD3-binding moiety
  • the activa competes with CD3 to specifically bind the CD3-binding moiety
  • the first antigen-binding fragment is a 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 multispecific antibody in an un-masked form (i.e., without the MM) , when used to determine the EC 50 .
  • the EC 50 is determined using the ELISA assay as described in Example 5.
  • the first antigen-binding fragment binds CD3 with a dissociation constant (Kd) of at least 50 nM.
  • the first antigen-binding fragment is a 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 an un-masked form (i.e., without the MM) , when used to determine the Kd.
  • the MM comprises the amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of the MM.
  • the MM comprises an amino acid sequence according to Formula (IX) : PYDDPDCPSHX 1 SDCDX 2 (SEQ ID NO: 668) , wherein X 1 is D or E, and X 2 is N or Q.
  • the MM comprises an amino acid sequence according to Formula (X) : X 1 X 2 X 3 DX 4 X 5 CX 6 X 7 DX 8 X 9 X 10 CX 11 X 12 (SEQ ID NO: 669) , wherein X 1 is A or D, X 2 is A, D, or P, X 3 is D, H, or P, X 4 is F or P, X 5 is D or P, X 6 is D or P, X 7 is A or P, X 8 is D, N, or P, X 9 is A, N, or P, X 10 is D, H, or S, X 11 is H, P, or Y, and X 12 is N, P, or Y.
  • Formula (X) : X 1 X 2 X 3 DX 4 X 5 CX 6 X 7 DX 8 X 9 X 10 CX 11 X 12 (SEQ ID NO: 669) , wherein X 1 is A or D, X 2 is
  • 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.
  • a masked antibody comprising, from N-terminus to C-terminus, a masking moiety (MM) , and a CD3-binding moiety
  • the CD3-binding moiety comprises a VL and the activatable antibody further comprises a second polypeptide comprising a VH
  • the CD3-binding moiety comprises a VH and the activatable antibody further comprises a second polypeptide comprising a VL
  • the CD3-binding moiety comprises from the N-terminus to the C-terminus, a VL and a VH
  • the CD3-binding moiety comprise from the N-terminus to the C-terminus, a VH and a VL
  • the MM competes with CD3 to specifically bind the CD3-binding moiety
  • the activatable antibody binds CD3 via the VH and the VL
  • the masked anti-CD3 antibody is an activatable antibody. In some embodiments, the masked anti-CD3 antibody comprises, from N-terminus to C-terminus, the masking moiety (MM) , a cleavable moiety (CM) , and the CD3-binding moiety. In some embodiments, the masked anti-CD3 antibody is a not an activatable antibody. In some embodiments, the masked anti-CD3 antibody comprises, from N-terminus to C-terminus, the masking moiety (MM) , a non-cleavable linker (NCL) , and the CD3-binding moiety.
  • NCL non-cleavable linker
  • a masked antibody comprising, from N-terminus to C-terminus, a masking moiety (MM) , a non-cleavable linker (NCL) , and a CD3-binding moiety
  • the CD3-binding moiety comprises a VL and the activatable antibody further comprises a second polypeptide comprising a VH
  • the CD3-binding moiety comprises a VH and the activatable antibody further comprises a second polypeptide comprising a VL
  • the CD3-binding moiety comprises from the N-terminus to the C-terminus, a VL and a VH
  • the CD3-binding moiety comprise from the N-terminus to the C-terminus, a VH and a VL
  • the MM competes with CD3 to specifically bind the CD3-binding moiety
  • the activa competes with CD3 to specifically bind the CD3-binding moiety
  • the activatable anti-CD3 antibody comprises an anti-CD3 antigen-binding fragment selected from the group consisting of a Fab, a Fv, a scFab and a scFv.
  • the anti-CD3 antigen-binding fragment is a scFv.
  • the scFv comprises from the N-terminus to the C-terminus, the VL, a linker and the VH.
  • the VH comprising a heavy chain complementarity determining region (CDR-H) 1 comprising the amino acid sequence according to Formula (I) : X 1 YAX 2 X 3 (SEQ ID NO: 382) , wherein X 1 is D, S, or T, X 2 is I, L, or M, and X 3 is N or T, a CDR-H2 comprising the amino acid sequence according to Formula (II) : RIRSKYNNYATYYAX 1 X 2 VKX 3 (SEQ ID NO: 383) , wherein X 1 is D or E, X 2 is S or T, and X 3 is D, G, or S, and a CDR-H3 comprising the amino acid sequence according to Formula (III) : HGNX 1 GX 2 SYVSX 3 X 4 AY (SEQ ID NO: 384) , wherein X 1 is F or Y, X 2 is N or
  • 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 3 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 3 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 3 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 3 amino acid substitutions, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SDR-L
  • 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 3 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 3 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 3 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 3 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 3 amino acid substitutions, and a CDR
  • the VH comprises a CDR-H1 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 the VL comprises a CDR-L1 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.
  • the VH 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 VL 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.
  • 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: 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.
  • 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: 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.
  • 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.
  • 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: 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.
  • 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: 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.
  • 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.
  • 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.
  • 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.
  • 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: 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: 381.
  • 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: 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.
  • 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: 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: 400.
  • 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: 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the VH comprises the amino acid sequence according to Formula (VII) : EVQLVESGGGLVX 1 PGGSLRLSCAASGFTFX 2 X 3 YAIX 4 WVRQAPGKGLEWVX 5 RIRSKY NNYATYYAX 6 SVKX 7 RFTISRDX 8 SKNTLYLQX 9 NSLRAEDTAVYYCX 10 RHGNX 11 GX 12 S YVSWFAYWGQGTLVTVSS (SEQ ID NO: 388) , wherein X 1 is K or Q, X 2 is N or S, X 3 is S or T, X 4 is H or N, X 5 is G or S, X 6 is D or E, X 7 is D or G, X 8 is D or N, X 9 is I or L, X 10 is A or V, X 11 is F or Y, X 12 is N or T; and the VL comprises the amino acid sequence according to Formula (VII) : EVQLVESGGGLVX
  • 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 thereof having at least about 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 thereof having at least about 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.
  • 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.
  • 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.
  • the VH comprises the amino acid sequence of SEQ ID NO: 402
  • the VL comprises the amino acid sequence of SEQ ID NO: 403.
  • the VH comprises the amino acid sequence of SEQ ID NO: 402
  • the VL comprises the amino acid sequence of SEQ ID NO: 404.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 moiety comprises the amino acid sequence of SEQ ID NO: 421 or SEQ ID NO: 422.
  • an activatable antibody comprising, from N-terminus to C-terminus, a masking moiety (MM) , a cleavable moiety (CM) , and a HER2-binding moiety
  • the HER2-binding moiety comprises a VL and the activatable antibody further comprises a second polypeptide comprising a VH
  • the HER2-binding moiety comprises a VH and the activatable antibody further comprises a second polypeptide comprising a VL
  • c) the HER2-binding moiety comprises from the N-terminus to the C-terminus, a VL and a VH
  • the HER2-binding moiety comprise from the N-terminus to the C-terminus, a VH and a VL
  • the CM comprises a cleavage site
  • the MM inhibits binding of the activatable antibody to HER
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515.
  • the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 127-129, 418, 420, 431 and 477-490, and 516-555.
  • the VH 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
  • the VL 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.
  • the VH comprises the amino acid sequence of SEQ ID NO: 75
  • the VL comprises the amino acid sequence of SEQ ID NO: 76.
  • a masked antibody comprising, from N-terminus to C-terminus, a masking moiety (MM) , and a HER2-binding moiety
  • the HER2-binding moiety comprises a VL and the activatable antibody further comprises a second polypeptide comprising a VH
  • the HER2-binding moiety comprises a VH and the activatable antibody further comprises a second polypeptide comprising a VL
  • c) the HER2-binding moiety comprises from the N-terminus to the C-terminus, a VL and a VH
  • the HER2-binding moiety comprise from the N-terminus to the C-terminus, a VH and a VL
  • the MM competes with HER2 to specifically bind the HER2-binding moiety
  • the activatable antibody binds HER2 via the VH and VL
  • the masked anti-HER2 antibody is an activatable antibody. In some embodiments, the masked anti-HER2 antibody comprises, from N-terminus to C-terminus, the masking moiety (MM) , a cleavable moiety (CM) , and the HER2-binding moiety. In some embodiments, the masked anti-HER2 antibody is a not an activatable antibody. In some embodiments, the masked anti-HER2 antibody comprises, from N-terminus to C-terminus, the masking moiety (MM) , a non-cleavable linker (NCL) , and the HER2-binding moiety.
  • NCL non-cleavable linker
  • a masked antibody comprising, from N-terminus to C-terminus, a masking moiety (MM) , a non-cleavable linker (NCL) , and a HER2-binding moiety
  • the HER2-binding moiety comprises a VL and the activatable antibody further comprises a second polypeptide comprising a VH
  • the HER2-binding moiety comprises a VH and the activatable antibody further comprises a second polypeptide comprising a VL
  • c) the HER2-binding moiety comprises from the N-terminus to the C-terminus, a VL and a VH
  • the HER2-binding moiety comprise from the N-terminus to the C-terminus, a VH and a VL
  • the MM competes with HER2 to specifically bind the HER2-binding moiety
  • the VH 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
  • the VL 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.
  • the VH comprises the amino acid sequence of SEQ ID NO: 75
  • the VL comprises the amino acid sequence of SEQ ID NO: 76.
  • One aspect of the present application provides one or more isolated nucleic acids encoding any one of the 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 described above.
  • a vector comprising the one or more nucleic acids according to any one of the nucleic acids described above.
  • a host cell comprising the one or more nucleic acids according to any one of the nucleic acids described above or any one of the vectors described above.
  • a method 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 one of the host cells under conditions that allow expression of the one or more nucleic acids or vector; and b) recovering the multispecific antibody, the activatable multispecific antibody, the 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.
  • compositions comprising any one of the 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 described above, and a pharmaceutically acceptable carrier.
  • the present application provides a method for treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of any one of the pharmaceutical compositions described above.
  • the pharmaceutical composition comprises an activatable multispecific antibody, wherein the CM1 and the CM2 are cleaved at a diseased site, thereby unblocking binding of the multispecific activatable antibody to CD3 and the target antigen at the diseased site.
  • the disease or condition is cancer such as liquid cancer and solid cancer.
  • the target antigen is HER2, the cancer is selected from the group consisting of breast cancer, ovarian cancer, and lung cancer.
  • the target antigen is CD20
  • the cancer is lymphoma or leukemia.
  • the target antigen is TROP2, and wherein the cancer is breast cancer or lymphoma.
  • compositions, kits and articles of manufacture comprising any one of the 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 described above.
  • FIGs. 1-5 provide schematic diagrams of exemplary antibody designs of the present application.
  • the antibodies may be converted to activatable antibodies by fusing one or more antigen-binding sites to masking peptide (s) .
  • FIG. 1 shows a Fab-Fc/Fc one-armed scaffold schematic.
  • FIG. 2 shows a schematic of a common light chain scaffold, in which the bispecific antibody has a first antibody heavy chain, a second antibody heavy chain and two copies of a commong light chain.
  • the first antibody heavy chain and a first common light chain form a first antigen-binding site
  • the second antibody heavy chain and a 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.
  • FIG. 3 shows a schematic of a Morrison format multispecific antibody scaffold, in which 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 a first common light chain form a first antigen-binding site
  • the second antibody heavy chain and a 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 may bind to the same target or different targets.
  • FIG. 4 shows ScFv bispecific scaffold schematics, including, at right, a HER2 and CD3 bispecific antibody schematic.
  • FIGS. 5A-5B show activatable scaffold schematics.
  • FIG. 5A shows at right a schematic of an activatable antibody targeting HER2 and CD3.
  • the masking peptide (represented as a ball) can be fused to the antigen-binding fragment via a cleavable linker.
  • FIG. 5B shows at right a schematic of an activatable antibody targeting HER2 and CD3 in which only the CD3-binding fragment is masked.
  • the masking peptide (represented as a ball) can be fused to the antigen-binding fragment via a cleavable linker.
  • FIG. 6 provides a characterization of bispecific antibodies through SDS-PAGE electrophoresis.
  • the left gel is a 12%SDS-PAGE gel under reducing conditions, and the right gel is a 4-15%SDS-PAGE gel under non-reducing conditions.
  • the MW lane shows molecular weight markers, which are labeled in kilodaltons to the left of each gel.
  • lane 1 shows antibody TY24051
  • lane 2 shows antibody TY24052
  • lane 3 shows antibody TY24053.
  • FIG. 7 provides size-exclusion high-performance liquid chromatography analyses of bispecific antibodies.
  • the upper plot shows antibody TY24051, the middle plot shows antibody TY24105, and the lower plot shows antibody TY24106.
  • time is on the x-axis, and relative protein abundance is on the y-axis. Peaks corresponding to heterodimeric proteins (major peak) , homodimeric proteins, and aggregates are indicated.
  • FIGs. 8A-8B provide enzyme-linked immunosorbent assay (ELISA) analyses of antibodies TY24051 and TY24052.
  • FIG. 8A shows binding of HER2 by TY24051 (squares) , TY24052 (triangles pointing up) , and TY24052 after activation (triangles pointing down) .
  • FIG. 8B shows binding of CD3 by TY24051 (squares) , TY24052 (triangles pointing up) , and TY24052 after activation (triangles pointing down) .
  • the concentration of antibody is on the x-axis in M, and the absorbance at 450 nm is on the y-axis.
  • FIG. 9 shows an assay of T-cell mediated cytotoxic killing upon treatment with bispecific antibodies.
  • the concentration of antibody (ng/ml) is shown on the x-axis, and the percentage of cell lysis is shown on the y-axis.
  • Target cells were incubated with T cells for 24 hours with TY24051 (circles) , TY24052 (squares) , an isotype control (triangles pointing up) , or without an antibody (triangles pointing down) .
  • FIGs. 10A-10B show activation of a nuclear factor of activated T-cells (NFAT) response element reporter in Jurkat cells in response to treatment with bispecific antibodies TY24051 (black circles) , TY24111 (squares) , TY24052 (white circles) , and TY24110 (triangles) .
  • the log-transformed concentration of antibody in ⁇ g/ml is indicated on the x-axis, and relative light units (RLU) of the reporter are indicated on the y-axis.
  • NFAT reporter activity was measured in the absence of target (SK-OV-3) cells.
  • NFAT reporter activity was measured in the presence of target cells.
  • FIGs. 11A-11B show secreted IFN ⁇ levels following administration of parental (TAC2245) or activatable (TY23104) anti-CD3 antibodies in a humanized peripheral blood mononuclear cell (PBMC) mouse model (huPBMC-NSG) .
  • the identity of the antibody and the time of sampling are indicated on the x-axis, including, from left to right, a blank, TAC2245 sampled after 0 hours of treatment, TAC2245 sampled after 3 hours of treatment, TAC2245 sampled after 24 hours of treatment, TY23104 sampled after 0 hours of treatment, TY23104 sampled after 3 hours of treatment, and TY23104 sampled after 24 hours of treatment.
  • the y-axis shows the concentration of IFN ⁇ in picograms/ml.
  • FIG. 11A shows the level of IFN ⁇ released in huPBMC-NSG mice that received fresh PBMCs.
  • FIG. 11B shows the level of IFN ⁇ released in huPBMC-NSG mice that received frozen PBMCs.
  • FIG. 12 shows secreted IFN ⁇ levels following administration of parental (TAC2245) or activatable (TY23115 and TY23118) cross-reactive anti-CD3 antibodies in a huPBMC-NSG mouse model.
  • the identity of the antibody and the time of sampling are indicated on the x-axis, including, from left to right, a blank, TAC2245 sampled after 0 hours of treatment, TAC2245 sampled after 3 hours of treatment, TAC2245 sampled after 24 hours of treatment, TY23115 sampled after 0 hours of treatment, TY23115 sampled after 3 hours of treatment, TY23115 sampled after 24 hours of treatment, TY23118 sampled after 0 hours of treatment, TY23118 sampled after 3 hours of treatment, and TY23118 sampled after 24 hours of treatment.
  • the y-axis shows the concentration of IFN ⁇ in picograms/ml.
  • FIG. 13 shows the level of Jurkat cell binding by parental anti-CD3 antibody TAC2245 (circles) and activatable anti-CD3 antibody TY23104 (squares) .
  • the log-transformed concentration of anti-CD3 antibody in nM is indicated on the x-axis, and mean fluorescence intensity (MFI) of the binding of a secondary anti-human IgG antibody is indicated on the y-axis.
  • MFI mean fluorescence intensity
  • FIG. 14 shows activation of a NFAT response element reporter in Jurkat cells in response to treatment with parental (TAC2225, circles) or activatable (TY23115, squares; and TY23118, triangles) cross-reactive anti-CD3 antibodies.
  • the log-transformed concentration of antibody in nM is indicated on the x-axis, and the relative light units (RLU) of the reporter is indicated on the y-axis.
  • FIGs. 15A-15B shows activation of a NFAT response element reporter in Jurkat cells in response to treatment with parental (TAC2245, circles) , or activatable (TY23100, black squares; TY23101, triangles pointing up; TY23102, triangles pointing down; and TY23104, white squares) anti-CD3 antibodies.
  • parental TAC2245, circles
  • activatable TY23100, black squares
  • TY23102 triangles pointing down
  • TY23104 white squares
  • FIGs. 16A-16B show analyses of the masking efficiencies of parental and activatable anti-CD3 antibodies.
  • FIG. 16A shows binding of parental (TAC2225, black circles) and activatable anti-CD3 antibodies (TY23110, squares; TY23115, triangles pointing up; and TY23118, triangles pointing down) to recombinant human CD3 ⁇ as determined by an ELISA.
  • the log-transformed concentration of antibody in M is indicated on the x-axis, and the absorbance at a wavelength of 450 nm is indicated on the y-axis.
  • FIG. 16B shows activation of a NFAT response element reporter in Jurkat cells in response to treatment with parental (TAC2225, black circles) and activatable anti-CD3 antibodies (TY23105, white circles; TY23110, squares; TY23115, triangles pointing up; and TY23118, triangles pointing down) .
  • parental TAC2225, black circles
  • activatable anti-CD3 antibodies TY23105, white circles; TY23110, squares; TY23115, triangles pointing up; and TY23118, triangles pointing down
  • the log-transformed concentration of antibody in ⁇ g/mL is indicated on the x-axis
  • the relative light units (RLU) of the reporter is indicated on the y-axis.
  • FIG. 17 shows activation of a NFAT response element reporter in Jurkat cells in response to treatment with parental (TAC2225, white circles) , or activatable anti-CD3 antibodies.
  • parental TAC2225, white circles
  • activatable anti-CD3 antibodies activatable anti-CD3 antibodies.
  • the log-transformed concentration of antibody in ⁇ g/mL is indicated on the x-axis
  • the relative light units (RLU) of the reporter is indicated on the y-axis
  • the identities of the activatable anti-CD3 antibodies are indicated by the shape of the data points, as shown in each legend.
  • Assays that were performed without FcRIIb crosslinking are indicated.
  • FIG. 18 shows the level of Jurkat cell binding by parental anti-CD3 antibody TAC2245 (TAC2225, circles) and activatable anti-CD3 antibodies.
  • TAC2245 parental anti-CD3 antibody
  • TAC2225 circles
  • MFI mean fluorescence intensity
  • FIG. 19 shows binding of parental and activatable anti-CD3 antibodies to recombinant human CD3 ⁇ as determined by an ELISA.
  • the log-transformed concentration of antibody in M is indicated on the x-axis
  • the absorbance at a wavelength of 450 nm is indicated on the y-axis
  • the identities of the anti-CD3 antibodies are indicated by the shape of the data points, as shown in the legend.
  • FIGs. 20A-20B show analyses of the masking efficiencies of parental and activatable SP34 variant anti-CD3/HER2 bispecific antibodies.
  • FIG. 20A shows binding of parental (TY25023, black circles) and activatable (TY25026, white circles) antibodies with low-anti-CD3 affinity, and comparison parental (TY24051, black squares) and activatable (TY24052, white squares) antibodies to recombinant human CD3 ⁇ , as determined by ELISA.
  • the log-transformed concentration of antibody in M is indicated on the x-axis, and the absorbance at a wavelength of 450 nm is indicated on the y-axis.
  • FIG. 20B shows the level of Jurkat cell binding by anti-CD3 antibodies TY24051 (black circles) , TY24052 (white circles) , and TY25023 (black squares) .
  • the log-transformed concentration of antibody in nM is indicated on the x-axis, and mean fluorescence intensity (MFI) of the binding of a secondary anti-human IgG antibody is indicated on the y-axis.
  • MFI mean fluorescence intensity
  • FIGs. 21A-21F show analyses of the masking efficiencies and functions of parental and activatable SP34 variant anti-CD3/HER2 bispecific antibodies.
  • FIG. 21A shows activation of a NFAT response element reporter in Jurkat cells in response to treatment with bispecific antibodies TY24051 (black circles) , TY24052 (white circles) , TY25023 (black squares) , and TY25026 (white squares) .
  • the log-transformed concentration of antibody in ⁇ g/ml is indicated on the x-axis, and relative light units (RLU) of the reporter are indicated on the y-axis.
  • NFAT reporter activity was measured in the presence of target (SK-OV-3) cells.
  • FIG. 21B shows an assay of T-cell mediated cytotoxic killing upon treatment with bispecific antibodies.
  • the log-transformed concentration of antibody (ng/ml) is shown on the x-axis, and the level of cytotoxicity as a percentage is shown on the y-axis.
  • SK-OV-3 target cells were incubated with T cells and TY24051 (black circles) , TY24052 (white circles) , TY25023 (black squares) , or TY25026 (white squares) .
  • FIG. 21C shows secreted IFN ⁇ levels in an activated CD8+ T cell assay 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 log-transformed concentration of antibody in nM is indicated on the x-axis, and concentration of IFN ⁇ in picograms/mL is indicated on the y-axis.
  • FIG. 21D shows the level of SK-OV3 tumor cell lysis in response to treatment with bispecific antibodies TY24051 (dark gray circles) , TY24052 (dark gray squares) , TY25023 (light gray triangles) , TY25026 (light gray squares) , and a reference CD3 x isotype control (dark gray triangles) .
  • the log-transformed concentration of antibody in ng/mL is indicated on the x-axis, and %cytotoxicity is indicated on the y-axis.
  • the EC 50 of cytotoxicity is indicated for each antibody in ng/mL in the table below the plot.
  • FIG. 21E shows secreted IFN ⁇ levels in an activated CD8+ T cell assay in response to treatment with bispecific antibodies TY24051 (dark gray circles) , TY24052 (dark gray squares) , TY25023 (light gray triangles) , TY25026 (light gray squares) , and a reference CD3 x isotype control (dark gray triangles) .
  • the log-transformed concentration of antibody in ng/mL is indicated on the x-axis, and concentration of IFN ⁇ in picograms/mL is indicated on the y-axis.
  • the EC 50 of IFN ⁇ secretion is indicated for each antibody in ng/mL in the table below the plot.
  • FIG. 21F shows results from an NFAT reporter assay (top) , secreted IFN ⁇ levels (bottom left) , and tumor cell lysis in response to treatment with parental (square) or activatable (circle) bispecific antibodies.
  • FIGs. 22A-22B show cytokine release in cynomolgus monkeys treated with parental or activatable bispecific antibodies.
  • FIG. 22A shows the level of cytokines IFN ⁇ , IL-2, IL-6, TNF ⁇ , 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) .
  • the x-axis shows the time following administration in hours, and the y-axis shows the concentration of the cytokine in picograms/mL.
  • the points in time at which 0.2, 0.5, and 0.9 mg/kg ( “mpk” ) doses of antibody were administered are indicated above each plot with arrows.
  • the level of IL-6 release is also provided in FIG. 24F, with a log-transformed y-axis.
  • FIG. 22B shows the level of cytokines IFN ⁇ , IL-2, IL-6, TNF ⁇ , IL-5, and IL-4 released in response to treatment with bispecific antibodies TY24051, TY24052, TY25023, and TY25026.
  • the x-axis shows the time following administration in hours
  • the y-axis shows the log-transformed concentration of the cytokine in picograms/mL. The points in time at which 0.2, 0.5, and 0.9 mg/kg doses of antibody were administered are indicated above each plot with arrows.
  • the level of IL-6 release is also provided in FIG. 24F, with a log-transformed y-axis.
  • FIG. 23 shows the level of CD4+ (left plot) and CD8+ (right plot) T cell activation 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 shows the time following administration in hours, and the y-axis shows the percentage of CD69+ T cells.
  • the points in time at which 0.2, 0.5, and 0.9 mg/kg ( “mpk” ) doses of antibody were administered are indicated above each plot with arrows.
  • FIGS. 24A-24G show results from a study of cynomolgus monkeys treated with parental or activatable bispecific antibodies.
  • FIG. 24A shows the level of T cells per ⁇ L for total T cells (top) , CD4+ T cells (bottom, left) , and CD8+ T cells (bottom, right) in monkeys 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 shows the time following administration in hours, and the y-axis shows the number of cells per ⁇ L.
  • the points in time at which 0.2, 0.5, and 0.9 mg/kg ( “mpk” ) doses of antibody were administered are indicated above each plot with arrows.
  • FIG. 24B shows the level of B cells (left) and NK cells (right) per ⁇ L in monkeys in response to treatment with bispecific antibodies TY24051 (circles) , TY24052 (squares) , TY25023 (triangles pointing up) , and TY25026 (triangles pointing down) .
  • the x-axis shows the time following administration in hours, and the y-axis shows the number of cells per ⁇ L.
  • the points in time at which 0.2, 0.5, and 0.9 mg/kg ( “mpk” ) doses of antibody were administered are indicated above each plot with arrows.
  • FIG. 24C shows the level of bispecific antibodies TY24051 (circles) , TY24052 (squares) , TY25023 (triangles pointing up) , and TY25026 (triangles pointing down) in cynomolgus monkeys.
  • the x-axis shows the time following administration in hours, and the y-axis shows the log-transformed concentration of antibody in ⁇ g/mL.
  • the points in time at which 0.2, 0.5, and 0.9 mg/kg ( “mpk” ) doses of antibody were administered are indicated above each plot with arrows.
  • FIG. 24D shows plasma concentrations of bispecific antibodies and pharmacokinetics parameters in monkeys treated with bispecific antibodies.
  • FIG. 24E provides a schematic diagram of the study design.
  • FIG. 24F shows IL-6 release in monkeys treated with bispecific antibodies.
  • the parental bispecific antibody is shown in squares, and the activatable bispecific antibody is shown in circles.
  • FIG. 24G shows absolute lymphocyte count in monkeys treated with bispecific antibodies.
  • the parental bispecific antibody is shown in squares, and the activatable bispecific antibody is shown in circles.
  • FIGs. 25A-25B provide a flow cytometry analysis of yeast cell surface display of anti-HER2 antibodies.
  • the x-axis shows the level of Fab or scFv displayed on the yeast cell (detected by the binding of an antibody to the affinity tag fused to the anti-HER2 antibody)
  • the y-axis indicates the level of HER2-binding (detected by the binding of PE conjugated streptavidin to biotinylated human HER2-Fc) .
  • FIG. 25A shows the binding of Fabs to HER2.
  • FIG. 25B shows the binding of scFvs to HER2.
  • FIG. 26 shows the results of four rounds (R1, R2, R3, and R4) of FACS to screen a CPL yeast library for masking peptides to mask binding to 10 nM of biotinylated HER2-Fc.
  • the x-axis indicates the level of myc-tagged anti-HER2 antibody
  • the y-axis indicates the level of indicates the level of HER2-binding.
  • FIGs. 27A-27B show FACS analyses of binding of the selected trastuzumab-derived activatable anti-HER2 antibodies.
  • samples were treated with the buffer PBSA (left) or TEV protease (right)
  • the x-axis shows the level of Fab or scFv displayed on the yeast cell (detected by the binding of an antibody to the affinity tag fused to the anti-HER2 antibody)
  • the y-axis indicates the level of HER2-binding (detected by the binding of PE conjugated streptavidin to biotinylated human HER2-Fc) .
  • the anti-HER2 antibody (B14126) is in the scFv format.
  • the anti-HER2 antibody (B14132) is in the Fab format.
  • FIG. 28 shows a Biolayer Interferometry analysis of binding of parental (trastuzumab) and activatable anti-HER2 antibodies (TY22841, TY22842, TY22839, TY22838, and TY22837) to His-tagged HER2, as a measurement of the masking efficiency of the activatable antibodies.
  • the x-axis indicates time in seconds, and the y-axis indicates the level of binding.
  • FIGs. 29A-29C show binding of parental (trastuzumab, black circles) and activatable anti-HER2 antibodies to recombinant HER2-Fc, as determined by an ELISA.
  • the log-transformed concentration of antibody in M is indicated on the x-axis, and the absorbance at a wavelength of 450 nm is indicated on the y-axis.
  • FIG. 29A shows results for TY22836, TY2237, TY2238, TY2239, TY2240, TY2241, TY2242, TY2243, and trastuzumab.
  • FIG. 29B shows results for TY22846, TY2247, TY2250, TY2251, TY2252, TY2253, TY2254, and trastuzumab.
  • FIG. 29C shows results for TY23523, TY23525, TY23526, TY23533, TY23536, TY23537, and trastuzumab.
  • FIG. 30 provides reduced a SDS-PAGE showing TY22837 alone (lane 1) or in the presence of the protease MMP-9 (lane 2) .
  • FIG. 31 shows binding of parental anti-HER2 antibody (trastuzumab, black circles) and TY22837 to recombinant HER2-Fc, as determined by an ELISA.
  • TY22837 binding is shown for TY22837 alone (triangles pointing down) or in the presence of the protease MMP-9 (triangles pointing up) .
  • the log-transformed concentration of antibody in M is indicated on the x-axis, and the absorbance at a wavelength of 450 nm is indicated on the y-axis.
  • FIG. 32 shows the level of SK-OV-3 cell binding by parental (trastuzumab, black circles) and activatable (TY22837, white circles; TY23536, squares) anti-HER2 antibodies.
  • the log-transformed concentration of antibody in nM is indicated on the x-axis, and mean fluorescence intensity (MFI) of the binding of a secondary anti-human IgG antibody is indicated on the y-axis.
  • FIGs. 33A-33C show the results of three stress tests of activatable anti-HER2 antibodies TY22837 (left column) and TY22838 (right column) .
  • the x-axis shows time in minutes
  • the y-axis shows the level of antibody aggregation, as indicated by absorbance units at 214 nm.
  • FIG. 33A shows results after the activatable antibodies underwent three or six freeze-thaw cycles.
  • FIG. 33B shows results after incubation of the activatable antibodies at 50°C for 7 days.
  • FIG. 33C shows results after incubation of the activatable antibodies at 40°C for 28 days.
  • FIGs. 34A-34B show binding of parental (trastuzumab) and activatable anti-HER2 antibodies to recombinant HER2-Fc, as determined by an ELISA.
  • the length of the masking peptides of the activatable antibodies was modified, as shown in Table 19.
  • the log-transformed concentration of antibody in M is indicated on the x-axis
  • the absorbance at a wavelength of 450 nm is indicated on the y-axis.
  • FIG. 34A shows the results for trastuzumab (circles) , TY23171 (triangles pointing up) , TY23172 (triangles pointing down) , and TY22836 (squares) .
  • FIG. 34B shows the results for trastuzumab (circles) , TY23173 (squares) , TY23174 (triangles pointing down) , and TY22837 (triangles pointing down) .
  • FIGs. 35A-35C show lymphocyte counts, T cell activation, and pharmacokinetic parameters in cynomolgus monkeys treated with the CD3 masked only bispecific antibody TY25362.
  • FIG. 35A shows the level of cells per ⁇ L 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 the CD3 masked only bispecific antibody TY25362.
  • the x-axis shows the time following administration in hours, and the y-axis shows the number of cells per ⁇ L.
  • the points in time at which 1, 10, and 30 mg/kg ( “mpk” ) doses of antibody were administered are indicated above each plot with arrows.
  • FIG. 35B shows the level of CD4+ (left plot) and CD8+ (right plot) T cell activation in response to treatment with bispecific antibody TY25362.
  • the x-axis shows the time following administration in hours, and the y-axis shows the percentage of CD69+ T cells.
  • the points in time at which 1, 10, and 30 mg/kg ( “mpk” ) doses of antibody were administered are indicated above each plot with arrows.
  • FIG. 35C shows the level of TY25362in cynomolgus monkeys.
  • the x-axis shows the time following administration in hours, and the y-axis shows the log-transformed concentration of antibody in ⁇ g/mL.
  • the points in time at which 1, 10, and 30 mg/kg ( “mpk” ) doses of antibody were administered are indicated above each plot with arrows.
  • FIGs. 36A-36E show binding affinity measurements of TY25023 and TY24051 to CD3.
  • FIG. 36A shows the EC 50 and Kd of TY25023 and TY24051 binding to human or monkey CD3 ⁇ as determined by an ELISA or Biacore interferometry, respectively.
  • FIG. 36B shows binding of TY25023 and TY24051 to human CD3 ⁇ as determined by ELISA.
  • the EC 50 of binding human CD3 ⁇ is indicated for each antibody in nM in the table to the right of the plot.
  • FIG. 36C shows binding of TY25023 and TY24051 to monkey CD3 ⁇ as determined by ELISA.
  • the EC 50 of binding monkey CD3 ⁇ is indicated for each antibody in nM in the table to the right of the plot.
  • FIG. 36D shows binding of TY25023 and TY24051 to human CD3 ⁇ as determined using Biacore interferometry.
  • FIG. 36E shows binding of TY25023 and TY24051 to monkey CD3 ⁇ as determined using Biacore interferometry.
  • FIGs. 37A-37D show the results of cytokine release assays in cynomolgus monkeys treated with parental or activatable anti-CD3 and anti-CD20 bispecific antibodies, as measured by ELISA.
  • FIG. 37A shows the level of IL-2 in cynomolgus monkey serum over time.
  • the x-axis shows the time following administration in hours, and the y-axis shows the level of IL-2 in pg/mL.
  • the point in time at which the 0.3 mg/kg dose of antibody was administered is indicated with an arrow.
  • TY25455 is shown as circles
  • TY25606 is shown as squares
  • TY25715 is shown as triangles pointing up
  • TY25816 is shown as triangles pointing down.
  • FIG. 37B shows the peak level of IL-2 in cynomolgus monkey serum.
  • the x-axis indicates the identity of the antibody, and the y-axis shows the peak level of IL-2 in pg/mL.
  • FIG. 37C shows the level of IFN- ⁇ in cynomolgus monkey serum over time.
  • the x-axis shows the time following administration in hours, and the y-axis shows the level of IFN- ⁇ in pg/mL.
  • the point in time at which the 0.3 mg/kg dose of antibody was administered is indicated with an arrow.
  • TY25455 is shown as circles
  • TY25606 is shown as squares
  • TY25715 is shown as triangles pointing up
  • TY25816 is shown as triangles pointing down.
  • FIG. 37D shows the peak level of IFN- ⁇ cynomolgus monkey serum.
  • the x-axis indicates the identity of the antibody, and the y-axis shows the peak level of IFN- ⁇ in pg/mL.
  • FIGs. 38A-38C show measurements of pharmacodynamics markers in cynomolgus monkeys treated with parental or activatable anti-CD3 and anti-CD20 bispecific antibodies, measured using FACS.
  • FIG. 38A shows lymphocyte (top left) , CD3+ T cell (top right) , and CD19+ B cell (bottom left) counts over the first 24 hours following antibody administration.
  • the x-axis shows the time following administration in hours
  • the y-axis shows the cell count in x10 9 cells/L.
  • the points in time at which the 0.3 mg/kg dose of antibody was administered is indicated with an arrow.
  • TY25455 is shown as circles
  • TY25606 is shown as squares
  • TY25715 is shown as triangles pointing up
  • TY25816 is shown as triangles pointing down.
  • FIG. 38B shows lymphocyte (top left) , CD3+ T cell (top right) , and CD19+ B cell (bottom left) counts over 14 days following antibody administration.
  • the x-axis shows the time following administration in hours
  • the y-axis shows the cell count in x10 9 cells/L.
  • the points in time at which the 0.3 mg/kg dose of antibody was administered is indicated with an arrow.
  • TY25455 is shown as circles
  • TY25606 is shown as squares
  • TY25715 is shown as triangles pointing up
  • TY25816 is shown as triangles pointing down.
  • FIG. 38C shows CD3+CD8+ T cell (top left) , CD3+CD4+ T cell (top right) , CD8+CD69+ T cell (bottom left) , and CD4+CD69+ T cell (bottom right) counts over 14 days following antibody administration.
  • the x-axis shows the time following administration in hours
  • the y-axis shows the percentage of cells vs. the level of lymphocytes.
  • the points in time at which the 0.3 mg/kg dose of antibody was administered is indicated with an arrow.
  • TY25455 is shown as circles
  • TY25606 is shown as squares
  • TY25715 is shown as triangles pointing up
  • TY25816 is shown as triangles pointing down.
  • FIGs. 39A-39B show measurements of pharmacodynamics markers in cynomolgus monkeys treated with the activatable anti-CD3 and anti-CD20 bispecific antibody TY25606, measuring using FACS.
  • FIG. 39A shows lymphocyte (top left) , CD3+ T cell (top right) , and CD19+ B cell (bottom left) counts over 50 days following antibody administration.
  • the x-axis shows the time following administration in hours
  • the y-axis shows the cell count in x10 9 cells/L. The points in time at which the 0.3 and 1 mg/kg doses of antibody were administered are indicated with arrows.
  • FIG. 39B shows CD3+CD8+ T cell (top left) , CD3+CD4+ T cell (top right) , CD8+CD69+ T cell (bottom left) , and CD4+CD69+ T cell (bottom right) counts over 50 days following antibody administration.
  • the x-axis shows the time following administration in hours
  • the y-axis shows the percentage of cells vs. the level of lymphocytes. The points in time at which the 0.3 and 1 mg/kg doses of antibody were administered are indicated with arrows.
  • FIG. 40 shows the level of total human IgG in cynomolgus monkeys treated with the activatable anti-CD3 and anti-CD20 bispecific antibody TY25606, measured using FACS.
  • the x-axis shows the time following administration in hours, and the y-axis shows the log-transformed level of total human IgG in ⁇ g/mL. The points in time at which the 0.3 and 1 mg/kg doses of antibody were administered are indicated with arrows.
  • FIGs. 41A-41B show the effect of parental or activatable anti-CD3 and anti-CD20 bispecific antibodies on a reporter assay with or without Raji tumor cells.
  • FIG. 41A shows the reporter assay with Raji tumor cells.
  • the x-axis shows the log-transformed concentration of antibody in nM
  • the y-axis shows the relative luminescence units ( “RLU” ) of the reporter.
  • the gray area represents the calculated peak concentration in cynomolgus serum at the 0.3 mg/kg dosage.
  • TAC2392 is shown as black circles
  • TAC2415 is shown as white circles
  • TY25455 is shown as black squares
  • TY25606 is shown as white squares
  • TY25715 is shown as triangles pointing up
  • TY25816 is shown as triangles pointing down
  • an isotype control is shown as diamonds.
  • FIG. 41B shows the reporter assay without Raji tumor cells.
  • the x-axis shows the log-transformed concentration of antibody in nM
  • the y-axis shows the relative luminescence units ( “RLU” ) of the reporter.
  • TAC2392 is shown as black circles
  • TAC2415 is shown as white circles
  • TY25455 is shown as black squares
  • TY25606 is shown as white squares
  • TY25715 is shown as triangles pointing up
  • TY25816 is shown as triangles pointing down
  • an isotype control is shown as diamonds.
  • FIGs. 42A-42B show the effect of parental or activatable anti-CD3 and anti-CD20 bispecific antibodies on a reporter assay with or without SU-DHL-4 tumor cells.
  • FIG. 42A shows the reporter assay with SU-DHL-4 tumor cells.
  • the x-axis shows the log-transformed concentration of antibody in nM
  • the y-axis shows the relative luminescence units ( “RLU” ) of the reporter.
  • TAC2392 is shown as black circles
  • TAC2415 is shown as white circles
  • TY25455 is shown as black squares
  • TY25606 is shown as white squares
  • TY25715 is shown as triangles pointing up
  • TY25816 is shown as triangles pointing down
  • an isotype control is shown as diamonds.
  • the gray area represents the calculated peak concentration in cyno serum at the 0.3 mg/kg dosage.
  • FIG. 42B shows the reporter assay without SU-DHL-4 tumor cells.
  • the x-axis shows the log-transformed concentration of antibody in nM
  • the y-axis shows the relative luminescence units ( “RLU” ) of the reporter.
  • TAC2392 is shown as black circles
  • TAC2415 is shown as white circles
  • TY25455 is shown as black squares
  • TY25606 is shown as white squares
  • TY25715 is shown as triangles pointing up
  • TY25816 is shown as triangles pointing down
  • an isotype control is shown as diamonds.
  • FIGs. 43A-43B show the effect of parental or activatable anti-CD3 and anti-CD20 bispecific antibodies on an in vitro B cell killing assay, using PBMCs.
  • FIG. 43A shows the level of endo B cell killing.
  • the x-axis shows the log-transformed concentration of antibody in nM
  • the y-axis shows the percentage of human endo B cell killing.
  • AC1281 is shown as black circles
  • TAC2415 is shown as white circles
  • TY25455 is shown as black squares
  • TY25606 is shown as white squares
  • TY25715 is shown as triangles pointing up
  • TY25816 is shown as triangles pointing down
  • an isotype control is shown as diamonds.
  • the EC 50 of B cell killing for each antibody is shown in nM.
  • FIG. 43B shows the level of CD8+ T cell activation.
  • the x-axis shows the log-transformed concentration of antibody in nM
  • the y-axis shows the percentage of CD69+ cells in the CD8+ T cell population.
  • TAC2392 is shown as black circles
  • TAC2415 is shown as white circles
  • TY25455 is shown as black squares
  • TY25606 is shown as white squares
  • TY25715 is shown as triangles pointing up
  • TY25816 is shown as triangles pointing down
  • an isotype control is shown as diamonds.
  • the EC 50 of T cell activation for each antibody is shown in nM.
  • FIG. 44 shows the level of T and B cell binding to antibodies TAC2392 (black circles) , TY2455 (black triangles pointing down) , and an isotype control (white circles) as measured using FACS, using PBMCs.
  • the x-axis shows the log-transformed concentration of antibody in nM
  • the y-axis shows the level of binding, as mean fluorescence intensity ( “MFI” ) .
  • Binding to human CD4+ T cells is shown on the upper left, binding to human CD8+ T cells is shown in the upper center, binding to human B cells is shown on the upper right, binding to monkey CD4+ T cells is shown on the lower left, binding to monkey CD8+ T cells is shown on the lower center, and binding to monkey B cells is shown on the lower right.
  • the EC 50 of TAC2392 and TY2455 binding to each cell type is shown in nM.
  • FIG. 45 shows tumor volume over time in female M-NSG immunodeficient mice with human PBMCs and EMT6 mouse breast cancer cells stably transfected with HER2.
  • the mice were administered 5 mg/kg of the antibodies TY24051 (black circles) , TY25023 (triangles pointing up) , TY25026 (squares) , TY25362 (triangles pointing down) , and an isotype control (white circles) .
  • the x-axis indicates the number of days post inoculation, with the points in time at which doses of antibody were administered indicated with arrows, and the y-axis shows tumor volume in mm 3 .
  • FIG. 46 shows a schematic diagram of a proposed SAFEbody mechanism of action.
  • a SAFEbody when a SAFEbody is in proximity to normal tissues (e.g., tissues lacking an epitope bound by the SAFEbody) , the SAFEbody remains masked.
  • normal tissues e.g., tissues lacking an epitope bound by the SAFEbody
  • path 1 a cleavable moiety is cleaved by a protease in proximity to the tumor tissue, thereby removing the masking moiety and unmasking the SAFEbody so that it can bind the target.
  • the cleavable moiety is not necessarily cleaved, and binding of the SAFEbody for the target is in competition for binding of the SAFEbody to the masking moiety.
  • the cleavable moiety can be cleaved by a protease, thereby unmasking the SAFEbody.
  • the present application provides multispecific antibodies (also referred herein as “masked multispecific antibodies” ) comprising a first antigen-binding fragment that specifically binds CD3 with weak affinity and a second antigen-binding fragment that specifically binds a target antigen, wherein the first antigen-binding fragment is fused to a first masking moiety.
  • the masking moiety may be fused to the first antigen-binding fragment via a cleavable linker or a non-cleavable linker.
  • a multispecific antibody comprising a first masking moiety can be in a state of dynamic equilibrium between a masked state in which the antigen-binding fragment that specifically binds CD3 is bound to the masking moiety, and a CD3-bound state in which the antigen-binding fragment that specifically binds CD3 is bound to CD3. Accordingly, the relative binding affinities of the masking moiety for the antigen-binding fragment and the antigen-binding fragment for CD3 determine the extent to which the antibody actually engages CD3.
  • the multispecific antibodies described herein provide a wide therapeutic window and reduce side effects associated with non-specific binding.
  • the multispecific antibodies described herein provide a safe and effective therapeutic approach for treatment of various diseases and conditions, including liquid and solid cancer that is associated with the target antigen.
  • one aspect of the present application provides a multispecific antibody 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; wherein the MM competes with CD3 to specifically bind the first antigen-binding fragment; and wherein the first antigen-binding fragment binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an enzyme-linked immunosorbent assay (ELISA, such as the ELISA assay of Example 3) .
  • the MM1 comprises an amino acid sequence of SEQ ID NO: 35 or 417.
  • the target antigen is HER2.
  • the target antigen is CD20.
  • the present application provides activatable multispecific antibodies (also referred to as “activatable multispecific T-cell engager” or “SAFEbody multispecific T-cell engager” ) comprising a first antigen-binding fragment that specifically binds CD3 with weak affinity and a second antigen-binding fragment that specifically binds a target antigen, wherein the first antigen-binding fragment is fused to a first masking moiety via a first cleavable moiety.
  • the second antigen-binding fragment is fused to a second masking moiety via a second cleavable moiety.
  • TAAxCD3 SAFEbody bispecific T-cell engager “SAFE-bsAb”
  • a TAAxCD3 SAFE-bsAb molecule comprises an antigen-binding fragment of an antibody that specifically binds to a tumor-associate antigen ( “TAA” ) , which may be masked or unmasked, and a masked anti-CD3 antigen-binding fragment.
  • TAA tumor-associate antigen
  • the activatable antibody is inactive because the masking moieties can block antigen binding.
  • the activatable antibody upon cleavage of the cleavable moieties at a target site (e.g., a disease site) , the activatable antibody is activated to bind 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 wide therapeutic window and reduce side effects associated with non-specific binding. For example, the exemplary TAAxCD3 SAFE-bsAbs in their activated forms have been observed to potently stimulate T-cell activation and TAA+ tumor cell killing.
  • the activatable multispecific antibodies described herein exhibit improved stability and more robust expression levels relative to parental antibodies.
  • the activatable multispecific antibodies described herein provide a safe and effective therapeutic approach for treatment of various diseases and conditions, including liquid and solid cancer that is associated with the target antigen.
  • an activatable multispecific antibody 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; wherein the CM1 comprises a first cleavage site; wherein the MM1 inhibits binding of the activatable antibody to CD3 when the CM1 is not cleaved; wherein the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment when the CM1 is cleaved; and wherein the first antigen-binding fragment binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an enzyme-linked immunosorbent assay (EL
  • isolated anti-CD3 antibodies include maksed anti-CD3 antibodies (including activatable anti-CD3 antibodies) , masked anti-HER2 antibodies (including activatable anti-HER2 antibodies) , compositions, methods of preparation and methods of use.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to (including full length monoclonal antibodies) , multispecific antibodies (e.g., bispecific antibodies) , and antibody fragments so long as they exhibit the desired biological activity.
  • antigen-binding fragment refers to one or more portions of an antibody that retain the ability to bind to the antigen of the antibody.
  • antigen-binding fragment of an antibody include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H1 domains; (ii) a F (ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a single chain Fv fragment comprising the VH and VL domains of an antibody, and the VH and VL domains are fused to each other; and (vi) a single chain Fab fragment comprising a single polypeptide comprising the V L , V H , C L and C H1 domains.
  • antibody includes, but is not limited to, fragments that are capable of binding 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, whose name reflects its ability to crystallize readily.
  • Pepsin treatment yields an F (ab’) 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, etc.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations 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.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, 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 making monoclonal antibodies being described herein.
  • hypervariable region refers to each of the regions of an antibody variable domain, which are hypervariable in sequence. HVRs may form structurally defined loops ( “hypervariable loops” ) . Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3) , and three in the VL (L1, L2, L3) . HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs) , CDRs being of highest sequence variability and/or involved in antigen recognition.
  • CDRs complementarity determining regions
  • Exemplary hypervariable loops occur at amino acid residues 26-32 (L1) , 50-52 (L2) , 91-96 (L3) , 26-32 (H1) , 53-55 (H2) , and 96-101 (H3) .
  • Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur 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.
  • CDRs generally comprise the amino acid residues that form the hypervariable loops.
  • CDRs also comprise “specificity determining residues, ” or “SDRs, ” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-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) occur 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.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four framework regions (FRs) and three hypervariable regions (HVRs) , arranged from amino-terminus to carboxy-terminus in the following order: FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4.
  • FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4 See, e.g., Kindt et al. Kuby Immunology, 6th ed., W. H. Freeman and Co., page 91 (2007) .
  • VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated 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) .
  • EU numbering or “amino acid position numbering based on EU numbering, ” and variations thereof, refers to the numbering system used for heavy chain constant domains of the compilation of antibodies in Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) .
  • the EU numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” EU numbered sequence.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) ) .
  • the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g.
  • residues 82a, 82b, and 82c, etc. according to Kabat after heavy chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • heterodimeric protein e.g., an activatable multispecific antibody
  • X a given amino acid position of a first CH3 domain
  • X the corresponding amino acid position of the second CH3 domain
  • N390C-S400’C refers to a heterodimeric protein (e.g., an activatable multispecific antibody) having a first CH3 domain having a N390C mutation and a second CH3 domain having a S400C mutation. All mutations or substitutions in the heterodimeric proteins (e.g., an activatable multispecific antibody) described herein are referred herein with respect to a wildtype, naturally occurring CH3 domain.
  • heavy chain constant region refers to a region comprising at least three heavy chain constant domains, CH1, CH2, and CH3, and a hinge region between CH1 and CH2.
  • Non-limiting exemplary heavy chain constant regions include ⁇ , ⁇ , and ⁇ .
  • Non-limiting exemplary heavy chain constant regions also include ⁇ and ⁇ .
  • Each heavy constant region corresponds to an antibody isotype.
  • an antibody comprising a ⁇ constant region is an IgG antibody
  • an antibody comprising a ⁇ constant region is an IgD antibody
  • an antibody comprising an ⁇ constant region is an IgA antibody.
  • an antibody comprising a ⁇ constant region is an IgM antibody
  • an antibody comprising an ⁇ constant region is an IgE antibody.
  • IgG antibodies include, but are not limited to, IgG1 (comprising a ⁇ 1 constant region) , IgG2 (comprising a ⁇ 2 constant region) , IgG3 (comprising a ⁇ 3 constant region) , and IgG4 (comprising a ⁇ 4 constant region) antibodies;
  • IgA antibodies include, but are not limited to, IgA1 (comprising an ⁇ 1 constant region) and IgA2 (comprising an ⁇ 2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.
  • CH2 domain of a human IgG Fc region usually extends from about residues 231 to about 340 of the IgG according to the EU numbering system.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain.
  • CH3 domain comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 of an IgG according to the EU numbering system) .
  • heavy chain refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence.
  • a heavy chain comprises at least a portion of a heavy chain constant region.
  • 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.
  • light chain constant region refers to a region comprising a light chain constant domain, CL.
  • Non-limiting exemplary light chain constant regions include ⁇ and ⁇ .
  • light chain refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence.
  • a light chain comprises at least a portion of a light chain constant region.
  • 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.
  • Affinity refers to the strength of the sum total of noncovalent interactions between a binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen) .
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K d ) .
  • K d dissociation constant
  • Affinity can be measured by common methods known in the art, including those described herein. In the context of a multispecific antibody (e.g., a bispecific or trispecific antibody) , affinity of the antibody with each binding specificity (i.e. target) can be measured.
  • binds refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • an antibody that binds or specifically binds a target is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds other targets.
  • 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 as measured, e.g., by a radioimmunoassay (RIA) .
  • RIA radioimmunoassay
  • an antibody that specifically binds a target has a dissociation constant (Kd) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
  • Kd dissociation constant
  • an antibody specifically binds an epitope on a protein that is conserved among the protein from different species.
  • specific binding can include, but does not require exclusive binding.
  • multispecific refers to an antibody having polyepitopic specificity (i.e., is capable of specifically binding to two, three, or more, different epitopes on one biological molecule or is capable of specifically binding to epitopes on two, three, or more, different biological molecules) .
  • an “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody, which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • an affinity-matured antibody refers to an antibody with one or more alterations in one or more complementarity determining regions (CDRs) compared to a parent antibody, which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • a “chimeric antibody” as used herein 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.
  • a chimeric antibody refers to an antibody comprising at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc. ) and at least one constant region from a second species (such as human, cynomolgus monkey, etc. ) .
  • a chimeric antibody comprises at least one mouse variable region and at least one human constant region.
  • a chimeric antibody comprises at least one cynomolgus variable region and at least one human constant region. In some embodiments, all of the variable regions of a chimeric antibody are from a first species and all of the constant regions of the chimeric antibody are from a second species.
  • a “humanized antibody” as used herein refers to an antibody in which at least one amino acid in a framework region of a non-human variable region has been replaced with the corresponding amino acid from a human variable region.
  • a humanized antibody comprises at least one human constant region or fragment thereof.
  • a humanized antibody is an Fab, a (Fab') 2 , etc.
  • 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 the framework regions (FRs) of a second (human) species.
  • CDR-grafted antibody refers to a humanized antibody in which one or more complementarity determining regions (CDRs) of a first (non-human) species have been grafted onto the framework regions (FRs) of a second (human) species.
  • human antibody refers to antibodies produced in humans, antibodies produced in non-human animals that comprise human immunoglobulin genes, such as and antibodies selected using in vitro methods, such as phage display, wherein the antibody repertoire is based on a human immunoglobulin sequence.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g. NK cells, neutrophils, and macrophages
  • NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
  • ADCC activity of a molecule of interest may be assessed in vitro, such as that described in US Pat. Nos. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta) .
  • Useful effector cells for such assays include PBMC and NK cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. (USA) 95: 652-656 (1998) .
  • polypeptide variants with altered Fc region amino acid sequences are described, e.g., in U.S. Pat. No. 7,923,538, and U.S. Pat. No. 7,994,290.
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell 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 antibodies (of the appropriate subclass) , which are bound to their cognate antigen.
  • C1q the first component of the complement system
  • a CDC assay e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) , may be performed.
  • Polypeptide variants with altered Fc region amino acid sequences polypeptides with a variant Fc region
  • increased or decreased C1q binding capability are described, e.g., in U.S. Pat. No.
  • a polypeptide variant with “altered” FcR binding affinity or ADCC activity is one which has either enhanced or diminished FcR binding activity and/or ADCC activity compared to a parent polypeptide or to a polypeptide comprising a native sequence Fc region.
  • the polypeptide variant which “displays increased binding” to an FcR binds at least one FcR with better affinity than the parent polypeptide.
  • the polypeptide variant which “displays decreased binding” to an FcR binds at least one FcR with lower affinity than a parent polypeptide.
  • Such variants which display decreased binding to an FcR may possess little or no appreciable binding to an FcR, e.g., 0-20%binding to the FcR compared to a native sequence IgG Fc region.
  • nucleic acid molecule refers to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or unnatural nucleotides, and include, but are not limited to, DNA, RNA, and PNA.
  • Nucleic acid sequence refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.
  • polypeptide and “peptide” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or unnatural amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • a “polypeptide” includes modifications, such as deletions, additions, and substitutions (generally conservative in nature) , to the native sequence, as long as the polypeptide maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts, which produce the proteins or errors due to PCR amplification.
  • a polypeptide “variant” means a biologically active polypeptide having at least 80%amino acid sequence identity with the native sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N-or C-terminus of the polypeptide.
  • a variant will have at least 80%amino acid sequence identity.
  • a variant will have at least 90%amino acid sequence identity.
  • a variant will have at least 95%amino acid sequence identity with the native sequence polypeptide.
  • Percent (%) amino acid sequence identity with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • amino acid substitution may include but are not limited to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table A. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, improved or reduced ADCC or CDC, or reduced crosslinking effects.
  • Amino acids may be grouped according to common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • vector is used to describe a polynucleotide that may be engineered to contain a cloned polynucleotide or polynucleotides that may be propagated in a host cell.
  • a vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that may be used in colorimetric assays, e.g., ⁇ -galactosidase) .
  • expression vector refers to a vector that is used to express a polypeptide of interest in a host cell.
  • a “host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide.
  • Host cells may be prokaryotic cells or eukaryotic cells.
  • Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells.
  • Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, cells (Crucell) , and 293 and CHO cells, and their derivatives, such as 293-6E and DG44 cells, respectively.
  • the term “cell” includes the primary subject cell and its progeny.
  • isolated refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced.
  • a polypeptide is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced.
  • a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be “isolating” the polypeptide.
  • a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, e.g., in the case of an RNA polynucleotide.
  • a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated” .
  • mammals including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets.
  • an “individual” or “subject” refers to an individual or subject in need of treatment for a disease or disorder.
  • treatment is an approach for obtaining beneficial or desired results including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease) , preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival.
  • treatment is a reduction of pathological consequence of cancer. The methods of the invention contemplate any one or more of these aspects of treatment.
  • prevention and similar words such as “prevented, ” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the recurrence of, a disease or condition, e.g., cancer. It also refers to delaying the recurrence of a disease or condition or delaying the recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to recurrence of the disease or condition.
  • “delaying” the development of cancer means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.
  • a method that “delays” development of cancer is a method that reduces probability of disease development in a given time frame and/or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of individuals.
  • Cancer development can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT Scan) , Magnetic Resonance Imaging (MRI) , abdominal ultrasound, clotting tests, arteriography, or biopsy. Development may also refer to cancer progression that may be initially undetectable and includes occurrence, recurrence, and onset.
  • CAT Scan computerized axial tomography
  • MRI Magnetic Resonance Imaging
  • abdominal ultrasound clotting tests
  • arteriography arteriography
  • biopsy biopsy.
  • cancer progression may be initially undetectable and includes occurrence, recurrence, and onset.
  • an effective amount refers to an amount of an agent or a combination of agents, sufficient to treat a specified disorder, condition or disease such as to ameliorate, palliate, lessen, and/or delay one or more of its symptoms.
  • an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other undesired cell proliferation.
  • an effective amount is an amount sufficient to delay disease development.
  • an effective amount is an amount sufficient to prevent or delay recurrence.
  • An effective amount can be administered in one or more administrations.
  • the effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.
  • references to "about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X” includes description of "X” .
  • reference to "not" a value or parameter generally means and describes "other than” a value or parameter.
  • the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.
  • a and/or B is intended to include both A and B; A or B; A (alone) ; and B (alone) .
  • the term “and/or” as used herein a phrase such as “A, B, and/or C” is 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) .
  • Certain aspects of the present application relate to multispecific antibodies, masked antibodies such as activatable antibodies (including activatable multispecific antibodies such as activatable bispecific T cell engager molecules) , antigen-binding fragments thereof, or derivatives of such antibodies.
  • activatable antibodies including activatable multispecific antibodies such as activatable bispecific T cell engager molecules
  • antigen-binding fragments thereof or derivatives of such antibodies.
  • multispecific antibodies that are capable of binding to both T cells and target cells such as tumor cells.
  • the multispecific antibody is bispecific.
  • the multispecific antibody is trispecific.
  • the multispecific antibody binds to CD3 on the surface of T cells. Because of their on-target off-tumor effects, traditional BiTE molecules are associated with high cytotoxicity, including toxicity to the central nervous system (CNS) and cytokine storms. Therefore, there is a need for antibodies capable of binding a T cell and a target cell such as a tumor cell with enhanced specificity and reduced side effects.
  • CNS central nervous system
  • a multispecific antibody 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 the MM1 competes with CD3 to specifically bind the CD3-binding moiety; wherein the multispecific antibody binds to CD3 via the first antigen-binding fragment; and wherein the first antigen-binding fragment binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) .
  • the first antigen-binding fragment binds CD3 with half-maximal binding
  • a multispecific antibody 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 the MM1 competes with CD3 to specifically bind the CD3-binding moiety; wherein the multispecific antibody binds to CD3 via the first antigen-binding fragment; wherein the first antigen-binding fragment binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is 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 the MM1 has a masking efficiency of at least
  • activatable multispecific antibodies that are capable of binding to both T cells and target cells such as tumor cells.
  • the activatable antibody is bispecific.
  • the activatable antibody is trispecific.
  • the activatable multispecific antibodies are activatable bispecific T cell engagers ( “BiTE” ) .
  • the activatable multispecific antibody binds to CD3 on the surface of T cells. Because of their on-target off-tumor effects, traditional BiTE molecules are associated with high cytotoxicity, including toxicity to the central nervous system (CNS) and cytokine storms. Therefore, there is a need for activatable BiTE molecules with enhanced specificity and reduced side effects.
  • FIG. 46 illustrates potential mechanisms of action of activatable BiTE molecules. Without wishing to be bound by theory, it is believed that activatable BiTE molecules with relatively weak affinities for CD3 and/or high masking efficiency for blocking CD3 binding have less severe side effects than traditional BiTE molecules. Due to this reduction in the severity of side effects, it is believed that the activatable BiTE molecules described herein allow for a greater therapeutic window.
  • activatable BiTE molecules described herein may be administered to treat disease effectively without producing toxic effects such as cytokine storm commonly associated with traditional BiTE molecules, e.g., BiTE molecules having stronger CD3 binding affinities.
  • the present application provides antibodies or antigen-binding fragments thereof, activatable antibodies, activatable multispecific antibodies, activatable antibody fragments, and polypeptides that bind specifically to human CD3 with a relatively weak binding affinity.
  • an activatable multispecific antibody 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 CM1 comprises a first cleavage site; wherein the MM1 inhibits binding of the activatable antibody to CD3 when the CM1 is not cleaved; wherein the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment when the CM1 is cleaved; and wherein the first antigen-binding fragment binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10
  • an activatable multispecific antibody 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 CM1 comprises a first cleavage site; wherein the MM1 inhibits binding of the activatable antibody to CD3 when the CM1 is not cleaved; wherein the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment when the CM1 is cleaved; wherein the first antigen-binding fragment binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 n
  • an activatable multispecific antibody comprising: a) a first antigen-binding fragment comprising a VH1 and a VL1 of an anti-CD3 antibody 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 comprising a VH2 and a VL2 of an antibody that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) ; wherein the MM1 is fused to the N-terminus of the VL1 via the CM1, wherein the CM1 comprises a first cleavage site; wherein the MM1 inhibits binding of the activatable antibody to CD3 when the CM1 is not cleaved; wherein the activatable multispecific antibody binds to CD3
  • a target antigen
  • the MM1 has a masking efficiency of at least 50 as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3) .
  • the first antigen-binding fragment is selected from the group consisting of a Fab, a Fv, a scFab and a scFv.
  • the first antigen-binding fragment is a scFv comprising, from N-terminus to C-terminus, VL1, an optional linker, and VH1.
  • an activatable multispecific antibody comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • first CH3 is a first immunoglobulin heavy chain constant domain 3;
  • second CH3 is a second immunoglobulin heavy chain constant domain 3;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a masking moiety
  • CM1 is a cleavable moiety comprising a cleavage site
  • VH1 and VL1 associate to form a scFv that specifically binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) , wherein VH2 and VL2 associate to form a Fv that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) , wherein the MM1 inhibits binding of the activatable antibody to CD3 when the CM1 is not cleaved; and wherein the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment when the CM1 is cleaved.
  • the MM1 has a masking efficiency of at least 50 as determined by a Jurkat NFAT reporter assay (e.g., the assay
  • an activatable multispecific antibody comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • the fourth polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a masking moiety
  • CM1 is a cleavable moiety comprising a cleavage site
  • VH1 and VL1 associate to form a first Fv that specifically binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) ; wherein VL2 and VH2 associate to form a second Fv that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) , wherein the MM1 inhibits binding of the activatable antibody to CD3 when the CM1 is not cleaved; and wherein the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment when the CM1 is cleaved.
  • the MM1 has a masking efficiency of at least 50 as determined by a Jurkat NFAT reporter assay (e.g., the assay in
  • an activatable multispecific antibody 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 a second masking moiety (MM2) via a second cleavable moiety (CM2) ; wherein the CM1 comprises a first cleavage site; wherein the MM1 inhibits binding of the activatable antibody to CD3 when the CM1 is not cleaved; wherein the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment when the CM1 is cleaved;
  • a target antigen
  • an activatable multispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • first CH3 is a first immunoglobulin heavy chain constant domain 3;
  • second CH3 is a second immunoglobulin heavy chain constant domain 3;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a first masking moiety
  • CM1 is a first cleavable moiety comprising a first cleavage site
  • MM2 is a second masking moiety
  • CM2 is a second cleavable moiety comprising a second cleavage site
  • VH1 and VL1 associate to form a scFv that specifically binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) , wherein VH2 and VL2 associate to form a Fv that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) , wherein the MM1 inhibits binding of the activatable antibody to CD3 when the CM1 is not cleaved; wherein the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment when the CM1 is cleaved; wherein the MM2 inhibits binding of the activatable antibody to the target antigen when the CM2 is not cleaved; and wherein the activatable multispecific
  • an activatable multispecific antibody comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • the fourth polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a first masking moiety
  • CM1 is a first cleavable moiety comprising a first cleavage site
  • MM2 is a second masking moiety
  • CM2 is a second cleavable moiety comprising a second cleavage site; wherein VH1 and VL1 associate to form a first Fv that specifically binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) ; wherein VL2 and VH2 associate to form a second Fv that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) , wherein the MM1 inhibits binding of the activatable antibody to CD3 when the CM1 is not cleaved; wherein the activatable multispecific antibody binds to CD3 via the first antigen-binding fragment when the CM1 is cleaved; wherein the MM2 inhibits binding of the activatable antibody to the
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with weak binding affinity. In some embodiments, the first antigen-binding fragment binds CD3 with a relatively weak binding affinity relative to the K D of a reference antibody for CD3. In some embodiments, the first antigen-binding fragment binds CD3 with a higher dissociation constant than the reference antibody for CD3. In some embodiments, the first antigen-binding fragment binds CD3 with a lower association constant than the reference antibody for CD3. In some embodiments, the reference antibody is SP34.
  • 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 an activated form of the activatable multispecific antibody, i.e., with CM1 cleaved and MM1 unbound to the first antigen-binding fragment.
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with half-maximal binding at a concentration of antibody (EC 50 ) that is 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 or more nM as determined by an enzyme-linked immunosorbent assay (ELISA) , including any value or range in between these values.
  • ELISA enzyme-linked immunosorbent assay
  • the first antigen-binding domain binds human CD3 with an EC 50 of about any one of 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 an enzyme-linked immunosorbent assay (ELISA) .
  • the EC 50 is determined by an ELISA measuring binding of an unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the first antigen binding fragment is a scFv
  • the EC 50 is determined by an ELISA measuring binding of an unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the EC 50 is determined by an ELISA measuring binding of a parental multispecific antibody that lacks a CM and an MM to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the first antigen binding fragment is a scFv
  • the EC 50 is determined by an ELISA measuring binding of a parental multispecific antibody that lacks a CM and an MM to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the EC 50 is determined by an ELISA measuring binding of an antigen-binding fragment that binds CD3 (e.g., an isolated anti-CD3 scFv or scFv-Fc fusion protein) to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • CD3 e.g., an isolated anti-CD3 scFv or scFv-Fc fusion protein
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with an EC 50 that is 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 times or more higher than the EC 50 of a reference antibody (e.g., SP34) , including any value or range in between these values.
  • a reference antibody e.g., SP34
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with an EC 50 that is about any one of 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 the EC 50 of a reference antibody (e.g., SP34) .
  • the EC 50 of the first antigen-binding fragment and the reference antibody are measured under the same experimental conditions.
  • the EC 50 of the first antigen-binding fragment and the reference antibody are measured in the same antibody format.
  • the EC 50 is determined by measuring binding of an unmasked multispecific antibody and an unmasked multispecific reference antibody to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the unmasked multispecific reference antibody comprises a CD3-binding moiety corresponding to SP34 (e.g., comprising the six CDRs of SP34) .
  • the EC 50 is determined by measuring binding of a parental multispecific antibody that lacks a CM and an MM and a reference parental multispecific antibody that lacks a CM and an MM to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the reference parental multispecific antibody that lacks a CM and an MM comprises a CD3-binding moiety corresponding to SP34 (e.g., comprising the six CDRs of SP34) .
  • the Kd of the first antigen-binding fragment binds CD3 and the EC 50 of the reference antibody are determined by an ELISA, such as the ELISA as described in Example 3.
  • the Kd of the first antigen-binding fragment binds CD3 and the EC 50 of the reference antibody are determined by a cell-based assay, such as a Jurkat NFAT reporter assay as described in Example 3.
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a relatively weak dissociation constant (Kd) compared to a reference antibody (e.g., SP34) , such as 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 times or more weaker than the Kd of the reference antibody, including any value or range in between these values.
  • a reference antibody e.g., SP34
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a dissociation constant (Kd) that is about any one of 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 a reference antibody (e.g., SP34) .
  • the Kd of the first antigen-binding fragment and the reference antibody are measured under the same experimental conditions.
  • the Kd of the first antigen-binding fragment and the reference antibody are measured in the same antibody format.
  • the Kd is determined by measuring binding of an unmasked multispecific antibody and an unmasked multispecific reference antibody to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the unmasked multispecific reference antibody comprises a CD3-binding moiety corresponding to SP34 (e.g., comprising the six CDRs of SP34) .
  • the Kd is determined by measuring binding of a parental multispecific antibody that lacks a CM and an MM and a reference parental multispecific antibody that lacks a CM and an MM to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the reference parental multispecific antibody that lacks a CM and an MM comprises a CD3-binding moiety corresponding to SP34 (e.g., comprising the six CDRs of SP34) .
  • the Kd of the first antigen-binding fragment binds CD3 and the Kd of the reference antibody are determined by an ELISA.
  • the first antigen-binding fragment binds 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 or more nM, including any value or range in between these values.
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a dissociation constant (Kd) of at least about any one of 1, 10, or 100 ⁇ M, including any value or range in between these values (when in the activated form) .
  • the first antigen-binding fragment binds 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.
  • CD3 e.g., human CD3
  • Kd dissociation constant
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a relatively fast off-rate (k off ) compared to a reference antibody (e.g., SP34) , such as at least about any one of 2, 5, 10, 20, 50, 100, 200 times or more faster than the k off of the reference antibody, including any value or range in between these values.
  • CD3 e.g., human CD3
  • k off a relatively fast off-rate
  • SP34 relatively fast off-rate
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a relatively slow on-rate (k on ) compared to a reference antibody (e.g., SP34) , such as at least about any one of 2, 5, 10, 20, 50, 100, 200 times or more slower than the k on of the reference antibody, including any value or range in between these values.
  • CD3 e.g., human CD3
  • SP34 relatively slow on-rate
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a relatively small dissociation constant (k d ) compared to a reference antibody (e.g., SP34) , such as at least about any one of 2, 5, 10, 20, 50, 100, 200 times or more smaller than the k d of the reference antibody, including any value or range in between these values.
  • CD3 e.g., human CD3
  • SP34 dissociation constant
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a relatively large association constant (k a ) compared to a reference antibody (e.g., SP34) , such as at least about any one of 2, 5, 10, 20, 50, 100, 200 times or more larger than the k a of the reference antibody.
  • CD3 e.g., human CD3
  • SP34 relatively large association constant
  • an antibody e.g., an activatable multispecific antibody
  • Methods of measuring the ability of an antibody to bind an antigen are known in the art, including, without limitation, via BIAcore analysis, surface plasmon resonance, ELISAs, flow cytometry, and cell-based assays (e.g., measuring binding to Jurkat cells) (See e.g., Example 5 and Table 6) .
  • the EC 50 , dissociation constant (k d ) , affinity constant (k a ) , off-rate (k off ) , and/or on-rate (k on ) of binding to CD3 (e.g., human CD3) may be measured in various contexts.
  • binding to CD3 is measured using an antigen-binding fragment that binds CD3 (e.g., a scFv or scFv-Fc fusion protein) .
  • binding to CD3 e.g., human CD3 is measured using an unmasked multispecific antibody.
  • binding to CD3 e.g., human CD3 is measured using an activatable antibody (e.g., activatable multispecific antibody) wherein the cleavable moiety associated with the anti-CD3 antigen-binding fragment is cleaved.
  • binding to human CD3 ⁇ is measured.
  • binding to human CD3 ⁇ fused with an Fc fragment is measured.
  • binding to Jurkat cells is measured.
  • an ELISA is performed using human CD3 ( ⁇ and ⁇ chain heterodimer) fused with human Fc fragment as a binding substrate.
  • An exemplary ELISA method is as follows:
  • serial diluted IgG e.g., a first antigen-binding fragment, an unmasked multispecific antibody, or an activatable antibody (e.g., activatable multispecific antibody) wherein the cleavable moiety associated with the anti-CD3 antigen-binding fragment is cleaved
  • IgG e.g., a first antigen-binding fragment, an unmasked multispecific antibody, or an activatable antibody (e.g., activatable multispecific antibody) wherein the cleavable moiety associated with the anti-CD3 antigen-binding fragment is cleaved
  • the concentration of each antibody that produced half-maximal binding to CD3 ⁇ is determined as the EC 50 in nM.
  • the first antigen-binding fragment and/or the second antigen-binding fragment may be of any suitable format, including, but are not limited to, a Fab, a Fv, a scFab and a scFv.
  • the antigen-binding fragment may have a single polypeptide chain, or two or more polypeptide chains.
  • the masking moiety e.g., MM1 or MM2 may be fused to the N-terminus of any one of the polypeptide chain of an antigen-binding fragment that has multiple polypeptide chains.
  • the masking moiety (e.g., MM1 or MM2) is fused to the N-terminus of a VL (e.g., VL1 or VL2) of the antigen-binding fragment. In some embodiments, the masking moiety (e.g., MM1 or MM2) is fused to the N-terminus of a VH (e.g., VH1 or VH2) of the antigen-binding fragment.
  • the first antigen-binding fragment may be derived from any one of the anti-CD3 antibodies described herein, which have an EC 50 of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) . Any one of the anti-CD3 antibodies and antigen-binding fragments described in Section i) “Anti-CD3 antibody” and Tables 5B-5H may be used.
  • the first antigen-binding fragment comprises one, two, three, four, five, or six CDRs of an antibody as shown in Table 7.
  • the first antigen-binding fragment of the multispecific antibody comprises one, two, three, four, five, or six CDRs of the 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.
  • the first antigen-binding fragment comprises a VH1 and/or a VL1 as shown in Table 8. In some embodiments, the first antigen-binding fragment comprises a VH1 and/or a VL1 of the 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.
  • 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 a VL of antibody TY25023 as shown in Table 8. In some embodiments, the first antigen-binding fragment comprises a 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.
  • 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.
  • a VH1 sequence contains substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 402, but retains the same ability to bind CD3 as the antibody comprising SEQ ID NO: 402.
  • a total of 1 to 13 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 402.
  • the VH1 comprises one, two or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 392, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395.
  • 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.
  • a VL1 sequence contains substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 403, but retains the same ability to bind CD3 as the antibody comprising SEQ ID NO: 403.
  • a total of 1 to 11 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 403.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs) .
  • 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.
  • the first antigen-binding fragment comprises a VH1 comprising 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 a VL1 comprising 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.
  • 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.
  • the first antigen-binding fragment comprises a VH1 comprising the CDR-H1, CDR-H2, and CDR-H3 of a VH having the sequence set forth in SEQ ID NO: 402; and a VL1 comprising the CDR-L1, CDR-L2, and CDR-L3 of a VL having the sequence set forth in SEQ ID NO: 403.
  • 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 a VH1 and/or a VL1 of antibody TY25238 as shown in Table 8. In some embodiments, the first antigen-binding fragment comprises a 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.
  • 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.
  • a VH1 sequence contains substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 410, but retains the same ability to bind human CD3 as the antibody comprising SEQ ID NO: 410.
  • a total of 1 to 13 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 410.
  • the VH1 comprises one, two or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395.
  • 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.
  • a VL1 sequence contains substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 411, but retains the same ability to bind human CD3 as the antibody comprising SEQ ID NO: 411.
  • a total of 1 to 11 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 411.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs) .
  • 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.
  • the first antigen-binding fragment comprises a VH1 comprising 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 a VL1 comprising 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.
  • 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.
  • the first antigen-binding fragment comprises a VH1 comprising the CDR-H1, CDR-H2, and CDR-H3 of a VH having the sequence set forth in SEQ ID NO: 410; and a VL1 comprising the CDR-L1, CDR-L2, and CDR-L3 of a VL having the sequence set forth in SEQ ID NO: 411.
  • the first antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 421 or SEQ ID NO: 422.
  • the masking moieties for anti-CD3 antibodies described herein may be used, including, for example, the masking moieties of section F. “Masking Moiety (MM) ” , Table B, and Table 13A.
  • the first masking moiety (MM1) comprises an amino acid sequence according to Formula (IX) : PYDDPDCPSHX 1 SDCDX 2 (SEQ ID NO: 668) , wherein X 1 is D or E, and X 2 is N or Q.
  • the first masking moiety (MM1) comprises an amino acid sequence according to Formula (X) : X 1 X 2 X 3 DX 4 X 5 CX 6 X 7 DX 8 X 9 X 10 CX 11 X 12 (SEQ ID NO: 669) , wherein X 1 is A or D, X 2 is A, D, or P, X 3 is D, H, or P, X 4 is F or P, X 5 is D or P, X 6 is D or P, X 7 is A or P, X 8 is D, N, or P, X 9 is A, N, or P, X 10 is D, H, or S, X 11 is H, P, or Y, and X 12 is N, P, or Y.
  • Formula (X) : X 1 X 2 X 3 DX 4 X 5 CX 6 X 7 DX 8 X 9 X 10 CX 11 X 12 (SEQ ID NO: 669) , wherein X 1 is A or
  • the first masking moiety (MM1) comprises the amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of the MM1. In some embodiments, the first masking moiety (MM1) comprises the amino acid sequence of SEQ ID NO: 417 (EVGSYPYDDPDCPSHESDCDQ) . In some embodiments, the first masking moiety (MM1) comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first masking moiety (MM1) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 585-588. In some embodiments, the first masking moiety (MM1) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 597-599.
  • the masking efficiency of the 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.
  • the masking efficiency of the MM1 is about any one of 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.
  • the masking efficiency of the MM1 is at least 50.
  • the masking efficiency of the 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.
  • the masking efficiency of the MM1 is 50-500. In some embodiments, the masking efficiency of the MM1 is 500. In some embodiments, masking efficiency is measured as the difference in affinity of an activatable antibody comprising the first masking moiety for binding its target (e.g., human CD3) before activation relative to the affinity of the affinity of a corresponding unmasked antibody ( “parental antibody” ) lacking the first masking moiety or the activatable antibody after activation for binding its target (e.g., human CD3) .
  • masking efficiency is measured as the difference in activity (e.g., activation of NFAT promoter) of an activatable antibody comprising the first masking moiety for binding its target (e.g., human CD3) before activation relative to the activity of the parental antibody or the activatable antibody after activation.
  • masking efficiency is measured as the difference in the level of binding a cell expressing its target (e.g., a cell expressing human CD3) for an activatable antibody comprising the first masking moiety before activation relative to the activity of the parental antibody or the activatable antibody after activation.
  • the masking efficiency is measured by dividing the EC 50 of an activatable antibody comprising the first masking moiety before activation by the EC 50 of the parental antibody.
  • the EC 50 values may be measured in an ELISA assay or a Jurkat NFAT reporter assay, see e.g., the methods of Example 3.
  • the masking efficiency is measured by dividing the k d of an activatable antibody comprising the first masking moiety before activation by the k d of the parental antibody.
  • CM Cyleavable Moiety
  • CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 418, 420, 431 and 477-490, and 516-555.
  • the first cleavable moiety (CM1) comprises the amino acid sequence of SEQ ID NO: 418 (GGGPLGLAGGS) .
  • the second antigen-binding fragment may specifically bind a target antigen, such as a tumor antigen.
  • the target antigen is a tumor antigen.
  • the target antigen is a tumor-associated antigen (TAA) .
  • TAA tumor-associated antigen
  • the target antigen is selected from the group consisting of 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, carcinoma associated antigen CTAA16, CA9, ENG, ACVRL1, CD80, CSPG4, EGFL7, FLT1, HAVCR1, HGF, HLA-DRB, IGF1R, TPBG, ERBB3, and STEAP2.
  • 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 (e.g., anti-HER2 antibodies and anti-CD20 antibodies) described in section H. “Target binding moiety (TBM) . ”
  • the second antigen-binding fragment is fused to a second masking moiety (MM2) via a second cleavable moiety (CM2) .
  • the second antigen-binding fragment is not masked.
  • the second antigen-binding fragment is not fused to a second masking moiety.
  • Any suitable masking moieties may be used, for example, anti-HER2 masking moieties described in Section F, “Masking Moiety (MM) . ”
  • Any suitable cleavable moieties may be used, for example, cleavable moieties described in Section G, “Cleavable Moiety (CM) . ”
  • 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 HER2.
  • the second antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs of trastuzumab.
  • the second antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs as shown in Table 10.
  • 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
  • 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.
  • the VH2 comprises the amino acid sequence of SEQ ID NO: 75
  • the VL2 comprises the amino acid sequence of SEQ ID NO: 76.
  • the second antigen-binding fragment is fused to a second masking moiety (MM2) via a second cleavable moiety (CM2) .
  • the MM2 comprises an amino acid sequence according to Formula (XI) : ESX1X 2 CX 3 X 4 DPFX 5 CQX 6 (SEQ ID NO: 670) , wherein X 1 is D or E, X 2 is A, F, V, or Y, X 3 is D or E, X 4 is A or L, X 5 is D or E, and X 6 is A, F, or Y.
  • the MM2 comprises an amino acid sequence according to Formula (XII) : X 1 X 2 X 3 X 4 X 5 X 6 CX 7 X 8 DPYECX 9 X 10 (SEQ ID NO: 671) , wherein X 1 is A, H, or S, X 2 is A, D, or S, X 3 is A, T, or V, X 4 is P, S, or T, X 5 is D or E, X 6 is A or V, X 7 is D or E, X 8 is A or L, X 9 is Q, S, or T, and X 10 is A, H, or V.
  • the MM2 comprises an amino acid sequence according to Formula (XIII) : YNSDDDCX 1 SX 2 YDPYTCYY (SEQ ID NO: 672) , wherein X 1 is A, I, or V, and X 2 is H or R.
  • the MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 419, 432-476, and 491-515.
  • the MM2 comprises the amino acid sequence of SEQ ID NO: 419 (ESDACDADPFDCQA) .
  • the MM2 comprises the amino acid sequence of SEQ ID NO: 36.
  • the CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 418, 420, 431 and 477-490, and 516-555. In some embodiments, the CM2 comprises the amino acid sequence of SEQ ID NO: 420.
  • 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 CD20.
  • the second antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs as shown in Table C.
  • 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
  • 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.
  • 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.
  • 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 as described in Section J, “Fc regions and CH3 domains. ” In some embodiments, the activatable multispecific antibody comprises any one of the CH3 domain mutations as described in Section J, “Fc regions and CH3 domains, ” including mutations as described in Tables D-F.
  • 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 the first CH3 domain comprises L351D, K370D, N390C and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K and S400C substitutions.
  • 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.
  • the activatable multispecific antibody is an activatable BiTE targeting human CD3 and HER2.
  • an activatable HER2xCD3 BiTE 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.
  • an activatable HER2xCD3 BiTE 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.
  • an activatable HER2xCD3 BiTE 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.
  • an activatable HER2xCD3 BiTE 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.
  • the activatable multispecific antibody is an activatable BiTE targeting human CD3 and CD20.
  • an activatable CD20xCD3 BiTE 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.
  • an activatable CD20xCD3 BiTE 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.
  • the activatable multispecific antibody is an activatable BiTE targeting human CD3 and TROP2.
  • the activatable multispecific antibody is an activatable BiTE targeting human CD3 and BCMA.
  • the activatable multispecific antibody is an activatable BiTE targeting human CD3 and CD19.
  • the activatable multispecific antibody is cross-reactive with a CD3 polypeptide from at least one non-human species selected from the group consisting of cynomolgus monkey, mouse, rat and dog.
  • a masked antibody comprises a masking moiety that binds to the target-binding moiety of the antibody, thus reducing binding of the antibody to the target when the masking moiety is bound to the target-binding moiety.
  • a masked antibody may contain a cleavable or a non-cleavable linker between the masking moiety and the antigen-binding fragment.
  • the masked antibody contains a non-cleavable linker
  • a masked antibody is in a state of 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.
  • the relative binding affinities of the masking moiety for the target-binding moiety and the target-binding moiety for the target, as well as the local concentrations of the target and the masked antibody determine the extent to which the antibody actually engages the target.
  • masked multispecific antibodies with relatively weak affinities for CD3 and/or high masking efficiency for blocking CD3 binding have less severe side effects than traditional BiTE molecules. Due to this reduction in the severity of side effects, it is believed that the masked multispecific antibodies described herein allow for a greater therapeutic window. That is, masked multispecific antibodies described herein may be administered to treat disease effectively without producing toxic effects such as cytokine storm commonly associated with traditional BiTE molecules, e.g., BiTE molecules having stronger CD3 binding affinities.
  • a multispecific antibody comprising: a) a first antigen-binding fragment comprising a VH1 and a VL1 of an anti-CD3 antibody 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 comprising a VH2 and a VL2 of an antibody that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) ; wherein the MM1 is fused to the N-terminus of the VL1; wherein the MM1 competes with CD3 to specifically bind the CD3-binding moiety; wherein the multispecific antibody binds to CD3 via the first antigen-binding fragment; and wherein the first antigen-binding fragment binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is
  • the MM1 has a masking efficiency of at least 50 as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3) .
  • the first antigen-binding fragment is selected from the group consisting of a Fab, a Fv, a scFab and a scFv.
  • the first antigen-binding fragment is a scFv comprising, from N-terminus to C-terminus, VL1, an optional linker, and VH1.
  • the masked multispecific antibody is an activatable antibody.
  • the multispecific antibody comprise a cleavable moiety. See, for example, activatable multispecific T cell engagers.
  • the multispecific antibody is a not an activatable multispecific antibody. In some embodiments, the multispecific antibody does not comprise a cleavable moiety.
  • 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 wherein the MM1 is fused to the N-terminus of the VL1 via a first non-cleavable linker (NCL1) .
  • the NCL1 is any one of the non-cleavable linkers known in the art. In some embodiments, the NCL1 is any one of the non-cleavable linkers described herein in Section I. Linker.
  • a multispecific antibody comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • first CH3 is a first immunoglobulin heavy chain constant domain 3;
  • second CH3 is a second immunoglobulin heavy chain constant domain 3;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a masking moiety
  • NCL1 is a non-cleavable linker
  • VH1 and VL1 associate to form a scFv that specifically binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) , wherein VH2 and VL2 associate to form a Fv that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) , wherein the MM1 competes with CD3 to specifically bind the CD3-binding moiety; and wherein the multispecific antibody binds to CD3 via the first antigen-binding fragment.
  • the MM1 has a masking efficiency of at least 50 as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3) .
  • a multispecific antibody comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • the fourth polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains.
  • MM1 is a masking moiety
  • NCL1 is a non-cleavable linker
  • VH1 and VL1 associate to form a first Fv that specifically binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) ; wherein VL2 and VH2 associate to form a second Fv that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) , wherein the MM1 competes with CD3 to specifically bind the CD3-binding moiety; and wherein the multispecific antibody binds to CD3 via the first antigen-binding fragment.
  • the MM1 has a masking efficiency of at least 50 as determined by a Jurkat NFAT reporter assay (e.g., the assay in Example 3) .
  • a multispecific antibody 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 the second antigen-binding fragment is fused to a second masking moiety (MM2) via a cleavable moiety (CM) ; wherein the MM1 competes with CD3 to specifically bind the CD3-binding moiety; wherein the CM comprises a cleavage site; wherein the MM2 inhibits binding of the multispecific antibody to the target antigen when the CM is not cleaved; wherein the multispecific antibody binds the target antigen via the second antigen-binding fragment when the CM is cleave
  • a multispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • first CH3 is a first immunoglobulin heavy chain constant domain 3;
  • second CH3 is a second immunoglobulin heavy chain constant domain 3;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a first masking moiety
  • NCL1 is a first non-cleavable linker
  • MM2 is a second masking moiety
  • NCL2 is a second non-cleavable linker
  • VH1 and VL1 associate to form a scFv that specifically binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) , wherein VH2 and VL2 associate to form a Fv that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) , wherein the MM1 competes with CD3 to specifically bind the CD3-binding moiety; wherein the multispecific antibody binds to CD3 via the first antigen-binding fragment; wherein the MM2 inhibits binding of the multispecific antibody to the target antigen; and wherein the multispecific antibody binds the target antigen via the second antigen-binding fragment.
  • a multispecific antibody comprises a first polypeptid
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • first CH3 is a first immunoglobulin heavy chain constant domain 3;
  • second CH3 is a second immunoglobulin heavy chain constant domain 3;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a first masking moiety
  • NCL1 is a non-cleavable linker
  • MM2 is a second masking moiety
  • CM is a cleavable moiety comprising a cleavage site
  • VH1 and VL1 associate to form a scFv that specifically binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) , wherein VH2 and VL2 associate to form a Fv that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) , wherein the MM1 competes with CD3 to specifically bind the CD3-binding moiety; wherein the multispecific antibody binds to CD3 via the first antigen-binding fragment; wherein the MM2 inhibits binding of the multispecific antibody to the target antigen when the CM is not cleaved; and wherein the multispecific antibody binds the target antigen via the second antigen-binding fragment when the CM is cleave
  • a multispecific antibody comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • the fourth polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a first masking moiety
  • NCL1 is a first non-cleavable linker
  • MM2 is a second masking moiety
  • NCL2 is a first non-cleavable linker
  • VH1 and VL1 associate to form a first Fv that specifically binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) ; wherein VL2 and VH2 associate to form a second Fv that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) , wherein the MM1 competes with CD3 to specifically bind the CD3-binding moiety; wherein the multispecific antibody binds to CD3 via the first antigen-binding fragment; wherein the MM2 inhibits binding of the multispecific antibody to the target antigen when the CM is not cleaved; and wherein the multispecific antibody binds the target antigen via the second antigen-binding fragment when the CM is cleaved
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • the fourth polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a first masking moiety
  • NCL1 is a non-cleavable linker
  • MM2 is a second masking moiety
  • CM is a cleavable moiety comprising a cleavage site
  • VH1 and VL1 associate to form a first Fv that specifically binds CD3 with half-maximal binding at a concentration of antibody (EC 50 ) that is at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) ; wherein VL2 and VH2 associate to form a second Fv that specifically binds a target antigen (e.g., a tumor antigen, such as HER2, CD20, TROP2, BCMA, or CD19) , wherein the MM1 competes with CD3 to specifically bind the CD3-binding moiety; wherein the multispecific antibody binds to CD3 via the first antigen-binding fragment; wherein the MM2 inhibits binding of the multispecific antibody to the target antigen when the CM is not cleaved; and wherein the multispecific antibody binds the target antigen via the second antigen-binding fragment when the CM is cleaved
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with weak binding affinity. In some embodiments, the first antigen-binding fragment binds CD3 with a relatively weak binding affinity relative to the K D of a reference antibody for CD3. In some embodiments, the first antigen-binding fragment binds CD3 with a higher dissociation constant than the reference antibody for CD3. In some embodiments, the first antigen-binding fragment binds CD3 with a lower association constant than the reference antibody for CD3. In some embodiments, the reference antibody is SP34.
  • 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.
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with half-maximal binding at a concentration of antibody (EC 50 ) that is 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 or more nM as determined by an enzyme-linked immunosorbent assay (ELISA) , including any value or range in between these values.
  • ELISA enzyme-linked immunosorbent assay
  • the first antigen-binding domain binds human CD3 with an EC 50 of about any one of 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 an enzyme-linked immunosorbent assay (ELISA) .
  • the EC 50 is determined by an ELISA measuring binding of an unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the first antigen binding fragment is a scFv
  • the EC 50 is determined by an ELISA measuring binding of an unmasked multispecific antibody to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the EC 50 is determined by an ELISA measuring binding of a parental multispecific antibody that lacks an MM to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the first antigen binding fragment is a scFv
  • the EC 50 is determined by an ELISA measuring binding of a parental multispecific antibody that lacks an MM to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the EC 50 is determined by an ELISA measuring binding of an antigen-binding fragment that binds CD3 (e.g., an isolated anti-CD3 scFv or scFv-Fc fusion protein) to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • CD3 e.g., an isolated anti-CD3 scFv or scFv-Fc fusion protein
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with an EC 50 that is 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 times or more higher than the EC 50 of a reference antibody (e.g., SP34) , including any value or range in between these values.
  • a reference antibody e.g., SP34
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with an EC 50 that is about any one of 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 the EC 50 of a reference antibody (e.g., SP34) .
  • the EC 50 of the first antigen-binding fragment and the reference antibody are measured under the same experimental conditions.
  • the EC 50 of the first antigen-binding fragment and the reference antibody are measured in the same antibody format.
  • the EC 50 is determined by measuring binding of an unmasked multispecific antibody and an unmasked multispecific reference antibody to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the unmasked multispecific reference antibody comprises a CD3-binding moiety corresponding to SP34 (e.g., comprising the six CDRs of SP34) .
  • the EC 50 is determined by measuring binding of a parental multispecific antibody that lacks an MM and a reference parental multispecific antibody that lacks an MM to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the reference parental multispecific antibody that an MM comprises a CD3-binding moiety corresponding to SP34 e.g., comprising the six CDRs of SP34
  • the Kd of the first antigen-binding fragment binds CD3 and the EC 50 of the reference antibody are determined by an ELISA, such as the ELISA as described in Example 3.
  • the Kd of the first antigen-binding fragment binds CD3 and the EC 50 of the reference antibody are determined by a cell-based assay, such as a Jurkat NFAT reporter assay as described in Example 3.
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a relatively weak dissociation constant (Kd) compared to a reference antibody (e.g., SP34) , such as 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 times or more weaker than the Kd of the reference antibody, including any value or range in between these values.
  • a reference antibody e.g., SP34
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a dissociation constant (Kd) that is about any one of 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 a reference antibody (e.g., SP34) .
  • the Kd of the first antigen-binding fragment and the reference antibody are measured under the same experimental conditions.
  • the Kd of the first antigen-binding fragment and the reference antibody are measured in the same antibody format.
  • the Kd is determined by measuring binding of an unmasked multispecific antibody and an unmasked multispecific reference antibody to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the unmasked multispecific reference antibody comprises a CD3-binding moiety corresponding to SP34 (e.g., comprising the six CDRs of SP34) .
  • the Kd is determined by measuring binding of a parental multispecific antibody that lacks an MM and a reference parental multispecific antibody that lacks an MM to CD3 (e.g., human CD3 or human CD3 ⁇ ) .
  • the reference parental multispecific antibody that lacks an MM comprises a CD3-binding moiety corresponding to SP34 (e.g., comprising the six CDRs of SP34) .
  • the Kd of the first antigen-binding fragment binds CD3 and the Kd of the reference antibody are determined by an ELISA.
  • the first antigen-binding fragment binds 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 or more nM, including any value or range in between these values.
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a dissociation constant (Kd) of at least about any one of 1, 10, or 100 ⁇ M, including any value or range in between these values (when in the activated form) .
  • the first antigen-binding fragment binds 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.
  • CD3 e.g., human CD3
  • Kd dissociation constant
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a relatively fast off-rate (k off ) compared to a reference antibody (e.g., SP34) , such as at least about any one of 2, 5, 10, 20, 50, 100, 200 times or more faster than the k off of the reference antibody, including any value or range in between these values.
  • CD3 e.g., human CD3
  • k off a relatively fast off-rate
  • SP34 relatively fast off-rate
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a relatively slow on-rate (k on ) compared to a reference antibody (e.g., SP34) , such as at least about any one of 2, 5, 10, 20, 50, 100, 200 times or more slower than the k on of the reference antibody, including any value or range in between these values.
  • CD3 e.g., human CD3
  • SP34 relatively slow on-rate
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a relatively small dissociation constant (k d ) compared to a reference antibody (e.g., SP34) , such as at least about any one of 2, 5, 10, 20, 50, 100, 200 times or more smaller than the k d of the reference antibody, including any value or range in between these values.
  • CD3 e.g., human CD3
  • SP34 dissociation constant
  • the first antigen-binding fragment binds CD3 (e.g., human CD3) with a relatively large association constant (k a ) compared to a reference antibody (e.g., SP34) , such as at least about any one of 2, 5, 10, 20, 50, 100, 200 times or more larger than the k a of the reference antibody.
  • CD3 e.g., human CD3
  • SP34 relatively large association constant
  • Methods of measuring the ability of an antibody (e.g., a masked multispecific antibody) to bind an antigen are known in the art, including, without limitation, via BIAcore analysis, surface plasmon resonance, ELISAs, flow cytometry, and cell-based assays (e.g., measuring binding to Jurkat cells) (See e.g., Example 5 and Table 6) .
  • the EC 50 , dissociation constant (k d ) , affinity constant (k a ) , off-rate (k off ) , and/or on-rate (k on ) of binding to CD3 (e.g., human CD3) may be measured in various contexts.
  • binding to CD3 is measured using an antigen-binding fragment that binds CD3 (e.g., a scFv or scFv-Fc fusion protein) .
  • binding to CD3 e.g., human CD3 is measured using an unmasked multispecific antibody.
  • binding to CD3 e.g., human CD3 is measured using an activatable antibody (e.g., activatable multispecific antibody) wherein the cleavable moiety associated with the anti-CD3 antigen-binding fragment is cleaved.
  • binding to human CD3 ⁇ is measured.
  • binding to human CD3 ⁇ fused with an Fc fragment is measured.
  • binding to Jurkat cells is measured.
  • an ELISA is performed using human CD3 ( ⁇ and ⁇ chain heterodimer) fused with human Fc fragment as a binding substrate.
  • An exemplary ELISA method is as follows:
  • serial diluted IgG e.g., a first antigen-binding fragment, an unmasked multispecific antibody, or an activatable antibody (e.g., activatable multispecific antibody) wherein the cleavable moiety associated with the anti-CD3 antigen-binding fragment is cleaved
  • IgG e.g., a first antigen-binding fragment, an unmasked multispecific antibody, or an activatable antibody (e.g., activatable multispecific antibody) wherein the cleavable moiety associated with the anti-CD3 antigen-binding fragment is cleaved
  • the concentration of each antibody that produced half-maximal binding to CD3 ⁇ is determined as the EC 50 in nM.
  • the first antigen-binding fragment and/or the second antigen-binding fragment may be of any suitable format, including, but are not limited to, a Fab, a Fv, a scFab and a scFv.
  • the antigen-binding fragment may have a single polypeptide chain, or two or more polypeptide chains.
  • the masking moiety e.g., MM1 or MM2 may be fused to the N-terminus of any one of the polypeptide chain of an antigen-binding fragment that has multiple polypeptide chains.
  • the masking moiety (e.g., MM1 or MM2) is fused to the N-terminus of a VL (e.g., VL1 or VL2) of the antigen-binding fragment. In some embodiments, the masking moiety (e.g., MM1 or MM2) is fused to the N-terminus of a VH (e.g., VH1 or VH2) of the antigen-binding fragment.
  • the first antigen-binding fragment may be derived from any one of the anti-CD3 antibodies described herein, which have an EC 50 of at least 10 nM (e.g., at least 100 nM) as determined by an ELISA assay (e.g., as described in Example 5) . Any one of the anti-CD3 antibodies and antigen-binding fragments described in Section i) “Anti-CD3 antibody” and Tables 5B-5H may be used.
  • the first antigen-binding fragment comprises one, two, three, four, five, or six CDRs of an antibody as shown in Table 7.
  • the first antigen-binding fragment of the multispecific antibody comprises one, two, three, four, five, or six CDRs of the 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.
  • the first antigen-binding fragment comprises a VH1 and/or a VL1 as shown in Table 8. In some embodiments, the first antigen-binding fragment comprises a VH1 and/or a VL1 of the 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.
  • 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 a VL of antibody TY25023 as shown in Table 8. In some embodiments, the first antigen-binding fragment comprises a 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.
  • 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.
  • a VH1 sequence contains substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 402, but retains the same ability to bind CD3 as the antibody comprising SEQ ID NO: 402.
  • a total of 1 to 13 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 402.
  • the VH1 comprises one, two or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 392, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395.
  • 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.
  • a VL1 sequence contains substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 403, but retains the same ability to bind CD3 as the antibody comprising SEQ ID NO: 403.
  • a total of 1 to 11 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 403.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs) .
  • 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.
  • the first antigen-binding fragment comprises a VH1 comprising 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 a VL1 comprising 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.
  • 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.
  • the first antigen-binding fragment comprises a VH1 comprising the CDR-H1, CDR-H2, and CDR-H3 of a VH having the sequence set forth in SEQ ID NO: 402; and a VL1 comprising the CDR-L1, CDR-L2, and CDR-L3 of a VL having the sequence set forth in SEQ ID NO: 403.
  • 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 a VH1 and/or a VL1 of antibody TY25238 as shown in Table 8. In some embodiments, the first antigen-binding fragment comprises a 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.
  • 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.
  • a VH1 sequence contains substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 410, but retains the same ability to bind human CD3 as the antibody comprising SEQ ID NO: 410.
  • a total of 1 to 13 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 410.
  • the VH1 comprises one, two or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 390, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 394, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 395.
  • 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.
  • a VL1 sequence contains substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 411, but retains the same ability to bind human CD3 as the antibody comprising SEQ ID NO: 411.
  • a total of 1 to 11 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 411.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs) .
  • 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.
  • the first antigen-binding fragment comprises a VH1 comprising 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 a VL1 comprising 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.
  • 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.
  • the first antigen-binding fragment comprises a VH1 comprising the CDR-H1, CDR-H2, and CDR-H3 of a VH having the sequence set forth in SEQ ID NO: 410; and a VL1 comprising the CDR-L1, CDR-L2, and CDR-L3 of a VL having the sequence set forth in SEQ ID NO: 411.
  • the first antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 421 or SEQ ID NO: 422.
  • the masking moieties for anti-CD3 antibodies described herein may be used, including, for example, the masking moieties of section F. “Masking Moiety (MM) ” , Table B, and Table 13A.
  • the first masking moiety (MM1) comprises an amino acid sequence according to Formula (IX) : PYDDPDCPSHX 1 SDCDX 2 (SEQ ID NO: 668) , wherein X 1 is D or E, and X 2 is N or Q.
  • the first masking moiety (MM1) comprises an amino acid sequence according to Formula (X) : X 1 X 2 X 3 DX 4 X 5 CX 6 X 7 DX 8 X 9 X 10 CX 11 X 12 (SEQ ID NO: 669) , wherein X 1 is A or D, X 2 is A, D, or P, X 3 is D, H, or P, X 4 is F or P, X 5 is D or P, X 6 is D or P, X 7 is A or P, X 8 is D, N, or P, X 9 is A, N, or P, X 10 is D, H, or S, X 11 is H, P, or Y, and X 12 is N, P, or Y.
  • Formula (X) : X 1 X 2 X 3 DX 4 X 5 CX 6 X 7 DX 8 X 9 X 10 CX 11 X 12 (SEQ ID NO: 669) , wherein X 1 is A or
  • the first masking moiety (MM1) comprises the amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of the MM1. In some embodiments, the first masking moiety (MM1) comprises the amino acid sequence of SEQ ID NO: 417 (EVGSYPYDDPDCPSHESDCDQ) . In some embodiments, the first masking moiety (MM1) comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the first masking moiety (MM1) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 585-588. In some embodiments, the first masking moiety (MM1) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 597-599.
  • the masking efficiency of the 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.
  • the masking efficiency of the MM1 is about any one of 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.
  • the masking efficiency of the MM1 is at least 50.
  • the masking efficiency of the 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.
  • the masking efficiency of the MM1 is 50-500. In some embodiments, the masking efficiency of the MM1 is 500. In some embodiments, masking efficiency is measured as the difference in affinity of a masked antibody comprising the first masking moiety for binding its target (e.g., human CD3) relative to the affinity of the affinity of a corresponding unmasked antibody ( “parental antibody” ) lacking the first masking moiety for binding its target (e.g., human CD3) . In some embodiments, masking efficiency is measured as the difference in activity (e.g., activation of NFAT promoter) of a masked antibody comprising the first masking moiety for binding its target (e.g., human CD3) relative to the activity of the parental antibody.
  • a masked antibody comprising the first masking moiety for binding its target e.g., human CD3
  • masking efficiency is measured as the difference in the level of binding a cell expressing its target (e.g., a cell expressing human CD3) for a masked antibody comprising the first masking moiety relative to the activity of the parental antibody.
  • the masking efficiency is measured by dividing the EC 50 of a masked antibody comprising the first masking moiety by the EC 50 of the parental antibody.
  • the EC 50 values may be measured in an ELISA assay or a Jurkat NFAT reporter assay, see e.g., the methods of Example 3.
  • the masking efficiency is measured by dividing the k d of a masked antibody comprising the first masking moiety before activation by the k d of the parental antibody.
  • the second antigen-binding fragment may specifically bind a target antigen, such as a tumor antigen.
  • the target antigen is a tumor antigen.
  • the target antigen is a tumor-associated antigen (TAA) .
  • TAA tumor-associated antigen
  • the target antigen is selected from the group consisting of 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, carcinoma associated antigen CTAA16, CA9, ENG, ACVRL1, CD80, CSPG4, EGFL7, FLT1, HAVCR1, HGF, HLA-DRB, IGF1R, TPBG, ERBB3, and STEAP2.
  • 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 (e.g., anti-HER2 antibodies and anti-CD20 antibodies) described in section H. “Target binding moiety (TBM) . ”
  • the second antigen-binding fragment is not masked. In some embodiments, the second antigen-binding fragment is not fused to a second masking moiety. Any suitable masking moieties may be used, for example, anti-HER2 masking moieties described in Section F, “Masking Moiety (MM) . ” In some embodiments, the second antigen-binding fragment is fused to a second masking moiety (MM2) via a second cleavable moiety (CM2) . Any suitable cleavable moieties may be used, for example, cleavable moieties described in Section G, “Cleavable Moiety (CM) .
  • the second antigen-binding fragment is fused to a second masking moiety (MM2) via a second non-cleavable linker (NCL2) .
  • MM2 masking moiety
  • NCL2 non-cleavable linker
  • Any suitable non-cleavable linker may be used, for example, non-cleavable linkers described in Section I, “Linker. ”
  • the second antigen-binding fragment is fused to a second masking moiety (MM2) via a second cleavable moiety (CM2) .
  • the second antigen-binding fragment is not masked.
  • the second antigen-binding fragment is not fused to a second masking moiety.
  • Any suitable masking moieties may be used, for example, anti-HER2 masking moieties described in Section F, “Masking Moiety (MM) . ”
  • Any suitable cleavable moieties may be used, for example, cleavable moieties described in Section G, “Cleavable Moiety (CM) . ”
  • the 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 HER2.
  • the second antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs of trastuzumab.
  • the second antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs as shown in Table 10.
  • 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
  • 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.
  • the VH2 comprises the amino acid sequence of SEQ ID NO: 75
  • the VL2 comprises the amino acid sequence of SEQ ID NO: 76.
  • the second antigen-binding fragment is fused to a second masking moiety (MM2) via a second cleavable moiety (CM2) .
  • the MM2 comprises an amino acid sequence according to Formula (XI) : ESX1X 2 CX 3 X 4 DPFX 5 CQX 6 (SEQ ID NO: 670) , wherein X 1 is D or E, X 2 is A, F, V, or Y, X 3 is D or E, X 4 is A or L, X 5 is D or E, and X 6 is A, F, or Y.
  • the MM2 comprises an amino acid sequence according to Formula (XII) : X 1 X 2 X 3 X 4 X 5 X 6 CX 7 X 8 DPYECX 9 X 10 (SEQ ID NO: 671) , wherein X 1 is A, H, or S, X 2 is A, D, or S, X 3 is A, T, or V, X 4 is P, S, or T, X 5 is D or E, X 6 is A or V, X 7 is D or E, X 8 is A or L, X 9 is Q, S, or T, and X 10 is A, H, or V.
  • the MM2 comprises an amino acid sequence according to Formula (XIII) : YNSDDDCX 1 SX 2 YDPYTCYY (SEQ ID NO: 672) , wherein X 1 is A, I, or V, and X 2 is H or R.
  • the MM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 419, 432-476, and 491-515.
  • the MM2 comprises the amino acid sequence of SEQ ID NO: 419 (ESDACDADPFDCQA) .
  • the MM2 comprises the amino acid sequence of SEQ ID NO: 36.
  • the CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 418, 420, 431 and 477-490, and 516-555. In some embodiments, the CM2 comprises the amino acid sequence of SEQ ID NO: 420.
  • the 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 CD20.
  • the second antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs as shown in Table C.
  • 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
  • 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.
  • 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.
  • the 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 multispecific antibody comprises any one of the Fc regions as described in Section J, “Fc regions and CH3 domains. ” In some embodiments, the multispecific antibody comprises any one of the CH3 domain mutations as described in Section J, “Fc regions and CH3 domains, ” including mutations as described in Tables D-F.
  • the 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 the first CH3 domain comprises L351D, K370D, N390C and K439D substitutions and the second CH3 domain comprises D356K, E357K, S364K and S400C substitutions.
  • the multispecific antibody is a bispecific antibody. In some embodiments, the multispecific antibody binds human CD3 and HER2. In some embodiments, the multispecific antibody binds human CD3 and CD20. In some embodiments, the multispecific antibody binds human CD3 and TROP2. In some embodiments, the multispecific antibody binds human CD3 and BCMA. In some embodiments, the multispecific antibody binds human CD3 and CD19.
  • the multispecific antibody is cross-reactive with a CD3 polypeptide from at least one non-human species selected from the group consisting of cynomolgus monkey, mouse, rat and dog.
  • activatable antibodies that target CD3 (e.g., human CD3) .
  • the activatable antibodies may be derived from any anti-CD3 antibodies known in the art, including, but not limited to, SP34, OKT3, as well as variants, mutants and derivatives thereof.
  • the present application provides activatable antibodies, activatable antibody fragments, and polypeptides that target CD3, comprising a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417, 585-588, and 597-599.
  • MM masking moiety
  • the present application also provides activatable antibodies, activatable antibody fragments, and polypeptides that target CD3, comprising a masking moiety (MM) comprising the amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of the MM. Further, the present application provides activatable antibodies, activatable antibody fragments, and polypeptides that target CD3, comprising a masking moiety (MM) comprising an amino acid sequence according to Formula (IX) : PYDDPDCPSHX 1 SDCDX 2 (SEQ ID NO: 668) , wherein X 1 is D or E, and X 2 is N or Q.
  • Formula (IX) PYDDPDCPSHX 1 SDCDX 2 (SEQ ID NO: 668) , wherein X 1 is D or E, and X 2 is N or Q.
  • the present application also provides activatable antibodies, activatable antibody fragments, and polypeptides that target CD3, comprising a masking moiety (MM) comprising an amino acid sequence according to Formula (X) : X 1 X 2 X 3 DX 4 X 5 CX 6 X 7 DX 8 X 9 X 10 CX 11 X 12 (SEQ ID NO: 669) , wherein X 1 is A or D, X 2 is A, D, or P, X 3 is D, H, or P, X 4 is F or P, X 5 is D or P, X 6 is D or P, X 7 is A or P, X 8 is D, N, or P, X 9 is A, N, or P, X 10 is D, H, or S, X 11 is H, P, or Y, and X 12 is N, P, or Y.
  • MM masking moiety
  • the activatable antibody comprises a MM comprising the amino acid sequence of EVGSY (SEQ ID NO: 667) at the N-terminus of the MM.
  • the activatable antibody comprises a MM comprising an amino acid sequence according to Formula (IX) : PYDDPDCPSHX 1 SDCDX 2 (SEQ ID NO: 668) , wherein X 1 is D or E, and X 2 is N or Q.
  • the MM comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417 and 597-599.
  • the MM comprises the amino acid sequence of SEQ ID NO: 35.
  • the MM comprises the amino acid sequence of SEQ ID NO: 35 or 417.
  • an antibody light chain comprising a polypeptide comprising, from N-terminus to C-terminus, a MM, a cleavable moiety (CM) , and a target binding moiety (TBM) , wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417, and 597-599; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises a VL of an anti-CD3 antibody.
  • an antibody heavy chain comprising a polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417, and 597-599; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises a VH of an anti-CD3 antibody.
  • an activatable antibody targeting CD3 comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417, and 597-599, wherein the MM inhibits binding of the activatable antibody to CD3 when the CM is not cleaved; wherein the CM comprises at least a first cleavage site; wherein the TBM comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; and wherein the activatable antibody binds CD3 via the VH and VL when the CM is cleaved.
  • an activatable antibody targeting CD3 comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417, and 597-599, wherein the MM inhibits binding of the activatable antibody to CD3 when the CM is not cleaved; wherein the CM comprises at least a first cleavage site; wherein the TBM comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; and wherein the activatable antibody binds CD3 via the VH and VL when the CM is cleaved.
  • an activatable antibody targeting CD3 comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a scFv of an anti-CD3 antibody, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417, and 597-599, wherein the MM inhibits binding of the activatable antibody to CD3 when the CM is not cleaved; wherein the CM comprises at least a first cleavage site; and wherein the activatable antibody binds CD3 via the scFv when the CM is cleaved.
  • an activatable antibody targeting CD3 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 the N-terminus to the C-terminus, a VL and a VH; or d) the CD3-binding moiety comprise from the N-terminus to the C-terminus, a VH and a VL; wherein the CM comprises a cleavage site; wherein the MM inhibits binding of the activatable antibody to CD3 when the CM is not cleave
  • the first antigen-binding fragment binds CD3 with a dissociation constant (Kd) of at least 50 nM.
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417, and 597-599.
  • any one of the anti-CD3 antibodies and antigen-binding fragments that competitively bind to the same epitope as SP34, including anti-CD3 antibodies described in Section i) “Anti-CD3 antibody” and Tables 5B-5H may be used.
  • the TBM (i.e., CD3 binding moiety) comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 63; and/or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 64, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 65, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 66.
  • the CD3 binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 67, and/or a VL comprising the amino acid sequence of SEQ ID NO: 68.
  • the CD3 binding moiety is a scFv comprising the amino acid sequence of SEQ ID NO: 79.
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417 and 597-599.
  • the TBM (i.e., CD3 binding moiety) comprises a VH comprising 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/or a VL comprising 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: 398, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 400.
  • the CD3 binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 402, and/or a VL comprising the amino acid sequence of SEQ ID NO: 403.
  • the CD3 binding moiety is a scFv comprising the amino acid sequence of SEQ ID NO: 421.
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417 and 597-599.
  • the TBM (i.e., CD3 binding moiety) comprises a VH comprising 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/or a VL comprising 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.
  • the CD3 binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 410, and/or a VL comprising the amino acid sequence of SEQ ID NO: 411.
  • the CD3 binding moiety is a scFv comprising the amino acid sequence of SEQ ID NO: 422.
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 417 and 597-599.
  • the MM comprises the amino acid sequence of SEQ ID NO: 417. 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: 597. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 598. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 599.
  • CM Cyleavable Moiety
  • Table 13A the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 418, 420, 431 and 477-490, and 516-555. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 418.
  • the activatable antibody targeting CD3 is a multispecific antibody, such as a bispecific antibody.
  • the activatable antibody targeting CD3 is a bispecific T cell engager (BiTE) molecule, which also targets a tumor antigen, such as HER2 or CD3.
  • BiTE bispecific T cell engager
  • the activatable antibody comprises a light chain comprising an amino acid sequence of TY23105, TY23110, TY23115, or TY23118, as shown in Table 3D. In some embodiments, the activatable antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 589, 591, 593, and 595. In some embodiments, the activatable antibody comprises a heavy chain comprising an amino acid sequence of TY23105, TY23110, TY23115, or TY23118, as shown in Table 3D. In some embodiments, the activatable antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 590, 592, 594, and 596.
  • MM masking moiety
  • the activatable antibody comprises a MM comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 585-588. In some embodiments, the MM comprises the amino acid of SEQ ID NO: 585. In some embodiments, the MM comprises the amino acid of SEQ ID NO: 586. In some embodiments, the MM comprises the amino acid of SEQ ID NO: 587. In some embodiments, the MM comprises the amino acid of SEQ ID NO: 588.
  • an antibody light chain comprising a polypeptide comprising, from N-terminus to C-terminus, a MM, a cleavable moiety (CM) , and a target binding moiety (TBM) , wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 585-588; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises a VL of an anti-CD3 antibody.
  • an antibody heavy chain comprising a polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 585-588; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises a VH of an anti-CD3 antibody.
  • an activatable antibody targeting CD3 comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 585-588, wherein the MM inhibits binding of the activatable antibody to CD3 when the CM is not cleaved; wherein the CM comprises at least a first cleavage site; wherein the TBM comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; and wherein the activatable antibody binds CD3 via the VH and VL when the CM is cleaved.
  • any one of the anti-CD3 antibodies and antigen-binding fragments that competitively bind to the same epitope as OKT3, including anti-CD3 antibodies described in Table 3B may be used.
  • an activatable antibody targeting CD3 comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 585-588, wherein the MM inhibits binding of the activatable antibody to CD3 when the CM is not cleaved; wherein the CM comprises at least a first cleavage site; wherein the TBM comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; and wherein the activatable antibody binds CD3 via the VH and VL when the CM is cleaved.
  • an activatable antibody targeting CD3 comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a scFv of an anti-CD3 antibody, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 585-588, wherein the MM inhibits binding of the activatable antibody to CD3 when the CM is not cleaved; wherein the CM comprises at least a first cleavage site; and wherein the activatable antibody binds CD3 via the scFv when the CM is cleaved.
  • an activatable 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 the N-terminus to the C-terminus, a VL and a VH; or d) the CD3-binding moiety comprises from the N-terminus to the C-terminus, a VH and a VL; and wherein the CM comprises a cleavage site; wherein the MM inhibits binding of the activatable antibody to CD3 when the CM is not cleaved; where
  • the anti-CD3 antigen-binding fragment is selected from the group consisting of a Fab, a Fv, a scFab and a scFv.
  • the TBM (i.e., CD3 binding moiety) comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 368, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 369, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 370; and/or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 371, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 372, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 373.
  • the CD3 binding moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 366, and/or a VL comprising the amino acid sequence of SEQ ID NO: 367.
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 585-588.
  • CM Cyleavable Moiety
  • Table 13A the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 418, 420, 431 and 477-490, and 516-555. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 431.
  • the activatable antibody comprises a light chain comprising an amino acid sequence of TY23100, TY23101, TY23102, or TY23104, as shown in Table 3C. In some embodiments, the activatable antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 577, 579, 581, and 583. In some embodiments, the activatable antibody comprises a heavy chain comprising an amino acid sequence of TY23100, TY23101, TY23102, or TY23104, as shown in Table 3C. In some embodiments, the activatable antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 578, 580, 582, and 584.
  • the present application provides activatable antibodies, activatable antibody fragments, and polypeptides that target HER2, comprising a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515.
  • MM masking moiety
  • the MM comprises the amino acid sequence of SEQ ID NO: 36 or 419.
  • an antibody light chain comprising a polypeptide comprising, from N-terminus to C-terminus, a MM, a cleavable moiety (CM) , and a target binding moiety (TBM) , wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises a VL of an anti-HER2 antibody.
  • the MM comprises the amino acid sequence of SEQ ID NO: 36 or 419.
  • an antibody heavy chain comprising a polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises a VH of an anti-HER2 antibody.
  • the MM comprises the amino acid sequence of SEQ ID NO: 36 or 419.
  • an activatable antibody targeting HER2 comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515, wherein the MM inhibits binding of the activatable antibody to HER2 when the CM is not cleaved; wherein the CM comprises at least a first cleavage site; wherein the TBM comprises a VL, and the activatable antibody further comprises a second polypeptide comprising a VH; and wherein the activatable antibody binds HER2 via the VH and VL when the CM is cleaved.
  • the MM comprises the amino acid sequence of SEQ ID NO: 36 or 419.
  • an activatable antibody targeting HER2 comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a TBM, wherein the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515, wherein the MM inhibits binding of the activatable antibody to HER2 when the CM is not cleaved; wherein the CM comprises at least a first cleavage site; wherein the TBM comprises a VH, and the activatable antibody further comprises a second polypeptide comprising a VL; and wherein the activatable antibody binds HER2 via the VH and VL when the CM is cleaved.
  • the MM comprises the amino acid sequence of SEQ ID NO: 36 or 419.
  • an activatable antibody targeting HER2 comprising a first polypeptide comprising, from N-terminus to C-terminus, a MM, a CM, and a scFv and an anti-HER2 antibody
  • the MM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 419, 432-476, and 491-515, wherein the MM inhibits binding of the activatable antibody to HER2 when the CM is not cleaved; wherein the CM comprises at least a first cleavage site; and wherein the activatable antibody binds HER2 via the scFv when the CM is cleaved.
  • the MM comprises the amino acid sequence of SEQ ID NO: 36 or 419.
  • the TBM (i.e., HER2-binding moiety) comprises a VH comprising an 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/or 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.
  • the TBM (i.e., HER2-binding moiety) comprises a VH comprising an CDR-H1 comprising the amino acid sequence of SEQ ID NO: 69 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 70 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and/or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73 or a variant thereof comprising a V
  • the TBM (i.e., HER2-binding moiety) comprises a VH comprising an 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/or 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.
  • the TBM (i.e., HER2-binding moiety) comprises a VH comprising an CDR-H1 comprising the amino acid sequence of SEQ ID NO: 423 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 424 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and/or a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 72 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 73 or a variant thereof
  • the TBM (i.e., HER2-binding moiety) comprises a VH comprising the amino acid sequence of SEQ ID NO: 75 and/or a VL comprising the amino acid sequence of SEQ ID NO: 76.
  • the TBM (i.e., HER2-binding moiety) comprises a VH comprising an amino acid sequence comprising at least 80% (e.g., at least any one of 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 75 and/or a VL comprising an amino acid sequence comprising at least 80%(e.g., at least any one of 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 76.
  • the activatable antibody targeting HER2 is a multispecific antibody, such as a bispecific antibody.
  • the activatable antibody targeting HER2 is a bispecific T cell engager (BiTE) molecule, which also targets CD3.
  • activatable antibodies including activatable multispecific antibodies, activatable anti-CD3 antibodies and activatable anti-HER2 antibodies described herein may have one or more of the general properties described in this Section E.
  • the activatable antibody comprises a polypeptide comprising a target-binding moiety (TBM) , a cleavable moiety (CM) , and a masking moiety (MM) .
  • TBM comprises an amino acid sequence that binds a target such as CD3, HER2, CD20, TROP2, BCMA, or CD19.
  • the TBM comprises an antigen-binding fragment (ABD) of an antibody.
  • the TBM is an antigen-binding fragment.
  • the TBM comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) , wherein the VH and VL forms a binding domain that binds the target in the absence of the MM.
  • the VH and VL are covalently linked, e.g., in a scFv.
  • the VH and VL form an Fv fragment.
  • the VH is linked to an antibody heavy chain constant region, and the VL is linked to an antibody light chain constant region.
  • the activatable antibody comprises an Fc region comprising any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the activatable antibody comprises an Fc region that does not comprise any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM) -cleavable moiety (CM) -VL, and the activatable antibody further comprises a second polypeptide comprising a VH (e.g., a Fab fragment) .
  • the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM) -cleavable moiety (CM) -VL-VH (e.g., a scFv) .
  • the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM) -cleavable moiety (CM) -VH, and the activatable antibody further comprises a second polypeptide comprising a VL (e.g., a Fab fragment) .
  • the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM) -cleavable moiety (CM) -VH-VL (e.g., a scFv) .
  • the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM) -L1-cleavable moiety (CM) -L2-VL, and the activatable antibody further comprises a second polypeptide comprising a VH (e.g., a Fab fragment) .
  • the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM) -L1-cleavable moiety (CM) -L2-VL-L3-VH (e.g., a scFv) .
  • the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM) -cleavable moiety (CM) -L1-VH, and the activatable antibody further comprises a second polypeptide comprising a VL (e.g., a Fab fragment) .
  • the activatable antibody comprises a polypeptide comprising the structure, from N-terminus to C-terminus, of: masking moiety (MM) -L1-cleavable moiety (CM) -L2-VH-L3-VL (e.g., a scFv) .
  • L1, L2, and/or L3 are linkers.
  • each of L1, L2, and L3 is a linker that can independently be either a bond or a peptide linker having an independently selected length of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids.
  • an activatable antibody comprising a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • VL is an immunoglobulin light chain variable domain
  • VH is an immunoglobulin heavy chain variable domain
  • scFv is a single-chain variable fragment
  • 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;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a first masking peptide
  • MM2 is a second masking peptide
  • CM1 is a first cleavable peptide
  • CM2 is a second cleavable peptide
  • VL and VH associate to form a first Fv that specifically binds a first target; wherein the scFv specifically binds a second target; wherein MM1 inhibits the binding of the scFv to the first target when CM1 is not cleaved; and wherein MM2 inhibits the binding of the first Fv to the second target when CM2 is not cleaved.
  • the first CH3 domain and the second CH3 domain do not comprise any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the first CH3 domain and the second CH3 domain comprise any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the first CH3 domain comprises N390C substitution and the second CH3 domain comprises S400C substitution, or the first CH3 domain comprises S400C substitution and the second CH3 domain comprises N390C substitution.
  • 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.
  • 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.
  • 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.
  • the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc having an N297A substitution.
  • the first target is a tumor antigen (e.g., HER2, CD20, TROP2, BCMA, or CD19) and the second target is CD3 (e.g., CD3e) .
  • the first target is CD3 (e.g., CD3e) and the second target is a tumor antigen (e.g., HER2, CD20, TROP2, BCMA, or CD19) .
  • an activatable antibody comprising a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, and a third polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • VL is an immunoglobulin light chain variable domain
  • VH is an immunoglobulin heavy chain variable domain
  • scFv is a single-chain variable fragment
  • 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;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a first masking peptide
  • MM2 is a second masking peptide
  • CM1 is a first cleavable peptide
  • CM2 is a second cleavable peptide
  • VL and VH associate to form a first Fv that specifically binds a first target; wherein the scFv specifically binds a second target; wherein MM1 inhibits the binding of the first Fv to the first target when CM1 is not cleaved; and wherein MM2 inhibits the binding of the scFv to the second target when CM2 is not cleaved.
  • the first CH3 domain and the second CH3 domain do not comprise any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the first CH3 domain and the second CH3 domain comprise any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the first CH3 domain comprises N390C substitution and the second CH3 domain comprises S400C substitution, or the first CH3 domain comprises S400C substitution and the second CH3 domain comprises N390C substitution.
  • 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.
  • 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.
  • 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.
  • the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc having an N297A substitution.
  • the first target is a tumor antigen (e.g., HER2, CD20, TROP2, BCMA, or CD19) and the second target is CD3 (e.g., CD3e) .
  • the first target is CD3 (e.g., CD3e) and the second target is a tumor antigen (e.g., HER2, CD20, TROP2, BCMA, or CD19) .
  • an activatable antibody comprising a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, a third polypeptide, and a fourth polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • the fourth polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a first masking peptide
  • MM2 is a second masking peptide
  • CM1 is a first cleavable peptide
  • CM2 is a second cleavable peptide
  • VL1 and VH1 associate to form a first Fv that specifically binds a first target; wherein VL2 and VH2 associate to form a second Fv that specifically binds a second target; wherein MM1 inhibits the binding of the first Fv to the first target when CM1 is not cleaved; and wherein MM2 inhibits the binding of the second Fv to the second target when CM2 is not cleaved.
  • the first CH3 domain and the second CH3 domain do not comprise any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the first CH3 domain and the second CH3 domain comprise any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the first CH3 domain comprises N390C substitution and the second CH3 domain comprises S400C substitution, or the first CH3 domain comprises S400C substitution and the second CH3 domain comprises N390C substitution.
  • 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.
  • 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.
  • 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.
  • the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc having an N297A substitution.
  • the first target is a tumor antigen (e.g., HER2, CD20, TROP2, BCMA, or CD19) and the second target is CD3 (e.g., CD3e) .
  • the first target is CD3 (e.g., CD3e) and the second target is a tumor antigen (e.g., HER2, CD20, TROP2, BCMA, or CD19) .
  • an activatable antibody comprising a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, a third polypeptide, and a fourth polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • the fourth polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a first masking peptide
  • MM2 is a second masking peptide
  • CM1 is a first cleavable peptide
  • CM2 is a second cleavable peptide; wherein VL1 and VH1 associate to form a first Fv that specifically binds a first target; wherein VL2 and VH2 associate to form a second Fv that specifically binds a second target; wherein MM1 inhibits the binding of the first Fv to the first target when CM1 is not cleaved; and wherein MM2 inhibits the binding of the second Fv to the second target when CM2 is not cleaved.
  • the first CH3 domain and the second CH3 domain do not comprise any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the first CH3 domain and the second CH3 domain comprise any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the first CH3 domain comprises N390C substitution and the second CH3 domain comprises S400C substitution, or the first CH3 domain comprises S400C substitution and the second CH3 domain comprises N390C substitution.
  • 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.
  • 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.
  • 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.
  • the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc having an N297A substitution.
  • the first target is a tumor antigen (e.g., HER2, CD20, TROP2, BCMA, or CD19) and the second target is CD3 (e.g., CD3e) .
  • the first target is CD3 (e.g., CD3e) and the second target is a tumor antigen (e.g., HER2, CD20, TROP2, BCMA, or CD19) .
  • an activatable antibody comprising a first polypeptide comprising a first CH3 domain, a second polypeptide comprising a second CH3 domain, a third polypeptide, and a fourth polypeptide, wherein:
  • the first polypeptide comprises a structure represented by the formula:
  • the second polypeptide comprises a structure represented by the formula:
  • the third polypeptide comprises a structure represented by the formula:
  • the fourth polypeptide comprises a structure represented by the formula:
  • VL1 is a first immunoglobulin light chain variable domain
  • VH1 is a first immunoglobulin heavy chain variable domain
  • VL2 is a second immunoglobulin light chain variable domain
  • VH2 is a second immunoglobulin heavy chain variable domain
  • 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;
  • hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains
  • MM1 is a first masking peptide
  • MM2 is a second masking peptide
  • CM1 is a first cleavable peptide
  • CM2 is a second cleavable peptide
  • VL1 and VH1 associate to form a first Fv that specifically binds a first target; wherein VL2 and VH2 associate to form a second Fv that specifically binds a second target; wherein MM1 inhibits the binding of the first Fv to the first target when CM1 is not cleaved; and wherein MM2 inhibits the binding of the second Fv to the second target when CM2 is not cleaved.
  • the first CH3 domain and the second CH3 domain do not comprise any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the first CH3 domain and the second CH3 domain comprise any one or combination of the engineered disulfide bonds or salt bridges described herein.
  • the first CH3 domain comprises N390C substitution and the second CH3 domain comprises S400C substitution, or the first CH3 domain comprises S400C substitution and the second CH3 domain comprises N390C substitution.
  • 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.
  • 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.
  • 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.
  • the activatable antibody comprises an IgG1 Fc region, such as an IgG1 Fc having an N297A substitution.

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