Improved protease-activatable T cell bispecific antibodies

Abstract

The present invention generally relates to improved protease-activatable antigen-binding molecules. The protease-activatable antigen-binding molecules contain an anti-idiotypic binding portion that reversibly masks the CD3 antigen-binding portion of the molecule. Furthermore, the present invention relates to polynucleotides encoding such protease-activatable T cell binding molecules, as well as vectors and host cells containing such polynucleotides. The present invention further relates to methods for producing the protease-activatable T cell binding molecules of the present invention and methods for using the protease-activatable T cell binding molecules, for example, in the treatment of diseases.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention generally relates to improved protease-activatable antigen-binding molecules, which contain an anti-idiotypic binding moiety that reversibly masks the CD3 antigen-binding portion of the molecule. Furthermore, the present invention relates to polynucleotides encoding such protease-activatable T cell binding molecules, as well as vectors and host cells containing such polynucleotides. The present invention further relates to methods for producing the protease-activatable T cell binding molecules of the present invention, and methods for using the protease-activatable T cell binding molecules, for example, in the treatment of diseases. [Background technology]

[0002] background The selective destruction of individual target cells or specific target cell types is often desirable in various clinical settings. For example, a primary goal of cancer therapy is the specific destruction of tumor cells while leaving healthy cells and tissues intact.

[0003] An attractive way to achieve this is to induce an immune response against tumors, using 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 target cell surface antigen with one "arm" and an 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 targets results in a transient interaction between the target cell and T cells, triggering the activation of cytotoxic T cells and subsequent lysis of the target cell. Thus, the immune response is redirected toward the target cell, independent of the presentation of peptide antigens by the target cell or the T cell specificity associated with the typical 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. To achieve efficient lysis of target cells, T cell-activating bispecific molecules that do not require lymphocyte preconditioning or costimulation are particularly desirable. Several bispecific antibody formats have been developed, and their suitability for T cell-mediated immunotherapy is being explored. 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. If a bispecific molecule targets an antigen expressed not only on tumor cells but also on normal tissues, on-target / off-tumor toxicity may occur. Therefore, there is a need for effective T cell-activating bispecific molecules that fully suppress T cell activation in the presence of target cells but not in the presence of normal cells or tissues. Summary of the Invention

[0006] Quick Overview The present invention provides improved T cell activating bispecific molecules. In particular, the present invention provides protease-activatable T cell activating bispecific molecules whose activity is reduced or eliminated before reaching the site of action, e.g., the tumor microenvironment. This results in an improved safety profile, e.g., reduced toxicity, and efficient activation of the molecule at the site of action.

[0007] In one embodiment, (a) a first antigen-binding moiety capable of binding to CD3; and (b) a second antigen-binding moiety capable of binding to a target cell antigen selected from the group consisting of IGF-1R, cMET, or TROP2; and (c) a masking moiety covalently attached to the T cell activating bispecific molecule via a peptide linker; wherein the masking moiety is capable of binding to the idiotype of the first antigen-binding moiety or the second antigen-binding moiety, thereby reversibly masking the first antigen-binding moiety or the second antigen-binding moiety; The peptide linker contains the protease recognition sequence XQARK (SEQ ID NO: 39), where X is histidine (H) or proline (P).

[0008] In one embodiment, the masking moiety is covalently attached to the first antigen-binding moiety and reversibly masks the first antigen-binding moiety.

[0009] In one embodiment, the masking moiety is covalently linked to the heavy chain variable region of the first antigen-binding moiety.

[0010] In one embodiment, the masking moiety is an scFv.

[0011] In one embodiment, (i) the second antigen-binding moiety is a conventional Fab, or (ii) the second antigen-binding moiety is a crossover Fab molecule in which either the variable or constant regions of the Fab light chain and the Fab heavy chain have been exchanged.

[0012] In one embodiment, the first antigen-binding moiety is a conventional Fab molecule.

[0013] In one embodiment, the protease-activatable T cell activating bispecific molecule comprises a third antigen-binding moiety that is a Fab molecule capable of binding to a target cell antigen.

[0014] In one embodiment, the third antigen-binding moiety is identical to the second antigen-binding moiety.

[0015] In one embodiment, the first antigen-binding moiety and the second antigen-binding moiety are fused to each other, optionally via a peptide linker.

[0016] In one embodiment, 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.

[0017] In one embodiment, the protease-activatable T cell activating bispecific molecule additionally comprises an Fc domain composed of a first and a second subunit capable of stable association.

[0018] In one embodiment, the Fc domain is an IgG, particularly an IgG1 or IgG4 Fc domain.

[0019] In one embodiment, the Fc domain exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain.

[0020] In one embodiment, the antigen binding moiety capable of binding to CD3 comprises: (a) the heavy chain complementarity determining region (HCDR) 1 amino acid sequence of SYAMN (SEQ ID NO: 1); (b) the HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 2); (c) HCDR3 amino acid sequence of ASNFPASYVSYFAY (SEQ ID NO: 3) a heavy chain variable (VH) region comprising: (d) the light chain complementarity-determining region (LCDR) 1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 7); (e) LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 8); (f) Selected LCDR3 amino acid sequence of ALWYSNLWV (SEQ ID NO: 9) a light chain variable (VL) region comprising Includes.

[0021] In one embodiment, the antigen-binding portion capable of binding to CD3 comprises a VH 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:5, and / or a VL 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:10.

[0022] In one embodiment, the antigen binding moiety capable of binding to CD3 comprises: (a) the heavy chain complementarity determining region (HCDR) 1 amino acid sequence of SYAMN (SEQ ID NO: 1); (b) the HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 2); (c) HCDR3 amino acid sequence of HTTFPSSYVSYYGY (SEQ ID NO: 4) a heavy chain variable (VH) region comprising: (d) the light chain complementarity-determining region (LCDR) 1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 7); (e) LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 8); (f) Selected LCDR3 amino acid sequence of ALWYSNLWV (SEQ ID NO: 9) a light chain variable (VL) region comprising Includes.

[0023] In one embodiment, the antigen-binding portion capable of binding to CD3 comprises a VH 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: 6, and / or a VL 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: 10.

[0024] In one embodiment, the masking moiety is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) an HCDR2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO: 16), WINTETGEPRYTDDFTG (SEQ ID NO: 17), and WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) a VH region comprising (d) an LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25) or KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) or QQSREFPYT (SEQ ID NO: 29) and a VL region comprising Includes.

[0025] In one embodiment, the masking moiety is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) a VH region comprising (d) the LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising Includes.

[0026] In one embodiment, the masking moiety is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) a VH region comprising (d) the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising Includes.

[0027] In one embodiment, the masking moiety is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO: 17); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) a VH region comprising (d) the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising Includes.

[0028] In one embodiment, the masking moiety is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) a VH region comprising (d) the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising Includes.

[0029] In one embodiment, the masking moiety is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) a VH region comprising (d) the LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) LCDR3 amino acid sequence of QQSREFPYT (SEQ ID NO: 29) and a VL region comprising Includes.

[0030] In one embodiment, the second antigen-binding moiety is capable of binding to IGF-1R and a) the HCDR1 amino acid sequence of SYGMH (SEQ ID NO: 61); b) the HCDR2 amino acid sequence of IIWFDGSSTYYADSVRG (SEQ ID NO: 62); c) HCDR3 amino acid sequence of ELGRRYFDL (SEQ ID NO: 63) a VH region comprising d) LCDR1 of RASQSVSSYLA (SEQ ID NO: 65); e) LCDR2 amino acid sequence of DASKRAT (SEQ ID NO: 66); f) LCDR3 amino acid sequence of QQRSKWPPWT (SEQ ID NO: 67) and a VL region comprising Includes.

[0031] In one embodiment, the antigen-binding portion capable of binding to IGF-1R comprises a VH 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:64, and / or a VL 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:68.

[0032] In one embodiment, the second antigen-binding moiety is capable of binding to cMET and a) the HCDR1 amino acid sequence of SYWLH (SEQ ID NO: 69); b) the HCDR2 amino acid sequence of MIDPSNSDTRFNPNFKD (SEQ ID NO: 70); c) HCDR3 amino acid sequence of YRSYVTPLDY (SEQ ID NO: 71) a VH region comprising d) LCDR1 of KSSQSLLYTSSQKNYLA (SEQ ID NO: 73); e) LCDR2 amino acid sequence of WASTRES (SEQ ID NO: 74); f) LCDR3 amino acid sequence of QQYYAYPWT (SEQ ID NO: 75) and a VL region comprising Includes.

[0033] In one embodiment, the antigen-binding portion capable of binding to cMET comprises a VH 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: 72, and / or a VL 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: 76.

[0034] In one embodiment, the second antigen-binding moiety is capable of binding to TROP2 and (a) the HCDR1 amino acid sequence of NYGMN (SEQ ID NO: 77); b) the HCDR2 amino acid sequence of WINTKTGEPTYAEEFKG (SEQ ID NO: 78); c) the HCDR3 amino acid sequence of GGYGSSYWYFDV (SEQ ID NO: 79); a VH region comprising d) LCDR1 of KASQDVSIAVA (SEQ ID NO: 81); e) the LCDR2 amino acid sequence of SASYRYT (SEQ ID NO: 82); f) LCDR3 amino acid sequence of QQHYITPLT (SEQ ID NO: 83) and a VL region comprising Includes.

[0035] In one embodiment, the antigen-binding portion capable of binding to TROP2 comprises a VH 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: 80, and / or a VL 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: 84.

[0036] In one embodiment, the protease-cleavable linker comprises the protease recognition sequence PQARK (SEQ ID NO: 41).

[0037] In one embodiment, an idiotype-specific polypeptide is provided for reversibly masking an anti-CD3 antigen binding site of a molecule, wherein the idiotype-specific polypeptide is covalently attached to the molecule via a peptide linker, the linker comprising the protease recognition sequence XQARK (SEQ ID NO: 39), where X is histidine (H) or proline (P).

[0038] In one embodiment, the idiotype-specific polypeptide is an anti-idiotype scFv.

[0039] In one embodiment, the molecule is a T cell activating bispecific molecule.

[0040] In one embodiment, the linker comprises the protease recognition sequence PQARK (SEQ ID NO: 41).

[0041] In one embodiment, a pharmaceutical composition is provided comprising a protease-activatable T cell activating bispecific molecule described herein and a pharmaceutically acceptable carrier.

[0042] In one embodiment, a pharmaceutical composition is provided comprising an idiotype-specific polypeptide described herein and a pharmaceutically acceptable carrier.

[0043] In one embodiment, an isolated polynucleotide encoding a protease-activatable T cell activating bispecific antigen binding molecule described herein is provided.

[0044] In one embodiment, an isolated polynucleotide encoding an idiotype-specific polypeptide described herein is provided.

[0045] In one embodiment, there are provided vectors, particularly expression vectors, that include the polynucleotides described herein.

[0046] In one embodiment, a host cell is provided comprising a vector described herein.

[0047] In one embodiment, provided is a method of producing a protease-activated T cell activating bispecific molecule, the method comprising the steps of a) culturing a host cell described herein under conditions suitable for expression of the protease-activated T cell activating bispecific molecule; and b) recovering the protease-activated T cell activating bispecific molecule.

[0048] In one embodiment, there is provided a protease-activatable T cell activating bispecific molecule as described herein for use as a medicament.

[0049] In one embodiment, 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.

[0050] In one embodiment, there is provided a use of a protease-activatable T cell activating bispecific molecule as described herein for the manufacture of a medicament for the treatment of a disease.

[0051] In one embodiment, there is provided a use of the protease-activatable T cell activating bispecific molecules described herein, wherein the disease is cancer.

[0052] In one embodiment, a method of treating a disease in an individual is provided comprising administering to the individual a therapeutically effective amount of a composition comprising a protease-activatable T cell activating bispecific molecule described herein.

[0053] In one embodiment, the method is for treating or delaying the progression of cancer.

[0054] Other technical features will be readily apparent to those skilled in the art from the following drawings, specifications, and claims.

[0055] Other technical features will be readily apparent to those skilled in the art from the following drawings, specifications, and claims. [Brief explanation of the drawings]

[0056] [Figure 1A] Schematic diagrams of exemplary protease-activated FolR1 proTCB molecules (SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 53) are shown. [Figure 1B] Schematic diagrams of exemplary IGF-1R proTCB molecules (SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89) are shown. [Figure 1C]Schematic diagrams of exemplary cMET proTCB molecules (SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94) are shown. [Figure 1D] Schematic diagrams of exemplary TROP2 proTCB molecules (SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100) are shown. [Figure 2] The study design for a single-dose PK and stability study is shown. Female NSG mice were intravenously injected with protease-activated FolR1 TCB molecules containing either the HQARK or PQARK linker (Groups A and B) and compared with the classical FolR1 TCB molecule (Group C). Blood was collected from the mice 24 hours, 7 days, and 10 days after injection. Serum was prepared and analyzed for the total and active versions of the FolR1 TCB molecule by ELISA. [Figure 3] Quantification of active pro-TCB in the serum of tumor-free mice is shown. Protease-activated FolR1 TCB or classical FolR1 TCB was administered intravenously once, and serum active and total TCB concentrations were measured over time by ELISA. Active and total TCB were quantified by ELISA using an anti-PG antibody (protease-activated FolR1 TCB) and an anti-idiotypic anti-CD3 antibody (active FolR1 TCB). The percentage of active TCB relative to total TCB is shown. Equimolar doses of protease-activated FolR1 TCB and classical FolR1 TCB were used in each study, so dose adjustment was not required. [Figure 4] The study design for the in vivo efficacy study is shown. Female NSG mice were subcutaneously injected with human breast cancer PDX (BC004) and received the first treatment when tumors reached approximately 200 mm3 in size (day 28). Mice were intravenously administered weekly protease-activated FolR1 TCB molecules containing PMAKK or PQARK cleavage sites (groups D and E), or the classical FolR1 TCB molecule (group B), as well as a masked FolR1 TCB molecule containing a non-cleavable linker (group C). One group received histidine buffer alone and served as a control (group A; vehicle). Tumor growth was measured by caliber measurement. The study was terminated on day 58, when tumors were harvested and weighed. [Figures 5A-5G] Tumor growth inhibition and tumor weight at the end of the study are shown. (5A) Tumor volumes over time are shown as the mean + / - standard error for all treatment groups. Protease-activated FolR1 TCB molecules containing the PQARK cleavage site resulted in tumor growth inhibition comparable to that seen with the classical FolR1 TCB. Masked FolR1 TCB molecules containing a non-cleavable linker and molecules containing a PMAKK cleavage site did not result in tumor growth inhibition. (5B-5F) Individual tumor growth kinetics for a single mouse are shown for vehicle (5B), classical FolR1 TCB (5C), protease-activated FolR1 TCB containing a PMAKK site (5D), masked FolR1 TCB containing a non-cleavable linker (5E), and protease-activated FolR1 TCB containing a PQARK cleavage site (5F). (5G) Tumor weight at the end of the study for all treatment groups. [Figure 6] Figure 1 shows binding of the indicated constructs to CD3 of Jurkat-NFAT T cells as determined by flow cytometry. Molecules were detected using a fluorescently labeled anti-human Fc-specific secondary antibody. Median MFI values ​​are shown, and each point represents the mean of triplicates. Standard deviations are indicated by error bars (n=1). [Figures 7A-7D] Binding of the indicated constructs to IGF1R of different human cancer cell lines as determined by flow cytometry is shown. 7A) Binding of TCB to IGF1R of T-47D cells, 7B) Binding of TCB to IGF1R of MKN-45 cells, 7C) Binding of TCB to IGF1R of OVMANA cells, and 7D) Binding of TCB to IGF1R of HPAF II cells. Molecules were detected using a fluorescently labeled anti-human Fcγ-specific secondary antibody. Median MFI values ​​are shown, and each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figure 8]Figure 1 shows Jurkat NFAT activation mediated by IGF1R TCB constructs. Quantification of luminescence (Jurkat NFAT activation) after 5 hours of incubation of hu-IGF1R-coated SA beads with IGF1R TCB constructs and Jurkat NFAT effector cells. Matriptase-precleaved IGF1R proTCB and classical IGF1R TCB (with and without matriptase) induced dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figures 9A-9D] Figure 1 shows Jurkat NFAT activation mediated by IGF1R pro-TCB. Quantification of luminescence after 5 hours of incubation of different target cells with IGF1R proTCB and Jurkat NFAT effector cells (Jurkat NFAT activation). Pre-cleaved IGF1R proTCB and classical IGF1R TCB induced dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figures 10A-10C] Figure 10 shows tumor cell killing by IGF1R proTCB. Tumor cell killing of target cell lines (T-47D, MKN-45, and HPAF II) by healthy donor PBMCs upon treatment with IGF1R proTCB was determined by LDH release after 72 hours (Figures 10A, 10B, and 10C). Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figures 11A-11F] Figure 6 shows CD4+ T cell activation by IGF1R proTCB. Upregulation of the T cell activation markers CD25 (11A-11C) and CD69 (11D-11F) was assessed using flow cytometry. Healthy donor PBMCs were incubated with different target cell lines (T-47D, MKN-45, and HPAF II) and treated for 72 hours with different concentrations of IGF1R TCB molecules as indicated. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figures 12A-12F]Figure 12 shows CD8+ T cell activation by IGF1R proTCB. Upregulation of the T cell activation markers CD25 (12A-12C) and CD69 (12D-12F) was assessed using flow cytometry. Healthy donor PBMCs were incubated with different target cell lines (T-47D, MKN-45, and HPAF II) and treated with different concentrations of IGF1R TCB molecules as indicated for 72 hours. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figure 13] Figure 1 shows binding of the indicated constructs to CD3 of Jurkat-NFAT T cells as determined by flow cytometry. Molecules were detected using a fluorescently labeled anti-human Fc-specific secondary antibody. Median MFI values ​​are shown, and each point represents the mean of triplicates. Standard deviations are indicated by error bars (n=1). [Figures 14A-14D] Binding of the indicated constructs to Trop2 in different human cancer cell lines as determined by flow cytometry is shown. 14A) TCB binding to Trop2 in T-47D cells, 14B) TCB binding to Trop2 in HPAF II cells, 14C) TCB binding to Trop2 in HeLa ST14 cells, and 14D) TCB binding to Trop2 in Ovmana cells. Molecules were detected using a fluorescently labeled anti-human Fc-specific secondary antibody. Median MFI values ​​are shown, and each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figure 15] Figure 1 shows Jurkat NFAT activation mediated by Trop2 proTCB. Quantification of luminescence (Jurkat NFAT activation) after 5 hours of incubation of hu-Trop2-coated SA beads with Trop2 proTCB and Jurkat NFAT effector cells. Pre-cleaved Trop2 proTCB and canonical Trop2TCB induced dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figures 16A-16C]Figure 1 shows Jurkat NFAT activation mediated by Trop2 proTCB. Quantification of luminescence after 5 hours of incubation of different target cells with Trop2 proTCB and Jurkat NFAT effector cells (Jurkat NFAT activation). Pre-cleaved Trop2 proTCB and canonical Trop2 TCB induced dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figures 17A-17C] Figure 1 shows tumor cell killing by Trop2 proTCB. Tumor cell killing of target cell lines (17A) T-47D, (17B) HPAF II, and (17C) OVMANA by healthy donor PBMCs upon treatment with Trop2 proTCB was determined by LDH release after 72 hours. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figures 18A-18F] Figure 18 shows CD4+ T cell activation by Trop2 proTCB. Upregulation of the T cell activation markers CD25 (18A-18C) and CD69 (18D-18F) was assessed using flow cytometry. Healthy donor PBMCs were incubated with different target cell lines (T-47D, HPAF II, and OVMANA) for 72 hours and then treated with different concentrations of Trop2 TCB molecules as indicated. [Figures 19A-19F] Figure 19 shows CD8+ T cell activation by Trop2 proTCB. Upregulation of the T cell activation markers CD25 (19A-19C) and CD69 (19D-19F) was assessed using flow cytometry. Healthy donor PBMCs were incubated with different target cell lines (T-47D, HPAF II, and OVMANA) for 72 hours and then treated with different concentrations of Trop2 TCB molecules as indicated. [Figure 20] Figure 1 shows binding of the indicated constructs to CD3 of Jurkat-NFAT T cells as determined by flow cytometry. Molecules were detected using a fluorescently labeled anti-human Fc-specific secondary antibody. Median MFI values ​​are shown, and each point represents the mean of triplicates. Standard deviations are indicated by error bars (n=1). [Figures 21A-21D] Binding of the indicated constructs to cMet in different human cancer cell lines as determined by flow cytometry is shown. 21A) TCB binding to cMet in OVMANA cells, 21B) TCB binding to cMet in HeLa ST14 cells, 21C) TCB binding to cMet in HPAF II cells, and 21D) TCB binding to cMet in T-47D cells. Molecules were detected using a fluorescently labeled anti-human Fc-specific secondary antibody. Median MFI values ​​are shown, and each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figure 22] Figure 1 shows Jurkat NFAT activation mediated by cMet proTCB. Quantification of luminescence (Jurkat NFAT activation) after 5 hours of incubation of hu-cMet-coated SA beads with cMet proTCB and Jurkat NFAT effector cells. Pre-cleaved cMet proTCB and classical cMet TCB induced dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figures 23A-23C] Figure 1 shows Jurkat NFAT activation mediated by cMet pro-TCB. Quantification of luminescence after 5 hours of incubation of different target cells with cMet proTCB and Jurkat NFAT effector cells (Jurkat NFAT activation). Pre-cleaved cMet proTCB and classical cMet TCB induced dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1). [Figures 24A-24C] Figure 1 shows tumor cell killing by cMet proTCB. Tumor cell killing of target cell lines (24A) OVMANA, (24B) HPAF II, and (24C) T-47D by healthy donor PBMCs upon treatment with cMet proTCB was determined by LDH release after 72 hours. [Figures 25A-25F]CD4+ T cell activation by cMet proTCB is shown. Upregulation of the T cell activation markers CD25 (25A-25C) and CD69 (25D-25F) was assessed using flow cytometry. Healthy donor PBMCs were incubated with different target cell lines (OVMANA, HPAF II, T-47D) and treated with different concentrations of cMet TCB molecule for 72 hours, as indicated. [Figures 26A-26F] CD8+ T cell activation by cMet proTCB is shown. Upregulation of the T cell activation markers CD25 (26A-26C) and CD69 (26D-26F) was assessed using flow cytometry. Healthy donor PBMCs were incubated with different target cell lines (OVMANA, HPAF II, T-47D) and treated with different concentrations of cMet TCB molecule for 72 hours, as indicated. DETAILED DESCRIPTION OF THE INVENTION

[0057] Detailed Description definition Unless otherwise defined below, terms are used herein as commonly used in the art.

[0058] For purposes herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0059] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative exemplary methods for measuring binding affinity are described below.

[0060] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more complementarity determining regions (CDRs) that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[0061] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0062] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds 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')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAbs), and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0063] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies," Harlow and Lane, Cold Spring Harbor Press, Cold Spring Harb., NY).

[0064] Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows multiple monoclonal antibodies that specifically bind to an antigen to be partitioned based on their respective binding profiles to chemically or enzymatically modified antigen surfaces (see, e.g., U.S. Patent Application Publication No. 2004 / 0101920). Each partitioned antibody binds to the same epitope, which may be distinct from epitopes represented by other partitions or may be a unique, overlapping epitope.

[0065] In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in competitive binding assays (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0066] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0067] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0068] The "class" of an antibody refers to the type of constant domain or constant region carried by its 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), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG1 isotype with P329G, L234A, and L235A mutations to reduce Fc region effector function. In other embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype with an S228P mutation in the hinge region to improve the stability of IgG4 antibodies. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated a, d, e, g, and m, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0069] The term "constant region of human origin" or "human constant region" as used herein refers to the constant heavy chain region and / or the constant light chain kappa or lambda region of a human antibody of the subclass IgG1, IgG2, IgG3, or IgG4. Such constant regions are known in the art and are described, for example, in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, e.g., Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A. et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, numbering of amino acid residues in the constant region follows the EU numbering system (also referred to as the Kabat EU index), as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0070] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which 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), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0071] An "effective amount" of an agent, eg, a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0072] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, 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 a full-length heavy chain or a truncated variant of the full-length heavy chain. This may be the case 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) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447), 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 indicated. In one embodiment, a heavy chain comprising an Fc region designated 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 one embodiment, a heavy chain comprising an Fc region designated 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 referred to as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0073] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in the VH (or VL) in the following order: FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0074] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0075] 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. The 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.

[0076] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or an antibody of non-human origin that utilizes human antibody-encoding sequences, such as the human antibody repertoire. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0077] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra. [[Modified as necessary to represent the actual subgroup of VH / VL of the invention]]

[0078] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDRs corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0079] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and determines antigen-binding specificity, e.g., the "complementarity-determining region" (CDR).

[0080] Generally, antibodies comprise six CDRs, three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)).

[0081] Unless otherwise specified, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that CDR nomenclature can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature.

[0082] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic agents.

[0083] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0084] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0085] The terms "nucleic acid molecule" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, whereby the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), particularly messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, and both single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases, including derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to increase the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP2101823B1).

[0086] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in cells that ordinarily contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0087] An "isolated nucleic acid" encoding a polypeptide refers to one or more nucleic acid molecules encoding, for example, antibody heavy and light chains (or fragments thereof) or idiotype-specific polypeptides, including one or more nucleic acid molecules in a single vector or separate vectors, and such one or more nucleic acid molecules are present in one or more locations within a host cell.

[0088] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for variant antibodies that contain, for example, naturally occurring mutations or that may arise during production of the monoclonal antibody preparation, in which such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0089] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0090] "Native antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, followed by a constant light chain (CL) domain.

[0091] The term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, concomitant treatments, contraindications and / or warnings for use of such therapeutic products.

[0092] "Amino acid sequence identity percentage (%)" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity for alignment.Alignment for determining amino acid sequence identity percentage can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package.Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.Alternatively, identity percentage values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code is on file in the user documentation at the U.S. Copyright Office, Washington, DC, 20559, registered under U.S. Copyright Registration No. TXU510087, and described in WO 2001 / 007611.

[0093] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, or the subsequent BLOSUM50 comparison matrix. The FASTA program package is described by W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; W.R. Pearson (1996) "Effective protein sequence comparison," Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch(global protein:protein) program and default options (BLOSUM50, open:-10, ext:-2, Ktup=2) to ensure a global, rather than local, alignment. The percent amino acid identity is shown in the output alignment header.

[0094] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredient contained therein is effective and that does not contain additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition may be administered.

[0095] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, 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.

[0096] As used herein, the term "FolR1" or "folate receptor 1," unless otherwise specified, refers to any native FolR1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed FolR1 and any form of FolR1 that results from processing within a cell. The term also encompasses naturally occurring variants of FolR1, such as splice variants or allelic variants.

[0097] As used herein, the term "IGF-1R" or "insulin-like growth factor type 1 receptor" refers to any native IGF-1R derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," unprocessed IGF-1R, and any form of IGF-1R resulting from processing within the cell. The term also encompasses naturally occurring variants of IGF-1R, such as splice variants or allelic variants.

[0098] As used herein, the term "cMET" or "tyrosine-protein kinase Met" refers to any native cMET from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," unprocessed cMET and any form of cMET that results from processing within the cell. The term also encompasses naturally occurring variants of cMET, such as splice variants or allelic variants.

[0099] As used herein, the term "TROP2" or "tumor-associated calcium signal transducer 2" refers to any native TROP2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," unprocessed TROP2 and any form of TROP2 resulting from processing within the cell. The term also encompasses naturally occurring variants of TROP2, such as splice variants or allelic variants.

[0100] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of disease in the treated individual and can be carried out for prophylaxis 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, attenuating the direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating disease symptoms, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of disease.

[0101] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain 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 similar structures, and each domain contains four conserved framework regions (FR) and three complementarity-determining regions (CDR). (See, for example, Kindt et al., Kuby Immunology, 6 thed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH or VL domain of an antibody that binds to that antigen and then used to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0102] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors that integrate into the genome of a host cell into which they are introduced, as well as vectors that act as self-replicating nucleic acid structures. Certain vectors are capable of directing the expression of a nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0103] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, 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 specific type of tumor cell or tumor stroma bearing the antigenic determinant. In another embodiment, 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 further defined herein. Particular antigen-binding moieties comprise the antigen-binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen-binding moiety may comprise 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.

[0104] 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-activated "T cell-activating bispecific molecules" of the present invention can induce T cell activation. Suitable assays for measuring T cell activation are known in the art and described herein.

[0105] As used herein, "target cell antigen" 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.

[0106] 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.

[0107] 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 a light chain (a "Fab light chain").

[0108] 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.

[0109] 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 linked by a peptide linker to form a single peptide chain. In certain such embodiments, the C-terminus of the Fab light chain in the single-chain Fab molecule is linked to the N-terminus of the Fab heavy chain.

[0110] A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which either the variable or constant regions of the Fab heavy and light chains have been exchanged; 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 regions of the Fab light chain and the Fab heavy chain have been exchanged, 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 regions of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0111] 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).

[0112] As used herein, an "idiotype-specific polypeptide" refers to a polypeptide that recognizes the idiotype of an antigen-binding portion, e.g., an antigen-binding portion specific for CD3. An "idiotype" can be defined as the specific combination of idiotopes present within the complement determining regions (CDRs) of an antigen-binding portion. An idiotype-specific polypeptide specifically binds to the variable region of an antigen-binding portion, thereby reducing or preventing specific binding of the antigen-binding portion to its cognate antigen. When associated with a molecule containing an antigen-binding portion, the idiotype-specific polypeptide can function as a masking portion 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.

[0113] 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 may remain associated with the target cell, for example, 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.

[0114] As used herein, "protease-activated" with respect to a T cell activating bispecific molecule refers to a T cell activating bispecific molecule that has reduced or eliminated its ability to activate T cells due to 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 and the T cell activating bispecific molecule is thereby activated.

[0115] As used herein, "reversibly concealing" 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 prevented from binding to its antigen, e.g., CD3. This concealing 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.

[0116] Protease-activated T cell activation bispecific molecule The present invention provides improved T cell activating bispecific molecules. In particular, the present invention provides protease-activatable T cell activating bispecific molecules whose activity is reduced or eliminated before reaching the site of action, e.g., the tumor microenvironment. This results in an improved safety profile, e.g., reduced toxicity, and efficient activation of the molecule at the site of action.

[0117] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: 1. a first antigen-binding moiety capable of binding to CD3; 2. A second antigen-binding moiety capable of binding to a target cell antigen; 3. a masking moiety covalently attached to the T cell bispecific binding molecule via a protease-cleavable linker; wherein the masking moiety is capable of binding to the idiotype of the first antigen-binding moiety or the second antigen-binding moiety, thereby reversibly masking the first antigen-binding moiety or the second antigen-binding moiety.

[0118] 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-activated T cell activation molecules that include such masking moieties, are described in detail below and in the Examples.

[0119] Exemplary antigen binding moieties The antigen-binding molecules of the present invention are bispecific, i.e., 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.

[0120] At least one of the antigen-binding moieties is a crossover Fab molecule. Such modification prevents mispairing of the heavy and light chains of different Fab molecules, thereby improving the yield and purity of the protease-activated T cell activating bispecific molecules of the invention during recombinant production. In certain crossover Fab molecules useful in the protease-activated T cell activating bispecific molecules of the invention, the constant regions of the Fab light chain and the Fab heavy chain are exchanged. In other crossover Fab molecules useful in the protease-activated T cell activating bispecific molecules of the invention, the variable regions of the Fab light chain and the Fab heavy chain are exchanged.

[0121] In particular 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 one embodiment, 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 a more particular embodiment, such simultaneous binding causes lysis of the target cell, particularly a tumor cell. In one embodiment, such simultaneous binding causes T cell activation. In other embodiments, such simultaneous binding causes 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 one embodiment, binding of the protease-activatable T cell activating bispecific molecule to CD3 without simultaneous binding to a target cell antigen does not cause T cell activation.

[0122] In one embodiment, the protease-activatable T cell activating bispecific molecule is capable of redirecting the cytotoxic activity of T cells to target cells, which in certain embodiments is independent of MHC-mediated peptide antigen presentation by the target cell and / or the specificity of the T cell.

[0123] 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.

[0124] CD3 binding part The protease-activated 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-activated T cell activating bispecific molecules comprise up to one antigen-binding moiety capable of binding to CD3. In one embodiment, the protease-activated 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 a protease-activated 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.

[0125] 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.

[0126] The CD3 antigen-binding portion comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0127] In a preferred embodiment, the CD3 antigen-binding portion comprises a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 7, a light chain CDR2 of SEQ ID NO: 8, and a light chain CDR3 of SEQ ID NO: 9.

[0128] In one embodiment, 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:5 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:10.

[0129] In a preferred embodiment, the CD3 antigen binding portion comprises the heavy chain variable region sequence of SEQ ID NO:5 and the light chain variable region sequence of SEQ ID NO:10.

[0130] In one embodiment, the CD3 antigen-binding portion comprises a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 4, a light chain CDR1 of SEQ ID NO: 7, a light chain CDR2 of SEQ ID NO: 8, and a light chain CDR3 of SEQ ID NO: 9.

[0131] In one embodiment, 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:6 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:10.

[0132] In one embodiment, the CD3 antigen binding portion comprises the heavy chain variable region sequence of SEQ ID NO:6 and the light chain variable region sequence of SEQ ID NO:10.

[0133] Target cell antigen binding moiety The protease-activated 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-activated T cell activating bispecific molecules comprise two antigen-binding moieties capable of binding to a target cell antigen. In certain such embodiments, each of the antigen-binding moieties specifically binds to the same antigenic determinant. In even more particular embodiments, the antigen-binding moieties are all identical. In one embodiment, the protease-activated T cell activating bispecific molecule comprises an immunoglobulin molecule capable of binding to a target cell antigen. In one embodiment, the protease-activated T cell activating bispecific molecule comprises up to two antigen-binding moieties capable of binding to a target cell antigen.

[0134] In a preferred embodiment, the target cell antigen binding moiety is a Fab molecule, particularly a conventional Fab molecule that binds to a specific antigenic determinant and can direct the protease-activatable T cell activating bispecific molecule to a target site (e.g., a specific type of tumor cell that bears the antigenic determinant).

[0135] 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 insulin-like growth factor 1 (IGF-1R) on the surface of the target cell. In another specific embodiment, the target cell antigen binding moiety specifically binds to tyrosine-protein kinase Met (cMET) on the surface of the target cell. In another specific embodiment, the target cell antigen binding moiety specifically binds to tumor-associated calcium signal transducer 2 (TROP2) on the surface of the target cell.

[0136] In certain embodiments, the target cell antigen-binding moiety is directed against an antigen associated with a pathological condition, such as an antigen presented 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 insulin-like growth factor 1 (IGF-1R). In another specific embodiment, the target cell antigen is tyrosine-protein kinase Met (cMET). In another specific embodiment, the target cell antigen is tumor-associated calcium signal transducer 2 (TROP2).

[0137] In certain embodiments, the protease-activated T cell activating bispecific molecule comprises at least one antigen-binding portion specific for IGF-1R. In one embodiment, the IGF-1R is human IGF-1R. In one embodiment, the antigen-binding portion specific for IGF-1R comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63, and at least one light chain CDR selected from the group of SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.

[0138] In one embodiment, the antigen-binding portion specific for IGF-1R comprises a heavy chain CDR1 of SEQ ID NO: 61, a heavy chain CDR2 of SEQ ID NO: 62, a heavy chain CDR3 of SEQ ID NO: 63, a light chain CDR1 of SEQ ID NO: 65, a light chain CDR2 of SEQ ID NO: 66, and a light chain CDR3 of SEQ ID NO: 67.

[0139] In a further embodiment, the antigen-binding portion specific for IGF-1R comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 64, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 68, or a variant thereof that retains functionality.

[0140] In one embodiment, the antigen-binding portion specific for IGF-1R comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:64 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:68.

[0141] In certain embodiments, the protease-activated T cell activating bispecific molecule comprises at least one antigen-binding portion specific for cMET. In one embodiment, the cMET is human cMET. In one embodiment, the antigen-binding portion specific for cMET comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71, and at least one light chain CDR selected from the group of SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75.

[0142] In one embodiment, the antigen-binding portion specific for cMET comprises a heavy chain CDR1 of SEQ ID NO: 69, a heavy chain CDR2 of SEQ ID NO: 70, a heavy chain CDR3 of SEQ ID NO: 71, a light chain CDR1 of SEQ ID NO: 73, a light chain CDR2 of SEQ ID NO: 74, and a light chain CDR3 of SEQ ID NO: 75.

[0143] In a further embodiment, the antigen-binding portion specific for cMET comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 72 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 76, or a variant thereof that retains functionality.

[0144] In one embodiment, an antigen-binding portion specific for cMET comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:76.

[0145] In certain embodiments, the protease-activated T cell-activating bispecific molecule comprises at least one antigen-binding portion specific for TROP2. In one embodiment, the TROP2 is human TROP2. In one embodiment, the antigen-binding portion specific for TROP2 comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79, and at least one light chain CDR selected from the group of SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83.

[0146] In one embodiment, the antigen-binding portion specific for TROP2 comprises a heavy chain CDR1 of SEQ ID NO: 77, a heavy chain CDR2 of SEQ ID NO: 78, a heavy chain CDR3 of SEQ ID NO: 79, a light chain CDR1 of SEQ ID NO: 81, a light chain CDR2 of SEQ ID NO: 82, and a light chain CDR3 of SEQ ID NO: 83.

[0147] In a further embodiment, the antigen-binding portion specific for TROP2 comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 80 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 84, or a variant thereof that retains functionality.

[0148] In one embodiment, an antigen-binding portion specific for PROT comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:80 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:84.

[0149] Masking part The protease-activated 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., International Publication No. 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 efficient 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 one specific embodiment, the masking moiety recognizes the idiotype of a first antigen-binding moiety capable of binding to CD3, e.g., human CD3. In one specific embodiment, the masking moiety recognizes the idiotype of a second antigen-binding moiety capable of binding to a target cell antigen.

[0150] In one embodiment, the masking portion masks the CD3 binding portion and comprises at least one of heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 15, HCDR2 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 17, HCDR2 of SEQ ID NO: 18, HCDR3 of SEQ ID NO: 19, light chain complementarity determining region (LCDR)1 of SEQ ID NO: 23, LCDR1 of SEQ ID NO: 26, LCDR2 of SEQ ID NO: 27, LCDR3 of SEQ ID NO: 28, and LCDR3 of SEQ ID NO: 29.

[0151] In one embodiment, the masking portion comprises a VH region comprising the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15), the HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16), and the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19); and a VL region comprising the LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25), the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27), and the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0152] In one embodiment, the masking portion comprises a VH region comprising the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15), the HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16), and the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19); and a VL region comprising the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26), the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27), and the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0153] In a preferred embodiment, the masking portion comprises a VH region comprising the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15), the HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO: 17), and the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19); and a VL region comprising the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26), the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27), and the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0154] In one embodiment, the masking portion comprises a VH region comprising the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15), the HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18), and the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19); and a VL region comprising the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26), the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27), and the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0155] In one embodiment, the masking portion comprises a VH region comprising the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15), the HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18), and the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19); and a VL region comprising the LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25), the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27), and the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 29).

[0156] In one embodiment, 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: 20. In one embodiment, the masking moiety masks the CD3 binding portion and comprises the polypeptide sequence of SEQ ID NO: 30.

[0157] In a preferred embodiment, the masking moiety is humanized. Methods for humanizing immunoglobulins are well known in the art and are described herein. Provided herein are humanized masking sites H1L1, H1L2, H2L2, H3L2, H3L3, and H7L5. The corresponding sequences are provided herein below.

[0158] In one embodiment, the masking portion comprises a heavy chain variable (VH) 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:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, and a light chain variable (VL) 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:31, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34.

[0159] In one embodiment, the masking portion comprises a VH region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31.

[0160] In a preferred embodiment, the masking moiety comprises a VH region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0161] In one embodiment, the masking portion comprises a VH region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0162] In one embodiment, the masking portion comprises a VH 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 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0163] In one embodiment, the masking portion comprises a VH 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 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 33.

[0164] In one embodiment, the masking portion comprises a VH region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 34.

[0165] In one embodiment, the masking moiety or idiotype-specific polypeptide for reversibly masking the antigen binding of the molecule is an scFc. Such an idiotype-specific polypeptide for reversibly masking the 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, an idiotype scF.

[0166] In one embodiment, the masking moiety comprises an idiotype-specific polypeptide for reversibly masking the antigen binding of the molecule. In one embodiment, the masking moiety comprises an idiotype-specific polypeptide. In a preferred embodiment, the idiotype-specific polypeptide is an scFv. In one preferred embodiment, the masking moiety is an scFv.

[0167] In one embodiment, the scFv comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35. In one embodiment, the anti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO:35.

[0168] In one embodiment, the scFv comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36. In a preferred embodiment, the anti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO:36.

[0169] In one embodiment, the scFv comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37. In one embodiment, the anti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO: 37.

[0170] In one embodiment, the scFv comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 38. In one embodiment, the anti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO: 38.

[0171] Protease-cleavable linker The protease-activated T cell activating bispecific molecules of the invention comprise at least one protease-activatable linker. Preferably, the protease-activated T cell activating bispecific molecules of the invention are inactive before the protease-activatable linker is cleaved, e.g., in the tumor microenvironment. In one embodiment, the masking moiety (e.g., idiotype-specific polypeptide) is covalently attached to the molecule via a linker. In one embodiment, the idiotype-specific polypeptide is covalently attached to the molecule via more than one linker. In one embodiment, the idiotype-specific polypeptide is covalently attached to the molecule via two linkers. In one embodiment, the linker is a peptide linker. In one embodiment, the linker is a protease-cleavable linker.

[0172] In one embodiment, the protease cleavable linker comprises a protease recognition site. In one embodiment, the protease is matriptase. In a preferred embodiment, the protease cleavable linker comprises a matriptase recognition site.

[0173] In one embodiment, the protease-activatable T cell activating bispecific molecule comprises a linker having a protease recognition site comprising the polypeptide sequence XQARK (SEQ ID NO: 39), where X is histidine (H) or proline (P). In one embodiment, the protease recognition site comprises the polypeptide sequence HQARK (SEQ ID NO: 43). In a preferred embodiment, the protease recognition site comprises the polypeptide sequence PQARK (SEQ ID NO: 41).

[0174] PQARK (SEQ ID NO: 41) and HQARK (SEQ ID NO: 43) are matriptase recognition sites with desirable and surprising properties. Ideally, protease-activatable (therapeutic) molecules should be inactive until they reach the site of action (e.g., a tumor). One desirable property of the matriptase recognition sites of the present invention (e.g., PQARK and HQARK) is that they are stable in vivo before reaching the site of action (see, e.g., Figure 3). Furthermore, such activated molecules must be efficiently activated at the site of action (e.g., a tumor). It is known that the tumor microenvironment can exhibit a pH as low as 5.6 compared to physiological pH (approximately 7.4) (see, e.g., Boedtkjer et al., 2020, Annual Review of Physiology, Volume 82, 2020, pp. 103-126).

[0175] Importantly, the matriptase recognition sites of the present invention (e.g., PQARK and HQARK) can be more strongly activated at physiological pH than the published matriptase recognition site PMAKK (see, e.g., Table 4). Surprisingly, the matriptase recognition sites of the molecules of the present invention (e.g., PQARK and cMET proTCB P1AI0623, TROP2 proTCB P1AI0690, IGF-1R proTCB P1AH0943) can be strongly activated at a pH as low as 5.6.

[0176] In one embodiment, the matriptase recognition site is embedded in a linker, e.g., an (unstructured) polypeptide. In one embodiment, the polypeptide comprises one or more unstructured peptide linkers. In one embodiment, the isolated polypeptide comprises at least one peptide linker, particularly, at least one peptide linker exhibits no secondary structure. In one embodiment, the peptide comprises an amino acid sequence having a length of at least 5 amino acids, preferably 5 to 100 amino acids, more preferably 10 to 50 amino acids, and most preferably 20 to 40 amino acids.

[0177] In one embodiment, the protease-cleavable linker is a polypeptide peptide having a length of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids. In a preferred embodiment, the protease-cleavable linker is a peptide having a length of 33 amino acids. In one embodiment, the polypeptide comprises a protease recognition site. In one embodiment, the protease recognition sequence is a substrate for matriptase. In one embodiment, the protease recognition site comprises or consists of the sequence PQARK (SEQ ID NO: 41) or HQARK (SEQ ID NO: 43).

[0178] In one embodiment, the protease-cleavable linker is an unstructured polypeptide. In one embodiment, the protease-cleavable linker exhibits no secondary structure. In one embodiment, the protease-cleavable linker comprises at least one linker that facilitates unstructured confirmation. In one embodiment, the linker comprises serine (S) and / or glycine (G). In one embodiment, the protease-cleavable linker is at least one linker comprising the amino acid sequence (GxS)n or (GxS)nGm (G=glycine, S=serine), where x=3, n=3, 4, 5, or 6, and m=0, 1, 2, or 3, or where x=4, n=2, 3, 4, or 5, and m=0, 1, 2, or 3, preferably where x=4 and n=2 or 3, and more preferably where x=4 and n=2. In one embodiment, the protease-cleavable linker comprises (G4S)2. In one embodiment, the protease-cleavable linker comprises (G4S)3. In one embodiment, the protease-cleavable linker comprises G2S. The protease-cleavable linker comprises a protease recognition site at any position (e.g., at the beginning, any position within, or at the end of the linker).

[0179] In one embodiment, the isolated polypeptide comprises or consists of the sequence SGGGSGGGGSPQARKGGGGSGGGGSGGGGSGGS (SEQ ID NO: 42). In one embodiment, the isolated polypeptide comprises or consists of the sequence SGGGSGGGGSHQARKGGGGSGGGGSGGGGSGGS (SEQ ID NO: 44).

[0180] Protease-activatable T cell activation bispecific molecule format The components of the protease-activatable T cell activating bispecific molecule can be fused to each other in a variety of configurations, exemplary configurations are shown in Figure 1.

[0181] In certain embodiments, the protease-activated T cell activating bispecific molecule comprises an Fc domain composed of a first and a second subunit capable of stable association, hi some embodiments, a 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.

[0182] 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.

[0183] 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, the Fc domain composed 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.

[0184] 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.

[0185] 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.

[0186] The antigen-binding portions may be fused to the Fc domain or to each other, 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 G4 (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 a first antigen-binding moiety and a second antigen-binding moiety to each other is (G4S)2. In addition, the linker may comprise (a portion of) an immunoglobulin hinge region. In particular, when the antigen-binding moiety is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

[0187] Protease-activatable T cell activating bispecific molecules with 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 can enhance internalization of the target cell antigen, thereby reducing its availability.

[0188] However, in many other cases, it will be advantageous to have a protease-activatable T cell activating bispecific molecule that contains two or more antigen-binding moieties specific for target cell antigens, for example, to optimize targeting to a target site or to allow cross-linking of target cell antigens.

[0189] Thus, in certain embodiments, the protease-activated T cell activating bispecific molecules of the invention further comprise a third antigen-binding moiety that is a Fab molecule capable of binding to a target cell antigen. In one embodiment, the third antigen-binding moiety is a conventional Fab molecule. In one embodiment, 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).

[0190] In certain embodiments, the first antigen-binding portion is capable of binding to CD3, the second and third antigen-binding portions are capable of binding to IGF-1R, and 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:61, SEQ ID NO:62, and SEQ ID NO:63, and at least one light chain CDR selected from the group of SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.

[0191] In certain embodiments, the first antigen binding portion is capable of binding to CD3, the second and third antigen binding portions are capable of binding to cMET, and 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:69, SEQ ID NO:70, and SEQ ID NO:71, and at least one light chain CDR selected from the group of SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75.

[0192] In certain embodiments, the first antigen-binding portion is capable of binding to CD3, the second and third antigen-binding portions are capable of binding to TROP2, and 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:77, SEQ ID NO:78 and SEQ ID NO:79, and at least one light chain CDR selected from the group of SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83.

[0193] 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:1, SEQ ID NO:2, and SEQ ID NO:3, and at least one light chain CDR selected from the group of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; and (ii) a second antigen-binding portion that is a Fab molecule capable of binding to a target cell antigen. Includes.

[0194] In one embodiment, the first 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:5, 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:10.

[0195] In one embodiment, the first antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:10.

[0196] In certain embodiments, the second antigen-binding portion is capable of binding to IGF-1R and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63, and at least one light chain CDR selected from the group of SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.

[0197] In another specific embodiment, the second antigen-binding portion is capable of binding to IGF-IR 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:64, 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:68.

[0198] In certain embodiments, the second antigen-binding portion is capable of binding to cMET and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, and at least one light chain CDR selected from the group of SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75.

[0199] In another specific embodiment, the second antigen-binding portion is capable of binding to IGF-IR 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:72, 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:76.

[0200] In certain embodiments, the second antigen-binding portion is capable of binding to TROP2 and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79, and at least one light chain CDR selected from the group of SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83.

[0201] In another specific embodiment, the second antigen-binding portion is capable of binding to TROP2 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: 80, 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: 84.

[0202] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a first antigen-binding moiety that is a Fab molecule capable of binding to CD3, the first antigen-binding moiety comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:30, and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; (ii) a second antigen-binding portion that is a Fab molecule capable of binding to IGF-1R, the second antigen-binding portion comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, and at least one light chain CDR selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0203] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a first antigen-binding moiety that is a Fab molecule capable of binding to CD3, the first antigen-binding moiety 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:5, 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:10; (ii) a second antigen-binding portion that is a Fab molecule capable of binding to IGF-1R, 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: 64, 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: 68.

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0204] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a first antigen-binding moiety that is a Fab molecule capable of binding to CD3, the first antigen-binding moiety comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:30, and at least one light chain CDR selected from the group of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; (ii) a second antigen-binding portion that is a Fab molecule capable of binding to cMET, comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, and at least one light chain CDR selected from the group consisting of SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75.

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0205] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a first antigen-binding moiety that is a Fab molecule capable of binding to CD3, the first antigen-binding moiety 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:5, 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:10; (ii) a second antigen-binding portion that is a Fab molecule capable of binding to cMET, 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: 72, 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: 76.

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0206] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a first antigen-binding moiety that is a Fab molecule capable of binding to CD3, the first antigen-binding moiety comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:30, and at least one light chain CDR selected from the group of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; (ii) a second antigen-binding portion that is a Fab molecule capable of binding to TROP2, comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO: 79, and at least one light chain CDR selected from the group consisting of SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83.

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0207] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a first antigen-binding moiety that is a Fab molecule capable of binding to CD3, the first antigen-binding moiety 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:5, 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:10; (ii) a second antigen-binding portion that is a Fab molecule capable of binding to TROP2, 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: 80, 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: 84.

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0208] In one embodiment, the second antigen-binding moiety is a conventional Fab molecule.

[0209] In certain embodiments, the first antigen-binding moiety is a crossover Fab molecule in which the constant regions of the Fab light and heavy chains are exchanged, 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.

[0210] 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.

[0211] 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).

[0212] 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:1, SEQ ID NO:2, and SEQ ID NO:3, and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; and the second and third antigen-binding portions are capable of binding to IGF-IR, and 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:61, SEQ ID NO:62, and SEQ ID NO:63, and at least one light chain CDR selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.

[0213] 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:1, SEQ ID NO:2, and SEQ ID NO:3, and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; and the second and third antigen-binding portions are capable of binding to IGF-IR, and 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:61, SEQ ID NO:62, and SEQ ID NO:63, and at least one light chain CDR selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.

[0214] 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:5 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:10; and the second and third antigen-binding portions are capable of binding to IGF-IR and 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:64 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:68.

[0215] 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:1, SEQ ID NO:2, and SEQ ID NO:3, and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; and the second and third antigen-binding portions are capable of binding to cMET, and 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:69, SEQ ID NO:70, and SEQ ID NO:71, and at least one light chain CDR selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75.

[0216] 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:1, SEQ ID NO:2, and SEQ ID NO:3, and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; and the second and third antigen-binding portions are capable of binding to cMET, and 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:69, SEQ ID NO:70, and SEQ ID NO:71, and at least one light chain CDR selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75.

[0217] 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:5 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:10; and the second and third antigen-binding portions are capable of binding to cMET, 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:72 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:76.

[0218] 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:1, SEQ ID NO:2, and SEQ ID NO:3, and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; and the second and third antigen-binding portions are capable of binding to TROP2, and 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:77, SEQ ID NO:78, and SEQ ID NO:79, and at least one light chain CDR selected from the group consisting of SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83.

[0219] 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:1, SEQ ID NO:2, and SEQ ID NO:3, and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; and the second and third antigen-binding portions are capable of binding to TROP2, and 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:77, SEQ ID NO:78, and SEQ ID NO:79, and at least one light chain CDR selected from the group consisting of SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83.

[0220] In certain embodiments, 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:5 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:10; and the second and third antigen-binding portions are capable of binding to TROP2 and 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:80 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:84.

[0221] 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 one embodiment, 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 of the immunoglobulin heavy chains.

[0222] 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 the first, second, and third antigen-binding moieties, an Fc domain composed 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.

[0223] In one embodiment, the present invention provides a method for producing a pharmaceutical composition 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: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 7, a light chain of SEQ ID NO: 8, and a light chain CDR3 of SEQ ID NO: 9, 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 IGF-1R, comprising a heavy chain CDR1 of SEQ ID NO: 61, a heavy chain CDR2 of SEQ ID NO: 62, a heavy chain CDR3 of SEQ ID NO: 63, a light chain CDR1 of SEQ ID NO: 65, a light chain CDR2 of SEQ ID NO: 66, and a light chain CDR3 of SEQ ID NO: 67.

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0224] In one embodiment, the present invention provides a method for producing a pharmaceutical composition 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: 5 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: 10, 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 IGF-1R, 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: 64, 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: 68.

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0225] In one embodiment, the present invention provides a method for producing a pharmaceutical composition 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: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 7, a light chain of SEQ ID NO: 8, and a light chain CDR3 of SEQ ID NO: 9, 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 cMET, comprising a heavy chain CDR1 of SEQ ID NO: 69, a heavy chain CDR2 of SEQ ID NO: 70, a heavy chain CDR3 of SEQ ID NO: 71, a light chain CDR1 of SEQ ID NO: 73, a light chain CDR2 of SEQ ID NO: 74, and a light chain CDR3 of SEQ ID NO: 75.

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0226] In one embodiment, the present invention provides a method for producing a pharmaceutical composition 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: 5 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: 10, 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 cMET, 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: 72, 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: 76.

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0227] In one embodiment, the present invention provides a method for producing a pharmaceutical composition 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: 1, a heavy chain CDR2 of SEQ ID NO: 2, a heavy chain CDR3 of SEQ ID NO: 3, a light chain CDR1 of SEQ ID NO: 7, a light chain of SEQ ID NO: 8, and a light chain CDR3 of SEQ ID NO: 9, 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 moieties, each of which is a Fab molecule capable of binding to TROP2, comprising a heavy chain CDR1 of SEQ ID NO: 77, a heavy chain CDR2 of SEQ ID NO: 78, a heavy chain CDR3 of SEQ ID NO: 79, a light chain CDR1 of SEQ ID NO: 81, a light chain CDR2 of SEQ ID NO: 82, and a light chain CDR3 of SEQ ID NO: 83;

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0228] In one embodiment, the present invention provides a method for producing a pharmaceutical composition 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: 5 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: 10, 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 moieties, each of which is a Fab molecule capable of binding to TROP2, 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: 80, 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: 84.

[0023] The present invention provides a protease-activatable T cell activating bispecific molecule comprising:

[0229] The protease-activated T cell activating bispecific molecule according to any of the above embodiments can 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.

[0230] In some of the protease-activated 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 antigen-binding moieties further reduces mispairing of mismatched Fab heavy and light chains and also reduces the number of plasmids required to express some of the protease-activated T cell activating bispecific molecules of the invention.

[0231] In certain embodiments, the protease-activated 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 is connected to a carboxy-terminal peptide bond with a Fab light chain constant region (VH (1) -CL (1) ) and a Fab light chain polypeptide (VL (2) -CL (2) ) In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0232] In an alternative embodiment, the protease-activated 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 (2) -CL (2) In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0233] In some embodiments, the protease-activated 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-activated T cell activating bispecific molecule comprises a polypeptide (VH) in which the Fab heavy chain variable region of a 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 a 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 the Fab light chain variable region of the first antigen-binding moiety, which in turn 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 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)).

[0234] In some of these embodiments, the protease-activatable T cell activating bispecific molecule comprises a crossover Fab light chain polypeptide (VH) of the 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) ), a polypeptide in which the Fab heavy chain variable region of the first antigen-binding portion shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first antigen-binding portion, which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the second antigen-binding portion (VH (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 includes.

[0235] 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 a third antigen-binding portion of the Fab light chain polypeptide (VL (3) -CL (3) In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0236] According to any of the above embodiments, the components of the protease-activatable T cell activating bispecific molecule (e.g., antigen-binding portion, Fc domain) may be fused directly or via a variety of 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, and n is usually a number from 1 to 10, typically from 2 to 4.

[0237] Fc domain The Fc domain of a protease-activated 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 the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment, a protease-activated T cell activating bispecific molecule of the invention comprises up to one Fc domain.

[0238] In an embodiment according to the invention, the Fc domain of the protease-activated 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 specific embodiment, 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.

[0239] Fc domain modifications that promote heterodimerization The protease-activated T cell activating bispecific molecules according to the present invention comprise different antigen-binding moieties fused to one or the other of the two subunits of the Fc domain, which are therefore typically comprised 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 protease-activated T cell activating bispecific molecules in recombinant production, it is advantageous to introduce modifications into the Fc domain of the protease-activated T cell activating bispecific molecule that promote the association of the desired polypeptides.

[0240] Thus, in a particular aspect, the protease-activatable T cell activating bispecific molecule according to the invention comprises a modification that promotes the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is within the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0241] 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.

[0242] This knob-into-hole technique has been described, for example, in U.S. Patent No. 5,731,168 and U.S. Patent 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, such that the protrusion can be positioned 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).

[0243] Thus, in certain embodiments, in the CH3 domain of a first subunit of the Fc domain of the protease-activated 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.

[0244] The protrusions and cavities can be created by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0245] 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, additionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0246] 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 further replaced with a serine residue (Y349C). The introduction of these two cysteine ​​residues creates a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0247] In certain embodiments, an antigen-binding moiety capable of binding to CD3 is fused to the first subunit of the Fc domain (including a "knob" modification) (optionally via an antigen-binding moiety capable of binding to a target cell antigen). Without wishing to be bound by theory, fusion of an antigen-binding moiety capable of binding to CD3 to the knob-containing subunit of the Fc domain will (further) minimize the generation of antigen-binding molecules comprising two antigen-binding moieties capable of binding to CD3 (steric clash of the two knob-containing polypeptides).

[0248] 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 replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues such that homodimer formation is electrostatically unfavorable, but heterodimerization is electrostatically favorable.

[0249] Fc domain modifications that reduce Fc receptor binding and / or effector function The Fc domain confers favorable pharmacokinetic properties to protease-activated T cell-activating bispecific molecules, such as a long serum half-life and favorable tissue-blood distribution ratio, which contribute to favorable accumulation in target tissues. However, it can also result in undesirable targeting of protease-activated 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 lead to cytokine release, which, combined with the T cell-activating properties and long half-life of the antigen-binding molecule, can result in 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-activated T cell-activating bispecific molecules due to the potential for T cell destruction by, for example, NK cells.

[0250] Thus, in certain embodiments, the Fc domain of a protease-activated 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-activated T cell activating bispecific molecule comprising said Fc domain) exhibits less than 50%, preferably less than 20%, even 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-activated T cell activating bispecific molecule comprising a native IgG1 Fc domain) and / or less than 50%, preferably less than 20%, even 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-activated T cell activating bispecific molecule comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or a protease-activated 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 one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly human FcγRIIIa. In one embodiment, 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 one embodiment, 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-activated 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-activated T cell activating bispecific molecule comprising a native IgG1 Fc domain).

[0251] In certain embodiments, the Fc domain is engineered 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-activated 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 one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor. In one embodiment, 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 where more than one amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor is present, the 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 one embodiment, a protease-activated T cell activating bispecific molecule comprising an altered Fc domain exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity to an Fc receptor compared to a protease-activated T cell activating bispecific molecule comprising an unmodified Fc domain. In a specific embodiment, 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 a specific embodiment, 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 one embodiment, binding affinity to 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-activated 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-activated T cell activating bispecific molecule comprising said unmodified Fc domain). An Fc domain or a protease-activated 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 a protease-activated T cell activating bispecific molecule is modified to have reduced effector function compared to the unmodified Fc domain. Reduced effector function can include, but is not limited to, one or more of the following: 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 one embodiment, the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a specific embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by an unmodified Fc domain (or a protease-activated T cell-activating bispecific molecule comprising an unmodified Fc domain).

[0252] In one embodiment, 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 one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329. In a more specific embodiment, 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 one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, 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 one embodiment, 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 a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235. In a further particular embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). In one such embodiment, 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 PCT Application Publication No. 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.

[0253] 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-activated T cell activating bispecific molecule of the present invention is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, 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 one embodiment, 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 particular 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.

[0254] 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.

[0255] In certain embodiments, the N-glycosylation of the Fc domain is ablated, in such embodiments, the Fc domain comprises an amino acid substitution at position N297, particularly replacing asparagine with alanine (N297A) or aspartic acid (N297D).

[0256] In addition to the Fc domains described above and in PCT Publication No. WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also include those with substitutions of one or more of 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, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0257] Mutant 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 correct nucleotide changes can be confirmed, for example, by screening.

[0258] Binding to Fc receptors can be readily determined, for example, by ELISA or surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare) and Fc receptors obtained by recombinant expression. Suitable 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 (e.g., human NK cells expressing the FcγIIIa receptor).

[0259] The effector function of Fc domain or the protease-activated T cell activating bispecific molecule comprising Fc domain can be measured by methods known in the art.Suitable assays for measuring ADCC are described herein.Other examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent No. 5,500,362; Hellstrom et al., Proc Natl Acad Sci USA 83,70597063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,14991502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166,13511361 (1987). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) 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)).

[0260] 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 engineered to have reduced effector function, the reduced effector function includes reduced CDC. To determine whether a protease-activatable T cell-activating bispecific molecule is capable of binding C1q and therefore has CDC activity, a C1q binding assay may be performed. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, 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)).

[0261] Exemplary Protease-Activated T Cell Activating Bispecific Molecules Capable of Binding to CD3 and IGF-1R, cMET, or TROP2 The first antigen-binding moiety capable of binding to CD3 as described herein, the second (and third) antigen-binding moiety capable of binding to IGF-1R, cMET, or TROP2 as described herein, the Fc domain as described herein, the masking moiety, and the protease-cleavable linker of the invention may be fused to each other in various configurations.

[0262] An exemplary configuration is shown in Figure 1. Exemplary sequences are provided herein below.

[0263] In one embodiment, the protease-activated T cell activating bispecific molecule comprising a first antigen-binding portion capable of binding to CD3 and a second and a third antigen-binding portion capable of binding to IGF-IR comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:85, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:87, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:88, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:89. In one embodiment, the protease-activated T cell activating bispecific molecule comprises a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 85, a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 87, a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 88, and a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 89. In one embodiment, the protease-activated T cell activating bispecific molecule comprises the polypeptide sequence of SEQ ID NO: 85, the polypeptide sequence of SEQ ID NO: 87, the polypeptide sequence of SEQ ID NO: 88, and the polypeptide sequence of SEQ ID NO: 89.

[0264] In one embodiment, a protease-activated T cell activating bispecific molecule comprising a first antigen-binding portion capable of binding to CD3 and a second antigen-binding portion capable of binding to cMET comprises a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 91, a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 93, and a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 94. In one embodiment, the protease-activated T cell activating bispecific molecule comprises a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 91, a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 93, and a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 94. In one embodiment, the protease-activated T cell activating bispecific molecule comprises the polypeptide sequence of SEQ ID NO: 91, the polypeptide sequence of SEQ ID NO: 93, and the polypeptide sequence of SEQ ID NO:94.

[0265] In one embodiment, a protease-activated T cell activating bispecific molecule comprising a first antigen-binding portion capable of binding to CD3 and a second and a third antigen-binding portion capable of binding to TROP2 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:98, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:99, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:100. In one embodiment, the protease-activated 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: 98, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 99, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 100. In one embodiment, the protease-activated T cell activating bispecific molecule comprises the polypeptide sequence of SEQ ID NO: 96, the polypeptide sequence of SEQ ID NO: 98, the polypeptide sequence of SEQ ID NO: 99, and the polypeptide sequence of SEQ ID NO: 100.

[0266] Polynucleotides The present invention further provides isolated polynucleotides encoding the protease-activated T cell activating bispecific molecules described herein, or fragments thereof, in some embodiments, wherein the fragments are antigen-binding fragments.

[0267] Polynucleotides encoding the protease-activated T cell activating bispecific molecules of the invention can be expressed as a single polynucleotide encoding the entire protease-activated T cell activating bispecific molecule, or as multiple (e.g., two or more) co-expressed polynucleotides. Polypeptides encoded by co-expressed polynucleotides can associate, for example, via disulfide bonds or other means, to form a functional protease-activated 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-activated 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 combines with the light chain polypeptide to form the antigen-binding moiety. In another example, the portion of a protease-activated 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-activated T cell activating bispecific molecule comprising the other of the two Fc domain subunits and, optionally, (a portion of) an antigen-binding moiety. When co-expressed, the Fc domain subunits associate to form an Fc domain.

[0268] In some embodiments, the isolated polynucleotide encodes the entire protease-activated T cell activating bispecific molecule according to the invention as described herein, hi other embodiments, the isolated polynucleotide encodes a polypeptide comprised in a protease-activated T cell activating bispecific molecule according to the invention as described herein.

[0269] In another embodiment, the present invention relates to an isolated polynucleotide encoding a protease-activated 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-activated T cell activating bispecific molecule or a fragment thereof, wherein the polynucleotide comprises a sequence encoding the polypeptide sequence set forth in SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, or a fragment thereof.

[0270] 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 one embodiment, 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.

[0271] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, for example in the form of messenger RNA (mRNA). The RNA of the invention can be single-stranded or double-stranded.

[0272] Recombinant methodsThe protease-activated 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-activated 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 one embodiment, 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-activated T cell activating bispecific molecules (fragments) along 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-activated T cell activating bispecific molecule (fragment) is cloned in operably linked to 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 translated into amino acids. A "stop codon" (TAG, TGA, or TAA) is not translated into amino acids, but is considered to be part of the coding region when present.However, any adjacent 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 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 into final proteins post- or co-translationally via 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-activated T cell activating bispecific molecule (fragment) or variant or derivative thereof of the invention. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. An operably linked region is one in which a coding region for a gene product, such as a polypeptide, is linked to one or more regulatory sequences in such a way that expression of the gene product is under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and its associated promoter) are "operably linked" 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 transcription of the DNA template. Thus, a promoter region is operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of that nucleic acid. A promoter can be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcriptional control elements besides a promoter, such as enhancers, operators, repressors, and transcription termination signals, can be operably linked to a polynucleotide to direct cell-specific transcription.Suitable promoters and other transcriptional regulatory regions are disclosed herein. Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as promoter and enhancer segments 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 α-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Further suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translational control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly, internal ribosome entry sites or IRESs, also known as CITE sequences). 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).

[0273] 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-activated T cell activating bispecific molecule is desired, DNA encoding a signal sequence can be located upstream of the nucleic acid encoding the protease-activated 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 growing protein chain across the rough endoplasmic reticulum. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells typically possess a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to generate the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, e.g., an immunoglobulin heavy or light chain signal peptide, is used, or a functional derivative of that sequence that retains the ability to direct the secretion of a polypeptide operably associated therewith is used. 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.

[0274] 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-activated T cell activating bispecific molecule may be included within or at the end of the protease-activated T cell activating bispecific molecule encoding polynucleotide (fragment).

[0275] In further embodiments, host cells are provided that comprise one or more polynucleotides of the invention. In particular embodiments, host cells are provided that comprise 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, either 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-activated 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-activated T cell activating bispecific molecule of the invention, or a fragment thereof. Suitable host cells for supporting the expression of protease-activated 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-activated T cell activating bispecific molecules for clinical applications. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, and the like. For example, polypeptides can be produced in bacteria, particularly when glycosylation is not required. After expression, the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains whose glycosylation pathways have 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 and 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 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293T cells described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells 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 (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (e.g., Mather et al., Annals NYAcad Sci 383, 44-68 (1982)), MRC5 cells, and FS4cells. Another useful mammalian host cell line is 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 cell lines 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 a few, as well as 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 (e.g., a Y0, NS0, Sp20 cell).

[0276] 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.

[0277] In one embodiment, there is provided a method for producing a protease-activated T cell activating bispecific molecule according to the invention, the method comprising culturing a host cell comprising a polynucleotide encoding a T cell activating bispecific antigen binding molecule under conditions suitable for expression of the protease-activated T cell activating bispecific molecule as provided herein, and recovering the protease-activated T cell activating bispecific molecule from the host cell (or host cell culture medium).

[0278] The components of the protease-activated T cell activating bispecific molecule are genetically fused to each other. The protease-activated T cell activating bispecific molecule can be designed so that its components are directly fused to each other or indirectly fused 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 different components of the protease-activated T cell activating bispecific molecule can be found in the sequences provided herein. Additional sequences, such as endopeptidase recognition sequences, can also be included as needed to incorporate cleavage sites for separating the individual components of the fusion.

[0279] In certain embodiments, one or more antigen-binding portions of the protease-activated T cell activating bispecific molecule comprise at least an antibody variable region capable of binding to an antigenic determinant. The variable region may form part of or be derived from naturally occurring or non-naturally occurring 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-naturally occurring 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 by McCafferty).

[0280] Antibodies, antibody fragments, antigen-binding domains, or variable regions of any animal species can be used in the protease-activated T cell activating bispecific molecules of the invention. Non-limiting antibodies, antibody fragments, antigen-binding domains, or variable regions useful in the present invention can be of murine, primate, or human origin. When the protease-activated 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. Patent 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) into the framework and constant regions of a human (e.g., recipient antibody) with or without retaining critical framework residues (e.g., those 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) into 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 are also described in, for example, 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).The human variable region can form part of and be derived from a human monoclonal antibody produced by the hybridoma method (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 modified 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. in Methods in Molecular Biology 178, 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments.

[0281] In certain embodiments, antigen-binding moieties useful in the present invention are engineered 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-activated 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 J17, 323-329 (2000)) and classical binding assays (Heeley, Endocr Res28, 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 Morris (1996) "Epitope Mapping Protocols" in Methods in Molecular Biology, vol. 66 (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 (e.g., V9 antibody, described in U.S. Pat. No. 6,054,297) that binds to the antigen 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, this indicates that the second antibody is competing with the first antibody for binding to the antigen.See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0282] Protease-activated 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-activated T cell-activating bispecific molecule binds. For example, for affinity chromatography purification of the protease-activated 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-activated T cell-activating bispecific molecules essentially as described in the Examples. The purity of the protease-activated 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 proteins of the protease-activatable T cell activating bispecific molecule.

[0283] 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.

[0284] Affinity assay The affinity of protease-activated 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, as described in the Examples. Alternatively, binding of protease-activated 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 and exemplary embodiments for measuring binding affinity are described below and in the Examples that follow.

[0285] According to one embodiment, K D is measured by surface plasmon resonance using a BIACORE® T100 instrument (GE Healthcare) at 25°C.

[0286] To analyze the interaction between the Fc moiety and Fc receptors, His-tagged recombinant Fc receptors are captured by anti-Penta His antibodies (Qiagen) immobilized on a CM5 chip, and bispecific constructs are used as analytes. Briefly, a carboxymethylated dextran biosensor chip (CM5, GE Healthcare) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The anti-Penta His antibody is diluted to 40 μg / ml in 10 mM sodium acetate (pH 5.0) and then injected at a flow rate of 5 μl / min, yielding approximately 6500 response units (RU) of bound protein. After ligand injection, 1 M ethanolamine is injected to block unreacted groups. The Fc receptor is then captured at 4 nM or 10 nM for 60 s. 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.

[0287] To determine affinity for the target antigen, the bispecific construct was captured by an anti-human Fab specific antibody (GE Healthcare) immobilized on an activated CM5 sensor chip surface, as described for the anti-Penta-His antibody. The final amount of bound protein was approximately 12,000 RU. The bispecific construct was captured at 300 nM for 90 seconds. The target antigen was passed through the flow cell for 180 seconds at a flow rate of 30 μl / min, ranging from 250 nM to 1,000 nM. Dissociation was monitored for 180 seconds.

[0288] 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. D The association rate (k on ) and dissociation rate (k off) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® T100 Evaluation Software Version 1.1.1). D ) is k off / k on It is calculated as a ratio. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0289] Activity assay The biological activity of the protease-activated 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 signal transduction 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.

[0290] Compositions, Formulations, and Routes of Administration In a further aspect, the present invention provides pharmaceutical compositions comprising any of the protease-activated T cell activating bispecific molecules provided herein, e.g., pharmaceutical compositions for use in any of the therapeutic methods described below. In one embodiment, the pharmaceutical composition comprises any of the protease-activated T cell activating bispecific molecules provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the protease-activated T cell activating bispecific molecules provided herein and at least one additional therapeutic agent, e.g., as described below.

[0291] Further provided is a method for producing a protease-activated T cell activating bispecific molecule of the invention in a form suitable for in vivo administration, the method comprising: (a) obtaining a protease-activated T cell activating bispecific molecule according to the invention; and (b) formulating the protease-activated T cell activating bispecific molecule with at least one pharmaceutically acceptable carrier, whereby the preparation of protease-activated T cell activating bispecific molecule is formulated for in vivo administration.

[0292] 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 as 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, it will be understood that for administration to animals (e.g., humans), the formulation must meet sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards or other national equivalents. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carriers" are 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), and include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such materials, and combinations thereof. Except insofar as a conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0293] 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 other methods or any combination of the above 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.

[0294] 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 aqueous solutions, preferably physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solutions may contain formulatory 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., sterile pyrogen-free 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. Typically, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and / or other ingredients. In the case of sterile powders for preparing sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying, which yields a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered liquid medium. The liquid medium should be appropriately buffered, if necessary, and the liquid diluent is first rendered isotonic with sufficient saline or glucose prior to 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 will be understood that endotoxin contamination should be kept to a minimum, for example, below 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 (less than about 10 residues) polypeptides; tannins, etc. Proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; 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 also contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, and the like. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow 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.

[0295] The active ingredient can also be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions, for example, by coacervation techniques or microcapsules prepared by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such 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 the use in the compositions of agents that delay absorption, such as aluminum monostearate, gelatin, or combinations thereof.

[0296] In addition to the above-mentioned compositions, the protease-activated T cell activating bispecific molecule can also be formulated as a depot preparation.Such long-acting preparations can be administered by implantation (for example, subcutaneously or intramuscularly) or intramuscular injection.Therefore, for example, the protease-activated T cell activating bispecific molecule can be formulated with suitable polymers or hydrophobic materials (for example, as an emulsion in acceptable oil) or ion exchange resins, or as a poorly soluble derivative, for example, as a poorly soluble salt.

[0297] Pharmaceutical compositions comprising the protease-activatable T cell activating bispecific molecules of the invention can be produced 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, additives, or adjuvants that facilitate processing of proteins into pharmaceutically usable preparations. The appropriate formulation depends on the chosen route of administration.

[0298] The protease-activatable T cell activating bispecific molecule can be formulated in the composition 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. These include acid addition salts, such as those formed with free amino groups of the proteinaceous composition, or those formed 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.

[0299] 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.

[0300] For use in therapeutic methods, the protease-activatable T cell activating bispecific molecules of the invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include 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 delivery of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners.

[0301] In one aspect, a protease-activated T cell activation bispecific molecule of the invention is provided for use as a pharmaceutical. In a further aspect, a protease-activated T cell activation bispecific molecule of the invention is provided for use in the treatment of a disease. In certain embodiments, a protease-activated T cell activation bispecific molecule of the invention is provided for use in a method of treatment. In one embodiment, the invention provides a protease-activated T cell activation 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-activated T cell activation 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-activated T cell activation 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, for example, an anti-cancer agent if 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.

[0302] In a further aspect, the present invention provides use of a protease-activated T cell activating bispecific molecule of the invention in the manufacture or preparation of a medicament. In one embodiment, 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 a particular embodiment, the disease being treated is a proliferative disorder. In a particular embodiment, the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, for example, an anti-cancer agent if the disease being treated is cancer. In a further embodiment, the medicament is for inducing lysis of target cells, particularly tumor cells. In a still 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 to the individual an effective amount of the medicament to induce lysis of the target cells. An "individual" according to any of the above embodiments may be a mammal, preferably a human.

[0303] In a further aspect, the present invention provides a method for treating a disease. In one embodiment, the method comprises administering to an individual having such a disease a therapeutically effective amount of a protease-activated T cell activating bispecific molecule of the invention. In one embodiment, a composition comprising a protease-activated 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, for example, an anti-cancer agent if the disease being treated is cancer. An "individual" according to any of the above embodiments may be a mammal, preferably a human.

[0304] 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.

[0305] In one embodiment, 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, gastric 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 present 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 gland), 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 can only provide partial benefit. In some embodiments, any physiological change that has any 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 specifically a human.

[0306] In some embodiments, an effective amount of a protease-activatable T cell activating bispecific molecule of the invention is administered to a cell. In 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.

[0307] The appropriate dosage of the protease-activated T cell activating bispecific molecule of the present invention (used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of a 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-activated T cell activating bispecific molecule, and the discretion of the attending physician. The physician responsible for administration will, in any event, determine the concentration of active ingredient in the composition and the appropriate dose for each individual subject. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0308] 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 to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg), whether by single or multiple individual administrations or by continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on symptoms, treatment is usually continued until a desired suppression of disease symptoms occurs. One exemplary dosage of the T cell-activating antigen-binding molecule would be in the range of about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, the dosage may be 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, including any range derivable 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, 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) may be administered to the patient. 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 higher loading dose, followed by one or more lower doses, may also be administered.However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0309] 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 disease symptoms, 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.

[0310] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. The IC 50 A dose may be formulated in animal models to achieve a circulating concentration range including 100 mg / kg / day. Such information can be used to more accurately determine useful doses in humans.

[0311] Initial dosages can also be estimated from in vivo data, e.g., from animal models, using techniques well known in the art. Those skilled in the art will readily be able to optimize administration to humans based on the animal data.

[0312] Dosage and administration intervals 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 for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma concentrations may be achieved by administering multiple doses each day. Plasma levels can be measured, for example, by HPLC.

[0313] 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. One skilled in the art will be able to optimize a therapeutically effective local dosage without undue experimentation.

[0314] 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. Using cell culture assays and animal studies, LD 50 (lethal dose for 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 Therapeutic indices can be expressed as a ratio. 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 to formulate a range of dosages appropriate for human use. Dosages are preferably within the ED50 range with little or no toxicity. 50 The blood concentration range includes: 0.05 to 0.15; ...

[0315] The attending physician of a patient being treated with a protease-activatable T cell activating bispecific molecule of the invention will know when and how 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 (barring toxicity). The magnitude of an administered dose in the management of a target disorder will vary depending on the severity of the condition being treated, the route of administration, etc. The severity of the condition can be assessed, for example, in part, by standard prognostic evaluation methods. Furthermore, the dose, and perhaps the frequency of administration, will also vary according to the age, weight, and response of the individual patient.

[0316] Other drugs and treatments In therapeutic settings, the protease-activated T cell activating bispecific molecules of the invention may be administered in combination with one or more other agents. For example, the protease-activated 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 of such treatment. Such additional therapeutic agents may 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.

[0317] Such other agents are suitably present in 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.

[0318] Such combination therapy encompasses combined administration (wherein the two or more therapeutic agents are in the same or separate compositions) and separate administration, where administration of the protease-activated 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-activated T cell activating bispecific molecules of the invention can also be used in combination with radiation therapy.

[0319] 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 solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds the composition by itself or in combination with another composition effective for treating, preventing, and / or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with 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 present invention. The label or package insert indicates that the composition is used for treating the condition of choice. 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 in 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 include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0320] Exemplary Implementations 1. (a) a first antigen-binding moiety capable of binding to CD3; and (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 peptide linker; wherein the masking moiety is capable of binding to the idiotype of the first antigen-binding moiety or the second antigen-binding moiety, thereby reversibly masking the first antigen-binding moiety or the second antigen-binding moiety; A protease-activatable T cell activating bispecific molecule, wherein the linker comprises the protease recognition sequence XQARK (SEQ ID NO: 39), wherein X is histidine (H) or proline (P).

[0321] 2. The protease-activatable T cell activating bispecific molecule of embodiment 1, wherein the masking moiety is covalently attached to and reversibly masks the first antigen-binding moiety.

[0322] 3. The protease-activatable T cell activating bispecific molecule of embodiment 1 or 2, wherein the masking moiety is covalently linked to the heavy chain variable region of the first antigen-binding moiety.

[0323] 4. The protease-activatable T cell activating bispecific molecule of embodiment 1 or 2, wherein the masking moiety is covalently linked to the light chain variable region of the first antigen-binding moiety.

[0324] 5. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 4, wherein the masking moiety is an scFv.

[0325] 6. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 5, comprising a second masking moiety that reversibly masks the second antigen-binding moiety.

[0326] 7. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 6, wherein the protease is expressed by the target cell.

[0327] 8. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 7, wherein (i) the second antigen-binding portion is a conventional Fab, or (ii) the second antigen-binding portion is a crossover Fab molecule in which either the variable or constant regions of the Fab light chain and the Fab heavy chain have been exchanged.

[0328] 9. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 8, 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 swapped.

[0329] 10. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 9, wherein the first antigen-binding portion is a conventional Fab molecule.

[0330] 11. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 10, comprising up to one antigen-binding moiety capable of binding to CD3.

[0331] 12. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 11, comprising a third antigen-binding portion which is a Fab molecule capable of binding to a target cell antigen.

[0332] 13. The protease-activatable T cell activating bispecific molecule of embodiment 12, wherein the third antigen-binding portion is identical to the second antigen-binding portion.

[0333] 14. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-13, wherein the second antigen-binding portion is capable of binding to IGF-1R, cMET, or TROP2.

[0334] 15. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 14, wherein the first antigen-binding portion and the second antigen-binding portion are fused to each other, optionally via a peptide linker.

[0335] 16. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-15, 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.

[0336] 17. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-15, 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.

[0337] 18. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 17, 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.

[0338] 19. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 18, additionally comprising an Fc domain composed of a first and a second subunit capable of stable association.

[0339] 20. The protease-activatable T cell activating bispecific molecule according to embodiment 19, wherein the Fc domain is an IgG, particularly an IgG1 or IgG4 Fc domain.

[0340] 21. The protease-activatable T cell activating bispecific molecule according to embodiment 19 or 20, wherein the Fc domain is a human Fc domain.

[0341] 22. The protease-activatable T cell activating bispecific molecule of any one of embodiments 19-21, wherein the Fc domain exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain.

[0342] 23. The protease-activatable T cell activating bispecific molecule of embodiment 22, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector function.

[0343] 24. The protease-activatable T cell activating bispecific molecule of embodiment 23, 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).

[0344] 25. The protease-activated T cell activating bispecific molecule of embodiment 24, 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.

[0345] 26. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 22 to 25, wherein the Fc receptor is an Fcγ receptor.

[0346] 27. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 22 to 26, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0347] 28. The moiety capable of binding to CD3 is (i) a heavy chain variable (VH) region comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, and an HCDR3 of SEQ ID NO: 3; and (ii) a light chain variable (VL) region comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 7, an LCDR2 of SEQ ID NO: 8, and an LCDR3 of SEQ ID NO: 9; The protease-activatable T cell activating bispecific molecule of claim 1 , comprising:

[0348] 29. The protease-activated T cell activating bispecific molecule of any one of claims 1 to 28, wherein the portion capable of binding to CD3 comprises a VH 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: 5, and / or a VL 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: 10.

[0349] 30. The moiety capable of binding to CD3 is (i) a heavy chain variable (VH) region comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, and an HCDR3 of SEQ ID NO: 4; and (ii) a light chain variable (VL) region comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 7, an LCDR2 of SEQ ID NO: 8, and an LCDR3 of SEQ ID NO: 9; The protease-activatable T cell activating bispecific molecule of claim 1 , comprising:

[0350] 31. The protease-activated T cell activating bispecific molecule of any one of claims 1 to 27 or 30, wherein the portion capable of binding to CD3 comprises a VH 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: 6, and / or a VL 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: 10.

[0351] 32.Masking part, (a) the heavy chain complementarity determining region (HCDR) 1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) an HCDR2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO: 16), WINTETGEPRYTDDFTG (SEQ ID NO: 17), and WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) 32. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 31, comprising a heavy chain variable region comprising at least one of:

[0352] 33.Masking part, (d) the light chain complementarity-determining region (LCDR) 1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25) or KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) an LCDR3 amino acid sequence selected from the group consisting of QHSREFPYT (SEQ ID NO: 28) or QQSREFPYT (SEQ ID NO: 29). 33. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 32, comprising a light chain variable region comprising at least one of:

[0353] 34.Masking part, (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) an HCDR2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO: 16), WINTETGEPRYTDDFTG (SEQ ID NO: 17), and WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60) a heavy chain variable region comprising (d) an LCDR1 amino acid sequence selected from the group consisting of RASKSVSTSSYSYMH (SEQ ID NO: 25) and KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) an LCDR3 amino acid sequence selected from the group consisting of QHSREFPYT (SEQ ID NO: 28) and QQSREFPYT (SEQ ID NO: 29). a light chain variable region comprising 34. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 33, comprising:

[0354] 35.Masking part, (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) a VH region comprising (d) the LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising 34. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 33, comprising:

[0355] 36.Masking part, (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) a VH region comprising (d) the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising 34. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 33, comprising:

[0356] 37.Masking part, (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO: 17); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) a VH region comprising (d) the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising 34. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 33, comprising:

[0357] 38.Masking part, (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) a VH region comprising (d) the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising 34. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 33, comprising:

[0358] 39.Masking part, (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) a VH region comprising (d) the LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) LCDR3 amino acid sequence of QQSREFPYT (SEQ ID NO: 29) and a VL region comprising 34. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 33, comprising:

[0359] 40. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 39, wherein the masking moiety is humanized.

[0360] 41. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-40, wherein the masking moiety comprises a VH 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: 21, and a VL 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: 32.

[0361] 42. The protease-activated T cell activating bispecific molecule according to any one of embodiments 1 to 33, wherein the masking moiety comprises a VH region comprising the amino acid sequence of SEQ ID NO: 21 and a VL region comprising the amino acid sequence of SEQ ID NO: 32.

[0362] 43. The protease-activated T cell activating bispecific molecule of any one of embodiments 1 to 42, wherein the second antigen-binding portion is capable of binding to IGF-IR and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63 and / or at least one light chain CDR selected from the group of SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67.

[0363] 44. The second antigen-binding moiety is capable of binding to IGF-1R, and a) the HCDR1 amino acid sequence of SYGMH (SEQ ID NO: 61); b) the HCDR2 amino acid sequence of IIWFDGSSTYYADSVRG (SEQ ID NO: 62); c) HCDR3 amino acid sequence of ELGRRYFDL (SEQ ID NO: 63) a heavy chain variable region comprising d) LCDR1 of RASQSVSSYLA (SEQ ID NO: 65); e) LCDR2 amino acid sequence of DASKRAT (SEQ ID NO: 66); f) LCDR3 amino acid sequence of QQRSKWPPWT (SEQ ID NO: 67) and a VL region comprising 43. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 42, comprising:

[0364] 45. The protease-activated T cell activating bispecific molecule of any one of embodiments 1 to 44, wherein the second antigen-binding portion is capable of binding to IGF-IR 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: 64, 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: 68.

[0365] 46. ​​The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 44, wherein the second antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 64 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 68.

[0366] 47. The protease-activated T cell activating bispecific molecule of any one of embodiments 1 to 42, wherein the second antigen-binding portion is capable of binding to cMET and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71 and / or at least one light chain CDR selected from the group of SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75.

[0367] 48. The second antigen-binding moiety is capable of binding to cMET, and a) the HCDR1 amino acid sequence of SYWLH (SEQ ID NO: 69); b) the HCDR2 amino acid sequence of MIDPSNSDTRFNPNFKD (SEQ ID NO: 70); c) HCDR3 amino acid sequence of YRSYVTPLDY (SEQ ID NO: 71) a heavy chain variable region comprising d) LCDR1 of KSSQSLLYTSSQKNYLA (SEQ ID NO: 73); e) LCDR2 amino acid sequence of WASTRES (SEQ ID NO: 74); f) LCDR3 amino acid sequence of QQYYAYPWT (SEQ ID NO: 75) and a VL region comprising 43. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 42, comprising:

[0368] 49. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1-42 or 46-47, wherein the second antigen-binding portion is capable of binding to cMET 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: 72, 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: 76.

[0369] 50. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 42 or 46 to 49, wherein the second antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 76.

[0370] 51. The protease-activated T cell activating bispecific molecule of any one of embodiments 1 to 42, wherein the second antigen-binding portion is capable of binding to TROP2 and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79 and / or at least one light chain CDR selected from the group of SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83.

[0371] 52. The second antigen-binding moiety is capable of binding to TROP2, and (a) the HCDR1 amino acid sequence of NYGMN (SEQ ID NO: 77); b) the HCDR2 amino acid sequence of WINTKTGEPTYAEEFKG (SEQ ID NO: 78); c) the HCDR3 amino acid sequence of GGYGSSYWYFDV (SEQ ID NO: 79); a heavy chain variable region comprising d) LCDR1 of KASQDVSIAVA (SEQ ID NO: 81); e) the LCDR2 amino acid sequence of SASYRYT (SEQ ID NO: 82); f) LCDR3 amino acid sequence of QQHYITPLT (SEQ ID NO: 83) and a VL region comprising 43. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 42, comprising:

[0372] 53. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 42 or 51 to 52, wherein the second antigen-binding portion is capable of binding to TROP2 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: 80, 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: 84.

[0373] 54. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 42 or 51 to 53, wherein the second antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 80 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 84.

[0374] 55. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1-46, wherein the protease-cleavable linker comprises the protease recognition sequence PQARK (SEQ ID NO: 41).

[0375] 56. (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 85; (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 89; (c) a first light chain comprising the amino acid sequence of SEQ ID NO: 87, and (c) a second light chain comprising the amino acid sequence of SEQ ID NO: 88. 1. A protease-activatable T cell-activating bispecific molecule comprising:

[0376] 57. (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 91; (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 94, and (c) a light chain comprising the amino acid sequence of SEQ ID NO: 93 1. A protease-activatable T cell-activating bispecific molecule comprising:

[0377] 58. (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: 100; (c) a light chain comprising the amino acid sequence of SEQ ID NO: 98, and (c) a second light chain comprising the amino acid sequence of SEQ ID NO: 99. 1. A protease-activatable T cell-activating bispecific molecule comprising:

[0378] 59. An isolated polynucleotide encoding a protease-activatable T cell-activating bispecific antigen-binding molecule according to any one of embodiments 1 to 58.

[0379] 60. A polypeptide encoded by the polynucleotide of embodiment 59.

[0380] 61. A vector, particularly an expression vector, comprising a polynucleotide according to embodiment 59.

[0381] 62. A host cell comprising a polynucleotide according to embodiment 59 or a vector according to embodiment 61.

[0382] 63. A method for producing a protease-activated T cell activating bispecific molecule, comprising the steps of: a) culturing a host cell according to embodiment 62 under conditions suitable for expression of the protease-activated T cell activating bispecific molecule; and b) recovering the protease-activated T cell activating bispecific molecule.

[0383] 64. A protease-activatable T cell-activating bispecific molecule produced by the method described in embodiment 63.

[0384] 65. A pharmaceutical composition comprising the protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 58 and a pharmaceutically acceptable carrier.

[0385] 66. A protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 58, or a composition according to embodiment 65, for use as a medicament.

[0386] 67. The protease-activatable T-cell activating bispecific molecule for use according to embodiment 66, wherein the medicament is for treating or delaying the progression of cancer, treating or delaying the progression of an immune-related disease, or improving or stimulating an immune response or function in an individual.

[0387] 68. A protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 58, for use in the treatment of a disease in an individual in need thereof.

[0388] 69. The protease-activatable T cell-activating bispecific molecule for use according to embodiment 68, wherein the disease is cancer.

[0389] 70. Use of a protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 58 for the manufacture of a medicament for the treatment of a disease.

[0390] 71. The use according to embodiment 70, wherein the disease is cancer.

[0391] 72. 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 to 58, or the composition according to embodiment 65.

[0392] 73. 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 to 58, or a composition according to embodiment 65, in the presence of T cells.

[0393] 74. The method of embodiment 73, wherein the target cells are cancer cells.

[0394] 75. The method of embodiment 73 or 74, wherein the target cell expresses a protease capable of activating the protease-activatable T cell activating bispecific molecule.

[0395] 76. The invention as herein described.

[0396] Exemplary Sequences [Table 2] TIFF2025533570000002.tif255169TIFF2025533570000003.tif252170TIFF2025533570000004.tif255170TIFF2025533570000005.tif255170 TIFF2025533570000006.tif250170TIFF2025533570000007.tif255170TIFF2025533570000008.tif255170TIFF2025533570000009.tif255170 TIFF2025533570000010.tif255170TIFF2025533570000011.tif252170TIFF2025533570000012.tif255170TIFF2025533570000013.tif255170 TIFF2025533570000014.tif255170TIFF2025533570000015.tif255170TIFF2025533570000016.tif255170TIFF2025533570000017.tif211170 [Example]

[0397] The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0398] Example 1. Generation of T cell bispecific molecules targeting FolR1 1.1 Expression constructs The T cell bispecific molecules were generated in a unique 2+1 heterodimer format based on knob-into-hole technology (two binding moieties for target antigens and one binding moiety for CD3). An anti-CD3 binder-blocking scFv (stabilized via a H44 / L100 disulfide bridge) in the VHVL order was fused to the N-terminus of the VH of a CD3-binding Fab (Figure 1A). The linker between the scFv and Fab was 33 amino acids long and consisted of a matriptase site embedded in a GS linker sequence.

[0399] The genes for each chain of proTCB were separately inserted into a mammalian expression vector. Expression of all genes was controlled by a human CMV promoter-intron A-5'UTR cassette. The BGH polyadenylation signal was located downstream of the genes.

[0400] 1.2 Preparation of FolR1proTCB containing a matriptase-cleavable linker Bispecific proTCB molecules with different matriptase linkers were identified as Expi293F TM The cells were generated by transient transfection of Expi293 cells. TM Culture medium (Gibco, Cat. No. 1435101) at a density of 2.5 x 10 6 The cells were seeded at 1 / ml. The expression vector and ExpiFectamine (Gibco, ExpiFectamine TM Transfection Kit, Catalog No. 13385544) in OptiMEM TM The cells were mixed separately in reduced serum medium (Gibco, Cat. No. 11520386). After 5 minutes, both solutions were combined, mixed by pipetting, and incubated at room temperature for 25 minutes. The cells were added to the expression vector / ExpiFectamine solution and incubated at 37°C in a shaking incubator with a 5% CO2 atmosphere for 24 hours. One day after transfection, supplements (Transfection Enhancer 1 and 2, ExpiFectamine) were added. TMAfter 4-5 days, the cell supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the collected supernatant by standard methods as follows.

[0401] 1.3 Purification of IgG-like proteins Proteins were purified from filtered cell culture supernatants according to standard protocols. Briefly, Fc-containing proteins were purified from 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). Elution was achieved at pH 3.0, followed immediately by neutralization of the sample pH. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15 (Art. Nr.: UFC903096)), and aggregated proteins were separated from monomeric proteins by size exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0 (or as otherwise described). [Table 3]

[0402] Example 2. Determining matriptase cleavage rates using SPR The cleavage kinetics of recombinant matriptase was investigated using surface plasmon resonance (SPR) on a Biacore T200 instrument (Cytiva). Biotinylated CD3ε was immobilized on a Series S Sensorchip SA (Cytiva, 29104992) at a final surface density of 2000-4000 response units (RU). FOLR1 proTCB at a concentration of 10 nM was incubated with 50 pM recombinant matriptase (R&D systems, 3946-SE) in PBS-T pH 7.4 and PBS-T pH 6.5 at 37°C. The CD3ε binding response, i.e., the proTCB activation kinetics, was monitored by continuous injections of the proTCB / matriptase mixture over the surface at a flow rate of 5 μl / min for 30 s for up to 10 h. After each injection, the CD3ε surface was regenerated by injection of 10 mM glycine (pH 1.5) at a flow rate of 5 μl / min for 60 s. Within the same experiment, a calibration line was generated by injecting a concentration series of 0.16, 0.31, 0.63, 1.25, and 2.5 nM FOLR1 TCB. The resulting binding responses of proTCB were converted from response units (RU) to molar concentrations (nM). The molar concentration of activated proTCB was plotted against incubation time, and the cleavage rate (pM / min) was calculated by determining the slope of each resulting line. [Table 4]

[0403] Example 3. Development potential of FOLR1 proTCB containing different variants of humanized masks Selected FOLR1 proTCB molecules with different humanized masks were produced as described in Example 1 and analyzed for stability and manufacturability. [Table 5]

[0404] 3.1 Thermal stability Thermal stability was investigated by static light scattering (SLS) using the UNcle platform (Unchained Labs). Briefly, 9 μl of a 1 mg / ml solution of proTCB was transferred to the instrument's sample chamber. A temperature gradient from 30°C to 90°C was applied at a rate of 0.1°C / min. Static light scattering was monitored at a wavelength of 266 nm to determine the aggregation temperature (Tagg).

[0405] 3.2 Apparent hydrophobicity Apparent hydrophobicity was examined by hydrophobic interaction chromatography (HIC) using high-pressure liquid chromatography (HPLC). Briefly, 20 μl of proTCB at a concentration of 1 mg / ml was injected onto a TSKgel Ether-5PW column (Tosoh Bioscience 0008641). A linear gradient of 0 to 1.5 M NH4SO4 in 25 mM sodium phosphate over 20 min at a flow rate of 0.8 ml / min was applied at a column temperature of 40°C. The detection wavelength was 214 nm. Relative retention times were calculated using appropriate reference antibodies.

[0406] 3.3 FcRn chromatography Relative FcRn binding affinity was determined by high-pressure liquid chromatography (HPLC). Briefly, 30 μl of proTCB at a concentration of 1 mg / ml was injected onto an FcRn streptavidin Sepharose column (Roche Diagnostics 08128057001). A step gradient using 20 mM MES sodium salt, 140 mM NaCl pH 5.5 and pH 8.8 was applied according to the manufacturer's recommendations at a column temperature of 25°C. The detection wavelength was set at 280 nm. Relative retention times were calculated using appropriate reference antibodies.

[0407] 3.4 Heparin chromatography Relative heparin binding affinities were determined by high-pressure liquid chromatography (HPLC). Briefly, 100 μl of proTCB at a concentration of 0.35 mg / ml was injected onto a TSK-Gel Heparin-5PW column (Tosoh Bioscience 13064). A step gradient was applied using 50 mM Tris pH 7.4 and 50 mM Tris, 1 M NaCl, pH 7.4, respectively. The flow rate was set to 0.8 ml / min, and the column temperature was set to 25°C. The detection wavelength was set to 280 nm. Relative retention times were calculated using appropriate reference antibodies. [Table 6]

[0408] All molecules tested showed good thermal stability (>60° C.) and showed fully acceptable values ​​for hydrophobicity and for FcRn and heparin chromatography.

[0409] Example 4. Measuring the cleavage rates of different proteases using SPR The cleavage kinetics of recombinant matriptase (R&D Systems 3946-SEB), matriptase-2 (Enzo ALX-201-752), hepsin (R&D Systems 4776-SE), uPA (Sigma-Aldrich 6273), legumain (R&D Systems 2199-CY), and furin (R&D Systems 1503-SE) were investigated using surface plasmon resonance (SPR) on a Biacore T200 instrument (Cytiva). Biotinylated CD3ε was immobilized on a Series S Sensorchip SA (Cytiva, 29104992) at a final surface density of 2000–4000 response units (RU). FOLR1 proTCB was incubated with the different proteases listed above at the following concentrations in PBS-T pH 7.4 at 37°C: i. 10 nM proTCB + 50 pM matriptase ii. 10 nM proTCB + 1 U matriptase-2 iii. 10 nM proTCB + 300 pM activated hepsin iv. 10nM proTCB + 5.5nM uPA v. 10nM proTCB + 5nM activated legumain vi. 10 nM proTCB + 3 nM furin (with 1 mM CaCl2)

[0410] The CD3ε binding response, i.e., the rate of proTCB activation, was monitored by sequentially injecting the proTCB / protease mixture over the surface for 30 seconds at a flow rate of 5 μl / min for up to 10 hours. After each injection, the CD3ε surface was regenerated by injecting 10 mM glycine (pH 1.5) at a flow rate of 5 μl / min for 60 seconds. Within the same experiment, a calibration line was generated by injecting a concentration series of FOLR1 TCB at 0.16, 0.31, 0.63, 1.25, and 2.5 nM. The resulting proTCB binding response was converted from response units (RU) to molar concentration (nM). The molar concentration of activated proTCB was plotted against incubation time, and the cleavage rate (pM / min) was calculated by determining the slope of each resulting line. [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12]

[0411] Example 5. Development potential of FOLR1proTCB (different matriptase cleavage site; humanized mask) Selected FOLR1 proTCB molecules were analyzed for stability and developability using the same methods as in Example 3. [Table 13]

[0412] All molecules tested showed good thermal stability (>60° C.) and showed fully acceptable values ​​for hydrophobicity and for FcRn and heparin chromatography.

[0413] Example 6. Development Potential of Humanized Anti-Idiotype IgG Anti-idiotype binding 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 examined by surface plasmon resonance using a T200 instrument (Cytiva). Briefly, biotinylated anti-human CD3ε antibody and biotinylated anti-human IgG (Capture Select, Thermoscientific, 7103302500) were immobilized on a Series s SensorChip CAP (Biotin CAPture Kit, Cytiva, 28920234) after injection of capture reagent according to the manufacturer's instructions. The resulting surface densities were approximately 1000 RU and 1500 RU, respectively. Anti-idiotype antibody was injected over the chip surface at a concentration of 1 μg / ml for 30 s at a flow rate of 5 μl / min. Dissociation was monitored for 30 s. After each injection, the chip surface was regenerated by injecting 2 M guanidine-HCl, 0.5 M NaOH for 120 s. Bulk refractive index differences were corrected by subtracting the response obtained from a mock surface.

[0414] To normalize the binding signal of the anti-idiotypic antibody, the binding response of the anti-human CD3ε antibody surface was divided by the binding response of the anti-human IgG 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. [Table 14]

[0415] Example 7. Development potential of proTCB The binding of proTCB 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 (Cytiva). Briefly, mouse anti-huIgG CH2 PG-LALA antibody (P1AE2335) and human CD3ε (P1AA6119) were immobilized on a Series s SensorChip CM5 (Cytiva) using standard amine coupling chemistry. The resulting ligand densities were approximately 8500 RU and 7000 RU, respectively. For FolR1 binding assessment, proTCB was captured on the anti-human IgG PG-LALA surface at a concentration of 2 μg / ml and a flow rate of 10 μl / min for 75 s. Subsequently, human FolR1 (P1AD6798) was injected at a concentration of 900 nM for 120 s at a flow rate of 10 μl / min. Dissociation was monitored for 120 seconds. After each human FolR1 injection, the surface was regenerated by injecting 20 mM NaOH for 35 seconds. For CD3ε binding assessment, proTCB was injected over the CD3ε surface at a concentration of 10 μg / ml for 90 seconds at a flow rate of 10 μg / ml. Dissociation was monitored for 90 seconds. After each injection, the surface was regenerated by injecting 10 mM glycine pH 2.1 for 70 seconds. The bulk refractive index difference for each interaction was corrected by subtracting the response obtained from the mock surface.

[0416] To normalize the binding signals of proTCB, the FolR1 and CD3ε binding responses were divided by the binding response of the anti-human IgG PG-LALA surface. Relative activity concentrations were obtained by dividing the normalized response of the stressed sample by the normalized response of the unstressed reference sample (FolR1) or unmasked control molecule (CD3ε). [Table 15] [Table 16]

[0417] Example 8. Stability and Pharmacokinetic Profiles of Different Linkers in Vivo After a Single Injection in NSG Mice NSG mice were injected with a single dose of 5 mg / kg of pro-FolR1-TCB molecules containing different matriptase selective cleavage sites. All mice were intravenously injected with 200 μl of the appropriate solution, as shown in Figure 2. The stock solution (Table 17) was diluted with histidine buffer to obtain the appropriate amount of compound per 200 μl. Two mice per time point and group were bled at 24 hours, 7 days, and 10 days. The injected compounds were analyzed in serum samples by ELISA.

[0418] Molecular detection was performed by LBA (ligand binding assay) as follows: Serum samples from mice treated with P1AF5419 (cleavage site: HQARK), P1AF5420 (cleavage site: PQARK), or P1AE6554 (classical FolR1 2+1 TCB) were analyzed using a Cobas e411 instrument with an ECLIA method specific for the human CH1 / PGLALA-containing domain ("total assay") and an ECLIA method using CD3 anti-ID antibody capture and anti-PGLALA-specific antibody detection ("activity assay").

[0419] In the "full assay," the test sample was P1AF5419 (cleavage site: HQARK) (004-09), P1AF5420 (cleavage site: PQARK) (004-06), or P1AE6554 (classical FolR1 2+1 TCB), a first detection antibody mAb <h-igg>11-1.19.31-IgG-Bi, second detection antibody mAb<H-Fc(PGLALA)> M-1.7.24-IgG-Ru and SA-beads were added stepwise to the detection vessel and incubated for 9 min at each step.

[0420] In the "activity assay," FolR1 TCB(007-19), the first detection antibody mAb <ch2527>rH-4.24.72-IgG()-Bi, second detection antibody mAb<H-Fc(PGLALA)> The test sample containing M-1.7.24-IgG-Ru and SA-beads was added stepwise to the detection vessel, and each step was incubated for 9 minutes. Finally, the complex bound to the SA-beads was detected by a measuring cell that repeatedly counted the number of SA-beads. The number of counts was proportional to the analyte concentration in the test sample. [Table 17]

[0421] Serum analysis of mice administered 5 mg / kg of FOLR1-TCB or FOLR1 pro-TCB revealed that active TCB was detected at low levels (<5% of total pro-TCB) at all time points for both FOLR1 pro-TCB molecules containing the HQARK or PQARK cleavage site. Active TCB was detected in 100% of the unmasked FOLR1-TCB (Fig. 3).

[0422] Example 9. Efficacy study in humanized mice BC004PDX using pro-FOLR1-TCB constructs containing different matriptase selective cleavage sites Human breast cancer patient-derived xenografts, HER2+ER-xenograft model BC004, were purchased from OncoTest (Freiburg, Germany). Tumor fragments were digested with collagenase D and DNase I (Roche), counted, and collected at 1 × 10 6 BC004 cells were injected in a total volume of 100 μl of a mixture of RPMI and Matrigel, subcutaneously injected into the flank of anesthetized mice with a 22 G–30 G needle.

[0423] Female NSG mice (Jackson Laboratory), 4-5 weeks old at the start of the experiment, were maintained in specific pathogen-free conditions with a 12-hour light / 12-hour dark daily cycle in accordance with relevant guidelines (GV-Solas; Felasa; TierschG). The experimental research protocol was reviewed and approved by the local government (P2011 / 128). After arrival, animals were maintained for one week to acclimate to their new environment and observed. Continuous health monitoring was performed regularly.

[0424] Female NSG mice were injected intraperitoneally with 15 mg / kg busulfan, and one day later, 1 x 10 5 Human hematopoietic stem cells were injected intravenously. Mice were bled sublingually 14–16 weeks after stem cell injection, and the blood was analyzed by flow cytometry for successful humanization. Successfully engrafted mice were randomized into different treatment groups according to the frequency of human T cells. At that time, mice were injected with tumor PDX cells and treated weekly with compound or histidine buffer (vehicle) when tumor size reached approximately 200 mm3 (day 28). All mice were injected intravenously with 200 μl of the appropriate solution. Stock solutions (Table 18) were diluted as needed with histidine buffer to obtain the appropriate amount of compound per 200 μl. Tumor growth was measured twice weekly using calipers (Figure 2), and tumor volume was calculated as follows: T v :(W 2 / 2)xL(W: width, L: length)

[0425] At termination (day 58), mice were sacrificed and tumors and spleens were removed and weighed.

[0426] Figure 5A shows tumor growth kinetics (mean, + standard error) in the most effective treatment group, as well as individual tumor growth per mouse. As described herein, FOLR1 pro-TCB containing one of the PQARK cleavage sites was identified as the best pro-TCB tested in this study. Furthermore, no efficacy was observed in the group treated with FOLR1 pro-TCB containing a non-cleavable linker. Figures 5A-5G show tumor weights at endpoint for all treatment groups. This readout clearly supports the findings in tumor growth kinetics and demonstrates that pro-TCBs containing the PQARK cleavage site result in comparable tumor weights at endpoint compared to the classic FolR1 TCB. [Table 18]

[0427] Example 10. Generation of T cell bispecific molecules targeting cMET, TROP2 or IGF-1R 10.1 Expression Constructs T cell bispecific molecules were generated in a unique heterodimeric format based on knob-into-hole technology (one or two binding moieties for target antigens and one binding moiety for CD3). An anti-CD3 binder-blocking scFv (stabilized via a H44 / L100 disulfide bridge) in the VHVL order was fused to the N-terminus of the VH of a CD3-binding Fab (Figures 1B-1D). The linker between the scFv and Fab was 33 amino acids long and consisted of a matriptase protease recognition site embedded in a GS linker sequence.

[0428] The genes for each chain of proTCB were separately inserted into a mammalian expression vector. Expression of all genes was controlled by a human CMV promoter-intron A-5'UTR cassette. A BGH polyadenylation signal was located downstream of the gene.

[0429] 10.2 Preparation of FolR1proTCB containing a matriptase-cleavable linker Bispecific proTCB molecules with different matriptase linkers were identified as Expi293F TM The cells were generated by transient transfection of Expi293 cells. TM Culture medium (Gibco, Cat. No. 1435101) at a density of 2.5 x 10 6 The cells were seeded at 1 / ml. The expression vector and ExpiFectamine (Gibco, ExpiFectamine TM Transfection Kit, Catalog No. 13385544) in OptiMEM TM The cells were mixed separately in reduced serum medium (Gibco, Cat. No. 11520386). After 5 minutes, both solutions were combined, mixed by pipetting, and incubated at room temperature for 25 minutes. The cells were added to the expression vector / ExpiFectamine solution and incubated at 37°C in a shaking incubator with a 5% CO2 atmosphere for 24 hours. One day after transfection, supplements (Transfection Enhancer 1 and 2, ExpiFectamine) were added. TM After 4-5 days, the cell supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the collected supernatant by standard methods as follows.

[0430] 10.3 Purification of IgG-like proteins Proteins were purified from filtered cell culture supernatants according to standard protocols. Briefly, Fc-containing proteins were purified from 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). Elution was achieved at pH 3.0, followed immediately by neutralization of the sample pH. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15 (Art. Nr.: UFC903096)), and aggregated proteins were separated from monomeric proteins by size exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0 (or as otherwise described). [Table 19] TIFF2025533570000035.tif116170

[0431] Example 11. Binding of IGF-1R proTCB constructs to T cells We evaluated IGF-1R proTCB (SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89) and its controls (unmasked classical IGF-1R TCB, uncleaved IGF-1R proTCB) for their ability to bind to CD3 on the human reporter T cell line Jurkat NFAT in comparison with the respective matriptase-precleaved, and therefore unmasked, molecules. Both matriptase-precleaved IGF-1R proTCB and classical IGF-1R TCB (with or without matriptase precleavage) bound CD3 equally well, whereas the uncleaved IGF-1R TCB construct and the non-matriptase-precleaved IGF-1R proTCB did not bind to CD3 on Jurkat T cells (Figure 6), demonstrating the effective CD3-blocking ability of the H1L2 mask.

[0432] 11.1 Method 11.1.1 Cell lines Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS176501) are a human acute lymphoblastic leukemia reporter cell line expressing human CD3 and carrying the NFAT promoter. Cells were cultured at 0.1–0.5 microcells / ml in RPMI 1640 medium containing 2 g / l glucose, 2 g / l NaHCO3, 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 1x NEAA, and 1x sodium pyruvate. Hygromycin B (Sigma, #10834555001) was added to a final concentration of 200 μg / ml after each cell passage.

[0433] 11.1.2 CD3 Binding by Flow Cytometry The day before the assay began, the TCB constructs were set up in duplicate. One vial of each molecule was left untreated, while the other was preactivated by adding 1 μl of human recombinant matriptase (Enzo ~2.5 U / μl, ALX-201-246-U25, Lot 12152015) overnight at room temperature. The following day, Jurkat cells were harvested and transferred to a 96-well round-bottom plate (100,000 cells per well). Cells were washed with FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.025% NaN3) and incubated with 25 μl of a 1:4 titration of the IGF-1R TCB construct (starting concentration: 50 nM) in FACS buffer for 30 minutes at 4°C. After staining, cells were washed twice with FACS buffer to remove unbound molecules. To distinguish live from dead cells, Jurkat cells were stained with 25 μl of diluted NIR Live / Dead dye (1:1000 dilution, lot: 2192282, Invitrogen) for 10 minutes at room temperature. Then, 25 μl of diluted Fitc AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment-specific secondary antibody (1:75 dilution, 109-116-170, Jackson ImmunoResearch) was added to the cells. After a 30-minute incubation at 4°C, the cells were washed twice with FACS buffer to remove unbound antibody. Finally, the cells were resuspended in 100 μl of FACS buffer and analyzed by flow cytometry using a BD Canto II.

[0434] Example 12. Binding of IGF-1R proTCB constructs to IGF-1R in different cancer cell lines We also evaluated IGF-1R proTCBs (SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89) and their controls (unmasked classical IGF-1R TCB and uncleaved IGF-1R proTCB) for their ability to bind to the target (IGF-1R) in human cancer cell lines expressing different levels of IGF-1R on the cell surface (T-47D>MKN-45>OVMANA>HPAF II). All three tested IGF-1R TCB constructs bind IGF-1R equally well (Figure 7).

[0435] 12.1 Method 12.1.1 Cell lines OVMANA is a human ovarian clear cell adenocarcinoma cell line (RNCB Accession ID: CL012520) cultured in RPMI1640 + 1x Glutamax + 10% FBS.

[0436] T-47D is a human carcinoma cell line (RNCB accession ID: CL000001) cultured in RPMI1640 + 1x Glutamax + 10% FBS.

[0437] HPAFII is a human pancreatic adenocarcinoma cell line (RNCB accession ID: CL010030) cultured in EMEM + 10% FBS + 1% Glutamax + 1% NEAA + 1% sodium pyruvate.

[0438] HeLa is a human epithelial adenocarcinoma cell line (RNCB accession ID: CL022232). The HeLa cells used were genetically modified (NLR-red nuclei, inactive ST14) and cultured in DMEM + 10% FBS + 1% Glutamax + 3 μg / ml puromycin (LabForce AG, #ant-pr-1) (0.6 μl / 10 ml) + 80 μg / ml hygromycin B (Sigma, #10834555001) (80 μl / 10 ml).

[0439] 12.1.2 Target Binding by Flow Cytometry Target cells were harvested and transferred to a 96-well round-bottom plate (100,000 cells per well). Cells were washed with FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.025% NaN3) and stained with 25 μl of the corresponding IGF-1R TCB construct in FACS buffer at 4°C for 30 minutes. After staining, cells were washed twice with FACS buffer to remove unbound molecules. To distinguish live from dead cells, Jurkat cells were stained with 25 μl of diluted NIR Live / Dead Dye (1:1000 dilution, lot: 2192282, Invitrogen) for 10 minutes at room temperature. Then, 25 μl of diluted Fitc AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment-specific secondary antibody (1:75 dilution, 109-116-170, Jackson ImmunoResearch) was added to the cells. After a 30-minute incubation at 4°C, the cells were washed twice with FACS buffer to remove unbound antibody. Finally, the cells were resuspended in 100 μl of FACS buffer and analyzed by flow cytometry using a BD Canto II.

[0440] Example 13. Functional activity of IGF-1R proTCB constructs - Jurkat NFAT reporter cell assay using beads The ability of IGF-1R proTCB (SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89) to induce TCR crosslinking and subsequent T cell activation was assessed using SA beads coated with biotinylated human IGF-1R antigen (biotinylated human IGF-1R / CD221 protein, His, Avitag™, BioCat) and Jurkat NFAT reporter cells (a human acute lymphoblastic leukemia reporter cell line harboring the NFAT promoter and expressing CD3, GloResponse Jurkat NFAT-RE-luc2P, Promega #CS176501). Simultaneous binding of the IGF-1R TCB construct to IGF-1R-coated SA beads and CD3 expressed on Jurkat NFAT reporter T cells activates the NFAT promoter, resulting in the expression of active firefly luciferase. The intensity of the luminescent signal (obtained upon addition of luciferase substrate) is proportional to the intensity of CD3 activation and signaling. Jurkat NFAT reporter T cells were grown in suspension and cultured at 0.1–0.5 mio cells / ml in RPMI 1640, 2 g / l glucose, 2 g / l NaHCO , 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 1× NEAA, and 1× sodium pyruvate, with 200 μg / ml hygromycin B.

[0441] Comparable Jurkat NFAT activation was observed for matriptase-precleaved (+M) IGF-1R proTCB and classical IGF-1R TCB (+M or untreated). Uncleaved IGF-1R proTCB (+M or untreated) and non-precleaved IGF-1R proTCB showed no NFAT activation (Figure 8). Thus, the CD3 mask H1L2 appears to efficiently block the CD3-binding agent P035.093.

[0442] 13.1 Method For the assay, 95 μl of SA beads (Streptavidin Dynabeads M-280 Streptavidin, Lubio Science #11205D) were diluted with 5 ml of DPBS. The beads were centrifuged at 400 rcf for 4 minutes, and the supernatant was aspirated. Biotinylated hu-IGF-1R antigen (final 46.90 nM, 5 μg / ml) was then added to 0.5 ml of DPBS and then added to the SA beads. The beads were resuspended in the antigen solution. The bead-antigen mixture was incubated at 25°C for 60 minutes with gentle rotation. After incubation, 5 ml of DPBS was added to the bead-ag conjugate, centrifuged, and the supernatant was discarded. The conjugate was resuspended in assay medium.

[0443] The day before starting the assay, the TCB constructs were set up in duplicate, with one vial of each molecule left untreated and the other preactivated by adding 1 μl of matriptase (Enzo ∼2.5 U / μl, ALX-201-246-U25, Lot 12152015) overnight at room temperature.

[0444] The next day, Jurkat cells were harvested and viability was measured using a ViCell XR cell counter (Beckman Coulter). After centrifugation at 300 rcf for 3 minutes, the medium was aspirated and the cells were resuspended in fresh assay medium (Sigma, #10834555001) without hygromycin B. The coated SA bead suspension (1x) was mixed with the effector cell suspension (2x) in a Falcon tube, and cAMP GloSensor (#E1291, Promega) was added (2% of the final volume). 30 μl of the mixture was plated into a flat-bottom, white-walled 96-well plate (#655098, Greiner Bio-One), and 10 μl / well of diluted TCB or medium (for negative controls) was added. The cells were incubated at 37°C for 5 hours in a humidified incubator. At the end of the incubation period, luminescence was detected using a TECAN Spark 10M.

[0445] Example 14. Functional activity of IGF-1R proTCB constructs - Jurkat NFAT reporter cell assay using cells IGF-1R-expressing cancer cells (T-47D>MKN-45>OVMANA>HPAF II) and Jurkat NFAT reporter cells (a human acute lymphoblastic leukemia reporter cell line containing the NFAT promoter and expressing CD3, GloResponse Jurkat NFAT-RE-luc2P, Promega #CS176501) were used to evaluate the ability of IGF-1R proTCB (SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89) to induce TCR crosslinking and subsequent T cell activation. Simultaneous binding of both arms of the TCB construct, the anti-IGF-1R and anti-CD3 arms, to CD3 on IGF-1R-expressing target cells and Jurkat-NFAT reporter cells activates the NFAT promoter in Jurkat cells, resulting in the expression of active firefly luciferase. The intensity of the luminescent signal (obtained upon addition of luciferase substrate) is proportional to the intensity of CD3 activation and signaling. Jurkat NFAT reporter cells were grown in suspension and cultured at 0.1–0.5 mio cells / ml and 200 μg / ml hygromycin B in RPMI1640, 2 g / l glucose, 2 g / l NaHCO , 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 1× NEAA, and 1× sodium pyruvate.

[0446] In this assay, we evaluated Jurkat NFAT activation induced by different IGF-1R proTCB constructs using four cell lines expressing different surface levels of IGF-1R (Figure 9). A) T-47D: medium-high, B) MKN-45: medium-high, C) OVMANA: medium, D) HPAF II: low. Upon stimulation with TCB constructs, comparable Jurkat NFAT activation was induced by unmasked classical TCB (with or without matriptase preactivation) and matriptase-precleaved IGF-1R proTCB. Uncleaved proTCB (negative control) and matriptase-unpreactivated IGF-1R proTCB did not induce NFAT activation, demonstrating the effective masking ability of H1L2. Maximal activation of Jurkat NFAT cells depends on the target level expressed on the tumor target cells (IGF-1R surface expression ranking: T-47D>MKN-45>OVMANA>HPAF II).

[0447] 14.1 Method For the assay, tumor target cells were harvested and viability was determined using a ViCell XR cell counter (Beckman Coulter). 10,000 target cells / well were plated in 100 μl of medium in a flat-bottom, white-walled 96-well plate (#655098, Greiner Bio-One), and 50 μl / well of diluted antibody or medium (for negative control) was added to the target cells. Jurkat NFAT reporter cells were then harvested and viability assessed. Cells were resuspended at 1.25 mio cells / ml in cell culture medium (Sigma, #10834555001) without hygromycin B and added to the tumor cells at 25,000 cells / well (50 μl / well), yielding a final E:T ratio of 2.5:1 and a final volume of 200 μl per well. Then, 4 μl of GloSensor (#E1291, Promega) was added to each well (2% of the final volume). The cells were incubated for 5 hours at 37°C in a humidified incubator. At the end of the incubation period, luminescence was detected using a TECAN Spark10M.

[0448] The day before the assay, the IGF-1R TCB constructs tested were activated by overnight incubation with human recombinant human matriptase (Enzo ∼2.5 U / μl, ALX-201-246-U25, lot 12152015) at room temperature.

[0449] Example 15. T cell-mediated tumor cell killing (LDH release) and T cell activation The potential of IGF-1R proTCB (SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89) to induce T cell-mediated tumor cell killing was assessed by quantification of LDH released into the supernatant by apoptotic / necrotic target cells (LDH detection kit, Roche Applied Science, #11644793001).

[0450] LDH release was only weakly induced by the highest concentration (50 nM) of matriptase-precleaved IGF-1R proTCB (Fig. 10), suggesting that IGF-1R proTCB-mediated target cell killing requires higher TCB concentrations.

[0451] However, within the same experiment, CD4 + and CD8 + Looking at the potential of the IGF-1R proTCB construct to activate T cells, we were able to show that both early (CD69) and late (CD25) T cell activation markers were upregulated in a dose-dependent manner by TCB (IGF-1R proTCB preactivated with matriptase) and IGF-1R TCB (with and without matriptase pretreatment) and target expression levels. CD69 was upregulated by CD25, CD4 + T cells (Figure 11) and CD8 + Compared to T cells (Figure 12), CD69 showed a stronger induced surface expression profile. Therefore, CD69 appears to be a more sensitive T cell activation marker than CD25 in this setting. The uncleaved proTCB construct showed CD69 induction with increasing TCB concentrations, but CD25 expression was not induced. This data suggests that the classical IGF-1R TCB and matriptase-precleaved IGF-1R proTCB induce CD4 T cell activation, even though LDH release is not fully sensitive. + (Figure 11) and CD8 + (Figure 12) This suggests that T cells can be activated.

[0452] 15.1 Method Target cells were harvested with cell dissociation buffer (Gibco, #13151-014), washed with PBS, and plated at a density of 20,000 cells / well in flat-bottom 96-well plates. Cells were allowed to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of fresh blood obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered on a Histopaque gradient (Sigma, #H8889). After centrifugation (450 × g, 30 min, room temperature), the plasma above the interface containing the PBMCs was discarded, and the PBMCs were then transferred to a new Falcon tube filled with 50 ml of PBS. The mixture was centrifuged (400 × g, 10 min, room temperature), the supernatant was discarded, and the PBMC pellet was washed twice with sterile PBS (centrifugation step 350 × g, 10 min). The resulting PBMC population was automatically counted (Beckman Coulter ViCell XR cell counter) and frozen in RPMI 1640 medium containing 10% FBS, 1% L-alanyl-L-glutamine (Biochrom, K0302), and 5% dimethyl sulfoxide (DMSO) until further use. For killing assays, antibodies were added in triplicate at the indicated concentrations. PBMCs were thawed the day before the assay and cultured at 2 mio cells / ml in RPMI 1640 medium containing 10% FBS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37°C in a humidified incubator. All TCB constructs were set up in duplicate on the same day. One vial of each molecule was left untreated, while the other was preactivated by adding 1 μl of matriptase (Enzo ~2.5 U / μl, ALX-201-246-U25, Lot 12152015) overnight at room temperature. The next day, PBMCs were added to the target cells at a final effector-to-target (E:T) ratio of 5:1 (100,000 cells / well). After 72 h of incubation at 37 °C and 5% CO2, target cell death was assessed by quantification of LDH released into the cell supernatant by apoptotic / necrotic cells (LDH detection kit, Roche Applied Science, #11644793001) according to the manufacturer's protocol.Maximum lysis of target cells (maximum release: MR) (=100%) was achieved by incubating target cells with 1% Triton X-100 for more than 1 h. Minimum lysis of target cells (spontaneous release: SR) (=0%) refers to target cells co-incubated with effector cells without bispecific constructs.

[0453] CD4 TCB construct-mediated T cell activation was analyzed by flow cytometry using antibodies that recognize the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker). + and CD8 + T cell activation was assessed after 72 hours of incubation. To this end, PBMCs were transferred to a round-bottom 96-well plate, centrifuged at 350 × g for 5 minutes, and washed twice with FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.025% NaN3). Surface staining for CD4APC (#300514, BioLegend), CD8FITC (#344704, BioLegend), CD25BV421 (#302630, BioLegend), and CD69PE (#310906, BioLegend) was performed according to the supplier's instructions. Cells were washed twice with 150 μl / well of FACS buffer and fixed overnight at 4°C with 50 μl / well of FACS buffer + 4% paraformaldehyde (PFA). After centrifugation, samples were washed with 150 μl / well of FACS buffer and resuspended in 100 μl / well of FACS buffer for FACS analysis. Samples were analyzed using a BD Fortessa.

[0454] Example 16. Binding of Trop2 proTCB constructs to T cells We evaluated Trop2 proTCB (SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100) and its controls (unmasked Trop2 TCB, uncleaved Trop2 proTCB) for their ability to bind to CD3 on the human reporter T cell line Jurkat NFAT, compared with the respective matriptase-precleaved molecules. Both the matriptase-precleaved Trop2 proTCB construct and the unmasked Trop2 TCB (with or without matriptase precleavage) bound equally well to CD3, indicating successful linker cleavage and subsequent release of the mask (Figure 13). Uncleaved Trop2 and masked Trop2 proTCB did not bind to CD3 on Jurkat T cells, indicating effective blocking of the CD3-binding agent by the mask (Figure 13).

[0455] 17.1 Method 17.1.1 Cell lines Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS176501) are a human acute lymphoblastic leukemia reporter cell line expressing human CD3 and carrying the NFAT promoter. Cells were cultured at 0.1–0.5 microcells / ml in RPMI 1640 medium containing 2 g / L glucose, 2 g / L NaHCO3, 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 1x NEAA, and 1x sodium pyruvate. Hygromycin B (Sigma, #10834555001) was added to a final concentration of 200 μg / ml after each cell passage.

[0456] 17.1.2 CD3 Binding by Flow Cytometry The day before starting the assay, the TCB constructs were set up in duplicate. One vial of each molecule was left untreated, while the other was preactivated by adding 1 μl of matriptase (Enzo ~2.5 U / μl, ALX-201-246-U25, Lot 12152015) overnight at room temperature. The following day, Jurkat cells were harvested and transferred to a 96-well round-bottom plate (100,000 cells per well). Cells were washed with FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.025% NaN3) and incubated with 25 μl of a 1:4 titration of Trop2 TCB constructs (starting concentration: 50 nM) in FACS buffer for 30 minutes at 4°C. After staining, cells were washed twice with FACS buffer to remove unbound molecules. To distinguish live from dead cells, Jurkat cells were stained with 25 μl of diluted NIR Live / Dead dye (1:1000 dilution, lot: 2192282, Invitrogen) for 10 minutes at room temperature. Then, 25 μl of diluted Fitc AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment-specific secondary antibody (1:75 dilution, 109-116-170, Jackson ImmunoResearch) was added to the cells. After a 30-minute incubation at 4°C, the cells were washed twice with FACS buffer to remove unbound antibody. Finally, the cells were resuspended in 100 μl of FACS buffer and analyzed by flow cytometry using a BD Canto II.

[0457] Example 17. Binding of Trop2 proTCB constructs to Trop2 in different cancer cell lines We also evaluated Trop2 proTCB (SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100) and its control (unmasked Trop2 TCB, non-cleaved Trop2 proTCB) for their ability to bind to target Trop2 in several human cancer cell lines expressing different levels of Trop2 on their surface (T-47D > HPAF II > HeLa NLR > OVMANA). All three tested Trop2 TCB constructs bind equally well to Trop2 (Figure 14).

[0458] 18.1 Method 18.1.1 Cell lines OVMANA is a human ovarian clear cell adenocarcinoma cell line (RNCB Accession ID: CL012520) cultured in RPMI1640 + 1x Glutamax + 10% FBS.

[0459] T-47D is a human carcinoma cell line (RNCB accession ID: CL000001) cultured in RPMI1640 + 1x Glutamax + 10% FBS.

[0460] HPAFII is a human pancreatic adenocarcinoma cell line (RNCB accession ID: CL010030) cultured in EMEM + 10% FBS + 1% Glutamax + 1% NEAA + 1% sodium pyruvate.

[0461] HeLa is a human epithelial adenocarcinoma cell line (RNCB accession ID: CL022232). The HeLa NLR cells used were genetically modified (NLR-red nuclei, ST14-overexpressing matriptase) and cultured in DMEM + 10% FBS + 1% Glutamax + 3 μg / ml puromycin (LabForce AG, #ant-pr-1) (0.6 μl / 10 ml) + 80 μg / ml hygromycin B (Sigma, #10834555001) (80 μl / 10 ml).

[0462] 18.1.2 Target Binding by Flow Cytometry Target cells were harvested and transferred to a 96-well round-bottom plate (100,000 cells per well). Cells were washed with FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.025% NaN3) and stained with 25 μl of the corresponding Trop2 TCB construct in FACS buffer at 4°C for 30 minutes. After staining, cells were washed twice with FACS buffer to remove unbound molecules. To distinguish live from dead cells, Jurkat cells were stained with 25 μl of diluted NIR Live / Dead Dye (1:1000 dilution, lot: 2192282, Invitrogen) for 10 minutes at room temperature. Then, 25 μl of diluted Fitc AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment-specific secondary antib...

Claims

1. (a) a first antigen-binding moiety capable of binding to CD3; and (b) a second antigen-binding moiety capable of binding to a target cell antigen selected from the group consisting of IGF-1R, cMET, or TROP2; and (c) a masking moiety covalently attached to the T cell activating bispecific molecule via a peptide linker, wherein the masking moiety is capable of binding to the idiotype of the first antigen-binding moiety or the second antigen-binding moiety, thereby reversibly masking the first antigen-binding moiety or the second antigen-binding moiety; and 1. A protease-activatable T cell activating bispecific molecule comprising:

1. A protease-activatable T cell activating bispecific molecule, wherein the peptide linker comprises the protease recognition sequence XQRK (SEQ ID NO: 39), wherein X is histidine (H) or proline (P).

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

3. 3. The protease-activatable T cell activating bispecific molecule of claim 1 or 2, wherein the masking moiety is covalently linked to the heavy chain variable region of the first antigen-binding moiety.

4. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 3, wherein the masking moiety is an scFv.

5. 5. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 4, wherein (i) the second antigen-binding moiety is a conventional Fab, or (ii) the second antigen-binding moiety is a crossover Fab molecule in which either the variable or constant regions of the Fab light chain and the Fab heavy chain have been exchanged.

6. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 5, wherein the first antigen-binding portion is a conventional Fab molecule.

7. 7. The protease-activatable T cell activating bispecific molecule of claim 1 , comprising a third antigen-binding moiety that is a Fab molecule capable of binding to a target cell antigen.

8. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 7, wherein the third antigen-binding portion is identical to the second antigen-binding portion.

9. 9. The protease-activatable T cell activating bispecific molecule of claim 1 , wherein the first antigen-binding portion and the second antigen-binding portion are fused to each other, optionally via a peptide linker.

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

11. 11. The protease-activatable T cell activating bispecific molecule of claim 1, further comprising an Fc domain composed of a first and a second subunit capable of stable association.

12. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 11, wherein the Fc domain is an IgG, specifically an IgG1 or IgG4 Fc domain.

13. 13. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 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 antigen-binding portion capable of binding to CD3, (a) the heavy chain complementarity determining region (HCDR) 1 amino acid sequence of SYAMN (SEQ ID NO: 1); (b) the HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 2); (c) the HCDR3 amino acid sequence of ASNFPASYVSYFAY (SEQ ID NO: 3) and a heavy chain variable (VH) region comprising: (d) the light chain complementarity-determining region (LCDR) 1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 7); (e) the LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 8); (f) Selected LCDR3 amino acid sequence of ALWYSNLWV (SEQ ID NO: 9) and a light chain variable (VL) region comprising: The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 13, comprising:

15. 15. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 14, wherein the antigen-binding portion capable of binding to CD3 comprises a VH 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: 5 and / or a VL 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:

10.

16. the antigen-binding portion capable of binding to CD3, (a) the heavy chain complementarity determining region (HCDR) 1 amino acid sequence of SYAMN (SEQ ID NO: 1); (b) the HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 2); (c) the HCDR3 amino acid sequence of HTTPSSYVSYYGY (SEQ ID NO: 4) and a heavy chain variable (VH) region comprising: (d) the light chain complementarity-determining region (LCDR) 1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 7); (e) the LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 8); (f) Selected LCDR3 amino acid sequence of ALWYSNLWV (SEQ ID NO: 9) and a light chain variable (VL) region comprising: The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 13, comprising:

17. 15. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 14, wherein the antigen-binding portion capable of binding to CD3 comprises a VH 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: 6 and / or a VL 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:

10.

18. The masking portion is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) an HCDR2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO: 16), WINTETGEPRYTDDFTG (SEQ ID NO: 17), and WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) and a VH region comprising: (d) the LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25) or KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) or QQSREFPYT (SEQ ID NO: 29) and a VL region comprising The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 17, comprising:

19. The masking portion is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16); (c) the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) and a VH region comprising: (d) the LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 18, comprising:

20. The masking portion is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16); (c) the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) and a VH region comprising: (d) the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 18, comprising:

21. The masking portion is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO: 17); (c) the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) and a VH region comprising: (d) the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 18, comprising:

22. The masking portion is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) and a VH region comprising: (d) the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) and a VL region comprising The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 18, comprising:

23. The masking portion is (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15); (b) the HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18); (c) the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19) and a VH region comprising: (d) the LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25); (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); (f) the LCDR3 amino acid sequence of QQSREFPYT (SEQ ID NO: 29) and a VL region comprising The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 18, comprising:

24. the second antigen-binding moiety is capable of binding to IGF-1R; and a) the HCDR1 amino acid sequence of SYGMH (SEQ ID NO: 61); b) the HCDR2 amino acid sequence of IIWFDGSSTYYADSVRG (SEQ ID NO: 62); c) the HCDR3 amino acid sequence of ELGRRYFDL (SEQ ID NO: 63) and a VH region comprising: d) LCDR1 of RASQSVSSYLA (SEQ ID NO: 65); e) the LCDR2 amino acid sequence of DASKRAT (SEQ ID NO: 66); f) LCDR3 amino acid sequence of QQRSKWPPWT (SEQ ID NO: 67) and a VL region comprising The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 23, comprising:

25. 25. The protease-activated T cell activating bispecific molecule of any one of claims 1 to 24, wherein the antigen-binding portion capable of binding to IGF-IR comprises a VH 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: 64 and / or a VL 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:

68.

26. the second antigen-binding moiety is capable of binding to cMET; and a) the HCDR1 amino acid sequence of SYWLH (SEQ ID NO: 69); b) the HCDR2 amino acid sequence of MIDPSNSDTRFNPNFKD (SEQ ID NO: 70); c) the HCDR3 amino acid sequence of YRSYVTPLDY (SEQ ID NO: 71) and a VH region comprising: d) LCDR1 of KSSQSLLYTSSQKNYLA (SEQ ID NO: 73); e) the LCDR2 amino acid sequence of WASTRES (SEQ ID NO: 74); f) LCDR3 amino acid sequence of QQYYAYPWT (SEQ ID NO: 75) and a VL region comprising The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 23, comprising:

27. 25. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 24, wherein the antigen-binding portion capable of binding to cMET comprises a VH 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: 72 and / or a VL 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:

76.

28. the second antigen-binding moiety is capable of binding to TROP2; and (a) the HCDR1 amino acid sequence of NYGMN (SEQ ID NO: 77); b) the HCDR2 amino acid sequence of WINTKTGEPTYAEEFKG (SEQ ID NO: 78); c) the HCDR3 amino acid sequence of GGYGSSYWYFDV (SEQ ID NO: 79); and a VH region comprising: d) LCDR1 of KASQDVSIAVA (SEQ ID NO: 81); e) the LCDR2 amino acid sequence of SASYRYT (SEQ ID NO: 82); f) the LCDR3 amino acid sequence of QQHYITPLT (SEQ ID NO: 83) and a VL region comprising The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 23, comprising:

29. 25. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 24, wherein the antigen-binding portion capable of binding to TROP2 comprises a VH 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: 80 and / or a VL 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:

84.

30. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 29, wherein the protease-cleavable linker comprises the protease recognition sequence PQARK (SEQ ID NO: 41).

31. 31. A pharmaceutical composition comprising the protease-activatable T cell activating bispecific molecule of any one of claims 1 to 30 and a pharmaceutically acceptable carrier.

32. An isolated polynucleotide encoding the protease-activatable T cell-activating bispecific antigen-binding molecule of any one of claims 1 to 30.

33. A vector, particularly an expression vector, comprising a polynucleotide according to claim 32.

34. A host cell comprising the vector of claim 33.

35. 35. A method for producing a protease-activated T cell activating bispecific molecule, comprising the steps of: a) culturing the host cell of claim 34 under conditions suitable for expression of the protease-activated T cell activating bispecific molecule; and b) recovering the protease-activated T cell activating bispecific molecule.

36. 31. The protease-activatable T cell activating bispecific molecule of any one of claims 1 to 30 for use as a medicament.

37. 37. The protease-activatable T cell activating bispecific molecule for use according to claim 36, 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.

38. 31. Use of the protease-activatable T cell activating bispecific molecule of any one of claims 1 to 30 for the manufacture of a medicament for the treatment of a disease.

39. 39. The use of a protease-activatable T cell activating bispecific molecule of claim 38, wherein the disease is cancer.

40. 31. 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 of any one of claims 1 to 30.

41. 41. The method of claim 40 for treating or delaying the progression of cancer.

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