Activatable Polypeptide Complexes

Abstract

The present disclosure relates to activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complexes (HBPCs) and methods of making and using same.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 256,410, filed October 15, 2021, and U.S. Provisional Application No. 63 / 370,895, filed August 9, 2022, which are incorporated by reference in their entireties herein.

[0002] Reference to sequence listing submitted electronically via EFS WEB The contents of the electronically submitted sequence listing (4681_001PC02_Seqlisting_ST26.xml, size: 179,444 bytes, and creation date: October 13, 2022) submitted with this application are incorporated by reference in their entirety into this specification.

[0003] The present disclosure relates to activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complexes (HBPCs) and methods of making and using same. [Background technology]

[0004] The development and activation of tumor antigen-specific T cells are involved in immune-mediated control of development and antitumor activity. This requires multiple T cell costimulatory receptors and T cell negative regulators, i.e., co-inhibitory receptors, to act in concert to control T cell activation, proliferation, and gain or loss of effector function. Tumor-specific T cell responses are difficult to initiate and maintain in cancer patients due to immune evasion mechanisms of tumor cells. However, attempts have been made to harness T cells for cancer therapy. Such approaches include using T cell-inducing bispecific antibodies that bind both surface target antigens on cancer cells and T cell surface antigens, such as CD3, on T cells. In general, by binding to each target, the T cell-inducing bispecific keeps the T cell in physical proximity to the cancer cell, allowing T cell proteins and enzymes to attack the tumor cell and cause apoptosis, thereby killing the cancer cell.

[0005] The epidermal growth factor receptor (EGFR), a receptor transmembrane glycoprotein that exhibits intrinsic tyrosine kinase activity, regulates numerous cellular processes, including but not limited to activation of signal transduction pathways that control cell proliferation, differentiation, cell survival, apoptosis, angiogenesis, mitosis, and metastasis (Atalay et al., Ann. Oncology 14:1346-1363 (2003); Tsao and Herbst, Signal 4:4-9 (2003); Herbst and Shin, Cancer 94:1593-1611 (2002); Modjtahedi et al., Br. J. Cancer 73:228-235 (1996)). Overexpression of EGFR is associated with numerous human cancers, including those of the bladder, brain, head and neck, pancreas, lung, breast, ovary, colon, prostate, and kidney. EGFR is also expressed on cells of normal tissues at lower levels than it is expressed on malignant cells.

[0006] Bispecific antibodies that bind to EGFR and CD3 suffer from drawbacks including T cell-mediated toxicity (i.e., cytokine release) and EGFR-related toxicity due to off-tumor binding. Furthermore, manufacturing challenges arise due to the complex structure of bispecific antibodies and high aggregation levels during production and scale-up. Thus, there is a need for immunotherapy options with improved manufacturability while at the same time improving safety profiles. Summary of the Invention

[0007] The present disclosure provides an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC), comprising: (a) a single chain variable fragment (scFv) comprising: (i) a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3) targeting domain that specifically binds to a CD3 polypeptide; (ii) a first masking moiety (MM1); (iii) a first cleavable moiety (CM1); (iv) a second heavy chain variable domain (VH1); (b) a first polypeptide comprising (i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, (ii) a second masking moiety (MM2), and (iii) a second cleavable moiety (CM2); and (c) a third polypeptide comprising (i) the second monomeric Fc domain (Fc2) and (ii) no immunoglobulin variable domain. In some aspects, the CD3 polypeptide is the epsilon chain of CD3.

[0008] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, the VH1 comprises (i) a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), and the VL1 comprises (i) a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), (ii) a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and (iii) a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8).

[0009] In some aspects, the scFv comprises a VH1 having an amino acid sequence at least 90% identical to SEQ ID NO: 9 and / or a VL1 having an amino acid sequence at least 90% identical to SEQ ID NO: 10. In some aspects, the scFv comprises a VH1 having the amino acid sequence of SEQ ID NO: 9 and a VL1 having the amino acid sequence of SEQ ID NO: 10.

[0010] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, the VH2 comprises (i) a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), (ii) a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and (iii) a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17). In some embodiments, the VH2 comprises an amino acid sequence at least 90% identical to SEQ ID NO: 21. In some embodiments, the VH2 comprises the amino acid sequence of SEQ ID NO: 21.

[0011] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide disclosed herein, Fc1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 23. In some embodiments, Fc1 comprises the amino acid sequence of SEQ ID NO: 23.

[0012] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, the first polypeptide further comprises a heavy chain CH1 domain between VH2 and Fc1. In some embodiments, the first polypeptide further comprises an immunoglobulin hinge region between VH2 and Fc1. In some embodiments, the first polypeptide comprises, from amino terminus to carboxy terminus, the structural arrangement MM1-CM1-scFv-VH2-CH1-hinge region-Fc1, where each "-" is independently a direct or indirect linkage.

[0013] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, the first polypeptide comprises one or more linkers. In some embodiments, the linker comprises from about 1 to about 20 amino acids.

[0014] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, the VL2 comprises (i) a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), (ii) a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and (iii) a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO: 20). In some embodiments, the VL2 comprises an amino acid sequence at least 90% identical to SEQ ID NO: 22. In some embodiments, the VL2 comprises the amino acid sequence of SEQ ID NO: 22.

[0015] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, the second polypeptide comprises, from amino terminus to carboxy terminus, the structural arrangement MM2-CM2-VL2, where each "-" is independently a direct or indirect linkage. In some embodiments, the second polypeptide comprises one or more linkers. In some embodiments, the linker comprises about 1 to about 20 amino acids.

[0016] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide disclosed herein, Fc2 binds to Fc1. In some embodiments, Fc2 comprises an amino acid sequence at least 90% identical to SEQ ID NO: 28. In some embodiments, Fc2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, Fc2 comprises the amino acid sequence of SEQ ID NO: 29.

[0017] In some aspects of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, at least one of the first and third polypeptides further comprises an immunoglobulin hinge region. In some aspects, the first and third polypeptides comprise an immunoglobulin hinge region. In some aspects, the immunoglobulin hinge region of the first polypeptide and the immunoglobulin hinge region of the third polypeptide comprise the same amino acid sequence. In some aspects, the immunoglobulin hinge region of the first polypeptide and the immunoglobulin hinge region of the third polypeptide comprise different amino acid sequences.

[0018] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, the third polypeptide comprises, from amino terminus to carboxy terminus, an immunoglobulin hinge region in the structural configuration hinge region-Fc2.

[0019] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, the first polypeptide, the second polypeptide, and / or the third polypeptide comprise one or more linkers. In some embodiments, MM1 is linked to CM1 via linker L1. In some embodiments, MM2 is linked to CM2 via linker L2. In some embodiments, the amino acid sequences of L1 and L2 are the same. In some embodiments, the amino acid sequences of L1 and L2 are different.

[0020] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, CM1 and CM2 each comprise a substrate for a protease present in the tumor microenvironment of a subject with cancer. In some embodiments, CM1 and CM2 each comprise a substrate for the same protease. In some embodiments, CM1 and CM2 comprise a substrate for a different protease. In some embodiments, CM1 and CM2 each independently comprise a substrate for a protease selected from the group of proteases shown in Table 2. In some embodiments, at least one of CM1 and CM2 comprises a substrate for a serine protease or a matrix metallopeptidase (MMP). In some embodiments, CM1 comprises the amino acid sequence of SEQ ID NO:2 and / or CM2 comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, CM1 comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, CM2 comprises the amino acid sequence of SEQ ID NO:14.

[0021] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, MM1 and / or MM2 comprise from about 5 amino acids to about 40 amino acids. In some embodiments, MM1 is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72. In some embodiments, MM2 comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, wherein MM1 comprises the amino acid sequence of SEQ ID NO:1.

[0022] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, at least one of the one or more linkers is (i) (GS)n, where n is an integer of at least 1; (GGS)n, where n is an integer of at least 1 (e.g., an integer of about 1 to about 20, or about 1 to about 10); (GGGS)n (SEQ ID NO:40), where n is an integer of at least 1 (e.g., an integer of about 1 to about 20, or about 1 to about 10); (GGGGS)n (SEQ ID NO:126), where n is an integer of at least 1 (e.g., an integer of about 1 to about 20, or about 1 to about 10); (GSGGS)n (SEQ ID NO:41) (wherein n is an integer of at least 1) (for example, an integer of about 1 to about 20, or about 1 to about 10), GSSGGSGGSG (SEQ ID NO:12), GGSG (SEQ ID NO:42), GGSGG (SEQ ID NO:43), GSGSG (SEQ ID NO:44), GSGGG (SEQ ID NO:45), GGGSG (SEQ ID NO:46), and GSSSG (SEQ ID NO:47), GGGGSGGGGSGGGGSGS (SEQ ID NO:48), GGGGSGS (SEQ ID NO:49), 9), a glycine-serine based linker selected from the group consisting of GGGSGGGGSGGGGS (SEQ ID NO:50), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:51), GGGGS (SEQ ID NO:52), GGGGSGGGGS (SEQ ID NO:53), GGGS (SEQ ID NO:54), GGGSGGGS (SEQ ID NO:55), GGGSGGGSGGGS (SEQ ID NO:56), GSSGGSGGSGG (SEQ ID NO:57), GGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:58), GGGSSGGS (SEQ ID NO:127), and GS. and (ii) glycine and serine, and lysine, threonine, selected from the group consisting of GSTSGSGKPGSSEGST (SEQ ID NO: 59), SKYGPPCPPCPAPEFLG (SEQ ID NO: 60), GGSLDPKGGGGS (SEQ ID NO: 61), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 62), GKSSGSGSESKS (SEQ ID NO: 63), GSTSGSGKSSEGKG (SEQ ID NO: 64), GSTSGSGKSSEGSGSTKG (SEQ ID NO: 65), and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 66).or a linker comprising at least one of proline.

[0023] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO:30, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO:31, and (3) the third polypeptide comprises the amino acid sequence of SEQ ID NO:32.

[0024] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides disclosed herein, (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 120, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO: 37, and (3) the third polypeptide comprises the amino acid sequence of SEQ ID NO: 32.

[0025] Disclosed herein are pharmaceutical compositions comprising an activatable bispecific polypeptide complex as disclosed herein and a pharma- ceutically acceptable carrier.

[0026] Also disclosed herein are kits that include a pharmaceutical composition comprising an activatable bispecific polypeptide complex as disclosed herein and a pharma- ceutically acceptable carrier.

[0027] Also disclosed herein are nucleic acids comprising nucleotide sequences encoding the first, second, and third polypeptides of the activatable bispecific polypeptides described herein.Further provided herein are vectors comprising the described nucleic acids and host cells comprising the vectors.

[0028] Also disclosed herein are methods of producing an activatable bispecific polypeptide complex, the methods comprising: (a) culturing a host cell in a liquid medium under conditions sufficient to produce an activatable bispecific polypeptide complex; and (b) recovering the activatable bispecific polypeptide complex.

[0029] Also disclosed herein is a method of treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an activatable bispecific polypeptide complex as described herein or a pharmaceutical composition as described herein. In some aspects, the subject is a human. In some aspects, the disease is cancer. In some aspects, the activatable bispecific polypeptide or pharmaceutical composition is for use in inhibiting tumor growth in a subject in need thereof.

[0030] Also disclosed herein is the use of an activatable bispecific polypeptide complex as described herein or a pharmaceutical composition as described herein in the manufacture of a medicament for treating cancer. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 is a schematic diagram of an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) described herein. [Figure 2A] Binding to EGFR by CI106 (activatable double-arm bivalent anti-CD3, anti-EGFR bispecific antibody control), complex-57 (activatable HBPC), and complex-67 (activatable HBPC), as well as activated CI106, activated complex-57, and activated complex-67, are shown. [Figure 2B] Binding to CD3 by CI106 (control), complex-57 (activatable HBPC), complex-67 (activatable HBPC), and activated CI106, activated complex-57, and activated complex-67 are shown. [Figure 3A] 1 shows the cytotoxic effect on HT29 cells following treatment with activated CI106 (control), activated complex-57, and activated complex-67, as well as CI106 (a double-arm bivalent bispecific control construct) and complex-57. [Figure 3B]Cytotoxicity against HT29 cells following treatment with CI106 (control), complex-67, activated CI106 (control), and activated complex-67 is shown. [Figure 4] 1 shows tumor volume in an HT29-luc2 xenograft tumor model as a function of time following treatment with vehicle, 1.0 mg / kg CI106 (control), and 0.2, 0.6, and 1.8 mg / kg conjugate-67. [Diagram 5] 1 shows tumor volume in a HCT116 xenograft tumor model as a function of time following treatment with vehicle, 0.3 mg / kg and 1 mg / kg of activated conjugate-67 and conjugate-67. [Figure 6] Percentage of monomer (%) versus concentration for CI106 (control), conjugate-57, and conjugate-67 are shown. [Figure 7] AC show flow cytometric assessment of CI107 binding to EGFR and CD3 expressed on the surface of HT29 cells (A), HCT116 cells (B), or Jurkat cells (C). Apparent Kd was calculated from duplicate experiments in HT29 cells and triplicate experiments in Jurkat cells. [Figure 8A] Figure 1 shows the percentage of cytotoxicity mediated by CI107 in HCT116-Luc2 cells. After 48 hours of culture, cell viability and cytotoxicity of HCT116-Luc2 were measured compared to untreated controls. [Figure 8B] Figure 1 shows the percentage of cytotoxicity mediated by CI107 in HT29-Luc2 cells. After 48 hours of culture, cell viability and cytotoxicity of HT29-Luc2 were measured relative to untreated controls. [Figure 8C] Figure 1 shows the percentage of cytotoxicity mediated by CI107 in HCT116-Luc2 cells. After 16 h of culture, CD69 expression was measured by flow cytometry. MFI, mean fluorescence intensity. [Figure 8D]Figure 1 shows the percentage of cytotoxicity mediated by CI107 in HT29-Luc2 cells. After 16 h of culture, CD69 expression was measured by flow cytometry. MFI, mean fluorescence intensity. [Figure 9A] Cytokine release following treatment with CI107 is shown (IFN-γ), measured after 16 hours of culture. [Figure 9B] Cytokine release following treatment with CI107 is shown (IL-2), measured after 16 hours of culture. [Figure 9C] Cytokine release following treatment with CI107 is shown (IL-6), measured after 16 hours of culture. [Figure 9D] Cytokine release following treatment with CI107 is shown (MCP-1), measured after 16 hours of culture. [Figure 9E] Cytokine release following treatment with CI107 is shown (TNF-α), measured after 16 hours of culture. [Figure 10] A-B show tumor volumes after treatment with test TCBs in mice bearing HT29-Luc2 tumors and engrafted with human PBMCs. (A) Mice were treated weekly for 3 weeks with vehicle (PBS) or 0.3 mg / kg CI020, CI011, CI040, or CI048 (n=8 per group). Tumor volumes were measured twice weekly. (B) NSG mice bearing HT29-Luc2 tumors and engrafted with human PBMCs were treated with vehicle or 1 mg / kg CI020, CI011, CI040, or CI048. Tumors were harvested 7 days after dosing and immunohistochemistry for CD3 was performed. Dark staining indicates CD3+ cells. [Figure 11] A-B show tumor volumes following weekly treatment with CI107 for 3 weeks in HT29 (A) and HCT116 (B) xenograft tumors. Tumor volumes were measured twice weekly. *p<0.5, **p<0.01, ****p<0.0001. [Figure 12A] IL-6 levels measured 8 hours after administration of CI107 are shown. [Figure 12B]IFN-γ levels measured 8 hours after administration of CI107 are shown. [Figure 12C] Aspartate aminotransferase (AST) levels measured by serum biochemistry analysis 48 hours after administration of CI107 are shown. [Figure 12D] 1 shows plasma concentrations of Act-CI107 and CI107 measured by ELISA using anti-idiotypic capture and anti-human Fc detection. The CI107 line represents data from three individual animals dosed with 2.0 mg / kg CI107, and the Act-TCB line represents a single animal dosed with 0.06 mg / kg or 0.18 mg / kg Act-TCB. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly defined herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0033] definition As used herein, the terms "heteromultimeric bispecific polypeptide complex" and "HBPC" are used interchangeably herein to refer to a set of polypeptides that together form a complex having binding domains capable of binding to two different biological targets.

[0034] The term "activatable" when used in connection with the term "heteromultimeric bispecific polypeptide complex" or "HBPC" refers herein to an HBPC whose binding activity is impaired by the presence of a masking moiety added to the structure of the HBPC. The terms "activated" and "act-" may each be used to refer to activated HBPC. The terms "activated" and "unmasked" are used interchangeably herein.

[0035] As used herein, the term "EGFR" refers to a member of the receptor, transmembrane glycoprotein, and protein kinase superfamily. Human epidermal growth factor receptor is a 170 kDa transmembrane receptor encoded by the c-erb B-1 proto-oncogene, which exhibits intrinsic tyrosine kinase activity (Modjtahedi et al., Br. J. Cancer 73:228-235 (1996); Herbst and Shin, Cancer 94:1593-1611 (2002)). There are also known isoforms and variants of EGFR (e.g., alternative RNA transcripts, truncated versions, polymorphisms, etc.), which are contemplated for use herein. EGFR regulates many cellular processes through tyrosine kinase-mediated signaling pathways, including but not limited to activation of signaling pathways that control cell proliferation, differentiation, cell survival, apoptosis, angiogenesis, mitosis, and metastasis (Atalay et al., Ann. Oncology 14:1346-1363 (2003); Tsao and Herbst, Signal 4:4-9 (2003); Herbst and Shin, Cancer 94:1593-1611 (2002); Modjtahedi et al., Br. J. Cancer 73:228-235 (1996)). Overexpression of EGFR is associated with many human cancers, including cancers of the bladder, brain, head and neck, pancreas, lung, breast, ovary, colon, prostate, and kidney. EGFR is also expressed in cells of normal tissues at lower levels than it is expressed in malignant cells.Exemplary anti-EGFR antigen binding proteins include human wild-type EGFR (NCBI accession no. NG_007726.E), human wild-type EGFR transcript variant 1 (NCBI accession no. NP_005219.2), human wild-type EGFR transcript variant 2 (NCBI accession no. NP_958439.1), human wild-type EGFR transcript variant 3 (NCBI accession no. NP_958440.1), human wild-type EGFR transcript variant 4 (NCBI accession no. NP_958441.1), and human wild-type EGFR transcript variant 5 (NCBI accession no. NP_958442.2). Ant5 (NCBI accession number NP_001333826.1), human wild-type EGFR transcript variant 6 (NCBI accession number NP_001333827.1), human wild-type EGFR transcript variant 7 (NCBI accession number NP_001333828.1), human wild-type EGFR transcript variant 8 (NCBI accession number NM_001346941.2), human wild-type EGFR transcript variant EGFRvIII (NCBI accession number NP_001333870.1), and the like.

[0036] As used herein, the term "CD3" or "cluster of differentiation 3" refers to a six-chain protein complex that is a subunit of the T cell receptor complex (Janeway et al., p. 166, 9). thed.). The TCR α:β heterodimer associates with the CD3 subunit to complete the TCR cell surface antigen receptor. Two CD3ε, CD3γ, and CD3δ chains, as well as a homodimer of the CD3ζ chain, complete the T cell receptor complex, which is involved in the recognition of peptides bound to major histocompatibility complex classes I and II, and influences T cell activation. The CD3 antigen is expressed on a subset of mature T lymphocytes and thymocytes. The CD3 targeting domains that specifically bind to the CD3 polypeptides disclosed herein can be 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 CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 resulting from processing in cells. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. The anti-CD3 targeting domains described herein are capable of specifically binding to human wild-type CD3E (NCBI Accession No. NM_000733.3).

[0037] As used herein, the term "T cells" is defined as thymus-derived lymphocytes that are involved in a variety of cell-mediated immune responses. As used herein, the term "regulatory T cells" refers to CD4 + CD25 + FoxP3 + "Treg" refers to regulatory T cells. "Treg" is the abbreviation used herein for regulatory T cells.

[0038] As used herein, the term "helper T cells" refers to CD4 + Helper T cells recognize antigens bound to MHC class II molecules. There are at least two types of helper T cells, Th1 and Th2, which produce different cytokines. Helper T cells express CD25 + However, FoxP3 + This is only a temporary phenomenon.

[0039] As used herein, the term "cytotoxic T cells" refers to CD8 + This refers to T cells. Cytotoxic T cells recognize antigens bound to MHC class I molecules.

[0040] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antigen-binding protein (e.g., an antibody) that is involved in binding the antigen-binding protein (e.g., an antibody) to an antigen. The variable regions or domains of the heavy and light chains (VH and VL, respectively) of an antigen-binding protein such as an antibody can be further subdivided into regions of hypervariability (or hypervariable regions (which can be highly variable in the sequence and / or configuration of structurally defined loops)), such as hypervariable regions (HVRs) or complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Generally, there are three HVRs (HVR-H1, HVR-H2, HVR-H3) or CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three HVRs (HVR-L1, HVR-L2, HVR-L3) or CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. The terms "framework region" and "FR" are known in the art to refer to the non-HVR or non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region. Within each VH and VL, the three HVRs or CDRs and four FRs are typically arranged in the following order from amino terminus to carboxy terminus: FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4 for HVRs, or FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 for CDRs (see also Chothia and Lesk J. Mot. Biol., 195, 901-917 (1987)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using a VH or VL domain from an antibody that binds to that antigen to screen a library of complementary VL or VH domains, respectively.See, e.g., Portolano et al. J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0041] The term "heavy chain variable region" (VH) as used herein refers to a region that comprises heavy chain HVR-H1, FR-H2, HVR-H2, FR-H3, and HVR-H3. For example, a heavy chain variable region may comprise heavy chain CDR-H1, FR-H2, CDR-H2, FR-H3, and CDR-H3. In some aspects, the heavy chain variable region also comprises at least a portion of FR-H1 and / or at least a portion of FR-H4.

[0042] As used herein, the term "heavy chain constant region" refers to at least three heavy chain constant domains, i.e., C H 1. C H 2, and C H 3. Non-limiting example heavy chain constant regions include gamma, delta, and alpha. Non-limiting example heavy chain constant regions also include epsilon and mu.

[0043] As used herein, the term "light chain variable region" (VL) refers to a region that comprises light chain HVR-L1, FR-L2, HVR-L2, FR-L3, and HVR-L3. In some aspects, the light chain variable region comprises light chain CDR-L1, FR-L2, CDR-L2, FR-L3, and CDR-L3. In some aspects, the light chain variable region also comprises FR-L1 and / or FR-L4.

[0044] As used herein, the term "light chain constant region" refers to a light chain constant domain, i.e., L Non-limiting example light chain constant regions include lambda and kappa.

[0045] The term "light chain" (LC) as used herein refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some aspects, a light chain comprises at least a portion of a light chain constant region. The term "full-length light chain" as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0046] The term "antibody" refers to an immunoglobulin molecule, or an immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen-binding site that specifically binds (immunoreacts with) an antigen. An "antigen-binding portion" (also called an "antigen-binding fragment") of an antibody or polypeptide refers to one or more portions of an antibody or polypeptide that specifically binds to a target antigen. Antibodies and antigen-binding portions include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, domain antibodies, single-chain antibodies, Fab and F(ab')2 fragments, scFv, Fd fragments, Fv fragments, single-domain antibody (sdAb) fragments, dual-affinity re-targeting antibodies (DART), dual variable domain immunoglobulins; isolated complementarity determining regions (CDRs), and combinations of two or more isolated CDRs, which may optionally be linked by synthetic linkers, and Fab expression libraries. Non-human antibodies, e.g., camelid antibodies, may be humanized by recombinant methods to reduce their immunogenicity in humans.

[0047] The CDR sequences designated herein are determined according to the Kabat numbering system as described in Abhinandan, KRand Martin, ACR (2008) "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains", Molecular Immunology, 45, 3832-3839, which is incorporated herein by reference in its entirety (i.e., the "Kabat CDRs"). The Kabat CDRs are defined as follows: CDR-L1: residues L24-L34; CDR-L2: residues L50-L56; CDR-L3: residues L89-L97; CDR-H1: residues H31-H35; CDR-H2: residues H50-H65; and CDR-H3: residues H95-H102, where "L" refers to the light chain variable domain and "H" refers to the heavy chain variable domain.

[0048] "Specifically binds" or "immunospecifically binds" means that a targeting domain, antibody or antigen-binding fragment reacts with one or more antigenic determinants of a desired antigen and does not react with other polypeptides or with a much lower affinity (Kd>10 -6 ) (a smaller Kd indicates a higher affinity). The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of antigen-binding site / antigen complexes, where the rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on-rate constant" (k on ) and the "off rate constant" (k off ) can be determined by calculation of the concentration and the actual rates of association and dissociation (see Nature 361:186-87 (1993)). off / k onThe ratio of Kd allows for the cancellation of all parameters not related to affinity and is equal to the dissociation constant Kd (see generally Davies et al. (1990) Annual Rev Biochem 59:439-473). In some embodiments, antigen targeting domains, antibodies, or antigen-binding fragments that specifically bind to a corresponding antigen exhibit a Kd for the target antigen of less than about 10 μM, and in some embodiments less than about 100 μM.

[0049] Immunoglobulins may be derived from any of the known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those of skill in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.

[0050] An "anti-antigen" antibody or polypeptide refers to an antibody or polypeptide that specifically binds to an antigen of interest. For example, an anti-CD3 polypeptide specifically binds to CD3.

[0051] As used herein, the terms "MM" and "masking moiety" are used interchangeably herein to refer to a peptide that prevents a targeting domain from binding to its corresponding antigen. For example, MM1 is a peptide that prevents a first targeting domain from binding to a first target, and MM2 is a peptide that prevents a second targeting domain from binding to a second target. The degree to which a masking moiety prevents a targeting domain from binding to its corresponding target is quantified by its "masking efficiency". The terms "masking efficiency" and "ME" are used interchangeably herein to refer to a ratio determined as follows: ME=EC50, activatable HBPC (i.e., not cleaved by proteases) JPEG2024539650000001.jpg3159EC50, activated HBPC

[0052] As used herein, the terms "CM" and "cleavable moiety" are used interchangeably to refer to a peptide substrate that is susceptible to cleavage by a protease that is upregulated in tumor cells. Protease-mediated cleavage of the CM results in the release of the MM from the structure of the activatable HBPC, thereby generating an "activated" (i.e., unmasked) product in which the corresponding "activated" (i.e., unmasked) first targeting domain and / or second targeting domain, respectively, are free to bind to their respective targets.

[0053] The term "isolated polynucleotide" as used herein refers to a polynucleotide of recombinant or synthetic origin that, by virtue of its origin, is not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide with which it is not linked in nature, or (3) is not found in nature as part of a larger sequence. Polynucleotides according to the present disclosure include nucleic acid molecules that encode a first polypeptide, a second polypeptide, and a third polypeptide.

[0054] As used herein, the term "operably linked" refers to the positioning of the components so described being in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0055] As discussed herein, minor changes in the amino acid sequences described herein (i.e., each reference sequence) are contemplated to be encompassed by the present disclosure as long as the resulting similar sequence maintains at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% sequence identity with the reference sequence. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids that are related in terms of the nature of the amino acid's side chain. Amino acids can be classified into the following families: (1) acidic amino acids are aspartic acid, glutamic acid; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) polar uncharged amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include: (i) the aliphatic hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. For example, within the HBPC polypeptides and polypeptide complexes described herein, it is reasonable to expect that single substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of an amino acid with a structurally related amino acid will not have a significant effect on the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within a CDR or framework region.Whether an amino acid change results in a functional polypeptide complex can be easily determined by assaying the specific activity of the resulting molecule, i.e., the resulting analog sequence. The assay is described in detail herein. The preferred amino and carboxy termini of the analogs are near the boundaries of the functional domains. Structural and functional domains can be identified by comparison of nucleotide and / or amino acid sequence data with public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that are present in other proteins of known structure and / or function. Methods are known for identifying protein sequences that fold into known three-dimensional structures (Bowie et al. Science 253:164 (1991)). Thus, the above examples show that one skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains according to the present disclosure.

[0056] Conservative amino acid substitutions should not substantially change the structural characteristics of the reference sequence (e.g., the replacement amino acid should not tend to break helices present in the reference sequence or disrupt other types of secondary structure that characterize the reference sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)), Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)), and Thornton et al. Nature 354:105 (1991).

[0057] Exemplary amino acid substitutions also include those that (1) reduce susceptibility to proteolysis in regions of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide other than the cleavable linker containing the CM, (2) reduce susceptibility to oxidation, (3) change the binding affinity for forming protein complexes, (4) change the binding affinity for antigen, and (4) confer or modify other physicochemical or functional properties of such analogs. Such amino acid substitutions may be identified using known mutagenesis and / or directed molecular evolution methods using the assays described herein. See, for example, International Publication No. WO2001 / 032712, U.S. Patent No. 7,432,083, U.S. Publication No. 2004 / 0180340, and U.S. Patent No. 6,297,053, each of which is incorporated herein by reference. Analogs may be prepared by introducing one or more mutations into a reference sequence within an activatable HBPC. For example, single or multiple amino acid substitutions may be made in the reference sequence, preferably in the part of the polypeptide that is outside the domain(s) that form intermolecular contacts.

[0058] As used herein, "pharmacologically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to an individual or subject without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition contained therein. A pharma-ceutically acceptable carrier or excipient is, for example, one that has met the required standards of toxicological and manufacturing testing and / or is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0059] As used herein, a "patient" includes any patient afflicted with cancer. The terms "subject" and "patient" are used interchangeably herein.

[0060] The terms "cancer," "cancerous," or "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include melanoma, such as unresectable or metastatic melanoma, leukemia, lymphoma, blastoma, carcinoma, and sarcoma. More specific examples of such cancers include chronic myelogenous leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer, gastric cancer, germ cell tumors, childhood sarcoma, sinonasal natural killer, multiple myeloma, acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CML).

[0061] As used herein, the term "tumor" refers to any mass of tissue resulting from excessive cell growth or proliferation, whether benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0062] "Administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In some aspects, the formulations are administered via parenteral routes, and in some aspects orally. Other parenteral routes include topical, epithelial, or mucosal routes of administration, for example, intranasal, vaginal, rectal, sublingual, or topical. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0063] "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject, or administration of an active agent to a subject, with the goal of reversing, alleviating, ameliorating, inhibiting, slowing the progression, onset, severity, or recurrence of symptoms, complications or pathology, or biochemical manifestations associated with a disease.

[0064] As used herein, "effective treatment" refers to a treatment that produces a beneficial effect, e.g., an improvement in at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement over baseline, i.e., an improvement over measurements or observations made before the start of therapy according to the method. A beneficial effect can also take the form of a halt, slowing, delay, or stabilization of the adverse progression of tumor markers. Effective treatment may refer to the alleviation of at least one symptom associated with cancer. Such effective treatment can, for example, reduce the patient's pain, reduce the size and / or number of lesions, reduce or prevent tumor metastasis, and / or slow tumor growth.

[0065] The term "effective amount" refers to an amount of an agent that produces a desired biological, therapeutic, and / or prophylactic result. The result can be a reduction, amelioration, palliative, relief, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to cause tumor shrinkage and / or a decrease in the rate of tumor growth (such as inhibiting tumor growth) or slow other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition may (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, slow to some extent, or stop the invasion of cancer cells into surrounding organs, (iv) inhibit, slow to some extent, or stop tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay the onset and / or recurrence of tumors, and / or (vii) alleviate to some extent one or more of the symptoms associated with cancer.

[0066] "Immune response" refers to the actions of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or by the liver, spleen, and / or bone marrow, resulting in the selective targeting, binding to, damaging, destroying, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune conditions or pathological inflammation, normal human cells or tissues.

[0067] The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any listed or cited components.

[0068] The term "and / or" as used herein should be construed as expressly disclosing each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0069] Wherever an embodiment is described herein in conjunction with the phrase "comprising," it is understood that otherwise similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0070] The term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially consisting of" can mean within one standard deviation or more than one standard deviation, as per convention in the art. Alternatively, "about" or "essentially consisting of" can mean within a range of up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (for 10%) or between 2.4 mg and 3.6 mg (for 20%). Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude, or up to 5 times, of the value. When a particular value or composition is provided in the present specification and claims, unless otherwise specified, the meaning of "about" should be assumed to be within an acceptable error range for that particular value or composition.

[0071] As described herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, should be understood to include any integer value within the recited range, and fractions thereof, where appropriate (such as tenths and hundredths of integers).

[0072] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press, and the Oxford Dictionary of Biochemistry and Molecular Biology, 2006, Oxford University Press provide those of ordinary skill in the art with a general dictionary for many of the terms used in this disclosure.

[0073] Units, prefixes, and symbols are expressed in their International System of Units (SI) accepted form. Numerical ranges are inclusive of the numbers that define the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Thus, the terms defined above are more fully defined by reference to the specification as a whole.

[0074] The schematic diagrams of activatable polypeptides of the present disclosure (e.g., FIG. 1) are not intended to be exhaustive. Other sequence elements, such as linkers, spacers, and signal sequences, may be present before, after, or between the sequence elements recited in such schematic diagrams. It should also be understood that the MM and CM may be linked to the VH of an antibody or polypeptide instead of the VL of the antibody or polypeptide, and vice versa.

[0075] Various aspects of the disclosure are described in further detail in the following subsections.

[0076] Activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complexes The present disclosure provides an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide (HBPC), comprising: (a) a first polypeptide comprising: (i) a single chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3) targeting domain that specifically binds to a CD3 polypeptide; (ii) a first masking moiety (MM1); (iii) a first cleavable moiety (CM1); (iv) a second heavy chain variable domain (VH2); and (v) a first monomeric Fc domain (Fc1). (b) a second polypeptide comprising (i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, (ii) a second masking portion (MM2), and (iii) a second cleavable portion (CM2); and (c) a third polypeptide comprising (i) a second monomeric Fc domain (Fc2) and (ii) no immunoglobulin variable domain, wherein MM1 is a peptide that prevents the CD3 targeting domain from binding to the CD3 polypeptide and MM2 is a peptide that prevents the EGFR targeting domain from binding to EGFR. As demonstrated in the Examples herein, the activatable HBPCs of the present disclosure offer advantages over activatable or masked molecules known in the art, including resistance to aggregation, low levels of concentration-dependent aggregation (which is particularly beneficial during purification where relatively high concentrations of activatable HBPC product may be generated), high potency when activated, and improved anti-tumor activity (when activated).

[0077] As described herein above, among the components present in the first polypeptide of an activatable anti-EGFR, anti-CD3 HBPC is a T cell CD3 targeting domain that comprises a single chain variable fragment (scFv) that specifically binds to a CD3 polypeptide. In some aspects, the CD3 polypeptide is the epsilon chain of CD3. In some aspects, the scFv (anti-CD3 scFv) used herein comprises a heavy chain variable domain (VH1) and a light chain variable domain (VL1).

[0078] VH1 comprises a variable heavy chain CDR1 (VH CDR1, also referred to herein as CDRH1), a variable heavy chain CDR2 (VH CDR2, also referred to herein as CDRH2), and a variable heavy chain CDR3 (VH CDR3, also referred to herein as CDRH3), and VL1 comprises a variable light chain CDR1 (VL CDR1, also referred to herein as CDRL1), a variable light chain CDR2 (VL CDR2, also referred to herein as CDRL2), and a variable light chain CDR3 (VL CDR3, also referred to herein as CDRL3).

[0079] The activatable anti-EGFR, anti-CD3 HBPCs provided herein include a masking moiety (MM). As used herein, the terms "masking moiety" and "MM" are used interchangeably herein to refer to a peptide that, when positioned proximal to a targeting domain, prevents the targeting domain from binding to its target. In some embodiments, the MM is an amino acid sequence that is linked or otherwise bound to the activatable anti-EGFR, anti-CD3 HBPC such that, upon binding to the activatable anti-EGFR, anti-CD3 HBPC, each MM reduces the ability of the activatable anti-EGFR, anti-CD3 HBPC to specifically bind to its target. In some embodiments, the MM1 blocks or reduces the ability of the activatable anti-EGFR, anti-CD3 HBPC to specifically bind to CD3. In some embodiments, the MM2 blocks or reduces the ability of the activatable anti-EGFR, anti-CD3 HBPC to specifically bind to EGFR. In some embodiments, the MM specifically binds to the antigen targeting domain(s). A suitable MM may be identified using any of a variety of known techniques.

[0080] For example, anti-EGFR masking moieties that are suitable for use in the practice of the present disclosure in connection with various antibody binding domains include any known in the art, including, for example, those described in PCT Publication Nos. WO2013 / 163631, WO2015 / 013671, WO2016 / 014974, WO2019 / 075405, and WO2019 / 213444, each of which is incorporated by reference in its entirety. Anti-CD3 masking moieties suitable for use in the practice of the present disclosure include any of those known in the art, including those described in WO2013 / 163631, WO2015 / 013671, WO2016 / 014974, WO2019 / 075405, and WO2019 / 213444, each of which is incorporated by reference in its entirety.

[0081] In some embodiments of the activatable anti-EGFR, anti-CD3 HBPCs provided herein, MM1 and / or MM2 comprise 5 amino acids to about 40 amino acids, or any range therebetween and including both 5 amino acids and 40 amino acids. As used herein, the term "MM1" refers to a masking moiety on the CD3 targeting domain. As used herein, the term "MM2" refers to a masking moiety on the EGFR targeting domain.

[0082] In some aspects of the activatable anti-EGFR, anti-CD3 HBPC provided herein, the MMI is selected from the group consisting of SEQ ID NO: 1, 67, 68, 69, 70, 71, and 72. In some aspects, MM1 comprises the amino acid sequence of SEQ ID NO: 1. In some aspects, MM2 comprises the amino acid sequence of SEQ ID NO: 13. In some aspects, MM1 comprises SEQ ID NO: 1 and MM2 is SEQ ID NO: 13. In some aspects, MM1 comprises SEQ ID NO: 72 and MM2 comprises SEQ ID NO: 13.

[0083] In some aspects of the disclosure, the single chain variable fragment comprises a heavy chain variable domain (VH1) comprising: (i) a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3); (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4); and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5); and a light chain variable domain (VL1) comprising: (i) a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6); (ii) a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7); and (iii) a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8).

[0084] In some aspects of the disclosure, VH1 comprises the amino acid sequence of SEQ ID NO: 9. In some aspects of the disclosure, VL1 comprises the amino acid sequence of SEQ ID NO: 10. In specific aspects of the disclosure, the scFv comprises the amino acid sequence of SEQ ID NO: 11 (which includes SEQ ID NOs: 9 and 10).

[0085] In some embodiments of the disclosure, VH1 comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9. In some embodiments of the disclosure, VL1 comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10.

[0086] In some embodiments of the disclosure, the first polypeptide single chain variable fragment comprises a heavy chain variable domain (VH1) comprising: (i) a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3); (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4); and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), and comprising a heavy chain variable domain at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9.

[0087] In some embodiments of the present disclosure, VL1 comprises an amino acid sequence comprising: (i) a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6); (ii) a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7); and (iii) a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8), wherein the amino acid sequence of VL1 is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10.

[0088] In some aspects, when VH1 comprises (i) a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO: 3), (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 4), and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 5), and VL1 comprises (i) a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO: 6), (ii) a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 7), and (iii) a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8), MM1 comprises the amino acid sequence of SEQ ID NO: 1.

[0089] In an alternative embodiment, the single chain variable fragment comprises a heavy chain variable domain (VH1) comprising: (i) a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128); (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129); and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130); and a light chain variable domain (VL1) comprising: (i) a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131); (ii) a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132); and (iii) a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133).

[0090] In some of these aspects of the disclosure, VH1 comprises the amino acid sequence of SEQ ID NO: 134. In certain aspects of the disclosure, VL1 comprises the amino acid sequence of SEQ ID NO: 135. In specific aspects of the disclosure, the scFv comprises the amino acid sequence of SEQ ID NO: 122 (which includes SEQ ID NOs: 134 and 135).

[0091] In some embodiments of the disclosure, VH1 comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 134. In some embodiments of the disclosure, VL1 comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 135.

[0092] In some embodiments of the disclosure, the first polypeptide single chain variable fragment comprises a heavy chain variable domain (VH1) comprising: (i) a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128); (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129); (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130); and a heavy chain variable domain at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 135.

[0093] In some embodiments of the present disclosure, the VL1 comprises an amino acid sequence comprising: (i) a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131); (ii) a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132); and (iii) a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133), wherein the amino acid sequence of VL1 is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 135.

[0094] In some of these aspects, when VH1 comprises (i) a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and VL1 comprises (i) a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), (ii) a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132), and (iii) a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133), MM1 comprises the amino acid sequence of SEQ ID NO: 72.

[0095] The EGFR targeting domain comprises a VH2 (located within a first polypeptide) and a VH1 (located within a second polypeptide), wherein VH2 comprises a variable heavy chain CDR1 (VH CDR1, also referred to herein as CDRH1), a variable heavy chain CDR2 (VH CDR2, also referred to herein as CDRH2), and a variable heavy chain CDR3 (VH CDR3, also referred to herein as CDRH3), and wherein VL2 comprises a variable light chain CDR1 (VL CDR1, also referred to herein as CDRL1), a variable light chain CDR2 (VL CDR2, also referred to herein as CDRL2), and a variable light chain CDR3 (VL CDR3, also referred to herein as CDRL3).

[0096] In some aspects of the disclosure, the EGFR-targeting heavy chain variable domain (VH2) comprises (i) a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), (ii) a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and (iii) a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17).

[0097] In some aspects of the disclosure, VH2 comprises (i) a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), (ii) a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and (iii) a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17), wherein the amino acid sequence of VH2 is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21.

[0098] In some aspects of the disclosure, VH2 comprises the amino acid sequence of SEQ ID NO:21.

[0099] In certain specific aspects of the disclosure, an activatable HBPC comprises a first polypeptide comprising: MM1 having the amino acid sequence of SEQ ID NO: 1; VH1 having a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 5; VL1 having a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 8; and VH2 comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some of these activatable HBPCs, the second polypeptide comprises MM2 having the amino acid sequence of SEQ ID NO: 13, and a VL2 comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 20.

[0100] In another specific embodiment of the disclosure, an activatable HBPC comprises a first polypeptide comprising: MM1 having the amino acid sequence of SEQ ID NO: 72; VH1 having a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 128, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 130; VL1 having a VL CDR1 having the amino acid sequence of SEQ ID NO: 131, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 132, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 133; and VH2 comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some of these activatable HBPCs, the second polypeptide comprises MM2 having the amino acid sequence of SEQ ID NO: 13, and a VL2 comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 20.

[0101] As noted above, the first polypeptide further comprises a monomeric Fc domain (Fc1). Fc domains known in the art are suitable for use in the activatable HBPCs of the present disclosure and are described in more detail herein below.

[0102] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides described herein, Fc1 comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23. In some embodiments, Fc1 comprises the amino acid sequence of SEQ ID NO: 23. In certain embodiments, Fc1 comprises the amino acid sequence of SEQ ID NO: 24.

[0103] In some embodiments of the activatable anti-EGFR, anti-CD3 HBPCs described herein, the first polypeptide further comprises a heavy chain CH1 domain disposed between the VH2 and Fc1.

[0104] In some embodiments of the activatable anti-EGFR, anti-CD3 HBPCs described herein, the first polypeptide further comprises an immunoglobulin hinge region disposed between the VH2 and Fc1. In some embodiments where a CH1 domain is present, the immunoglobulin hinge region is disposed between the CH1 and Fc1 domains.

[0105] In some embodiments of the activatable anti-EGFR, anti-CD3 HBPCs described herein, the first polypeptide comprises, from amino terminus to carboxy terminus: It includes the structural arrangement MM1-CM1-scFv-VH2-CH1-hinge region-Fc1, where each "-" is independently a direct linkage or an indirect linkage (eg, via a linker).

[0106] In some embodiments of the activatable anti-EGFR, anti-CD3 HBPCs described herein, the first polypeptide further comprises one or more optional linkers, as described in more detail herein below.

[0107] In some embodiments of the disclosure, an activatable anti-EGFR, anti-CD3 HBPC comprises a first polypeptide that includes an Fc1 having the amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 24. In some embodiments of the disclosure, an activatable anti-EGFR, anti-CD3 HBPC comprises a first polypeptide that includes a hinge region having the sequence of hinge-1 (SEQ ID NO: 34) or hinge-2 (SEQ ID NO: 35).

[0108] In some aspects of the disclosure, the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a second polypeptide comprising an EGFR-targeting light chain variable domain (VL2) comprising a VL CDR1, a VL CDR2, and a VL CDR3.

[0109] In some aspects, the disclosure provides an activatable anti-EGFR, anti-CD3 HBPC comprising a second polypeptide comprising an EGFR-targeting light chain variable domain (VL2) comprising: (i) a CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18); (ii) a CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19); and (iii) a CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO: 20).

[0110] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides described herein, the second polypeptide comprises a VL2 having an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:22.

[0111] In some aspects of the disclosure, the VL2 comprises the amino acid sequence set forth in SEQ ID NO:22.

[0112] In certain of the above-mentioned aspects of the disclosure, MM2 comprises the amino acid sequence of SEQ ID NO:13.

[0113] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides described herein, the second polypeptide can comprise, from amino terminus to carboxy terminus, the structural arrangement MM2-CM2-VL2, where each "-" is independently a direct linkage or an indirect linkage (e.g., via a linker).

[0114] In some embodiments of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides described herein, the second polypeptide comprises one or more linkers. In some embodiments, MM2 is linked to CM2 via a linker.

[0115] In some embodiments, the second polypeptide of an activatable anti-EGFR, anti-CD3 HBPC described herein further comprises a linker comprising from about 1 to about 20 amino acids. Linkers suitable for use in the present disclosure are discussed in more detail below.

[0116] In some embodiments, the second polypeptide further comprises a constant light chain domain (CL). Exemplary CLs include any known in the art. In some embodiments, the second polypeptide comprises a CL having the amino acid sequence of SEQ ID NO: 25. In certain of these embodiments, the second polypeptide comprises, from amino terminus to carboxy terminus, the structural arrangement of MM2-CM2-VL2-CL, where each "-" is independently a direct or indirect (e.g., via a linker) linkage.

[0117] In some aspects, the third polypeptide of an activatable HBPC described herein comprises a monomeric Fc domain (Fc2) and does not comprise an immunoglobulin variable domain.

[0118] In some aspects, an activatable anti-EGFR, anti-CD3 HBPC disclosed herein comprises a third polypeptide comprising, from amino terminus to carboxy terminus, the structural arrangement of hinge region-Fc2, where each "-" is independently a direct or indirect (e.g., via a linker) linkage. In some aspects, the third polypeptide comprises an Fc2 having an amino acid sequence comprising SEQ ID NO:28 (optionally with a C-terminal lysine, i.e., SEQ ID NO:29). In one aspect, the third polypeptide comprises a hinge comprising an amino acid sequence of SEQ ID NO:35 and an Fc2 comprising an amino acid sequence of SEQ ID NO:28 (optionally with a C-terminal lysine, i.e., SEQ ID NO:29). In certain aspects, the first polypeptide comprises a hinge comprising an amino acid sequence of SEQ ID NO:34 and an Fc1 comprising an amino acid sequence of SEQ ID NO:23 (optionally with a C-terminal lysine, i.e., SEQ ID NO:24).

[0119] As defined above, in some embodiments, the third polypeptide may include a linker, for example, between the hinge region and the second Fc domain. The linker may include any of the linkers discussed herein.

[0120] The activatable anti-EGFR, anti-CD3 HBPCs of the present disclosure are activated when the cleavable moiety is cleaved by a protease, thereby generating an activated (i.e., unmasked) anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) capable of binding to EGFR and CD3. In comparison, the activatable anti-EGFR, anti-CD3 HBPCs exhibit greatly reduced binding to EGFR and CD3 compared to the activated heteromultimeric bispecific polypeptide because the activatable HBPC remains masked until activated by proteases in the tumor environment. Without wishing to be bound by theory or mechanism, typical protease activity levels in healthy tissues are likely reduced due to the presence of endogenous inhibitors and / or protease-unfavorable pH conditions, whereas within the tumor environment protease activity is generally upregulated via upregulation of protease expression, activation of zymogens, downregulation of inhibitor expression, or a combination of these effects (see Desnoyers et al., ScienceTranslationalMedicine.org, vol. 5, Issue 207 (October 2013), incorporated herein by reference).

[0121] Thus, these activatable anti-EGFR, anti-CD3 HBPCs may be useful in treating subjects with cancers in which proteolytic activity in the tumor microenvironment is upregulated compared to normal tissues and controlled in normal tissues. The significant reduction in binding of EGFR and CD3 by activatable HBPCs in normal tissues may allow for a reduction in side effects associated with binding to anti-EGFR and anti-CD3 outside of the tumor. In some embodiments, the activatable anti-EGFR, anti-CD3 HBPCs comprise a first CM and a second CM (CM1 and CM2, respectively).

[0122] In some embodiments, CM comprises a substrate for a protease that is upregulated in tumor cells. In some embodiments of the HBPC disclosed herein, CM may comprise a substrate for two or more proteases that are upregulated in tumor cells (i.e., a first protease, a second protease, a third protease, etc.). There are reports in the literature that protease levels are increased in some cancers, e.g., liquid tumors or solid tumors. See, e.g., La Rocca et al, (2004) British J. of Cancer 90(7):1414-1421. Numerous studies have demonstrated the correlation of abnormal protease levels in solid tumors, e.g., uPA, legumain, MT-SP1, matrix metalloproteases (MMPs) (see, e.g., Murthy RV, et al. “Legumain expression in relation to clinicopathologic and biological variables in colorectal cancer,” Clin Cancer Res. 11 (2005): 2293-2299; Nielsen BS, et al. “Urokinase plasminogen activator is localized in stromal cells in ductal breast cancer,” Lab Invest 81 (2001): 1485-1501; Look OR, et al. “In situ localization of gelatinolytic activity in the extracellular matrix of metastases of colon cancer in rat liver using quenched fluorogenic DQ-gelatin,” J Histochem Cytochem. 51 (2003): 821-829). The CM may include substrates for multiple proteases, e.g., a substrate for a serine protease and a second, different protease, e.g., an MMP. In some embodiments, the CM may include substrates for more than one serine protease, e.g., matriptase and / or uPA.In some aspects, the CM may contain substrates for more than one MMP, for example, MMP9 and MMP14.

[0123] In certain embodiments, CM1 and CM2 each independently comprise an amino acid sequence that is a substrate for a protease defined in Table 1 below. [Table 1] JPEG2024539650000003.jpg31159

[0124] In some embodiments of the activatable anti-EGFR, anti-CD3 HBPC described herein, CM1 and / or CM2 comprise from about 3 amino acids to about 15 amino acids. In some embodiments, CM1 and / or CM2 may comprise two or more cleavage sites. In some embodiments, the two or more cleavage sites on CM1 may comprise a substrate for one protease. In some embodiments, the two or more cleavage sites on CM2 may comprise a substrate for two or more proteases. In some embodiments, the first protease and the second protease are the same protease. In some embodiments, CM1 and CM2 comprise different substrates for the same protease. In some embodiments, CM1 and CM2 comprise the same amino acid sequence. In some embodiments, CM1 and CM2 comprise different amino acid sequences. In some embodiments, CM1 comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, CM1 comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, CM2 comprises the amino acid sequence of SEQ ID NO: 14. In certain aspects, an activatable anti-EGFR, anti-CD3 HBPC described herein comprises a CM1 comprising the amino acid sequence of SEQ ID NO: 2 and a CM2 comprising the amino acid sequence of SEQ ID NO: 14. In some aspects, an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex described herein comprises a CM1 comprising the amino acid sequence of SEQ ID NO: 73 and a CM2 comprising the amino acid sequence of SEQ ID NO: 14.

[0125] Exemplary CMs suitable for use in the activatable anti-EGFR, anti-CD3 HBPCs described herein include CMs known in the art. Exemplary CMs include, but are not limited to, those described in Table 2, as well as International Publication Nos. WO2009 / 025846, WO2010 / 081173, WO2015 / 013671, WO2015 / 048329, WO2015 / 116933, WO2016 / 014974, and WO2016 / 118629, each of which is incorporated herein by reference in its entirety.

[0126] In some embodiments, CM1 and / or CM2 comprise an amino acid sequence set forth below in Table 2. In certain embodiments, CM1 and CM2 each independently comprise an amino acid sequence set forth below in Table 2. [Table 2] JPEG2024539650000005.jpg56159

[0127] In some aspects of the disclosure, when an activatable HBPC comprises (i) a heavy chain variable domain (VH1) comprising a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), and a VL1 comprising a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8), CM1 comprises the amino acid sequence of SEQ ID NO:2. In certain of these activatable HBPCs, MM1 comprises the amino acid sequence of SEQ ID NO:1.

[0128] In some aspects of the disclosure, when an activatable HBPC comprises (i) a heavy chain variable domain (VH1) comprising a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), and a VL1 comprising a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8), the CM1 comprises the amino acid sequence of SEQ ID NO:73. In certain of these activatable HBPCs, the MM1 comprises the amino acid sequence of SEQ ID NO:1. In some of these activatable HBPCs, the MM1 comprises the amino acid sequence of SEQ ID NO:1 and the CM1 comprises the amino acid sequence of SEQ ID NO:73.

[0129] In a specific embodiment of the present disclosure, the activatable HBPC comprises: (a) a first polypeptide comprising a first heavy chain variable domain (VH1), a first light chain variable domain (VL1), and a second heavy chain variable domain (VH2), a first masking moiety (MM1), a first cleavable moiety (CM1), and a first Fc domain (Fc1), The VH1 is (i) a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO: 3); (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 4), and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5); The VL1 is (i) a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO: 6); (ii) a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 7), and (iii) a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8); The VH2 is (i) a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15); (ii) a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and (iii) said first polypeptide comprising a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17); (b) a second polypeptide comprising a second light chain variable domain (VL2) and a second masking portion (MM2) and a second cleavable portion (CM2), The VL2 is (i) a VL CDR1 comprising RASQSIGTNIH (SEQ ID NO: 18); (ii) a VL CDR2 comprising YASESIS (SEQ ID NO: 19), and (iii) the second polypeptide comprising a VL CDR3 comprising QQNNNWPTT (SEQ ID NO: 20); and (c) a third polypeptide comprising a second Fc domain (Fc2), The Fc1 binds to Fc2, the VH1 and the VL1 together form a targeting domain that specifically binds to a CD3 polypeptide, the VH2 and the VL2 together form a targeting domain that specifically binds to EGFR; the third polypeptide does not contain an immunoglobulin variable domain; MM1 is a peptide that prevents the first targeting domain from binding to the first target; MM2 is a peptide that prevents the second targeting domain from binding to a second target; The CM1 and the CM2 each independently comprise a substrate for a protease, and the third polypeptide does not comprise an immunoglobulin variable domain. In certain aspects of the disclosure, VH1 and VL1 are arranged within an scFv. In some of these aspects, MM1 comprises SEQ ID NO: 1, CM1 comprises SEQ ID NO: 73, MM2 comprises SEQ ID NO: 13, and CM2 comprises SEQ ID NO: 14. In some of these aspects, the second polypeptide further comprises a constant light domain (CL) and the third polypeptide further comprises a hinge (HR).

[0130] In some aspects of the disclosure, when an activatable HBPC comprises (i) a VH1 comprising a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and (ii) a VL1 comprising a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence of ALWYSNLWV (SEQ ID NO: 133), the CM1 comprises the amino acid sequence of SEQ ID NO: 73. In some of these activatable HBPCs, the MM1 comprises the amino acid sequence of SEQ ID NO: 72.

[0131] In some of the above-mentioned activatable HBPCs, the first polypeptide further comprises a VH2 having a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In certain of these activatable HBPCs, the second polypeptide comprises a VL2 comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 20. In some of these HBPCs, the third polypeptide comprises the amino acid sequence of SEQ ID NO: 28 (and does not comprise an immunoglobulin variable domain).

[0132] In a specific embodiment of the present disclosure, the activatable HBPC comprises: (i) a first polypeptide comprising a first heavy chain variable domain (VH1), a first light chain variable domain (VL1), and a second heavy chain variable domain (VH2), a first masking moiety (MM1), a first cleavable moiety (CM1), and a first Fc domain (Fc1), The VH1 is (i) a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128); (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129); (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130); The VL1 is (i) a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131); (ii) a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132), and (iii) a VL CDR3 comprising the amino acid sequence of ALWYSNLWV (SEQ ID NO: 133); The VH2 is (ii) a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15); (ii) a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and (iii) said first polypeptide comprising a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17); (b) a second polypeptide comprising a second light chain variable domain (VL2) and a second masking portion (MM2) and a second cleavable portion (CM2), wherein the VL2 comprises: (i) a VL CDR1 comprising RASQSIGTNIH (SEQ ID NO: 18); (ii) a VL CDR2 comprising YASESIS (SEQ ID NO: 19), and (iii) the second polypeptide comprising a VL CDR3 comprising QQNNNWPTT (SEQ ID NO: 20); and (c) a third polypeptide comprising a second Fc domain (Fc2), wherein the Fc1 binds to the Fc2; the VH1 and the VL1 together form a targeting domain that specifically binds to a CD3 polypeptide, the VH2 and the VL2 together form a targeting domain that specifically binds to EGFR; the third polypeptide does not contain an immunoglobulin variable domain; MM1 is a peptide that prevents the first targeting domain from binding to the first target; MM2 is a peptide that prevents the second targeting domain from binding to a second target; said CM1 and said CM2 each independently comprise a substrate for a protease; The third polypeptide does not comprise an immunoglobulin variable domain. In certain aspects of the disclosure, VH1 and VL1 are arranged in an scFv. In some of these aspects, MM1 comprises SEQ ID NO: 72, CM1 comprises SEQ ID NO: 73, MM2 comprises SEQ ID NO: 13, and CM2 comprises SEQ ID NO: 22. In some of these aspects, the second polypeptide further comprises a constant light domain (CL) and the third polypeptide further comprises a hinge (HR).

[0133] In some embodiments of the activatable anti-EGFR, anti-CD3 HBPCs of the present disclosure, the first polypeptide comprises one or more linkers between MM and CM. In some embodiments, MM1 is linked to CM1 via a linker. In some embodiments, the first polypeptide comprises a linker between CM1 and VH2. In certain embodiments, the first polypeptide comprises a linker between VH2 and Fc1. In some embodiments, the first polypeptide comprises at least one linker disposed between a pair of members selected from the group consisting of MM1 and CM1, CM1 and scFv, scFv and VH2, and VH2 and Fc1. Linkers suitable for use in the activatable anti-EGFR, anti-CD3 HBPCs described herein are generally those that provide flexibility to the activatable anti-EGFR, anti-CD3 HBPC to facilitate inhibition of the activatable polypeptide from binding to a target. Such linkers are collectively referred to as flexible linkers. Suitable linkers can be readily selected and can be of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, including from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0134] Exemplary flexible linkers include glycine polymers (G), glycine-serine polymers (e.g., (GS), (GSGGS) and (GGGS) (SEQ ID NO: 41 and SEQ ID NO: 40, respectively, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively structure-indefinite and may therefore be capable of functioning as neutral tethers between components. Glycine has access to significantly more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those skilled in the art will recognize that HBPC polypeptides can be designed to include fully or partially flexible linkers, such that the linker can include one or more moieties that confer a less flexible structure while at the same time being a flexible linker to allow for desired structures.

[0135] In some aspects, the activatable anti-EGFR, anti-CD3 HBPC comprises one or more linker sequences disposed in the first polypeptide, the second polypeptide, and / or the third polypeptide. For example, in the first polypeptide, the linker is disposed between MM1 and CM1, between the heavy chain variable domain and the CH1 domain, between the CH1 domain and the hinge region (if both are present), and / or between the hinge region (if present) and the first Fc domain. In the second polypeptide (described elsewhere herein), the linker can be present, for example, between MM2 and CM2, between CM2 and the light chain variable domain, and / or between the light chain variable domain and the CL. In the third polypeptide (described elsewhere herein), the linker can be present, for example, between the CH1 domain and the second Fc domain, between the CH1 domain and the hinge region, and / or between the hinge region and the second Fc domain.

[0136] In some embodiments of the activatable anti-EGFR, anti-CD3 HBPCs described herein, MM1 is linked to CM1 via a linker L1. In some embodiments, MM2 is linked to CM2 via a linker L2. In some embodiments, the amino acid sequences of L1 and L2 are the same.In some embodiments, the linker is selected from the group consisting of (i) (GS)n (wherein n is an integer of at least 1), (GGS)n (wherein n is an integer of at least 1) (e.g., an integer of about 1 to about 20, or about 1 to about 10), (GGGS)n (SEQ ID NO:40) (wherein n is an integer of at least 1) (e.g., an integer of about 1 to about 20, or about 1 to about 10), (GGGGS)n (SEQ ID NO:126) (wherein n is an integer of at least 1) (e.g., an integer of about 1 to about 20, or about 1 to about 10), (GSGGS)n (SEQ ID NO:41) (wherein n is an integer of at least 1) (e.g., an integer of about 1 to about 20, or about 1 to about 10), In the column, n is an integer of at least 1) (e.g., an integer from about 1 to about 20, or from about 1 to about 10), GSSGGSGGSG (SEQ ID NO: 12), GGSG (SEQ ID NO: 42), GGSGG (SEQ ID NO: 43), GSGSG (SEQ ID NO: 44), GSGGG (SEQ ID NO: 45), GGGSG (SEQ ID NO: 46), and GSSSG (SEQ ID NO: 47), GGGGSGGGGSGGGGSGS (SEQ ID NO: 48), GGGGSGS (SEQ ID NO: 49), GGGGSGGGGSGGGGS (SEQ ID NO: 50), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 51), no. 51), GGGGS (SEQ ID NO: 52), GGGGSGGGGS (SEQ ID NO: 53), GGGS (SEQ ID NO: 54), GGGSGGGS (SEQ ID NO: 55), GGGSGGGSGGGS (SEQ ID NO: 56), GSSGGSGGSGG (SEQ ID NO: 57), GGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 58), GGGSSGGS (SEQ ID NO: 127), and GS; and (ii) a glycine-serine based linker selected from the group consisting of GSTSGSGKPGSSEGST (SEQ ID NO: 59), SKYGPPCPP CPAPEFLG (SEQ ID NO: 60), GGSLDPKGGGGS (SEQ ID NO: 61), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 62), GKSSGSGSESKS (SEQ ID NO: 63), GSTSGSGKSSEGKG (SEQ ID NO: 64), GSTSGSGKSSEGSGSTKG (SEQ ID NO: 65), and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 66), and linkers comprising glycine and serine, and at least one of lysine, threonine, or proline are selected from the group consisting of CPAPEFLG (SEQ ID NO: 60), GGSLDPKGGGGS (SEQ ID NO: 61), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 62), GKSSGSGSESKS (SEQ ID NO: 63), GSTSGSGKSSEGKG (SEQ ID NO: 64), GSTSGSGKSSEGSGSTKG (SEQ ID NO: 65), and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 66).

[0137] In some aspects of the present disclosure, the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide may include components in addition to those described above. Such components may include a spacer. The term "spacer" as used herein refers to an amino acid residue or peptide incorporated at the free end of the first polypeptide, the second polypeptide, and / or the third polypeptide. Spacers suitable for use in the practice of the present disclosure include any single amino acid residue or any peptide. Suitable spacers include, for example, any of those described in International Publication Nos. WO2016 / 014974, WO2019 / 075405, and WO2019 / 213444, each of which is incorporated herein by reference in its entirety.

[0138] In some embodiments, the spacer can comprise from about 1 amino acid to about 10 amino acids (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids) or any number therebetween. In some embodiments of the activatable anti-EGFR, anti-CD3 HBPCs described herein, the spacer is positioned N-terminal to MM1 and / or MM2. In some embodiments, the spacer has a sequence of QGQSGS (SEQ ID NO: 116). In some embodiments, the spacer has a sequence of QGQSGQG (SEQ ID NO: 117). In some embodiments, the spacer has a sequence of QGQSGS (SEQ ID NO: 118). In some embodiments, the spacer has a sequence of QGQSGQG (SEQ ID NO: 117).

[0139] In some embodiments, the Fc domains used as Fc1 and / or Fc2 are native Fc domains (e.g., human IgG1 Fc domains or human IgG4 Fc domains). In some embodiments of the present disclosure, the Fc domains used as Fc1 and / or Fc2 are mutants of native Fc amino acid sequences. The mutations can confer desirable beneficial properties to the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides (and correspondingly, the activated HBPCs). For example, certain mutations in the FcRn binding site are known to modulate effector functions (see, e.g., Petkova et al., Intl. Immunol. 18:1759-1769, 2006; Deng et al., MAbs 4:101-109, 2012; and Olafson et al., Methods Mol. Biol. 907:537-556, 2012). Incorporation of any known mutation in the Fc domain that can modulate effector function is suitable. For example, N297A or N297G mutations in the Fc amino acid sequence may be used to reduce the effector function (e.g., ADCC and CDC) of IgG, which can reduce target-independent toxicity (see, e.g., Lund et al., Mol. Immunol. 29:35-39, 1992). Suitable Fc domains for use in the context of the present disclosure include any Fc domain known in the art, including, but not limited to, any known heterodimeric Fc, such as knob-in-hole, etc.

[0140] In some aspects, the activatable anti-EGFR, anti-CD3 HBPC disclosed herein further comprises an immunoglobulin hinge region. Suitable hinge regions include any known hinge region. For example, hinge regions from any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) are suitable for use in the present disclosure. Different classes of immunoglobulins have different, well-known subunit structures and three-dimensional configurations.

[0141] In some aspects, the hinge regions of Fc1 of the first polypeptide and Fc2 of the third polypeptide comprise the same sequence. In some aspects, the first and second Fc domains (Fc1 and Fc2, respectively) of the activatable anti-EGFR, anti-CD3 HBPC described herein are IgG1 or IgG4 Fc domains (e.g., human IgG1 or IgG4 Fc domains), or variants thereof. In some aspects, Fc1 and / or Fc2 are modified variants of a native (e.g., human) IgG1 Fc domain. In some aspects, Fc1 and / or Fc2 are modified variants of a native (e.g., human) IgG4 Fc domain.

[0142] In some embodiments of the activatable anti-EGFR, anti-CD3 HBPCs described herein, Fc1 comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23. In some embodiments, Fc1 comprises the amino acid sequence of SEQ ID NO: 23, optionally with a C-terminal lysine (i.e., SEQ ID NO: 24).

[0143] In some embodiments, the third polypeptide further comprises a monomeric Fc domain (Fc2) that binds Fc1. In some embodiments, Fc2 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 28. In some embodiments, Fc2 comprises SEQ ID NO: 28, optionally with a terminal lysine (i.e., SEQ ID NO: 29).

[0144] In some aspects, the third polypeptide comprises a hinge region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 35.

[0145] As provided elsewhere herein, the formats or structures of the activatable anti-EGFR, anti-CD3 HBPCs disclosed herein may include any number of optional additional components, including linkers and spacers. By way of example only, the structures defined below are among the contemplated embodiments. However, the embodiments set forth below are not intended to limit the present disclosure in any way.

[0146] In some aspects, the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a first polypeptide having the structure (I): First Polypeptide Structure (I): (S1)-MM1-(L1)-CM1-L2-VH1-L3-VL1-(L4)-VH2-(L5)-(CH11)-(L6)-(Hinge 1)-(L7)-Fc1 In the structure, (S1) is an optional spacer, (L1), (L4), (L5), (L6), and (L7) are each independently an optional linker; L2 and L3 are linkers, (CH11) is an optional CH1 domain, (hinge 1) is an optional hinge region; · Fc1 is as described herein above.

[0147] In some embodiments, the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a second polypeptide having the structure (II). Second Polypeptide Structure (II): (S2)-(L8)-MM2-(L9)-CM2-(L10)-VL2-(CL) In the structure, (S2) is an optional spacer, (L8), (L9), and (L10) are each independently an optional linker; MM2 is an anti-EGFR masking moiety; VL2 is as described herein above, ·(CL) is an optional light chain constant domain.

[0148] In some embodiments, the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a third polypeptide having the structure (III). Third Polypeptide Structure (III): (S3)-(CH12)-(L11)-(hinge 2)-(L12)-Fc2 In the structure, (S3) is an optional spacer, (CH12) is an optional CH1 domain, (L11) and (L12) are each independently an optional linker; · Fc2 is as described herein above.

[0149] Linkers, spacers, MM, CM, Fc domains, CH1 (i.e., CH11 and CH12) domains, hinge regions, and CLs suitable for use in structures (I), (II), and (III) include any known in the art or described herein.

[0150] In some aspects of the disclosure, an activatable anti-EGFR, anti-CD3 HBPC comprises a first polypeptide, a second polypeptide, and a third polypeptide, where (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 (optionally with a C-terminal lysine and / or optionally without a spacer (e.g., SEQ ID NO:120 (with a terminal lysine and without a spacer)), (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO:31 (or SEQ ID NO:37 (without a spacer)), and (3) the third polypeptide comprises the amino acid sequence of SEQ ID NO:32 (optionally with a C-terminal lysine (i.e., SEQ ID NO:36) In some aspects of the disclosure, the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a first polypeptide, a second polypeptide, and a third polypeptide, where, as defined in the sequences below, (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 120, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO: 37, and (3) the third polypeptide comprises the amino acid sequence of SEQ ID NO: 32 (and does not comprise an immunoglobulin variable domain).

[0151] In some aspects of the disclosure, an activatable anti-EGFR, anti-CD3 HBPC comprises a first polypeptide, a second polypeptide, and a third polypeptide, where (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 30, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO: 31, and (3) the third polypeptide consists of or consists essentially of the amino acid sequence of SEQ ID NO: 32. In some aspects of the disclosure, an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a first polypeptide, a second polypeptide, and a third polypeptide, where, as set forth in the sequences below, (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 120, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO: 37, and (3) the third polypeptide consists of or consists essentially of the amino acid sequence of SEQ ID NO: 32, and does not include an immunoglobulin variable domain.

[0152] In some aspects of the disclosure, the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a first polypeptide, a second polypeptide, and a third polypeptide, where, as defined in the sequences below, (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 144, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO: 37, and (3) the third polypeptide consists of or consists essentially of the amino acid sequence of SEQ ID NO: 32, and does not comprise an immunoglobulin variable domain.

[0153] In the first polypeptide shown below, the spacer sequence is in brackets, the mask sequence is underlined, the linker is bolded (the linker in the scFv is also italicized and underlined), the substrate (i.e., the cleavable moiety) is italicized, and the scFv (which binds to the CD3 polypeptide) is italicized and underlined. First Polypeptide [QGQSGS] VSTTCWWDPPCTPNT GSSGGSGGSGGLSGRSDDHGGGS EVQLVESGGGLVQ PGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDD SKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGG GGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPG TPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL GGGGSQVQLKQ SGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGST YRCVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRKEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:30) (optionally with a C-terminal lysine and / or without a spacer (e.g., SEQ ID NO:137)). In some aspects, the first polypeptide has the amino acid sequence of SEQ ID NO:120 (without a spacer but with a C-terminal lysine) or the amino acid sequence of SEQ ID NO:144 (without a spacer and without a C-terminal lysine).

[0154] In the second polypeptide shown below, the spacer sequence is in brackets, the mask sequence is underlined, the linker is in bold, and the substrate (ie, the cleavable moiety) is in italics. Second Polypeptide [QGQSGQG ]LSCEGWAMNREQCRA GGGSGGSISSGLLSGRSDQHGGGSQILLTQSPVIL SVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLS INSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:31) (optionally without the spacer (SEQ ID NO:37)).

[0155] In the third polypeptide shown below, the hinge region is bold and underlined, and the remainder of the sequence is Fc2. Third Polypeptide DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPP VLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32) (optionally with a C-terminal lysine (SEQ ID NO: 36)).

[0156] kit Provided herein is a kit comprising one or more activatable anti-EGFR, anti-CD3 HBPCs or HBPCs thereof as described herein, the kit being for diagnosis or treatment. In certain embodiments, provided herein is a pack or kit comprising one or more containers filled with one or more of the components of the compositions described herein, such as one or more activatable anti-EGFR, anti-CD3 HBPCs or antigen-binding fragments thereof as provided herein, and optional instructions for use. In some embodiments, the kit contains the compositions described herein and any diagnostic, prophylactic, or therapeutic agents, such as those described herein.

[0157] Therapeutic Uses and Methods of Treatment In some embodiments, a method for treating a disease, such as cancer, is provided herein, comprising administering to a subject in need thereof an activatable anti-EGFR, anti-CD3 HBPC or HBPC thereof, or a pharmaceutical composition thereof as described herein. In some embodiments, a method for inhibiting tumor growth in a subject in need thereof is provided herein, comprising administering to a subject in need thereof an activatable anti-EGFR, anti-CD3 HBPC or HBPC thereof, or a pharmaceutical composition thereof as described herein. In some embodiments, the present disclosure relates to an activatable anti-EGFR, anti-CD3 HBPC or HBPC thereof, or a pharmaceutical composition thereof as provided herein, for use as a medicament. Usually, the subject is a human, but non-human mammals, including transgenic mammals, can also be treated.

[0158] The amount of activatable HBPC or a composition thereof that will be effective in treating a condition will depend on the nature of the disease. The precise dose to be used in the composition will also depend on the route of administration, and the severity of the disease.

[0159] Non-limiting examples of diseases include cancer, rheumatoid arthritis, Crohn's disease, SLE, cardiovascular injury, ischemia, etc. For example, indications may include leukemias, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic diseases, including multiple myeloma, and solid tumors, including lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, and breast cancer, including triple-negative breast cancer. For example, indications may include bone disease or metastasis in cancer, regardless of primary tumor origin; breast cancer, including but not limited to ER / PR+ breast cancer, Her2+ breast cancer, and triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as head and neck squamous cell carcinoma; esophageal cancer; lung cancer, including but not limited to non-small cell lung cancer; multiple myeloma, ovarian cancer; pancreatic cancer; prostate cancer; sarcomas, such as osteosarcoma; kidney cancer, including but not limited to renal cell carcinoma; and / or skin cancer, including but not limited to squamous cell carcinoma, basal cell carcinoma, or melanoma.

[0160] Polynucleotides In some aspects, provided herein are polynucleotides (correspondingly referred to herein as "first polynucleotides," "second polynucleotides," and "third polynucleotides," respectively) that comprise a nucleotide sequence encoding a first polypeptide, a second polypeptide, and / or a third polypeptide of an activatable anti-EGFR, anti-CD3 HBPC of the present disclosure. Suitable polynucleotides include any that encode any of the first polypeptides, second polypeptides, and / or third polypeptides, or portions thereof, described herein. Exemplary sets of polynucleotide sequences encoding first polypeptides, second polypeptides, and third polypeptides are described herein below.

[0161] The polynucleotides of the present disclosure may be sequence-optimized for optimal production from the host organism selected for expression, for example, by codon / RNA optimization, substitution with a heterologous signal sequence, and elimination of mRNA instability elements. Methods for generating optimized nucleic acids encoding activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides or antigen-binding fragments thereof (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., codon changes that code for the same amino acid due to the degeneracy of the genetic code) in the mRNA and / or eliminating inhibitory regions can be performed by adapting, as appropriate, the optimization methods described in, for example, U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498. nucleic acid Polynucleotide encoding the first polypeptide (SEQ ID NO:112) CAAGGACAATCTGGCTCTGTGTCCACCACCTGTTGGTGGGACCCTCCATGCACAC CTAATACCGGCAGCTCTGGTGGCTCTGGCGGAAGCGGAGGACTGTCTGGCAGAT CCGATGATCACGGCGGAGGATCTGAGGTGCAGCTGGTTGAATCTGGTGGCGGAC TGGTTCAGCCTGGCGGATCTCTGAAACTGAGCTGTGCCGCCAGCGGCTTCACCTT CAACAAATACGCCATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATG GGTCGCCAGAATCAGAAGCAAGTACAACAACTATGCCACCTACTACGCCGACAG CGTGAAGGACAGATTCACCATCAGCCGGGACGACAGCAAGAACACCGCCTACCT GCAGATGAACAACCTGAAAACCGAGGACACCGCCGTGTACTACTGTGTGCGGCA CGGCAACTTCGGCAACAGCTACATCAGCTACTGGGCCTATTGGGGCCAGGGCAC ACTGGTCACAGTTTCTAGTGGCGGAGGCGGATCTGGCGGCGGTGGAAGTGGCGG CGGAGGTTCTCAAACAGTGGTCACCCAAGAGCCTAGCCTGACCGTTTCTCCTGGC GGAACCGTGACACTGACATGCGGATCTTCTACAGGCGCCGTGACCAGCGGCAAC TACCCTAATTGGGTGCAGCAGAAGCCAGGCCAGGCTCCTAGAGGACTGATCGGC GGCACAAAGTTTCTGGCTCCCGGAACACCAGCCAGATTCAGCGGTTCTCTGCTCG GAGGAAAGGCCGCTCTGACACTTTCTGGCGTGCAGCCTGAGGATGAGGCCGAGT ACTATTGCGTGCTGTGGTACAGCAACAGATGGGTGTTCGGCGGAGGCACCAAGC TGACAGTTCTTGGAGGTGGCGGTAGCCAGGTCCAGCTGAAACAATCTGGACCCG GACTCGTGCAGCCAAGCCAGAGCCTGTCTATCACCTGTACCGTGTCCGGCTTCAG CCTGACCAATTACGGCGTGCACTGGGTTCGACAATCTCCCGGCAAGGGACTCGA ATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCATTCAC CAGCAGACTGAGCATCAACAAGGACAACAGCAAGTCCCAGGTGTTCTTCAAGAT GAACTCCCTGCAGAGCCAGGATACCGCCATCTATTACTGCGCTCGGGCCCTGACC TACTATGACTACGAGTTTGCCTACTGGGGACAGGGAACCCTCGTGACAGTGTCTG CTGCTAGCACAAAGGGCCCTAGCGTTTTCCCACTGGCTCCCAGCAGCAAGTCTAC ATCCGGTGGAACAGCCGCTCTGGGCTGCCTGGTCAAGGATTACTTTCCCGAGCCA GTGACCGTGTCCTGGAATAGCGGAGCACTGACATCTGGCGTGCACACATTTCCAG CCGTGCTGCAGTCTAGCGGCCTGTACTCTCTGTCCAGCGTTGTGACAGTGCCCAG CAGCTCTCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAA CACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGATAAGACACACACCT GTCCTCCATGTCCTGCTCCAGAGCTGCTCGGAGGCCCTTCCGTGTTTCTGTTCCCT CCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTG GTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTCGAC GGCGTGGAAGTGCACAATGCCAAGACCAAGCCTTGCGAGGAACAGTACGGCAGC ACCTACAGATGCGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGC AAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAA ACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAACCCCAGGTGTACACACTGCCT CCAAGCCGGAAAGAGATGACCAAGAATCAGGTGTCCCTGACCTGCCTGGTCAAG GGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGACAGCCCGAG AACAACTACAAGACAACCCCTCCTGTGCTGAAGTCCGACGGCTCATTCTTCCTGT ACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCT GCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCT GAGCCCCGGCAAA

[0162] The terminal lysine may not be present in the purified protein, regardless of its presence in the gene. In a variation of this exemplary polynucleotide, the codon encoding the C-terminal lysine may be absent (i.e., SEQ ID NO: 139). A polynucleotide encoding a second polypeptide (SEQ ID NO:113) CAAGGCCAGTCTGGCCAAGGTCTTAGTTGTGAAGGTTGGGCGATGAATAGAGAA CAATGTCGAGCCGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGT CCGGCAGATCCGACCAGCACGGCGGAGGATCCCAAATCCTGCTGACACAGTCTC CTGTCATACTGAGTGTCTCCCCCGGCGAGAGAGTCTCTTTCTCATGTCGGGCCAG TCAGTCTATTGGGACTAACATACACTGGTACCAGCAACGCACCAACGGAAGCCC GCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTT AGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGC GAAGATATTGCGGATTATTATTGCCAGCAGAAACAACAACTGGCCGACCACCTTTG GCGCGGGCACCAAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCA TCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCT GCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGC CCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA GAGCTTCAACAGGGGAGAGTGT The second polypeptide is also encoded by a polynucleotide having the sequence of SEQ ID NO:115. Polynucleotide encoding the third polypeptide (SEQ ID NO:114) GATAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTT CCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCC TGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTT CAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCCTGCGA GGAACAGTACGGCAGCACCTACAGATGCGTGTCCGTGCTGACAGTGCTGCACCA GGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCC TGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAACCCCA GGTGTACACACTGCCTCCAAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCT GACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGC AATGGACAGCCCGAGAACAACTACGACACCACACCTCCAGTGCTGGACAGCGAC GGCTCATTCTTCCTGTACAGCGACCTGACCGTGGACAAGAGCAGATGGCAGCAG GGCAACGGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACC CAGAAGTCCCTGAGCCTGTCTCCTGGCAAA

[0163] In a variation of this exemplary polynucleotide, the codon encoding the C-terminal lysine may be absent (ie, SEQ ID NO:141).

[0164] Further exemplary activatable HBPCs of the disclosure are described in Example 1 and include a first polypeptide having the amino acid sequence of SEQ ID NO:30 (encoded by a polynucleotide sequence that includes the polynucleotide sequence of SEQ ID NO:139 or 112 (the terminal lysine is absent in the purified protein whether or not present in the gene)), a second polypeptide having the amino acid sequence of SEQ ID NO:31 (encoded by the polynucleotide sequence of SEQ ID NO:113 or SEQ ID NO:115), and a third polypeptide having the amino acid sequence of SEQ ID NO:32 (encoded by the polynucleotide sequence of SEQ ID NO:114 (the terminal lysine is absent in the purified protein whether or not present in the gene) or SEQ ID NO:141).

[0165] In another exemplary activatable HBPC of the disclosure described in Example 1, the activatable HBPC comprises a first polypeptide having the amino acid sequence of SEQ ID NO:38 (encoded by a polynucleotide sequence that includes the polynucleotide sequence of SEQ ID NO:142 or 143 (the terminal lysine is absent in the purified protein whether or not it is present in the gene)), a second polypeptide having the amino acid sequence of SEQ ID NO:31 (encoded by the polynucleotide sequence of SEQ ID NO:113 or SEQ ID NO:115), and a third polypeptide having the amino acid sequence of SEQ ID NO:32 (encoded by the polynucleotide sequence of SEQ ID NO:114 (the terminal lysine is absent in the purified protein whether or not it is present in the gene) or SEQ ID NO:141 (the terminal lysine is absent in the purified protein whether or not it is present in the gene)).

[0166] Polynucleotides encoding the polypeptides or antigen-binding fragments thereof or domains thereof described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods), synthesized using techniques well known in the art, etc. Polynucleotides encoding the first, second, and third polypeptides can be cloned into one or more vectors for expression in a host cell and for further cloning to generate, for example, chimeric and humanized antibodies or antigen-binding fragments thereof.

[0167] The polynucleotides provided herein may be RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and DNA may be single-stranded or double-stranded. If single-stranded, DNA may be the coding strand or the non-coding (antisense) strand. In some aspects, the polynucleotide is a cDNA, i.e., DNA lacking one additional endogenous intron. In some aspects, the polynucleotide is a non-naturally occurring polynucleotide. In some aspects, the polynucleotide is recombinantly produced. In some aspects, the polynucleotide is isolated. In some aspects, the polynucleotide is substantially pure. In some aspects, the polynucleotide is purified from natural components.

[0168] In some aspects, the polynucleotides described herein encode an activatable anti-EGFR, anti-CD3 HBPC or an antigen-binding fragment thereof, comprising a heavy chain (VH) and a light chain (VL) and CDRs provided herein.

[0169] Vectors, host cells, and methods of production Provided herein are one or more vectors comprising polynucleotides (corresponding to the first polynucleotide, the second polynucleotide, and the third polynucleotide, respectively) encoding the first, second, and / or third polypeptides of the present disclosure. In some aspects, such vectors may be used to recombinantly produce polypeptides of HBPC from host cells, as further detailed herein below. In some aspects, the vector comprises the first polynucleotide, the second polynucleotide, and / or the third polynucleotide operably linked to one or more promoter sequences. In certain aspects, the present disclosure provides a plurality of vectors comprising polynucleotides (i.e., the first polynucleotide, the second polynucleotide, and the third polynucleotide) encoding the first, second, and third polypeptides together, where the plurality includes at least one vector comprising no more than two, or only one, of the first, second, and third polynucleotides. In these embodiments, the first polynucleotide, the second polynucleotide, and the third polynucleotide sequences in the multiple vectors are typically operably linked to one or more promoter sequences.

[0170] Also provided herein are recombinant host cells comprising any of the above-mentioned polynucleotides and / or vectors for recombinantly expressing a polynucleotide encoding an activatable anti-EGFR, anti-CD3 HBPC polypeptide of the present disclosure. A variety of host-expression vector systems can be utilized to express the polypeptides described herein (see, for example, U.S. Pat. No. 5,807,715). Such host-expression systems represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequence, can express in situ an antibody or antigen-binding fragment thereof described herein. Exemplary host cells suitable for use as recombinant expression hosts for the above-described polynucleotides include mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 cells, etc.). Vectors used in constructing recombinant mammalian host cells may include promoters derived from the genome of a mammalian cell (e.g., metallothionein promoter) or from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter). In some embodiments, the recombinant host cell is a CHO cell or an NS0 cell.

[0171] In some aspects, recombinant expression of the polypeptides described herein, e.g., the first polypeptide, the second polypeptide, and / or the third polypeptide, involves the construction of an expression vector containing a polynucleotide encoding an activatable anti-EGFR, anti-CD3 HBPC. The vector(s) containing the polynucleotide encoding the HBPC can be readily generated by recombinant DNA technology using techniques well known in the art. Methods well known to those skilled in the art can be used to construct expression vectors containing one or more polynucleotides encoding the polypeptides described herein, e.g., the first polypeptide, the second polypeptide, and / or the third polypeptide, and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors containing a nucleotide sequence operably linked to a promoter. Such vectors can include, for example, nucleotide sequences encoding the constant regions of a polypeptide described herein, e.g., a first polypeptide, a second polypeptide, and / or a third polypeptide (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036, and U.S. Pat. No. 5,122,464), and the variable domains of the polypeptides can be cloned into such vectors for expression of the entire VH, the entire VL, or both the entire VH and VL.

[0172] The expression vector can be transfected into a cell (e.g., a host cell) by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce an HBPC described herein (e.g., the CDRs, VH1, VH2, VL1, and VL2 of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides provided herein). Thus, provided herein is a host cell containing a polynucleotide encoding an HBPC described herein operably linked to a promoter for expression of such sequence in the host cell. In some aspects, the host cell contains a vector comprising a polynucleotide encoding an HBPC or a domain thereof described herein. In some aspects, the host cell contains three different vectors, a first vector comprising a first polynucleotide encoding a first polypeptide described herein, a second vector comprising a second polynucleotide encoding a second polypeptide described herein, and a third vector comprising a third polynucleotide encoding a third polypeptide described herein.

[0173] In some aspects, provided herein is a population of vectors that together comprise polynucleotides encoding a first polypeptide, a second polypeptide, and a third polypeptide, where each vector comprises only one or two of the polynucleotides encoding the first polypeptide, the second polypeptide, or the third polypeptide. In certain aspects, provided herein is a single vector that comprises polynucleotides encoding the first polypeptide, the second polypeptide, and the third polypeptide (i.e., the first polynucleotide, the second polynucleotide, and the third polynucleotide, respectively).

[0174] In some aspects, the disclosure provides a method for producing an activatable HBPC, the method comprising: (a) culturing a host cell comprising one or more polynucleotides encoding a polypeptide of the disclosure (e.g., a first polynucleotide, a second polynucleotide, and / or a third polynucleotide, and vector(s) comprising the foregoing polynucleotides) in a liquid medium under conditions sufficient to produce an activatable HBPC; and (b) recovering the activatable HBPC.

[0175] In certain aspects, methods are provided herein for producing an activatable anti-EGFR, anti-CD3 HBPC, comprising expressing such a polypeptide in a host cell. More specifically, methods are provided herein for producing an activatable HBPC, comprising (a) culturing a host cell comprising one or more polynucleotides encoding a polypeptide of the present disclosure in a liquid medium under conditions sufficient to produce an HBPC, and (b) recovering the activatable HBPC.

[0176] In another embodiment, the method further comprises purifying a bioharvest (cell-free expression product) of activatable HBPC or other in-process composition comprising subjecting the aqueous composition comprising activatable HBPC to a unit operation such as, for example, affinity chromatography, size exclusion chromatography, ion exchange chromatography, ceramic hydroxyapatite chromatography, etc. In one particular embodiment, the unit operation is ceramic hydroxyapatite chromatography.

[0177] composition In some embodiments, the activatable HBPC of the present disclosure or its HBPCs can be utilized in pharmaceutical compositions useful for any of the therapeutic applications disclosed herein. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more activatable HBPCs together with a pharma- ceutically acceptable diluent or carrier. In other embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more activatable HBPCs, a pharma- ceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. Acceptable formulation materials are non-toxic to recipients at the dosages and concentrations used. The pharmaceutical composition can be formulated as a liquid, frozen, or lyophilized composition.

[0178] In certain embodiments, the pharmaceutical composition may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeability of the composition. Suitable formulation materials include, but are not limited to, amino acids; antimicrobial agents; antioxidants; buffers; bulking agents; chelating agents; complexing agents; fillers; carbohydrates, such as monosaccharides or disaccharides; proteins; colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers; low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives; solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols; suspending agents; surfactants or wetting agents; stability enhancers; isotonicity enhancers; delivery vehicles; and / or pharmaceutical adjuvants. Additional details and options for suitable agents that may be incorporated into the pharmaceutical composition may be found, for example, in Remington's Pharmaceutical Sciences, 22 nd Edition, (Loyd V. Allen, ed.) Pharmaceutical Press (2013), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 thed., Lippencott Williams and Wilkins (2004), and Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd ed., Pharmaceutical Press (2000).

[0179] The components of the pharmaceutical composition are selected depending on, for example, the intended route of administration, the delivery format, and the desired dosage. nd Edition, (Loyd V. Allen, ed.) Pharmaceutical Press (2013). The composition is selected to affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the disclosed antigen binding protein. The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier can be water for injection or saline. In certain aspects, the antigen binding protein composition can be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing the selected composition having the desired purity with optional formulating agents. Furthermore, in certain aspects, the antigen binding protein can be formulated as a lyophilizate using appropriate excipients.

[0180] In certain formulations, the concentration of activatable HBPC is at least 2 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, or 150 mg / ml. In other formulations, the activatable HBPC has a concentration of 10-20 mg / ml, 20-40 mg / ml, 40-60 mg / ml, 60-80 mg / ml, or 80-100 mg / ml.

[0181] Some compositions include a buffering agent or pH adjusting agent. Representative buffering agents include, but are not limited to, organic acid salts (such as salts of citric acid, acetic acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, or phthalic acid), Tris, phosphate buffers, and in some cases, amino acids as described below. In certain embodiments, a buffering agent is used to maintain the composition at or slightly below physiological pH, typically within a pH range of about 5 to about 8. Some compositions have a pH of about 5 to 6, 6 to 7, or 7 to 8. In other embodiments, the pH is 5.5 to 6.5, 6.5 to 7.5, or 7.5 to 8.5.

[0182] Free amino acids or proteins are used in some compositions as bulking agents, stabilizers, and / or antioxidants. As examples, lysine, proline, serine, and alanine may be used to stabilize proteins in the formulation. Glycine is useful in lyophilization to ensure proper cake structure and properties. Arginine may be useful to inhibit protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant. Glutamine and asparagine are included in some embodiments. Amino acids are included in some formulations for their buffering capacity. Such amino acids include, for example, alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Certain formulations also include protein excipients such as serum albumin (e.g., human serum albumin (HSA) and recombinant human albumin (rHA)), gelatin, casein, and the like.

[0183] Some compositions include polyols. Polyols include sugars (e.g., mannitol, sucrose, trehalose, and sorbitol) and polyhydric alcohols, such as, for example, glycerol and propylene glycol, and polyethylene glycol (PEG) and related substances. Polyols are cosmotropic. They are useful stabilizers in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols are also useful for adjusting the isotonicity of the formulation.

[0184] Certain compositions contain mannitol as a stabilizer. It is generally used with cryoprotectants, such as sucrose. Sorbitol and sucrose are useful as stabilizers to adjust isotonicity and protect against freeze-thaw stress during transportation or during preparation of bulk products during manufacturing process. PEG is useful for stabilizing proteins and as a cryoprotectant, and can be used in the present disclosure in this regard.

[0185] Some formulations may include sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derived sugars, such as alditols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers. For example, suitable carbohydrate excipients include monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myo-inositol, and the like.

[0186] Certain formulations may contain surfactants.Surfactants are typically used to prevent, minimize or reduce protein adsorption and subsequent aggregation on surfaces at air-liquid, solid-liquid and liquid-liquid interfaces, and to control protein conformational stability.Suitable surfactants include, for example, polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan esters, Triton surfactants, lecithin, tyloxapol and poloxamer 188.

[0187] In some embodiments, one or more antioxidants are included in the pharmaceutical composition.Antioxidant excipients can be used to prevent oxidative degradation of proteins.Reducing agents, oxygen / free radical scavengers, and chelating agents are useful antioxidants in this regard.Antioxidants are typically water-soluble and maintain their activity throughout the shelf life of the product.EDTA is another useful antioxidant.

[0188] Certain formulations contain metal ions, which are protein cofactors and necessary to form protein coordination complexes. Metal ions can also inhibit some processes that break down proteins. For example, magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartic acid to isoaspartic acid.

[0189] Certain formulations may also include isotonicity enhancing agents. Examples of such agents include alkali metal halides, preferably sodium or potassium chloride, mannitol, and sorbitol.

[0190] Certain formulations may contain one or more preservatives. Preservatives are necessary when developing parenteral formulations for multiple doses with more than one extraction from the same container. Their primary function is to inhibit microbial growth and ensure sterility of the product throughout the shelf life or shelf life of the drug. Suitable preservatives include phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, phenyl alcohol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, thimerosal, benzoic acid, salicylic acid, chlorhexidine, or mixtures thereof in aqueous diluents.

[0191] A pharmaceutical composition is formulated to be compatible with its intended route of administration, examples of which are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, and rectal administration.

[0192] Formulation components suitable for parenteral administration (e.g., intravenous, subcutaneous, intraocular, intraperitoneal, intramuscular) include a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates, or phosphates; and agents for adjusting isotonicity such as sodium chloride or dextrose.

[0193] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and preserved against microorganisms. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0194] Further guidance on appropriate formulations depending on the mode of delivery can be found, for example, in Remington's Pharmaceutical Sciences, 22 nd Edition, (Loyd V. Allen, ed.) Pharmaceutical Press (2013).

[0195] The pharmaceutical formulation can be sterile. Sterilization can be accomplished by any suitable method, for example, filtration through a sterile filtration membrane. If the composition is lyophilized, sterilization by filtration can be performed before or after lyophilization and reconstitution. As demonstrated in Examples 7 and 8, the activatable HBPC described herein appears to be relatively resistant to aggregation, even at relatively high concentrations. Thus, in another aspect, a composition is provided herein that includes any of the activatable HBPCs described herein and water, wherein the activatable HBPC is present at a concentration of at least 1 mg / mL, and the composition comprises at least about 95% monomeric activatable HBPC, or at least about 96% monomeric activatable HBPC, or at least about 97% monomeric activatable HBPC, or at least about 98% monomeric activatable HBPC, or at least about 99% monomeric activatable HBPC. As used herein, the term "monomeric activatable HBPC" refers to the non-aggregated form of activatable HBPC. In certain of these embodiments, the composition comprises at least about 2 mg / ml, and at least about 95% monomeric activatable HBPC, or at least about 96% monomeric activatable HBPC, or at least about 97% monomeric activatable HBPC, or at least about 98% monomeric activatable HBPC, or at least about 99% monomeric activatable HBPC. In some embodiments, the composition comprises at least about 3 mg / ml, and at least about 95% monomeric activatable HBPC, or at least about 96% monomeric activatable HBPC, or at least about 97% monomeric activatable HBPC, or at least about 98% monomeric activatable HBPC, or at least about 99% monomeric activatable HBPC. In some embodiments, the composition comprises at least about 4 mg / ml, and at least about 95% monomeric activatable HBPC, or at least about 96% monomeric activatable HBPC, or at least about 97% monomeric activatable HBPC, or at least about 98% monomeric activatable HBPC, or at least about 99% monomeric activatable HBPC.The percentage of monomeric activatable HBPC can be readily determined, for example, by size exclusion (SE)-HPLC as exemplified in Example 7, where the percent of monomeric activatable HBPC is determined as the peak area percentage corresponding to monomeric activatable HBPC based on the total peak area. EXAMPLES

[0196] The examples in this Examples section are offered by way of illustration and not by way of limitation.

[0197] Example 1: Construction and expression of an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide In this example, two exemplary activatable anti-EGFR, anti-CD3 HBPCs, Conjugate-57 and Conjugate-67, were prepared, having the structures shown in Figure 1. With reference to Figure 1, each of the activatable anti-EGFR, anti-CD3 HBPCs was constructed as three polypeptides as described below. (a) a first polypeptide comprising a CD3 masking portion (MM1) (100), a first cleavable portion (CM1) (101), an anti-CD3 scFv (102) (comprising VH1 and VL1 sequences connected via a linker), an anti-EGFR heavy chain variable domain (VH2) (top) and a CH1 domain (bottom) shown together as (103), which are linked via a hinge region (109) to a first Fc domain (Fc1) (104); and (b) a second polypeptide comprising an EGFR masking portion (MM2) (105), a second cleavable portion (CM2) (106), and an anti-EGFR light chain variable domain (VL2) (top) and a constant light chain domain (CL) (bottom), together shown as (107); and (c) a third polypeptide comprising a hinge region (110) and a second Fc domain (Fc2) (108). As can be seen in FIG. 1, the first Fc domain and the second Fc domain bind to each other and the anti-EGFR heavy and light chain variable domains form an EGFR targeting domain that specifically binds to EGFR. Conjugate-57 and Conjugate-67 contained the same anti-EGFR targeting domain but different anti-CD3 scFvs. The components of Conjugate-67 are listed in Tables 4A-4C and the components of Conjugate-57 are listed in Tables 5A-5C. [Table 3] * The corresponding polynucleotide sequence is SEQ ID NO:112 (the terminal lysine is not present in the purified protein whether present or absent in the gene) or SEQ ID NO:139. ++ Contains an N-terminal spacer, SEQ ID NO:33. Δ Fc1 is located at the C-terminus of the CH1 (SEQ ID NO: 26)-hinge (SEQ ID NO: 34) sequence. [Table 4] * The corresponding polynucleotide sequence is SEQ ID NO:113 or alternatively SEQ ID NO:115. ++ Contains an N-terminal spacer, SEQ ID NO:117. [Table 5] * The corresponding polynucleotide sequence is SEQ ID NO:114 (the terminal lysine is not present in the purified protein whether present or absent in the gene) or SEQ ID NO:141. ++ Contains a hinge (SEQ ID NO: 35) located at the N-terminus of Fc2. [Table 6] *The corresponding polynucleotide sequence is SEQ ID NO:143 (the terminal lysine is not present in the purified protein whether present or absent in the gene) or SEQ ID NO:142. ++ Contains an N-terminal spacer (SEQ ID NO: 117). Δ Fc1 is located at the C-terminus of the CH1 (SEQ ID NO: 26)-hinge (SEQ ID NO: 34) sequence. [Table 7] * The corresponding polynucleotide sequence is SEQ ID NO:113 or alternatively SEQ ID NO:115. ++ Contains an N-terminal spacer, SEQ ID NO:117. [Table 8] * The corresponding polynucleotide sequence is SEQ ID NO:114 (the terminal lysine is absent in the purified protein despite its presence in the gene) or SEQ ID NO:141. ++ Contains a hinge (SEQ ID NO: 35) located at the N-terminus of Fc2.

[0198] Construction of an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide as a control A control activatable bispecific construct (herein referred to as "CI106") was prepared as described in International Patent Application Publication No. WO2019 / 075405, which is incorporated herein by reference. CI106 is an activatable dual-arm bivalent bispecific construct consisting of four polypeptides corresponding to two identical heavy chains (two first polypeptides) and identical light chains (two second polypeptides), each heavy and light chain forming an arm of the bispecific construct. CI106 is "bivalent" in that it has two binding domains of each type (i.e., two EGFR binding domains and two CD3 binding domains). The amino acid sequence of the light chain is identical to that of the second polypeptide of complex-67 and complex-57. The heavy chain of CI106 and the first polypeptide of complex-67 have identical spacer, cleavable moiety, anti-EGFR VH, and cleavable moiety components. The heavy chain of CI106 and the first polypeptide of complex-57 have the same spacer, anti-CD3 MM / MM1, cleavable moiety, and anti-CD3 VL / VH (and the same anti-CD3 scFv), as well as anti-EGFR VH components. For CI106, all four targeting domains (two anti-CD3 binding domains and two anti-EGFR binding domains) were masked. The components of CI106 are provided in Tables 6A-6B. [Table 9] * The corresponding polynucleotide sequence is SEQ ID NO: 125 (the protein appears to lose a terminal lysine during expression / purification). ++Contains an N-terminal spacer (SEQ ID NO: 116). Δ The Fc domain is located at the C-terminus of the CH1 (SEQ ID NO: 26)-hinge (SEQ ID NO: 34) sequence. [Table 10] * The corresponding polynucleotide sequence is SEQ ID NO:113 or alternatively SEQ ID NO:115. ++Contains N-terminal spacer, SEQ ID NO:117.

[0199] Example 2. EGFR + HT-29 cells and CD3ε + Binding of activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptides to Jurkat cells To assess whether the described anti-EGFR and anti-CD3 masking peptides could inhibit the binding of activatable heteromultimeric bispecific polypeptides to EGFR and CD3, flow cytometry-based binding assays were performed.

[0200] HT-29-luc2 (Perkin Elmer, Inc., Waltham, MA (formerly Caliper Life Sciences, Inc.) and Jurkat (clone E6-1, ATCC, TIB-152) cells were cultured in RPMI 1640 + glutamax (Life Technologies, catalog 10438-026) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, Life Technologies, catalog 10438-026). Technologies, catalog 72400-047). As indicated, the "activating" (herein appended with "act") molecule was produced as a masked HBPC and proteolytically cleaved to produce the activated form. Activatable HBPC was produced but not proteolytically cleaved prior to the experiment. The following polypeptide complexes were tested: act-CI106 (bivalent double-arm bispecific construct), act-complex-57 (HBPC), and act-complex-67 (HBPC), as well as activatable (masked) HBPC complex-57, (masked) HBPC complex-67, and dual masked bivalent double-arm bispecific construct CI106. As mentioned in Example 1, one combination of CD3 binder (anti-CD3 scFv v16) and mask (MM H20GG) was utilized in CI106 and complex-57, and a different combination of CD3 binder (anti-CD3 scFv I2C) and mask (ML15) was utilized in complex-67.

[0201] HT29-luc2 cells were detached with Versene™ (Life Technologies, Catalog 15040-066), washed, plated at approximately 150,000 cells / well in 96-well plates, and resuspended in 50 μL of activated HBPC or activatable (masked) HBPC. Jurkat cells were counted and plated as described for HT29-luc2 cells. Titration of activated (unmasked) HBPC or activatable (masked) HBPC started at the concentrations shown in Figures 2A and 2B, followed by 3-fold serial dilutions in FACS stain buffer + 2% FBS (BD Pharmingen, Catalog 554656). Cells were incubated with shaking for approximately 1 hour at 4°C, harvested, and washed with 2 x 200 μL of FACS stain buffer. Cells were resuspended in 50 μL of Alexa Fluor 488-conjugated anti-human IgG Fc (10 μg / ml, Jackson ImmunoResearch) and incubated for approximately 1 hour at 4° C. with shaking. Cells were harvested, washed, and resuspended in a final volume of 200 μL of FACS staining buffer containing 2.5 μg / mL of 7-AAD (BD Biosciences, catalog 559925). Cells stained with secondary antibody alone were used as negative control. Data were acquired on an Attune NxT flow cytometer, and median fluorescence intensity (MFI) of live cells was calculated using FlowJo® V10 (Treestar). Background-subtracted MFI data was graphed in GraphPad Prism using curve-fitting analysis.

[0202] As shown in Figures 2A-2B, both activatable HBPCs, complex-57 and complex-67, as well as the control CI106, showed reduced binding to both EGFR and CD3 targets compared to activated (unmasked) complex-57, activated complex-67, and activated CI106. The reduced binding is represented by a rightward shift in the binding curves. The EGFR masking efficiencies in this on-cell binding experiment were 105 for complex-57, 338 for complex-67, and 594 for CI106.

[0203] Example 3. Biological activity of activatable and activated HBPC The biological activity of activatable (masked) HBPC and activated (unmasked) HBPC was assayed using a cytotoxicity assay. Human PBMCs were purchased from Stemcell Technologies (Vancouver, Canada) and co-cultured with the EGFR-expressing cancer cell line HT29-luc2 (Perkin Elmer, Inc., Waltham, MA (formerly Caliper Life Sciences, Inc.)) at an E(CD3+):T ratio of 5:1 in RPMI-1640+glutamax supplemented with 5% heat-inactivated human serum (Sigma, Cat. H3667). Titrations of act-CI106, act-complex-57 and act-complex-67, as well as activatable (masked) CI106, complex-57 and complex-67 were tested. After 48 hours, cytotoxicity was assessed using the ONE-Glo™ Luciferase Assay System (Promega, Madison, WI, Cat. E6130). Luminescence was measured with an Infinite® M200 Pro (Tecan Trading AG, Switzerland). Percent cytotoxicity was calculated and plotted in GraphPad PRISM using curve fitting analysis. The potency of activated molecules was compared by calculating EC50. Masking efficiency was calculated for each molecule as the ratio of unchanged EC50 to activated EC50.

[0204] As shown in Figures 3A and 3B, activatable (masked) HBPC has a shifted dose response curve compared to activated (unmasked) bispecific antibodies. In this assay, the data in Figure 3A shows a masking efficiency of 29,650 for CI106 and a masking efficiency of 1,034 for conjugate-57. The data in Figure 3B shows a masking efficiency of 26,537 for CI106 and a masking efficiency of 7,141 for conjugate-67. Conjugate-57 generally showed a 10-42 fold reduced potency compared to conjugate-67 based on multiple experiments using this assay.

[0205] Example 4. HBPC-induced regression of established HT29 tumors in mice In this example, activatable (masked) HBPC complex-67 and control CI106 were analyzed for their ability to induce regression or reduce the growth of established HT29 xenograft tumors in human PBMC-engrafted NSG mice.

[0206] The human colon cancer cell line HT29-luc2 (Perkin Elmer, Inc., Waltham, MA) was cultured according to established procedures. Purified frozen human PBMCs were obtained from Hemacare, Inc. (Van Nuys, CA). NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ) mice were obtained from The Jackson Laboratories (Bar Harbor, ME).

[0207] On day 0, each mouse received 2 × 10 6 HT29-luc2 cells were inoculated subcutaneously. On day 3, previously frozen PBMCs from a single donor were administered (i.p.) at a 1:1 ratio of CD3+ T cells to tumor cells. 3 When tumor mass reached 0.01 mg / kg / day (approximately day 12), mice were randomized into treatment groups and dosed according to Table 7. Tumor volumes and body weights were measured twice weekly. Dose levels of conjugate-67 were adjusted to account for the molecular weight difference between CI106 and conjugate-67. [Table 11]

[0208] As shown in Figure 4, which depicts a plot of tumor volume versus days after the first treatment dose (day 0), there is a dose-dependent effect of conjugate-67 on the growth of HT29-luc2 xenograft tumors. Conjugate-67 demonstrated more potent antitumor activity than the control, CI106, at equivalent doses (1 mg / kg CI106 and 0.6 mg / kg conjugate-67) (p=0.0099, Dunnett's random-average OVA).

[0209] Example 5. Tumor regression following treatment of established HCT116 tumors in mice with activatable HBPC The activated (unmasked) HBPC act-complex-67 and the activatable (masked) HBPC complex-67 were analyzed for their ability to induce regression or reduce the growth of established HCT116 xenograft tumors in human T cell engrafted NSG mice. Human colon cancer cell line HCT116 (ATCC) was cultured in RPMI+Glutamax+10%FBS according to established procedures. The tumor model was performed as described in Example 4. Mice were dosed according to Table 8. [Table 12]

[0210] Example 6: Evaluation of Percent Monomer after Purification by Ceramic Hydroxyapatite Chromatography (CHT) Dual masked CI106 control and activatable (masked) HBPC complex-67 were purified using ceramic hydroxyapatite chromatography columns to compare the amount of dimerization at high concentrations during purification, which was assessed by analyzing the percentage of monomer at each step of the purification process.

[0211] The samples were loaded onto a CHT type I, 40 μm bead column (Biorad catalog 157-0040 and number 157-0041) loaded with 20 g / L resin. The column was washed with equilibration buffer 10 mM NaPO4, 100 mM histidine buffer, pH 6.5, then eluted in 2 mL fractions with 10 mM NaPO4, 100 mM histidine, 200 mM lysine-HCl buffer, pH 6.5 for CI106 and 10 mM NaPO4, 100 mM histidine 100 mM lysine-HCl buffer, pH 6.5 for complex-67. CI106 was collected in 2 mL fractions, and then five fractions were pooled to form the eluate. For CI106, peak collection started around 25 mAU and stopped around 300 mAU. Complex-67 was collected in one tube and peak collection started at 100 mAU and stopped at 500 mAU. This was followed by a strip buffer step of 500 mM NaPO4 (pH 7.0). Protein concentration of each fraction was quantified by UV absorbance at a wavelength of 280 nm. Percent monomer in each fraction was determined by SE-HPLC (analytical scale size exclusion chromatography) based on total peak area.

[0212] During the binding step of chromatography, proteins bind to the top of the column first and move down the column only after the top sites are filled. This leads to a high concentration of molecules on the column. The multimeric forms of CI106 and complex-67 bind to the column with stronger affinity than the monomeric forms and therefore require stronger buffers to be completely removed from the column. Thus, when eluted from the column with a weaker buffer and then stripped with a stronger buffer, the eluate has a lower percentage of dimers (higher percentage of monomers) than the stripping solution. As shown in Table 10, delivery of complex-67 (an activatable HBPC) resulted in a 7.6% increase in the percent monomer in the eluate, leaving the high molecular weight material on the column until the stripping step, with a 77% recovery in the eluate. This is in contrast to the CI106 delivery, which resulted in a 5.4% decrease in percent monomer in the eluate to 65.0%, resulting in an 81% recovery in the eluate, even though more dimeric material (only 30.6% monomer) remained on the column until stripping. [Table 13]

[0213] These results suggest that complex-67 does not undergo additional dimerization when highly concentrated on the column, resulting in the removal of nearly all high molecular weight species, with 98.5% monomer in the eluate compared to only 65% ​​for CI106. In the case of CI106, there are more high molecular weight species in the eluate pool than in the original load. The improved behavior of complex-67 allows for the purification of highly monomeric complex-67 via CHT type 1 chromatography. However, CI106 could not be purified by this bind / elute chromatography method or any of the bind / elute chromatography methods evaluated due to the dimerization that occurs when CI106 is highly concentrated on the column.

[0214] Example 7: Assessment of concentration-dependent dimerization via concentration in a centrifugal concentrator Protein A and SEC purified preparations of conjugate-67, conjugate-57, and the dual masked control CI106 were compared for percent monomer after centrifugal concentration and overnight incubation at the highest concentration.

[0215] Conjugate-67, Conjugate-57, and CI106 were purified with Protein A and SEC, then formulated in low pH buffer (10 mM acetate, 100 mM lysine, pH 6). Samples were diluted 1:15 in PBS (753-45-01) and concentrated at each concentration by centrifugation at 14,000 RPM for 2 minutes using a Pierce™ Protein Concentrator PES 10K MWCO 0.5 ml (Thermo Fisher Catalog No. 88513). The highest concentration sample was stored overnight and assessed for percent monomer. The resulting concentrations and percent monomer amounts are shown in Table 10 and Figure 8. [Table 14]

[0216] Figure 6 and Table 10 show that Conjugate-67 maintains a high monomer percentage (98%-99%) and very low aggregation in solution as the concentration is increased. This is in contrast to CI106, which shows significant concentration-dependent dimerization as the concentration is increased. Conjugate-57 shows negligible concentration-dependent dimerization as the concentration is increased and maintained a stable monomer percentage. Conjugate-67 also maintained the monomer percentage during overnight incubation at the highest concentration, demonstrating the stability of the monomer percentage at higher concentrations.

[0217] Example 8: Safety and efficacy of the activatable anti-EGFR, anti-CD3 TCB construct CI107 In this study, the safety and efficacy of CI107, an anti-EGFR, anti-CD3 TCB construct with the same structural format as the CI106 control (described above), was evaluated in a preclinical model to assess its therapeutic potential for treating tumors expressing EGFR. CI107 was prepared as described in International Patent Application Publication No. WO2019 / 075405, which is incorporated herein by reference. The CI107 TCB construct is alternatively referred to in this example as "T cell-inducing bispecific antibody" or "TCB."

[0218] method Animal testing All animal studies were performed in compliance with the regulations of the Institutional Animal Care and Use Committee governing the facility in which each study was performed. Mouse xenograft studies were performed by CytomX Therapeutics, Inc. (CytomX) and cynomolgus monkey studies were performed by Altasciences (Everett, WA). All animal studies followed regulations set forth by the USDA Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals.

[0219] material All TCBs and other constructs described in this study, including CI107, CI128, CI020, CI011, CI040, CI048, and CI104, were produced by CytomX Therapeutics, Inc. (see WO2016 / 014974 and WO2019 / 075405). CI107, CI128, CI020, CI011, CI040, and CI104 have the same structural format as CI106. CI048 corresponds to activated CI011. Activated TCBs were produced by in vitro treatment with urokinase-type plasminogen activator (uPA), followed by SEC purification (Desnoyers 2013). HT29-Luc2 cells were obtained from Caliper Life Sciences (Hopkinton, MA), HCT116 and Jurkat cells were obtained from the American Type Culture Collection (ATCC). Human peripheral blood mononuclear cells (PBMCs) were obtained as cryovials of cells from individual donors from HemaCare Corporation (Northridge, CA), AllCells (Alameda, CA), or STEMCELL Technologies (Seattle, WA). NOD.Cg-Prkcdscid Il2rg tm1Wjl / SzJ (NSG) mice were obtained from Jackson Laboratories (Sacramento, CA).

[0220] Cell binding assay HT29 and Jurkat cells were maintained in complete medium. HT29 cells were harvested using Versene™ cell dissociation buffer. Cells were centrifuged at 250×g for 5-10 min and resuspended in FACS buffer (BD Pharminogen) containing 2% FBS. Cells were plated at 150,000 / well in V-bottom 96-well plates and treated with Complex-07 or in vitro protease-activated CI104 at various concentrations obtained by 3-fold serial dilutions in FACS buffer starting with 1.5 μM CI107 for both HT29 and Jurkat cells, 0.05 μM activated CI104 for HT29 cells, and 0.5 μM activated CI104 for Jurkat cells. Cells were incubated for 1 h at 4° C., washed twice with FACS buffer, and resuspended in 10 μg / ml Alexa Fluor 647 anti-human Fc secondary antibody. Cells were then incubated for 30–60 min at 4°C protected from light, washed twice with FACS buffer, resuspended in FACS buffer containing 7-AAD, and analyzed on a MACSQuant flow cytometer (Miltenyi Biotech). Mean fluorescence intensity data were corrected for background signal of the secondary antibody, graphed in Graphpad Prism, and EC50 values ​​were calculated.

[0221] Cytotoxicity assay HCT116-Luc2 or HT29-Luc2 were plated at 10,000 cells / well in 96-well white flat-bottom tissue culture treated plates (Costar #3917) in RPMI + 5% human serum. Human PBMCs were freshly thawed and washed twice with RPMI + 5% human serum, and 100,000 PBMCs were added to wells containing HCT116-Luc2 or HT29-Luc2 in RPMI + 5% human serum. Protease-activated TCB or CI107 was then added to the wells at various concentrations obtained by 3-fold serial dilutions. Control wells contained untreated target + effector cells, target cells only, effector cells only, or media only. Plates were then incubated at 37°C and 5% CO2 for approximately 48 hours. Cell viability was measured using the ONE-Glo Luciferase Assay System (Promega, #E6120) and a Tecan plate reader. Percent cytotoxicity was calculated as follows: (1-(experimental RLU / mean untreated RLU))x100.

[0222] In vitro T cell activation and cytokine analysis T cell activation was measured by induction of CD69 expression in PBMCs cocultured with HT29-Luc2 or HCT116-Luc2 cells. HT29-Luc2 or HCT116-Luc2 cells were plated at 10,000 cells / well in U-bottom non-adherent plates. Human PBMCs were freshly thawed and washed twice with RPMI containing serum, and 100,000 PBMCs / well were added to plates containing tumor cells. Duplicate plates containing PBMCs only were seeded for flow cytometry fluorescence compensation controls. Three-fold serial dilutions of CI107, activated CI107, or CI128 were prepared in medium and added to the plated cells. Cells were incubated for 16 h at 37 °C and 5% CO2. Plates were centrifuged at 250 × g for 10–15 min in preparation for flow cytometry analysis. Supernatants were removed for cytokine analysis, Fc block (Human TruStain FcX, BioLegend) was added to each well, and plates were incubated for 10 min. An antibody cocktail containing anti-CD45-FITC (BioLegend), anti-CD3-Pacific Blue (BioLegend), anti-CD8a-APC (BioLegend), and anti-CD69-PE-Cy7 (BioLegend), or appropriate fluorescence compensation controls, was added to the wells, and plates were incubated for 30-60 min at 4 °C with shaking, protected from light. Plates were then washed with FACS buffer and resuspended in FACS buffer containing 7-AAD. Luminescence was measured using an Attune flow cytometer, and 15,000 events representing PBMCs were collected.

[0223] For cytokine analysis, the Meso Scale Discovery U-PLEX plate assay (Meso Scale Diagnostics, Rockville, MA) was used. U-PLEX plates were prepared according to the manufacturer's protocol to assess levels of MCP-1, TNF-α, IL-6, IL-2, and IFN-γ. Supernatant samples collected from HT29-Luc2 or HCT116-Luc2 cells co-cultured with PBMCs and treated with masked (activatable) CI107, activated (also referred to herein as "act-") CI107, or CI128 were diluted, added to the plates, and processed according to the manufacturer's instructions.

[0224] In vivo efficacy studies For in vivo experiments, the effect of TCB on tumor growth was measured in mice bearing HT29-Luc2 or HCT116 tumors and engrafted with human T cells resulting from intraperitoneal (IP) injection of human PBMCs. On day 0, 2 million HT29-Luc2 or HCT116 cells were injected subcutaneously into the flank of female NSG mice in 100 μl serum-free RPMI. On day 3, frozen PBMCs from a single donor were freshly thawed and administered via IP injection in 100–200 μL RPMI+Glutamax serum-free medium. PBMCs were previously characterized for CD3+ T cell percentage, and the number of PBMCs used for in vivo administration was based on a 1:1 CD3+ T cell to tumor cell ratio. Tumor measurements at approximately day 12 were used to randomize mice prior to intravenous (IV) administration of TCB, control substance, or vehicle. Animals were administered test substances weekly for three weeks, and tumor volume and body weight were recorded twice weekly. Activated TCBCI104 was used for in vivo studies. The CI104 construct differs from CI107 only in that the CD3 mask was tethered to the scFv using a cleavable linker. Once the mask was completely removed by in vitro protease activation, activated CI104 was identical to activated CI107 and could be used to assess the activity of activated CI107, which was then verified by in vitro cytotoxicity studies to be the same as that of activated CI107.

[0225] Safety studies in non-human primates Male cynomolgus monkeys were given a slow IV bolus injection of the test article once on day 1 or once on days 1 and 15 depending on the test article. Clinical observations were performed twice daily after administration of the test article. Blood samples were collected at various times after administration for analysis of cytokine release, serum biochemistry, hematology, and toxicokinetics. Cytokine analysis was performed on serum samples using a Life Technologies Monkey Magnetic 29-Plex Panel Kit (Thermo Fisher Scientific, Waltham, MA). For toxicokinetic analysis, samples were processed to plasma and stored at -60 to -86°C prior to shipping for analysis by AIT Bioscience (Indianapolis IN) or CytomX. Plasma concentrations of the test article were measured by ELISA using anti-idiotypic capture antibodies and anti-human IgG (Fc) capture antibodies. Toxicokinetic analysis was performed by Northwest PK Solutions using noncompartmental analysis utilizing Phoenix WinNonlin v6.4 (Certara, Princeton, NJ).

[0226] result CI107 was designed as a dual-masked (activatable) dual-arm bivalent bispecific molecule containing an anti-EGFR domain and an anti-CD3 domain. CI107 was generated using a cetuximab-derived antibody with an anti-CD3ε scFv from SP34 fused to the N-terminus of the heavy chain. CI107 has a human IgG1 Fc domain containing mutations that silence Fc function. To generate CI107, a masking peptide specific for the anti-EGFR antibody element was fused to the N-terminus of the light chain using a protease-cleavable substrate linker flanked by flexible Gly-Ser-rich peptide linkers as previously described (Desnoyers 2013). A masking peptide specific for the anti-CD3 element was similarly added to the scFv using a protease-cleavable substrate linker. CI107 minimized cross-linking to cells expressing FcγR by impairing Fc-effector function. This design is intended to maximize target binding and activity in the protease-rich tumor microenvironment while minimizing binding and activity in normal tissues. All comparative TCBs used throughout this example contain EGFR and CD3 binding domains, masks, and linker peptides with varying degrees of cleavability. CI011 and CI040 are first generation versions of CI104 and CI107. CI104 and CI107 molecules contain optimized CD3 scFv, next generation cleavable linkers, and additional Fc silencing mutations. CI104 and CI107 have the same mask and EGFR and CD3 binding domains, but different CD3 protease linkers. However, after protease activation, the activated TCBs are the same. CI128, in which the EGFR binding agent is replaced by an unrelated antibody (anti-RSV), was used as a non-targeting control.

[0227] The masking impairs binding to EGFR on the cell surface. To assess whether masking of the EGFR-binding domain impairs binding to EGFR expressed on the cell surface, binding of CI107 and a comparative activated TCB construct (i.e., act-TCB) to EGFR-expressing HT29 and HCT116 cells was measured.

[0228] Target cells were incubated with increasing concentrations of CI107 or comparative activated constructs, and binding was assessed by flow cytometry. As shown in Figures 7A and 7B, the presence of the EGFR mask in CI107 substantially attenuated binding to EGFR expressed on the cell surface compared to the activated TCB CI107. The activated TCB construct bound to HT29 cells with a calculated Kd of 0.17 nM, while the Kd for CI107 binding was 91.28 nM, which corresponds to a more than 500-fold reduction in binding compared to the activated TCB. Similar results were obtained using HCT116 cells. The binding of CI128, a non-targeting control TCB that contains the same anti-CD3 module as CI107 but lacks EGFR targeting, was also assessed. This control did not bind to either HT29 or HCT116 cells (see Figures 7A and 7B).

[0229] The masking impairs binding to CD3 on the surface of lymphocytes. To determine whether masking the anti-CD3 binding domain impairs the binding of CI107 to CD3 on the surface of lymphocytes, binding of CI107 and activated CI107 (i.e., activated TCB) to Jurkat cells was measured. As shown in Figure 7C, activated TCB bound to Jurkat cells with a Kd of 0.62 nM. However, no binding of CI107 was detected and the Kd could not be calculated. The activated control CI128 bound to Jurkat cells with a similar affinity as activated TCB.

[0230] Taken together, these data demonstrate that dual masking of the anti-EGFR and anti-CD3 binding domains in CI107 attenuates binding to cells expressing EGFR or CD3.

[0231] Masking attenuates cytotoxicity and T cell activation in PBMC co-cultures. To address whether targeting EGFR with CI107 could lead to anti-tumor cell effects, an in vitro cytotoxicity assay was performed. Luciferase-expressing HT29 or HCT116 cells were co-cultured with human PBMCs and incubated with increasing concentrations of CI107, activated TCB, or non-targeting control CI128. After 48 h of culture, the viability of HCT116-Luc2 or HT29-Luc2 cells was measured via luciferase assay. As shown in Figure 8A, treatment with control CI128 resulted in minimal cytotoxic effects on HCT116-Luc2 cells co-cultured with PBMCs, demonstrating that binding of both EGFR and CD3 is required for cytotoxic activity. In contrast, both masked CI107 and activated CI107 (i.e., act-TCB) had cytotoxic effects on HCT116-Luc2 cells. However, activated TCB produced cytotoxic effects at much lower concentrations compared to the masked forms, with EC50 values ​​of 0.44 pM and 7297 pM, respectively. Similar results were observed in HT29-Luc2 cells, with EC50 values ​​of 0.25 pM for activated TCB versus 3678 pM for CI107 (Figure 8B). Thus, dual masking of the anti-EGFR and anti-CD3 domains in CI107 resulted in an approximately 15,000-fold reduction in cytotoxic activity mediated by PBMCs in the absence of protease activation.

[0232] Treatment with CI107 results in the induction of CD69 expression, a marker of T cell activation. To determine whether CI107 results in T cell activation, CD69 levels in PBMCs co-cultured with HCT116-Luc2 or HT29-Luc2 cells were measured after treatment with masked CI107, activated CI107 (i.e., act-TCB), and control CI128. CD69 serves as a marker of T cell activation, and CD69 expression is rapidly induced on the surface of T lymphocytes following TCR / CD3 engagement, serving as a costimulatory molecule for T cell activation and proliferation. Human PBMCs co-cultured with HCT116-Luc2 or HT29-Luc2 cells were treated with increasing concentrations of CI107, activated TCB (i.e., activated CI107), or control CI128 for 16 hours, and CD69 expression levels were measured by flow cytometry. As shown in Figure 8C, CI107 resulted in induction of CD69 expression on CD8+ T cells co-cultured with HCT116-Luc2 cells with an EC50 of 14178 pM. In contrast, treatment with activated CI107 resulted in CD69 induction with an EC50 of 7.65 pM, reflecting an approximately 18,000-fold shift in the T cell activation curve compared to CI107. No T cell activation was observed with the non-EGFR-targeted control CI128, indicating that CD3 binding alone is not sufficient for T cell activation. Similarly, treatment of PBMC from the same donor co-cultured with HT29-Luc2 cells resulted in CD69 induction, with EC50 values ​​of 65971 pM for masked CI107 versus 8.75 pM for activated TCB, reflecting an approximately 7500-fold difference in CD69 induction capacity (Figure 8D).

[0233] Treatment with CI107 results in cytokine release. To further assess T cell activation upon treatment with TCB in PBMCs co-cultured with EGFR-expressing cancer cells, cytokine release was assessed following treatment with CI107, activated TCB (i.e., activated CI107), or control CI128. Levels of IFN-γ, IL-2, IL6, MCP-1, and TNF-α were measured 16 hours after treatment with increasing concentrations of TCB. As shown in Figures 9A-9E, treatment with CI107 at concentrations ranging from 104 pM resulted in the release of each of the measured cytokines. In contrast, activated TCB resulted in cytokine release upon treatment at concentrations ranging from 1 to 100 pM. These results were generally consistent between the different PBMC donor cells and cancer cell lines (HCT116-Luc2 vs. HT29-Luc2).

[0234] Taken together, these data demonstrate that dual masking of the EGFR- and CD3-binding domains in CI107 attenuates T cell activation in the absence of protease activation.

[0235] Susceptibility of TCB to protease cleavage correlates with antitumor efficacy and intratumoral T cells in vivo. The antitumor efficacy of TCB was evaluated in vivo. Immunodeficient mice bearing HT29-Luc2 tumors and engrafted with human PBMCs were treated once a week for 3 weeks with vehicle (PBS) or 0.3 mg / kg TCB containing linkers with different protease sensitivities (CI011, CI040), non-cleavable linkers (CI020), or the unmasked bispecific therapeutic CI048. CI020 is expected to have the least antitumor activity due to the non-cleavable linker, while unmasked CI048 is expected to have the greatest efficacy. CI011 and CI040, both of which contain EGFR and CD3 masks, have different protease sensitivities due to different linker peptides, with the protease sensitivity of CI040 being higher than that of CI011.

[0236] As shown in Figure 10A, treatment with unmasked TCB CI048 resulted in tumor regression within one week of initiating treatment. Similarly, treatment with masked CI011 and CI040 also resulted in tumor regression or stasis. The regression seen with CI040 correlates with the higher cleavability of the linker in this molecule compared to CI011. In contrast, treatment with CI020, which contains a non-cleavable linker, did not affect tumor growth, indicating that protease cleavability is required for the antitumor activity of TCB in vivo.

[0237] To determine whether the antitumor efficacy mediated by the tested TCBs correlated with the presence of T cells in the tumors, tumors were harvested 1 week after animals received a 1 mg / kg dose of masked or activated TCB, and immunohistochemistry for CD3 was performed. As shown in Figure 10B, minimal numbers of T cells were observed in tumor tissues after treatment with vehicle or non-cleavable CI020. In contrast, increased numbers of T cells were observed upon treatment with TCB CI040 or in vitro protease-activated TCB CI048. Again, the TCB with higher protease sensitivity (CI040) resulted in a higher number of T cells in the tumors.

[0238] Taken together, these data suggest that TCB may confer intratumoral T cell and in vivo antitumor efficacy that correlates with the susceptibility of the EGFR- and CD3-binding domain masks to protease cleavage.

[0239] Treatment with CI107 induces dose-dependent regression of established xenograft tumors. The effect of CI107 on tumor growth in vivo was evaluated. NSG mice were subcutaneously implanted with HT29 cells, followed by IP injection of PBMCs, and PBMCs were allowed to engraft for approximately 11 days. Animals were then treated with vehicle, 0.5mg / kg CI107, or 1.5mg / kg CI107 once a week for 3 weeks. As shown in Figure 11A, starting approximately 1 week after treatment initiation, treatment with 0.5mg / kg CI107 resulted in tumor stasis, and 1.5mg / kg CI107 resulted in tumor regression.

[0240] The in vivo efficacy of CI107 was also evaluated in HCT116 tumors. After tumor and PBMC implantation, animals were treated with vehicle, 0.3 mg / kg CI107, 1 mg / kg CI107, or 0.3 mg / kg activated TCB. As shown in Figure 11B, 0.3 mg / kg CI107 delayed HCT116 tumor growth, while 1 mg / kg CI107 and 0.3 mg activated TCB caused similar levels of tumor regression and stasis over the duration of treatment.

[0241] These data demonstrate that CI107 induces dose-dependent inhibition of tumor growth and regression in HT29 and HCT116 xenograft tumors, and that the antitumor activity of a three-fold higher dose of CI107 is similar to that of activated TCB.

[0242] Masked CI107 provides increased safety compared to activated CI107 in cynomolgus monkeys. The preclinical tolerability of CI107 was evaluated in a cynomolgus monkey study. Animals were given a single dose of 0.06 mg / kg or 0.18 mg / kg activated CI107 (i.e., act-TCB) and 0.6 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 6.0 mg / kg CI107, and animals were followed for clinical observations. Animals treated with 0.18 mg / kg activated TCB experienced severe clinical effects including vomiting, anorexia, weak appearance, hunched posture, and thin appearance, with adverse effects noted as early as 2 hours and up to 10 days after dosing. Animals treated with 0.06 mg / kg activated TCB experienced moderate, transient clinical effects including vomiting and hunched posture on day 1 after dosing. Based on the rapid resolution of these effects, 0.06 mg / kg was defined as the maximum tolerated dose (MTD) for activated TCB. In contrast, animals treated with 2.0 mg / kg CI107 experienced only transient and mild clinical effects (vomiting on day 2), and animals treated with 0.6 mg / kg CI107 did not experience any adverse effects. Animals treated with 4.0 mg / kg CI107 experienced moderate clinical effects (including vomiting at 4, 8, and 24 hours after dosing and anorexia on day 2). Animals treated with 6.0 mg / kg CI107 were found dead on day 2. Clinical signs observed prior to death included hunched posture, weak appearance, vomiting, and liquid stools after dosing. Therefore, 4.0 mg / kg was considered the MTD for CI107. Overall, masked CI107 achieved a greater than 60-fold improvement in tolerability compared to activated TCB.

[0243] Cytokine levels after treatment with activated or masked CI107 were also investigated. As shown in Figure 12, levels of IL-6 (12A) and IFN-γ (12B) were elevated 8 hours after administration in animals treated with activated TCB. In contrast, minimal changes in IL-6 or IFN-γ were observed after treatment with 0.6 mg / kg or 2.0 mg / kg CI107. Increases in the levels of these cytokines were only seen after treatment with 4.0 mg / kg CI107. Consistent with clinical observations, CI107 shifts the dose response of cytokine release by more than 60-fold.

[0244] Analysis of serum biochemistry also demonstrated differences between activated TCB and CI107. As shown in Figure 12C, treatment with activated TCB resulted in a dose-dependent increase in aspartate aminotransferase (AST), a marker of hepatocellular injury, 48 hours after administration. In contrast, no changes in AST were observed after treatment with CI107 at any of the tolerated dose levels, demonstrating improved tolerability of this masked TCB.

[0245] To address whether masking of the EGFR- and CD3-binding domains affects pharmacokinetics, plasma concentrations were measured after administration of activated TCB (i.e., activated CI107) and masked CI107. As shown in Figure 12D, activated TCB was rapidly cleared from the circulation within 24 hours after administration. In contrast, CI107 was maintained in plasma for up to 7 days after administration, suggesting that masking may increase exposure compared to activated TCB. The AUC(0-7) after a single dose of 0.06 mg / kg activated TCB was 0.04 nM x day (n=1), while the AUC(0-7) after a single dose of 2 mg / kg CI107 was 331.7 nM x day (mean n=3), demonstrating a more than 8,000-fold increase in tolerable exposure.

[0246] This demonstrates that the improved tolerability and pharmacokinetics observed with masked CI107 are consistent with the expected attenuated binding to EGFR and CD3 in the normal tissue environment. [Table 15] JPEG2024539650000019.jpg216159 JPEG2024539650000020.jpg219159 JPEG2024539650000021.jpg218159 JPEG2024539650000022.jpg219159 JPEG2024539650000023.jpg198159 JPEG2024539650000024.jpg178159 JPEG2024539650000025.jpg170159 JPEG2024539650000026.jpg194159 JPEG2024539650000027.jpg219159 JPEG2024539650000028.jpg196159 JPEG2024539650000029.jpg216159 JPEG2024539650000030.jpg172159 JPEG2024539650000031.jpg192159 JPEG2024539650000032.jpg162159 JPEG2024539650000033.jpg203159 JPEG2024539650000034.jpg205159 JPEG2024539650000035.jpg180159 JPEG2024539650000036.jpg106159

[0247] The present disclosure is not limited in scope by the embodiments described herein. Indeed, various modifications of the present disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0248] All references (e.g., publications or patents or patent applications) cited in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0249] Certain aspects are within the scope of the following claims.

Claims

1. 1. An activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC), comprising: (a) a first polypeptide comprising: (i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a cluster of differentiation (CD3) targeting domain that specifically binds to a CD3 polypeptide; (ii) a first masking moiety (MM1); (iii) a first cleavable moiety (CM1); (iv) a second heavy chain variable domain (VH2); and (v) a first monomeric Fc domain (Fc1); (b) a second polypeptide comprising: (i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds to EGFR; (ii) a second masking moiety (MM2); and (iii) a second cleavable moiety (CM2); (c) a third polypeptide comprising (i) a second monomeric Fc domain (Fc2), and (ii) no immunoglobulin variable domain.

2. 2. The activatable bispecific polypeptide complex of claim 1, wherein the CD3 polypeptide is the epsilon chain of CD3.

3. The VH1 is (i) a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO: 3); (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 4); and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 5), wherein the VL1 comprises: (i) a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO: 6); (ii) a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 7); and 2. The activatable bispecific polypeptide complex of claim 1 , comprising: (iii) a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8).

4. 4. The activatable bispecific polypeptide complex of claim 3, wherein the scFv comprises a VH1 having an amino acid sequence that is at least 90% identical to SEQ ID NO: 9 and / or a VL1 having an amino acid sequence that is at least 90% identical to SEQ ID NO: 10, and optionally the scFv comprises a VH1 having the amino acid sequence of SEQ ID NO: 9 and a VL1 having the amino acid sequence of SEQ ID NO:

10.

5. The VH2 is (i) a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15); (ii) a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16); and (iii) a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17).

6. The VL2 is (i) a VL CDR1 comprising RASQSIGTNIH (SEQ ID NO: 18); (ii) a VL CDR2 comprising YASESIS (SEQ ID NO: 19), and 5. The activatable bispecific polypeptide complex of claim 1 , comprising (iii) a VL CDR3 comprising QQNNNWPTT (SEQ ID NO: 20).

7. The VH2 is (a) comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 21; or (b) comprising the amino acid sequence of SEQ ID NO: 21; The activatable bispecific polypeptide complex of claim 5 .

8. 5. The activatable bispecific polypeptide complex of claim 1, wherein the Fc1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 23, and optionally the Fc1 comprises the amino acid sequence of SEQ ID NO:

23. (a) the first polypeptide further comprises a heavy chain CH1 domain between the VH2 and the Fc1; (b) the first polypeptide further comprises an immunoglobulin hinge region between the VH2 and the Fc1; (c) the first polypeptide comprises, from amino terminus to carboxy terminus, the structural arrangement: MM1-CM1-scFv-VH2-CH1-hinge region-Fc1, wherein each "-" is independently a direct or indirect linkage; and / or (d) the first polypeptide comprises one or more linkers, and optionally comprises from about 1 to about 20 amino acids; 5. The activatable bispecific polypeptide complex of any one of claims 1 to 4.

10. The VL2 is (i) a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18); (ii) a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and (iii) a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO: 20).

11. 11. The activatable bispecific polypeptide complex of claim 10, wherein the VL2 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 22, and optionally the VL2 comprises the amino acid sequence of SEQ ID NO:

22. (a) the second polypeptide comprises, from the amino terminus to the carboxy terminus, the structural arrangement MM2-CM2-VL2, wherein each "-" is independently a direct or indirect linkage; and / or (b) the second polypeptide comprises one or more linkers, optionally wherein the linkers comprise from about 1 to about 20 amino acids.

13. The activatable bispecific polypeptide complex of any one of claims 1 to 4, wherein said Fc2 binds to said Fc1.

14. 5. The activatable bispecific polypeptide complex of any one of claims 1 to 4, wherein the Fc2 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:28, and optionally the Fc2 comprises the amino acid sequence of SEQ ID NO:

28.

15. (a) at least one of the first polypeptide and the third polypeptide further comprises an immunoglobulin hinge region; (b) the first polypeptide and the third polypeptide each comprise an immunoglobulin hinge region, and optionally (i) the immunoglobulin hinge region of the first polypeptide and the immunoglobulin hinge region of the third polypeptide comprise the same amino acid sequence; or (ii) the immunoglobulin hinge region of the first polypeptide and the immunoglobulin hinge region of the third polypeptide comprise different amino acid sequences; (c) the third polypeptide comprises, from amino terminus to carboxy terminus, an immunoglobulin hinge region in a hinge region-Fc2 structural configuration; and / or (d) the first polypeptide, the second polypeptide, and / or the third polypeptide comprise one or more linkers, and optionally (i) MM1 is linked to CM1 via a linker L1, and / or 5. The activatable bispecific polypeptide complex of claim 1, wherein (ii) MM2 is linked to CM2 via a linker L2.

16. (a) wherein the CM1 and the CM2 each comprise a substrate for a protease present in the tumor microenvironment of a subject with cancer. (b) the CM1 and the CM2 each contain a substrate for the same protease; (c) the CM1 and the CM2 contain substrates for different proteases; (d) said CM1 and CM2 each independently comprise a substrate for a protease selected from the group of proteases shown in Table 2; (e) at least one of the CM1 and the CM2 comprises a substrate for a serine protease or a matrix metallopeptidase (MMP); (f) the CM1 comprises the amino acid sequence of SEQ ID NO: 2, and the CM2 comprises the amino acid sequence of SEQ ID NO: 14; (g) the CM1 comprises the amino acid sequence of SEQ ID NO: 2; (h) the CM2 comprises the amino acid sequence of SEQ ID NO: 14; (i) the CM1 comprises the amino acid sequence of SEQ ID NO: 73, and the CM2 comprises the amino acid sequence of SEQ ID NO: 14; (j) the CM1 comprises the amino acid sequence of SEQ ID NO: 73; or (k) The activatable bispecific polypeptide complex of any one of claims 1 to 4, wherein the CM2 comprises the amino acid sequence of SEQ ID NO:

14. (a) the MM1 and / or the MM2 comprise from about 5 amino acids to about 40 amino acids; or (b) the activatable bispecific polypeptide complex of any one of claims 1 to 4, wherein the MM1 is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:

72.

18. 5. The activatable bispecific polypeptide complex of claim 1, wherein MM2 comprises the amino acid sequence of SEQ ID NO:

13.

19. 5. The activatable bispecific polypeptide complex of claim 1, wherein MM1 comprises the amino acid sequence of SEQ ID NO:

1.

20. 2. The activatable bispecific polypeptide complex of claim 1, wherein (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 30, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO: 31, and (3) the third polypeptide comprises the amino acid sequence of SEQ ID NO:

32.

21. 2. The activatable bispecific polypeptide complex of claim 1, wherein (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 120, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO: 37, and (3) the third polypeptide comprises the amino acid sequence of SEQ ID NO:

32.

22. 2. The activatable bispecific polypeptide complex of claim 1, wherein (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 144, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO: 37, and (3) the third polypeptide comprises the amino acid sequence of SEQ ID NO:

32.

23. A pharmaceutical composition comprising the activatable bispecific polypeptide complex of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

24. 24. A kit comprising the pharmaceutical composition of claim 23.

25. 5. A nucleic acid comprising a nucleotide sequence encoding the first polypeptide, the second polypeptide, and the third polypeptide of the activatable bispecific polypeptide of any one of claims 1 to 4.

26. A vector comprising the nucleic acid of claim 25.

27. 26. A host cell comprising the nucleic acid of claim 25.

28. 1. A method for producing an activatable heteromultimeric bispecific polypeptide complex (HBPC), comprising: (a) culturing the host cell of claim 27 in a liquid medium under conditions sufficient to produce said HBPC; (b) recovering the HBPCs.

29. 24. The pharmaceutical composition of claim 23, for treating a disease in a subject, optionally wherein the subject is a human.

30. 30. The method of claim 29, wherein the disease is cancer.

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