Improved protease activatable t cell bispecific antibodies

Abstract

The present invention generally relates to improved protease activatable antigen binding molecules comprising an anti-idiotype binding moiety that reversibly masks the CD3 antigen binding moiety of the molecule. In addition, the present invention relates to polynucleotides encoding such protease activatable T cell binding molecules, as well as vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the protease activatable T cell binding molecules of the invention, and to methods of using these molecules, for example in the treatment of disease.

Description

Technical Field

[0001] The present invention generally relates to improved protease-activatable antigen binding molecules comprising an anti-idiotypic binding portion that reversibly masks the CD3 antigen binding portion of the molecule. In addition, the present invention relates to polynucleotides encoding such protease-activatable T cell binding molecules, and vectors and host cells comprising such polynucleotides. The present invention further relates to methods for producing the protease-activatable T cell binding molecules of the present invention, and to methods of using these molecules, for example, in the treatment of diseases. Background Art

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

[0003] An attractive way to achieve this goal is by inducing an immune response against the tumor, causing 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 surface antigens on target cells with one "arm" and to the activated, invariant component of the T cell receptor (TCR) complex with a second "arm" have attracted interest in recent years. The simultaneous binding of such an antibody to its two targets will force a temporary interaction between the target cell and the T cell, leading to the activation of any cytotoxic T cells and subsequent lysis of the target cell. The immune response is thus redirected to the target cell and is independent of the peptide antigen presentation by the target cell or the specificity of the T cell, which is associated with normal MHC-restricted CTL activation.

[0004] In this context, it is crucial that CTLs are activated only when in close proximity to target cells, i.e., mimicking an immune synapse. Particularly needed are T cell activating bispecific molecules that do not require lymphocyte preconditioning or co-stimulation to elicit efficient lysis of target cells. Several bispecific antibody formats have been developed and their suitability for T cell-mediated immunotherapy has also been investigated. These include BiTE (bispecific T cell engager) molecules (Nagorsen and 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 redirected) molecules (Moore et al., Blood 117, 4542-51 (2011)) and triomabs (Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)).

[0005] The task of generating bispecific molecules suitable for therapy presents several technical challenges related to efficacy, toxicity, applicability, and manufacturability that must be met. In the case of bispecific molecules targeting antigens that are expressed in tumor cells but are also expressed in normal tissues, on-target / off-tumor toxicity may occur. Therefore, there is a need for effective T cell activating bispecific molecules that unleash full T cell activation in the presence of target cells, but not in the presence of normal cells or tissues. Summary of the invention

[0006] The present invention provides improved T cell activation bispecific molecules. Specifically, the present invention provides T cell activation bispecific molecules that can be activated by proteases with reduced or missing activity before reaching the site of action, such as the tumor microenvironment. This leads to improved safety, such as less toxicity, and effective activation of molecules at the site of action.

[0007] In one embodiment, a protease-activatable T cell activating bispecific molecule is provided, comprising

[0008] (a) a first antigen binding portion that is capable of binding to CD3;

[0009] (b) a second antigen binding moiety capable of binding to a target cell antigen selected from the group consisting of IGF-1R, cMET and TROP2; and

[0010] (c) a masking moiety covalently linked 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 concealing the first antigen binding moiety or the second antigen binding moiety,

[0011] The peptide linker comprises a protease recognition sequence XQARK (SEQ ID NO: 39), wherein X is histidine (H) or proline (P).

[0012] In one embodiment, the masking moiety is covalently attached to the first antigen binding moiety and reversibly conceals the first antigen binding moiety.

[0013] In one embodiment, the masking moiety is covalently linked to the heavy chain variable region of the first antigen binding moiety.

[0014] In one embodiment, the masking moiety is a scFv.

[0015] 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, wherein the variable or constant regions of the Fab light chain and Fab heavy chain are exchanged.

[0016] In one embodiment, the first antigen binding moiety is a covalent Fab molecule.

[0017] In one embodiment, the protease-activatable T cell activating bispecific molecule comprises a third antigen binding portion of a Fab molecule capable of binding to a target cell antigen.

[0018] In one embodiment, the third antigen binding moiety is identical to the second antigen binding moiety.

[0019] In one embodiment, the first antigen binding moiety and the second antigen binding moiety are fused to each other, optionally via a peptide linker.

[0020] In one embodiment, 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.

[0021] In one embodiment, the protease-activatable T cell activating bispecific molecule further comprises an Fc domain composed of a first subunit and a second subunit capable of stably associating.

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

[0023] In one embodiment, the Fc domain exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to a native IgG1 Fc domain.

[0024] In one embodiment, the antigen binding portion capable of binding to CD3 comprises: a heavy chain variable (VH) region

[0025] (a) heavy chain complementarity determining region (HCDR) 1 amino acid sequence of SYAMN (SEQ ID NO: 1);

[0026] (b) HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 2);

[0027] (c) the HCDR3 amino acid sequence of ASNFPASYVSYFAY (SEQ ID NO: 3);

[0028] and a light chain variable (VL) region comprising:

[0029] (d) the light chain complementarity determining region (LCDR) 1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 7);

[0030] (e) LCDR2 amino acid sequence of GTNCRAP (SEQ ID NO: 8); and

[0031] (f) LCDR3 amino acid sequence of ALWYSNLWV (SEQ ID NO: 9).

[0032] 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.

[0033] In one embodiment, the antigen binding portion capable of binding to CD3 comprises a heavy chain variable (VH) region comprising

[0034] (a) heavy chain complementarity determining region (HCDR) 1 amino acid sequence of SYAMN (SEQ ID NO: 1);

[0035] (b) HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 2);

[0036] (c) HCDR3 amino acid sequence of HTTFPSSYVSYYGY (SEQ ID NO: 4);

[0037] and a light chain variable (VL) region comprising:

[0038] (d) the light chain complementarity determining region (LCDR) 1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 7);

[0039] (e) LCDR2 amino acid sequence of GTNCRAP (SEQ ID NO: 8); and

[0040] (f) LCDR3 amino acid sequence of ALWYSNLWV (SEQ ID NO: 9).

[0041] 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.

[0042] In one embodiment, the masking portion comprises: a VH region comprising:

[0043] (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15),

[0044] (b) a 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);

[0045] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19);

[0046] and a VL region comprising:

[0047] (d) LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25) or KSSKSVSTSSYSYMH (SEQ ID NO: 26);

[0048] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0049] (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) or QQSREFPYT (SEQ ID NO: 29).

[0050] In one embodiment, the masking portion comprises: a VH region comprising:

[0051] (a) HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0052] (b) HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16);

[0053] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19);

[0054] and a VL region comprising:

[0055] (d) LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25);

[0056] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0057] (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0058] In one embodiment, the masking portion comprises: a VH region comprising:

[0059] (a) HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0060] (b) HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16);

[0061] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19);

[0062] and a VL region comprising:

[0063] (d) LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26);

[0064] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0065] (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0066] In one embodiment, the masking portion comprises: a VH region comprising:

[0067] (a) HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0068] (b) HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO: 17);

[0069] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19);

[0070] and a VL region comprising:

[0071] (d) LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26);

[0072] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0073] (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0074] In one embodiment, the masking portion comprises: a VH region comprising:

[0075] (a) HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0076] (b) HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18);

[0077] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19);

[0078] and a VL region comprising:

[0079] (d) LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26);

[0080] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0081] (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0082] In one embodiment, the masking portion comprises: a VH region comprising:

[0083] (a) HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0084] (b) HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18);

[0085] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19);

[0086] and a VL region comprising:

[0087] (d) LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25);

[0088] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0089] (f) LCDR3 amino acid sequence of QQSREFPYT (SEQ ID NO: 29).

[0090] In one embodiment, the second antigen binding moiety is capable of binding to IGF-1R and comprises: a VH region comprising:

[0091] a) HCDR1 amino acid sequence of SYGMH (SEQ ID NO: 61);

[0092] b) a HCDR2 amino acid sequence of IIWFDGSSTYYADSVRG (SEQ ID NO: 62); and

[0093] c) the HCDR3 amino acid sequence of ELGRRYFDL (SEQ ID NO: 63);

[0094] and a VL region comprising:

[0095] d) LCDR1 of RASQSVSSYLA (SEQ ID NO: 65);

[0096] e) the LCDR2 amino acid sequence of DASKRAT (SEQ ID NO: 66); and

[0097] f) LCDR3 amino acid sequence of QQRSKWPPWT (SEQ ID NO: 67).

[0098] 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.

[0099] In one embodiment, the second antigen binding moiety is capable of binding to cMET and comprises: a VH region comprising:

[0100] a) HCDR1 amino acid sequence of SYWLH (SEQ ID NO: 69);

[0101] b) the HCDR2 amino acid sequence of MIDPSNSDTRFNPNFKD (SEQ ID NO: 70); and

[0102] c) HCDR3 amino acid sequence of YRSYVTPLDY (SEQ ID NO: 71);

[0103] and a VL region comprising:

[0104] d) LCDR1 of KSSQSLLYTSSQKNYLA (SEQ ID NO:73);

[0105] e) the LCDR2 amino acid sequence of WASTRES (SEQ ID NO: 74); and

[0106] f) LCDR3 amino acid sequence of QQYYAYPWT (SEQ ID NO: 75).

[0107] 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.

[0108] In one embodiment, the second antigen binding portion is capable of binding to TROP2 and comprises: a VH region comprising:

[0109] a) HCDR1 amino acid sequence of NYGMN (SEQ ID NO: 77);

[0110] b) the HCDR2 amino acid sequence of WINTKTGEPTYAEEFKG (SEQ ID NO: 78); and

[0111] c) HCDR3 amino acid sequence of GGYGSSYWYFDV (SEQ ID NO: 79);

[0112] and a VL region comprising:

[0113] d) LCDR1 of KASQDVSIAVA (SEQ ID NO:81);

[0114] e) the LCDR2 amino acid sequence of SASYRYT (SEQ ID NO: 82); and

[0115] f) LCDR3 amino acid sequence of QQHYITPLT (SEQ ID NO: 83).

[0116] 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.

[0117] In one embodiment, the protease-cleavable linker comprises the protease recognition sequence PQARK (SEQ ID NO: 41).

[0118] In one embodiment, an idiotype-specific polypeptide for reversibly concealing an anti-CD3 antigen binding site of a molecule is provided, wherein the idiotype-specific polypeptide is covalently linked to the molecule via a peptide linker, wherein the linker comprises a protease recognition sequence XQARK (SEQ ID NO: 39), wherein X is histidine (H) or proline (P).

[0119] In one embodiment, the idiotype-specific polypeptide is an anti-idiotype scFv.

[0120] In one embodiment, the molecule is a T cell activating bispecific molecule.

[0121] In one embodiment, the linker comprises the protease recognition sequence PQARK (SEQ ID NO: 41).

[0122] In one embodiment, a pharmaceutical composition is provided, comprising a protease-activatable T cell activating bispecific molecule as described herein and a pharmaceutically acceptable carrier.

[0123] In one embodiment, a pharmaceutical composition is provided, comprising an idiotype-specific polypeptide as described herein and a pharmaceutically acceptable carrier.

[0124] In one embodiment, an isolated polynucleotide encoding a protease-activatable T cell activating bispecific antigen binding molecule as described herein is provided.

[0125] In one embodiment, an isolated polynucleotide encoding an idiotype-specific polypeptide as described herein is provided.

[0126] In one embodiment, a vector, particularly an expression vector, comprising a polynucleotide as described herein is provided.

[0127] In one embodiment, a host cell is provided, comprising a vector as described herein.

[0128] In one embodiment, a method for producing a protease-activatable T cell activating bispecific molecule is provided, the method comprising the steps of: a) culturing a host cell as described herein under conditions suitable for expressing the protease-activatable T cell activating bispecific molecule, and b) recovering the protease-activatable T cell activating bispecific molecule.

[0129] In one embodiment, a protease-activatable T cell activating bispecific molecule as described herein is provided for use as a medicament.

[0130] 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 an immune response or function in an individual.

[0131] 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 treating a disease.

[0132] In one embodiment, there is provided a use of a protease-activatable T cell activating bispecific molecule as described herein, wherein the disease is cancer.

[0133] In one embodiment, a method of treating a disease in an individual is provided, the method comprising administering to the individual a therapeutically effective amount of a composition comprising a protease-activatable T cell activating bispecific molecule as described herein.

[0134] In one embodiment, the method is used to treat or delay progression of cancer.

[0135] Other technical features will be apparent to those skilled in the art from the following drawings, description and claims.

[0136] Other technical features will be apparent to those skilled in the art from the following drawings, description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1A . Schematic diagram depicting exemplary protease-activatable FolR1 proTCB molecules (SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:53). Figure 1B Schematic representations of exemplary IGF-1R proTCB molecules (SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89) are depicted. Figure 1C Schematic representations of exemplary cMET proTCB molecules (SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94) are depicted. Figure 1D Schematic representations of exemplary TROP2 proTCB molecules (SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100) are depicted.

[0138] Figure 2 .Describes the study design for single-dose PK and stability studies. Female NSG mice were injected intravenously with protease-activatable FolR1 TCB molecules containing HQARK or PQARK (Groups A and B) linkers and compared with the classical FolR1 TCB molecule (Group C). Mice were bled 24 hours, 7 days, and 10 days after injection. Serum was prepared and analyzed by ELISA for total and active forms of FolR1 TCB.

[0139] Figure 3 .Depicted is the quantification of active pro-TCB in the serum of non-tumor bearing mice. Active and total TCB concentrations in serum were measured over time by ELISA after a single intravenous injection of protease-activatable FolR1 TCB or classical FolR1 TCB. Active and total TCB were quantified by ELISA using anti-PG antibody (protease-activatable FolR1 TCB) and anti-idiotypic anti-CD3 antibody (active FolR1 TCB). The percentage of active TCB to total TCB is shown. No dose correction was required because equimolar doses of protease-activatable FolR1 TCB and classical FolR1 TCB were used in the corresponding studies.

[0140] Figure 4The study design of the in vivo efficacy study is described. Female NSG mice were injected subcutaneously with human breast cancer PDX (BC004) and received the first treatment when the tumor size reached approximately 200 mm3 (day 28). Mice were treated intravenously once a week with protease-activatable FolR1 TCB molecules containing PMAKK or PQARK (groups D and E) cleavage sites or classic FolR1 TCB molecules (group B) and masked FolR1 TCB containing non-cleavable linkers (group C). One group received only histidine buffer and served as a control (group A; vehicle). Tumor growth was measured by caliber, and the study was terminated at day 58, and tumors were harvested and weighed.

[0141] Figures 5A to 5G Tumor growth inhibition and tumor weight at the end of the study are depicted. (5A) Changes in tumor volume over time for all treatment groups are depicted as mean + / - SEM. Protease-activatable FolR1 TCB molecules containing a PQARK cleavage site produce comparable tumor growth inhibition as observed with the classical FolR1 TCB. Masked FolR1 TCBs containing a non-cleavable linker and molecules containing a PMAKK cleavage site do not result in tumor growth inhibition. (5B to 5F) Individual tumor growth kinetics of a single mouse in vehicle (5B), classical FolR1 TCB (5C), protease-activatable FolR1 TCB containing a PMAKK site (5D), masked FolR1 TCB containing a non-cleavable linker (5E), and protease-activatable FolR1 TCB containing a PQARK cleavage site (5F). (5G) Tumor weights for all treatment groups at the end of the study.

[0142] Figure 6 Depicted is the binding of the constructs shown to CD3 on Jurkat-NFAT T cells as determined by flow cytometry. Molecules were detected using a fluorescently labeled anti-human Fc specific secondary antibody. Median MFI is shown, and each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0143] Figure 7 A to 7D. depicts the binding of the constructs shown to IGF1R on different human cancer cell lines determined by flow cytometry. 7A) TCB binds to IGF1R on T-47D cells, 7B) TCB binds to IGF1R on MKN-45 cells, 7C) TCB binds to IGF1R on OVMANA cells, 7D) TCB binds to IGF1R on HPAF II cells. Molecules were detected using fluorescently labeled anti-human Fcγ specific secondary antibodies. Median MFI is shown; each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0144] Figure 8 .Depicts Jurkat NFAT activation mediated by IGF1R TCB constructs. Quantification of luminescence (Jurkat NFAT activation) after 5 h of incubation of hu-IGF1R coated SA beads with IGF1R TCB constructs and Jurkat NFAT effector cells. Protease pre-cleaved IGF1RproTCB and classic IGF1R TCB (with and without protease) induce dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0145] Fig. 9 A to 9D. depict Jurkat NFAT activation mediated by IGF1R pro-TCB. Quantification of luminescence (Jurkat NFAT activation) after incubation of different target cells with IGF1R proTCB and Jurkat NFAT effector cells for 5 h. Pre-cut IGF1R proTCB and classical IGF1R TCB induce dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0146] Fig.10 A to C. depicts tumor cell killing by IGF1R proTCB. Tumor cell killing of target cell lines (T-47D, MKN-45 and HPAF II) by healthy donor PBMCs after treatment with IGF1R proTCB was determined by LDH release after 72 h (10A, 10B, 10C). Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0147] Fig.11 A to 11F. depicts Figure 6 : CD4 through IGF1R proTCB + T cell activation. Flow cytometry was used to assess the upregulation of T cell activation markers CD25 (11A to 11C) and CD69 (11D to 11F). 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 for 72 h as shown. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0148] Fig.12A to 12F. depicts CD8+T cell activation by IGF1R proTCB. Flow cytometry was used to assess the upregulation of T cell activation markers CD25 (12A to 12C) and CD69 (12D to 12F). 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 for 72 h as shown. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0149] Fig.13 .Depicts the binding of the indicated constructs to CD3 on Jurkat-NFAT T cells as determined by flow cytometry. Molecules were detected using a fluorescently labeled anti-human Fc specific secondary antibody. Median MFI is shown, and each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0150] Fig.14 A to 14D. depicts the binding of the constructs shown to Trop2 on different human cancer cell lines determined by flow cytometry. 14A) TCB binds to Trop2 on T-47D cells, 14B) TCB binds to Trop2 on HPAF II cells, 14C) TCB binds to Trop2 on HeLa ST14 cells, 14D) TCB binds to Trop2 on OVMANA cells. Molecules were detected using fluorescently labeled anti-human Fc specific secondary antibodies. Median MFI is shown, and each point represents the average of triplicates. Standard deviation is indicated by error bars (n=1).

[0151] Fig.15 .Depicts Jurkat NFAT activation mediated by Trop2 pro-TCB. Quantification of luminescence (Jurkat NFAT activation) after 5 h incubation of hu-Trop2 coated SA beads with Trop2 proTCB and Jurkat NFAT effector cells. Pre-cut Trop2 proTCB and classical Trop2 TCB induce dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0152] Fig.16A to 16C. depict Jurkat NFAT activation mediated by Trop2 pro-TCB. Quantification of luminescence (Jurkat NFAT activation) after incubation of different target cells with Trop2 proTCB and Jurkat NFAT effector cells for 5 h. Pre-cut Trop2 proTCB and classic Trop2 TCB induce dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0153] Fig.17 A to 17C. Tumor cell killing by Trop2 proTCB is depicted. Tumor cell killing of target cell lines (17A) T-47D, (17B) HPAF II and (17C) OVMANA by healthy donor PBMCs after treatment with Trop2 proTCB was determined by LDH release after 72 h. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0154] Fig.18 A to 18F. CD4+ T cell activation by Trop2 proTCB is depicted. Flow cytometry was used to assess the upregulation of T cell activation markers CD25 (18A to 18C) and CD69 (18D to 18F). Healthy donor PBMCs were incubated with different target cell lines (T-47D, HPAF II and OVMANA) for 72 h and treated with different concentrations of Trop2 TCB molecules as indicated.

[0155] Fig.19 A to 19F. CD8+ T cell activation by Trop2 proTCB is depicted. Flow cytometry was used to assess the upregulation of T cell activation markers CD25 (19A to 19C) and CD69 (19D to 19F). Healthy donor PBMCs were incubated with different target cell lines (T-47D, HPAF II and OVMANA) for 72 h and treated with different concentrations of Trop2 TCB molecules as indicated.

[0156] Fig. 20 Depicted is the binding of the constructs shown to CD3 on Jurkat-NFAT T cells as determined by flow cytometry. Molecules were detected using a fluorescently labeled anti-human Fc specific secondary antibody. Median MFI is shown, and each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0157] Fig.21A to 21D. depicts the binding of the indicated constructs to cMet on different human cancer cell lines as determined by flow cytometry. 21A) TCB binding to cMet on OVMANA cells, 21B) TCB binding to cMet on HeLa ST14 cells, 21C) TCB binding to cMet on HPAF II cells, 21D) TCB binding to cMet on T-47D cells. Molecules were detected using a fluorescently labeled anti-human Fc specific secondary antibody. The median MFI is shown, and each point represents the mean of triplicates. The standard deviation is indicated by the error bars (n=1).

[0158] Fig. 22 .Depicts Jurkat NFAT activation mediated by cMet proTCB. Quantification of luminescence (Jurkat NFAT activation) after 5 h incubation of hu-cMet coated SA beads with cMet proTCB and Jurkat NFAT effector cells. Pre-cut cMet proTCB and classical cMet TCB induce dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0159] Fig.23 A to C. depict Jurkat NFAT activation mediated by cMet pro-TCB. Quantification of luminescence (Jurkat NFAT activation) after 5 h incubation of different target cells with cMet proTCB and Jurkat NFAT effector cells. Pre-cut cMet proTCB and classical cMet TCB induce dose-dependent Jurkat NFAT activation. Each point represents the mean of triplicates. Standard deviation is indicated by error bars (n=1).

[0160] Fig.24 A to 24C. Tumor cell killing by cMet proTCB is depicted. Tumor cell killing of target cell lines (24A) OVMANA, (24B) HPAF II, (24C) T-47D by healthy donor PBMCs after treatment with cMet proTCB was determined by LDH release after 72 h.

[0161] Fig.25 A to 25F. CD4+ T cell activation by cMet proTCB is depicted. Upregulation of T cell activation markers CD25 (25A to 25C) and CD69 (25D to 25F) was assessed using flow cytometry. Healthy donor PBMCs were incubated with different target cell lines (OVMANA, HPAFII, T-47D) and treated with different concentrations of cMet TCB molecules for 72 h.

[0162] Fig.26 A to 26F. CD8+ T cell activation by cMet proTCB is depicted. Upregulation of T cell activation markers CD25 (26A to 26C) and CD69 (26D to 26F) was assessed using flow cytometry. Healthy donor PBMCs were incubated with different target cell lines (OVMANA, HPAFII, T-47D) and treated with different concentrations of cMet TCB molecules for 72 h. DETAILED DESCRIPTION

[0163] definition

[0164] Unless otherwise defined below, the terms used herein are the same as those commonly used in the art.

[0165] "Acceptor human framework" for the purposes of this paper is such a framework, which 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. The acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the amino acid sequence identical to the human immunoglobulin framework or a human consensus framework, or it can contain amino acid sequence changes. In some aspects, 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 aspects, the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or a human consensus framework sequence in sequence.

[0166] "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 intrinsic binding affinity, which 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 can be generally described by the dissociation constant (K D ) represents. Affinity can be measured by conventional methods known in the art (including those described herein). Specific illustrative and exemplary methods for measuring binding affinity are described below.

[0167] An "affinity matured" antibody is one with one or more alterations in one or more complementarity determining regions (CDRs) which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess such alterations.

[0168] The term "antibody" herein is used in the broadest sense and encompasses various 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.

[0169] "Antibody fragment" refers to a molecule that contains a portion of an intact antibody in addition to an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).

[0170] Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using methods routine in the art, such as, for example, but not limited to, alanine scanning, peptide blotting (see 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)).

[0171] Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows the classification of a variety of monoclonal antibodies that specifically bind to an antigen based on the binding profile of each antibody from a large number of antibodies to a chemically or enzymatically modified antigen surface (see, e.g., US 2004 / 0101920). The antibodies in each group bind to the same epitope, which may be a unique epitope that is significantly different from or partially overlaps with the epitope represented by another group.

[0172] In some aspects, 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 the binding of the other antibody by at least 50%, at least 75%, at least 90%, or even 99% or more as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0173] In some aspects, two antibodies are considered to bind 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. [End Epitope Section]]

[0174] 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.

[0175] The "class" of an antibody refers to the type of constant domain or region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG. 1 IgG 2 IgG 3 IgG 4 , IgA 1 and IgA 2 In some aspects, the antibody is an IgG 1 Isotype. In some aspects, the antibody is an IgG 1 isotype, which has P329G, L234A and L235A mutations to reduce Fc region effector function. In other aspects, the antibody is an IgG 2 Isotype. In some aspects, the antibody is an IgG 4 isotype, which has an S228P mutation in the hinge region to improve IgG 4 Stability of antibodies. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called a, d, e, g, and m. The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0176] As used in the present application, the term "constant region derived from human origin" or "human constant region" refers to the constant heavy chain region and / or constant light chain kappa or lambda region of a human antibody of subclass IgG1, IgG2, IgG3 or IgG4. Such constant regions are well known in the prior art and are described, for example, by Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson, G. and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218; Kabat, EA et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, the numbering of amino acid residues in the constant region is according to the EU numbering system, also known as the EU index of Kabat, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0177] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with 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; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0178] An "effective amount" of a pharmaceutical agent (eg, a pharmaceutical composition) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0179] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. On the one hand, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the antibody produced by the host cell can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Therefore, the antibody produced by the host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain can include a full-length heavy chain, or the antibody can include a cleavage variant of the full-length heavy chain. This may be a situation where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may or may not be present. If not otherwise indicated, the amino acid sequence of the heavy chain including the Fc region is represented herein as the absence of a C-terminal glycine-lysine dipeptide. On the one hand, the heavy chain including the Fc region as specified herein is included in the antibody according to the present invention, and the heavy chain includes an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). On the one hand, the heavy chain including the Fc region as specified herein is included in the antibody according to the present invention, and the heavy chain includes an additional C-terminal glycine residue (G446, numbered according to the EU index). Unless otherwise specified herein, the numbering of amino acid residues in the 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 Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0180] "Framework" or "FR" refers to the variable domain residues excluding the complementarity determining regions (CDRs). The FR of the variable domain is generally composed of the following four FR domains: FR1, FR2, FR3 and FR4. Therefore, CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0181] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0182] 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 the progeny derived from the primary transformed cell (regardless of the number of passages). The progeny may not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny with the same function or biological activity as screened or selected in the original transformed cell are included herein.

[0183] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source utilizing a full repertoire of human antibodies or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0184] 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. In general, the selection of human immunoglobulin VL or VH sequences comes from a subset of variable domain sequences. In general, the subset of sequences is as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1 to 3. On the one hand, for VL, the subgroup is as in Kabat et al., subgroup κI, supra. On the one hand, for VH, the subgroup is as in Kabat et al., supra subgroup III. [[Adjust as needed to refer to the actual subgroup of VH / VL of the present invention]]

[0185] "Humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some aspects, a humanized antibody will substantially comprise at least one and typically two variable domains, wherein all or substantially all CDRs correspond to those of non-human antibodies, and all or substantially all FRs correspond to those of human antibodies. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody in "humanized form", such as a non-human antibody, refers to an antibody that has undergone humanization.

[0186] As used herein, the term "hypervariable region" or "HVR" refers to individual regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, such as the "complementarity determining region" ("CDR").

[0187] In general, antibodies contain six CDRs: three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include:

[0188] (a) the hypervariable loops present 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));

[0189] (b) the CDRs present 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

[0190] (c) Antigenic contacts present 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)).

[0191] Unless otherwise indicated, CDRs are identified according to Kabat et al., supra. One skilled in the art will appreciate that CDR names may also be identified according to Chothia, supra, McCallum, supra, or any other scientifically acceptable nomenclature system.

[0192] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0193] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, 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 aspects, an individual or subject is a human.

[0194] An "isolated" antibody is one that has been separated from a component of its natural environment. In some aspects, the antibody is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-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).

[0195] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance comprising a nucleotide polymer. Each nucleotide is composed of a base (particularly 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. Typically, nucleic acid molecules are described by base sequences, wherein the bases represent the primary structure (linear structure) of nucleic acid molecules. Typically, the sequence of bases is represented from 5' to 3'. In this article, the term nucleic acid molecule encompasses synthetic forms of deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense strands and antisense strands, as well as single-stranded and double-stranded forms. In addition, nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases or chemically modified residues with derivatized sugar or phosphate backbone bonds. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for direct expression in vitro and / or in vivo (e.g., in a host or patient) of the antibodies of the present invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of RNA carriers and / or the expression of coding molecules so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online on June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0196] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0197] 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 such nucleic acid molecules in a single vector or in different vectors, and one or more such nucleic acid molecules present at one or more locations in a host cell.

[0198] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants are usually present in small amounts), the individual antibodies comprising the population have identity and / or bind to the same epitope. Contrary to polyclonal antibody preparations typically including different antibodies for different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents that the feature of an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibody to be produced by any ad hoc method. For example, monoclonal antibodies according to the present invention can be prepared 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 human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0199] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical compositions.

[0200] "Native antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, 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 referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain.

[0201] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0202] "Percentage (%) of amino acid sequence identity" relative 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 a reference polypeptide sequence, after aligning the sequences and introducing spaces (if necessary) to achieve maximum percentage of sequence identity, and for the purpose of alignment without considering any conservative substitutions as components of sequence identity. Alignment for determining percentage of amino acid sequence identity 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 packages. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate percentage identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code has been filed with user documentation in the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087 and is described in WO 2001 / 007611.

[0203] Unless otherwise indicated, for the purposes of this article, the values ​​of percent amino acid sequence identity are generated using the BLOSUM50 comparison matrix using the ggsearch program of the FASTA package version 36.3.8c or higher. The FASTA package was written by W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; W.R. Pearson (1996) "Effective proteins sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using a 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 global rather than local alignment. Amino acid identity percentages are given in the output alignment header.

[0204] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0205] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0206] Unless otherwise indicated, the term "FoR1" or "folate receptor 1" as used herein 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, as well as any form of FolR1 produced by processing in a cell. The term also encompasses naturally occurring variants of FolR1, such as splice variants or allelic variants.

[0207] Unless otherwise indicated, the term "IGF-1R" or "insulin-like growth factor receptor type 1" as used herein refers to any native IGF-1R from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length," unprocessed IGF-1R, as well as any form of IGF-1R produced by processing in a cell. The term also encompasses naturally occurring variants of IGF-1R, such as splice variants or allelic variants.

[0208] Unless otherwise indicated, the term "cMET" or "tyrosine-protein kinase Met" as used herein refers to any native cMET 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 cMET, as well as any form of cMET produced by processing in cells. The term also encompasses naturally occurring variants of cMET, such as splice variants or allelic variants.

[0209] Unless otherwise indicated, the term "TROP2" or "tumor-associated calcium signaling protein 2" as used herein refers to any native TROP2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length" unprocessed TROP2, as well as any form of TROP2 produced by processing in cells. The term also encompasses naturally occurring variants of TROP2, such as splice variants or allelic variants.

[0210] As used herein, "treatment" (and grammatical variants thereof such as "treat" or "treating") refers to an attempt to alter the natural course of a disease in the individual being treated, and may be performed for prevention or clinical interventions that may be performed during clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0211] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using VH or VL domains from antibodies that bind to the antigen 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).

[0212] The term "vector" as used herein refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0213] As used herein, the term "antigen binding portion" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen binding portion is capable of directing the entity to which it is attached (e.g., a second antigen binding portion) to a target site, for example, to a specific type of tumor cell or tumor stroma having an antigenic determinant. In another embodiment, the antigen binding portion is capable of activating signal transduction by its target antigen (e.g., a T cell receptor complex antigen). The antigen binding portion includes antibodies and fragments thereof, as further defined herein. Specific antigen binding portions include the antigen binding domains of antibodies, including antibody heavy chain variable regions and antibody light chain variable regions. In certain embodiments, the antigen binding portion may include an antibody constant region, as further defined herein and known in the art. Useful heavy chain constant regions include any of the following five isotypes: α, δ, ε, γ or μ. Useful light chain constant regions include any of the following two isotypes: κ and λ.

[0214] As used herein, "T cell activation" refers to one or more cellular responses 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. The protease-activatable "T cell activation bispecific molecules" of the present invention are capable of inducing T cell activation. Suitable assays for measuring T cell activation are known in the art as described herein.

[0215] As used herein, "target cell antigen" refers to an antigenic determinant presented on the surface of a target cell (eg, a cell in a tumor, such as a cancer cell or a cell of a tumor stroma).

[0216] As used herein, the terms "first" and "second" with respect to antigen binding moieties and the like are used for convenient distinction when there is more than one of each type of moiety. Unless explicitly stated, the use of these terms is not intended to confer a particular order or orientation of the protease-activatable T cell activating bispecific molecules.

[0217] "Fab molecule" refers to a protein composed of the VH and CH1 domains of the heavy chain ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain") of an immunoglobulin.

[0218] "Fusion" means that the components (eg, a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0219] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly connected 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 a Fab light chain and a Fab heavy chain are connected by a peptide linker to form a single peptide chain. In specific such embodiments, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0220] "Cross" Fab molecules (also referred to as "Crossfab") refer to a Fab molecule in which the variable or constant regions of the Fab heavy and light chains are exchanged, i.e., the cross-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 cross-Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the cross-Fab molecule. Conversely, in a cross-Fab molecule in which the constant regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the cross-Fab molecule.

[0221] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native form, ie, comprising a heavy chain consisting of a heavy chain variable region and a constant region (VH-CH1), and a light chain consisting of a light chain variable region and a constant region (VL-CL).

[0222] As used herein, "idiotype-specific polypeptide" refers to a polypeptide that recognizes the idiotype of an antigen-binding portion (e.g., an antigen-binding portion that is specific for CD3). "Idiotype" can be defined as a specific combination of idiotypes present within the complement determining regions (CDRs) of an antigen-binding portion. Idiotype-specific polypeptides are capable of specifically binding to the variable region of an antigen-binding portion, and thereby reducing or preventing specific binding of the antigen-binding portion to its cognate antigen. When associated with a molecule comprising an antigen-binding portion, an idiotype-specific polypeptide can act as a masking portion of the molecule. Specifically disclosed herein are anti-idiotype antibodies or anti-idiotype binding antibody fragments that are specific for the idiotype of an anti-CD3 binding molecule.

[0223] As used herein, "protease" or "proteolytic enzyme" refers to any proteolytic enzyme that cuts a linker at a recognition site and is expressed by a target cell. Such proteases may be secreted by the target cell or remain associated with the target cell, for example, on the surface of the target cell. Examples of proteases include, but are not limited to, metalloproteinases, such as matrix metalloproteinases 1-28 and disintegrin metalloproteinases (ADAM) 2, 7-12, 15, 17-23, 28-30 and 33, serine proteases, such as urokinase-type plasminogen activator, and matriptase, cysteine ​​protease, aspartic protease and members of the cathepsin family.

[0224] As used herein, "protease activatable" with respect to a T cell activating bispecific molecule refers to a T cell activating bispecific molecule that has reduced or eliminated 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. When the masking moiety is dissociated by proteolytic cleavage, for example, by proteolytic cleavage of a linker connecting the masking moiety to the T cell activating bispecific molecule, binding to CD3 is restored, and the T cell activating bispecific molecule is thereby activated.

[0225] As used herein, "reversible concealment" refers to the binding of a masking moiety or idiotype-specific polypeptide to an antigen-binding moiety or molecule, such as to prevent the antigen-binding moiety or molecule from binding to its antigen (e.g., CD3). Such concealment is reversible because the idiotype-specific polypeptide can be released from the antigen-binding moiety or molecule (e.g., by protease cleavage), thereby releasing the antigen-binding moiety or molecule to bind to its antigen.

[0226] Protease-activatable T cell-activating bispecific molecules

[0227] The present invention provides improved T cell activation bispecific molecules. Specifically, the present invention provides T cell activation bispecific molecules that can be activated by proteases with reduced or missing activity before reaching the site of action, such as the tumor microenvironment. This leads to improved safety, such as less toxicity, and effective activation of molecules at the site of action.

[0228] In one aspect, the invention relates to a protease-activatable T cell activating bispecific molecule comprising

[0229] 1. a first antigen binding portion that is capable of binding to CD3;

[0230] 2. a second antigen binding moiety capable of binding to a target cell antigen; and

[0231] 3. A masking moiety covalently linked 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 concealing the first antigen binding moiety or the second antigen binding moiety.

[0232] The first antigen binding moiety capable of binding to CD3 includes an idiotype. In one embodiment, the masking portion of the protease-activatable T cell activation bispecific molecule is covalently attached to the first antigen binding moiety. In one embodiment, the masking portion is covalently attached to the heavy chain variable region of the first antigen binding moiety. In one embodiment, the masking portion is covalently attached to the light chain variable region of the first antigen binding moiety. This covalent bond is distinguished from the specific binding (which is preferably non-covalent) of the masking portion to the idiotype first antigen binding site. The idiotype of the first antigen binding moiety includes its variable region. In one embodiment, when the first antigen binding moiety binds to CD3, the masking portion binds to the amino acid residues in contact with CD3. In a preferred embodiment, the masking portion is not a cognate antigen or a fragment thereof of the first antigen binding moiety, i.e., the masking portion is not CD3 or a fragment thereof. In one embodiment, the masking portion is an anti-idiotype antibody or a fragment thereof. In one embodiment, the masking portion is an anti-idiotype scFv. Exemplary embodiments of the masking portion as an anti-idiotype scFv and the protease-activatable T cell activation molecule comprising such a masking portion are described in detail below and in the embodiments.

[0233] Exemplary Antigen Binding Moieties

[0234] The antigen binding molecules of the present invention are bispecific, i.e., they include at least two antigen binding moieties that can specifically bind to two different antigenic determinants. According to the present invention, the antigen binding moiety is a Fab molecule (i.e., an antigen binding domain consisting of a heavy chain and a light chain, each comprising 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 includes a human heavy chain constant region and a light chain constant region.

[0235] At least one antigen binding moiety is a cross Fab molecule. This modification prevents mispairing of heavy chains and light chains from different Fab molecules, thereby improving the output and purity of the protease-activatable T cell activation bispecific molecule of the present invention in recombinant production. In a specific cross Fab molecule that can be used for the protease-activatable T cell activation bispecific molecule of the present invention, the constant region of the Fab light chain and the Fab heavy chain is exchanged. In another cross Fab molecule that can be used for the protease-activatable T cell activation bispecific molecule of the present invention, the variable region of the Fab light chain and the Fab heavy chain is exchanged.

[0236] In a specific embodiment according to the present invention, the protease-activatable T cell activation bispecific molecule is capable of binding to a target cell antigen (particularly a tumor cell antigen) and CD3 simultaneously. In one embodiment, the protease-activatable T cell activation bispecific molecule is capable of crosslinking T cells and target cells by binding to a target cell antigen and CD3 simultaneously. In an even more specific embodiment, this simultaneous binding results in the lysis of target cells, particularly tumor cells. In one embodiment, this simultaneous binding results in the activation of T cells. In other embodiments, this simultaneous binding results in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, which is selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. In one embodiment, the binding of the protease-activatable T cell activation bispecific molecule to CD3 without simultaneously binding to a target cell antigen does not result in T cell activation.

[0237] In one embodiment, the protease-activatable T cell activating bispecific molecule is capable of redirecting the cytotoxic activity of T cells to target cells. In a specific embodiment, the redirection is independent of MHC-mediated peptide antigen presentation by the target cell and / or the specificity of the T cell.

[0238] In particular, the T cells according to any embodiment of the present invention are cytotoxic T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells, especially CD8 + T cells.

[0239] CD3 binding part

[0240] The protease-activatable T cell activation bispecific molecule of the present invention comprises at least one antigen binding portion capable of binding to CD3 (also referred to herein as "CD3 antigen binding portion" or "first antigen binding portion"). In a specific embodiment, the protease-activatable T cell activation bispecific molecule comprises no more than one antigen binding portion capable of binding to CD3. In one embodiment, the protease-activatable T cell activation bispecific molecule provides monovalent binding to CD3. 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 are exchanged. In embodiments in which the protease-activatable T cell activation bispecific molecule comprises more than one antigen binding portion capable of binding to a target cell antigen, the antigen binding portion capable of binding to CD3 is preferably a crossover Fab molecule, and the antigen binding portion capable of binding to a target cell antigen is a conventional Fab molecule.

[0241] In a specific embodiment, CD3 is human CD3 or cynomolgus CD3, most particularly human CD3. In a specific embodiment, the CD3 antigen binding portion is cross-reactive to (i.e., specifically binds to) human and cynomolgus CD3. In some embodiments, the first antigen binding portion is capable of binding to the epsilon subunit of CD3.

[0242] 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, SEQ ID NO: 9.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] Target cell antigen binding moiety

[0250] The protease-activatable T cell activation bispecific molecule of the present invention comprises at least one antigen binding portion capable of binding to a target cell antigen (also referred to herein as "target cell antigen binding portion" or "second" or "third" antigen binding portion). In certain embodiments, the protease-activatable T cell activation bispecific molecule comprises two antigen binding portions capable of binding to a target cell antigen. In specific such embodiments, each of these antigen binding portions specifically binds to the same antigenic determinant. In even more specific embodiments, all of these antigen binding portions are the same. In one embodiment, the protease-activatable T cell activation bispecific molecule comprises an immunoglobulin molecule capable of binding to a target cell antigen. In one embodiment, the protease-activatable T cell activation bispecific molecule comprises no more than two antigen binding portions capable of binding to a target cell antigen.

[0251] In a preferred embodiment, the target cell antigen binding portion is a Fab molecule, particularly a conventional Fab molecule, which binds to a specific antigenic determinant and is capable of directing the protease-activatable T cell activating bispecific molecule to a target site, such as a specific type of tumor cell having the antigenic determinant.

[0252] In certain embodiments, the target cell antigen binding portion specifically binds to a cell surface antigen. In a specific embodiment, the target cell antigen binding portion 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 portion specifically binds to tyrosine protein kinase Met (cMET) on the surface of the target cell. In another specific embodiment, the target cell antigen binding portion specifically binds to tumor-associated calcium signaling protein 2 (TROP2) on the surface of the target cell.

[0253] In certain embodiments, the target cell antigen binding moiety is directed to an antigen associated with a pathological condition, such as an antigen presented on a tumor cell or a virally infected cell. Suitable antigens are cell surface antigens, such as but not limited to cell surface receptors. In a particular embodiment, the antigen is a human antigen. In a specific embodiment, 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 transduction protein 2 (TROP2).

[0254] In a specific embodiment, the protease-activatable T cell activating bispecific molecule comprises at least one antigen binding portion that is specific for IGF-1R. In one embodiment, IGF-1R is human IGF-1R. In one embodiment, the antigen binding portion that is specific for IGF-1R comprises at least one heavy chain complementary determining region (CDR) selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 62, 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, SEQ ID NO: 67.

[0255] 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.

[0256] In additional embodiments, the antigen binding portion that is 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 variants thereof that retain functionality.

[0257] 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.

[0258] In a specific embodiment, the protease-activatable T cell activating bispecific molecule comprises at least one antigen binding portion that is specific for cMET. In one embodiment, cMET is human cMET. In one embodiment, the antigen binding portion that is specific for cMET comprises at least one heavy chain complementary determining region (CDR) selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 70, 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, SEQ ID NO: 75.

[0259] 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.

[0260] In additional embodiments, the antigen binding portion that is 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 variants thereof that retain functionality.

[0261] In one embodiment, the 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.

[0262] In a specific embodiment, the protease-activatable T cell activating bispecific molecule comprises at least one antigen binding portion specific for TROP2. In one embodiment, TROP2 is human TROP2. In one embodiment, the antigen binding portion specific for TROP2 comprises at least one heavy chain complementary 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.

[0263] 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.

[0264] In additional embodiments, 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 variants thereof that retain functionality.

[0265] In one embodiment, the 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.

[0266] Masking part

[0267] The protease-activatable T cell activation bispecific molecule of the present invention comprises at least one masking part. Others have attempted to mask the binding of antibodies by capping the binding part with a fragment of the antigen recognized by the binding part (e.g., WO2013128194). This approach has several limitations. For example, the use of antigens has less flexibility in reducing the affinity of the binding part. This is so because the affinity must be high enough to be reliably masked by the antigen mask. In addition, the dissociated antigen may bind and interact with one or more of its cognate receptors in vivo and produce unwanted signals for cells expressing such receptors. In contrast, the method described herein uses an anti-idiotypic antibody or a fragment thereof as a mask. Two opposing considerations for designing an effective masking part are 1. the effectiveness of the masking and 2. the reversibility of the masking. If the affinity is too low, the masking efficiency will be low. However, if the affinity is too high, the masking process may not be easily reversible. It is impossible to predict which high-affinity anti-idiotypic mask or low-affinity anti-idiotypic mask works better. As described herein, higher affinity masking moieties generally perform better in terms of masking the antigen binding side and at the same time can be effectively removed to activate the molecule. In one embodiment, the anti-idiotypic mask has a KD of 1 to 8 nM. In one embodiment, the anti-idiotypic mask has a KD of 2 nM at 37°C. In a specific embodiment, the masking moiety recognizes the idiotype of a first antigen binding moiety that is capable of binding to CD3 (e.g., human CD3). In a specific embodiment, the masking moiety recognizes the idiotype of a second antigen binding moiety that is capable of binding to a target cell antigen.

[0268] In one embodiment, the masking portion masks the CD3 binding portion and comprises at least one of the following: heavy chain complementary 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 complementary 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.

[0269] In one embodiment, the masking portion comprises: a VH region comprising a HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15), a HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16), and a HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19); and a VL region comprising a LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25), a LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27), and a LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0270] In one embodiment, the masking portion comprises: a VH region comprising a HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15), a HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16), and a HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19); and a VL region comprising a LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26), a LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27), and a LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0271] In a preferred embodiment, the masking portion comprises: a VH region comprising a HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15), a HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO: 17), and a HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19); and a VL region comprising a LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26), a LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27), and a LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0272] In one embodiment, the masking portion comprises: a VH region comprising a HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15), a HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18), and a HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19); and a VL region comprising a LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26), a LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27), and a LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0273] In one embodiment, the masking portion comprises: a VH region comprising a HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15), a HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18), and a HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19); and a VL region comprising a LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25), a LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27), and a LCDR3 amino acid sequence of QQSREFPYT (SEQ ID NO: 29).

[0274] In one embodiment, the masking moiety masks the CD3 binding moiety 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 moiety and comprises a polypeptide sequence of SEQ ID NO: 30.

[0275] In a preferred embodiment, the masking moiety is humanized. Methods for humanizing immunoglobulins are well known in the art and are described herein. Humanized masking moieties H1L1, H1L2, H2L2, H3L2, H3L3 and H7L5 are provided herein. The corresponding sequences are provided below.

[0276] 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.

[0277] 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.

[0278] In preferred embodiments, 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:32.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] In one embodiment, the antigen binding masking moiety or idiotype-specific polypeptide used to reversibly conceal the antigen binding of the molecule is a scFc. Such idiotype-specific polypeptides used to reversibly conceal the anti-CD3 antigen binding site must be able to bind to the idiotype of the anti-CD3 antigen binding site and thereby reduce or eliminate the binding of the anti-CD3 antigen binding site to CD3. In one embodiment, the idiotype scF.

[0284] In one embodiment, the masking moiety comprises an idiotype-specific polypeptide for reversibly concealing 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 a scFv. In a preferred embodiment, the masking moiety is a scFv.

[0285] In one embodiment, the scFv comprises a polypeptide sequence 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.

[0286] In one embodiment, the scFv comprises a polypeptide sequence 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.

[0287] In one embodiment, the scFv comprises a polypeptide sequence 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.

[0288] In one embodiment, the scFv comprises a polypeptide sequence 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.

[0289] Protease cleavable linker

[0290] The protease-activatable T cell activation bispecific molecule of the present invention comprises at least one protease-activatable linker. Preferably, the protease-activatable T cell activation bispecific molecule of the present invention is inactive before the protease-activatable linker cleavage (e.g., in a tumor microenvironment). In one embodiment, the masking portion (e.g., an 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.

[0291] In one embodiment, the protease cleavable linker comprises a protease recognition site. In one embodiment, the protease is a proteinase. In a preferred embodiment, the protease cleavable linker comprises a proteinase recognition site.

[0292] In one embodiment, the protease-activatable T cell activating bispecific molecule comprises a linker having a protease recognition site, the protease recognition site comprising the polypeptide sequence XQARK (SEQ ID NO: 39), wherein 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).

[0293] PQARK (SEQ ID NO: 41) and HQARK (SEQ ID NO: 43) are protease recognition sites with advantageous and unexpected properties. Ideally, a protease-activatable (therapeutic) molecule should be inactive before reaching the site of action (e.g., a tumor). An advantageous property of the protease recognition sites of the invention (e.g., PQARK and HQARK) is that they are stable in vivo before reaching the site of action (see, e.g., Figure 3 ). In addition, such activatable molecules should be effectively activated at the site of action (e.g., tumor). It is known that the tumor microenvironment may exhibit a pH as low as 5.6 compared to physiological pH (about pH 7.4) (see, e.g., Boedtkjer et al. 2020, Annual Review of Physiology, Vol. 82, 2020, pp. 103-126).

[0294] Importantly, the protease recognition sites of the invention (e.g., PQARK and HQARK) can be more strongly activated at physiological pH than the disclosed protease recognition site PMAKK (see, e.g., Table 4). Unexpectedly, the protease recognition sites of the molecules of the invention (e.g., PQARK and cMET proTCB P1AI0623, TROP2 proTCBP1AI0690, IGF-1R proTCB P1AH0943) can be strongly activated at pH as low as pH 5.6.

[0295] In one embodiment, the protease recognition site is embedded in a joint such as a (unstructured) polypeptide. In one embodiment, the polypeptide comprises one or more unstructured peptide joints. In one embodiment, the isolated polypeptide comprises at least one peptide joint, particularly wherein the at least one peptide joint does not exhibit 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, more preferably 10 to 50 amino acids, most preferably 20 to 40 amino acids.

[0296] In one embodiment, the protease cleavable linker is a polypeptide peptide with 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 with 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 a protease. In one embodiment, the protease recognition site comprises, or consists of, the sequence PQARK (SEQ ID NO: 41) or HQARK (SEQ ID NO: 43).

[0297] In one embodiment, the protease-cleavable linker is an unstructured polypeptide. In one embodiment, the protease-cleavable linker does not exhibit a secondary structure. In one embodiment, the protease-cleavable linker comprises at least one linker that promotes an unstructured conformation. In one embodiment, the linker comprises serine (S) and / or glycine (G). In one embodiment, at least one linker of the protease-cleavable linker comprises an amino acid sequence (GxS)n or (GxS)nGm, wherein G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3), preferably x=4 and n=2 or 3, more preferably x=4, n=2. In one embodiment, the protease-cleavable linker comprises (G 4 S) 2 In one embodiment, the protease-cleavable linker comprises (G 4 S)3 In one embodiment, the protease-cleavable linker comprises G 2 S. The protease-cleavable linker comprises a protease recognition site at any position (eg, at the beginning of the linker, anywhere within, or at the end).

[0298] 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).

[0299] Protease-activatable T cell-activating bispecific molecule formats

[0300] 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 depicted in Figure 1.

[0301] In specific embodiments, the protease-activatable T cell activating bispecific molecule comprises an Fc domain consisting of a first subunit and a second subunit capable of stably associating. In some embodiments, the second antigen binding moiety is fused to the N-terminus of the first subunit or the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

[0302] In one such embodiment, the first 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 a specific such embodiment, the protease-activatable T cell activation bispecific molecule consists essentially of the first and second antigen binding moieties, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the first antigen binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen binding moiety at the C-terminus of the Fab heavy chain, and the second 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 can be additionally fused to each other.

[0303] In another such embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit or the second subunit of the Fc domain. In a specific such embodiment, the protease-activatable T cell activating bispecific molecule consists essentially of a first and a second antigen binding moiety, an Fc domain consisting of a first subunit and a second subunit, and optionally one or more peptide linkers, wherein each of the first and second antigen binding moieties is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain.

[0304] 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.

[0305] In specific 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 specific such embodiments, the protease-activatable T cell activation bispecific molecule consists essentially of the first and second antigen binding moieties, the Fc domain consisting of the 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 can be additionally fused to each other.

[0306] The antigen binding portion can be fused to the Fc domain or to each other directly or through a peptide linker comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic peptide linkers include, for example (G 4 S) n , (SG 4 ) n , (G 4 S) n or G 4 (SG 4 ) n Peptide linker. "n" is usually a number between 1 and 10, typically between 2 and 4. A particularly suitable peptide linker for fusing the Fab light chains of the first and second antigen binding moieties to each other is (G 4 S) 2 In addition, the linker may comprise (a portion of) an immunoglobulin hinge region. In particular, when the antigen binding portion is fused to the N-terminus of the Fc domain subunit, it may be fused via the immunoglobulin hinge region or a portion thereof with or without an additional peptide linker.

[0307] Protease-activatable T cell activating bispecific molecules having a single antigen binding portion capable of binding to a target cell antigen are useful, particularly where internalization of the target cell antigen is expected following binding of the high affinity antigen binding portion. In this case, the presence of more than one antigen binding portion specific for the target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availability.

[0308] However, in many other situations it would be advantageous to have a protease-activatable T cell activating bispecific molecule comprising two or more antigen binding portions specific for target cell antigens, for example to optimize targeting to a target site or to allow cross-linking of target cell antigens.

[0309] Therefore, in certain embodiments, the protease-activatable T cell activation bispecific molecule of the present invention comprises a third antigen binding portion of a Fab molecule that can bind to a target cell antigen. In one embodiment, the third antigen binding portion is a conventional Fab molecule. In one embodiment, the third antigen binding portion can bind to the same target cell antigen as the second antigen binding portion. In a specific embodiment, the first antigen binding portion can bind to CD3, and the second and third antigen binding portions can bind to the target cell antigen. In a specific embodiment, the second and third antigen binding portions are identical (i.e., they comprise the same amino acid sequence).

[0310] In a specific embodiment, the first antigen binding portion is capable of binding to CD3, and the second and third antigen binding portions are capable of binding to IGF-1R, wherein the second and third antigen binding portions comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: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.

[0311] In a specific embodiment, the first antigen binding moiety is capable of binding to CD3, and the second and third antigen binding moieties are capable of binding to cMET, wherein the second and third antigen binding moieties 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.

[0312] In a specific embodiment, the first antigen binding portion is capable of binding to CD3, and the second and third antigen binding portions are capable of binding to TROP2, wherein the second and third antigen binding portions comprise at least one heavy chain complementary 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 SEQID NO:81, SEQ ID NO:82 and SEQ ID NO:83.

[0313] In one embodiment, the protease-activatable T cell activating bispecific molecule comprises

[0314] (i) a first antigen binding portion, which is a Fab molecule capable of binding to CD3 and which 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;

[0315] (ii) a second antigen binding moiety which is a Fab molecule capable of binding to a target cell antigen.

[0316] 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.

[0317] 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.

[0318] In a specific embodiment, 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 consisting of SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67.

[0319] In another specific embodiment, the second antigen binding portion is capable of binding to IGF-1R 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.

[0320] In a specific embodiment, 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 consisting of SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75.

[0321] In another specific embodiment, the second antigen binding portion is capable of binding to IGF-1R 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.

[0322] In a specific embodiment, 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 consisting of SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83.

[0323] 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.

[0324] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0325] (i) a first antigen binding portion, which is a Fab molecule capable of binding to CD3, comprising at least one heavy chain complementary 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;

[0326] (ii) a second antigen binding portion, which is a Fab molecule capable of binding to IGF-1R, comprising at least one heavy chain complementary 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.

[0327] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0328] (i) a first antigen binding portion, which 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,

[0329] (ii) a second antigen binding portion, which is a Fab molecule capable of binding to IGF-1R, 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.

[0330] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0331] (i) a first antigen binding portion, which is a Fab molecule capable of binding to CD3, comprising at least one heavy chain complementary 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, SEQ ID NO: 9;

[0332] (ii) a second antigen binding portion, which 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.

[0333] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0334] (i) a first antigen binding portion, which 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,

[0335] (ii) a second antigen binding portion, which is a Fab molecule capable of binding to cMET, 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.

[0336] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0337] (i) a first antigen binding portion, which is a Fab molecule capable of binding to CD3, comprising at least one heavy chain complementary 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, SEQ ID NO: 9;

[0338] (ii) a second antigen binding portion, which is a Fab molecule capable of binding to TROP2, comprising at least one heavy chain complementary 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.

[0339] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0340] (i) a first antigen binding portion, which 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,

[0341] (ii) a second antigen binding portion, 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.

[0342] In one embodiment, the second antigen binding moiety is a conventional Fab molecule.

[0343] In a specific embodiment, the first antigen binding moiety is a crossover Fab molecule, wherein the constant regions of the Fab light chain and the Fab heavy chain are exchanged, and the second antigen binding moiety is a conventional Fab molecule. In another specific embodiment, the first and second antigen binding moieties are fused to each other, optionally fused via a peptide linker.

[0344] In specific embodiments, the protease-activatable T cell activating bispecific molecule further comprises an Fc domain composed of a first subunit and a second subunit capable of stably associating.

[0345] In further specific embodiments, no more than one antigen binding moiety capable of binding to CD3 is present in the protease-activatable T cell activating bispecific molecule (ie, the protease-activatable T cell activating bispecific molecule provides monovalent binding to CD3).

[0346] In a specific embodiment, the first antigen binding portion is capable of binding to CD3 and comprises at least one heavy chain complementary 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, SEQ ID NO: 9; and the second and third antigen binding portions are capable of binding to IGF-1R, wherein the second and third antigen binding portions comprise at least one heavy chain complementary 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.

[0347] In a specific embodiment, the first antigen binding portion is capable of binding to CD3 and comprises at least one heavy chain complementary 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, SEQ ID NO: 9; and the second and third antigen binding portions are capable of binding to IGF-1R, wherein the second and third antigen binding portions comprise at least one heavy chain complementary 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.

[0348] In a specific embodiment, the first antigen binding moiety is capable of binding to CD3 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: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, and the second and third antigen binding moieties are capable of binding to IGF-1R, wherein the second and third antigen binding moieties comprise: 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.

[0349] In a specific embodiment, the first antigen binding portion is capable of binding to CD3 and comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 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, SEQ ID NO: 9, and the second and third antigen binding portions are capable of binding to cMET, wherein the second and third antigen binding portions comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 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.

[0350] In a specific embodiment, the first antigen binding portion is capable of binding to CD3 and comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 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, SEQ ID NO: 9; and the second and third antigen binding portions are capable of binding to cMET, wherein the second and third antigen binding portions comprise at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 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.

[0351] In a specific embodiment, the first antigen binding moiety is capable of binding to CD3 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: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, and the second and third antigen binding moieties are capable of binding to cMET, wherein the second and third antigen binding moieties comprise: 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.

[0352] In a specific embodiment, the first antigen binding portion is capable of binding to CD3 and comprises at least one heavy chain complementary 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, SEQ ID NO:9; and the second and third antigen binding portions are capable of binding to TROP2, wherein the second and third antigen binding portions comprise at least one heavy chain complementary 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.

[0353] In a specific embodiment, the first antigen binding portion is capable of binding to CD3 and comprises at least one heavy chain complementary 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, SEQ ID NO:9; and the second and third antigen binding portions are capable of binding to TROP2, wherein the second and third antigen binding portions comprise at least one heavy chain complementary 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.

[0354] 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 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, and the second and third antigen binding portions are capable of binding to TROP2, wherein the second and third antigen binding portions comprise: 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.

[0355] The second and third antigen binding moieties can be fused to the Fc domain directly or via a peptide linker. In a specific embodiment, 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 human IgG. 1 Hinge region. In one embodiment, the second and third antigen binding moieties and the Fc domain are part of an immunoglobulin molecule. In a specific embodiment, the immunoglobulin molecule is an IgG class immunoglobulin. In an even more specific embodiment, the immunoglobulin is an IgG 1 In another embodiment, the immunoglobulin is IgG. 4 Subclass immunoglobulin. In another 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, wherein the antigen binding portion is a Fab molecule, particularly a cross-Fab molecule, fused to the N-terminus of one of the immunoglobulin heavy chains, optionally via a peptide linker.

[0356] In a specific embodiment, the first and third 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, and 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 a specific embodiment of this type, the protease-activatable T cell activation bispecific molecule consists essentially of the first, second and third antigen binding moieties, an Fc domain consisting of a first and a second subunit, 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 subunit of the Fc domain at the C-terminus of the Fab heavy chain, and wherein the third antigen binding moiety is fused to the N-terminus of the 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 can be additionally fused to each other.

[0357] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0358] (i) a first antigen binding moiety, which is a Fab molecule capable of binding to CD3, comprising a heavy chain complementary determining region (CDR) 1 of SEQ ID NO: 1, a heavy chain CDR 2 of SEQ ID NO: 2, a heavy chain CDR 3 of SEQ ID NO: 3, a light chain CDR 1 of SEQ ID NO: 7, a light chain CDR of SEQ ID NO: 8 and a light chain CDR 3 of SEQ ID NO: 9, wherein the first antigen binding moiety is a crossover Fab molecule in which the variable regions or constant regions, in particular the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;

[0359] (ii) the second and third antigen binding portions, each of which is a Fab molecule capable of binding to IGF-1R, comprising heavy chain CDR 1 of SEQ ID NO:61, heavy chain CDR 2 of SEQ ID NO:62, heavy chain CDR 3 of SEQ ID NO:63, light chain CDR 1 of SEQ ID NO:65, light chain CDR 2 of SEQ ID NO:66 and light chain CDR3 of SEQ ID NO:67.

[0360] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0361] (i) a first antigen binding moiety, which 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, wherein the variable regions or constant regions, in particular the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;

[0362] (ii) a second and a third antigen binding portion, each of which is a Fab molecule capable of binding to IGF-1R, 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.

[0363] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0364] (i) a first antigen binding moiety, which is a Fab molecule capable of binding to CD3, comprising a heavy chain complementary determining region (CDR) 1 of SEQ ID NO: 1, a heavy chain CDR 2 of SEQ ID NO: 2, a heavy chain CDR 3 of SEQ ID NO: 3, a light chain CDR 1 of SEQ ID NO: 7, a light chain CDR of SEQ ID NO: 8 and a light chain CDR 3 of SEQ ID NO: 9, wherein the first antigen binding moiety is a crossover Fab molecule in which the variable regions or constant regions, in particular the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;

[0365] (ii) the second and third antigen binding portions, each of which is a Fab molecule capable of binding to cMET, comprising heavy chain CDR 1 of SEQ ID NO:69, heavy chain CDR 2 of SEQ ID NO:70, heavy chain CDR 3 of SEQ ID NO:71, light chain CDR 1 of SEQ ID NO:73, light chain CDR 2 of SEQ ID NO:74 and light chain CDR3 of SEQ ID NO:75.

[0366] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0367] (i) a first antigen binding moiety, which 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, wherein the variable regions or constant regions, in particular the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;

[0368] (ii) a second and a third antigen binding portion, each of which is a Fab molecule capable of binding to cMET, 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.

[0369] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0370] (i) a first antigen binding moiety, which is a Fab molecule capable of binding to CD3, comprising a heavy chain complementary determining region (CDR) 1 of SEQ ID NO: 1, a heavy chain CDR 2 of SEQ ID NO: 2, a heavy chain CDR 3 of SEQ ID NO: 3, a light chain CDR 1 of SEQ ID NO: 7, a light chain CDR of SEQ ID NO: 8 and a light chain CDR 3 of SEQ ID NO: 9, wherein the first antigen binding moiety is a crossover Fab molecule in which the variable regions or constant regions, in particular the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;

[0371] (ii) the second and third antigen binding portions, each of which is a Fab molecule capable of binding to TROP2, comprising heavy chain CDR 1 of SEQ ID NO:77, heavy chain CDR 2 of SEQ ID NO:78, heavy chain CDR 3 of SEQ ID NO:79, light chain CDR 1 of SEQ ID NO:81, light chain CDR 2 of SEQ ID NO:82 and light chain CDR3 of SEQ ID NO:83.

[0372] In one embodiment, the present invention provides a protease-activatable T cell activating bispecific molecule comprising

[0373] (i) a first antigen binding moiety, which 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, wherein the variable regions or constant regions, in particular the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;

[0374] (ii) a second and a third antigen binding portion, 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.

[0375] The protease-activatable T cell activating bispecific molecule according to any of the above embodiments may further comprise (iii) an Fc domain composed of a first and a second subunit 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, and 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 wherein 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.

[0376] In some protease-activatable T cell activation bispecific molecules of the present 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 linker peptide. Depending on the configuration of the first and second antigen binding moieties, the Fab light chain of the first antigen binding moiety can be fused to the N-terminus of the Fab light chain of the second antigen binding moiety at its C-terminus, or the Fab light chain of the second antigen binding moiety can be fused to the N-terminus of the Fab light chain of the first antigen binding moiety at its C-terminus. The fusion of the Fab light chains of the first and second antigen binding moieties further reduces the mismatch of the unmatched Fab heavy and light chains, and also reduces the number of plasmids required to express some protease-activatable T cell activation bispecific molecules of the present invention.

[0377] In certain embodiments, the protease-activatable T cell activating bispecific molecule comprises a polypeptide wherein the Fab light chain variable region of the first antigen binding moiety shares a carboxyl-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 by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond (VL ) with an Fc domain subunit. (1) -CH1 (1) -CH2-CH3(-CH4)); and a polypeptide in which the Fab heavy chain of the second antigen binding moiety shares a carboxyl-terminal peptide bond (VH (2) -CH1 (2) -CH2-CH3(-CH4)). In some embodiments, the protease-activatable T cell activating bispecific molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the first antigen binding moiety shares a carboxyl-terminal peptide bond (VH (1) -CL (1) ), and shares a carboxyl-terminal peptide bond (VL (2) -CL (2) ). In certain embodiments, the polypeptides are covalently linked, for example, via a disulfide bond.

[0378] In alternative embodiments, the protease-activatable T cell activating bispecific molecule comprises a polypeptide wherein the Fab heavy chain variable region of the first antigen binding moiety shares a carboxyl-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 by a light chain constant region), which in turn shares a carboxyl-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 moiety shares a carboxyl-terminal peptide bond (VH (2) -CH1 (2) -CH2-CH3(-CH4)). In some embodiments, the protease-activatable T cell activating bispecific molecule further comprises a polypeptide wherein the Fab light chain variable region of the first antigen binding moiety shares a carboxyl-terminal peptide bond (VL (1) -CH1 (1) ), and shares a carboxyl-terminal peptide bond (VL (2) -CL (2) ). In certain embodiments, the polypeptides are covalently linked, for example, via a disulfide bond.

[0379] In some embodiments, the protease-activatable T cell activating bispecific molecule comprises a polypeptide wherein the Fab light chain variable region of a first antigen binding moiety shares a carboxyl-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 by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the second antigen binding moiety, which in turn shares a carboxyl-terminal peptide bond with an Fc domain subunit (VL (1) -CH1 (1) -VH (2) -CH1 (2) -CH2-CH3(-CH4)). In other embodiments, the protease-activatable T cell activating bispecific molecule comprises a polypeptide wherein the Fab heavy chain variable region of the first antigen binding moiety shares a carboxyl-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 by a light chain constant region), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the second antigen binding moiety, which in turn shares a carboxyl-terminal peptide bond with the Fc domain subunit (VH(1) -CL (1) -VH (2) -CH1 (2) -CH2-CH3(-CH4)). In still other embodiments, the protease-activatable T cell activating bispecific molecule comprises a polypeptide wherein the Fab heavy chain of the second antigen binding moiety shares a carboxyl-terminal peptide bond with the Fab light chain variable region of the first antigen binding moiety, which in turn shares a carboxyl-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 by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) -VL (1) -CH1 (1) -CH2-CH3(-CH4)). In other embodiments, the protease-activatable T cell activating bispecific molecule comprises a polypeptide wherein the Fab heavy chain of the second antigen binding moiety shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of the first antigen binding moiety, which in turn shares a carboxyl-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 by a light chain constant region), which in turn shares a carboxyl-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) -VH (1) -CL (1) -CH2-CH3(-CH4)).

[0380] In some of these embodiments, the protease-activatable T cell activating bispecific molecule further comprises a crossover Fab light chain polypeptide of the first antigen binding moiety, wherein the Fab heavy chain variable region of the first antigen binding moiety shares a carboxyl-terminal peptide bond (VH (1) -CL (1) ), and shares a carboxyl-terminal peptide bond (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, wherein the Fab light chain variable region of the first antigen binding moiety shares a carboxyl-terminal peptide bond (VL (1) -CH1 (1)), and shares a carboxyl-terminal peptide bond (VL (2) -CL (2) In still other of these embodiments, the protease-activatable T cell activating bispecific molecule further comprises a polypeptide wherein the Fab light chain variable region of the first antigen binding moiety shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the first antigen binding moiety, which in turn shares a carboxyl-terminal peptide bond (VL (1) -CH1 (1) -VL (2) -CL (2) ); a polypeptide in which the Fab heavy chain variable region of the first antigen binding moiety shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the first antigen binding moiety, which in turn shares a carboxyl-terminal peptide bond with the Fab light chain polypeptide of the second antigen binding moiety (VH (1) -CL (1) -VL (2) -CL (2) ); a polypeptide in which the Fab light chain polypeptide of the second antigen binding moiety shares a carboxyl-terminal peptide bond with the Fab light chain variable region of the first antigen binding moiety, which in turn shares a carboxyl-terminal peptide bond (VL (2) -CL (2) -VL (1) -CH1 (1) ); or a polypeptide wherein the Fab light chain polypeptide of the second antigen binding moiety shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of the first antigen binding moiety, which in turn shares a carboxyl-terminal peptide bond (VL (2) -CL (2) -VH (1) -CL (1) ).

[0381] The protease-activatable T cell activating bispecific molecule according to these embodiments may further 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 carboxyl-terminal peptide bond (VH (3) -CH1 (3) -CH2-CH3(-CH4)), and shares a carboxyl-terminal peptide bond (VL (3) -CL (3)). In certain embodiments, the polypeptides are covalently linked, for example, via a disulfide bond.

[0382] 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) can be fused directly or through various linkers (particularly peptide linkers comprising one or more amino acids, typically about 2 to 20 amino acids, described herein or known in the art). Suitable non-immunogenic peptide linkers include, for example (G 4 S) n , (SG 4 ) n , (G 4 S) n or G 4 (SG 4 ) n A peptide linker wherein n is generally a number between 1 and 10, typically between 2 and 4.

[0383] Fc domain

[0384] The Fc domain of the protease-activatable T cell activation bispecific molecule is composed of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises a CH2 and CH3 IgG heavy chain constant domain. The two subunits of the Fc domain can stably associate with each other. In one embodiment, the protease-activatable T cell activation bispecific molecule of the present invention comprises no more than one Fc domain.

[0385] In one embodiment according to the present invention, the Fc domain of the protease-activatable T cell activating bispecific molecule is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG 1 In another embodiment, the Fc domain is IgG 4 In a more specific embodiment, the Fc domain is an IgG comprising an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. 4 Fc domain. This amino acid substitution reduces IgG 4 In vivo Fab arm exchange of antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In further specific embodiments, the Fc domain is human.

[0386] Fc domain modifications that promote heterodimerization

[0387] The protease-activatable T cell activation bispecific molecules according to the present invention comprise different antigen binding moieties, which are fused to one or the other of the two subunits of the Fc domain, so that the two subunits of the Fc domain are typically contained in two different polypeptide chains. The recombinant co-expression of these polypeptides and the subsequent dimerization result in several possible combinations of the two polypeptides. In order to improve the yield and purity of the protease-activatable T cell activation bispecific molecules in recombinant production, it is therefore advantageous to introduce modifications that promote the association of the desired polypeptides in the Fc domain of the protease-activatable T cell activation bispecific molecules.

[0388] Therefore, in a specific embodiment, the Fc domain of the protease-activatable T cell activating bispecific molecule according to the present invention comprises a modification that promotes the association of the first subunit and the second subunit of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is located in the CH3 domain of the Fc domain.

[0389] In a specific embodiment, the modification is a so-called "knob-to-hole" modification, which comprises a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

[0390] Knob-hole structure technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). In general, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide so that the protrusion can be positioned in the cavity to promote heterodimer formation and hinder 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 compensatory cavity having the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0391] Thus, in a specific embodiment, in the CH3 domain of the first subunit of the Fc domain of the protease-activatable T cell activating bispecific molecule, an amino acid residue is substituted with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion can be positioned in the cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is substituted with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit can be positioned in the cavity.

[0392] Protrusions and cavities can be produced by altering the nucleic acid encoding the polypeptide (eg, by site-specific mutagenesis or by peptide synthesis).

[0393] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced by 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 by a valine residue (Y407V). In one embodiment, in addition, in the second subunit of the Fc domain, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A).

[0394] In yet another embodiment, in addition in the first subunit of the Fc domain, the serine residue at position 354 is substituted by a cysteine ​​residue (S354C), and in addition in the second subunit of the Fc domain, the tyrosine residue at position 349 is substituted by a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0395] In a specific embodiment, the antigen binding moiety capable of binding to CD3 is fused (optionally via an antigen binding moiety capable of binding to a target cell antigen) to the first subunit of the Fc domain (comprising a "knob" modification). Without wishing to be bound by theory, the fusion of the 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 hindrance of the two knob-containing polypeptides).

[0396] In alternative embodiments, modifications that promote association of the first and second subunits of the Fc domain include modifications that mediate an electrostatic steering effect, such as described in PCT Publication WO 2009 / 089004. In general, this approach involves replacing one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues such that homodimer formation becomes electrostatically unfavorable, but heterodimerization is electrostatically favorable.

[0397] Fc domain modifications that reduce Fc receptor binding and / or effector function

[0398] The Fc domain gives the protease-activatable T cell activation bispecific molecule favorable pharmacokinetic properties, including a long serum half-life and a favorable tissue-blood distribution ratio that contribute to good accumulation in the target tissue. However, at the same time, it may cause the protease-activatable T cell activation bispecific molecule to undesirably target cells expressing Fc receptors, rather than cells that preferably carry antigens. In addition, the co-activation of the Fc receptor signaling pathway may lead to cytokine release, which, combined with the long half-life of the T cell activation characteristics and the antigen binding molecule, leads to excessive activation of cytokine receptors and serious side effects after systemic administration. The activation of immune cells (carrying Fc receptors) other than T cells may even reduce the efficacy of the protease-activatable T cell activation bispecific molecule, because T cells may, for example, be destroyed by NK cells.

[0399] Thus, in a specific embodiment, the Fc domain of the protease-activatable T cell activating bispecific molecule according to the present invention is conjugated to a native IgG 1 The Fc domain exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to the Fc domain. In one such embodiment, the Fc domain (or a protease-activatable T cell activating bispecific molecule comprising the Fc domain) is tagged with native IgG. 1 Fc domain (or containing native IgG 1 The protease-activatable T cell activating bispecific molecule containing an Fc domain exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% binding affinity to an Fc receptor compared to a protease-activatable T cell activating bispecific molecule containing an Fc domain; and / or the Fc domain has a binding affinity to a native IgG 1 Fc domain (or containing native IgG 1In one embodiment, the Fc domain domain (or the protease-activatable T cell activating bispecific molecule comprising the Fc domain) does not significantly bind to Fc receptors and / or induce effector functions. In a specific 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 activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically 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 specific embodiment, the effector function is ADCC. In one embodiment, the Fc domain domain is similar to natural IgG. 1 The Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the Fc domain. When the Fc domain (or a protease-activatable T cell activating bispecific molecule comprising the Fc domain) exhibits native IgG 1 Fc domain (or containing native IgG 1 Substantially similar binding to FcRn is achieved when the protease-activatable T cell activating bispecific molecule containing an Fc domain has a binding affinity to FcRn of greater than about 70%, particularly greater than about 80%, more particularly greater than about 90%.

[0400] In certain embodiments, the Fc domain is engineered to have a reduced binding affinity and / or reduced effector function to Fc receptors compared to an unengineered Fc domain. In a specific embodiment, the Fc domain of a protease-activatable T cell activation bispecific molecule comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to Fc receptors. 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 mutation reduces the binding affinity of the Fc domain to Fc receptors. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to Fc receptors by at least 2 times, at least 5 times or at least 10 times. In the embodiment where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to Fc receptors, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to Fc receptors by at least 10 times, at least 20 times or even at least 50 times. In one embodiment, the protease-activatable T cell activating bispecific molecule comprising an engineered Fc domain exhibits a binding affinity to an Fc receptor of less than 20%, specifically less than 10%, and more specifically less than 5%, compared to a protease-activatable T cell activating bispecific molecule comprising an unengineered 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 activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some embodiments, the binding affinity to complementary components, specifically the binding affinity to C1q, is also reduced. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. When the Fc domain (or a protease-activatable T cell activating bispecific molecule comprising the Fc domain) exhibits a binding affinity of greater than about 70% of the unengineered form of the Fc domain (or a protease-activatable T cell activating bispecific molecule comprising the unengineered form of the Fc domain) to FcRn, substantially similar binding to FcRn, i.e., retention of the binding affinity of the Fc domain to the receptor, is achieved. The Fc domain or the protease-activatable T cell activating bispecific molecule of the present invention comprising the Fc domain may exhibit such affinity greater than about 80% or even greater than about 90%. In certain embodiments, the Fc domain of the protease-activatable T cell activating bispecific molecule is engineered to have reduced effector function compared to an unengineered Fc domain.The reduced effector function may 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 of antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced cross-linking 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 following group: 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 unengineered Fc domain (or a protease-activatable T cell activating bispecific molecule comprising an unengineered Fc domain).

[0401] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to the 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 IgG 1 Fc domain, especially human IgG 1 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 further amino acid substitutions at positions 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 specific embodiment, the Fc domain comprises an amino acid substitution at position P329, L234 and L235. In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A and P329G ("P329G LALA"). In one such embodiment, the Fc domain is IgG 1 Fc domain, especially human IgG1 Fc domain. The amino acid substitution "P329G LALA" combination almost completely eliminated human IgG 1 Fcγ receptor (and complement) binding of Fc domains, as described in PCT Publication No. WO 2012 / 130831 (incorporated herein by reference in its entirety). WO 2012 / 130831 also describes methods of making such mutant Fc domains and methods for determining their properties such as Fc receptor binding or effector function.

[0402] IgG 4 Antibodies and IgG 1 Therefore, in some embodiments, the Fc domain of the protease-activatable T cell activating bispecific molecule of the present invention is IgG 4 Fc domain, especially human IgG 4 In one embodiment, the Fc domain of IgG 4 The Fc domain comprises an amino acid substitution at position S228, specifically an amino acid substitution S228P. In order to further reduce its binding affinity to Fc receptors and / or its effector function, in one embodiment, IgG 4 The Fc domain comprises an amino acid substitution at position L235, specifically an amino acid substitution L235E. In another embodiment, IgG 4 The Fc domain comprises an amino acid substitution at position P329, specifically an amino acid substitution P329G. In a specific embodiment, IgG 4 The Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G. Such IgG 4 Fc domain mutations and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012 / 130831 (incorporated herein by reference in its entirety).

[0403] In a specific embodiment, with natural IgG 1 An Fc domain that exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to an Fc domain is a human IgG comprising the amino acid substitutions L234A, L235A and optionally P329G 1 Fc domain or human IgG comprising amino acid substitutions S228P, L235E and optionally P329G 4 Fc domain.

[0404] In certain embodiments, the N-glycosylation of the Fc domain has been eliminated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, in particular an amino acid substitution replacing asparagine with alanine (N297A) or aspartic acid (N297D).

[0405] 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 in which one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted to alanine (U.S. Pat. No. 7,332,581).

[0406] 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-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The correct nucleotide changes can be verified, for example, by sequencing.

[0407] Binding to Fc receptors can be readily determined using standard instruments such as BIAcore instruments (GE Healthcare), for example by ELISA or by surface plasmon resonance (SPR), and Fc receptors can be obtained, for example, by recombinant expression. Suitable such binding assays are described herein. Alternatively, cell lines known to express specific Fc receptors, such as human NK cells expressing FcγIIIa receptors, can be used to evaluate the binding affinity of the Fc domain or cell-activated bispecific antigen binding molecules comprising the Fc domain to the Fc receptor.

[0408] The effector function of the T cell activation bispecific molecule that can be activated by the Fc domain, or the protease containing the 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 molecules of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays can be used (see, for example, ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA); and CytoTox 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 a molecule of interest can 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).

[0409] In some embodiments, the binding of the Fc domain to a complement component (particularly C1q) is reduced. Therefore, in some embodiments in which the Fc domain is engineered to have reduced effector functions, the reduced effector functions include reduced CDC. C1q binding assays can be performed to determine whether the protease-activatable T cell activation bispecific molecule is able to bind to C1q and therefore has CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. In order to assess complement activation, CDC assays can 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)).

[0410] Exemplary protease-activatable T cell activating bispecific molecules capable of binding to CD3 and IGF-1R, cMET or TROP2

[0411] The first antigen binding portion capable of binding to CD3 as described above, the second (and third) antigen binding portion capable of binding to IGF-1R, cMET or TROP2 as described above, the Fc domain as described above, the masking portion of the present invention and the protease-cleavable linker can be fused to each other in a variety of configurations.

[0412] Exemplary configurations are shown in Figure 1. Exemplary sequences are shown below.

[0413] In one embodiment, the protease-activatable T cell activating bispecific molecule comprising a first antigen binding moiety capable of binding to CD3 and second and third antigen binding moieties capable of binding to IGF-1R 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-activatable T cell activating bispecific molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 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-activatable 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.

[0414] In one embodiment, the protease-activatable T cell activating bispecific molecule comprising a first antigen binding moiety capable of binding to CD3 and a second antigen binding moiety 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-activatable T cell activating bispecific molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 91, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 93, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 94. In one embodiment, the protease-activatable 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.

[0415] In one embodiment, the protease-activatable T cell activating bispecific molecule comprising a first antigen binding portion capable of binding to CD3 and second and third antigen binding portions capable of binding to TROP2 comprises a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:96, a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:98, a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:99, and a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:100. In one embodiment, the protease-activatable T cell activating bispecific molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 96, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 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-activatable 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.

[0416] Polynucleotide

[0417] The present invention further provides an isolated polynucleotide encoding a protease-activatable T cell activating bispecific molecule as described herein or a fragment thereof. In some embodiments, the fragment is an antigen binding fragment.

[0418] The polynucleotides encoding the protease-activatable T cell activation bispecific molecules of the present invention can be expressed as a single polynucleotide encoding a complete protease-activatable T cell activation bispecific molecule, or as a plurality of (e.g., two or more) polynucleotides co-expressed. The polypeptides encoded by the co-expressed polynucleotides can be associated by, for example, disulfide bonds or other means to form functional protease-activatable T cell activation bispecific molecules. For example, the light chain portion of the antigen binding portion can be encoded by a polynucleotide separate from the heavy chain portion, Fc domain subunits and optionally another antigen binding portion (a part) of the protease-activatable T cell activation bispecific molecule of the antigen binding portion. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an antigen binding portion. In another example, the portion of the protease-activatable T cell activation bispecific molecule comprising one of the two Fc domain subunits and optionally one or more antigen binding portions (a part) can be encoded by a polynucleotide separate from the portion of the protease-activatable T cell activation bispecific molecule comprising another of the two Fc domain subunits and optionally an antigen binding portion (a part). When co-expressed, the Fc domain subunits will associate to form an Fc domain.

[0419] In some embodiments, the isolated polynucleotides encode a complete protease-activatable T cell activating bispecific molecule according to the present invention as described herein. In other embodiments, the isolated polynucleotides encode a polypeptide contained in a protease-activatable T cell activating bispecific molecule according to the present invention as described herein.

[0420] In another embodiment, the present invention relates to an isolated polynucleotide encoding a protease-activatable T cell activation bispecific molecule of the present invention or a fragment thereof, wherein the polynucleotide comprises a sequence encoding a variable region sequence. In another embodiment, the present invention relates to an isolated polynucleotide encoding a protease-activatable T cell activation bispecific molecule or a fragment thereof, wherein the polynucleotide comprises a sequence encoding a polypeptide sequence as shown 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 or SEQ ID NO: 100 or a fragment thereof.

[0421] The polynucleotide encoding the idiotype-specific polypeptide of the present invention can be expressed as a single polynucleotide encoding a complete idiotype-specific polypeptide, or as a plurality of (e.g., two or more) polynucleotides of co-expression. The polypeptide encoded by the co-expressed polynucleotide can be associated to form a functional idiotype-specific polypeptide, such as a masking portion, by, for example, a disulfide bond or other means. For example, in one embodiment, the idiotype-specific polypeptide is an anti-idiotype scFv (single-chain variable fragment), wherein the light chain variable portion of the anti-idiotype scFv can be encoded by a polynucleotide separated from the part of the anti-idiotype scFv comprising the heavy chain variable portion of the anti-idiotype scFv. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an anti-idiotype scFv. In certain embodiments, the isolated polynucleotide encoding is as described herein according to the idiotype-specific polypeptide of the present invention.

[0422] 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.

[0423] Recombination methods

[0424] The protease-activatable T cell activation bispecific molecules of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding, for example, protease-activatable T cell activation bispecific molecules (fragments) as described above are separated and inserted into one or more vectors for further cloning and / or expression in host cells. Such polynucleotides can be easily separated and sequenced using conventional procedures. In one embodiment, a vector comprising one or more polynucleotides of the present invention is provided, preferably an expression vector. Methods well known to those skilled in the art can be used to construct an expression vector containing a coding sequence of a protease-activatable T cell activation bispecific molecule (fragment) and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo recombination / genetic recombination. See, for example, the technology described in the following documents: 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). The expression vector can be a part of a plasmid, a virus, or a nucleic acid fragment. The expression vector includes a polynucleotide encoding a protease-activatable T cell activation bispecific molecule (fragment) (i.e., a coding region) to be operably associated with a promoter and / or other transcription or translation control elements and cloned into an expression cassette therein. As used herein, a "coding region" is a part of a nucleic acid consisting of codons translated into amino acids. Although "stop codons" (TAG, TGA or TAA) are not translated into amino acids, if present, it can be considered as a part of the coding region, but any flanking sequence (e.g., promoter, ribosome binding site, transcription terminator, intron, 5' and 3' untranslated region, etc.) is not a part of the coding region. Two or more coding regions can be present in a single polynucleotide construct, for example, on a single vector, or in a separate polynucleotide construct, for example, on a separate (different) vector. In addition, any vector can contain a single coding region, or can contain two or more coding regions, for example, the vector of the present invention can encode one or more polypeptides, and the one or more polypeptides are separated into final proteins via proteolytic cleavage after translation or in co-translation.In addition, the vectors, polynucleotides or nucleic acids of the present invention may encode heterologous coding regions that are fused or unfused to polynucleotides encoding the protease-activatable T cell activation bispecific molecules (fragments) of the present invention or variants or derivatives thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association refers to when the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in such a manner that the expression of the gene product is placed under the influence or control of the regulatory sequences. If the induction of promoter function leads to the transcription of mRNA encoding the desired gene product and if the nature of the connection between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or the ability of the DNA template to transcribe, then the two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated". Therefore, if the promoter is able to affect the transcription of a nucleic acid encoding a polypeptide, the promoter region will be operably associated with the nucleic acid. The promoter may be a cell-specific promoter that directs a large amount of transcription of DNA only in a predetermined cell. In addition to the promoter, other transcription control elements (e.g., enhancers, operators, repressors, and transcription termination signals) can also be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., immediate early promoter associated with intron-A), simian virus 40 (e.g., early promoter), and retroviruses (such as, Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit. -globulin) transcriptional control regions, and other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., promoters that can induce tetracycline). Similarly, a variety of translational control elements are known to those of ordinary skill 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 IRES, also referred to 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 virus (AAV) inverted terminal repeats (ITRs).

[0425] The polynucleotides and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides of the present invention. For example, if it is desired to secrete a protease-activatable T cell activation bispecific molecule, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the protease-activatable T cell activation bispecific molecule of the present invention or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cut from the mature protein after the growing protein chain begins to be exported to the rough endoplasmic reticulum. Those of ordinary skill in the art understand that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide that is cut from the translated polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, a natural signal peptide is used, such as an immunoglobulin heavy chain or light chain signal peptide, or a functional derivative of the sequence that retains the ability to direct the secretion of a polypeptide operably associated therewith. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence can be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0426] DNA encoding a short protein sequence that can be used to facilitate subsequent purification (e.g., a histidine tag) or to help tag the protease-activatable T cell activating bispecific molecule can be included within or at the end of the protease-activatable T cell activating bispecific molecule (fragment) encoding polynucleotide.

[0427] In further embodiments, a host cell comprising one or more polynucleotides of the present invention is provided. In certain embodiments, a host cell comprising one or more vectors of the present invention is provided. Polynucleotides and vectors may be incorporated into any of the features described herein for polynucleotides and vectors, respectively, either alone or in combination. In one such embodiment, the host cell comprises such a vector (e.g., having been transformed or transfected with the vector) comprising a polynucleotide encoding a protease-activatable T cell activation bispecific molecule (a portion thereof) of the present invention. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce a protease-activatable T cell activation bispecific molecule of the present invention or a fragment thereof. Host cells suitable for replication and support for expression of protease-activatable T cell activation bispecific molecules are well known in the art. Such cells can be appropriately transfected or transduced with specific expression vectors, and a large number of cells containing the vector can be grown for inoculation of large-scale fermenters to obtain sufficient amounts of protease-activatable T cell activation bispecific molecules for clinical applications. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli (E. coli); or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, polypeptides can be produced in bacteria, particularly when glycosylation is not required. The polypeptide can be separated from the bacterial cell paste in the soluble fraction after expression, and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungi and yeast strains whose glycosylation pathways have been "humanized" so that antibodies with partial or complete human glycosylation patterns are produced. See Gerngross, Nat Biotech 22, 1409-1414 (2004); and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for expressing (glycosylated) polypeptides also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains that can be used with insect cells have been identified, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TMTechniques). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells, as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals NY Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, which include dhfr - 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 certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name a few, and also include cells contained in transgenic animals, transgenic plants or cultured plants 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 lymphoid cell (e.g., Y0, NS0, Sp20 cell).

[0428] Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing polypeptides comprising antigen binding domains such as heavy or light chains of antibodies can be engineered to also express the other antibody chain so that the expressed product is an antibody having heavy and light chains.

[0429] In one embodiment, a method for producing a protease-activatable T cell activating bispecific molecule according to the present invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding a protease-activatable T cell activating bispecific molecule as provided herein under conditions suitable for expressing the protease-activatable T cell activating bispecific molecule, and recovering the protease-activatable T cell activating bispecific molecule from the host cell (or host cell culture medium).

[0430] The components of the protease-activatable T cell activation bispecific molecule are genetically fused to each other. The protease-activatable T cell activation bispecific molecule can be designed so that its components are directly fused to each other or indirectly fused through a linker sequence. The composition and length of the linker can be determined according to methods well known in the art and can test efficacy. Examples of linker sequences between the different components of the protease-activatable T cell activation bispecific molecule can be found in the sequences provided herein. If necessary, additional sequences can also be included to incorporate cleavage sites to separate the various components of the fusion, such as endopeptidase recognition sequences.

[0431] In certain embodiments, one or more antigen binding portions of the protease-activatable T cell activation bispecific molecule at least include an antibody variable region capable of binding an antigenic determinant. The variable region can form a part of a natural or non-naturally occurring antibody and its fragment and be derived from a natural or non-naturally occurring antibody and its fragment. The method for producing polyclonal antibodies and monoclonal antibodies is well known in the art (see, for example, Harlow and Lane, "Antibodies, alaboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced (e.g., as described in U.S. Patent No. 4,186,567) or can be obtained, for example, by screening a combinatorial library comprising a variable heavy chain and a variable light chain (see, for example, U.S. Patent No. 5,969,108 granted to McCafferty).

[0432] Antibodies, antibody fragments, antigen binding domains or variable regions of any animal species can be used in the protease-activatable T cell activation bispecific molecules of the present invention. Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions that can be used in the present invention can be of mouse, primate or human origin. If the protease-activatable T cell activation bispecific molecule is intended for human use, a chimeric form of the antibody can be used, wherein the constant region of the antibody is from a human. "Humanization" or fully human forms of antibodies can also be prepared according to methods well known in the art (see, for example, U.S. Patent No. 5,565,332 to Winter). Humanization can be achieved by a variety of methods, including but not limited to (a) grafting non-human (e.g., donor antibody) CDRs onto human (e.g., recipient antibody) frameworks and constant regions with or without retaining key framework residues (e.g., those important for retaining good antigen binding affinity or antibody function), (b) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues important for antibody-antigen interactions) onto human frameworks and constant regions, or (c) grafting entire non-human variable domains but "hiding" them with human-like portions by replacing surface residues.Humanized antibodies and methods for making them are reviewed in, e.g., Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and further described in, e.g., Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Pat. 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 the "guided selection" method for FR shuffling). Various techniques known in the art can be used to produce human antibodies and human variable regions. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regions can form a part of human monoclonal antibodies prepared by hybridoma methods and are derived from these human monoclonal antibodies (see, e.g., Monoclonal Antibody Production Techniques and Applications, pp. 51 to 63 (Marcel Dekker, Inc., New York, 1987)).Human antibodies and human variable regions can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic stimulation (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 phage display libraries (see, e.g., Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments.

[0433] In certain embodiments, the antigen binding moiety that can be used in the present invention is engineered to have enhanced binding affinity according to methods such as those described in U.S. Patent Application Publication No. 2004 / 0132066 (the entire contents of which are hereby incorporated by reference). The ability of the protease-activatable T cell activation bispecific molecules of the present invention to bind to specific antigenic determinants can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance technology (analyzed on a BIACORE T100 system)) (Liljeblad, et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays can be used to identify antibodies, antibody fragments, antigen binding domains or variable domains that compete with reference antibodies for binding to specific antigens, such as antibodies that compete with V9 antibodies for binding to CD3. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitopes) that is bound by the reference antibody. Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ). In an exemplary competition assay, an immobilized antigen (e.g., CD3) is incubated in a solution comprising a first labeled antibody that binds to the antigen (e.g., the V9 antibody described in US 6,054,297) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution comprising the first labeled antibody (but not the second unlabeled antibody). After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed and the amount of label associated with the immobilized antibody is measured. If the amount of label associated with the immobilized antigen is substantially reduced in the test sample relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0434] The protease-activatable T cell activation 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 for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens to which the protease-activatable T cell activation bispecific molecules bind can be used. For example, for affinity chromatography purification of the protease-activatable T cell activation bispecific molecules of the present invention, a matrix with protein A or protein G can be used. Basically as described in the examples, sequential protein A or G affinity chromatography and size exclusion chromatography can be used to separate the protease-activatable T cell activation bispecific molecules. The purity of the protease-activatable T cell activation bispecific molecules can be determined by any of a variety of well-known analytical methods (including gel electrophoresis, high performance liquid chromatography, etc.). For example, the heavy chain fusion protein expressed as described in the examples is shown to be complete and correctly assembled, as demonstrated by reduced SDS-PAGE (see, e.g. Figures 8 to 12 ). Three bands were resolved at approximately Mr 25,000, Mr 50,000 and Mr 75,000, corresponding to the predicted molecular weights of the protease-activatable T cell activating bispecific molecule light chain, heavy chain and heavy chain / light chain fusion protein.

[0435] Determination

[0436] The physical / chemical properties and / or biological activities of the protease-activatable T cell activating bispecific molecules provided herein can be identified, screened or characterized by various assays known in the art.

[0437] Affinity determination

[0438] The affinity of the protease-activatable T cell activation bispecific molecule to Fc receptors or target antigens can be determined by surface plasmon resonance (SPR) according to the method described in the examples using standard instruments such as BIAcore instruments (GE Healthcare) and receptors or target proteins such as those obtainable by recombinant expression. Alternatively, cell lines expressing specific receptors or target antigens can be used, for example, by flow cytometry (FACS) to evaluate the binding of the protease-activatable T cell activation bispecific molecule to different receptors or target antigens. Specific illustrative and exemplary embodiments for measuring binding affinity are described below and in the following examples.

[0439] According to one embodiment, the T100 machine (GE Healthcare) measures K by surface plasmon resonanceD .

[0440] In order to analyze the interaction between the Fc part and the Fc receptor, the recombinant Fc receptor of His tag is captured by the anti-Penta His antibody (Qiagen) fixed on the CM5 chip, and the bispecific construct is used as an analyte. In brief, according to the instructions of the supplier, the carboxymethylated dextran biosensor chip (CM5, GE Healthcare) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Penta-His antibody is diluted to 40 μg / ml with 10mM sodium acetate (pH 5.0), and then injected with a flow rate of 5 μl / min to obtain about 6500 response units (RU) of coupled protein. After the injection of the ligand, 1M ethanolamine is injected to block the unreacted group. Subsequently, the Fc receptor is captured for 60s with 4 or 10nM. For kinetic measurements, four-fold serial dilutions of the bispecific constructs (ranging between 500 nM and 4000 nM) in HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4) were injected at 25°C at a flow rate of 30 μl / min for 120 s.

[0441] To determine the affinity with 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 anti-Penta-His antibodies. The final amount of coupled protein was approximately 12000RU. The bispecific construct was captured at 300nM for 90s. The target antigen was passed through a flow cell at a flow rate of 30μl / min at a concentration range of 250 to 1000nM for 180s. Dissociation was monitored for 180s.

[0442] Bulk refractive index differences were corrected by subtracting the response obtained on a reference flow cell. The steady-state response was used to derive the dissociation constant, K, by nonlinear curve fitting of the Langmuir binding isotherm. D Using a simple one-to-one Langmuir binding model ( T100 Evaluation Software Version 1.1.1) was used to calculate the association rate (k) by simultaneously fitting the association sensorgram and the dissociation sensorgram. on ) and dissociation rate (k off ). The equilibrium dissociation constant (K D ) is calculated as the ratio k off / k onSee, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).

[0443] Activity assay

[0444] The biological activity of the protease-activatable T cell activation bispecific molecules of the present invention can be measured by various assays as described in the examples. Biological activity can, for example, include induction of T cell proliferation, induction of signal transduction in T cells, induction of activation marker expression in T cells, induction of cytokine secretion by T cells, induction of target cell (such as tumor cell) lysis, and induction of tumor regression and / or an increase in survival rate.

[0445] Compositions, formulations and routes of administration

[0446] In other aspects, the invention provides a pharmaceutical composition comprising any of the protease-activatable T cell activation bispecific molecules provided herein, for example, for use in any of the following treatment methods. In one embodiment, the pharmaceutical composition comprises any of the protease-activatable T cell activation bispecific molecules provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the protease-activatable T cell activation bispecific molecules provided herein and at least one additional therapeutic agent, for example, as described below.

[0447] Further provided is a method for producing a protease-activatable T cell activating bispecific molecule of the present invention in a form suitable for in vivo administration, the method comprising (a) obtaining a protease-activatable T cell activating bispecific molecule according to the present invention, and (b) formulating the protease-activatable T cell activating bispecific molecule with at least one pharmaceutically acceptable carrier, thereby formulating a preparation of the protease-activatable T cell activating bispecific molecule for in vivo administration.

[0448] The 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 pharmaceutical carrier. The phrase "pharmaceutical / pharmaceutically acceptable or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to the recipient at the doses and concentrations employed (i.e., do not produce adverse, allergic or other undesirable reactions when administered to animals (e.g., such as humans) as appropriate). The preparation of pharmaceutical compositions containing at least one protease-activatable T cell activating bispecific molecule and optionally additional active ingredients will be known to those skilled in the art in view of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Edition Mack Printing Company, 1990 (incorporated herein by reference). In addition, for animal (e.g., human) administration, it should be understood that the preparation should meet the sterility, pyrogenicity, overall safety and purity standards required by the FDA Office of Biological Standards or the corresponding agencies of other countries. The preferred composition is a lyophilized formulation or an aqueous solution. As used herein, "pharmaceutically acceptable carriers" include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes and the like materials and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, it is contemplated for use in a therapeutic or pharmaceutical composition.

[0449] The composition may contain different types of carriers depending on whether the composition is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for administration routes such as injection. The protease-activatable T cell activating bispecific molecules of the invention (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrasplenicly, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, topically, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, local perfusion directly into target cells, via a catheter, via lavage, in a cream, in a lipid composition (e.g., liposomes), or by other methods or any combination of the foregoing as known to one of ordinary 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 injection) is most commonly used to administer polypeptide molecules, such as the protease-activatable T cell activating bispecific molecules of the invention.

[0450] Parenteral compositions include those designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection). For injection, the protease-activatable T cell activation bispecific molecules of the present invention can be formulated in aqueous solution, preferably in a physiologically compatible buffer (such as Hanks solution, Ringer's solution or saline buffer). The solution may contain a formulation agent (formulatory agent), such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the protease-activatable T cell activation bispecific molecules can be in powder form for construction with a suitable vehicle (e.g., sterile pyrogen-free water) before use. Sterile injectable solutions are prepared by incorporating the protease-activatable T cell activation bispecific molecules of the present invention in the desired amount with various other ingredients (as required) enumerated below into a suitable solvent. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane. In general, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and / or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred preparation method is vacuum drying or freeze drying techniques, which produce a powder of the active ingredient plus any additional required ingredients from a previously sterile filtered liquid medium. If necessary, the liquid medium should be appropriately buffered, and sufficient saline or glucose should first be used to make the liquid diluent isotonic before injection. The composition must be stable under the conditions of manufacture and storage, and is preserved to resist the contaminating effects of microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a minimum, for example, at a safety level below 0.5 ng / mg protein. Suitable pharmaceutical excipients include, but are not limited to, buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium 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; m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as Such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinyl pyrrolidone; 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., zinc protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, etc.Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound to allow the preparation of a highly concentrated solution. In addition, suspensions of the active compound may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil; or synthetic fatty acid esters such as ethyl oleate or triglycerides; or liposomes.

[0451] The active ingredient can be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization; embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or embedded in coarse emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Printing Company, 1990). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing polypeptides in the form of shaped articles such as films or microcapsules. In particular embodiments, prolonged absorption of injectable compositions can be achieved by using an agent that delays absorption (e.g., such as aluminum monostearate, gelatin, or a combination thereof) in the composition.

[0452] In addition to the compositions described previously, the protease-activatable T cell activation bispecific molecules can also be formulated into long-acting preparations. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular implantation) or by intramuscular injection. Thus, for example, the protease-activatable T cell activation bispecific molecules can be formulated with suitable polymeric or hydrophobic materials (e.g., formulated into an emulsion in an acceptable oil) or ion exchange resins, or formulated into slightly soluble derivatives (e.g., formulated into slightly soluble salts).

[0453] Pharmaceutical compositions comprising the protease-activatable T cell activating bispecific molecules of the invention can be manufactured by conventional mixing, dissolving, emulsifying, encapsulating, embedding or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that assist in processing the protein into a pharmaceutically usable preparation. Appropriate formulations depend on the selected route of administration.

[0454] Protease-activatable T cell activating bispecific molecules can be formulated into compositions in the form of free acids or bases, neutral or salts. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of free acids or bases. These pharmaceutically acceptable salts include acid addition salts, such as acid addition salts formed with free amino groups of protein compositions, or acid addition salts 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 hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases, such as isopropylamine, trimethylamine, histidine or procaine. Compared with the corresponding free base form, pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents.

[0455] Methods of treatment and compositions

[0456] 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, to treat cancer.

[0457] 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 be considered in this context include the specific disorder being treated, the specific 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 schedule of administration, and other factors known to medical practitioners.

[0458] In one aspect, a protease-activatable T cell activation bispecific molecule of the present invention is provided for use as a medicament. In another aspect, a protease-activatable T cell activation bispecific molecule of the present invention for treating a disease is provided. In certain embodiments, a protease-activatable T cell activation bispecific molecule of the present invention for use in a method of treatment is provided. In one embodiment, the present invention provides a protease-activatable T cell activation bispecific molecule as described herein for use in treating a disease of an individual in need. In certain embodiments, the present invention provides a protease-activatable T cell activation bispecific molecule for use in a method of treating an individual suffering from a disease, the method comprising administering to the individual a therapeutically effective amount of a protease-activatable T cell activation bispecific molecule. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, if the disease to be treated is cancer, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anticancer agent. In another embodiment, the present invention provides a protease-activatable T cell activation bispecific molecule as described herein for use in inducing target cells, particularly tumor cell lysis. In certain embodiments, the present invention provides a protease-activatable T cell activating bispecific molecule for use in a method for inducing lysis of a target cell, particularly a tumor cell, in an individual, the method comprising administering to the individual an effective amount of a protease-activatable T cell activating bispecific molecule to induce lysis of the target cell. The "individual" according to any of the above embodiments is a mammal, preferably a human.

[0459] In another aspect, the present invention provides the use of the protease-activatable T cell activation bispecific molecule of the present invention in the manufacture or preparation of a drug. In one embodiment, the drug is used to treat a disease in an individual in need thereof. In another embodiment, the drug is used in a method for treating a disease, the method comprising administering a therapeutically effective amount of the drug to an individual suffering from the disease. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In one embodiment, if the disease to be treated is cancer, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent, such as an anticancer agent, to the individual. In another embodiment, the drug is used to induce the lysis of target cells, particularly tumor cells. In still another embodiment, the drug is used in a method for inducing the lysis of target cells, particularly tumor cells, in an individual, the method comprising administering an effective amount of the drug to the individual to induce the lysis of the target cells. The "individual" according to any of the above embodiments can be a mammal, preferably a human.

[0460] In another aspect, the present invention provides a method for treating a disease. In one embodiment, the method comprises administering a therapeutically effective amount of a protease-activatable T cell activation bispecific molecule of the present invention to an individual suffering from such a disease. In one embodiment, a composition is administered to the individual, which comprises a protease-activatable T cell activation bispecific molecule of the present invention in a pharmaceutical form. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, if the disease to be treated is cancer, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent, such as an anticancer agent, to the individual. According to any of the above embodiments, the "individual" can be a mammal, preferably a human.

[0461] In a further aspect, the invention provides a method for inducing the lysis of a target cell, particularly a tumor cell. 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 a T cell, particularly a cytotoxic T cell. In a further aspect, a method for inducing the lysis of a target cell, particularly a tumor cell, in an individual is provided. In one such embodiment, the method comprises administering to the individual an effective amount of a protease-activatable T cell activating bispecific molecule to induce the lysis of the target cell. In one embodiment, an "individual" is a human.

[0462] In certain embodiments, the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer and kidney cancer. Other cell proliferative disorders that can be treated with the protease-activatable T cell-activating bispecific molecules of the present invention include, but are not limited to, tumors located in the abdomen, bones, breasts, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary glands, testicles, ovaries, thymus, thyroid glands), eyes, head and neck, nervous system (central nervous system and peripheral nervous system), lymphatic system, pelvis, skin, soft tissue, spleen, chest and urogenital system. Precancerous conditions or lesions and cancer metastasis are also included. In certain embodiments, the cancer is selected from the group consisting of the following items: renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. Those skilled in the art will readily recognize that in many cases, protease-activatable T cell activation bispecific molecules may not provide a cure, but may only provide partial benefits. In certain embodiments, physiological changes with some benefits are also considered to be therapeutically beneficial. Therefore, in certain embodiments, the amount of protease-activatable T cell activation bispecific molecules providing physiological changes is considered to be an "effective amount" or a "therapeutically effective amount". The subject, patient or individual in need of treatment is typically a mammal, more specifically a human.

[0463] 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.

[0464] For the prevention or treatment of a disease, an appropriate dose of the protease-activatable T cell activating bispecific molecule of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be 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 preventive or therapeutic purposes, prior or concurrent therapeutic interventions, the patient's clinical history and response to the protease-activatable T cell activating bispecific molecule, and the judgment of the attending physician. In any case, the practitioner responsible for administration will determine the concentration of one or more active ingredients in the composition and one or more appropriate doses for individual subjects. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations, bolus administrations, and pulse infusions at various time points.

[0465] The protease-activatable T cell activation bispecific molecule is appropriately applied to the patient once or in a series of treatments.Depending on the type and severity of the disease, the protease-activatable T cell activation bispecific molecule of about 1 μg / kg to 15mg / kg (e.g., 0.1mg / kg to 10mg / kg) can be, for example, an initial candidate dose applied to the patient by one or more separate administrations or by continuous infusion.Depending on the above factors, a typical daily dose can range from about 1 μg / kg to 100mg / kg or more.For repeated administration for several days or longer, depending on the disease, treatment will generally continue until the desired disease symptoms are suppressed.An exemplary dosage of a T cell activation bispecific antigen binding molecule can be in the range of from about 0.005mg / kg to about 10mg / kg. In other non-limiting examples, dosages can also include per administration from 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 to about 1000 mg / kg body weight or more, and any range derivable therein. In non-limiting examples of ranges derivable from the numbers listed herein, ranges from 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, etc. can be administered based on the above numbers. Therefore, 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 (e.g., weekly or every three weeks (e.g., so that the patient receives about twice to about twenty times, or, for example, about six doses of a protease-activatable T cell activating bispecific molecule)). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may be available. The progress of this therapy may be easily monitored by conventional techniques and assays.

[0466] The protease-activatable T cell activating bispecific molecules of the present invention are generally used in an amount effective to achieve the intended purpose. For use in treating or preventing disease conditions, the protease-activatable T cell activating bispecific molecules of the present invention or their pharmaceutical compositions are administered or applied in a therapeutically effective amount. The determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosure provided herein.

[0467] For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays (such as cell culture assays). A dose can then be formulated in animal models to achieve an IC including that determined in cell culture. 50 Such information can be used to more accurately determine useful doses in humans.

[0468] Initial dosages can also be estimated based on in vivo data (eg, animal models) using techniques well known in the art. One of ordinary skill in the art can easily optimize administration to humans based on animal data.

[0469] Dosage and interval can be adjusted individually to provide plasma levels of the protease-activatable T cell activating bispecific molecule sufficient to maintain the therapeutic effect. Common patient dosage ranges for administration by injection are from about 0.1 to 50 mg / kg / day, typically from about 0.5 to 1 mg / kg / day. Therapeutic effective plasma levels can be achieved by administering multiple doses per day. Levels in plasma can be measured, for example, by HPLC.

[0470] In cases 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 dose without undue experimentation.

[0471] The therapeutically effective dose of the protease-activatable T cell activating bispecific molecules described herein will generally provide therapeutic benefit without causing significant toxicity. The toxicity and therapeutic efficacy of the protease-activatable T cell activating bispecific molecules can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (the dose that causes 50% of the population to die) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 / ED 50 Protease-activatable T cell activating bispecific molecules that exhibit a large therapeutic index are preferred. In one embodiment, the protease-activatable T cell activating bispecific molecules according to the present invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of doses suitable for use in humans. The dose is preferably within the range of ED including little or no toxicity. 50The dosage may vary within this range depending on a variety of factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, etc. The exact formulation, route of administration, and dosage may be selected by an individual physician based on the patient's condition (see, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, p. 1, incorporated herein by reference in its entirety).

[0472] The attending physician of a patient treated with a protease-activatable T cell activating bispecific molecule of the present invention will know how and when to terminate, interrupt or adjust administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is insufficient (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. The size of the dose administered in the management of the disorder of interest will vary with the severity of the condition to be treated, the route of administration, etc. For example, the severity of the condition can be evaluated, in part, by standard prognostic evaluation methods. In addition, the dosage and possible dosage frequency will also vary according to the age, weight, and response of the individual patient.

[0473] Other medicines and treatments

[0474] The protease-activatable T cell activation bispecific molecules of the present invention can be administered in combination with one or more other agents in therapy. For example, the protease-activatable T cell activation bispecific molecules of the present invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent that is administered to treat the symptoms or diseases of an individual that needs such treatment. Such additional therapeutic agents may include any active ingredients suitable for the specific indications treated, preferably those active ingredients with complementary activities that do not adversely affect each other. In certain embodiments, additional therapeutic agents are immunomodulators, cell growth inhibitors, cell adhesion inhibitors, cytotoxic agents, apoptosis activators, or agents that increase the sensitivity of cells to apoptosis inducing agents. In specific embodiments, additional therapeutic agents are anticancer agents, such as microtubule disruptors, antimetabolites, topoisomerase inhibitors, DNA intercalators, alkylating agents, hormone therapy, kinase inhibitors, receptor antagonists, tumor cell apoptosis activators, or anti-angiogenic agents.

[0475] Such other agents are appropriately present in combination in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of the protease-activatable T cell activating bispecific molecule used, the type of disorder or treatment, and other factors discussed above. The protease-activatable T cell activating bispecific molecule is generally used in the same dosage and administration route as described herein, or about 1% to 99% of the dosage described herein, or in any dosage and by empirically / clinically determined to be a suitable route.

[0476] Such combination therapies described above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions) and separate administration, in which case the administration of the protease-activatable T cell activation bispecific molecules of the invention can be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant. The protease-activatable T cell activation bispecific molecules of the invention can also be administered in combination with radiotherapy.

[0477] Products

[0478] In another aspect of the present invention, an article of manufacture is provided, which contains materials that can be used to treat, prevent and / or diagnose the above-mentioned disorders. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, etc. The container can be formed of a variety of materials such as glass or plastic. The container holds a composition, which can be effectively used to treat, prevent and / or diagnose the condition by itself or in combination with another composition, and the container can have a sterile access port (for example, the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a protease-activatable T cell activation bispecific molecule of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the article of manufacture may include (a) a first container containing a composition, wherein the composition comprises a protease-activatable T cell activation bispecific molecule of the present invention; and (b) a second container containing a composition, wherein the composition comprises a further cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the present invention may further include a package insert indicating that these compositions can be used to treat specific conditions. Alternatively, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0479] Exemplary Embodiments

[0480] 1. A protease-activatable T cell-activating bispecific molecule comprising

[0481] (a) a first antigen binding portion that is capable of binding to CD3;

[0482] (b) a second antigen binding moiety capable of binding to a target cell antigen; and

[0483] (c) a masking moiety covalently linked 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 concealing the first antigen binding moiety or the second antigen binding moiety,

[0484] The linker comprises a protease recognition sequence XQARK (SEQ ID NO: 39), wherein X is histidine (H) or proline (P).

[0485] 2. The protease-activatable T cell activating bispecific molecule according to embodiment 1, wherein the masking moiety is covalently linked to the first antigen binding moiety and reversibly conceals the first antigen binding moiety.

[0486] 3. The protease-activatable T cell activating bispecific molecule according to embodiment 1 or 2, wherein the masking moiety is covalently linked to the heavy chain variable region of the first antigen binding moiety.

[0487] 4. The protease-activatable T cell activating bispecific molecule according to embodiment 1 or 2, wherein the masking moiety is covalently linked to the light chain variable region of the first antigen binding moiety.

[0488] 5. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 4, wherein the masking moiety is a scFv.

[0489] 6. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 5, wherein the protease-activatable T cell activating bispecific molecule comprises a second masking portion that reversibly conceals the second antigen binding portion.

[0490] 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.

[0491] 8. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 7, 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 the variable regions or constant regions of the Fab light chain and the Fab heavy chain are exchanged.

[0492] 9. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 8, wherein the second antigen binding moiety is a crossover Fab molecule, wherein the constant regions of the Fab light chain and the Fab heavy chain are exchanged.

[0493] 10. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 9, wherein the first antigen binding moiety is a conventional Fab molecule.

[0494] 11. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 10, comprising no more than one antigen binding portion capable of binding to CD3.

[0495] 12. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 11, comprising a third antigen binding portion of a Fab molecule capable of binding to a target cell antigen.

[0496] 13. The protease-activatable T cell activating bispecific molecule according to embodiment 12, wherein the third antigen binding moiety is the same as the second antigen binding moiety.

[0497] 14. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 13, wherein the second antigen binding portion is capable of binding to IGF-1R, cMET or TROP2.

[0498] 15. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 14, wherein the first antigen binding moiety and the second antigen binding moiety are fused to each other, optionally via a peptide linker.

[0499] 16. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 15, 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.

[0500] 17. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 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 first antigen binding moiety.

[0501] 18. The protease-activatable T cell activating bispecific molecule according to 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.

[0502] 19. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 18, further comprising an Fc domain composed of a first subunit and a second subunit capable of stably associating.

[0503] 20. The protease-activatable T cell activating bispecific molecule according to embodiment 19, wherein the Fc domain is an IgG Fc domain, specifically an IgG1 Fc domain or an IgG4 Fc domain.

[0504] 21. The protease-activatable T cell activating bispecific molecule according to embodiment 19 or 20, wherein the Fc domain is a human Fc domain.

[0505] 22. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 19 to 21, wherein the Fc domain exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to a native IgG1 Fc domain.

[0506] 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.

[0507] 24. The protease-activatable T cell activating bispecific molecule according to embodiment 23, wherein the one or more amino acid substitutions are located at one or more positions selected from the group consisting of L234, L235 and P329 (Kabat numbering).

[0508] 25. The protease-activatable T cell activating bispecific molecule of embodiment 24, wherein each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and / or effector function, wherein the amino acid substitutions are L234A, L235A and P329G.

[0509] 26. The protease-activatable T cell activating bispecific molecule of any one of embodiments 22 to 25, wherein the Fc receptor is an Fcγ receptor.

[0510] 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).

[0511] 28. The protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 27, wherein the portion capable of binding to CD3 comprises

[0512] (i) a heavy chain variable (VH) region comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3, and

[0513] (ii) a light chain variable (VL) region comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO:7, LCDR 2 of SEQ ID NO:8, and LCDR 3 of SEQ ID NO:9.

[0514] 29. The protease-activatable T cell activating bispecific molecule according to 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.

[0515] 30. The protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 27, wherein the portion capable of binding to CD3 comprises

[0516] (i) a heavy chain variable (VH) region comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 4, and

[0517] (ii) a light chain variable (VL) region comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO:7, LCDR 2 of SEQ ID NO:8, and LCDR 3 of SEQ ID NO:9.

[0518] 31. The protease-activatable T cell activating bispecific molecule according to 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.

[0519] 32. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 31, wherein the masking moiety comprises a heavy chain variable region comprising at least one of:

[0520] (a) heavy chain complementarity determining region (HCDR) 1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0521] (b) a 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);

[0522] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19).

[0523] 33. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 32, wherein the masking moiety comprises a light chain variable region comprising at least one of:

[0524] (d) the light chain complementarity determining region (LCDR) 1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25) or KSSKSVSTSSYSYMH (SEQ ID NO: 26);

[0525] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0526] (f) a LCDR3 amino acid sequence selected from the group consisting of QHSREFPYT (SEQ ID NO: 28) or QQSREFPYT (SEQ ID NO: 29).

[0527] 34. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 33, wherein the masking moiety comprises: a heavy chain variable region comprising:

[0528] (a) HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0529] (b) a 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);

[0530] (c) a HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60); and a light chain variable region comprising:

[0531] (d) a LCDR1 amino acid sequence selected from the group consisting of RASKSVSTSSYSYMH (SEQ ID NO: 25) and KSSKSVSTSSYSYMH (SEQ ID NO: 26);

[0532] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0533] (f) a LCDR3 amino acid sequence selected from the group consisting of QHSREFPYT SEQ ID NO: 28 and QQSREFPYT (SEQ ID NO: 29).

[0534] 35. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 33, wherein the masking moiety comprises a VH region comprising:

[0535] (a) HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0536] (b) HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16);

[0537] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19);

[0538] and a VL region comprising:

[0539] (d) LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25);

[0540] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0541] (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0542] 36. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 33, wherein the masking moiety comprises a VH region comprising:

[0543] (a) HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0544] (b) HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO: 16);

[0545] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19);

[0546] and a VL region comprising:

[0547] (d) LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26);

[0548] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0549] (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0550] 37. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 33, wherein the masking moiety comprises a VH region comprising:

[0551] (a) HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0552] (b) HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO: 17);

[0553] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19);

[0554] and a VL region comprising:

[0555] (d) LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26);

[0556] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0557] (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0558] 38. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 33, wherein the masking moiety comprises a VH region comprising:

[0559] (a) HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0560] (b) HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18);

[0561] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19);

[0562] and a VL region comprising:

[0563] (d) LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO: 26);

[0564] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0565] (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28).

[0566] 39. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 33, wherein the masking moiety comprises a VH region comprising:

[0567] (a) HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15);

[0568] (b) HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO: 18);

[0569] (c) HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 19);

[0570] and a VL region comprising:

[0571] (d) LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 25);

[0572] (e) the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO: 27); and

[0573] (f) LCDR3 amino acid sequence of QQSREFPYT (SEQ ID NO: 29).

[0574] 40. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 39, wherein the masking moiety is humanized.

[0575] 41. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 40, wherein the masking portion 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.

[0576] 42. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 33, wherein the masking portion 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.

[0577] 43. The protease-activatable 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-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 / or 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.

[0578] 44. The protease-activatable 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-1R and comprises: a heavy chain variable region comprising:

[0579] a) HCDR1 amino acid sequence of SYGMH (SEQ ID NO: 61);

[0580] b) a HCDR2 amino acid sequence of IIWFDGSSTYYADSVRG (SEQ ID NO: 62); and

[0581] c) the HCDR3 amino acid sequence of ELGRRYFDL (SEQ ID NO: 63);

[0582] and a VL region comprising:

[0583] d) LCDR1 of RASQSVSSYLA (SEQ ID NO: 65);

[0584] e) the LCDR2 amino acid sequence of DASKRAT (SEQ ID NO: 66); and

[0585] f) LCDR3 amino acid sequence of QQRSKWPPWT (SEQ ID NO: 67).

[0586] 45. The protease-activatable 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-1R 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.

[0587] 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.

[0588] 47. The protease-activatable 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 consisting of SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75.

[0589] 48. The protease-activatable 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: a heavy chain variable region comprising:

[0590] a) HCDR1 amino acid sequence of SYWLH (SEQ ID NO: 69);

[0591] b) the HCDR2 amino acid sequence of MIDPSNSDTRFNPNFKD (SEQ ID NO: 70); and

[0592] c) HCDR3 amino acid sequence of YRSYVTPLDY (SEQ ID NO: 71);

[0593] and a VL region comprising:

[0594] d) LCDR1 of KSSQSLLYTSSQKNYLA (SEQ ID NO:73);

[0595] e) the LCDR2 amino acid sequence of WASTRES (SEQ ID NO: 74); and

[0596] f) LCDR3 amino acid sequence of QQYYAYPWT (SEQ ID NO: 75).

[0597] 49. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 42 or 46 to 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.

[0598] 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.

[0599] 51. The protease-activatable 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 consisting of SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83.

[0600] 52. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 42, wherein the second antigen binding portion is capable of binding to TROP2 and comprises: a heavy chain variable region comprising:

[0601] a) HCDR1 amino acid sequence of NYGMN (SEQ ID NO: 77);

[0602] b) the HCDR2 amino acid sequence of WINTKTGEPTYAEEFKG (SEQ ID NO: 78); and

[0603] c) HCDR3 amino acid sequence of GGYGSSYWYFDV (SEQ ID NO: 79);

[0604] and a VL region comprising:

[0605] d) LCDR1 of KASQDVSIAVA (SEQ ID NO:81);

[0606] e) the LCDR2 amino acid sequence of SASYRYT (SEQ ID NO: 82); and

[0607] f) LCDR3 amino acid sequence of QQHYITPLT (SEQ ID NO: 83).

[0608] 53. The protease-activatable T cell activating bispecific molecule according to 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.

[0609] 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.

[0610] 55. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 46, wherein the protease-cleavable linker comprises the protease recognition sequence PQARK (SEQ ID NO: 41).

[0611] 56. A protease-activatable T cell-activating bispecific molecule comprising

[0612] (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 85,

[0613] (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 89,

[0614] (c) a first light chain comprising the amino acid sequence of SEQ ID NO: 87, and

[0615] (d) a second light chain comprising the amino acid sequence of SEQ ID NO:88.

[0616] 57. A protease-activatable T cell-activating bispecific molecule comprising

[0617] (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 91,

[0618] (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 94, and

[0619] (c) a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0620] 58. A protease-activatable T cell-activating bispecific molecule comprising

[0621] (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 96,

[0622] (b) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 100,

[0623] (c) a light chain comprising the amino acid sequence of SEQ ID NO: 98, and

[0624] (d) a second chain comprising the amino acid sequence of SEQ ID NO:99.

[0625] 59. An isolated polynucleotide encoding the protease-activatable T cell activating bispecific antigen binding molecule according to any one of embodiments 1 to 58.

[0626] 60. A polypeptide encoded by the polynucleotide of embodiment 59.

[0627] 61. A vector, in particular an expression vector, comprising the polynucleotide according to embodiment 59.

[0628] 62. A host cell comprising the polynucleotide of embodiment 59 or the vector of embodiment 61.

[0629] 63. A method for producing a protease-activatable T cell activating bispecific molecule, the method comprising the following steps: a) culturing the host cell according to embodiment 62 under conditions suitable for expressing the protease-activatable T cell activating bispecific molecule, and b) recovering the protease-activatable T cell activating bispecific molecule.

[0630] 64. A protease-activatable T cell activating bispecific molecule produced by the method according to embodiment 63.

[0631] 65. A pharmaceutical composition comprising the protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 58 and a pharmaceutically acceptable carrier.

[0632] 66. The protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 58 or the composition according to embodiment 65 for use as a medicament.

[0633] 67. The protease-activatable T cell activating bispecific molecule for use according to Example 66, wherein the medicament is used to treat cancer or delay its progression, treat immune-related diseases or delay its progression, or enhance or stimulate immune responses or functions in an individual.

[0634] 68. The protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 58, for use in treating a disease in an individual in need thereof.

[0635] 69. The protease-activatable T cell activating bispecific molecule for use according to embodiment 68, wherein the disease is cancer.

[0636] 70. Use of the protease-activatable T cell activating bispecific molecule according to any one of embodiments 1 to 58 for the manufacture of a medicament for treating a disease.

[0637] 71. The use according to embodiment 70, wherein the disease is cancer.

[0638] 72. A method of treating a disease in an individual, the method 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 embodiments 1 to 58 or the composition of embodiment 65.

[0639] 73. A method for inducing lysis of a target cell, the method comprising contacting the target cell with the protease-activatable T cell activating bispecific molecule of any one of embodiments 1 to 58 or the composition of embodiment 65 in the presence of a T cell.

[0640] 74. The method of embodiment 73, wherein the target cell is a cancer cell.

[0641] 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.

[0642] 76. The present invention is as described above.

[0643] Exemplary sequences

[0644] Table 2: CDR definitions according to Kabat

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664] Examples

[0665] The following are examples of methods and compositions of the present invention.It should be understood that various other embodiments may be practiced given the general description provided above.

[0666] Example 1. Generation of T cell bispecific molecules targeting FolR1

[0667] 1.1 Expression constructs

[0668] T cell bispecific molecules are produced in a proprietary 2+1 heterodimeric format based on knob-in-hole technology (two binding moieties for the target antigen and one for CD3). The anti-CD3 binder blocking scFv (stabilized via H44 / L100 disulfide bridges) is fused to the N-terminus of the VH of the CD3 binding Fab in the order of VHVL ( Figure 1A The linker between the scFv and Fab is 33 amino acids in length and consists of a protease site embedded in the GS linker sequence.

[0669] The genes of each chain of proTCB were inserted into mammalian expression vectors separately. The expression of all genes was controlled by the human CMV promoter-intron A-5'UTR box. The BGH polyadenylation signal was located downstream of the gene.

[0670] 1.2 Generation of FolR1proTCB with a protease-cleavable linker

[0671] By transient transfection of Expi293F TM The cells produced bispecific proTCB molecules with different protease linkers. The cells were cultured at 2.5x10 6 / ml density was inoculated in Expi293 TM Culture medium (Gibco, catalog number 1435101). The expression vector and ExpiFectamine (Gibco, ExpiFectamine TM Transfection kit, catalog number 13385544) in OptiMEM TM The cells were added to the expression vector / ExpiFectamine solution and incubated at 37°C in a 5% CO atmosphere. 2 Incubate in a shaking incubator with a 4% CO atmosphere for 24 hours. One day after transfection, add supplements (transfection enhancers 1 and 2, ExpiFectamine TM Transfection kit). After 4 to 5 days, the cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the harvested supernatant by standard methods as shown below.

[0672] 1.3 Purification of IgG-like proteins

[0673] Proteins were purified from filtered cell culture supernatants with reference 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 by immediate pH neutralization of the sample. The elution was performed by centrifugation (Millipore Proteins were concentrated using ULTRA-15 (Cat. No. 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 otherwise indicated).

[0674] Table 3: Production and purification

[0675]

[0676] Example 2. Determination of protease cleavage rates using SPR

[0677] The cleavage rate of recombinant protein lysing enzymes was studied using surface plasmon resonance (SPR) on a Biacore T200 instrument (Cytiva). Biotinylated CD3ε was fixed on a Series S Sensorchip SA (Cytiva, 29104992) at a final surface density of 2000-4000 resonance units (RU). FOLR1 proTCB at a concentration of 10nM was incubated with 50pM of recombinant protein lysing enzyme (R&Dsystems, 3946-SE) at 37°C in PBS-T pH 7.4 and PBS-T pH 6.5. CD3ε binding sequence and thus proTCB activation rate were monitored by continuously injecting the proTCB / protein lysing enzyme mixture onto the surface at a flow rate of 5μl / min for 30s for up to 10 hours. After each injection, the CD3e surface was regenerated by injecting 10mM glycine pH 1.5 at a flow rate of 5μl / min for 60s. In the same experiment, a concentration series of 0.16, 0.31, 0.63, 1.25 and 2.5 nM FOLR1 TCB was injected to generate a calibration line and the obtained proTCB binding response was converted from resonance units (RU) to molar concentrations (nM). The molar concentration of activated proTCB was plotted against the incubation time and the cleavage rate (pM / min) was calculated by determining the slope of each derived straight line.

[0678] Table 4: Initial cleavage rate at different pH values:

[0679]

[0680]

[0681] Example 3. Developability of FOLR1 proTCBs containing different variants of humanized masks

[0682] FOLR1 proTCB molecules with different humanization masks generated as described in Example 1 were selected and analyzed for stability and developability.

[0683] Table 5: Production and purification

[0684]

[0685] 3.1 Thermal stability

[0686] Thermal stability was studied 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 sample device of the instrument. 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, and the aggregation temperature (Tagg) was determined thereby.

[0687] 3.2 Apparent hydrophobicity

[0688] The apparent hydrophobicity was investigated by hydrophobic interaction chromatography (HIC) using high performance 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 0 to 1.5 M NH 2+ solution in 25 mM sodium phosphate was applied at a column temperature of 40°C at a rate of 0.8 ml / min over 20 min. 4 SO 4 The detection wavelength was set to 214 nm. Relative retention times were calculated using appropriate reference antibodies.

[0689] 3.3 FcRn Chromatography

[0690] Relative FcRn binding affinity was determined by high performance liquid chromatography (HPLC). Briefly, 30 μl of proTCB at a concentration of 1 mg / ml was injected onto an FcRn streptavidin agarose column (Roche Diagnostics 08128057001). A step gradient of 20 mM MES sodium salt, 140 mM NaCl pH 5.5 and pH 8.8 was applied at a column temperature of 25°C according to the manufacturer's recommendations. The detection wavelength was set to 280 nm. Relative retention times were calculated using appropriate reference antibodies.

[0691] 3.4 Heparin chromatography

[0692] Relative heparin binding affinity was determined by high performance 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.

[0693] Table 6: Results

[0694] parameter P1AF5794 P1AF65795 P1AF5796 <![CDATA[Thermal stability (SLS T agg )]]> 63.5 62.4 62.5 Apparent hydrophobicity (HIC) 0.22 0.21 0.21 FcRn chromatography 0.25 0.25 0.29 Heparin chromatography 0.64 0.64 0.69

[0695] All tested molecules showed good thermal stability (>60°C) and had good acceptable values ​​for hydrophobicity as well as FcRn and heparin chromatography.

[0696] Example 4. Determination of cleavage rates of different proteases using SPR

[0697] Surface plasmon resonance (SPR) on a Biacore T200 instrument (Cytiva) was used to study the cleavage rates of recombinant proteases (R&D Systems 3946-SEB), proteases-2 (Enzo ALX-201-752), serine transmembrane proteases (R&D Systems 4776-SE), uPA (Sigma-Aldrich 6273), leguminous aspartate proteinase (R&D Systems 2199-CY) and furin (R&D Systems 1503-SE). Biotinylated CD3ε was immobilized on a Series S Sensorchip SA (Cytiva, 29104992) at a final surface density of 2000-4000 resonance units (RU). FOLR1 proTCB was incubated in PBS-T pH 7.4 at 37°C with the different proteases listed above at the following concentrations:

[0698] i.10nM proTCB+50pM proteinase

[0699] ii.10nM proTCB+1U proteinase-2

[0700] iii.10nM proTCB+300pM activated serine transmembrane protease

[0701] iv.10nM proTCB+5.5nM uPA

[0702] v.10nM proTCB+5nM activated legume aspartate protein endoproteinase vi.10nM proTCB+3nM furin (1mM CaCl2 added)

[0703] The CD3ε binding sequence and thus the proTCB activation rate were monitored by continuously injecting the proTCB / protease mixture onto the surface at a flow rate of 5 μl / min for 30 s 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 s. In the same experiment, the injection concentration series was 0.16, 0.31, 0.63, 1.25 and 2.5 nM FOLR1 TCB to generate a calibration line and the obtained proTCB binding response was converted from resonance units (RU) to molar concentrations (nM). The molar concentration of activated proTCB was plotted against the incubation time, and the cleavage rate (pM / min) was calculated by determining the slope of each derived straight line.

[0704] Table 7: Cleavage rate proteinases

[0705]

[0706] Table 8: Cleavage rate matriptase-2

[0707]

[0708] Table 9: Cleavage rate of serine transmembrane proteases

[0709]

[0710] Table 10: Cutting rate uPA

[0711]

[0712] Table 11: Cleavage rate of legume aspartate protein endoenzyme

[0713]

[0714] Table 12: Cleavage rate of furin

[0715]

[0716] Example 5. Developability of FOLR1proTCB (different protease cleavage sites; humanized mask)

[0717] The stability and developability of the selected FOLR1proTCB molecules were analyzed. The same method as in Example 3 was used.

[0718] Table 13: Results

[0719]

[0720]

[0721] All tested molecules showed good thermal stability (>60°C) and had good acceptable values ​​for hydrophobicity as well as FcRn and heparin chromatography.

[0722] Example 6. Developability of humanized anti-idiotypic IgG

[0723] Biacore T200 instrument (Cytiva) was used to study the binding of anti-idiotype after incubation for 14d in 20mM His / HCl, 140mM NaCl pH 6.0 (at 40°C) or 1xPBS pH 7.4 (at 37°C) by surface plasmon resonance. In brief, according to the manufacturer's instructions, after injection of capture reagent, biotinylated anti-human CD3ε antibody and biotinylated anti-human IgG (Capture Select, Thermoscientific, 7103302500) were fixed on a series of sensor chips CAP (Biotin CAPture kit, Cytiva 28920234). The surface density obtained was approximately 1000RU and 1500RU respectively. Anti-idiotype antibodies were injected onto the chip surface at a concentration of 1 μg / ml at a flow rate of 5 μl / min for 30s. Dissociation was monitored for 30s. After each injection, the chip surface was regenerated by injecting 2 M guanidine hydrochloride, 0.5 M NaOH for 120 s. Bulk refractive index differences were corrected by subtracting the response obtained from the simulated surface.

[0724] 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. The relative activity concentration was obtained by dividing the normalized response of the stressed sample by the normalized response of the unstressed reference sample for each molecule.

[0725] Table 14: Results

[0726]

[0727]

[0728] Example 7. Developability of proTCB

[0729] The binding of proTCB after incubation for 14d in 20mM His / HCl, 140mM NaCl pH 6.0 (at 40°C) or 1xPBS pH 7.4 (at 37°C) was studied by surface plasmon resonance using a Biacore T200 instrument (Cytiva). In brief, mouse anti-huIgG CH2 PG-LALA antibody (P1AE2335) and human CD3ε (P1AA6119) were immobilized on a serial sensor chip CM5 (Cytiva) using standard amine coupling chemistry. The ligand densities obtained were approximately 8500RU and 7000RU, respectively. For FolR1 binding assessment, proTCB was captured onto the anti-human IgGPG-LALA surface at a concentration of 2μg / ml and a flow rate of 10μl / min for 75s. Subsequently, human FolR1 (P1AD6798) was injected at a concentration of 900nM and a flow rate of 10μl / min for 120s. Dissociation was monitored for 120 s. After each human FolR1 injection, the surface was regenerated by injecting 20 mM NaOH for 35 s. For CD3ε binding assessment, proTCB was injected onto the CD3ε surface at a concentration of 10 μg / ml, at a flow rate of 10 μg / ml for 90 s. Dissociation was monitored for 90 s. After each injection, the surface was regenerated by injecting 10 mM glycine pH 2.1 for 70 s. The bulk refractive index differences for each injection were corrected by subtracting the response obtained from the simulated surface.

[0730] To normalize the binding signal of proTCB, the FolR1 and CD3ε binding responses were divided by the binding response of the anti-human IgG PG-LALA surface. The relative activity concentration was obtained by dividing the normalized response of the stressed sample by the normalized response of the unstressed reference sample (FolR1) or the unmasked control molecule (CD3ε).

[0731] Table 15: Relative FolR1 binding

[0732]

[0733]

[0734] Table 16: Relative CD3 binding

[0735]

[0736] Example 8. In vivo stability and pharmacokinetic characteristics of different linkers after a single injection in NSG mice

[0737] A single dose of 5 mg / kg of pro-FolR1-TCB molecules containing selective cleavage sites for different proteases was injected into NSG mice. Figure 2As shown in, all mice were intravenously injected with 200 μl of appropriate solution. In order to obtain the appropriate amount of compound per 200 μl, the stock solution (Table 17) was diluted with histidine buffer. Blood was collected for each time point and two mice per group at 24 hr, 7 days and 10 days. The injected compound in the serum sample was analyzed by ELISA.

[0738] 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+1TCB) were analyzed using an ECLIA method specific for the human CH1 / PGLALA domain ("total assay") and an ECLIA method captured with CD3 anti-ID antibody and detected with anti-PGLALA specific antibody ("activity assay") using a cobas e411 instrument.

[0739] For the “total assay”, test samples of P1AF5419 (cleavage site: HQARK) (004-09), P1AF5420 (cleavage site: PQARK) (004-06), or P1AE6554 (classical FolR1 2+1TCB), primary 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 minutes in each step.

[0740] For the "activity assay", the test sample of FolR1 TCB (007-19), the primary detection antibody mAb <ch2527>rH-4.24.72-IgG()-Bi, second detection antibody mAb<H-Fc(PGLALA)> M-1.7.24-IgG-Ru and SA beads were added to the detection container stepwise and incubated for 9 minutes in each step. Finally, the complex bound by the SA beads was detected by a measuring pool, which repeatedly counted the number of SA beads. The count was proportional to the analyte concentration in the test sample.

[0741] Table 17: Preparation of stock solution

[0742]

[0743] Analysis of serum from mice dosed with 5 mg / kg FOLR1-TCB or FOLR1 pro-TCB showed low levels of active TCB at all time points, less than 5% of total pro-TCB, when FOLR1 pro-TCB molecules containing either the HQARK or PQARK cleavage sites were measured. Classical (unmasked) FOLR1-TCB detected 100% of active TCB ( Figure 3 ).

[0744] Example 9. Efficacy study of pro-FOLR1-TCB constructs containing selective cleavage sites for different proteases in BC004 PDX of humanized mice

[0745] The human breast cancer patient-derived xenograft HER2+ER- xenograft model BC004 was purchased from OncoTest (Freiburg, Germany). Tumor fragments were digested with collagenase D and DNase I (Roche), counted, and injected at 1×10 6 BC004 cells were injected subcutaneously into the flank of anesthetized mice using a 22G to 30G needle.

[0746] Female NSG mice (Jackson Laboratory) aged 4-5 weeks at the start of the experiment were maintained under specific pathogen-free conditions with a 12 h light / 12 h dark day cycle according to regulatory guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local authorities (P 2011 / 128). After arrival, the animals were maintained for one week to acclimate to the new environment and observed. Continuous health monitoring was performed regularly.

[0747] Female NSG mice were injected intraperitoneally with 15 mg / kg of busulfan, followed by an intravenous injection of 1x10 5 The human hematopoietic stem cells separated from umbilical cord blood. At the 14th to 16th week after stem cell injection, mice were bled sublingually and analyzed for successful humanization by flow cytometry. The mice effectively transplanted were randomly assigned to different treatment groups according to their human T cell frequencies. At this time, mice were injected with tumor PDX cells, and when the tumor size reached about 200mm3 (the 28th day), they were treated once a week with compounds or histidine buffer (vehicle). All mice were injected intravenously with 200 μl of appropriate solution. In order to obtain an appropriate amount of compound per 200 μl, the stock solution (Table 18) was diluted with histidine buffer (if necessary).

[0748] Tumor growth was measured twice weekly using calipers ( Figure 2 ), and the tumor volume was calculated as follows:

[0749] T v :(W 2 / 2) x L (W: width, L: length)

[0750] At termination (day 58), mice were sacrificed and tumors and spleens were removed and weighed.

[0751] Figure 5A Tumor growth kinetics (mean, + SEM) of the most effective treatment group as well as individual tumor growth per mouse are shown. As described herein, FOLR1 pro-TCB containing the PQARK cleavage site was identified as the best pro-TCB tested in this study. In addition, no efficacy was observed in the group treated with FOLR1 pro-TCB containing a non-cleavable linker. Figures 5A to 5G Tumor weights at termination are depicted in all treatment groups. This result clearly supports the findings of tumor growth kinetics and indicates that pro-TCBs containing the PQARK cleavage site resulted in comparable tumor weights at termination compared to the classical FolR1TCBs.

[0752] Table 18: Preparation of stock solution

[0753]

[0754]

[0755] Example 10. Generation of T cell bispecific molecules targeting cMET, TROP2 or IGF-1R 10.1 Expression constructs:

[0756] T cell bispecific molecules are produced in a proprietary heterodimeric format based on knob-in-hole technology (one or two binding moieties for the target antigen and one for CD3). The anti-CD3 binder blocking scFv (stabilized via H44 / L100 disulfide bridges) is fused to the N-terminus of the VH of the CD3 binding Fab in the order VHVL ( Figures 1B to 1D The linker between the scFv and Fab is 33 amino acids in length and consists of a protease recognition site for the mab proteinase embedded in the GS linker sequence.

[0757] The genes of each chain of proTCB were inserted into mammalian expression vectors separately. The expression of all genes was controlled by the human CMV promoter-intron A-5'UTR box. The BGH polyadenylation signal was located downstream of the gene.

[0758] 10.2 Generation of FolR1proTCB with a protease-cleavable linker

[0759] By transient transfection of Expi293F TM The cells produced bispecific proTCB molecules with different protease linkers. The cells were cultured at 2.5x10 6 / ml density was inoculated in Expi293 TM Culture medium (Gibco, catalog number 1435101). The expression vector and ExpiFectamine (Gibco, ExpiFectamine TM Transfection kit, catalog number 13385544) in OptiMEM TM The cells were added to the expression vector / ExpiFectamine solution and incubated at 37°C in a 5% CO atmosphere. 2 Incubate in a shaking incubator with a 4% CO atmosphere for 24 hours. One day after transfection, add supplements (transfection enhancers 1 and 2, ExpiFectamine TM Transfection kit). After 4 to 5 days, the cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the harvested supernatant by standard methods as shown below.

[0760] 10.3 Purification of IgG-like proteins

[0761] Proteins were purified from filtered cell culture supernatants with reference 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 by immediate pH neutralization of the sample. The elution was performed by centrifugation (Millipore Proteins were concentrated using ULTRA-15 (Cat. No. 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 otherwise indicated).

[0762] Table 19: Production and purification

[0763]

[0764]

[0765] Example 11. Binding of IGF-1R proTCB constructs to T cells

[0766] We evaluated the ability of IGF-1RproTCBs (SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89) and their controls (unmasked classical IGF-1R TCBs, non-cleavable IGF-1RproTCBs) to bind to CD3 on the human reporter T cell line Jurkat NFAT compared to the corresponding protease pre-cleaved (thus unmasked) molecules. Both protease pre-cleaved IGF-1R proTCBs and classical IGF-1R TCBs (with or without protease pre-cleavage) bound CD3 comparably well, whereas non-cleavable IGF-1R TCB constructs and non-protease pre-cleaved IGF-1RproTCBs did not bind to CD3 on Jurkat T cells ( Figure 6 ), indicating the effective CD3 blocking ability of H1L2 masking.

[0767] 11.1 Methods

[0768] 11.1.1 Cell lines

[0769] Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS176501) are a human acute lymphoblastic leukemia reporter cell line with a NFAT promoter that expresses human CD3. The cells were cultured at 0.1-0.5 mio cells / ml in RPMI1640, 2 g / l glucose, 2 g / l NaHCO 3 The cells were cultured in 1% FBS, 25 mM HEPES, 2 mM L-glutamine, 1x NEAA, and 1x sodium pyruvate. Whenever the cells were passaged, hygromycin B (Sigman, #10834555001) was added at a final concentration of 200 μg / ml.

[0770] 11.1.2 CD3 Binding by Flow Cytometry

[0771] One day before the start of the assay, the TCB constructs were set up twice, one vial of each molecule was untreated, and the other vial was pre-activated overnight at room temperature by adding 1 μl of human recombinant protein lysing enzyme (Enzo about 2.5U / μl, ALX-201-246-U25, batch number 12152015). The next day, Jurkat cells were harvested and transferred to 96-well round-bottom plates (100'000 cells / well). The cells were washed with FACS buffer (PBS, 2% FBS, 5mM EDTA, 0.025% NaN 3 ) and incubated with 25 μl of 1:4 titrated IGF-1R TCB construct (starting concentration: 50 nM) in FACS buffer at 4°C for 30 min. After staining, cells were washed twice with FACS buffer to remove unbound molecules. To distinguish live cells from dead cells, Jurkat cells were stained with 25 μl of diluted NIR live / dead dye (1:1000 dilution, batch number: 2192282, Invitrogen) for 10 min 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 incubation at 4°C for 30 min, the cells were washed twice with FACS buffer to remove unbound antibodies. Finally, the cells were resuspended in 100 μl FACS buffer and analyzed by flow cytometry using BD Canto II.

[0772] Example 12. Binding of IGF-1R proTCB constructs to IGF-1R on different cancer cell lines

[0773] We also evaluated the ability of 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 uncleavable IGF-1R proTCB) to bind to the target (IGF-1R) on human cancer cell lines expressing different levels of IGF-1R on the cell surface (T-47D>MKN-45>OVMANA>HPAF II). All three IGF-1R TCB constructs tested bound IGF-1R comparably well ( Figure 7 ).

[0774] 12.1 Methods

[0775] 12.1.1 Cell lines

[0776] OVMANA is a human ovarian clear cell adenocarcinoma cell line (RNCB accession number: CL012520), which was cultured in RPMI1640+1x Glutamax+10% FBS.

[0777] T-47D is a human cancer cell line (RNCB accession number: CL000001), which was cultured in RPMI1640+1x Glutamax+10% FBS.

[0778] HPAF II is a human pancreatic adenocarcinoma cell line (RNCB accession number: CL010030), which was cultured in EMEM+10% FBS+1% Glutamax+1% NEAA+1% sodium pyruvate.

[0779] HeLa is a human epithelial adenocarcinoma cell line (RNCB accession number: CL022232). The HeLa cells used have been genetically modified (NLR-red nucleus, inactivated ST14) and cultured in DMEM+10% FBS+1% Glutamax, +3μg / ml puromycin (LabForce AG, #ant-pr-1) (0.6μl / 10ml), +80μg / ml hygromycin B (Sigman, #10834555001) (80μl / 10ml).

[0780] 12.1.2 Target Binding by Flow Cytometry

[0781] Target cells were harvested and transferred to 96-well round-bottom plates (100,000 cells / well). Cells were incubated with FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.025% NaN 3 ) and stained with 25 μl of the corresponding IGF-1R TCB construct in FACS buffer for 30 min at 4°C. After staining, the cells were washed twice with FACS buffer to remove unbound molecules. To distinguish live cells from dead cells, Jurkat cells were stained with 25 μl of diluted NIR live / dead dye (1:1000 dilution, batch number: 2192282, Invitrogen) for 10 min 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 incubation at 4°C for 30 min, the cells were washed twice with FACS buffer to remove unbound antibodies. Finally, the cells were resuspended in 100 μl FACS buffer and analyzed by flow cytometry using BD Canto II.

[0782] Example 13. Functional activity of IGF-1R proTCB constructs - Jurkat NFAT reporter cell assay using beads

[0783] Biotinylated human IGF-1R antigen (biotinylated human IGF-1R / CD221 protein, His, Avitag TM , BioCat) coated SA beads and Jurkat NFAT reporter cells (human acute lymphoblastic leukemia reporter cell line expressing CD3 with NFAT promoter, GloResponse Jurkat NFAT-RE-luc2P, Promega #CS176501) evaluate the ability of IGF-1RproTCB (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. After the IGF-1R TCB construct is combined with the CD3 expressed on the SA beads coated with IGF-1R and Jurkat NFAT reporter T cells at the same time, the NFAT promoter is activated and causes the expression of active firefly luciferase. The intensity of the luminescent signal (obtained after adding luciferase substrate) is proportional to the intensity of CD3 activation and signal transduction. Jurkat NFAT reporter T cells are grown in suspension and in RPMI1640, 2g / l glucose, 2g / l NaHCO 3 The cells were cultured in 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 1xNEAA, 1x sodium pyruvate at 0.1-0.5 mio cells / ml and 200 μg / ml hygromycin B.

[0784] Comparable Jurkat NFAT activation was observed for protease pre-cleaved (+M) IGF-1R proTCB and classical IGF-1R TCB (+M or untreated). Non-cleavable IGF-1R proTCB (+M or untreated) and non-pre-cleaved IGF-1R proTCB showed no NFAT activation ( Figure 8 ). Thus, CD3-masking H1L2 appears to effectively block the CD3 binder P035.093.

[0785] 13.1 Methods

[0786] For determination, 95 μl SA beads (Streptavidin Dynabeads M-280 streptavidin, LubioScience #11205D) are diluted in 5 ml DPBS. The beads are centrifuged at 400 rcf for 4 min, and the supernatant is aspirated. Then the biotinylated hu-IGF-1R antigen (final 46.90 nM, 5 μg / ml) is added to 0.5 ml DPBS and then added to the SA beads. The beads are resuspended with antigen solution. The beads-antigen mixture is slowly rotated and incubated at 25 ° C for 60 min. After incubation, 5 ml DPBS is added to the beads-ag conjugate, centrifuged and the supernatant is discarded. The conjugate is resuspended in the assay culture medium.

[0787] One day before the start of the assay, TCB constructs were set up twice, one vial of each molecule was untreated and the other vial was preactivated by adding 1 μl of protease (Enzo approximately 2.5 U / μl, ALX-201-246-U25, batch number 12152015) overnight at room temperature.

[0788] On the second day, Jurkat cells were harvested and their viability was determined using a ViCell XR cell counter (Beckman Coulter). Cells were centrifuged at 300rcf for 3min, then the culture medium was aspirated, and cells were resuspended in a fresh assay culture medium (Sigman, #10834555001) that did not contain hygromycin B. SA beads suspension (1 times) coated was mixed with effector cell suspension (2 times) in a falcon tube, and cAMP GloSensor (#E1291, Promega) (2% final volume) was added. 30 μl of mixture was plated in a flat white wall 96-well plate (#655098, Greiner bio-one), and diluted TCB or culture medium (for negative control) of 10 μl / wells were added. Cells were cultured at 37 °C for 5 hours in a humidified incubator. At the end of the incubation time, TECAN Spark 10M was used to detect luminescence.

[0789] Example 14. Functional activity of IGF-1R proTCB constructs - Jurkat NFAT reporter assay using cells

[0790] IGF-1R proTCB (SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89) was used to evaluate the ability of inducing TCR cross-linking and subsequent T cell activation using IGF-1R expressing cancer cells (T-47D>MKN-45>OVMANA>HPAF II) and Jurkat NFAT reporter cells (human acute lymphoblastic leukemia reporter cell line expressing CD3 with NFAT promoter, GloResponseJurkat NFAT-RE-luc2P, Promega #CS176501). After the two arms (anti-IGF-1R and anti-CD3 arms) of the TCB construct are simultaneously combined with CD3 on target cells expressing IGF-1R and Jurkat-NFAT reporter cells, the NFAT promoter of Jurkat cells is activated and causes the expression of active firefly luciferase. The intensity of the luminescent signal (obtained after adding luciferase substrate) is proportional to the intensity of CD3 activation and signal transduction. Jurkat NFAT reporter cells were grown in suspension and cultured in RPMI1640, 2 g / l glucose, 2 g / l NaHCO 3 , 10% FBS, 25mM HEPES, 2mM L-glutamine, 1x NEAA, 1x sodium pyruvate at 0.1-0.5mio cells / ml, 200μg / ml hygromycin B.

[0791] 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 ( Fig. 9 ). A) T-47D: medium-high, B) MKN-45: medium-high, C) OVMANA: medium, D) HPAF II: low. Unmasked classical TCBs (with and without protease pre-activation) and protease pre-cleaved IGF-1R proTCBs induced comparable Jurkat NFAT activation after stimulation with TCB constructs. Uncleavable proTCBs (negative control) and non-protease pre-activated IGF-1R proTCBs did not induce NFAT activation, indicating that H1L2 masking has effective masking capabilities. Maximal activation of Jurkat NFAT cells depends on the level of target expressed on tumor target cells (IGF-1R surface expression ranking: T-47D>MKN-45>OVMANA>HPAF II).

[0792] 14.1 Methods

[0793] For determination, tumor target cells are harvested and vitality is determined using ViCell XR cell counter (Beckman Coulter).10.000 target cells / well are plated in 100 μl culture medium of flat white wall 96-well plate (#655098, Greiner bio-one), and the antibody or culture medium (for negative control) diluted by 50 μl / well is added to the target cell.Subsequently, Jurkat NFAT reporter cells are harvested and vitality is assessed.Cell is resuspended in the cell culture medium without hygromycin B (Sigman, #10834555001) with 1.25mio cells / ml, and is added to tumor cells with 25.000 cells / well (50 μl / well), to obtain the final E:T of 2.5:1 and the final volume of 200 μl / every hole.Then 4 μl GloSensor (#E1291, Promega) is added to each hole (2% of final volume). The cells were incubated for 5 hours at 37° C. in a humidified incubator. At the end of the incubation time, luminescence was detected using a TECAN Spark 10M.

[0794] The day before the assay, the tested IGF-1R TCB constructs had been activated by incubation with human recombinant matriptase (Enzo approximately 2.5 U / μl, ALX-201-246-U25, batch number 12152015) overnight at room temperature.

[0795] Example 15. T cell-mediated tumor cell killing (LDH release) and T cell activation

[0796] 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 quantifying LDH released into the supernatant by apoptotic / necrotic target cells (LDH detection kit, Roche Applied Science, #11 644 793 001).

[0797] Precleavage of IGF-1R proTCB by the highest concentration (50 nM) of proteases only weakly induced LDH release ( Fig.10 ). This suggests that target cell killing mediated by IGF-1R proTCB requires higher TCB concentrations.

[0798] However, the IGF-1R proTCB construct was observed to activate CD4 + and CD8 + In order to investigate the potential of T cells, we could demonstrate that both early (CD69) and late (CD25) T cell activation markers were upregulated in a dose-dependent manner in TCBs (IGF-1R proTCBs pre-activated with matriptase as well as IGF-1R TCBs (with or without matriptase pre-treatment)) and target expression levels. + T cells ( Fig.11 ) and CD8 + T cells ( Fig.12 ). Thus, CD69 appears to be a more sensitive T cell activation marker than CD25 in this context. The non-cleavable proTCB construct showed induction of CD69 with increasing TCB concentrations, whereas CD25 expression was not induced. This data suggests that both classical IGF-1R TCBs and protease-pre-cleaved IGF-1R proTCBs are able to activate CD4 + ( Fig.11 ) and CD8 + ( Fig.12 )T cells, even though LDH release is not sensitive enough.

[0799] 15.1 Methods

[0800] Harvest target cells with cell dissociation buffer (Gibco, #13151-014), wash with PBS, and use flat-bottom 96-well plates with a density of 20.000 cells / well for plating. Make cells adhere overnight. Prepare peripheral blood mononuclear cells (PBMC) from fresh blood obtained from healthy donors by Histopaque density centrifugation. Fresh blood is diluted with sterile PBS and layered on Histopaque gradient (Sigma, #H8889). After centrifugation (450x g, 30 minutes, room temperature), discard the plasma above the PBMC interphase, and transfer PBMC to a new falcon tube, then fill with 50ml PBS. The mixture is centrifuged (400x g, 10 minutes, room temperature), discard the supernatant, and the PBMC precipitate is washed twice with sterile PBS (centrifugation step 350x g, 10 minutes). The obtained PBMC colony is automatically counted (from Beckman Coulter's ViCell XR cell counter) and frozen in RPMI1640 culture medium containing 10% FBS and 1% L-alanyl-L-glutamine (Biochrom, K0302) and 5% dimethyl sulfoxide (DMSO) until further use. For killing determination, antibodies are added in triplicate at a specified concentration. PBMCs are thawed the day before the determination and cultured at 37°C in a humidified incubator with 2mio cells / ml RPMI1640 culture medium containing 10% FBS and 1% L-alanyl-L-glutamine (Biochrom, K0302). On the same day, all TCB constructs were set up twice, one vial of each molecule was untreated, and the other vial was pre-activated overnight at room temperature by adding 1 μl of protein lysate (Enzo about 2.5U / μl, ALX-201-246-U25, batch number 12152015). The next day, PBMCs were added to the target cells with a final effector to target (E:T) ratio of 5:1 (100.000 cells / well). According to the manufacturer's protocol (LDH detection kit, Roche Applied Science, #11 644 793001), after incubation for 72 hours at 37°C, 5% CO2, target cell killing was assessed by quantitatively releasing LDH from apoptotic / necrotic cells into the cell supernatant. The maximum lysis of target cells (maximum release: MR) (=100%) was achieved by incubating target cells with 1% Triton X-100 for more than 1 hour. Minimal lysis (spontaneous release: SR) of target cells (=0%) refers to target cells co-incubated with effector cells without bispecific construct.

[0801] CD4 TCB-mediated activation was assessed by flow cytometry after 72 h of incubation using antibodies recognizing the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker). + and CD8 + Activation of T cells.For this reason, PBMC is transferred to a round-bottom 96-well plate, centrifuged at 350x g for 5 min, and washed twice with FACS buffer (PBS, 2% FBS, 5mMEDTA, 0.025% NaN3). Surface staining of CD4 APC (#300514, BioLegend), CD8FITC (#344704, BioLegend), CD25BV421 (#302630, BioLegend) and CD69 PE (#310906, BioLegend) is carried out according to the instructions of the supplier. Cells are washed twice with 150 μl / well FACS buffer, and 50 μl / well FACS buffer + 4% paraformaldehyde (PFA) is used to fix overnight at 4 ° C. After centrifugation, samples are washed with 150 μl / well FACS buffer, and are resuspended in 100 μl / well FACS buffer for FACS analysis. Samples are analyzed using BD Fortessa.

[0802] Example 16. Binding of Trop2 proTCB constructs to T cells

[0803] We evaluated the ability of Trop2 proTCB (SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100) and their controls (unmasked Trop2 TCB, non-cleavable Trop2 proTCB) to bind to CD3 on the human reporter T cell line Jurkat NFAT compared to the corresponding protease pre-cleaved molecules. Both protease pre-cleaved Trop2 proTCB constructs and unmasked Trop2 TCB (with or without protease pre-cleavage) bound CD3 comparably well, indicating successful linker cleavage and subsequent release of the mask ( Fig.13 ). Non-cleavable Trop2 and masked Trop2proTCB did not bind CD3 on Jurkat T cells, indicating effective blocking of CD3 binding by the masking agent. ( Fig.13 ).

[0804] 17.1 Methods

[0805] 17.1.1 Cell lines

[0806] Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS176501) are a human acute lymphoblastic leukemia reporter cell line with a NFAT promoter that expresses human CD3. The cells were cultured at 0.1-0.5 mio cells / ml in RPMI1640, 2 g / l glucose, 2 g / l NaHCO 3 The cells were cultured in 1% FBS, 25 mM HEPES, 2 mM L-glutamine, 1x NEAA, and 1x sodium pyruvate. Whenever the cells were passaged, hygromycin B (Sigman, #10834555001) was added at a final concentration of 200 μg / ml.

[0807] 17.1.2 CD3 Binding by Flow Cytometry

[0808] One day before the start of the assay, the TCB constructs were set up twice, one vial of each molecule was untreated, and the other vial was pre-activ...

Claims

1. A protease-activatable T cell-activating bispecific molecule comprising (a) a first antigen binding portion that is capable of binding to CD3; (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 linked 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 concealing the first antigen binding moiety or the second antigen binding moiety, The peptide linker comprises a protease recognition sequence XQARK (SEQ ID NO: 39), wherein X is histidine (H) or proline (P).

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 conceals the first antigen binding moiety.

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 according to any one of claims 1 to 3, wherein the masking moiety is a scFv.

5. The protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 4, 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 the variable regions or constant regions of the Fab light chain and the Fab heavy chain are exchanged.

6. The protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 5, wherein the first antigen binding moiety is a conventional Fab molecule. 7 . The protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 6 , comprising a third antigen binding portion which is a Fab molecule capable of binding to a target cell antigen.

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

9. The protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 8, wherein the first antigen binding moiety and the second antigen binding moiety are fused to each other, optionally via a peptide linker.

10. The protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 9, 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 protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 10, further comprising an Fc domain composed of a first subunit and a second subunit capable of stably associating. 12 . The protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 11 , wherein the Fc domain is an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain.

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

15. The protease-activatable T cell activating bispecific molecule according to 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 protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 13, wherein the antigen binding portion capable of binding to CD3 comprises: a heavy chain variable (VH) region comprising (a) heavy chain complementarity determining region (HCDR) 1 amino acid sequence of SYAMN (SEQ ID NO: 1); (b) HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 2); (c) HCDR3 amino acid sequence of HTTFPSSYVSYYGY (SEQ ID NO: 4); and a light chain variable (VL) 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 GTNCRAP (SEQ ID NO: 8); and (f) LCDR3 amino acid sequence of ALWYSNLWV (SEQ ID NO: 9).

17. The protease-activatable T cell activating bispecific molecule according to 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 protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 17, wherein the masking moiety comprises: a VH region comprising: (a) the HCDR1 amino acid sequence of DYSMN (SEQ ID NO: 15), (b) a 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); and a VL region comprising: (d) 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); and (f) LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO: 28) or QQSREFPYT (SEQ ID NO: 29).

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

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

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

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

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

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

25. The protease-activatable T cell activating bispecific molecule according to any one of claims 1 to 24, wherein 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.

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

27. The protease-activatable T cell activating bispecific molecule according to 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 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.

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

29. The protease-activatable T cell activating bispecific molecule according to 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. A pharmaceutical composition comprising the protease-activatable T cell activating bispecific molecule according to 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 according to any one of claims 1 to 30.

33. A vector, in particular an expression vector, comprising the polynucleotide according to claim 32.

34. A host cell comprising the vector according to claim 33.

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

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

37. The protease-activatable T cell activating bispecific molecule for use according to claim 36, wherein the medicament is for treating cancer or delaying its progression, treating immune-related diseases or delaying their progression, or enhancing or stimulating an immune response or function in an individual.

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

39. Use of the protease-activatable T cell activating bispecific molecule according to claim 38, wherein the disease is cancer.

40. A method of treating a disease in an individual, the method 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. The method of claim 40, for treating or delaying the progression of cancer.

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