Protease activated T-cell dual-specific antibody

Abstract

The present invention relates generally to novel protease-activatable T cell-activating bispecific molecules and idiotype-specific polypeptides. The invention also relates to polynucleotides encoding such protease-activatable T cell-activating bispecific molecules and idiotype-specific polypeptides, as well as vectors and host cells comprising such polynucleotides. Furthermore, the invention relates to methods for producing the protease-activatable T cell-activating bispecific molecules and idiotype-specific polypeptides of the invention, and to methods of using these protease-activatable T cell-activating bispecific molecules and idiotype-specific polypeptides in the treatment of disease.

Description

[Technical Field]

[0001] The present invention generally relates to novel protease-activatable antigen-binding molecules. The protease-activatable antigen-binding molecules contain an anti-idiotype antigen-binding portion that reversibly masks its antigen binding. Specifically, the present invention relates to T cell binding molecules having an anti-idiotype antigen-binding portion that masks the CD3-binding portion until cleaved by a protease. This renders the CD3-binding portion inaccessible or "masked" until it approaches a target tissue, such as a tumor, e.g., tumor-infiltrating T cells. Additionally, the present invention relates to polynucleotides encoding such protease-activated T cell antigen-binding molecules and idiotype-specific polypeptides, as well as vectors and host cells comprising such polynucleotides. The present invention further relates to methods for producing the protease-activatable T cell binding molecules of the present invention and methods of using the molecules, e.g., in the treatment of disease. [Background technology]

[0002] The selective destruction of individual target cells or specific target cell types is often desired in various clinical settings. For example, a major goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and tissues intact and undamaged.

[0003] An attractive way to achieve this is to induce an immune response against tumors, allowing immune effector cells, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs), to attack and destroy tumor cells. In this regard, bispecific antibodies, designed to bind to a surface antigen on a target cell with one "arm" and to the activation invariant component of the T cell receptor (TCR) complex with a second "arm," have recently attracted attention. Simultaneous binding of such antibodies to both of their targets results in a transient interaction between the target cell and T cells, triggering activation of any cytotoxic T cells and subsequent lysis of the target cell. Thus, the immune response is redirected to the target cell, independent of peptide antigen presentation by the target cell or T cell specificity, as is appropriate for normal MHC-restricted activation of CTLs.

[0004] From this perspective, it is crucial that CTLs are activated only when they are in close proximity to target cells, i.e., when the immune synapse is mimicked. Particularly desirable are T cell-activating bispecific molecules that do not require lymphocyte preconditioning or costimulation to induce efficient target cell lysis. Several bispecific antibody formats have been developed, and their suitability for T cell-mediated immunotherapy is being investigated. These include BiTE (bispecific T cell engager) molecules (Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)), diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies (Kipriyanov et al., J Mol Biol 293, 41-66 (1999)), DART (dual affinity retargeting) molecules (Moore et al., Blood 117, 4542-51 (2011)) and triomab (Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)).

[0005] The task of generating therapeutically suitable bispecific molecules presents several technical challenges that must be met, related to efficacy, toxicity, applicability, and productivity. Toxicity can occur when bispecific molecules target antigens on target cells (e.g., cancer cells) that are also expressed in non-target tissues. Thus, there is a need for effective T cell-activating bispecific molecules that fully reverse T cell activation suppression in the presence of target cells but not in the presence of normal cells or tissues. Summary of the Invention

[0006] The present invention relates generally to T cell activating bispecific molecules that are selectively activated in the presence of target cells.

[0007] In one aspect, provided is a method for producing a pharmaceutical composition comprising: (a) a first antigen-binding moiety capable of binding to CD3, comprising: (i) a first antigen-binding moiety capable of binding to CD3; (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, an HCDR 2 of SEQ ID NO: 4, and an HCDR 3 of SEQ ID NO: 10; (ii) a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, an LCDR 2 of SEQ ID NO: 21, and an LCDR 3 of SEQ ID NO: 22; (b) a second antigen-binding moiety capable of binding to a target cell antigen; and (c) a masking moiety covalently attached to the T cell bispecific binding molecule via a protease-cleavable linker, the masking moiety being capable of binding to the idiotype of the first antigen-binding moiety, thereby reversibly masking the first or second antigen-binding moiety.

[0039] The present invention is a protease-activatable T cell activating bispecific molecule comprising:

[0008] In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16, and / or the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0009] In certain embodiments, the masking moiety is covalently attached to the first antigen-binding moiety and reversibly masks the first antigen-binding moiety.

[0010] In certain embodiments, the masking moiety is covalently attached to the heavy chain variable region of the first antigen-binding moiety.

[0011] In one embodiment, the masking moiety is an anti-idiotypic scFv.

[0012] In one embodiment, the second antigen-binding portion is a crossover Fab molecule in which the variable or constant regions of the Fab light chain and Fab heavy chain have been swapped.

[0013] In one embodiment, the first antigen-binding portion is a generic Fab molecule.

[0014] In one aspect, provided is a protease-activatable T cell activating bispecific molecule as described herein above, comprising up to one antigen-binding portion capable of binding to CD3.

[0015] In one embodiment, provided is a protease-activatable T cell activating bispecific molecule as described herein above, comprising a third antigen-binding portion that is a Fab molecule capable of binding to a target cell antigen.

[0016] In certain embodiments, the third antigen-binding portion is identical to the second antigen-binding portion.

[0017] In certain embodiments, the second antigen-binding moiety is capable of binding to a target cell antigen selected from the group consisting of FolR1 and TYRP1.

[0018] In certain embodiments, the first antigen-binding moiety and the second antigen-binding moiety are fused to each other, optionally via a peptide linker.

[0019] In certain embodiments, the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain.

[0020] In one embodiment, a first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second antigen-binding moiety.

[0021] In one aspect, provided is a protease-activatable T cell activating bispecific molecule as described herein above, further comprising an Fc domain composed of a first subunit and a second subunit that are capable of stably associating.

[0022] In one embodiment, the Fc domain is an IgG, specifically an IgG1 or IgG4 Fc domain.

[0023] In certain embodiments, the Fc domain exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain.

[0024] In one embodiment, the masking moiety is (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of DYSMN (SEQ ID NO: 58); (b) a CDR H2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO: 59), WINTETGEPRYTDDFTG (SEQ ID NO: 84), and WINTETGEPRYTQGFKG (SEQ ID NO: 86); (c) CDR H3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) a light chain (CDR L) 1 amino acid sequence selected from the group consisting of RASKSVSTSSYSYMH (SEQ ID NO: 62) and KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of YVSYLES (SEQ ID NO: 63); and (f) a CDR L3 amino acid sequence selected from the group consisting of QHSREFPYT (SEQ ID NO: 64) and QQSREFPYT (SEQ ID NO: 88). and a light chain variable region comprising:

[0025] In one embodiment, the masking moiety is (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of DYSMN (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 59); (c) CDR H3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 62); (e) the CDR L2 amino acid sequence of YVSYLES (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHSREFPYT (SEQ ID NO: 64) and a light chain variable region comprising:

[0026] In one embodiment, the masking moiety is (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID NO: 59); (c) CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising:

[0027] In one embodiment, the masking moiety is (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO: 84); (c) CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising:

[0028] In one embodiment, the masking moiety is (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 86); (c) CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising:

[0029] In certain embodiments, the protease-cleavable linker comprises at least one protease recognition sequence.

[0030] In one aspect, the protease recognition sequence is (a) RQARVVNG (SEQ ID NO: 100); (b) VHMPLGFLGPGRSRGSFP (SEQ ID NO: 101); (c) RQARVVNGXXXXXVPLSLYSG (SEQ ID NO: 102); (d) RQARVVNGVPLSLYSG (SEQ ID NO: 103); (e) PLGLWSQ (SEQ ID NO: 104); (f) VHMPLGFLGPRQARVVNG (SEQ ID NO: 105); (g) FVGGTG (SEQ ID NO: 106); (h) KKAAPVNG (SEQ ID NO: 107); (i) PMAKKVNG (SEQ ID NO: 108); (j) QARAKVNG (SEQ ID NO: 109); (k) VHMPLGFLGP (SEQ ID NO: 110); (l) QARAK (SEQ ID NO: 111); (m) VHMPLGFLGPPMAKK (SEQ ID NO: 112); (n) KKAAP (SEQ ID NO: 113); and (o) PMAKK (SEQ ID NO: 114) and X is any amino acid.

[0031] In some embodiments, the protease-cleavable linker comprises the protease recognition sequence PMAKK (SEQ ID NO: 114).

[0032] In one embodiment, the second antigen-binding moiety is capable of binding to FolR1 and a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of NAWMS (SEQ ID NO: 54); b) the CDR H2 amino acid sequence of RIKSKTDGGTTDYAAPVKG (SEQ ID NO: 55); and c) CDR H3 amino acid sequence of PWEWSWYDY (SEQ ID NO: 56) a heavy chain variable region comprising d) the light chain (CDR L) 1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 20); e) the CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO: 21); and (f) CDR L3 amino acid sequence of ALWYSNLWV (SEQ ID NO: 22) and a light chain variable region comprising:

[0033] In one aspect, the second antigen-binding moiety is capable of binding to TYRP1 and a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of DYFLH (SEQ ID NO: 24); b) the CDR H2 amino acid sequence of WINPDNGNTVYAQKFQG (SEQ ID NO: 25); and c) CDR H3 amino acid sequence of RDYTYEKAALDY (SEQ ID NO: 26) a heavy chain variable region comprising d) light chain (CDR L) 1 amino acid sequence of RASGNIYNYLA (SEQ ID NO: 28); e) the CDR L2 amino acid sequence of DAKTLAD (SEQ ID NO: 29); and f) CDR L3 amino acid sequence of QHFWSLPFT (SEQ ID NO: 30) and a light chain variable region comprising:

[0034] In another aspect, provided is a method for producing a pharmaceutical composition comprising: (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of DYSMN (SEQ ID NO: 58); (b) a CDR H2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO: 59), WINTETGEPRYTDDFTG (SEQ ID NO: 84), and WINTETGEPRYTQGFKG (SEQ ID NO: 86); (c) CDR H3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) a light chain (CDR L) 1 amino acid sequence selected from the group consisting of RASKSVSTSSYSYMH (SEQ ID NO: 62) and KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of YVSYLES (SEQ ID NO: 63); and (f) a CDR L3 amino acid sequence selected from the group consisting of QHSREFPYT (SEQ ID NO: 64) and QQSREFPYT (SEQ ID NO: 88). and a light chain variable region comprising:

[0035] In one aspect, the idiotype-specific polypeptide is (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of DYSMN (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 59); (c) CDR H3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 62); (e) the CDR L2 amino acid sequence of YVSYLES (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHSREFPYT (SEQ ID NO: 64) and a light chain variable region comprising:

[0036] In one aspect, the idiotype-specific polypeptide is (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID NO: 59); (c) CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising:

[0037] In one aspect, the idiotype-specific polypeptide is (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO: 84); (c) CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising:

[0038] In one aspect, the idiotype-specific polypeptide is (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 86); (c) CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising:

[0039] In one embodiment, the idiotype-specific polypeptide is an anti-idiotype scFv.

[0040] In some embodiments, the idiotype-specific polypeptide is covalently attached to the molecule via a linker.

[0041] In some embodiments, the linker is a peptide linker.

[0042] In some embodiments, the linker is a protease-cleavable linker.

[0043] In certain embodiments, the peptide linker comprises at least one protease recognition site.

[0044] In one aspect, the protease recognition sequence is (a) RQARVVNG (SEQ ID NO: 100); (b) VHMPLGFLGPGRSRGSFP (SEQ ID NO: 101); (c) RQARVVNGXXXXXVPLSLYSG (SEQ ID NO: 102); (d) RQARVVNGVPLSLYSG (SEQ ID NO: 103); (e) PLGLWSQ (SEQ ID NO: 104); (f) VHMPLGFLGPRQARVVNG (SEQ ID NO: 105); (g) FVGGTG (SEQ ID NO: 106); (h) KKAAPVNG (SEQ ID NO: 107); (i) PMAKKVNG (SEQ ID NO: 108); (j) QARAKVNG (SEQ ID NO: 109); (k) VHMPLGFLGP (SEQ ID NO: 110); (l) QARAK (SEQ ID NO: 111); (m) VHMPLGFLGPPMAKK (SEQ ID NO: 112); (n) KKAAP (SEQ ID NO: 113); and (o) PMAKK (SEQ ID NO: 114) and X is any amino acid.

[0045] In some embodiments, the protease-cleavable linker comprises the protease recognition sequence PMAKK (SEQ ID NO: 114).

[0046] In certain embodiments, the idiotype-specific polypeptide is part of a T cell activating bispecific molecule.

[0047] In another aspect, provided is a pharmaceutical composition comprising the protease-activatable T cell activating bispecific molecule described above or the idiotype-specific polypeptide described above and a pharmaceutically acceptable carrier.

[0048] In another aspect, provided is an isolated polynucleotide encoding the aforementioned protease-activatable T cell-activating bispecific antigen-binding molecule or the aforementioned idiotype-specific polypeptide.

[0049] In one aspect, provided are vectors, particularly expression vectors, that include the aforementioned polynucleotides.

[0050] In one aspect, provided is a host cell comprising the above-described polynucleotide or the above-described vector.

[0051] In another aspect, provided is a method of producing a protease activatable T cell activating bispecific molecule, comprising the steps of a) culturing the aforementioned host cell under conditions suitable for expression of the protease activatable T cell activating bispecific molecule; and b) recovering the protease activatable T cell activating bispecific molecule.

[0052] In another aspect, provided is the aforementioned protease-activatable T cell activating bispecific molecule, the aforementioned idiotype-specific polypeptide, or the aforementioned pharmaceutical composition for use as a medicament.

[0053] In one aspect, the medicament is for treating or delaying the progression of cancer, treating or delaying the progression of an immune-related disease, or enhancing or stimulating immune response or function in an individual.

[0054] In another aspect, provided is the use of an aforementioned protease-activatable T cell activating bispecific molecule or an aforementioned idiotype-specific polypeptide for the manufacture of a medicament for the treatment of a disease.

[0055] In some embodiments, the disease is cancer.

[0056] In another aspect, provided is a method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising the protease-activatable T cell activating bispecific molecule described above.

[0057] In some aspects, the method is for treating or delaying the progression of cancer, treating or delaying the progression of an immune-related disease, or enhancing or stimulating an immune response or function in an individual. [Brief explanation of the drawings]

[0058] [Figure 1A-F] Figure 1 shows exemplary conformations of (multispecific) antibodies of the invention. (A, D) Diagram of a "1+1 CrossMab" molecule. (B, E) Diagram of a "2+1 IgG Crossfab" molecule with the Crossfab and Fab components in an alternative order ("flipped"). (C, F) Diagram of a "2+1 IgG Crossfab" molecule. [Figure 1G-N] (G,K) Diagram of a "1+1 IgG Crossfab" molecule with the Crossfab and Fab components in an alternative order ("flipped"). (H,L) Diagram of a "1+1 IgG Crossfab" molecule. (I,M) Diagram of a "2+1 IgG Crossfab" molecule with two CrossFabs. (J,N) Diagram of a "2+1 IgG Crossfab" molecule with two CrossFabs and the Crossfab and Fab components in an alternative order ("flipped"). [Figure 1O-V] (O, S) Diagram of "Fab-Crossfab" molecule. (P, T) Diagram of "Crossfab-Fab" molecule. (Q, U) Diagram of "(Fab)2-Crossfab" molecule. (R, V) Diagram of "Crossfab-(Fab)2" molecule. [Figure 1W-Z] (W, Y) Diagram of "Fab-(Crossfab)2" molecule. (X, Z) Diagram of "(Crossfab)2-Fab" molecule. Black dots: optional modifications in the Fc domain that promote heterodimerization. ++, --: amino acids of opposite charge optionally introduced in the CH1 and CL domains. Although Crossfab molecules are depicted as including swapped VH and VL domains, in embodiments where no charge modifications have been introduced in the CH1 and CL domains, they alternatively include swapped CH1 and CL domains. [Figure 2A-E] (A) Schematic diagram of the T cell bispecific antibody (TCB) molecules used in the examples. All TCB antibody molecules tested were generated as "2 + 1 IgG CrossFab, inverted" with charge modifications (VH / VL exchange of the CD3 binder, charge modifications of the target cell antigen binder, EE = 147E, 213E; RK = 123R, 124K). (B-E) Components for TCB assembly: light chain of an anti-TYRP1 Fab molecule with charge modifications in CH1 and CL (B), light chain of an anti-CD3 crossover Fab molecule (C), heavy chain with knob and PG-LALA mutations in the Fc region (D), and heavy chain with hole and PG-LALA mutations in the Fc region (E). [Figure 3] Schematic diagram of the surface plasmon resonance (SPR) setup used in Example 3. Anti-PG antibody bound to a C1 sensor chip. Human and cynomolgus monkey CD3 (fused to the Fc region) was passed over the surface to analyze the interaction between the anti-CD3 antibody and CD3 in the TCB. [Figure 4A-B] TCB containing the optimized anti-CD3 antibody was tested in a Jurkat NFAT reporter assay using CHO-K1 TYRP1 clone 76 as target cells and compared to TCB containing CD3orig. Activation of Jurkat NFAT reporter cells was determined by measuring luminescence 4 hours (A) and 24 hours (B) after treatment. [Figure 5A-B]Tumor cell killing of the melanoma cell line M150543 using PBMCs from healthy donors was assessed upon treatment with TCB containing either the optimized anti-CD3 antibody or the parental binder CD3orig. Tumor cell killing was assessed by quantification of LDH release after 24 hours (A) and 48 hours (B). [Figure 6A-D] CD25 and CD69 upregulation on CD8 T cells (A, B) and CD4 T cells (C, D) was analyzed in PBMCs from healthy donors treated with TCB containing either the optimized anti-CD3 antibody or the parental binder CD3orig in the presence of the M150543 melanoma cell line as target cells. Analysis was performed by flow cytometry 48 hours later. [Figure 7A-B] CD25 expression on CD8 (A) and CD4 T cells (B) was analyzed in PBMCs from healthy donors treated with TCB containing either the optimized anti-CD3 antibody or the parental binder CD3orig in the absence of tumor target cells. Analysis was performed by flow cytometry 48 hours later. [Figure 8A-D] (A) Schematic diagram of the monovalent IgG molecule generated in Example 19. This monovalent IgG molecule was produced as a human IgG1 with VH / VL swapping on a CD3 binder. (B-E) Components for assembly of the monovalent IgG: light chain of an anti-CD3 crossover Fab molecule (B), heavy chain with knob and PG LALA mutations in the Fc region (C), heavy chain with hole and PG LALA mutations in the Fc region (D). [Figure 9A-C](A): A classic 2+1 TCB molecule with a CD3 Fab fused to the VH of the inner FOLR1 Fab via a (G4S)2 linker (L1). Heterodimerization was achieved using knob-into-hole technology, with a PGLALA mutation in the Fc. (B): A FOLR1 proTCB in which a CD3 anti-idiotype scFv (VH-VL orientation) is fused to the CD3 VH. The linker (L2; 33 aa in total) contains a specific protease-cleavable sequence. A (G4S)4 linker (L3) is located between the VH and VL of the scFv. (C): The same proTCB as in B, but without a protease cleavage site in the linker between the scFv and CD3 Fab. The light chains in each molecule are identical (common light chain). [Figures 10A-E](A): Jurkat NFAT activation mediated by TYRP1 TCB containing different CD3 binders. Figure 1 shows Jurkat NFAT activation mediated by TYRP1 TCB containing different CD3 binders. TYRP1 TCB (used at the EC90 concentration determined in the previous assay) was incubated with TYRP1-positive target cells (CHO-huTYRP1 clone 76) and Jurkat NFAT effector cells (E:T 2.5:1) at 37°C for 22 hours. The dotted line indicates NFAT activation in Jurkats incubated with target cells without TCB. DP47 non-targeting TCB was used as a negative control. Each point represents the mean of triplicates. Standard deviation is indicated by error bars. (B): Blocking ability of anti-idiotype 4.24.72 IgG as measured by the reduction of Jurkat NFAT activation mediated by TYRP1 TCB. Figure 1 shows Jurkat NFAT activation mediated by TYRP1 TCB with different CD3 binders. TYRP1 TCB (used at the EC90 concentration determined in the previous assay) was incubated with TYRP1-positive target cells (CHO-huTYRP1 clone 76) and Jurkat NFAT effector cells (E:T 2.5:1) for 22 hours at 37°C. Dose-dependent blocking of CD3 binders by anti-idiotypic (anti-ID) 4.24.72 IgG is shown. Each point represents the mean of triplicates. Standard deviations are indicated by error bars. The dotted line indicates NFAT activation in Jurkats incubated with target cells without TCB. DP47 non-targeting TCB was used as a negative control. EC50 values ​​were calculated using a nonlinear fit: "log(agonist) vs. response—variable slope (4 parameters)" (GraphPad Prism 6). (C): Blocking ability of anti-ID4.32.63 IgG measured by reduction of Jurkat NFAT activation mediated by TYRP1 TCB. Jurkat NFAT activation mediated by TYRP1 TCB with different CD3 binders is shown.TYRP1 TCB (used at the EC90 concentration determined in the previous assay) was incubated with TYRP1-positive target cells (CHO-huTYRP1 clone 76) and Jurkat NFAT effector cells (E:T 2.5:1) for 22 hours at 37°C. Dose-dependent blocking of CD3 binders by anti-idiotype 4.32.63 IgG is shown. Each point represents the mean of triplicates. Standard deviations are indicated by error bars. The dotted line indicates NFAT activation in Jurkats incubated with target cells without TCB. DP47 non-targeting TCB was used as a negative control. EC50 values ​​were calculated using a nonlinear fit "log(agonist) vs. response—variable slope (4 parameters)" (GraphPad Prism 6). (D): Blocking potency of anti-ID4.15.64 IgG measured by the reduction of Jurkat NFAT activation mediated by TYRP1 TCB. Figure 1 shows Jurkat NFAT activation mediated by TYRP1 TCB with different CD3 binders. TYRP1 TCB (used at the EC90 concentration determined in the previous assay) was incubated with TYRP1-positive target cells (CHO-huTYRP1 clone 76) and Jurkat NFAT effector cells (E:T 2.5:1) for 22 hours at 37°C. Dose-dependent blocking of CD3 binders by anti-idiotype 4.15.64 IgG is shown. Each point represents the mean of triplicates. Standard deviations are indicated by error bars. The dotted line indicates NFAT activation in Jurkats incubated with target cells without TCB. DP47 non-targeting TCB was used as a negative control. EC50 values ​​were calculated using a nonlinear fit: "log(agonist) vs. response—variable slope (4 parameters)" (GraphPad Prism 6). (E): Blocking ability of anti-ID 4.21 IgG as measured by reduction of Jurkat NFAT activation mediated by TYRP1 TCB. Jurkat NFAT activation mediated by TYRP1 TCB with different CD3 binders is shown.TYRP1 TCB (used at the EC concentration determined in the previous assay) was incubated with TYRP1-positive target cells (CHO-huTYRP1 clone 76) and Jurkat NFAT effector cells (E:T 2.5:1) for 22 hours at 37°C. Dose-dependent blocking of CD3 binders by anti-idiotype 4.21 IgG is shown. Each point represents the mean of triplicates. Standard deviations are indicated by error bars. The dotted line indicates NFAT activation in Jurkats incubated with target cells without TCB. DP47 non-targeting TCB was used as a negative control. EC50 values ​​were calculated using a nonlinear fit: "log(agonist) vs. response—variable slope (4 parameters)" (GraphPad Prism 6). [Figure 11A] (A): Jurkat NFAT activation mediated by FOLR1 TCB or FOLR1 pro-TCB containing different CD3 binders. FOLR1 (pro-)TCB was incubated with huFOLR1-coated beads and Jurkat NFAT effector cells for 5 hours at 37°C. FOLR1 pro-TCB containing anti-ID mask 4.24.72 does not mediate Jurkat NFAT activation over the indicated concentration range. However, FOLR1 TCB mediates Jurkat NFAT activation in a dose-dependent manner. Each point represents the mean of triplicates. Standard deviation is indicated by error bars. The dotted line indicates NFAT activation in Jurkats incubated with target cells without TCB. [Figure 11B](B): After 48 hours of incubation with huPBMCs, TCB, and FOLR1-positive target cells (E:T = 10:1, effector: human PBMC), dose-dependent target cell killing (HeLa cells, which highly express FOLR1) was measured. FOLR1 TCB and activated FOLR1 pro-TCB induce dose-dependent target cell killing with an EC50 of approximately 0.29 pM. Masked FOLR1 pro-TCB (CD3 P035.093, masked 4.24.72 scFv), which contains a non-cleavable linker, reduces target cell lysis approximately 239-fold compared to the EC50 values. Each point represents the mean of triplicates. Standard deviations are indicated by error bars. The dotted line indicates spontaneous release of target cells incubated with huPBMCs without TCB. EC50 values ​​were calculated using a nonlinear fit, "log(agonist) vs. response—variable slope (4 parameters)" (GraphPad Prism 6). [Figure 11C-D](C): Dose-dependent T cell activation of CD8+ T cells was analyzed by CD69 quantification. Median CD69 fluorescence intensity was blotted for CD8+ T cells. Target cells (HeLa cells, which express highly elevated FOLR1) were incubated with huPBMCs and TCB at 37°C for 48 hours (E:T = 10:1, human PBMCs as effectors). FOLR1 TCB and activated FOLR1 pro-TCB induced T cell activation in a dose-dependent manner. Masked FOLR1 pro-TCB (CD3 P035.093, mask 4.24.72 scFv) containing a non-cleavable linker showed no T cell activation (CD69 for CD8+ T cells) within the indicated concentration range. Each point represents the mean of triplicates. Standard deviations are indicated by error bars. For calculation of EC50 values, a nonlinear fit "log(agonist) vs. response—variable slope (4 parameters)" was calculated (GraphPad Prism 6). (D): Dose-dependent T cell activation of CD8 T cells was measured by CD69 quantification. The percentage of CD69-positive CD8 T cells is shown. Target cells (HeLa cells, which highly express FOLR1) were incubated with huPBMCs and TCB at 37°C for 48 hours (E:T = 10:1, human PBMCs as effectors). FOLR1 TCB and activated FOLR1 pro-TCB induce T cell activation in a dose-dependent manner. Masked FOLR1 pro-TCB (CD3 P035.093, masked 4.24.72 scFv) containing a non-cleavable linker exhibits reduced T cell activation (CD69 for CD8 T cells) over the indicated concentration range. However, starting from 5 nM, some CD69-positive CD8 T cells were detected, increasing to approximately 30% of the highest concentration used here. Each point represents the mean of triplicates. Standard deviation is indicated by error bars. For calculation of EC50 values, a nonlinear fit "log(agonist) vs. response--variable slope (4 parameters)" was calculated (GraphPad Prism6). [Figure 12A-B](A): To analyze the masking efficiency of anti-ID 4.24.72 in the pro-TCB format with CD3 P035.093, dose-dependent target cell killing (Hela, with high FOLR1 expression; Ovcar-3 and Skov-3, with medium FOLR1 expression; and HT-29, with low FOLR1 expression) was measured after 48 h of incubation with huPBMCs. TCB and FOLR1-positive target cells (E:T = 10:1, effector: human PBMCs). FOLR1 TCB induces dose-dependent target cell killing in all cell lines (Hela, Skov-3, and Ovcar-3), while masked FOLR1 pro-TCB exhibits reduced target cell killing. (B): Dose-dependent target cell killing (Skov-3, with medium FOLR1 expression; and HT-29, with low FOLR1 expression) was measured after 48 h of incubation with huPBMCs (E:T = 10:1). FOLR1 TCB induces dose-dependent target cell killing in both cell lines (Skov-3, HT-29), whereas masked FOLR1 pro-TCB exhibits reduced target cell killing. Each point represents the mean of triplicates. Standard deviations are indicated by error bars. The dotted line indicates spontaneous release of target cells incubated with huPBMCs without TCB. For calculation of EC50 values, a nonlinear fit "log(agonist) vs. response—variable slope (4 parameters)" was calculated (GraphPad Prism 6). [Figure 13] A humanized variant of the anti-idiotype-masked 4.24.72 antibody in a one-arm IgG format. Heterodimerization is achieved using knob-into-hole technology. [Figure 14A-B]Figure 14 shows Jurkat NFAT activation mediated by TYRP1 TCB with different CD3 binders. TYRP1 TCB (used at the EC concentration determined in the previous assay) was incubated with TYRP1-positive target cells (M150543) and Jurkat NFAT effector cells (E:T 2.5:1) for 5 hours at 37°C. Dose-dependent blocking of CD3 binders by anti-idiotypic (anti-ID) 4.24.72 IgG (parental and humanized variants) is shown for CD3 CH2527 (Figure 14A) and CD3 P035.093 (Figure 14B). Each point represents the mean of triplicates. Standard deviations are indicated by error bars. For calculation of EC50 values, a nonlinear fit "log(agonist) vs. response—variable slope (4 parameters)" was calculated (GraphPad Prism 6). [Figure 15A-C](A): To analyze the masking efficiency of a humanized variant of anti-ID 4.24.72 in the pro-TCB format with CD3 P035.093, dose-dependent target cell killing (Ovcar-3 cells expressing FOLR1) was measured after 48 hours of incubation with huPBMCs. TCB and FOLR1-positive target cells (E:T = 10:1, effector: human PBMCs). FOLR1 TCB induces dose-dependent target cell killing in Ovcar-3 cells, whereas masked FOLR1 pro-TCB exhibits reduced target cell killing. (B) and (C): Dose-dependent T cell activation of CD8 T cells was measured by quantification of CD69. The percentage of CD69-positive CD8 T cells (Figure 15B) and median fluorescence intensity (Figure 15C) are shown. Target cells (Ovcar-3 cells expressing FOLR1) were incubated with huPBMCs and TCB at 37°C for 48 hours (E:T = 10:1, effector: human PBMCs). FOLR1 TCB induces dose-dependent T cell activation. Masked FOLR1 pro-TCB (a humanized variant of the CD3 P035.093, masked 4.24.72 scFv) containing a non-cleavable linker exhibits reduced T cell activation (CD69 for CD8 T cells) over the indicated concentration range. No difference in masking efficiency was detectable for T cell activation with the humanized variants. Each point represents the mean of triplicates. Standard deviations are indicated by error bars. For EC50 calculations, a nonlinear fit "log(agonist) vs. response—variable slope (4 parameters)" was calculated (GraphPad Prism 6). [Figure 16A] Figure 16 is a schematic diagram of different protease-activatable FolR1 TCB molecules with humanized masking moieties. Figure 16A: Anti-CD3 with common light chain P035.093 masked scFv H1L1 matriptase site anti-FolR1 16D5 P329G LALA 2+1 Fc(hole) Fc(knob), SEQ ID NOs: 95, 66, 67. [Figure 16B]Figure 16B: Anti-CD3 with common light chain P035.093 masked scFv H1L2 matriptase site anti-FolR1 16D5 P329G LALA 2+1 Fc(hole) Fc(knob), SEQ ID NOs: 96, 66, 67. [Figure 16C] Figure 16C: Anti-CD3 with common light chain P035.093 masked scFv H2L2 matriptase site anti-FolR1 16D5 P329G LALA 2+1 Fc(hole) Fc(knob), SEQ ID NOs: 97, 66, 67. [Figure 16D] Figure 16D: Anti-CD3 with common light chain P035.093 masked scFv H3L2 matriptase site anti-FolR1 16D5 P329G LALA 2+1 Fc(hole) Fc(knob), SEQ ID NOs: 98, 66, 67. DETAILED DESCRIPTION OF THE INVENTION

[0059] definition Terms herein are used as commonly used in the art unless otherwise defined below.

[0060] As used herein, the term "antigen-binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and derivatives, such as fragments thereof.

[0061] The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two separate cells.

[0062] As used herein, the term "valency" indicates that a specific number of antigen-binding sites are present in an antigen-binding molecule. Thus, the term "monovalent binding to an antigen" indicates that one (and up to one) antigen-binding site specific for the antigen is present in the antigen-binding molecule.

[0063] "Antigen-binding site" refers to the site of an antigen-binding molecule, i.e., one or more amino acid residues, that interacts with an antigen. For example, the antigen-binding site of an antibody comprises amino acid residues of the complementarity-determining regions (CDRs). A naturally occurring immunoglobulin molecule typically has two antigen-binding sites, while a Fab molecule typically has a single antigen-binding site.

[0064] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In certain embodiments, an antigen-binding moiety can direct the entity to which it binds (e.g., a second antigen-binding moiety) to a target site, such as a particular type of tumor cell or tumor stroma bearing the antigenic determinant. In other embodiments, an antigen-binding moiety can activate signaling through its target antigen, e.g., a T-cell receptor complex antigen. Antigen-binding moieties include antibodies and fragments thereof, as described below. Particular antigen-binding moieties include the antigen-binding domain of an antibody, including an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, an antigen-binding moiety can include an antibody constant region, as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of the two isotypes: κ and λ.

[0065] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a contiguous stretch of amino acids or a conformational structure composed of different regions of non-contiguous amino acids) to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise specified, proteins referred to herein as antigens (e.g., FolR1, HER1, HER2, CD3, mesothelin) refer to the native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. When a particular protein is referred to herein, the term encompasses not only the "full-length," unprocessed protein, but also any form of the protein resulting from processing within the cell. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants. Examples of human proteins useful as antigens include, but are not limited to, FolR1, HER1, and CD3, particularly the epsilon subunit of CD3 (human sequence available from UniProt no. P07766 (version 130), NCBI RefSeq no. Np_000724.1, SEQ ID NO: 54; or for the cynomolgus monkey [Macaca fascicularis] sequence see UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1). In certain embodiments, the protease-activatable T cell activating bispecific molecules of the invention bind to an epitope of CD3 or a target cell antigen that is conserved among CD3 or target antigens from different species. In certain embodiments, the protease-activatable T cell activating bispecific molecules of the invention bind to CD3 and FolR1 but not FolR2 or FolR3.In certain embodiments, the protease-activatable T cell-activating bispecific molecules of the invention bind to CD3 and HER1. In certain embodiments, the protease-activatable T cell-activating bispecific molecules of the invention bind to CD3 and mesothelin. In certain embodiments, the protease-activatable T cell-activating bispecific molecules of the invention bind to CD3 and HER2. "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding moiety to bind to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) technology (analysis on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and classical binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In some embodiments, the extent of binding of the antigen-binding portion to an unrelated protein is less than about 10% of the binding of the antigen-binding portion to the antigen, as measured, for example, by SPR. In certain embodiments, the antigen-binding portion, or an antigen-binding molecule comprising the antigen-binding portion, that binds to the antigen has a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M to 10 -13 M, e.g. 10 -9 M to 10 -13 Dissociation constant (K D )

[0066] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise specified, "binding affinity," as used herein, refers to the intrinsic binding affinity, which reflects the one-to-one interaction between members of a binding pair (e.g., an antigen-binding site and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y is typically measured by the dissociation constant (K D ) and this dissociation constant (K D) are the dissociation rate constant and the association rate constant (k off and k on ) is the ratio of the rate constants. Thus, equivalent affinities may involve different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by established methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0067] "Decreased binding," e.g., reduced binding to an Fc receptor, refers to a decrease in the affinity of the respective interaction, as measured, e.g., by SPR. For clarity, the term also includes a decrease in affinity to zero (or below the detection limit of the analytical method), i.e., a complete cessation of the interaction. Conversely, "increased binding" refers to an increase in the binding affinity of the respective interaction.

[0068] As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. The protease-activatable T cell activating bispecific molecules of the invention are capable of inducing T cell activation. Suitable assays for measuring T cell activation are known in the art and described herein.

[0069] "Target cell antigen," as used herein, refers to an antigenic determinant displayed on the surface of a target cell, e.g., a cell within a tumor, such as a cancer cell or a cell of the tumor stroma.

[0070] As used herein, the terms "first" and "second," with respect to antigen-binding moieties, etc., are used for convenience to distinguish when there is more than one of each type of moiety. The use of these terms is not intended to confer a particular order or orientation of the protease-activatable T cell activating bispecific molecules, unless explicitly stated.

[0071] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of an immunoglobulin light chain (a "Fab light chain").

[0072] By "fused" is meant that the components (e.g., a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0073] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen-binding moieties is a single-chain Fab molecule, i.e., a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In certain such embodiments, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0074] A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which the variable regions or constant regions of the Fab heavy and light chains have been swapped; i.e., the crossover Fab molecule comprises a peptide chain consisting of a light chain variable region and a heavy chain constant region, and a peptide chain consisting of a heavy chain variable region and a light chain constant region. For clarity, in a crossover Fab molecule in which the variable domain of a Fab light chain and the variable domain of a Fab heavy chain have been swapped, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a crossover Fab molecule in which the constant domain of a Fab light chain and the constant domain of a Fab heavy chain have been swapped, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0075] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native format, i.e., a Fab molecule comprising a heavy chain composed of a heavy chain variable and constant region (VH-CH1) and a light chain composed of a light chain variable and constant region (VL-CL).

[0076] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region. Immunoglobulin heavy chains can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and some of these types can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain connected via an immunoglobulin hinge region.

[0077] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0078] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab'), diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). See also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. See U.S. Patent No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have increased in vivo half-lives. "Diabodies" are antibody fragments that have two antigen-binding sites and may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med. 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med. 9, 129-134 (2003). Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Waltham, Massachusetts; see, e.g., U.S. Patent No. 6,248,516). Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0079] The term "antigen-binding domain" refers to a portion of an antibody comprising an area that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain may be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). In particular, an antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0080] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of a natural antibody generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6 th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain is sufficient to confer antigen-binding specificity.

[0081] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop ("hypervariable loop"). Naturally occurring four-chain antibodies generally contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Generally, HVRs contain amino acid residues from the hypervariable loops and / or complementarity-determining regions (CDRs), the latter of which are most highly sequence variable and / or involved in antigen recognition. With the exception of CDR1 in the VH, CDRs generally contain amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity-determining regions" (CDRs), and these terms are used interchangeably herein with respect to the portions of the variable domain that form the antigen-binding region. This particular region is described in Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J Mol Biol 196:901-917 (1987), and the definitions therein include overlapping or subsets of amino acid residues when compared with each other. However, application of either definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs defined by each of the above references are set forth in Table 1 below for comparison. The exact residue numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR. One of ordinary skill in the art can routinely determine which residues comprise a particular CDR, given the amino acid sequence of the variable region of an antibody.

[0082] Table 1. CDR definitions 1 JPEG0007815150000001.jpg41170 1 All numbering of the CDR definitions in Table 1 follows the numbering convention established by Kabat et al. (see below). 2As used in Table 1, "AbM" with a lowercase "b" refers to the CDRs as defined by Oxford Molecular's "AbM" antibody modeling software.

[0083] Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system defined by Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in antibody variable regions are in accordance with the Kabat numbering system.

[0084] The polypeptide sequences in the sequence listing have not been numbered according to the Kabat numbering system, however, it is well within the ordinary skill of one in the art to convert the numbering of the sequences in the sequence listing to Kabat numbering.

[0085] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. Typically, the FR of a variable domain consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0086] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by the antibody or immunoglobulin heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0087] The term "Fc region" is used herein to refer to the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term encompasses native-sequence Fc regions and variant Fc regions. In certain embodiments, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Thus, antibodies produced by host cells by expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain the full-length heavy chain or a truncated variant of the full-length heavy chain. This is true when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. The amino acid sequence of a heavy chain comprising an Fc region is shown herein without the C-terminal glycine-lysine dipeptide unless otherwise specified. In certain embodiments, a heavy chain comprising an Fc region as specified herein and comprised in an antibody according to the invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In certain embodiments, a heavy chain comprising an Fc region as specified herein and comprised in an antibody according to the invention comprises an additional C-terminal glycine residue (G446, EU index numbering). Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system (also known as the EU index) as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide that comprises the C-terminal constant region of an immunoglobulin heavy chain and has the ability to stably self-associate.For example, the subunits of the IgG Fc domain include the IgG CH2 and IgG CH3 constant domains.

[0088] By "fused" is meant that the components (e.g., a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0089] A "modification that promotes association between a first subunit and a second subunit of an Fc domain" refers to a peptide backbone modification or a post-translational modification of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising an Fc domain subunit with an identical polypeptide to form a homodimer. As used herein, a modification that promotes association specifically includes separate modifications made to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, where these modifications are complementary to each other to promote the association of the two Fc domain subunits. For example, a modification that promotes association can alter the structure or charge of one or both of these Fc domain subunits to favor their association sterically or electrostatically, respectively. Thus, (hetero)dimerization can occur between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, and these subunits may not be identical, in the sense that the additional components (e.g., antigen-binding moieties) fused to each of these subunits may not be the same. In some embodiments, the modifications that promote association comprise amino acid mutations, particularly amino acid substitutions, within the Fc domain, hi certain embodiments, the modifications that promote association comprise separate amino acid mutations, particularly amino acid substitutions, in each of the two subunits of the Fc domain.

[0090] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody and varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0091] As used herein, the terms "engineer, engineered, engineering" are intended to include any alteration of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Modifications include modifications of the amino acid sequence, glycosylation pattern, or side groups of individual amino acids, as well as combinations of these techniques.

[0092] As used herein, the term "amino acid mutation" is intended to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., reduced binding to Fc receptors or increased association with another peptide). Amino acid sequence deletions and insertions include amino- and / or carboxy-terminal deletions and amino acid insertions. In particular, amino acid mutations are amino acid substitutions. For example, to alter the binding characteristics of the Fc region, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the 20 common amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is believed that methods other than genetic engineering, such as changing the side chain group of amino acids by chemical modification, may also be useful.In this specification, various notations are used to indicate the same amino acid mutation.For example, the substitution of proline to glycine at position 329 of the Fc domain is represented by 329G, G329, G 329 , P329G or Pro329Gly.

[0093] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amino bonds (also known as peptide bonds). The term "polypeptide" refers to a chain of two or more amino acids, not a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term referring to a chain of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be obtained from natural biological sources or produced by recombinant technology, but are not necessarily translated from a designated nucleic acid sequence. Polypeptides can be produced by any means, including chemical synthesis. Polypeptides of the invention are of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids. Polypeptides may have a defined three-dimensional structure, but do not necessarily have such a structure. Polypeptides that have a defined three-dimensional structure are said to be "folded," while polypeptides that do not have a defined three-dimensional structure and can adopt a number of different conformations are said to be "unfolded."

[0094] An "isolated" polypeptide or variant or derivative thereof refers to a polypeptide that is not in its natural environment. Purification is not required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the present invention, as are naturally occurring or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0095] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be obtained by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including the algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. However, as used herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office (Washington, DC 20559), where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, CA), or can be compiled from its source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparisons, the percent amino acid sequence identity of a given amino acid sequence A with (or relative to) a given amino acid sequence B (alternatively, given amino acid sequence A can be said to have or contain a particular percent amino acid sequence identity with (or relative to) a given amino acid sequence B) is calculated as follows: Fraction X / Y x 100 where X is the number of amino acid residues scored as perfect matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be recognized that if the length of amino acid sequence A differs from the length of amino acid sequence B, the % amino acid sequence identity of A to B will differ from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0096] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral-derived RNA, or plasmid DNA (pDNA). A polynucleotide can contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0097] An "isolated" nucleic acid molecule or polynucleotide is a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain the polynucleotide molecule, but where the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its native chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive-stranded, negative-stranded, and double-stranded forms. Isolated polynucleotides or nucleic acids of the present invention further include such molecules produced synthetically. In addition, polynucleotides or nucleic acids may or may not contain regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0098] A nucleic acid or polynucleotide having, for example, a nucleotide sequence at least 95% "identical" to a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the nucleotide sequence of the polynucleotide may contain up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. Such alterations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between these terminal positions, interspersed individually among residues in the reference sequence, or interspersed in one or more contiguous groups within the reference sequence. In practice, whether a particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be routinely determined using known computer programs such as those described above for polypeptides (e.g., ALIGN-2).

[0099] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide with a set of specific nucleic acid elements that allow transcription of a specific nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector contains, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In a specific embodiment, an expression cassette of the invention comprises a polynucleotide sequence encoding a bispecific antigen-binding molecule of the invention, or a fragment thereof.

[0100] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce and direct the expression of a specific gene with which it is operably associated in a target cell. This term encompasses not only vectors integrated into the genome of the host cell into which it is introduced, but also vectors as self-replicating nucleic acid structures. The expression vector of the present invention comprises an expression cassette. The expression vector is capable of transcribing large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence or a fragment thereof encoding a bispecific antigen-binding molecule of the present invention.

[0101] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. Host cells are any type of cell line that can be used to produce the bispecific antigen-binding molecules of the invention. Host cells include cultured cells, such as cultured mammalian cells such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, and PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, to name a few, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0102] An "activating Fc receptor" is an Fc receptor that, upon binding to the Fc domain of an antibody, initiates a signal transduction event that stimulates receptor-bearing cells to exert effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0103] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. These target cells are cells to which an antibody or its derivative containing an Fc region specifically binds, usually via a protein portion located at the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as either a decrease in the number of target cells lysed by the ADCC mechanism defined above at a given antibody concentration in the medium surrounding the target cells within a given time period and / or an increase in the antibody concentration in the medium surrounding the target cells required to achieve lysis of a given number of target cells within a given time period by the ADCC mechanism. Reduced ADCC is compared to ADCC mediated by the same unmodified antibody produced by the same type of host cell using the same standard manufacturing, purification, formulation, and storage methods (known to those skilled in the art). For example, reduced ADCC mediated by an antibody containing an ADCC-reducing amino acid substitution in the Fc domain is compared to ADCC mediated by the same antibody without such amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, WO 2006 / 082515 or WO 2012 / 130831).

[0104] An "effective amount" of a drug is the amount necessary to produce a physiological change in the cells or tissue to which it is administered.

[0105] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent may, for example, eliminate, reduce, delay, minimize, or prevent the adverse effects of a disease.

[0106] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice, rats). In particular, the individual or subject is a human.

[0107] The term "pharmaceutical composition" refers to a preparation that is in a form that effectively potentiates the biological activity of the active ingredient contained therein and that does not contain any additional ingredients that are unacceptably toxic to the subject to whom the formulation will be administered.

[0108] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0109] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of disease in the individual being treated and may be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, diminishing the direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, remission or palliation of disease symptoms, and remission or improved prognosis. In some embodiments, the protease-activatable T cell activating bispecific molecules of the invention are used to delay the onset of disease or slow the progression of disease.

[0110] The term "package insert" is used to refer to instructions customarily included in commercial packaging of a therapeutic product, which contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or precautions regarding the use of such therapeutic product.

[0111] As used herein, an "idiotype-specific polypeptide" refers to a polypeptide that recognizes the idiotype of an antigen-binding moiety (e.g., an antigen-binding moiety for CD3). An idiotype-specific polypeptide can specifically bind to the variable region of an antigen-binding moiety, thereby reducing or preventing specific binding of the antigen-binding moiety to its cognate antigen. When associated with a molecule that contains an antigen-binding moiety, an idiotype-specific polypeptide can function as a masking moiety of the molecule. Specifically disclosed herein are anti-idiotype antibodies or anti-idiotype-binding antibody fragments specific for the idiotype of an anti-CD3 binding molecule.

[0112] As used herein, "protease" or "protease" refers to any proteolytic enzyme expressed by a target cell that cleaves a linker at a recognition site. Such proteases may be secreted by the target cell or remain associated with the target cell (e.g., on the target cell surface). Examples of proteases include, but are not limited to, metalloproteinases, such as matrix metalloproteinases 1-28, and A Disintegrin and Metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30, and 33; serine proteases, such as urokinase-type plasminogen activator and matriptase; cysteine ​​proteases; aspartic acid proteases; and members of the cathepsin family.

[0113] "Protease-activatable," as used herein with respect to a T cell activating bispecific molecule, refers to a T cell activating bispecific molecule whose ability to activate T cells is reduced or eliminated by a masking moiety that reduces or eliminates the ability of the T cell activating bispecific molecule to bind to CD3. Upon release of the masking moiety by proteolytic cleavage (e.g., by proteolytic cleavage of a linker connecting the masking moiety and the T cell activating bispecific molecule), binding to CD3 is restored, thereby activating the T cell activating bispecific molecule.

[0114] As used herein, "reversibly masking" refers to attaching a masking moiety or idiotype-specific polypeptide to an antigen-binding moiety or molecule such that the antigen-binding moiety or molecule is masked from its antigen, e.g., CD3. This masking is reversible in that the idiotype-specific polypeptide can be released from the antigen-binding moiety or molecule, e.g., by protease cleavage, thereby freeing the antigen-binding moiety or molecule to bind to its antigen.

[0115] Detailed Description In one aspect, the present invention provides a protease-activatable T cell activating bispecific molecule comprising: (a) a first antigen-binding moiety capable of binding to CD3; (b) a second antigen-binding moiety capable of binding to a target cell antigen; and (c) a masking moiety covalently attached to the T cell bispecific binding molecule via a protease-cleavable linker, the masking moiety being capable of binding to the idiotype of the first or second antigen-binding moiety, thereby reversibly masking the first or second antigen-binding moiety. The present invention relates to a protease-activatable T cell activating bispecific molecule comprising:

[0116] The first antigen-binding moiety capable of binding to CD3 comprises an idiotype. In one embodiment, the masking moiety of the protease-activatable T cell activating bispecific molecule is covalently linked to the first antigen-binding moiety. In one embodiment, the masking moiety is covalently linked to the heavy chain variable region of the first antigen-binding moiety. In one embodiment, the masking moiety is covalently linked to the light chain variable region of the first antigen-binding moiety. This covalent linkage is separate from the specific binding (preferably non-covalent binding) of the masking moiety to the first antigen-binding site of the idiotype. The idiotype of the first antigen-binding moiety comprises its variable region. In one embodiment, the masking moiety binds to amino acid residues that contact CD3 when the first antigen-binding moiety binds to CD3. In a preferred embodiment, the masking moiety is not the cognate antigen of the first antigen-binding moiety or a fragment thereof, i.e., the masking moiety is not CD3 or a fragment thereof. In one embodiment, the masking moiety is an anti-idiotype antibody or a fragment thereof. In one embodiment, the masking moiety is an anti-idiotype scFv. Exemplary embodiments of masking moieties that are anti-idiotypic scFvs and protease-activatable T cell activation molecules comprising such masking moieties are detailed in the Examples.

[0117] In one embodiment, the protease activatable T cell activating bispecific molecule comprises: (i) a first antigen-binding portion that is a Fab molecule capable of binding to CD3, the first antigen-binding portion comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:10, and at least one light chain CDR selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; and (ii) a second antigen-binding portion that is a Fab molecule capable of binding to a target cell antigen. Includes.

[0118] In one embodiment, the first antigen-binding portion comprises a heavy chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16, and a light chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0119] In certain embodiments, the first antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23.

[0120] In certain embodiments, the second antigen-binding portion is capable of binding to FolR1 and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, and at least one light chain CDR selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

[0121] In another specific embodiment, the second antigen-binding portion is capable of binding to FolR1 and comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0122] In another specific embodiment, the second antigen-binding portion is capable of binding to TYRP1 and comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26 and at least one light chain CDR selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.

[0123] In another specific embodiment, the second antigen-binding portion is capable of binding to TYRP1 and comprises a heavy chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27, and a light chain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31.

[0124] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising: (i) a first antigen-binding portion that is a Fab molecule capable of binding to CD3, the first antigen-binding portion comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:10, and at least one light chain CDR selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; and (ii) a second antigen-binding portion that is a Fab molecule capable of binding to FolR1, the second antigen-binding portion comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, and at least one light chain CDR selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

[0023] In one aspect, a protease-activatable T cell activating bispecific molecule is provided, comprising:

[0125] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising: (i) a first antigen-binding portion that is a Fab molecule capable of binding to CD3, the first antigen-binding portion comprising a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23; and (ii) a second antigen-binding portion that is a Fab molecule capable of binding to FolR1, the second antigen-binding portion comprising a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0023] In one aspect, a protease-activatable T cell activating bispecific molecule is provided, comprising:

[0126] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising: (i) a first antigen-binding portion that is a Fab molecule capable of binding to CD3, the first antigen-binding portion comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:10, and at least one light chain CDR selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; and (ii) a second antigen-binding portion that is a Fab molecule capable of binding to TYRP1, the second antigen-binding portion comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, and at least one light chain CDR selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0023] In one aspect, a protease-activatable T cell activating bispecific molecule is provided, comprising:

[0127] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising: (i) a first antigen-binding portion that is a Fab molecule capable of binding to CD3, the first antigen-binding portion comprising a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23; and (ii) a second antigen-binding portion that is a Fab molecule capable of binding to TYRP1, the second antigen-binding portion comprising a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31.

[0023] In one aspect, a protease-activatable T cell activating bispecific molecule is provided, comprising:

[0128] In one embodiment, the second antigen-binding moiety is a conventional Fab molecule.

[0129] In certain embodiments, the first antigen-binding moiety is a crossover Fab molecule in which the constant regions of the Fab light chain and the Fab heavy chain have been swapped, and the second antigen-binding moiety is a conventional Fab molecule. In further certain embodiments, the first and second antigen-binding moieties are fused to each other, optionally via a peptide linker.

[0130] In certain embodiments, the protease activatable T cell activating bispecific molecule further comprises an Fc domain comprised of a first and a second subunit capable of stable association.

[0131] In a further specific embodiment, no more than one antigen-binding moiety capable of binding to CD3 is present in the protease-activatable T cell activating bispecific molecule (i.e., the protease-activatable T cell activating bispecific molecule provides monovalent binding to CD3).

[0132] Protease-activatable T cell-activating bispecific molecule format The components of the protease-activatable T cell activating bispecific molecules can be fused to each other in a variety of configurations, with exemplary configurations shown in Figures 1A-1Z, 2, 9A-9C, and 17A-17DH.

[0133] In certain embodiments, the protease-activatable T cell activating bispecific molecule comprises an Fc domain composed of first and second subunits capable of stable association, hi some embodiments, the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

[0134] In such embodiments, the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety. In certain such embodiments, the protease-activatable T cell activating bispecific molecule consists essentially of first and second antigen-binding moieties, an Fc domain composed of first and second subunits, and optionally one or more peptide linkers, wherein the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Optionally, the Fab light chain of the first antigen-binding moiety and the Fab light chain of the second antigen-binding moiety may further be fused to each other.

[0135] In another such embodiment, the first antigen-binding moiety is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. In certain such embodiments, the protease-activatable T cell activating bispecific molecule consists essentially of the first and second antigen-binding moieties, an Fc domain comprised of the first and second subunits, and optionally one or more peptide linkers, wherein the first and second antigen-binding moieties are each fused to the N-terminus of one subunit of the Fc domain at the C-terminus of the Fab heavy chain.

[0136] In other embodiments, the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

[0137] In certain such embodiments, the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain. In certain such embodiments, the protease-activatable T cell activating bispecific molecule consists essentially of first and second antigen-binding moieties, an Fc domain composed of first and second subunits, and optionally one or more peptide linkers, wherein the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain, and the first antigen-binding moiety is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. Optionally, the Fab light chain of the first antigen-binding moiety and the Fab light chain of the second antigen-binding moiety may further be fused to each other.

[0138] The antigen-binding moieties may be fused to the Fc domain, either directly or via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or 4 (SG4) n Peptide linkers are included. "n" is generally an integer between 1 and 10, typically between 2 and 4. A particularly suitable peptide linker for fusing the first and second antigen-binding moieties to each other is (G4S)2. An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and second antigen-binding moieties is EPKSC(D)-(G4S)2 (SEQ ID NOs: 105 and 106). In addition, the linker can comprise (a portion of) an immunoglobulin hinge region. In particular, when the antigen-binding moiety is fused to the N-terminus of the Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

[0139] Protease-activatable T cell activating bispecific molecules having a single antigen-binding moiety capable of binding to a target cell antigen are useful, particularly when internalization of the target cell antigen is expected following binding of the high-affinity antigen-binding moiety. In such cases, the presence of more than one antigen-binding moiety specific for the target cell antigen may promote internalization of the target cell antigen, thereby reducing its availability.

[0140] However, in many other cases, it will be advantageous to have a T cell-activating bispecific antigen-binding molecule that comprises two or more antigen-binding moieties specific for target cell antigens (see examples shown in Figures 1B, 1C, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, 1Q, 1R, 1U, 1V), e.g., to optimize targeting to the target site or to allow cross-linking of target cell antigens.

[0141] Thus, in certain embodiments, the protease-activatable T cell activating bispecific molecules of the invention further comprise a third antigen-binding moiety capable of binding to a target cell antigen. In some embodiments, the third antigen-binding moiety is a conventional Fab molecule. In some embodiments, the third antigen-binding moiety is capable of binding to the same target cell antigen as the second original binding moiety. In certain embodiments, the first antigen-binding moiety is capable of binding to CD3, and the second and third antigen-binding moieties are capable of binding to the target cell antigen. In certain embodiments, the second and third antigen-binding moieties are identical (i.e., they comprise the same amino acid sequence).

[0142] In a specific embodiment, the first antigen-binding portion is capable of binding to CD3, and the second and third antigen-binding portions are capable of binding to FolR1, wherein the second and third antigen-binding portions comprise at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56, and at least one light chain CDR selected from the group of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0143] In certain embodiments, the first antigen-binding portion is capable of binding to CD3 and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:10, and at least one light chain CDR selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; and the second and third antigen-binding portions are capable of binding to FolR1, wherein the second and third antigen-binding portions comprise at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56, and at least one light chain CDR selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0144] In certain embodiments, the first antigen-binding portion is capable of binding to CD3 and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:10, and at least one light chain CDR selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; and the second and third antigen-binding portions are capable of binding to FolR1, wherein the second and third antigen-binding portions comprise at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56, and at least one light chain CDR selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0145] In a specific embodiment, the first antigen-binding portion is capable of binding to CD3 and comprises a heavy chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23; and the second and third antigen-binding portions are capable of binding to FolR1, wherein the second and third antigen-binding portions comprise a heavy chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53 and a light chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0146] In one embodiment, the first antigen binding portion is capable of binding to CD3, and the second and third antigen binding portions are capable of binding to TYRP1, wherein the second and third antigen binding portions comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:10, and at least one light chain CDR selected from the group of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0147] In one embodiment, the first antigen binding portion is capable of binding to CD3 and comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:10, and at least one light chain CDR selected from the group of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; and the second and third antigen binding portions are capable of binding to TYRP1, wherein the second and third antigen binding portions comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, and at least one light chain CDR selected from the group of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0148] In one embodiment, the first antigen binding portion is capable of binding to CD3 and comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:10, and at least one light chain CDR selected from the group of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; and the second and third antigen binding portions are capable of binding to TYRP1, wherein the second and third antigen binding portions comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, and at least one light chain CDR selected from the group of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0149] In one embodiment, the first antigen-binding portion is capable of binding to CD3 and comprises a heavy chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23; and the second and third antigen-binding portions are capable of binding to TYRP1, wherein the second and third antigen-binding portions comprise a heavy chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31.

[0150] The second and third antigen-binding moieties may be fused directly or via a peptide linker to the Fc domain. In certain embodiments, the second and third antigen-binding moieties are each fused to the Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region. In some embodiments, the second and third antigen-binding moieties and the Fc domain are part of an immunoglobulin molecule. In certain embodiments, the immunoglobulin molecule is an IgG class immunoglobulin. In a more specific embodiment, the immunoglobulin is an IgG1 subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG4 subclass immunoglobulin. In a more specific embodiment, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the protease-activatable T cell activating bispecific molecule consists essentially of an immunoglobulin molecule capable of binding to a target cell antigen and an antigen-binding portion capable of binding to CD3, where the antigen-binding portion is a Fab molecule fused (optionally via a peptide linker) to the N-terminus of one immunoglobulin heavy chain.

[0151] In certain embodiments, the first and third antigen-binding moieties are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety. In certain embodiments, the protease-activatable T cell activating bispecific molecule consists essentially of first, second, and third antigen-binding moieties, an Fc domain comprised of the first and second subunits, and optionally one or more peptide linkers, wherein the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety, the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Optionally, the Fab light chain of the first antigen-binding moiety and the Fab light chain of the second antigen-binding moiety may further be fused to each other.

[0152] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising: (i) a first antigen-binding moiety that is a Fab molecule capable of binding to CD3, comprising a heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 2, a heavy chain CDR 2 of SEQ ID NO: 4, a heavy chain CDR 3 of SEQ ID NO: 10, a light chain CDR 1 of SEQ ID NO: 20, a light chain CDR 2 of SEQ ID NO: 21, and a light chain CDR 3 of SEQ ID NO: 22, wherein the first antigen-binding moiety is a crossover Fab molecule in which the variable or constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain have been exchanged; (ii) second and third antigen-binding portions, each of which is a Fab molecule capable of binding to FolR1, comprising a heavy chain CDR1 of SEQ ID NO: 54, a heavy chain CDR2 of SEQ ID NO: 55, a heavy chain CDR3 of SEQ ID NO: 56, a light chain CDR1 of SEQ ID NO: 20, a light chain CDR2 of SEQ ID NO: 21, and a light chain CDR3 of SEQ ID NO: 22;

[0023] In one aspect, a protease-activatable T cell activating bispecific molecule is provided, comprising:

[0153] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising: (i) a first antigen-binding moiety that is a Fab molecule capable of binding to CD3, comprising a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, wherein the first antigen-binding moiety is a crossover Fab molecule in which the variable or constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain have been exchanged; (ii) second and third antigen-binding portions, each of which is a Fab molecule capable of binding to FolR1, comprising a heavy chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53, and a light chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23;

[0023] In one aspect, a protease-activatable T cell activating bispecific molecule is provided, comprising:

[0154] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising: (i) a first antigen-binding moiety that is a Fab molecule capable of binding to CD3, comprising a heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 2, a heavy chain CDR 2 of SEQ ID NO: 4, a heavy chain CDR 3 of SEQ ID NO: 10, a light chain CDR 1 of SEQ ID NO: 20, a light chain CDR 2 of SEQ ID NO: 21, and a light chain CDR 3 of SEQ ID NO: 22, wherein the first antigen-binding moiety is a crossover Fab molecule in which the variable or constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain have been exchanged; (ii) second and third antigen-binding portions, each of which is a Fab molecule capable of binding to TYRP1, comprising a heavy chain CDR1 of SEQ ID NO: 24, a heavy chain CDR2 of SEQ ID NO: 25, a heavy chain CDR3 of SEQ ID NO: 26, a light chain CDR1 of SEQ ID NO: 28, a light chain CDR2 of SEQ ID NO: 29, and a light chain CDR3 of SEQ ID NO: 30;

[0023] In one aspect, a protease-activatable T cell activating bispecific molecule is provided, comprising:

[0155] The protease activatable T cell activating bispecific molecule according to any of the ten embodiments above may further comprise (iii) an Fc domain composed of first and second subunits capable of stably associating, wherein the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety, the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0156] In some of the protease-activatable T cell activating bispecific molecules of the invention, the Fab light chain of the first antigen-binding moiety and the Fab light chain of the second antigen-binding moiety are fused to each other, optionally via a peptide linker. Depending on the configuration of the first and second antigen-binding moieties, the Fab light chain of the first antigen-binding moiety may be fused at its C-terminus to the N-terminus of the Fab light chain of the second antigen-binding moiety, or the Fab light chain of the second antigen-binding moiety may be fused at its C-terminus to the N-terminus of the Fab light chain of the first antigen-binding moiety. Fusing the Fab light chains of the first and second Fab molecules further reduces mispairing of mismatched Fab heavy and light chains and also reduces the number of plasmids required to express some of the protease-activatable T cell activating bispecific molecules of the invention.

[0157] In certain embodiments, the protease-activatable T cell activating bispecific molecule comprises a Fab light chain variable region of a first antigen-binding moiety that shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the first antigen-binding moiety (i.e., the first antigen-binding moiety comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region), which in turn is linked to a polypeptide that shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (1) -CH1 (1) -CH2-CH3(-CH4)) and a polypeptide in which the Fab heavy chain of the second antigen-binding portion shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) In some embodiments, the protease activatable T cell activating bispecific molecule comprises a Fab heavy chain variable region of the first antigen binding moiety that has a carboxy-terminal peptide bond and a Fab light chain constant region (VH (1) -CL (1) ) and the Fab light chain polypeptide (VL (2) -CL (2) ) In certain embodiments, these polypeptides are covalently linked, for example, by a disulfide bond.

[0158] In an alternative embodiment, the protease-activatable T cell activating bispecific molecule comprises a Fab heavy chain variable region of a first antigen-binding moiety that shares a carboxy-terminal peptide bond with a Fab light chain constant region of the first antigen-binding moiety (i.e., the first antigen-binding moiety comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced with a light chain constant region), which in turn is linked to a polypeptide that shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CL (1) -CH2-CH3(-CH4)) and a polypeptide in which the Fab heavy chain of the second antigen-binding portion shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2)In some embodiments, the protease activatable T cell activating bispecific molecule comprises a polypeptide in which the Fab light chain variable region of the first antigen binding moiety shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first antigen binding moiety (VL (1) -CH1 (1) ) and a Fab light chain polypeptide (VL) consisting of a second antigen-binding portion. (2) -CL (2) In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0159] In some embodiments, the protease activatable T cell activating bispecific molecule comprises a polypeptide in which the Fab light chain variable region of a first antigen-binding moiety shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first antigen-binding moiety (i.e., the first antigen-binding moiety comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a second antigen-binding moiety, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (1) -CH1 (1) -VH (2) -CH1 (2) In other embodiments, the protease activatable T cell activating bispecific molecule comprises a polypeptide (VH) in which the Fab heavy chain variable region of the first antigen-binding moiety shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first antigen-binding moiety (i.e., the first antigen-binding moiety comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second antigen-binding moiety, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CL (1) -VH (2) -CH1 (2)In yet another embodiment, the protease-activatable T cell activating bispecific molecule comprises a polypeptide (VH) in which the Fab heavy chain of the second antigen-binding moiety shares a carboxy-terminal peptide bond with a Fab light chain variable region of the first antigen-binding moiety, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the first antigen-binding moiety (i.e., the first antigen-binding moiety comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) -VL (1) -CH1 (1) In other embodiments, the protease-activatable T cell activating bispecific molecule comprises a polypeptide (VH) in which the Fab heavy chain of the second antigen-binding moiety shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of the first antigen-binding moiety, which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region of the first antigen-binding moiety (i.e., the first antigen-binding moiety comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) -VH (1) -CL (1) -CH2-CH3(-CH4)).

[0160] In some of these embodiments, the protease-activatable T cell activating bispecific molecule comprises a crossover Fab light chain polypeptide (VH) of a first antigen-binding moiety, in which the Fab heavy chain variable region of the first antigen-binding moiety shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first antigen-binding moiety. (1) -CL (1) ) and a Fab light chain polypeptide (VL (2) -CL (2)In other of these embodiments, the protease-activatable T cell activating bispecific molecule further comprises a crossover Fab light chain polypeptide (VL) in which the Fab light chain variable region of the first antigen-binding moiety shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first antigen-binding moiety. (1) -CH1 (1) ) and a Fab light chain polypeptide (VL (2) -CL (2) In still other of these embodiments, the protease activatable T cell activating bispecific molecule further comprises a polypeptide (VL) in which the Fab light chain variable region of the first antigen-binding moiety shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the first antigen-binding moiety, which in turn shares a carboxy-terminal peptide bond with a Fab light chain polypeptide of the second antigen-binding moiety. (1) -CH1 (1) -VL (2) -CL (2) ) polypeptide, a polypeptide (VH) in which the Fab heavy chain variable region of a first antigen-binding portion shares a carboxy-terminal peptide bond with the Fab light chain constant region of a first antigen-binding portion, which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of a second antigen-binding portion. (1) -CL (1) -VL (2) -CL (2) ), a polypeptide in which the Fab light chain polypeptide of the second antigen-binding portion shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first antigen-binding portion, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first antigen-binding portion (VL (2) -CL (2) -VL (1) -CH1 (1) ), or a polypeptide in which the Fab light chain polypeptide of the second antigen-binding moiety shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first antigen-binding moiety, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first antigen-binding moiety (VL (2) -CL (2) -VH (1) -CL (1) ) further included.

[0161] The protease-activatable T cell activating bispecific molecules according to these embodiments comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of the third antigen-binding moiety shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third antigen-binding portion (VL (3) -CL (3) In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0162] In accordance with these above embodiments, the components of the protease-activatable T cell activating bispecific molecules (e.g., antigen-binding portions, Fc domains) may be fused directly or via various linkers, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, as described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or G4 (SG4) n A peptide linker is included, where n is usually a number from 1 to 10, typically from 2 to 4.

[0163] Fc domain The Fc domain of a protease-activatable T cell activating bispecific molecule consists of a pair of polypeptide chains comprising the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises a CH2 IgG heavy chain constant domain and a CH3 IgG heavy chain constant domain. The two subunits of the Fc domain can stably associate with each other. In one embodiment, a protease-activatable T cell activating bispecific molecule of the invention comprises up to one Fc domain.

[0164] In an embodiment according to the invention, the Fc domain of the protease-activatable T cell activating bispecific molecule is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more particular aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P. This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment, the Fc domain is human.

[0165] Fc domain modifications that promote heterodimerization The protease-activatable T cell activating bispecific molecules according to the invention comprise two subunits of the Fc domain, and therefore the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. To improve the yield and purity of the protease-activatable T cell activating bispecific molecules in recombinant production, it is advantageous to introduce modifications into the Fc domain of the protease-activatable T cell activating bispecific molecule that promote the association of the desired polypeptides.

[0166] Thus, in certain embodiments, the protease-activatable T cell activating bispecific molecules according to the invention comprise a modification that promotes the association of the first and second subunits of the Fc domain. The most extensive site of protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is within the CH3 domain of the Fc domain.

[0167] In a specific embodiment, the modification is a so-called "knob-into-hole" modification, comprising a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0168] This knob-into-hole technique is described, for example, in U.S. Pat. No. 5,731,168 and U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, positioning the protrusion within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0169] Thus, in certain embodiments, in the CH3 domain of a first subunit of the Fc domain of the protease-activatable T cell activating bispecific molecule, amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be accommodated in a cavity in the CH3 domain of the second subunit, and in the CH3 domain of a second subunit of the Fc domain, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit that can accommodate the protrusion in the CH3 domain of the first subunit.

[0170] The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or peptide synthesis.

[0171] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is additionally replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0172] In yet a further embodiment, the serine residue at position 354 in the first subunit of the Fc domain is replaced with a cysteine ​​residue (S354C), and the tyrosine residue at position 349 in the second subunit of the Fc domain is replaced with a serine residue (Y349C). Introduction of these two cysteine ​​residues allows for the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0173] In certain embodiments, the antigen-binding moiety capable of binding to CD3 is fused (optionally via an antigen-binding moiety capable of binding to a target cell antigen) to the first subunit (comprising a "knob" modification) of the Fc domain. Without wishing to be bound by theory, fusing the antigen-binding moiety capable of binding to CD3 to the knob-containing subunit of the Fc domain (further) minimizes the generation of antigen-binding molecules comprising two antigen-binding moieties capable of binding to CD3 (steric clashes between the two knob-containing polypeptides).

[0174] In an alternative embodiment, the modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates an electrostatic steering effect, e.g., as described in WO 2009 / 089004. Generally, this method involves substituting one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues, such that homodimerization is electrostatically unfavorable but heterodimerization is electrostatically favored.

[0175] Fc domain modifications that reduce Fc receptor binding and / or effector function The Fc domain confers favorable pharmacokinetic properties to protease-activatable T cell-activating bispecific molecules, such as a long serum half-life and a favorable tissue-blood distribution ratio, which contribute to favorable accumulation in target tissues. However, it can also result in undesirable targeting of protease-activatable T cell-activating bispecific molecules to cells expressing Fc receptors rather than to preferred antigen-bearing cells. Furthermore, coactivation of Fc receptor signaling pathways can result in cytokine release, which, combined with the T cell-activating properties and long half-life of the antigen-binding molecule, can lead to excessive cytokine receptor activation and severe side effects upon systemic administration. Activation of immune cells other than T cells (Fc receptor-bearing) may even reduce the efficacy of protease-activatable T cell-activating bispecific molecules due to the potential for T cell destruction by, for example, NK cells.

[0176] Thus, in certain embodiments, the Fc domain of a protease activatable T cell activating bispecific molecule according to the invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to the Fc domain of a native IgG1. In such embodiments, the Fc domain (or a protease activatable T cell activating bispecific molecule comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to Fc receptors compared to a native IgG1 Fc domain (or a protease activatable T cell activating bispecific molecule comprising a native IgG1 Fc domain) and / or less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or a protease activatable T cell activating bispecific molecule comprising a native IgG1 Fc domain). In certain embodiments, the Fc domain (or a protease-activatable T cell activating bispecific molecule comprising said Fc domain) does not substantially bind to an Fc receptor and / or does not induce effector function. In a particular embodiment, the Fc receptor is an Fcγ receptor. In certain embodiments, the Fc receptor is a human Fc receptor. In certain embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly human FcγRIIIa. In certain embodiments, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In certain embodiments, the Fc domain exhibits substantially similar binding affinity for the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain.Substantially similar binding to FcRn is achieved when the Fc domain (or a protease activatable T cell activating bispecific molecule comprising said Fc domain) exhibits more than about 70%, particularly more than about 80%, and even more particularly more than about 90% of the binding affinity for FcRn of a native IgG1 Fc domain (or a protease activatable T cell activating bispecific molecule comprising a native IgG1 Fc domain).

[0177] In certain embodiments, the Fc domain is modified to reduce binding affinity to an Fc receptor and / or effector function compared to an unmodified Fc domain. In certain embodiments, the Fc domain of the protease-activatable T cell activating bispecific molecule comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In certain embodiments, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor. In certain embodiments, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments in which there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor, a combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In certain embodiments, a protease-activatable T cell-activating bispecific molecule comprising an altered Fc domain exhibits less than 20%, particularly less than 10%, and even more particularly less than 5% of the binding affinity to an Fc receptor compared to a protease-activatable T cell-activating bispecific molecule comprising an unaltered Fc domain. In certain embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In specific embodiments, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complement components, particularly C1q, is also reduced. In certain embodiments, binding affinity to the neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain for the receptor, is achieved when the Fc domain (or a protease-activatable T cell activating bispecific molecule comprising said Fc domain) exhibits greater than about 70% of the binding affinity for FcRn of the unmodified Fc domain (or a protease-activatable T cell activating bispecific molecule comprising said unmodified Fc domain). The Fc domain or a protease-activatable T cell activating bispecific molecule of the invention comprising said Fc domain may exhibit greater than about 80%, or even greater than about 90%, of such affinity. In certain embodiments, the Fc domain of the protease-activatable T cell activating bispecific molecule is modified to have a reduced effector function compared to the unmodified Fc domain. Reduced effector function can include, but is not limited to, one or more of: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In certain embodiments, the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In certain embodiments, the reduced effector function is reduced ADCC. In certain embodiments, the reduced ADCC is less than 20% of the ADCC induced by an unmodified Fc domain (or a protease-activatable T cell-activating bispecific molecule comprising an unmodified Fc domain).

[0178] In certain embodiments, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or the effector function is an amino acid substitution. In certain embodiments, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329. In more specific embodiments, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235, and P329. In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A. In such embodiments, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In certain embodiments, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G. In certain embodiments, the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297, and P331. In more specific embodiments, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In particular embodiments, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235. In even more particular embodiments, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). In such embodiments, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor (and complement) binding of human IgG1 Fc domains, as described in WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their properties (such as Fc receptor binding or effector function).

[0179] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function compared to IgG1 antibodies. Accordingly, in some embodiments, the Fc domain of the protease-activatable T cell activating bispecific molecule of the present invention is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In certain embodiments, the IgG4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P. To further reduce its binding affinity to Fc receptors and / or effector function, in certain embodiments, the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E. In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G. In a specific embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235, and P329, specifically the amino acid substitutions S228P, L235E, and P329G. Such IgG4 Fc domain variants and their Fcγ receptor binding properties are described in WO 2012 / 130831, which is incorporated herein by reference in its entirety.

[0180] In certain embodiments, the Fc domain that exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain is a human IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G.

[0181] In certain embodiments, N-glycosylation of the Fc domain is eliminated, in such embodiments, the Fc domain comprises an amino acid substitution at position N297, particularly replacing asparagine with alanine (N297A) or aspartic acid (N297D).

[0182] In addition to the Fc domains described herein above and in WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also include those with one or more substitutions at Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, such as the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0183] Variant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The exact nucleotide changes can be verified, for example, by sequencing.

[0184] Binding to Fc receptors can be readily determined, for example, by ELISA or surface plasmon resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors can be obtained by recombinant expression. Suitable such binding assays are described herein. Alternatively, the binding affinity of an Fc domain or a cell-activating bispecific antigen-binding molecule containing an Fc domain to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor, such as human NK cells expressing the FcγIIIa receptor.

[0185] The effector function of an Fc domain or a protease-activatable T cell-activating bispecific molecule comprising an Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al., Proc Natl Acad Sci USA 83, 7059-7063 (1986); and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays may be used (e.g., ACTI for flow cytometry). TM Non-radioactive cytotoxicity assays (see CellTechnology, Mountain View, CA; and CytoTox 96® Non-radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998)).

[0186] In some embodiments, binding of the Fc domain to complement components, particularly C1q, is reduced. Thus, in some embodiments in which the Fc domain is modified to have reduced effector function, the reduced effector function includes reduced CDC. A C1q binding assay can be performed to determine whether a protease-activatable T cell activating bispecific molecule is capable of binding C1q and therefore has CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, et al., Blood 101:1045-1052 (2003); and Cragg and Glennie, Blood 103:2738-2743 (2004)).

[0187] antigen binding part The antigen-binding molecules of the present invention are bispecific, i.e., they contain at least two antigen-binding portions capable of specifically binding to two different antigenic determinants. According to the present invention, the antigen-binding portions are Fab molecules (i.e., antigen-binding domains composed of a heavy chain and a light chain, each of which contains a variable region and a constant region). In one embodiment, the Fab molecule is human. In another embodiment, the Fab molecule is humanized. In yet another embodiment, the Fab molecule contains a human heavy chain constant region and a human light chain constant region.

[0188] At least one of the antigen-binding moieties is a crossover Fab molecule. Such a modification prevents mispairing of heavy and light chains of different Fab molecules, thereby improving the yield and purity of the protease-activatable T cell activating bispecific molecules of the invention during recombinant production. In certain crossover Fab molecules useful in the protease-activatable T cell activating bispecific molecules of the invention, the constant regions of the Fab light chain and the Fab heavy chain are swapped. In other crossover Fab molecules useful in the protease-activatable T cell activating bispecific molecules of the invention, the variable regions of the Fab light chain and the Fab heavy chain are swapped.

[0189] In certain embodiments according to the invention, the protease-activatable T cell activating bispecific molecule is capable of simultaneous binding to a target cell antigen (particularly a tumor cell antigen) and CD3. In certain embodiments, the protease-activatable T cell activating bispecific molecule is capable of crosslinking a T cell to a target cell by simultaneous binding to a target cell antigen and CD3. In further specific embodiments, such simultaneous binding results in lysis of the target cell, particularly a tumor cell. In certain embodiments, such simultaneous binding results in T cell activation. In other embodiments, such simultaneous binding results in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. In certain embodiments, binding of the protease-activatable T cell activating bispecific molecule to CD3 without simultaneous binding to a target cell antigen does not result in T cell activation.

[0190] In certain embodiments, the protease-activatable T cell activating bispecific molecule is capable of redirecting the cytotoxic activity of a T cell to a target cell, which in certain embodiments is independent of MHC-mediated peptide antigen presentation by the target cell and / or the specificity of the T cell.

[0191] In particular, T cells according to embodiments of the present invention are cytotoxic T cells. In some embodiments, the T cells are CD4 +or CD8 + T cells, especially CD8 + T cells.

[0192] CD3 binding part The protease-activatable T cell activating bispecific molecules of the invention comprise at least one antigen-binding moiety capable of binding to CD3 (also referred to herein as a "CD3 antigen-binding moiety" or "first antigen-binding moiety"). In certain embodiments, the protease-activatable T cell activating bispecific molecules comprise up to one antigen-binding moiety capable of binding to CD3. In some embodiments, the protease-activatable T cell activating bispecific molecules provide monovalent binding to CD3. The CD3 antigen binding is a crossover Fab molecule, i.e., a Fab molecule in which the variable or constant regions of the Fab heavy and light chains have been swapped. In embodiments in which there is more than one antigen-binding moiety capable of binding to a target cell antigen comprised in the protease-activatable T cell activating bispecific molecule, the antigen-binding moiety capable of binding to CD3 is preferably a crossover Fab molecule, and the antigen-binding moiety capable of binding to the target cell antigen is a conventional Fab molecule.

[0193] In certain embodiments, the CD3 is human CD3 or cynomolgus CD3, particularly human CD3. In certain embodiments, the CD3 antigen-binding portion is cross-reactive with (i.e., specifically binds to) human CD3 and cynomolgus CD3. In some embodiments, the first antigen-binding portion can bind to the epsilon subunit of CD3.

[0194] The CD3 antigen-binding portion comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:10, and at least one light chain CDR selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0195] In one embodiment, the CD3 antigen-binding portion comprises a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 4, a heavy chain CDR3 of SEQ ID NO: 10, a light chain CDR1 of SEQ ID NO: 20, a light chain CDR2 of SEQ ID NO: 21, and a light chain CDR3 of SEQ ID NO: 22.

[0196] In certain embodiments, the CD3 antigen-binding portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0197] In one embodiment, the CD3 antigen binding portion comprises the heavy chain variable region sequence of SEQ ID NO:16 and the light chain variable region sequence of SEQ ID NO:23.

[0198] Target cell antigen binding moiety The protease-activatable T cell activating bispecific molecules of the invention comprise at least one antigen-binding moiety capable of binding to a target cell antigen (also referred to herein as a "target cell antigen-binding moiety" or a "second" or "third" antigen-binding moiety). In certain embodiments, the protease-activatable T cell activating bispecific molecules comprise two antigen-binding moieties capable of binding to a target cell antigen. In certain such embodiments, each of these antigen-binding moieties specifically binds to the same antigenic determinant. In further particular embodiments, these antigen-binding moieties are all identical. In some embodiments, the protease-activatable T cell activating bispecific molecules comprise an immunoglobulin molecule capable of binding to a target cell antigen. In some embodiments, the protease-activatable T cell activating bispecific molecules comprise up to two antigen-binding moieties capable of binding to a target cell antigen.

[0199] In a preferred embodiment, the target cell antigen binding moiety is a Fab molecule, particularly a conventional Fab molecule that can bind to a specific antigenic determinant and direct the protease-activatable T cell activating bispecific molecule to a target site (e.g., a specific type of tumor cell bearing the antigenic determinant).

[0200] In certain embodiments, the target cell antigen binding moiety specifically binds to a cell surface antigen. In certain embodiments, the target cell antigen binding moiety specifically binds to folate receptor 1 (FolR1) on the surface of a target cell. In another such embodiment, the target cell antigen binding moiety specifically binds to tyrosinase-related protein 1 (TYRP1), particularly human TYRP1.

[0201] In certain embodiments, the target cell antigen-binding moiety is directed against an antigen associated with a pathological condition, such as an antigen displayed on tumor cells or virus-infected cells. Suitable antigens include, but are not limited to, cell surface antigens, such as cell surface receptors. In certain embodiments, the antigen is a human antigen. In certain embodiments, the target cell antigen is selected from folate receptor 1 (FolR1) and tyrosinase-related protein 1 (TYRP1).

[0202] In certain embodiments, the protease-activatable T cell-activating bispecific molecule comprises at least one antigen-binding portion specific for FolR1. In some embodiments, the FolR1 is human FolR1. In some embodiments, the protease-activatable T cell-activating bispecific molecule comprises at least one antigen-binding portion specific for human FolR1 and that does not bind to human FolR2 or human FolR3. In some embodiments, the antigen-binding portion specific for FolR1 comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56, and at least one light chain CDR selected from the group of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0203] In one embodiment, the antigen-binding portion specific for FolR1 comprises a heavy chain CDR1 of SEQ ID NO: 54, a heavy chain CDR2 of SEQ ID NO: 55, a heavy chain CDR3 of SEQ ID NO: 56, a light chain CDR1 of SEQ ID NO: 20, a light chain CDR2 of SEQ ID NO: 21, and a light chain CDR3 of SEQ ID NO: 22.

[0204] In a further embodiment, the antigen-binding portion specific for FolR1 comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 53 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 23, or a variant thereof that retains functionality.

[0205] In one embodiment, the antigen-binding portion specific for FolR1 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:53 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:23.

[0206] Masking part The protease-activatable T cell activating bispecific molecules of the present invention comprise at least one masking moiety. Other researchers have attempted to mask antibody binding by capping the binding moiety with a fragment of the antigen recognized by the binding moiety (e.g., WO 2013128194). This approach has several limitations. For example, the use of an antigen reduces flexibility in lowering the affinity of the binding moiety because the affinity must be high enough to ensure masking by the antigen mask. Furthermore, dissociated antigens may bind and interact with their cognate receptors in vivo, potentially causing undesirable signaling in cells expressing the receptor. In contrast, the approach described herein uses an anti-idiotypic antibody or a fragment thereof as a mask. Two competing considerations for designing an effective masking moiety are 1. masking efficacy and 2. masking reversibility. If the affinity is too low, masking will be ineffective. However, if the affinity is too high, the masking process may not be easily reversible. It was not possible to predict whether a high-affinity or low-affinity anti-idiotype mask would perform better. As described herein, high-affinity masking moieties performed better overall in masking the antigen-binding side, while at the same time allowing for effective removal for molecule activation. In one embodiment, the anti-idiotype mask has a KD of 1-8 nM. In one embodiment, the anti-idiotype mask has a KD of 2 nM at 37°C. In certain embodiments, the masking moiety recognizes the idiotype of a first antigen-binding moiety capable of binding to CD3, e.g., human CD3. In certain embodiments, the masking moiety recognizes the idiotype of a second antigen-binding moiety capable of binding to a target cell antigen.

[0207] In one embodiment, the masking moiety masks the CD3 binding moiety and comprises at least one of a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 4, a heavy chain CDR3 of SEQ ID NO: 10, a light chain CDR1 of SEQ ID NO: 20, a light chain CDR2 of SEQ ID NO: 21, and a light chain CDR3 of SEQ ID NO: 22. In one embodiment, the masking moiety comprises a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 4, a heavy chain CDR3 of SEQ ID NO: 10, a light chain CDR1 of SEQ ID NO: 20, a light chain CDR2 of SEQ ID NO: 21, and a light chain CDR3 of SEQ ID NO: 22.

[0208] In certain embodiments, the masking moiety masks the CD3 binding portion and comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 16. In certain embodiments, the masking moiety masks the CD3 binding portion and comprises the polypeptide sequence of SEQ ID NO: 23.

[0209] In one embodiment, the masking portion comprises at least one of a heavy chain CDR1 of SEQ ID NO: 58, a heavy chain CDR2 selected from the group consisting of SEQ ID NO: 59, SEQ ID NO: 84 and SEQ ID NO: 86, a heavy chain CDR3 of SEQ ID NO: 60, a light chain CDR1 selected from the group consisting of SEQ ID NO: 62 and SEQ ID NO: 82, a light chain CDR2 of SEQ ID NO: 63, and a light chain CDR3 selected from the group consisting of SEQ ID NO: 64 and SEQ ID NO: 88.

[0210] In one embodiment, the masking moiety comprises at least one of a heavy chain CDR1 of SEQ ID NO: 58, a heavy chain CDR2 of SEQ ID NO: 59, a heavy chain CDR3 of SEQ ID NO: 60, a light chain CDR1 of SEQ ID NO: 62, a light chain CDR2 of SEQ ID NO: 63, and a light chain CDR3 of SEQ ID NO: 64. In one embodiment, the masking moiety comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 57, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 61, or a variant thereof that retains functionality.

[0211] In one embodiment, the masking portion comprises at least one of a heavy chain CDR1 of SEQ ID NO: 58, a heavy chain CDR2 of SEQ ID NO: 59, a heavy chain CDR3 of SEQ ID NO: 60, a light chain CDR1 of SEQ ID NO: 82, a light chain CDR2 of SEQ ID NO: 63, and a light chain CDR3 of SEQ ID NO: 64.

[0212] In one embodiment, the masking portion comprises at least one of a heavy chain CDR1 of SEQ ID NO: 58, a heavy chain CDR2 of SEQ ID NO: 84, a heavy chain CDR3 of SEQ ID NO: 60, a light chain CDR1 of SEQ ID NO: 82, a light chain CDR2 of SEQ ID NO: 63, and a light chain CDR3 of SEQ ID NO: 64.

[0213] In one embodiment, the masking portion comprises at least one of a heavy chain CDR1 of SEQ ID NO: 58, a heavy chain CDR2 of SEQ ID NO: 86, a heavy chain CDR3 of SEQ ID NO: 60, a light chain CDR1 of SEQ ID NO: 82, a light chain CDR2 of SEQ ID NO: 63, and a light chain CDR3 of SEQ ID NO: 64.

[0214] In one embodiment, the masking portion comprises at least one of a heavy chain CDR1 of SEQ ID NO: 59, a heavy chain CDR2 of SEQ ID NO: 86, a heavy chain CDR3 of SEQ ID NO: 60, a light chain CDR1 of SEQ ID NO: 62, a light chain CDR2 of SEQ ID NO: 63, and a light chain CDR3 of SEQ ID NO: 88.

[0215] In a preferred embodiment, the masking moiety is humanized. In a preferred embodiment, the idiotype-specific polypeptide for reversibly masking the anti-CD3 antigen-binding site of the molecule is humanized. Methods for humanizing immunoglobulins are well known in the art and are described herein.

[0216] In certain embodiments, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, the idiotype-specific polypeptide comprising a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:89, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:87 and SEQ ID NO:90.

[0217] In a preferred embodiment, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, the idiotype-specific polypeptide comprising a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:79, SEQ ID NO:83 and SEQ ID NO:85, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:80 and SEQ ID NO:81.

[0218] In certain embodiments, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, the idiotype-specific polypeptide comprising a heavy chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 79 and a light chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 80. In a preferred embodiment, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, the idiotype-specific polypeptide comprising a heavy chain variable region sequence of SEQ ID NO: 79 and a light chain variable region sequence of SEQ ID NO: 80.

[0219] In certain embodiments, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, the idiotype-specific polypeptide comprising a heavy chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 79 and a light chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81. In a preferred embodiment, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, the idiotype-specific polypeptide comprising a heavy chain variable region sequence of SEQ ID NO: 79 and a light chain variable region sequence of SEQ ID NO: 81.

[0220] In certain embodiments, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, the idiotype-specific polypeptide comprising a heavy chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 83 and a light chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81. In a preferred embodiment, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, the idiotype-specific polypeptide comprising a heavy chain variable region sequence of SEQ ID NO: 83 and a light chain variable region sequence of SEQ ID NO: 81.

[0221] In certain embodiments, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, the idiotype-specific polypeptide comprising a heavy chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 85 and a light chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81. In a preferred embodiment, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, the idiotype-specific polypeptide comprising a heavy chain variable region sequence of SEQ ID NO: 85 and a light chain variable region sequence of SEQ ID NO: 81.

[0222] In certain embodiments, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, wherein the idiotype-specific polypeptide comprises a heavy chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 84 and a light chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 87. In certain embodiments, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, wherein the idiotype-specific polypeptide comprises a heavy chain variable region sequence of SEQ ID NO: 84 and a light chain variable region sequence of SEQ ID NO: 87.

[0223] In certain embodiments, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, wherein the idiotype-specific polypeptide comprises a heavy chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 89 and a light chain variable region sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 90. In certain embodiments, provided is an idiotype-specific polypeptide for reversibly masking an anti-CD3 antigen binding site of a molecule, wherein the idiotype-specific polypeptide comprises a heavy chain variable region sequence of SEQ ID NO: 89 and a light chain variable region sequence of SEQ ID NO: 90.

[0224] In certain embodiments, the masking moiety is an anti-idiotypic scFv comprising a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 91. In certain embodiments, the anti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO: 91.

[0225] In certain embodiments, the masking moiety is an anti-idiotypic scFv comprising a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 92. In certain embodiments, the anti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO:92.

[0226] In certain embodiments, the masking moiety is an anti-idiotypic scFv comprising a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 93. In certain embodiments, the anti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO:93.

[0227] In certain embodiments, the masking moiety is an anti-idiotypic scFv comprising a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 94. In certain embodiments, the anti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO:94.

[0228] A protease-activatable T cell-activating bispecific molecule capable of binding to CD3 and FolR1 The first antigen-binding moiety capable of binding to CD3, the second antigen-binding moiety capable of binding to FolR1, the Fc domain, and the masking moiety can be fused to each other in various configurations, exemplary configurations and sequences of which are disclosed below.

[0229] In one embodiment, the protease activatable T cell activating bispecific molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:65, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:66, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:67.

[0230] In one embodiment, the protease activatable T cell activating bispecific molecule comprises the polypeptide sequence of SEQ ID NO:65, the polypeptide sequence of SEQ ID NO:66, and the polypeptide sequence of SEQ ID NO:67.

[0231] In one embodiment, the protease activatable T cell activating bispecific molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 74, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 66, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 67.

[0232] In one embodiment, the protease activatable T cell activating bispecific molecule comprises the polypeptide sequence of SEQ ID NO:74, the polypeptide sequence of SEQ ID NO:66, and the polypeptide sequence of SEQ ID NO:67.

[0233] In one embodiment, the protease activatable T cell activating bispecific molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:76, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:66, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:67.

[0234] In one embodiment, the protease activatable T cell activating bispecific molecule comprises the polypeptide sequence of SEQ ID NO:76, the polypeptide sequence of SEQ ID NO:66, and the polypeptide sequence of SEQ ID NO:67.

[0235] In one embodiment, the protease activatable T cell activating bispecific molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:95, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:66, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:67.

[0236] In one embodiment, the protease activatable T cell activating bispecific molecule comprises the polypeptide sequence of SEQ ID NO: 95, the polypeptide sequence of SEQ ID NO: 66, and the polypeptide sequence of SEQ ID NO: 67. In one embodiment, the protease activatable T cell activating bispecific molecule comprises one polypeptide sequence of SEQ ID NO: 95, one polypeptide sequence of SEQ ID NO: 66, and two polypeptide sequences of SEQ ID NO: 67.

[0237] In one embodiment, the protease activatable T cell activating bispecific molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:96, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:66, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:67.

[0238] In one embodiment, the protease activatable T cell activating bispecific molecule comprises the polypeptide sequence of SEQ ID NO: 96, the polypeptide sequence of SEQ ID NO: 66, and the polypeptide sequence of SEQ ID NO: 67. In one embodiment, the protease activatable T cell activating bispecific molecule comprises one polypeptide sequence of SEQ ID NO: 96, one polypeptide sequence of SEQ ID NO: 66, and two polypeptide sequences of SEQ ID NO: 67.

[0239] In certain embodiments, the protease activatable T cell activating bispecific molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:97, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:66, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:67.

[0240] In one embodiment, the protease activatable T cell activating bispecific molecule comprises the polypeptide sequence of SEQ ID NO: 97, the polypeptide sequence of SEQ ID NO: 66, and the polypeptide sequence of SEQ ID NO: 67. In one embodiment, the protease activatable T cell activating bispecific molecule comprises one polypeptide sequence of SEQ ID NO: 97, one polypeptide sequence of SEQ ID NO: 66, and two polypeptide sequences of SEQ ID NO: 67.

[0241] In one embodiment, the protease activatable T cell activating bispecific molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:98, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:66, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:67.

[0242] In one embodiment, the protease activatable T cell activating bispecific molecule comprises the polypeptide sequence of SEQ ID NO: 98, the polypeptide sequence of SEQ ID NO: 66, and the polypeptide sequence of SEQ ID NO: 67. In one embodiment, the protease activatable T cell activating bispecific molecule comprises one polypeptide sequence of SEQ ID NO: 98, one polypeptide sequence of SEQ ID NO: 66, and two polypeptide sequences of SEQ ID NO: 67.

[0243] Linker In one aspect, the present invention relates to idiotype-specific polypeptides for reversibly masking antigen binding of a molecule. In one embodiment, the present invention relates to idiotype-specific polypeptides for reversibly masking an anti-CD3 antigen-binding site of a molecule. Such idiotype-specific polypeptides for reversibly masking an anti-CD3 antigen-binding site should be capable of binding to the idiotype of the anti-CD3 antigen-binding site, thereby reducing or eliminating binding of the anti-CD3 antigen-binding site to CD3. In one embodiment, the idiotype-specific polypeptide is an anti-idiotype scFv. In one embodiment, the idiotype-specific polypeptide is covalently linked to a molecule via a linker. In one embodiment, the idiotype-specific polypeptide is covalently linked to a molecule via more than one linker. In one embodiment, the idiotype-specific polypeptide is covalently linked to a molecule via two linkers. In one embodiment, the linker is a peptide linker. In one embodiment, the linker is a protease-cleavable linker.

[0244] In one embodiment, the protease-activated T cell activating bispecific molecule comprises a linker comprising a protease recognition site comprising a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 68, 70, 75, 99, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or 127. In one embodiment, the protease recognition site comprises the polypeptide sequence of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113 or 114. In a preferred embodiment, the protease recognition site comprises the polypeptide sequence of SEQ ID NO: 114.

[0245] In certain embodiments, the proteinase is selected from the group consisting of metalloproteinases (e.g., matrix metalloproteinases (MMPs) 1-28, and A Disintegrin And Metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30, and 33); serine proteases (e.g., urokinase-type plasminogen activator and matriptase); cysteine ​​proteases; aspartic acid proteases; and cathepsin proteases. In certain embodiments, the protease is MMP9 or MMP2. In further specific embodiments, the protease is matriptase.

[0246] Polynucleotides The present invention further provides isolated polynucleotides encoding the protease-activatable T cell activating bispecific molecules described herein, or fragments thereof. In some embodiments, the fragments are antigen-binding fragments.

[0247] Polynucleotides encoding the protease-activatable T cell activating bispecific molecules of the invention can be expressed as a single polynucleotide encoding the entire protease-activatable T cell activating bispecific molecule, or as multiple (e.g., two or more) co-expressed polynucleotides. Polypeptides encoded by co-expressed polynucleotides can associate, e.g., via disulfide bonds or other means, to form a functional protease-activatable T cell activating bispecific molecule. For example, the light chain portion of the antigen-binding moiety can be encoded by a separate polynucleotide from the portion of the protease-activatable T cell activating bispecific molecule comprising the heavy chain portion of the antigen-binding moiety, an Fc domain subunit, and, optionally, (a portion of) another antigen-binding moiety. When co-expressed, the heavy chain polypeptide associates with the light chain polypeptide to form the antigen-binding moiety. In another example, the portion of a protease activatable T cell activating bispecific molecule comprising one of the two Fc domain subunits and optionally (a portion of) one or more antigen binding moieties can be encoded by a separate polynucleotide from the portion of a protease activatable T cell activating bispecific molecule comprising the other of the two Fc domain subunits and optionally (a portion of) the antigen binding moieties. When co-expressed, the Fc domain subunits associate to form an Fc domain.

[0248] In some embodiments, the isolated polynucleotide encodes the entire protease activatable T cell activating bispecific molecule according to the invention as described herein. In other embodiments, the isolated polynucleotide encodes a polypeptide comprised in a protease activatable T cell activating bispecific molecule according to the invention as described herein.

[0249] In another embodiment, the present invention relates to an isolated polynucleotide encoding a protease activatable T cell activating bispecific molecule of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence encoding a variable region sequence. In another embodiment, the present invention relates to an isolated polynucleotide encoding a protease activatable T cell activating bispecific molecule of the invention or a fragment thereof, wherein the polynucleotide comprises a sequence encoding the polypeptide sequence set forth in SEQ ID NOs: 65, 66, 67, 69, 74, 76, 91, 92, 93, 94, 95, 96, 97, 98, or a fragment thereof.

[0250] Polynucleotides encoding idiotype-specific polypeptides of the invention may be expressed as a single polynucleotide encoding the entire idiotype-specific polypeptide, or as multiple (e.g., two or more) co-expressed polynucleotides. Polypeptides encoded by co-expressed polynucleotides can associate, e.g., by disulfide bonds or other means, to form a functional idiotype-specific polypeptide, e.g., a masking moiety. For example, in certain embodiments, the idiotype-specific polypeptide is an anti-idiotype scFv (single-chain variable fragment), and the light chain variable portion of the anti-idiotype scFv may be encoded by a separate polynucleotide from the portion comprising the heavy chain variable portion of the anti-idiotype scFv. When co-expressed, the heavy chain polypeptide associates with the light chain polypeptide to form the anti-idiotype scFv. In some embodiments, an isolated polynucleotide encodes an idiotype-specific polypeptide according to the invention described herein.

[0251] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, such as RNA in the form of messenger RNA (mRNA). The RNA of the invention can be single-stranded or double-stranded.

[0252] Recombinant methods The protease-activatable T cell activating bispecific molecules of the invention may be obtained, for example, by solid-phase peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the protease-activatable T cell activating bispecific molecules (fragments), e.g., as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such polynucleotides can be readily isolated and sequenced using conventional procedures. In certain embodiments, vectors, preferably expression vectors, are provided that contain one or more of the polynucleotides of the invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence for the protease-activatable T cell activating bispecific molecules (fragments) together with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY (1989). An expression vector can be part of a plasmid, a virus, or a nucleic acid fragment. An expression vector contains an expression cassette into which a polynucleotide (i.e., coding region) encoding a protease-activatable T cell activating bispecific molecule (fragment) has been cloned in operative association with a promoter and / or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids.A "stop codon" (TAG, TGA, or TAA) is not translated into amino acids but can be considered part of the coding region (if present); however, flanking sequences such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc. are not part of the coding region. Two or more coding regions can be present in a single polynucleotide construct (e.g., on a single vector) or in separate polynucleotide constructs (e.g., on separate (different) vectors). Furthermore, any vector can contain a single coding region or two or more coding regions; for example, a vector of the invention can encode one or more polypeptides that are separated post- or co-translationally into the final protein by proteolytic cleavage. In addition, the vectors, polynucleotides, or nucleic acids of the invention can encode heterologous coding regions, which may or may not be fused to the polynucleotide encoding the protease-activatable T cell activating bispecific molecule (fragment) of the invention or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs such as secretory signal peptides or heterologous functional domains. An operably associated state is when a coding region for a gene product, such as a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and its associated promoter) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region is operably associated with a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells.In addition to promoters, other transcriptional regulatory elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional regulatory regions are disclosed herein. A variety of transcriptional regulatory regions are known to those of skill in the art. These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, such as promoter and enhancer segments derived from cytomegalovirus (e.g., the immediate early promoter linked to intron A), Simian Virus 40 (e.g., the early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcriptional regulatory regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit alpha globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., tetracycline-inducible promoters). Similarly, a variety of translational regulatory elements are known to those of skill in the art. These include, but are not limited to, a ribosome binding site, translation initiation and termination codons, and elements from viral systems (particularly an internal ribosome entry site or IRES, also known as a CITE sequence). The expression cassette may also include other features such as an origin of replication and / or chromosomal integration elements such as retroviral long terminal repeats (LTRs) or adeno-associated viral (AAV) inverted terminal repeats (ITRs).

[0253] Polynucleotide and nucleic acid coding regions of the invention can be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of a polypeptide encoded by a polynucleotide of the invention. For example, if secretion of a protease-activatable T cell activating bispecific molecule of the invention or a fragment thereof is desired, DNA encoding a signal sequence can be located upstream of the nucleic acid encoding the protease-activatable T cell activating bispecific molecule of the invention or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells possess a signal peptide or secretory leader sequence that is cleaved from the mature protein upon initiation of transport of the nascent protein chain across the rough endoplasmic reticulum. Those skilled in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide is used, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of that sequence that retains the ability to direct the secretion of a polypeptide operably associated with it. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0254] DNA encoding a short protein sequence (e.g., a histidine tag) that can be used to facilitate subsequent purification or to serve to label the protease-activatable T cell activating bispecific molecule may be included within or at the end of the protease-activatable T cell activating bispecific molecule (fragment) encoding polynucleotide.

[0255] In further embodiments, host cells are provided comprising one or more polynucleotides of the invention. In particular embodiments, host cells are provided comprising one or more vectors of the invention. The polynucleotides and vectors can incorporate any of the features described herein in connection with the polynucleotides and vectors, respectively, alone or in combination. In such embodiments, the host cell comprises (e.g., is transformed or transfected with) a vector comprising a polynucleotide encoding (a portion of) a protease-activatable T cell-activating bispecific molecule of the invention. As used herein, the term "host cell" refers to any type of cell line that can be engineered to produce a protease-activatable T cell-activating bispecific molecule of the invention or a fragment thereof. Host cells suitable for supporting the expression of protease-activatable T cell-activating bispecific molecules are well known in the art. Such cells can be appropriately transfected or transduced with a particular expression vector, and large quantities of the vector-containing cells can be grown to inoculate large-scale fermenters, yielding sufficient quantities of protease-activatable T cell-activating bispecific molecules for clinical applications. Suitable host cells include prokaryotic microorganisms (e.g., Escherichia coli) or various eukaryotic cells (Chinese hamster ovary cells (CHO), insect cells, etc.). For example, polypeptides can be produced in bacteria, particularly if glycosylation is not required. After expression, the polypeptide can be isolated from the cell paste as a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable as cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of polypeptides with partially or fully human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES FOR PRODUCING ANTIBODIES IN TRANSGENIC PLANTS). TM See the description of the technique. Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293T cells as described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (e.g., Mather et al., Annals of NY Acad Sci 383, 44-68 (1982), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include dhfr -These include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. For a review of specific mammalian host cells suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells, and plant cells, to name just a few, but also cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocytic cell (eg, a Y0, NS0, Sp20 cell).

[0256] Standard techniques for expressing foreign genes in these systems are known in the art. Cells that express a polypeptide containing either the heavy or light chain of an antigen-binding domain, such as an antibody, may also be engineered to express the other antibody chain, such that the expression product is an antibody having both a heavy and a light chain.

[0257] In one embodiment, a method of producing a protease activatable T cell activating bispecific molecule according to the invention is provided, the method comprising culturing a host cell comprising a polynucleotide encoding a T cell activating bispecific antigen binding molecule as provided herein under conditions suitable for expression of the protease activatable T cell activating bispecific molecule, and recovering the protease activatable T cell activating bispecific molecule from the host cell (or host cell culture medium).

[0258] The components of the protease-activatable T cell activating bispecific molecule are genetically fused to each other. The protease-activatable T cell activating bispecific molecule can be designed so that its components are fused to each other directly or indirectly via a linker sequence. The composition and length of the linker can be determined according to methods well known in the art and tested for effectiveness. Examples of linker sequences between the different components of the protease-activatable T cell activating bispecific molecule can be found in the sequences provided herein. Also, if desired, additional sequences (e.g., endopeptidase recognition sequences) can be included to incorporate cleavage sites to separate the individual components of the fusion.

[0259] In certain embodiments, one or more antigen-binding portions of the protease-activatable T cell activating bispecific molecule comprise at least an antibody variable region capable of binding to an antigenic determinant. The variable region can form part of, and can be derived from, natural or non-natural antibodies and fragments thereof. Methods for producing polyclonal and monoclonal antibodies are well known in the art (see, e.g., Harlow and Lane, "Antibodies, a laboratory manual," Cold Spring Harbor Laboratory, 1988). Non-natural antibodies can be constructed using solid-phase peptide synthesis, produced recombinantly (e.g., as described in U.S. Pat. No. 4,186,567), or obtained, for example, by screening combinatorial libraries containing variable heavy and light chains (see, e.g., U.S. Pat. No. 5,969,108 to McCafferty).

[0260] Antibodies, antibody fragments, antigen-binding domains, or variable regions of any animal species can be used in the protease-activatable T cell activating bispecific molecules of the invention. Non-limiting antibodies, antibody fragments, antigen-binding domains, or variable regions useful in the invention can be of murine, primate, or human origin. When the protease-activatable T cell activating bispecific molecules are intended for use in humans, chimeric forms of antibodies in which the antibody constant regions are human-derived can be used. "Humanized" or fully human forms of antibodies can also be prepared according to methods well known in the art (see, e.g., U.S. Pat. No. 5,565,332 to Winter). Humanization can be achieved by various methods, including, but not limited to, (a) grafting CDRs of a non-human (e.g., donor antibody) onto the framework and constant regions of a human (e.g., recipient antibody), with or without preserving critical framework residues (e.g., residues important for maintaining good antigen-binding affinity or antibody function); (b) grafting only non-human specificity-determining regions (SDRs or a-CDRs; residues important for antibody-antigen interactions) onto human framework and constant regions; or (c) grafting entire non-human variable domains but "cloaking" them with human-like sections by replacing surface residues.Humanized antibodies and methods for their production are reviewed, for example, by Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000) (describing a "guided selection" approach to FR shuffling). Human antibodies and human variable regions can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).Human variable regions can form part of, and can be derived from, human monoclonal antibodies produced by hybridoma technology (see, e.g., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions can also be prepared by administering an immunogen to transgenic animals that have been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge (see, e.g., Lonberg, Nat Biotech 23, 1117-1125 (2005)). Human antibodies and human variable regions can also be produced by isolating Fv clone variable region sequences selected from human-derived phage display libraries (see, e.g., Hoogenboom et al., Methods in Molecular Biology 178, 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments.

[0261] In certain embodiments, antigen-binding moieties useful in the present invention are modified to have enhanced binding affinity, for example, according to the methods disclosed in U.S. Patent Application Publication No. 2004 / 0132066, the entire contents of which are incorporated herein by reference. The ability of the protease-activatable T cell-activating bispecific molecules of the present invention to bind to a particular antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance technology (analysis on a BIACORE T100 system) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and classical binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays can be used to identify antibodies, antibody fragments, antigen-binding domains, or variable domains that compete with a reference antibody for binding to a particular antigen, for example, an antibody that competes with the V9 antibody for binding to CD3. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) bound by the reference antibody. Detailed exemplary methods for mapping antibody-binding epitopes are provided in "Epitope Mapping Protocols" in Methods in Molecular Biology, Vol. 66, by Morris (1996) (Humana Press, Totowa, NJ). In an exemplary competitive assay, an immobilized antigen (e.g., CD3) is incubated in a solution containing a first labeled antibody that binds to the antigen (e.g., the V9 antibody, described in U.S. Pat. No. 6,054,297) and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to the antigen. The second antibody may be present in hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to the antigen, excess unbound antibody is removed and the amount of label associated with the immobilized antigen is measured.If the amount of label associated with the immobilized antigen is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody competes with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0262] Protease-activatable T cell activating bispecific molecules prepared as described herein can be purified by techniques known in the art, such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography, size-exclusion chromatography, etc. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those of skill in the art. Affinity chromatography purification can use an antibody, ligand, receptor, or antigen to which the protease-activatable T cell activating bispecific molecule binds. For example, for affinity chromatography purification of protease-activatable T cell activating bispecific molecules of the invention, a matrix containing Protein A or Protein G can be used. Sequential Protein A or G affinity chromatography and size-exclusion chromatography can be used to isolate protease-activatable T cell activating bispecific molecules essentially as described in the Examples. The purity of the protease-activatable T cell activating bispecific molecules can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high-pressure liquid chromatography, etc. For example, the heavy chain fusion proteins expressed as described in the Examples were shown to be intact and properly assembled as demonstrated by reducing SDS-PAGE (see Figures 8-12). Three bands were resolved, approximately Mr 25,000, Mr 50,000, and Mr 75,000, corresponding to the predicted molecular weights of the light chain, heavy chain, and heavy / light chain fusion protein of the protease-activatable T cell activating bispecific molecule.

[0263] Assay The protease-activatable T cell activating bispecific molecules provided herein can be identified, screened or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0264] Affinity assay The affinity of protease-activatable T cell-activating bispecific molecules for Fc receptors or target antigens can be determined by surface plasmon resonance assays (SPR) using standard instrumentation, such as a BIAcore instrument (GE Healthcare), and receptors or target proteins that can be obtained by recombinant expression, according to the methods described in the Examples. Alternatively, binding of protease-activatable T cell-activating bispecific molecules to different receptors or target antigens can be assessed, for example, by flow cytometry (FACS) using cell lines expressing the particular receptors or target antigens. Specific illustrative exemplary embodiments for measuring binding affinity are described below and in the Examples that follow.

[0265] In one embodiment, K D is measured by surface plasmon resonance using a BIACORE® T100 machine (GE Healthcare) at 25°C.

[0266] To analyze the interaction between the Fc moiety and Fc receptors, His-tagged recombinant Fc receptors were captured by an anti-pentaHis antibody (Qiagen) immobilized on a CM5 chip, and the bispecific construct was used as the analyte. Briefly, a carboxymethylated dextran biosensor chip (CM5, GE Healthair) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The anti-pentaHis antibody was diluted to 40 μg / ml in 10 mM sodium acetate (pH 5.0) and injected at a flow rate of 5 μl / min, yielding approximately 6500 response units (RU) of bound protein. After ligand injection, 1 M ethanolamine was injected to block unreacted groups. The Fc receptor was then captured at 4 or 10 nM for 60 seconds. For kinetic measurements, four-fold serial dilutions of the bispecific constructs (ranging between 500 nM and 4000 nM) are injected for 120 seconds in HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20, pH 7.4) at 25°C and a flow rate of 30 μl / min.

[0267] To determine affinity for the target antigen, the bispecific construct is captured by an anti-human Fab specific antibody (GE Healthcare) immobilized on an activated CM5 sensor chip surface, as described for the anti-pentaHis antibody. The final amount of bound protein is approximately 12,000 RU. The bispecific construct is captured at 300 nM for 90 seconds. The target antigen is passed through the flow cell for 180 seconds at a flow rate of 30 μl / min, ranging from 250 to 1,000 nM. Dissociation is monitored for 180 seconds.

[0268] Bulk refractive index differences are corrected for by subtracting the response obtained with a reference flow cell. The steady-state response is used to determine the dissociation constant, K, by nonlinear curve fitting of the Langmuir binding isotherm. DThe association rate (k on ) and dissociation rate (k off ) to calculate the equilibrium dissociation constant (K D ) is the ratio k off / k on See, for example, Chen et al., J Mol Biol 293, 865-881 (1999).

[0269] Activity assay The biological activity of the protease-activatable T cell activating bispecific molecules of the invention can be measured by various assays described in the Examples, including, for example, inducing T cell proliferation, inducing signaling in T cells, inducing expression of activation markers in T cells, inducing cytokine secretion by T cells, inducing lysis of target cells such as tumor cells, and inducing tumor regression and / or improving survival.

[0270] Compositions, Formulations, and Routes of Administration In a further aspect, the present invention provides pharmaceutical compositions comprising any of the protease-activatable T cell activating bispecific molecules provided herein, e.g., pharmaceutical compositions for use in any of the therapeutic methods described below. In embodiments, the pharmaceutical composition comprises any of the protease-activatable T cell activating bispecific molecules provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the protease-activatable T cell activating bispecific molecules provided herein and at least one additional therapeutic agent, e.g., as described below.

[0271] Further provided is a method of producing a protease-activatable T cell activating bispecific molecule of the invention in a form suitable for in vivo administration, comprising: (a) obtaining a protease-activatable T cell activating bispecific molecule according to the invention; and (b) combining the protease-activatable T cell activating bispecific molecule with at least one pharmaceutically acceptable carrier, whereby a preparation of protease-activatable T cell activating bispecific molecule is formulated for in vivo administration.

[0272] Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more protease-activatable T cell-activating bispecific molecules dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic, or other untoward reactions when administered as needed to animals, e.g., humans. The preparation of pharmaceutical compositions containing at least one protease-activatable T cell-activating bispecific molecule, and optionally additional active ingredients, is exemplified by Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990 (incorporated herein by reference) and will be known to those of skill in the art in light of the present disclosure. Furthermore, for administration to animals (e.g., humans), preparations must meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA's Office of Biological Standards or equivalent agencies in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except where conventional carriers are incompatible with the active ingredient, their use in therapeutic or pharmaceutical compositions is contemplated.

[0273] The composition can contain different types of carriers depending on whether it is to be administered in solid, liquid, or aerosol form, and whether it needs to be sterile for the route of administration, such as injection. The protease activatable T cell activating bispecific molecules of the invention (and any additional therapeutic agents) can be administered intravenously, intradermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrasplenicly, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, local perfusion bathing directly in target cells, via a catheter, via a lavage solution, in creams, in lipid compositions (e.g., liposomes), or by any combination of the above, or by any other method that would be known to one of skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). Parenteral administration, particularly intravenous infusion, is most commonly used to administer polypeptide molecules such as the protease-activatable T cell activating bispecific molecules of the invention.

[0274] Parenteral compositions include those designed for administration by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, the protease-activatable T cell-activating bispecific molecules of the invention can be formulated in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulating agents, such as suspending, stabilizing, and / or dispersing agents. Alternatively, the protease-activatable T cell-activating bispecific molecules may be in powder form for constitution with a suitable vehicle (e.g., pyrogen-free sterile water) before use. Sterile injectable solutions are prepared by incorporating the required amount of the protease-activatable T cell-activating bispecific molecules of the invention in an appropriate solvent with various other ingredients, as listed below, as needed. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. Dispersions are usually prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and / or other ingredients. In the case of sterile powders for preparing sterile injections, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying, which obtains a powder of the active ingredient plus any additional desired ingredients from a liquid medium that has previously been sterilized by filtration. If necessary, the liquid medium should be appropriately buffered, and the liquid diluent should first be made isotonic with sufficient saline or glucose before injection. The composition must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. It is understood that endotoxin contamination must be kept to a minimum safe level, for example, less than 0.5 ng / mg protein.Suitable pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (fewer than about 10 residues) polypeptides; proteins (such as serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (polyvinylpyrrolidone; amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine); glucose, mannose, or dextrose; Examples of suitable surfactants include monosaccharides, disaccharides, and other carbohydrates, including sucrose; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds, allowing for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.

[0275] The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules, poly(methylmethacylate) microcapsules, respectively) prepared by coacervation techniques or interfacial polymerization methods, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films or microcapsules. In certain embodiments, sustained absorption of injectable compositions can be achieved by using agents delaying absorption in the compositions (e.g., aluminum monostearate, gelatin, or combinations thereof).

[0276] In addition to the compositions already described, the protease-activatable T cell activating bispecific molecules may be formulated as depot preparations. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the protease-activatable T cell activating bispecific molecules may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, such as a sparingly soluble salt.

[0277] Pharmaceutical compositions comprising the protease-activatable T cell activating bispecific molecules of the invention can be manufactured by common mixing, dissolving, emulsifying, encapsulating, entrapment, or lyophilization processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliary agents that facilitate processing of the protein into pharmaceutically usable preparations. Suitable formulations depend on the chosen route of administration.

[0278] The protease-activatable T cell activating bispecific molecules can be formulated in the compositions in free acid or free base, neutral, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. Such salts include acid addition salts, for example, those formed with free amino groups of the proteinaceous composition, or with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0279] Therapeutic methods and compositions Any of the protease-activatable T cell activating bispecific molecules provided herein can be used in therapeutic methods. The protease-activatable T cell activating bispecific molecules of the invention can be used as immunotherapeutic agents, for example, in the treatment of cancer.

[0280] For use in therapeutic methods, the protease-activatable T cell activating bispecific molecules of the invention will be formulated, dispensed, and administered in a manner consistent with the principles of good medicine, with factors to consider in this regard including the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to medical practitioners.

[0281] In certain aspects, protease-activatable T cell activating bispecific molecules of the invention are provided for use as a medicament. In a further aspect, protease-activatable T cell activating bispecific molecules of the invention are provided for use in the treatment of a disease. In certain embodiments, protease-activatable T cell activating bispecific molecules of the invention are provided for use in a method of treatment. In certain embodiments, the invention provides a protease-activatable T cell activating bispecific molecule described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, the invention provides a protease-activatable T cell activating bispecific molecule for use in a method of treating an individual having a disease, the method comprising administering to the individual a therapeutically effective amount of the protease-activatable T cell activating bispecific molecule. In certain embodiments, the disease being treated is a proliferative disorder. In certain embodiments, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent (e.g., an anticancer agent when the disease being treated is cancer). In further embodiments, the present invention provides a protease-activatable T cell activating bispecific molecule as described herein for use in inducing lysis of target cells, particularly tumor cells. In a particular embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule for use in a method of inducing lysis of target cells, particularly tumor cells, in an individual, the method comprising administering to the individual an effective amount of the protease-activatable T cell activating bispecific molecule to induce lysis of target cells. An "individual" according to any of the above embodiments is a mammal, preferably a human.

[0282] In a further aspect, the invention provides the use of a protease-activatable T cell activating bispecific molecule of the invention in the manufacture or preparation of a medicament. In certain embodiments, the medicament is for the treatment of a disease in an individual in need thereof. In a further embodiment, the medicament is for use in a method of treating a disease, comprising administering a therapeutically effective amount of the medicament to an individual having the disease. In certain embodiments, the disease being treated is a proliferative disorder. In certain embodiments, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent (e.g., an anti-cancer agent when the disease being treated is cancer). In a further embodiment, the medicament is for inducing lysis of target cells, particularly tumor cells. In yet a further embodiment, the medicament is for use in a method of inducing lysis of target cells, particularly tumor cells, in an individual, the method comprising administering an effective amount of the medicament to the individual to induce lysis of the target cells. The "individual" according to any of the above embodiments may be a mammal, preferably a human.

[0283] In a further aspect, the present invention provides a method for treating a disease. In certain embodiments, the method comprises administering to an individual having such a disease an effective amount of a protease-activatable T cell activating bispecific molecule of the invention. In certain embodiments, a composition comprising a protease-activatable T cell activating bispecific molecule of the invention in a pharmaceutically acceptable form is administered to the individual. In certain embodiments, the disease being treated is a proliferative disorder. In certain embodiments, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent (e.g., an anti-cancer agent when the disease being treated is cancer). An "individual" according to any of the above embodiments may be a mammal, preferably a human.

[0284] In a further aspect, the present invention provides a method for inducing lysis of target cells, particularly tumor cells. In one embodiment, the method comprises contacting the target cell with a protease-activatable T cell activating bispecific molecule of the invention in the presence of T cells, particularly cytotoxic T cells. In a further aspect, a method is provided for inducing lysis of target cells, particularly tumor cells, in an individual. In such an embodiment, the method comprises administering to the individual an effective amount of an antibody of the invention to induce lysis of the target cell. In one embodiment, the "individual" is a human.

[0285] In certain embodiments, the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferative disorders that can be treated using the protease-activatable T cell-activating bispecific molecules of the invention include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included. In certain embodiments, the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, and head and neck cancer. Those skilled in the art will readily recognize that protease-activatable T cell activating bispecific molecules often do not provide a cure but may only provide a partial benefit. In some embodiments, any physiological change that has some benefit is also considered therapeutically beneficial. Thus, in some embodiments, the amount of protease-activatable T cell activating bispecific molecule that results in a physiological change is considered an "effective amount" or a "therapeutically effective amount." The subject, patient, or individual in need of treatment is typically a mammal, and more particularly a human.

[0286] In some embodiments, an effective amount of a protease-activatable T cell activating bispecific molecule of the invention is administered to a cell, hi other embodiments, a therapeutically effective amount of a protease-activatable T cell activating bispecific molecule of the invention is administered to an individual to treat a disease.

[0287] The appropriate dosage of the protease-activatable T cell activating bispecific molecules of the invention (used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will be determined by the type of disease being treated, the route of administration, the patient's weight, the type of T cell activating bispecific antigen-binding molecule, the severity and course of the disease, whether the T cell activating bispecific antigen-binding molecule is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's medical history and response to the protease-activatable T cell activating bispecific molecule, and the discretion of the attending physician. In any event, the physician responsible for administration will determine the amount of the protease-activatable T cell activating bispecific molecule in the composition. The concentration of the active ingredient and appropriate dose for the individual subject is determined. A variety of administration schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0288] The protease-activatable T cell-activating bispecific molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, an initial candidate dose for administration to the patient may be about 1 μg / kg-15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) of the protease-activatable T cell-activating bispecific molecule, whether by single or multiple individual administrations or by continuous infusion. A typical daily dosage may range from about 1 μg / kg-100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, treatment is usually continued until a desired suppression of disease symptoms occurs, depending on the condition. One exemplary dosage of the T cell-activating antigen-binding molecule would be in the range of about 0.005 mg / kg-about 10 mg / kg. In other non-limiting examples, dosages include about 1 microgram / kg body weight, about 5 micrograms / kg body weight, about 10 micrograms / kg body weight, about 50 micrograms / kg body weight, about 100 micrograms / kg body weight, about 200 micrograms / kg body weight, about 350 micrograms / kg body weight, about 500 micrograms / kg body weight, about 1 milligram / kg body weight, about 5 milligrams / kg body weight, about 10 milligrams / kg body weight, about 50 milligrams / kg body weight, about 100 milligrams / kg body weight, about 200 milligrams / kg body weight, about 350 milligrams / kg body weight, about 500 milligrams / kg body weight, about 1000 mg / kg body weight or more per administration, and any ranges derived therein. Non-limiting examples of ranges derived from the numbers recited herein include ranges such as about 5 mg / kg body weight to about 100 mg / kg body weight, about 5 micrograms / kg body weight to about 500 milligrams / kg body weight, based on the above numbers. Thus, the patient may be administered one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof). Such doses may be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about 2 to about 20 doses, or for example about 6 doses of the protease-activatable T cell activating bispecific molecule). An initial high loading dose may be followed by one or more lower doses. However, other dosing regimens are also useful.The progress of this therapy is easily monitored by conventional techniques and assays.

[0289] The protease-activatable T cell activating bispecific molecules of the invention are typically used in an amount effective to achieve the intended purpose. When used to treat or prevent a disease state, the protease-activatable T cell activating bispecific molecules of the invention or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0290] For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, e.g., cell culture assays. The IC determined in cell culture can then be used to 50 A dose can also be formulated in animal models to achieve a circulating concentration range that includes 100 mg / kg of the active ingredient in a compound of formula (I) or (II). Such information can be used to more accurately determine useful doses in humans.

[0291] Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques well known in the art. Those skilled in the art will be able to readily optimize human administration based on animal data.

[0292] Dosage and interval can be individually adjusted to achieve plasma concentrations of the protease-activatable T cell-activating bispecific molecule sufficient to maintain therapeutic efficacy. Typical patient dosages useful for administration by injection range from about 0.1-50 mg / kg / day, typically about 0.5-1 mg / kg / day. Therapeutically effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by HPLC.

[0293] In the case of local administration or selective uptake, the effective local concentration of the protease-activatable T cell activating bispecific molecule may not be related to plasma concentration, and one of skill in the art would be able to optimize a therapeutically effective local dose without undue experimentation.

[0294] Typically, a therapeutically effective dose of the protease-activatable T cell activating bispecific molecules described herein will provide a therapeutic effect without causing substantial toxicity. The toxicity and therapeutic effect of the protease-activatable T cell activating bispecific molecules can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies have demonstrated that LD 50 (the dose that causes death in 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50 Protease-activatable T cell activating bispecific molecules that exhibit large therapeutic indices are preferred. In one embodiment, the protease-activatable T cell activating bispecific molecules according to the invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating various dosages suitable for use in humans. Dosages are preferably administered at or above the ED with little or no toxicity. 50 The blood concentration range includes: (a) a dose of 100 mg / kg / day or more ...

[0295] The attending physician of a patient being treated with a protease-activatable T cell activating bispecific molecule of the invention will know how and when to terminate, interrupt, or adjust administration due to toxicity, organ failure, etc. Conversely, the attending physician will also know to adjust treatment to higher levels if the clinical response is not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary depending on the severity of the condition being treated, the route of administration, etc. For example, the severity of the condition may be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dose, and perhaps dosing frequency, will also vary depending on the age, weight, and response of the individual patient.

[0296] Other Medications and Treatments In a therapeutic setting, the protease-activatable T cell activating bispecific molecules of the invention may be administered in combination with one or more other agents. For example, the protease-activatable T cell activating bispecific molecules of the invention may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a condition or disease in an individual in need thereof. Such additional therapeutic agents can include any active ingredients appropriate for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that sensitizes cells to apoptosis-inducing factors. In certain embodiments, the additional therapeutic agent is an anti-cancer agent, such as a microtubule-disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.

[0297] Such other agents are preferably present in the combination in amounts effective for the intended purpose. The effective amount of such other agents will depend on the amount of protease-activatable T cell activating bispecific molecule used, the type of disorder or treatment, and other factors discussed above. Typically, the protease-activatable T cell activating bispecific molecule is used in the same dosages and by the same routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at any dosage and route determined empirically / clinically to be appropriate.

[0298] Such combination therapy encompasses combined administration (two or more therapeutic agents in the same or separate compositions) and separate administration, where administration of the protease-activatable T cell activating bispecific molecules of the invention occurs before, simultaneously with, and / or after administration of the additional therapeutic agent(s) and / or adjuvant. The protease-activatable T cell activating bispecific molecules of the invention can also be used in combination with radiation therapy.

[0299] manufactured goods In another aspect of the present invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders is provided. The article of manufacture comprises a container and a label or package insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV infusion bags, and the like. The container can be made from a variety of materials, such as glass or plastic. The container holds the composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing a medical condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a protease-activatable T-cell activating bispecific molecule of the invention. The label or package insert indicates that the composition is used for treating the selected medical condition. Additionally, the article of manufacture may comprise (a) a first container containing a composition comprising the protease-activatable T cell activating bispecific molecule of the invention; and (b) a second container containing a composition comprising an additional cytotoxic or other therapeutic agent. The article of manufacture of this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0300] Exemplary Implementations 1. A protease-activatable T cell activating bispecific molecule comprising: (a) a first antigen-binding moiety capable of binding to CD3, comprising: (i) a first antigen-binding moiety capable of binding to CD3; (a) a first antigen-binding moiety capable of binding to CD3, comprising: (i) a first antigen-binding moiety capable of binding to CD3; (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, an HCDR 2 of SEQ ID NO: 4, and an HCDR 3 of SEQ ID NO: 10; (ii) a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, an LCDR 2 of SEQ ID NO: 21, and an LCDR 3 of SEQ ID NO: 22; (b) a second antigen-binding moiety capable of binding to a target cell antigen; and (c) a masking moiety covalently attached to the T cell bispecific binding molecule via a protease-cleavable linker, the masking moiety being capable of binding to the idiotype of the first or second antigen-binding moiety, thereby reversibly masking the first antigen-binding moiety. 1. A protease-activatable T cell-activating bispecific molecule comprising: 2. The protease activatable T cell activating bispecific molecule of embodiment 1, wherein the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16, and / or the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23. 3. The protease activatable T cell activating bispecific molecule of embodiment 1 or 2, wherein the masking moiety is covalently linked to the first antigen-binding moiety and reversibly masks the first antigen-binding moiety. 4. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-3, wherein the masking moiety is covalently linked to the heavy chain variable region of the first antigen-binding moiety. 5. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-3, wherein the masking moiety is covalently linked to the light chain variable region of the first antigen-binding moiety. 6. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-5, wherein the masking moiety is an scFv. 7. The protease-activatable T cell activating bispecific molecule of any one of embodiments 2-6, comprising a second masking moiety that reversibly masks the second antigen-binding moiety. 8. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-7, wherein the protease is expressed by the target cell. 9. The protease activatable T cell activating bispecific molecule of any one of embodiments 1-8, wherein the second antigen-binding portion is a crossover Fab molecule in which the variable or constant regions of the Fab light chain and Fab heavy chain have been exchanged. 10. The protease activatable T cell activating bispecific molecule of any one of embodiments 1-9, wherein the second antigen-binding portion is a crossover Fab molecule in which the constant regions of the Fab light chain and the Fab heavy chain have been exchanged. 11. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-10, wherein the first antigen-binding portion is a conventional Fab molecule. 12. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-11, comprising up to one antigen-binding portion capable of binding to CD3. 13. The protease activatable T cell activating bispecific molecule of any one of embodiments 1-12, comprising a third antigen-binding portion which is a Fab molecule capable of binding to a target cell antigen. 14. The protease-activatable T cell activating bispecific molecule of embodiment 13, wherein the third antigen-binding portion is identical to the second antigen-binding portion. 15. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-14, wherein the second antigen-binding portion is capable of binding to FolR1 or TYRP1. 16. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-14, wherein the second antigen-binding portion is capable of binding to FolR1. 17. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-14, wherein the second antigen-binding portion is capable of binding to TYRP1. 18. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-17, wherein the first and second antigen-binding moieties are fused to each other, optionally via a peptide linker. 19. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-18, wherein the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety. 20. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-18, wherein the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety. 21. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-20, wherein the Fab light chain of the first antigen-binding moiety and the Fab light chain of the second antigen-binding moiety are fused to each other, optionally via a peptide linker. 22. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-21, additionally comprising an Fc domain composed of a first and a second subunit capable of stable association. 23. The protease-activatable T cell activating bispecific molecule according to embodiment 22, wherein the Fc domain is an IgG, particularly an IgG1 or IgG4 Fc domain. 24. The protease-activatable T cell activating bispecific molecule of embodiment 22 or 23, wherein the Fc domain is a human Fc domain. 25. The protease activatable T cell activating bispecific molecule according to any one of embodiments 22-24, wherein the Fc domain exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain. 26. The protease activatable T cell activating bispecific molecule of embodiment 25, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector function. 27. The protease activatable T cell activating bispecific molecule of embodiment 26, wherein said one or more amino acid substitutions are at one or more positions selected from the group of L234, L235 and P329 (Kabat numbering). 28. The protease activatable T cell activating bispecific molecule of embodiment 27, wherein each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to activating Fc receptors and / or effector function, said amino acid substitutions being L234A, L235A and P329G. 29. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 25-28, wherein the Fc receptor is an Fcγ receptor. 30. The protease-activatable T cell-activating bispecific molecule according to any one of embodiments 25-28, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). 31.Masking part, (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of DYSMN (SEQ ID NO: 58); (b) a CDR H2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO: 59), WINTETGEPRYTDDFTG (SEQ ID NO: 84), and WINTETGEPRYTQGFKG (SEQ ID NO: 86); (c) CDR H3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60) 31. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-30, comprising a heavy chain variable region comprising at least one of: 32.Masking part, (d) a light chain (CDR L) 1 amino acid sequence selected from the group consisting of RASKSVSTSSYSYMH (SEQ ID NO: 62) and KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of YVSYLES (SEQ ID NO: 63); and (f) a CDR L3 amino acid sequence selected from the group consisting of QHSREFPYT (SEQ ID NO: 64) and QQSREFPYT (SEQ ID NO: 88). 32. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-31, comprising a light chain variable region comprising at least one of: 33.Masking part, (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of DYSMN (SEQ ID NO: 58); (b) a CDR H2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO: 59), WINTETGEPRYTDDFTG (SEQ ID NO: 84), and WINTETGEPRYTQGFKG (SEQ ID NO: 86); (c) CDR H3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) a light chain (CDR L) 1 amino acid sequence selected from the group consisting of RASKSVSTSSYSYMH (SEQ ID NO: 62) and KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of YVSYLES (SEQ ID NO: 63); and (f) a CDR L3 amino acid sequence selected from the group consisting of QHSREFPYT (SEQ ID NO: 64) and QQSREFPYT (SEQ ID NO: 88). and a light chain variable region comprising: 34.Masking part, (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of DYSMN (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 59); (c) CDR H3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 62); (e) the CDR L2 amino acid sequence of YVSYLES (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHSREFPYT (SEQ ID NO: 64) and a light chain variable region comprising: 35.Masking part, (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID NO: 59); (c) CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising: 36.Masking part, (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO: 84); (c) CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising: 37.Masking part, (a) the heavy chain complementarity-determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) CDR H2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 86); (c) CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L)1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising: 38. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-37, wherein the masking moiety is humanized. 39. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-38, wherein the masking moiety is human. 40. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-39, wherein the protease-cleavable linker comprises at least one protease recognition sequence. 41. The protease-activatable T cell activating bispecific molecule according to embodiment 40, wherein the protease-cleavable linker comprises a protease recognition sequence. 42. A protease recognition sequence is (a) RQARVVNG (SEQ ID NO: 100); (b) VHMPLGFLGPGRSRGSFP (SEQ ID NO: 101); (c) RQARVVNGXXXXXVPLSLYSG (SEQ ID NO: 102) (wherein X is any amino acid); (d) RQARVVNGVPLSLYSG (SEQ ID NO: 103); (e) PLGLWSQ (SEQ ID NO: 104); (f) VHMPLGFLGPRQARVVNG (SEQ ID NO: 105); (g) FVGGTG (SEQ ID NO: 106); (h) KKAAPVNG (SEQ ID NO: 107); (i) PMAKKVNG (SEQ ID NO: 108); (j) QARAKVNG (SEQ ID NO: 109); (k) VHMPLGFLGP (SEQ ID NO: 110); (l) QARAK (SEQ ID NO: 111); (m) VHMPLGFLGPPMAKK (SEQ ID NO: 112); (n) KKAAP (SEQ ID NO: 113); and (o) PMAKK (SEQ ID NO: 114) 42. The protease activatable T cell activating bispecific molecule according to embodiment 41, selected from the group consisting of: 43. The protease activatable T cell activating bispecific molecule according to embodiment 41 or 42, wherein the protease cleavable linker comprises the protease recognition sequence PMAKK (SEQ ID NO: 114). 44. The protease activatable T cell activating bispecific molecule according to embodiment 40 or 41, wherein the protease cleavable linker comprises the protease recognition sequence VHMPLGFLGPPMAKK (SEQ ID NO: 112). 45. The protease activatable T cell activating bispecific molecule according to embodiment 40 or 41, wherein the protease cleavable linker comprises the protease recognition sequence VHMPLGFLGPRQARVVNG (SEQ ID NO: 105). 46. ​​The protease-activated T cell activating bispecific molecule according to embodiment 40 or 41, wherein the protease-cleavable linker comprises the protease recognition sequence RQARVVNG (SEQ ID NO: 100) or the protease recognition sequence VHMPLGFLGPRQARVVNG (SEQ ID NO: 105). 47. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-46, wherein the protease is selected from the group consisting of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic acid proteases, and cathepsin proteases. 48. The protease-activatable T cell activating bispecific molecule according to embodiment 47, wherein the metalloproteinase is a matrix metalloproteinase (MMP), preferably MMP9 or MMP2. 49. The protease-activatable T cell activating bispecific molecule according to embodiment 47, wherein the serine protease is matriptase. 50. The protease activatable T cell activating bispecific molecule of any one of embodiments 1-49, wherein the second antigen-binding portion is capable of binding to FolR1 and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56 and / or at least one light chain CDR selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. 51. The protease activatable T cell activating bispecific molecule of any one of embodiments 1-49, wherein the second antigen-binding portion is capable of binding to FolR1 and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, and at least one light chain CDR selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. 52. The second antigen-binding moiety is capable of binding to FolR1, and a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of NAWMS (SEQ ID NO: 54); b) the CDR H2 amino acid sequence of RIKSKTDGGTTDYAAPVKG (SEQ ID NO: 55); and c) CDR H3 amino acid sequence of PWEWSWYDY (SEQ ID NO: 56) a heavy chain variable region comprising d) the light chain (CDR L) 1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 20); e) the CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO: 21); and (f) CDR L3 amino acid sequence of ALWYSNLWV (SEQ ID NO: 22) and a light chain variable region comprising: 53. The protease activatable T cell activating bispecific molecule of any one of embodiments 1-52, wherein the second antigen-binding portion comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53, and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23. 54. The protease-activatable T cell-activating bispecific molecule of any one of embodiments 1-53, wherein the second antigen-binding portion is capable of binding to FolR1 and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23. 55. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-49, wherein the second antigen-binding portion is capable of binding to TYRP1 and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and / or at least one light chain CDR selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30. 56. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-49 and 55, wherein the second antigen-binding portion is capable of binding to TYRP1 and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and at least one light chain CDR selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30. 57. The second antigen-binding moiety is capable of binding to TYRP1, and a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of NAWMS (SEQ ID NO: 24); b) the CDR H2 amino acid sequence of RIKSKTDGGTTDYAAPVKG (SEQ ID NO: 25); and c) CDR H3 amino acid sequence of PWEWSWYDY (SEQ ID NO: 26) a heavy chain variable region comprising d) the light chain (CDR L) 1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 28); e) the CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO: 29); and f) CDR L3 amino acid sequence of ALWYSNLWV (SEQ ID NO: 30) 57. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-49 and 55-56, comprising a light chain variable region comprising: 58. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-49 and 55-57, wherein the second antigen-binding portion comprises a heavy chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27, and a light chain variable region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31. 59. The protease-activatable T cell-activating bispecific molecule of any one of embodiments 1-53, wherein the second antigen-binding portion is capable of binding to TYRP1 and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31. 60. A protease-activatable T cell-activating bispecific molecule, comprising: a) at least one heavy chain comprising the amino acid sequence of SEQ ID NO: 66; b) at least one light chain comprising the amino acid sequence of SEQ ID NO: 67 55. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-54, comprising: 61. A protease-activatable T cell-activating bispecific molecule, comprising: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 65; (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66; and (c) a light chain comprising the amino acid sequence of SEQ ID NO: 67 51. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-50, comprising: 62. A protease-activatable T cell-activating bispecific molecule, comprising: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 69; (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66; and (c) a light chain comprising the amino acid sequence of SEQ ID NO: 67 55. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-54, comprising: 63. A protease-activatable T cell-activating bispecific molecule, comprising: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 74; (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66; and (c) a light chain comprising the amino acid sequence of SEQ ID NO: 67 55. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-54, comprising: 64. A protease-activatable T cell-activating bispecific molecule, comprising: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 76; (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66; and (c) a light chain comprising the amino acid sequence of SEQ ID NO: 67 55. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-54, comprising: 65. A protease-activatable T cell-activating bispecific molecule, comprising: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 95; (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66; and (c) a light chain comprising the amino acid sequence of SEQ ID NO: 67 55. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-54, comprising: 66. A protease-activatable T cell-activating bispecific molecule, comprising: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 96; (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66; and (c) a light chain comprising the amino acid sequence of SEQ ID NO: 67 55. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-54, comprising: 67. A protease-activatable T cell-activating bispecific molecule, comprising: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 97; (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66; and (c) a light chain comprising the amino acid sequence of SEQ ID NO: 67 55. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-54, comprising: 68. A protease-activatable T cell-activating bispecific molecule, comprising: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 98; (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 66; and (c) a light chain comprising the amino acid sequence of SEQ ID NO: 67 55. The protease activatable T cell activating bispecific molecule according to any one of embodiments 1-54, comprising: 69. A protease-activatable T cell-activating bispecific molecule, comprising: (c) two light chains comprising the amino acid sequence of SEQ ID NO: 67 69. The protease activatable T cell activating bispecific molecule according to any one of embodiments 60-68, comprising: 70. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-69, wherein the masking moiety comprises an scFv comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 91. 71. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-69, wherein the masking moiety comprises an scFv comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 92. 72. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-69, wherein the masking moiety comprises an scFv comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 93. 73. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-69, wherein the masking moiety comprises an scFv comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 94. 74. The protease activatable T cell activating bispecific molecule according to any one of embodiments 70-73, wherein the binding affinity of the masking moiety to the first antigen-binding moiety, as measured by SPR, is approximately the same as or higher than the binding affinity of a masking moiety comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94. 75. An idiotype-specific polypeptide capable of reversibly masking an anti-CD3 antigen binding site of a molecule, comprising a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:79, SEQ ID NO:83 and SEQ ID NO:85, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:80 and SEQ ID NO:81. 76. The idiotype-specific polypeptide of claim 28, comprising a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 79, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 80. 77. The idiotype-specific polypeptide of claim 28, comprising a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 79, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81. 78. The idiotype-specific polypeptide of claim 28, comprising a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 83, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81. 79. The idiotype-specific polypeptide of claim 28, comprising a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 85, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81. 80. The idiotype-specific polypeptide according to embodiment 75, which is an anti-idiotype scFv, an anti-idiotype Fab or an anti-idiotype scFab. 81. An idiotype-specific polypeptide according to any one of embodiments 75-80, which is an scFv. 82. The idiotype-specific polypeptide according to any one of embodiments 75-80, which is covalently linked to the molecule via a linker. 83. The idiotype-specific polypeptide according to embodiment 82, wherein the linker is a peptide linker. 84. The idiotype-specific polypeptide according to embodiment 82 or 83, wherein the linker is a protease-cleavable linker. 85. The idiotype-specific polypeptide according to any one of embodiments 82-84, wherein the peptide linker comprises at least one protease recognition sequence. 86. The idiotype-specific polypeptide of embodiment 85, wherein the protease is selected from the group consisting of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic acid proteases, and cathepsin proteases. 87. The idiotype-specific polypeptide according to embodiment 91, wherein the metalloproteinase is a matrix metalloproteinase (MMP), preferably MMP9 or MMP2. 88. The idiotype-specific polypeptide according to embodiment 86, wherein the serine protease is matriptase. 89. A protease recognition sequence is (a) RQARVVNG (SEQ ID NO: 100); (b) VHMPLGFLGPGRSRGSFP (SEQ ID NO: 101); (c) RQARVVNGXXXXXVPLSLYSG (SEQ ID NO: 102) (wherein X is any amino acid); (d) RQARVVNGVPLSLYSG (SEQ ID NO: 103); (e) PLGLWSQ (SEQ ID NO: 104); (f) VHMPLGFLGPRQARVVNG (SEQ ID NO: 105); (g) FVGGTG (SEQ ID NO: 106); (h) KKAAPVNG (SEQ ID NO: 107); (i) PMAKKVNG (SEQ ID NO: 108); (j) QARAKVNG (SEQ ID NO: 109); (k) VHMPLGFLGP (SEQ ID NO: 110); (l) QARAK (SEQ ID NO: 111); (m) VHMPLGFLGPPMAKK (SEQ ID NO: 112); (n) KKAAP (SEQ ID NO: 113); and (o) PMAKK (SEQ ID NO: 114) 89. The idiotype-specific polypeptide according to any one of embodiments 85-88, selected from the group consisting of: 90. The idiotype-specific polypeptide according to any one of embodiments 80-83, wherein the protease-cleavable linker comprises the protease recognition sequence PMAKK (SEQ ID NO: 114). 91. The idiotype-specific polypeptide according to any one of embodiments 80-90, which is part of a T cell activating bispecific molecule. 92. The idiotype-specific polypeptide of any one of embodiments 75-92, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 80. 93. The idiotype-specific polypeptide of any one of embodiments 75-92, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 81. 94. The idiotype-specific polypeptide of any one of embodiments 75-92, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 83, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 84. 95. The idiotype-specific polypeptide of any one of embodiments 75-92, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 85, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 86. 96. The idiotype-specific polypeptide of any one of embodiments 75-92, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 84, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 87. 97. The idiotype-specific polypeptide of any one of embodiments 75-92, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 89, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 90. 98. The idiotype-specific polypeptide according to any one of embodiments 75-97, wherein the anti-CD3 antigen-binding site comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23. 99. The idiotype-specific polypeptide according to any one of embodiments 75-98, which is humanized. 100. An isolated polynucleotide encoding a protease-activatable T cell-activating bispecific antigen-binding molecule according to any one of embodiments 1-74 or an idiotype-specific polypeptide according to any one of embodiments 75-99. 101. A polypeptide encoded by the polynucleotide of embodiment 100. 102. A vector, particularly an expression vector, comprising a polynucleotide according to embodiment 100. 103. A host cell comprising a polynucleotide according to embodiment 99 or a vector according to embodiment 102. 104. A method for producing a protease-activatable T cell activating bispecific molecule, comprising the steps of: a) culturing a host cell according to embodiment 103 under conditions suitable for expression of the protease-activatable T cell activating bispecific molecule; and b) recovering the protease-activatable T cell activating bispecific molecule. 105. A protease-activatable T cell-activating bispecific molecule produced by the method of embodiment 104. 106. A method for producing an idiotype-specific polypeptide, comprising the steps of: a) culturing a host cell according to embodiment 103 under conditions suitable for expression of the idiotype-specific polypeptide; and b) recovering the idiotype-specific polypeptide. 107. An idiotype-specific polypeptide produced by the method of embodiment 106. 108. A pharmaceutical composition comprising a protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-74 and a pharmaceutically acceptable carrier. 109. A pharmaceutical composition comprising an idiotype-specific polypeptide according to any one of embodiments 75-99 and a pharmaceutically acceptable carrier. 110. A protease-activatable T cell-activating bispecific molecule according to any one of embodiments 1-74, an idiotype-specific polypeptide according to any one of embodiments 75-99, or a composition according to embodiment 108, for use as a medicament. 111. The protease-activatable T-cell activating bispecific molecule of embodiment 110, wherein the medicament is for treating or delaying the progression of cancer, treating or delaying the progression of an immune-related disease, or enhancing or stimulating an immune response or function in an individual. 112. A protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-74 or an idiotype-specific polypeptide according to any one of embodiments 75-99 for use in the treatment of a disease in an individual in need thereof. 113. The protease-activatable T-cell activating bispecific molecule or idiotype-specific polypeptide according to embodiment 112 for use in treating a disease in an individual in need thereof, wherein the disease is cancer. 114. Use of a protease-activatable T cell-activating bispecific molecule according to any one of embodiments 1-74 or an idiotype-specific polypeptide according to any one of embodiments 75-99 for the manufacture of a medicament for the treatment of a disease. 115. The use according to embodiment 114, wherein the disease is cancer. 116. A method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising a protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-74, or a composition according to embodiment 108. 117. A method for inducing lysis of target cells, comprising contacting the target cells with a protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-74 or a composition according to embodiment 108, in the presence of T cells. 118. The method of embodiment 117, wherein the target cells are cancer cells. 119. The method of embodiment 117 or 118, wherein the target cell expresses a protease capable of activating the protease-activatable T cell activating bispecific molecule. 120. A humanized anti-idiotype CD3 antibody or antigen-binding fragment thereof specific for the idiotype of an anti-CD3 antigen-binding molecule, which, when bound to the anti-CD3 antigen-binding molecule, specifically blocks the binding of the anti-CD3 antigen-binding molecule to CD3. 121. The anti-idiotype CD3 antibody or antigen-binding fragment thereof according to embodiment 120, wherein the anti-idiotype CD3 antibody or fragment thereof is reversibly associated with the anti-CD3 antigen-binding molecule via a peptide linker comprising a protease recognition site. 122. The anti-idiotypic CD3 antibody or antigen-binding fragment thereof according to embodiment 120 or 121, wherein CD3 is murine, monkey or human CD3. 123. A method for reducing the in vivo toxicity of a T cell activating bispecific molecule, comprising linking an idiotype-specific polypeptide according to any one of embodiments 75-99 to a T cell activating bispecific molecule via a protease-cleavable linker to form a protease-activatable T cell activating bispecific molecule, wherein the in vivo toxicity of the protease-activatable T cell activating bispecific molecule is reduced compared to the toxicity of the T cell activating bispecific molecule. 124. The above mentioned invention.

[0301] Exemplary Sequences JPEG0007815150000002.jpg255170JPEG0007815150000003.jpg255170JPEG00078151500 00004.jpg255170JPEG0007815150000005.jpg255170JPEG0007815150000006.jpg245170

[0302] Protease-activatable T-cell activating bispecific molecules containing improved anti-CD3 (P035.093) binders JPEG0007815150000007.jpg250170JPEG0007815150000008.jpg255170JPEG0007815150000009.jpg255170 JPEG0007815150000010.jpg255170JPEG0007815150000011.jpg255170JPEG0007815150000012.jpg118170

[0303] JPEG0007815150000013.jpg245170JPEG0007815150000014.jpg207170

[0304] JPEG0007815150000015.jpg146170

[0305] JPEG0007815150000016.jpg252170JPEG0007815150000017.jpg255170JPEG0007815150000018.jpg179170

[0306] JPEG0007815150000019.jpg251170JPEG0007815150000020.jpg167170 [Example]

[0307] The following are examples of the methods and compositions of the present invention. Given the above general description, it will be understood that various other embodiments may be practiced.

[0308] Example 1 - Preparation of optimized anti-CD3 (multispecific) antibodies The optimized anti-CD3 antibodies (clones P033.078, P035.093, P035.064, P021.045, and P004.042) were all generated by a phage display selection campaign using a library derived from a previously described CD3 binder (also referred to herein as "CD3orig") (see, e.g., WO 2014 / 131712, incorporated herein by reference), and comprise the VH and VL sequences of SEQ ID NOs: 14 and 23, respectively. In these libraries, positions N97 and N100 (Kabat numbering) in the CDR3 region of the heavy chain were silenced or deleted. For direct comparison, all molecules were converted into a T cell bispecific antibody (TCB) format using an anti-TYRP1 antibody as an exemplary target cell antigen binding site (SEQ ID NOs: 24-31), as depicted in Figure 2A.

[0309] As shown in Figures 2B-E, the variable regions of the heavy and light chain DNA sequences were subcloned in frame with the constant heavy or constant light chain previously inserted into the respective recipient mammalian expression vector.

[0310] The sequences of the optimized anti-CD3 antibodies are shown in the SEQ ID NOs shown in Table 1.

[0311] Table 1. Sequences of optimized anti-CD3 antibodies generated in examples of the present invention JPEG0007815150000021.jpg77170

[0312] To further improve the correct pairing of the light chain with the corresponding heavy chain, mutations were introduced into the human CL (E123R, Q124K) and human CH1 (K147E, K213E) of the TYRP1-binding Fab molecule.

[0313] To ensure precise heavy chain pairing (heterodimeric molecule formation), knob-into-hole mutations were introduced into the constant region of each antibody heavy chain (T366W / S354C, T366S / L368A / Y407V / Y349C, respectively).

[0314] Furthermore, P329G, L234A, and L235A mutations were introduced into the constant region of each antibody heavy chain to abolish binding to Fcγ receptors.

[0315] The complete sequences of the prepared TCB molecules are shown in SEQ ID NOs: 32, 33, 34 and 36 (P033.078), 32, 33, 34 and 37 (P035.093), 32, 33, 34 and 38 (P035.064), 32, 33, 34 and 39 (P021.045), and 32, 33, 34 and 40 (P004.042).

[0316] CD3 orig A corresponding molecule containing as a CD3 binder was also prepared.

[0317] TCB was prepared by Evitria (Switzerland) using a proprietary vector system and conventional (non-PCR-based) cloning techniques in suspension-adapted CHO K1 cells (originally acquired from ATCC and adapted for serum-free culture in suspension at Evitria). For production, Evitria used proprietary animal-component-free serum-free media (eviGrow and eviMake2) and a proprietary transfection reagent (eviFect). These cells were transfected with the corresponding expression vectors in a 1:1:2:1 ratio (vector knob heavy chain:vector hole heavy chain:vector CD3 light chain:vector TYRP1 light chain). The supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the collected supernatant using standard methods.

[0318] Briefly, Fc-containing proteins were purified from filtered cell culture supernatants by Protein A affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). After elution at pH 3.0, the pH of the sample was immediately neutralized. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15, #UFC903096), and aggregated proteins were separated from monomeric proteins by size-exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0319] The concentration of purified protein was determined by measuring absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423. Protein purity and molecular weight were analyzed by CE-SDS in the presence and absence of reducing agents using a LabChip GXII (Perkin Elmer). Aggregate content was measured by HPLC chromatography at 25°C using an analytical size-exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated with running buffer (25 mM KHPO, 125 mM NaCl, 200 mM L-arginine monohydrochloride, pH 6.7 or 200 mM KHPO, 250 mM KCl, pH 6.2, respectively).

[0320] The results of biochemical and biophysical analysis of the prepared TCB molecules are shown in Table 2.

[0321] All TCB molecules could be produced with good quality.

[0322] Table 2. Biochemical and biophysical analysis of anti-CD3 antibodies in TCB format. JPEG0007815150000022.jpg81170

[0323] Example 2 - Determining the thermal stability of optimized anti-CD3 (multispecific) antibodies The thermal stability of the anti-CD3 antibody (TCB format) prepared in Example 1 was monitored by dynamic light scattering (DLS) using an Optim2 instrument (Avacta Analytical, UK) and by monitoring the temperature-dependent intrinsic protein fluorescence by applying a temperature ramp.

[0324] A 10 μg filtered protein sample at a protein concentration of 1 mg / ml was run in duplicate through Optim2. The temperature was ramped from 25°C to 85°C at 0.1°C / min, and the ratio of fluorescence intensity at 350 nm / 330 nm to scattering intensity at 266 nm was collected.

[0325] The results are shown in Table 3. The aggregation temperatures (T agg ) and the midpoint of the observed temperature-induced unfolding transition (T m ) is the CD3 binder CD3 orig It is equal to or better than that of

[0326] Table 3. Thermal stability of anti-CD3 antibodies in TCB format as measured by dynamic light scattering and temperature-dependent changes in intrinsic protein fluorescence. JPEG0007815150000023.jpg73170

[0327] Example 3 - Functional characterization of optimized anti-CD3 (multispecific) antibodies by surface plasmon resonance (SPR) Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 at 25°C using HBS-EP+ (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20; Biacore, Freiburg, Germany) as the running buffer.

[0328] For affinity measurements, TCB molecules were captured on a C1 sensor chip (GE Healthcare) surface immobilized with anti-Fc(P329G) IgG (an antibody that specifically binds to human IgG1Fc(P329G); "anti-PG antibody"—see WO 2017 / 072210, incorporated herein by reference). The experimental setup is shown schematically in Figure 3. Capture IgG was coupled to the sensor chip surface by direct immobilization of approximately 400 resonance units (RU) using a standard amine coupling kit (GE Healthcare Life Sciences).

[0329] To analyze interactions with CD3, TCB molecules were captured at 25 nM for 80 s at a flow rate of 10 μl / min. Human and cynomolgus CD3ε stalk-Fc(knob)-Avi / CD3δ stalk-Fc(hole) (CD3ε / δ, see SEQ ID NOs: 41 and 42 (human) and SEQ ID NOs: 43 and 44 (cynomolgus)) were passed through the flow cell at concentrations of 0.122–125 nM at a flow rate of 30 μl / min for 300 s. Dissociation was monitored for 800 s.

[0330] Bulk refractive index differences were corrected by subtracting the response obtained with a reference flow cell, in which the antigen flew over a surface on which anti-PG antibodies were immobilized but on which HBS-EP was injected instead of TCB molecules.

[0331] The reaction rate constants were derived using Biacore T200 Evaluation Software (GE Healthcare Life Sciences), and the reaction rate equation was fitted to a 1:1 Langmuir binding equation by numerical integration. The half-life of the interaction (t 1 / 2 ) into the equation t 1 / 2 =ln2 / k off was calculated using

[0332] Table 4 shows the binder CD3 orig All kinetic parameters for the binding of the optimized anti-CD3 antibody compared to the TCB format are listed. The optimized anti-CD3 antibody (TCB format) exhibits K values ​​ranging from low nM to high pM. D binds to CD3ε / δ with K values ​​ranging from 600 pM to 1.54 nM for human CD3ε / δ and from 200 pM to 700 pM for cynomolgus monkey CD3ε / δ D CD3 orig In comparison, the binding affinity of the optimized anti-CD3 antibody to human CD3ε / δ is improved by 7 to 10-fold when measured under identical conditions by SPR.

[0333] The half-life of monovalent binding to human CD3ε / δ was 11.6 minutes for the anti-CD3 antibody clone P033.078, and 11.6 minutes for CD3 orig The binding half-life of

[0334] Table 4. Affinity of anti-CD3 antibodies (TCB format) to human CD3ε / δ and cynomolgus monkey CD3ε / δ JPEG0007815150000024.jpg127170

[0335] Example 4 - Characterization of optimized anti-CD3 (multispecific) antibodies after stress by surface plasmon resonance (SPR) To evaluate the removal of deamidation sites and its effect on antibody stability, optimized anti-CD3 antibodies (TCB format) were incubated at 37°C, pH 7.4 and 40°C, pH 6 for 14 days and further analyzed for their binding ability to human CD3ε / δ by SPR. Samples stored at -80°C, pH 6 were used as reference. The reference sample and the 40°C-stressed sample were in 20 mM His, 140 mM NaCl, pH 6.0, while the 37°C-stressed sample was in PBS, pH 7.4, all at a concentration of 1.0 mg / ml. After the stress period (14 days), the samples were dialyzed back into 20 mM His, 140 mM NaCl, pH 6.0 in PBS for further analysis.

[0336] All SPR experiments were performed on a Biacore T200 instrument (GE Healthcare) at 25 °C using HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) as the running and dilution buffer. Biotinylated human CD3ε / δ (see Example 3, SEQ ID NOs: 41 and 42) and biotinylated anti-huIgG (Capture Select, Thermo Scientific, #7103262100) were immobilized on a Series S Sensor Chip SA (GE Healthcare, #29104992) to obtain a surface density of at least 1000 resonance units (RU). Anti-CD3 antibody at a concentration of 2 μg / ml was injected for 30 s at a flow rate of 5 μl / min, and dissociation was monitored for 120 s. The surface was regenerated by injecting 10 mM glycine pH 1.5 for 60 s. Bulk refractive index differences were corrected by subtracting a blank injection and the response obtained from a blank control flow cell. The binding response was evaluated 5 seconds after the end of the injection. To normalize the binding signal, CD3 binding was divided by the anti-huIgG response (signal (RU) obtained upon capture of CD3 antibodies on immobilized anti-huIgG antibodies). Relative binding activity was calculated by benchmarking each temperature-stressed sample to the corresponding unstressed sample.

[0337] As shown in Table 5, all of the anti-CD3 antibodies prepared in Example 1 were CD3 orig It can be seen that binding to CD3ε / δ under stress is improved compared to

[0338] Table 5. Binding activity of anti-CD3 antibodies (TCB format) to human CD3ε / δ after 2 weeks of incubation at pH 6 / 40°C or pH 7.4 / 37°C. JPEG0007815150000025.jpg81170

[0339] Example 5 - Jurkat NFAT reporter cell assay using optimized anti-CD3 (multispecific) antibodies The optimized anti-CD3 antibody-containing TCB (targeting TYRP1) was tested in a Jurkat NFAT reporter cell assay in the presence of CHO-K1 TYRP1 clone 76 (generated by stable transfection of CHO-K1 cells) as target cells. Jurkat NFAT reporter cells (Promega) were cultured at 0.1-0.5 microcells / ml in RPMI 1640 (Gibco) containing 10% FBS, 2 g / L glucose (Sigma), 2 g / L NaHCO₃ (Sigma), 25 mM HEPES (Gibco), 1% GlutaMax (Gibco), 1x NEAA (Sigma), and 1% SoPyr (Sigma) (Jurkat NFAT medium). CHO-K1 TYRP1 clone 76 cells were cultured in DMEM / F12 + GlutaMAX (1x) (Gibco) containing 10% FBS and 6 μg / ml puromycin (Invivogen). The assay was performed in Jurkat NFAT medium.

[0340] CHO-K1 TYRP1 clone 76 cells were detached using trypsin (Gibco). Cells were counted and viability confirmed. Target cells were resuspended in assay medium and seeded at 10,000 cells per well in a white, flat-bottom 384-well plate. TCB was then added at the indicated concentrations. Jurkat NFAT reporter cells were counted and viability confirmed, and 20,000 cells were seeded per well, corresponding to an effector-to-target (E:T) ratio of 2:1. 2% final volume of GloSensor cAMP Reagent (E1291, Promega) was also added to each well. After the indicated incubation period, luminescence was measured using a Tecan Spark10M instrument.

[0341] As shown in Figure 4A-B, the optimized anti-CD3 antibody-containing TCB inhibited the parent binder CD3 orig The TCBs tested induced CD3 activation in a concentration-dependent manner.

[0342] Example 6 - Tumor cell killing of primary melanoma cells by optimized anti-CD3 (multispecific) antibodies The optimized anti-CD3 antibody (targeting TYRP1) in TCB format was tested in a tumor cell killing assay using freshly isolated human PBMCs co-incubated with the human melanoma cell line M150543 (a primary melanoma cell line obtained from the Dermatology Cell Bank at the University of Zurich). Tumor cell lysis was determined by quantification of LDH released into the cell supernatant by apoptotic or necrotic cells after 24 and 48 hours. CD4 and CD8 T cell activation was analyzed by upregulation of CD69 and CD25 in both cell subsets after 48 hours.

[0343] On the day before the start of the assay, target cells (M150543) were detached using trypsin (Gibco), washed once with PBS, and resuspended in growth medium (RPMI1640 (Gibco) containing 10% FBS, 1% GlutaMax (Gibco), and 1% SoPyr (Sigma)) at a density of 0.3 mio cells / ml. 100 μl of this cell suspension (containing 30,000 cells) was seeded into a 96-well flat-bottom plate. The cells were incubated overnight at 37°C in an incubator.

[0344] The next day, PBMCs were isolated from healthy donor blood and confirmed for viability. The medium was removed from the plated target cells, and 100 μl of assay medium (RPMI 1640 (Gibco) containing 2% FBS and 1% GlutaMax (Gibco)) was added to each well. Antibodies were diluted to the indicated concentrations in assay medium and added to the target cells at 50 μl per well. Assay medium was added to control wells. Isolated PBMCs were resuspended at a density of 6 mio cells / ml and added at 50 μl per well, for a total of 300,000 cells / well (E:T 10:1). To measure spontaneous LDH release (minimum lysis = 0%), PBMCs were co-cultured with target cells alone. To measure maximum LDH release (maximum lysis = 100%), target cells were added with assay medium alone. Control wells containing PBMCs and TCB in the absence of target cells were used to test the specificity of the TCB. To determine whether CD8 and CD4 T cells were activated in the absence of target-expressing tumor cells, CD25 expression was analyzed after 48 hours.

[0345] A few hours before the first LDH measurement, 50 μl of assay medium containing 4% Triton X-100 (Bio-Rad) was added to wells containing only target cells (resulting in a final concentration of 1% Triton X-100 per well) to maximize LDH release. The assay was incubated for a total of 48 hours at 37°C in an incubator. The first LDH measurement was performed 24 hours after the start of the assay. For this purpose, a Cytotoxicity Detection Kit (LDH) (Roche / Sigma, #11644793001) was allowed to warm to room temperature before measurement. The assay plate was centrifuged at 420 × g for 4 minutes, and 50 μl of supernatant per well was transferred to a 96-well flat-bottom plate for analysis. A reaction mixture of 1.25 μl of LDH catalyst and 56.25 μl of LDH substrate was then prepared per well. 50 μl of the LDH reaction mixture was then added to each well, and the absorbance was immediately measured using a TECAN Infinite F50 instrument. This measurement was repeated 48 hours after the start of the assay.

[0346] PBMCs were then harvested and analyzed for upregulation of CD25 and CD69 activation. Specifically, 100 μl of FACS buffer was added to each well, and the cells were transferred to a 96-well U-bottom plate for FACS staining. The plate was centrifuged at 400 × g for 4 minutes, the supernatant was removed, and the cells were washed with 150 μl of FACS buffer per well. The plate was centrifuged again at 400 × g for 4 minutes, and the supernatant was removed. An antibody mix containing CD4 APC (clone RPA-T4, BioLegend), CD8 FITC (clone SK1, BioLegend), CD25 BV421 (clone BC96, BioLegend), and CD69 PE (clone FN50, BioLegend) was then added at 30 μl per well. The cells were then incubated in a refrigerator for 30 minutes. The cells were then washed twice with FACS buffer and resuspended in 100 μl of FACS buffer containing 1% PFA per well. Before measurement, the cells were resuspended in 150 μl of FACS buffer. Analysis was performed using a BD LSR Fortessa instrument.

[0347] Treatment with TCB containing anti-CD3 antibody clones P035.093 and P021.045 resulted in the greatest tumor cell killing, while clones P033.078 and P035.064 resulted in intermediate tumor cell killing, followed by clone P004.042, which suppressed the parental binder CD3 orig T cell activation was highest when treated with TCB containing anti-CD3 antibody clones P035.093 and P021.045, whereas TCB containing other anti-CD3 antibody clones induced comparable tumor cell killing compared with TCB containing the parental binder CD3 (Figure 5A-B). orig The TCB induced T cell activation comparable to that of TCB containing α-glucan (Figure 6A-D).

[0348] As shown in Figures 7A-B, the tested TCBs did not induce CD25 upregulation on CD8 and CD4 T cells in the absence of tumor target cells, indicating that the tested CD3 binders induce T cell activation dependent on cross-linking, e.g., via binding to tumor cells, and are unable to induce T cell activation in a monovalent format.

[0349] Example 7 - Preparation of optimized anti-CD3 antibodies Optimized anti-CD3 antibody clones P033.078, P035.093 and P004.042 were converted into a monovalent human IgG1 format with cross-over VH and VL domains on the CD3 binding moiety as shown in Figure 8A.

[0350] As shown in Figures 8B-D, the variable regions of the heavy and light chain DNA sequences were subcloned in frame with the constant heavy or constant light chain previously inserted into the respective recipient mammalian expression vector.

[0351] To ensure precise heavy chain pairing (heterodimeric molecule formation), knob-into-hole mutations were introduced into the constant region of each antibody heavy chain (T366W / S354C, T366S / L368A / Y407V / Y349C, respectively).

[0352] Furthermore, P329G, L234A, and L235A mutations were introduced into the constant region of each antibody heavy chain to abolish binding to Fcγ receptors.

[0353] CD3 orig A corresponding molecule containing as a CD3 binder was also prepared.

[0354] Monovalent IgG molecules were prepared at Evitria (Switzerland) and purified and analyzed as described for the TCB molecules in Example 1. For transfection of these cells, the corresponding expression vectors were used in a 1:1:1 ratio ("vector knob heavy chain":"vector hole heavy chain":"vector light chain").

[0355] The results of the biochemical and biophysical analysis of the prepared monovalent IgG molecules are shown in Table 6.

[0356] All monovalent IgG molecules could be produced with good quality.

[0357] Table 6. Biochemical and biophysical characterization of anti-CD3 antibodies in monovalent IgG format JPEG0007815150000026.jpg65170

[0358] Example 8 - Determining the thermal stability of optimized anti-CD3 antibodies The thermal stability of the anti-CD3 antibody in monovalent IgG format (prepared in Example 19) was monitored by dynamic light scattering (DLS) and by monitoring temperature-dependent intrinsic protein fluorescence as described in Example 2.

[0359] The results are shown in Table 7. The aggregation temperatures (T agg ) and the midpoint of the observed temperature-induced unfolding transition (T m ) is the CD3 binder CD3 orig It is equal to or better than that of

[0360] Table 7. Thermal stability of anti-CD3 antibodies in monovalent IgG format measured by dynamic light scattering and temperature-dependent changes in intrinsic protein fluorescence. JPEG0007815150000027.jpg57170

[0361] Example 9 - Functional characterization of optimized anti-CD3 antibodies by surface plasmon resonance (SPR) SPR experiments were performed as described in Example 3 using the monovalent IgG molecules prepared in Example 7.

[0362] To analyze interactions with CD3, monovalent IgG molecules were captured at 50 nM for 240 s at a flow rate of 5 μl / min. Human and cynomolgus CD3ε stalk-Fc(knob)-Avi / CD3δ stalk-Fc(hole) were passed through the flow cell at concentrations ranging from 0.061 to 250 nM at a flow rate of 30 μl / min for 300 s. Dissociation was monitored for 800 s.

[0363] Table 8 shows the binder CD3 orig All kinetic parameters for binding of the optimized anti-CD3 antibody compared to the IgG are listed. The optimized anti-CD3 antibody (monovalent IgG format) exhibits K values ​​ranging from the low nM to high pM range. D binds to CD3ε / δ with K values ​​ranging from 770 pM to 1.36 nM for human CD3ε / δ and from 200 pM to 400 pM for cynomolgus monkey CD3ε / δ D CD3 orig In comparison, the binding affinity of the optimized anti-CD3 antibodies to human CD3ε / δ is improved by 3.5 to 15-fold when measured under identical conditions by SPR.

[0364] The half-life of monovalent binding to human CD3ε / δ was 8.69 min for the anti-CD3 antibody clone P033.078, and 8.69 min for CD3 orig This is more than twice the binding half-life of

[0365] Table 8. Affinity of anti-CD3 antibodies (monovalent IgG format) to human CD3ε / δ and cynomolgus monkey CD3ε / δ. Data from triplicate determinations. JPEG0007815150000028.jpg108170

[0366] Example 10 - Production of an anti-idiotype mask Preparation and evaluation of anti-idiotype masks as chimeric IgG The chimeric IgG described here was produced by Evitria using a proprietary vector system and conventional (non-PCR-based) cloning technology in suspension-adapted CHO K1 cells (originally obtained from ATCC and adapted by Evitria for serum-free growth in suspension culture). For production, Evitria used proprietary animal-component-free serum-free media (eviGrow and eviMake2) and a proprietary transfection reagent (eviFect). Supernatants were harvested by centrifugation and subsequent filtration (0.2 μm filter) and purified using standard methods.

[0367] Characterization of anti-idiotype mask-binding with different CD3 mAbs SPR experiments were performed on a Biacore T200 using HBS-EP+ as the running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20 (BR-1006-69, GE Healthcare)). Three anti-idiotypic antibodies were directly immobilized on a CM5 chip (GE Healthcare) by amine coupling. A three-fold dilution series of different T cell bispecifics (TCBs) was passed over the ligand at 30 μl / min for 180 s, and the association phase was recorded. The dissociation phase was monitored for 600 s and triggered by switching from the sample solution to HBS-EP+. The chip surface was regenerated after each cycle by one 60 s injection of 10 mM glycine pH 2.1, followed by two 30 s injections. Bulk refractive index differences were corrected by subtracting the response obtained with reference flow cell 1. Affinity constants were derived from the kinetic constants by fitting to 1:1 Langmuir binding using Biaeval software (GE Healthcare). Measurements were performed in one dilution series.

[0368] JPEG0007815150000029.jpg62170Table 9: Binding affinity of different masks to different CD3 binders. SPR analysis was evaluated using masks as IgG (immobilized on a CM5 chip) and TCB with different CD3 binding Fabs as analytes.

[0369] Characterization of anti-idiotype masks - development potential As one of the anti-idiotype masks (4.15.64) represents the N-glycosylation site of CDRL1 (NYS), this molecule was not considered further, and only the 4.24.72 and 4.32.63 masks were further evaluated as they could be used to block different CD3 binders.

[0370] The binding of anti-idiotypic antibodies 4.32.63 and 4.24.72 after 14 days of incubation in either 20 mM His / HCl, 140 mM NaCl pH 6.0, at 40°C or 1x PBS pH 7.4, at 37°C, was investigated by surface plasmon resonance using a T200 instrument (GE Healthcare). Briefly, monomeric FolR1-Fc (flow cell 2) and anti-PGLALA antibody (flow cell 4) were immobilized on a Series S SensorChip CM5 (CE Healthcare) using standard amine coupling chemistry, yielding a surface density of over 10,000 resonance units (RU). Flow cells 1 and 3 were used as mock controls. FolR1 TCB-D-16D5, containing the CD3-CH2527 binding domain, was injected over the FolR1-Fc surface alone at a concentration of 10 μg / ml at a flow rate of 5 μl / min for 120 s, yielding a surface density of over 1,000 RU. Anti-idiotypic antibodies were then injected into all flow cells at a concentration of 1 μg / ml for 60 and 120 s at a flow rate of 5 μl / min. Dissociation was monitored for 60 s. The FolR1-Fc surface was regenerated by injecting 10 mM glycine pH 1.7 for 60 s, and the anti-PGLALA surface was regenerated by injecting 10 mM NaOH for 60 s. Bulk refractive index differences were corrected by subtracting the responses obtained from flow cells 1 and 3 (mock surfaces).

[0371] To normalize the binding signal of the anti-idiotypic antibody, the binding response of the FOLR1 TCB-D-16D5 surface was divided by the binding response of the anti-PGLALA surface. Relative activity concentrations were obtained for each molecule by dividing the normalized response of the stressed sample by the normalized response of the unstressed reference sample.

[0372] JPEG0007815150000030.jpg103170Table 10: Comparison of molecular stability of parental chimeric anti-idiotype masks (thermostability and molecular integrity / activity after stress conditions such as 14 days of incubation in different buffers).

[0373] A significant decrease in relative activity concentration (73% remaining target binding) was observed for the 4.32.63 mask after 14 days of incubation at 40°C and pH 6.0, whereas 4.24.72 was stable under these conditions with 96% remaining target binding activity.

[0374] Example 11 - Screening of anti-idiotypic clones against CD3 binder P035.093 The binding and blocking abilities of anti-idiotypic (anti-ID) clones (4.24.72, 4.32.63, 4.21, and 4.15.64) to CD3 binders were tested in a Jurkat NFAT activation assay using TYRP1 TCB (a distinct CD3 binder). Blocking of anti-ID IgG can be seen in the reduced Jurkat NFAT activation resulting from blocked CD3 binders (Figure 10).

[0375] TYRP1-targeting T cell bispecific antibody (TCB) simultane...

Claims

1. 1. A protease-activatable T cell activating bispecific molecule comprising: (a) one first antigen-binding moiety capable of binding to CD3, comprising: (i) and (ii) (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, a HCDR 2 of SEQ ID NO: 4, and a HCDR 3 of SEQ ID NO: 10; (ii) a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, a LCDR 2 of SEQ ID NO: 21, and a LCDR 3 of SEQ ID NO: 22; (b) one second antigen-binding moiety capable of binding to a target cell antigen; and (c) a masking moiety covalently attached to the T cell bispecific binding molecule via a protease-cleavable linker, the masking moiety being capable of binding to the idiotype of the first antigen-binding moiety, thereby reversibly masking the first antigen-binding moiety. Including, The masking moiety is an scFv. Protease activatable T cell activation bispecific molecule.

2. 2. The protease-activatable T cell activating bispecific molecule of claim 1 , wherein the VH comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16 and / or the VL comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

23.

3. 3. The protease-activatable T cell activating bispecific molecule of claim 1 or 2, wherein the masking moiety is covalently attached to the first antigen-binding moiety and reversibly masks the first antigen-binding moiety.

4. 4. The protease-activatable T cell activating bispecific molecule of claim 1 , wherein the masking moiety is covalently linked to the heavy chain variable region of the first antigen-binding moiety.

5. 5. The protease-activatable T cell activating bispecific molecule of claim 1 , wherein the second antigen-binding portion is a crossover Fab molecule in which the variable or constant regions of the Fab light chain and the Fab heavy chain have been exchanged.

6. 6. The protease-activatable T cell activating bispecific molecule of claim 1 , wherein the first antigen-binding portion is a conventional Fab molecule.

7. 7. The protease-activatable T cell activating bispecific molecule of claim 1 , wherein the second antigen-binding portion is capable of binding to a target cell antigen selected from the group consisting of FolR1 and TYRP1.

8. 8. The protease-activatable T cell activating bispecific molecule of claim 1 , wherein the first and second antigen-binding moieties are fused to each other.

9. 9. The protease-activatable T cell activating bispecific molecule of claim 1 , wherein the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety.

10. 10. The protease activatable T cell activating bispecific molecule of claim 1 , wherein the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety.

11. A protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 10, additionally comprising an Fc domain.

12. The protease-activatable T cell activating bispecific molecule of claim 11 , wherein the Fc domain is IgG.

13. 13. The protease-activatable T cell activating bispecific molecule of claim 11 or 12, wherein the Fc domain exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to a native IgG1 Fc domain.

14. The masking part is (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of DYSMN (SEQ ID NO: 58); (b) a CDR H2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO:59), WINTETGEPRYTDDFTG (SEQ ID NO:84), and WINTETGEPRYTQGFKG (SEQ ID NO:86); (c) the CDR H3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) a light chain (CDR L) 1 amino acid sequence selected from the group consisting of RASKSVSTSSYSYMH (SEQ ID NO: 62) and KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of YVSYLES (SEQ ID NO: 63); and (f) a CDR L3 amino acid sequence selected from the group consisting of QHSREFPYT (SEQ ID NO: 64) and QQSREFPYT (SEQ ID NO: 88). and a light chain variable region comprising:

15. The masking part is (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of DYSMN (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 59); (c) the CDR H3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) the light chain (CDR L) 1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 62); (e) the CDR L2 amino acid sequence of YVSYLES (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHSREFPYT (SEQ ID NO: 64) and a light chain variable region comprising:

16. The masking part is (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID NO:59); (c) a heavy chain variable region comprising a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60); and (d) the light chain (CDR L) 1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising:

17. The masking part is (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO: 84); (c) a heavy chain variable region comprising a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60); and (d) the light chain (CDR L) 1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising:

18. The masking part is (a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO: 58); (b) the CDR H2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 86); (c) a heavy chain variable region comprising a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO: 60); and (d) the light chain (CDR L) 1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 82); (e) the CDR L2 amino acid sequence of AATFLAD (SEQ ID NO: 63); and (f) CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO: 64) and a light chain variable region comprising:

19. 19. The protease-activatable T cell activating bispecific molecule of claim 1 , wherein the protease-cleavable linker comprises at least one protease recognition sequence.

20. The protease recognition sequence is (a) RQARVVNG (SEQ ID NO: 100); (b) VHMPLGFLGPGRSRGSFP (SEQ ID NO: 101); (c) RQARVVNGXXXXXXVPLSLYSG (SEQ ID NO: 102), where X is any amino acid; (d) RQARVVNGVPLSLYSG (SEQ ID NO: 103); (e) PLGLWSQ (SEQ ID NO: 104); (f) VHMPLGFLGPRQARVVNG (SEQ ID NO: 105); (g) FVGGTG (SEQ ID NO: 106); (h) KKAAPVNG (SEQ ID NO: 107); (i) PMAKKVNG (SEQ ID NO: 108); (j) QARAKVNG (SEQ ID NO: 109); (k) VHMPLGFLGP (SEQ ID NO: 110); (l) QARAK (SEQ ID NO: 111); (m) VHMPLGFLGPPMAKK (SEQ ID NO: 112); (n) KKAAP (SEQ ID NO: 113); and (o) PMAKK (SEQ ID NO: 114) 20. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 19, selected from the group consisting of:

21. 21. The protease-activatable T cell activating bispecific molecule of claim 19 or 20, wherein the protease-cleavable linker comprises the protease recognition sequence PMAKK (SEQ ID NO: 114).

22. the second antigen-binding moiety is capable of binding to FolR1; and a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of NAWMS (SEQ ID NO: 54); b) the CDR H2 amino acid sequence of RIKSKTDGGTTDYAAPVKG (SEQ ID NO: 55); and c) CDR H3 amino acid sequence of PWEWSWYDY (SEQ ID NO: 56) a heavy chain variable region comprising d) the light chain (CDR L) 1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 20); e) the CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO: 21); and f) CDR L3 amino acid sequence of ALWYSNLWV (SEQ ID NO: 22) and a light chain variable region comprising:

23. the second antigen-binding moiety is capable of binding to TYRP1; and a) the heavy chain complementarity determining region (CDR H) 1 amino acid sequence of DYFLH (SEQ ID NO: 24); b) the CDR H2 amino acid sequence of WINPDNGNTVYAQKFQG (SEQ ID NO: 25); and c) CDR H3 amino acid sequence of RDYTYEKAALDY (SEQ ID NO: 26) a heavy chain variable region comprising d) the light chain (CDR L) 1 amino acid sequence of RASGNIYNYLA (SEQ ID NO: 28); e) the CDR L2 amino acid sequence of DAKTLAD (SEQ ID NO: 29); and f) CDR L3 amino acid sequence of QHFWSLPFT (SEQ ID NO: 30) and a light chain variable region comprising:

24. 24. A pharmaceutical composition comprising the protease-activatable T cell activating bispecific molecule of any one of claims 1 to 23 and a pharmaceutically acceptable carrier.

25. 24. An isolated polynucleotide encoding the protease-activatable T cell activating bispecific molecule of any one of claims 1 to 23.

26. A vector, particularly an expression vector, comprising a polynucleotide according to claim 25.

27. 27. A host cell comprising the polynucleotide of claim 25 or the vector of claim 26.

28. 28. A method for producing a protease-activatable T cell activating bispecific molecule, comprising the steps of: a) culturing the host cell of claim 27 under conditions suitable for expression of the protease-activatable T cell activating bispecific molecule; and b) recovering the protease-activatable T cell activating bispecific molecule.

29. 25. A protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 23, or a pharmaceutical composition according to claim 24, for use as a medicament.

30. 30. The protease-activatable T cell activating bispecific molecule for use according to claim 29, wherein the medicament is for treating or delaying the progression of cancer, treating or delaying the progression of an immune related disease, or enhancing or stimulating immune response or function in an individual.

31. 24. Use of a protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 23 for the manufacture of a medicament for the treatment of a disease.

32. 32. The use according to claim 31, wherein the disease is cancer.

33. A medicament for treating a disease in an individual, comprising a composition comprising the protease-activatable T cell activating bispecific molecule of any one of claims 1 to 23; A therapeutically effective amount of the composition is administered to said individual.

34. 34. The medicament according to claim 33, for treating or delaying the progression of cancer, treating or delaying the progression of an immune-related disease, or improving or stimulating immune response or function in an individual.

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