Antibodies, activatable antibodies, bispecific antibodies, and bispecific activatable antibodies and methods of use thereof

Abstract

To provide antibodies that mimic desirable characteristics of small molecule prodrugs in the field of antibody-based therapeutics.SOLUTION: Antibodies, activatable antibodies (AA), bispecific antibodies, and bispecific activatable antibodies (BAA) are provided. Methods for making and using these antibodies, AAs, bispecific antibodies, and BAAs are also provided. In one aspect, an activatable antibody (AA) is provided, comprising: a) an antibody or antigen-binding fragment (AB) thereof that specifically binds to the epsilon chain of CD3 (CD3ε); b) a masking moiety (MM) coupled to the AB, the MM reducing or inhibiting binding of the AB to the CD3ε when the AA is in an uncleaved state, and the MM comprising a specific amino acid sequence; and c) a cleavable moiety (CM) coupled to the AB, the CM being a polypeptide that functions as a substrate for a protease.SELECTED DRAWING: Figure 17

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 572,468, filed October 14, 2017; U.S. Provisional Patent Application No. 62 / 577,140, ​​filed October 25, 2017; U.S. Provisional Patent Application No. 62 / 613,358, filed January 3, 2018; U.S. Provisional Patent Application No. 62 / 666,065, filed May 2, 2018; and U.S. Provisional Patent Application No. 62 / 731,622, filed September 14, 2018, the contents of each of which are incorporated by reference in their entirety. FIELD OF THE INVENTION

[0002] Provided herein are antibodies, activatable antibodies (AA), bispecific antibodies, and bispecific activatable antibodies (BAA). Also provided herein are methods of making and using these antibodies, AAs, bispecific antibodies, and BAA.

[0003] Sequence Listing Reference The "Sequence Listing," which was submitted electronically contemporaneously herewith in computer-readable form (CRF) via EFS-Web in accordance with 37 CFR § 1.821 under the filename CYTX-045-US_SEQLIST_10-12-18_ST25.txt, is incorporated herein by reference. The electronic copy of the Sequence Listing was created on December 10, 2018, and is 440 kilobytes in size on disk. [Background technology]

[0004] background Although antibody-based therapeutics have proven effective treatments for several diseases, in some cases their therapeutic efficacy is limited by toxicity due to the broad target spectrum they express. Furthermore, antibody-based therapeutics also exhibit other limitations, such as rapid clearance from the circulation after administration. Summary of the Invention [Means for solving the problem]

[0005] In the area of ​​small molecule therapeutics, strategies have been developed to provide prodrugs of active chemicals. Such prodrugs are administered in a relatively inactive (or significantly less active) form. Upon administration, the prodrug is metabolized in vivo to the active compound. Such prodrug strategies can increase the selection of drugs aimed at their intended target and reducing adverse effects.

[0006] Thus, there remains a need in the field of antibody-based therapeutics for antibodies that mimic the desirable characteristics of small molecule prodrugs. Summary of the Invention

[0007] Provided herein are antibodies, bispecific antibodies, activatable antibodies, and bispecific activatable antibodies, methods for making and using them, which have applications in therapeutics and diagnostics. The activatable antibodies and bispecific activatable antibodies of the present disclosure can be used to reduce damage to healthy tissues typically resulting from an antibody binding to its target on healthy tissues as well as diseased tissues.

[0008] Thus, according to one embodiment, there is provided herein a bispecific activatable antibody (BAA), wherein said BAA, when activated, specifically binds to two targets and has the following structure: a) an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 is a. two heavy chains (AB1 HC) and two light chains (AB1 LC); and b. a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form an MM1-CM1 construct, wherein the carboxyl terminus of said MM1-CM1 construct is linked to each amino terminus of each light chain of said AB1; where: c. said MM1 inhibits the binding of said AB1 to its target; d. CM1 is a polypeptide that functions as a substrate for the first protease, an IgG antibody (AB1); b) two scFvs (AB2) each specifically binding to a second target, wherein each AB2 is a. a light chain variable region linked to a heavy chain variable region, wherein the carboxyl terminus of each of said AB2 is linked to the amino terminus of each of said AB1 heavy chains; and b. a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of said MM2-CM2 construct is linked to the amino terminus of each said AB2; where: c. said MM2 inhibits the binding of said AB2 to its target; d. The aforementioned CM2 is a polypeptide that functions as a substrate for a second protease, two scFvs (AB2) Including, c) wherein said BAA has the following characteristics: a. MM2 comprises the amino acid sequence of SEQ ID NO: 12; b. MM1 comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7; c. AB2 comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3 or a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4; and d. AB1 comprises an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced. It has at least one of the following.

[0009] In some embodiments, the BAAs provided herein are A bispecific activatable antibody (BAA), wherein said BAA, when activated, specifically binds to two targets and has the following structure: a. an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein said AB1 is linked to a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl termini of said MM1-CM1 construct are linked to each amino terminus of each light chain of said AB1, wherein said MM1 inhibits the binding of said AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, an IgG antibody (AB1), b. two scFvs (each AB2) each specifically binding to a second target, wherein each AB2 comprises a light chain variable region linked to a heavy chain variable region, wherein the carboxyl terminus of each of said AB2 is linked to the amino terminus of each of said AB1 heavy chains; wherein each AB2 is linked to a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each of said MM2-CM2 constructs is linked to the amino terminus of each of said AB2, wherein said MM2 inhibits the binding of said AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for a second protease, two scFvs (each AB2) Including, Here, the aforementioned BAA has the following characteristics: i. MM2 comprises the amino acid sequence of SEQ ID NO: 12; ii. MM1 comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7; iii. AB2 comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3 or a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4; and iv. AB1 comprises an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced. The present invention also includes a bispecific activatable antibody (BAA) having at least one of the following:

[0010] In some embodiments, the BAA comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3 and a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4. In some embodiments, AB1 binds a tumor target and AB2 binds an immune effector target. In some embodiments, the BAA is a T cell-inducing bispecific (TCB) AA (TCBAA). In some embodiments, AB1 binds EGFR and AB2 binds CD3. In some embodiments, MM1 comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7. In some embodiments, MM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:85 and SEQ ID NO:78. In some embodiments, MM1 comprises SEQ ID NO:78. In some embodiments, MM2 comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14 and SEQ ID NO:16. In some embodiments, CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 17. In some embodiments, provided herein is BAA CI106, which comprises the layout and sequence provided in Table 11 and Example 1. In some embodiments, provided herein is BAA CI107, which comprises the layout and sequence provided in Table 11 and Example 1. In some embodiments, provided herein is BAA CI079, which comprises the layout and sequence provided in Table 11 and Example 1. In some embodiments, provided herein is BAA CI090, which comprises the layout and sequence provided in Table 11 and Example 1. In some embodiments, provided herein is BAA CI135, which comprises the layout and sequence provided in Table 11 and Example 1. In some embodiments, provided herein is BAA CI136, which comprises the layout and sequence provided in Table 11 and Example 1. In some embodiments, AB1 comprises amino acid substitutions at at least two of amino acid positions L234, L235, and P331.In some embodiments, AB1 comprises amino acid substitutions at amino acid positions L234, L235, and P331. In some embodiments, AB1 comprises amino acid substitutions at L234F, L235E, and P331S. In some embodiments, AB1 comprises an Fc region comprising an amino acid substitution at N297. In some embodiments, AB1 comprises amino acid substitutions at L234F, L235E, P331S, and N297Q. In some embodiments, the heavy chain of AB1 comprises any one of SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76 in Table 6.

[0011] In another embodiment, a bispecific activatable antibody (BAA) comprising: a) an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 is i. two heavy chains (AB1 HC) and two light chains (AB1 LC); and ii. a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of said MM1-CM1 construct is linked to each amino terminus of each light chain of said AB1, wherein: said MM1 inhibits the binding of said AB1 to its target; The aforementioned CM1 is a first masking moiety (MM1), which is a polypeptide that functions as a substrate for a first protease. IgG antibody (AB1), b) two scFvs (AB2) each specifically binding to a second target, wherein each AB2 is i. a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each of said AB2 heavy chains is linked to the amino terminus of each of said AB1 heavy chains; and ii. a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of said MM2-CM2 construct is linked to the amino terminus of each said AB2, wherein: said MM2 inhibits the binding of said AB2 to its target; The aforementioned CM2 is a second masking moiety (MM2), which is a polypeptide that serves as a substrate for a second protease. Two scFvs (AB2) containing Including, Provided herein is a bispecific activatable antibody (BAA), wherein said AB1 comprises an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, as numbered according to the EU index as set forth in Kabat, such that said BAA has a reduced effector function. In some embodiments, the Fc region comprises amino acid substitutions at at least amino acid positions L234, L235, N297, and P331, as numbered according to the EU index as set forth in Kabat, such that said BAA has a reduced effector function. In some embodiments, the Fc region comprises amino acid substitutions at at least amino acid positions L234, L235, and P331, as numbered according to the EU index as set forth in Kabat, such that said BAA has a reduced effector function. In some embodiments, the Fc region comprises amino acid substitutions at at least amino acid positions L234, L235, and P331, as numbered according to the EU index as set forth in Kabat, such that said BAA has a reduced effector function. In some embodiments, the first target is selected from the group consisting of the targets set forth in Table 9, and the second target is selected from the group consisting of the targets set forth in Table 9.

[0012] In some embodiments, the BAAs provided herein are a) a bispecific activatable antibody (BAA), i) an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein said AB1 is linked to a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl termini of said MM1-CM1 construct are linked to each amino terminus of each light chain of said AB1, wherein said MM1 inhibits the binding of said AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, an IgG antibody (AB1), ii) two scFvs (each AB2) each specifically binding to a second target, wherein each AB2 comprises a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each of said AB2 is linked to the amino terminus of each of said AB1 heavy chains; wherein each AB2 is linked to a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each of said MM2-CM2 constructs is linked to the amino terminus of each of said AB2, wherein said MM2 inhibits the binding of said AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for a second protease, a polypeptide that binds two scFvs. Including, wherein said AB1 comprises a bispecific activatable antibody (BAA) comprising an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, as numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced. In some embodiments, the Fc region comprises amino acid substitutions at at least amino acid positions L234, L235, N297, and P331, as numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced. In some embodiments, the Fc region comprises amino acid substitutions at at least amino acid positions L234, L235, and P331, as numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced. In some embodiments, the first target is selected from the group consisting of the targets set forth in Table 9, and the second target is selected from the group consisting of the targets set forth in Table 9.

[0013] In another aspect, provided herein is an activatable antibody (AA), comprising: (a) an antibody (AB) that specifically binds to epidermal growth factor receptor (EGFR), wherein the AB is an IgG1 antibody, and wherein the Fc region of the AB comprises an amino acid substitution at at least one of amino acid positions L234, L235, and P331, as numbered according to the EU index as set forth in Kabat, such that the effector function of the AA is reduced; (b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits binding of the AB to EGFR when the AA is in an uncleaved state; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, AB comprises amino acid substitutions at at least two of amino acid positions L234, L235, and P331. In some embodiments, AB comprises amino acid substitutions at amino acid positions L234, L235, and P331. In some embodiments, AB comprises amino acid substitutions at L234F, L235E, and P331S. In some embodiments, AB comprises an Fc region comprising an amino acid substitution at N297. In some embodiments, the Fc region comprises an N297Q mutation. In some embodiments, AB comprises amino acid substitutions at L234F, L235E, P331S, and N297Q. In some embodiments, MM comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7. In some embodiments, MM comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, MM comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, CM comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 4. In some embodiments, CM comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, AA is a portion of BAA.

[0014] In another aspect, provided herein is an activatable antibody (AA) comprising: (a) an antibody or antigen-binding fragment thereof (AB) that specifically binds to epidermal growth factor receptor (EGFR); (b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits binding of the AB to EGFR when the AA is in an uncleaved state, and wherein the MM comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the CM comprises a substrate cleavable by a serine protease or an MMP. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-56. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the AA is part of a BAA.

[0015] In another embodiment, an activatable antibody (AA) comprising: Provided herein is an activatable antibody (AA) comprising: (a) an antibody or antigen-binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3, wherein the antibody comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3, or a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4; (b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits binding of the AB to CD3 when the AA is in an uncleaved state; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments, the AB comprises a heavy chain variable domain set forth in SEQ ID NO:2. In some embodiments, the AB comprises a heavy chain variable domain set forth in SEQ ID NO:3. In some embodiments, the AB comprises a light chain variable domain set forth in SEQ ID NO:1. In some embodiments, the AB comprises a light chain variable domain set forth in SEQ ID NO:4. In some embodiments, the AB comprises a heavy chain variable domain set forth in SEQ ID NO:2 and a light chain variable domain set forth in SEQ ID NO:1. In some embodiments, the AB comprises a heavy chain variable domain set forth in SEQ ID NO: 3 and a light chain variable domain set forth in SEQ ID NO: 1. In some embodiments, the AB comprises a heavy chain variable domain set forth in SEQ ID NO: 2 and a light chain variable domain set forth in SEQ ID NO: 4. In some embodiments, the AB comprises a heavy chain variable domain set forth in SEQ ID NO: 3 and a light chain variable domain set forth in SEQ ID NO: 4. In some embodiments, the MM comprises any one of the sequences set forth in Table 3. In some embodiments, the CM comprises any one of the sequences set forth in Table 4. In some embodiments, the AA is a portion of a BAA.

[0016] In another aspect, provided herein is an activatable antibody (AA) comprising: (a) an antibody or antigen-binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3; (b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits binding of the AB to CD3 when the AA is in an uncleaved state, and wherein the MM comprises the amino acid sequence of SEQ ID NO: 12; and (b) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments, the CM comprises any one of the sequences set forth in Table 4. In some embodiments, the CM comprises a substrate cleavable by a serine protease or an MMP. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-56. In some embodiments, the protease is an MMP. In some embodiments, the protease is a serine protease. In some embodiments, the AA is part of a BAA.

[0017] In another aspect, provided herein is an activatable antibody (AA), comprising: (a) an antibody (AB) that specifically binds to a target, wherein the antibody is an IgG1 antibody, and wherein an Fc region of the antibody comprises an amino acid substitution at at least one of amino acid positions L234, L235, and P331, numbered according to the EU index as set forth in Kabat, such that an effector function of the AA is reduced; (b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits binding of the AB to a target when the AA is in an uncleaved state; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments, the Fc region comprises amino acid substitutions at least at amino acid positions L234, L235, N297, and P331, as numbered by the EU index as set forth in Kabat, such that the effector function of the aforementioned AA is reduced. In some embodiments, the target is selected from the group consisting of the targets set forth in Table 9. In some embodiments, the AA is part of a BAA.

[0018] In another aspect, provided herein is an antibody or antigen-binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3, wherein said antibody comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3 or a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4. In some embodiments, the antibody comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3 and a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4. In some embodiments, AB comprises a heavy chain variable domain set forth in SEQ ID NO:2. In some embodiments, AB comprises a heavy chain variable domain set forth in SEQ ID NO:3. In some embodiments, AB comprises a light chain variable domain set forth in SEQ ID NO:1. In some embodiments, AB comprises a light chain variable domain set forth in SEQ ID NO:4. In some embodiments, AB comprises a heavy chain variable domain set forth in SEQ ID NO:2 and a light chain variable domain set forth in SEQ ID NO:1. In some embodiments, AB comprises a heavy chain variable domain set forth in SEQ ID NO:3 and a light chain variable domain set forth in SEQ ID NO:1. In some embodiments, AB comprises a heavy chain variable domain set forth in SEQ ID NO:3 and a light chain variable domain set forth in SEQ ID NO:4. In some embodiments, the AB comprises a heavy chain variable domain set forth in SEQ ID NO:2 and a light chain variable domain set forth in SEQ ID NO:4. In some embodiments, the antibody is a bispecific AB. In some embodiments, the antibody is an scFv. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is part of an AA or part of a BAA.

[0019] In another aspect, provided herein is an antibody (AB) that specifically binds to EGFR or CD3, wherein the antibody is an IgG1 antibody or an scFv linked to an Fc domain, and wherein the antibody comprises an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, and P331, numbered according to the EU index as set forth in Kabat, such that the antibody has reduced effector function. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, the antibody comprises amino acid substitutions at at least two of amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises amino acid substitutions at amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises amino acid substitutions at L234F, L235E, and P331S. In some embodiments, the antibody comprises amino acid substitutions at L234F, L235E, and P331S. In some embodiments, the antibody comprises an Fc region comprising an amino acid substitution at N297. In some embodiments, the Fc region comprises an N297Q mutation. In some embodiments, the antibody comprises amino acid substitutions L234F, L235E, P331S, and N297Q. In some embodiments, the heavy chain of the antibody comprises any one of SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76 set forth in Table 6. In some embodiments, the heavy chain variable domain of the antibody comprises any one of SEQ ID NO:2 or SEQ ID NO:3, and the light chain variable domain of AB comprises any one of SEQ ID NO:1 or SEQ ID NO:4. In some embodiments, the antibody is part of AA or part of BAA.

[0020] In another aspect, also provided herein is a pharmaceutical composition comprising any one of the above BAAs, AAs, and antibodies, and an optional carrier. In another aspect, also provided herein is a pharmaceutical composition comprising any one of the above BAAs, AAs, and antibodies, and a carrier. In some embodiments, the composition comprises an additional agent, for example, the additional agent can be a therapeutic agent.

[0021] In another aspect, also provided herein is an isolated nucleic acid molecule encoding any one of the above BAA, AAs, and antibodies. Also provided is a vector comprising the nucleic acid. In some embodiments, the vector comprises the nucleic acid sequence of pLW289. In some embodiments, the vector comprises the nucleic acid sequence of pLW246. In some embodiments, the vector comprises the nucleic acid sequence of pLW307. In some embodiments, the vector comprises the nucleic acid sequence of pLW291. In some embodiments, the vector comprises the nucleic acid sequence of pLW352. In some embodiments, the vector comprises the nucleic acid sequence of pLW246. In some embodiments, the vector comprises the nucleic acid sequence of pLW353.

[0022] In another aspect, also provided herein are cells comprising any one of the above vectors. In some embodiments, provided herein are cells comprising pLW289 and pLW246. In some embodiments, provided herein are cells comprising pLW307 and pLW291. In some embodiments, provided herein are cells comprising pLW352 and pLW246. In some embodiments, provided herein are cells comprising pLW353 and pLW246.

[0023] In another aspect, provided herein is a method of producing an antibody, AA, or BAA provided above by culturing cells under conditions that allow expression of the antibody, AA, or BAA, wherein the cells contain the relevant nucleic acid molecule or vector provided herein.

[0024] In another aspect, provided herein are methods of treating, alleviating symptoms of, or delaying progression of a disorder or disease, comprising administering a therapeutically effective amount of the above-described antibody / AA / BAA / pharmaceutical composition to a subject in need thereof. In some embodiments, the disorder or disease involves disease cells that express EGFR. In some embodiments, the disorder or disease is cancer. In some embodiments, the cancer is anal cancer, basal cell carcinoma, brain cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, intrahepatic bile duct cancer, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, small intestine carcinoma, skin cancer, testicular cancer, thyroid cancer, or uterine cancer. In some embodiments, the disorder is lymphoma, e.g., Epstein-Barr virus-associated lymphoma, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is cutaneous squamous cell carcinoma. In some embodiments, the cancer is esophageal squamous cell carcinoma. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is lung squamous cell carcinoma.

[0025] In another aspect, provided herein are methods of inhibiting angiogenesis in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody / AA / BAA / pharmaceutical composition as described above. In some embodiments, the method comprises administering an additional agent. In some embodiments, the additional agent is a therapeutic agent.

[0026] In another aspect, provided herein is a method for reducing damage to healthy tissue caused by an antibody binding to its target on diseased tissue (e.g., cancerous tissue) as well as on healthy tissue, comprising administering an AA or BAA or a pharmaceutical composition comprising an AA or BAA to a subject in need thereof, wherein the AA or BAA is an AA or BAA of any one of the embodiments provided herein.

[0027] In another aspect, provided herein is a method for improving the tolerability of antibody treatment, comprising administering an AA or BAA or a pharmaceutical composition comprising an AA or BAA to a subject in need thereof (e.g., a subject suffering from cancer), wherein the AA or BAA is an AA or BAA of any one of the embodiments provided herein.

[0028] In another aspect, provided herein is a method for recruiting T cells to tumor tissue, comprising administering to a subject in need thereof an AA or BAA or a pharmaceutical composition comprising an AA or BAA, wherein the AA or BAA is an AA or BAA of any one of the embodiments provided herein.

[0029] In another aspect, the present invention provides an antibody, AA, BAA, or pharmaceutical composition of any one of the embodiments provided herein for use as a medicament.The medicament can be used in a method for reducing the damage to healthy tissue caused by the antibody binding to its target on diseased tissue and healthy tissue.The medicament can be used to improve the tolerability of antibody treatment.

[0030] In another aspect, provided herein is an antibody, AA, BAA, or pharmaceutical composition of any one of the embodiments provided herein for use in a method of treating, alleviating a symptom of, or slowing the progression of a disorder or disease, wherein said disorder or disease comprises diseased cells that express EGFR.

[0031] In another aspect, provided herein is any one of the antibodies, AA, BAA, or pharmaceutical compositions of the embodiments provided herein for use in a method for treating cancer; wherein optionally, the cancer is anal cancer, basal cell carcinoma, brain cancer, bladder cancer, breast cancer, bone cancer, cervical cancer, intrahepatic bile duct cancer, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, small intestine carcinoma, squamous cell carcinoma, skin cancer, testicular cancer, thyroid cancer, or uterine cancer. Use can include recruiting T cells to tumor tissue.

[0032] In another aspect, provided herein is an antibody, AA, BAA, or pharmaceutical composition of any one of the embodiments provided herein for use in a method comprising inhibiting angiogenesis.

[0033] The antibody, AA, BAA, or pharmaceutical composition of any one of the embodiments provided herein may be for use in a method of treatment that includes administering an additional agent; optionally, the additional agent is a therapeutic agent. [Brief explanation of the drawings]

[0034] [Figure 1A] Figure 1A demonstrates that incorporation of the h20GG CD3ε masking peptide into the EGFR-masked BAAs CI106 and CI107 significantly reduced binding to Jurkat cells compared to CI011. Reduced binding to EGFR+ HT29-luc2 cells was also evident for CI106 and CI107 compared to CI011 (Figure 1B). [Figure 2A]Figure 2A demonstrates that EGFR+ HT29-luc2 cell killing is further attenuated by CI106 and CI107 compared to CI011 and CI040. Figure 2B shows that no detectable cytotoxicity was observed when cells were treated with CI127 and CI128, demonstrating that cell killing is dependent on EGFR targeting. Figure 2B also demonstrates a >300,000-fold EC50 shift for the doubly masked antibodies (i.e., BAAs in which both the EGFR and CD3 target binding domains are masked) CI106 and CI107 compared to the protease-activated bispecific antibody (i.e., BAA activated by protease treatment) act-104 (interchangeably referred to as CI104). Figure 2C shows the number of EGFR receptors on a panel of cell lines, including HT29. The approximate number of EGFR receptors on HT29 cells is 75,000, indicating that a high density of antigen is not required for the potent cytotoxicity of the tested BAAs. [Figure 3A] Figure 3A demonstrates that activation of primary CD8+ T cells was attenuated by CI106 and CI107 compared to CI011 and CI040. Figure 3B shows a shift in the dose-response curve for T cell activation with doubly masked antibodies compared to the protease-activated bispecific antibody act-104, demonstrating that masking weakens T cell activation. [Figure 4] FIG. 4 plots tumor volume versus days after first treatment dose and demonstrates the dose-dependent effect of the doubly masked bispecific AAs CI106 and CI107 on HT29-luc2 xenograft tumor growth. [Figure 5] FIG. 5 plots tumor volume versus days after first treatment dose and demonstrates the dose-dependent effect of the doubly masked bispecific AAs CI106 and CI107 on HCT116 xenograft tumor growth. [Figure 6A] Figures 6A-6B demonstrate that the EC50s of the tested doubly masked and protease-activated bispecific antibodies are similar when using either human (6A) or cynomolgus monkey (6B) effector cells. [Figure 6B] Figures 6A-6B demonstrate that the EC50s of the tested doubly masked and protease-activated bispecific antibodies are similar when using either human (6A) or cynomolgus monkey (6B) effector cells. [Figure 6C] Figures 6C-6D demonstrate similar binding of protease-activated and doubly masked antibodies to human (6C) and cynomolgus monkey (6D) T cells. [Figure 6D] Figures 6C-6D demonstrate similar binding of protease-activated and doubly masked antibodies to human (6C) and cynomolgus monkey (6D) T cells. [Figure 7A] 7A-7C show pre-dose, 48 hours post-dose, and 7 days post-dose serum concentrations of ALT (7A), AST (7B), and total bilirubin (7C) in cynomolgus monkeys treated with CI106 or CI107. [Figure 7B] 7A-7C show pre-dose, 48 hours post-dose, and 7 days post-dose serum concentrations of ALT (7A), AST (7B), and total bilirubin (7C) in cynomolgus monkeys treated with CI106 or CI107. [Figure 7C] 7A-7C show pre-dose, 48 hours post-dose, and 7 days post-dose serum concentrations of ALT (7A), AST (7B), and total bilirubin (7C) in cynomolgus monkeys treated with CI106 or CI107. [Figure 8A] 8A-8C plot the increase in serum cytokine levels of IL-2 (8A), IL-6 (8B), and IFN-g (8C) in cynomolgus monkeys treated with CI106 or CI107. [Figure 8B] 8A-8C plot the increase in serum cytokine levels of IL-2 (8A), IL-6 (8B), and IFN-g (8C) in cynomolgus monkeys treated with CI106 or CI107. [Figure 8C]8A-8C plot the increase in serum cytokine levels of IL-2 (8A), IL-6 (8B), and IFN-g (8C) in cynomolgus monkeys treated with CI106 or CI107. [Figure 9A] 9A-9C show T cell activation as measured by expression of CD69 (9A), Ki67 (9B), and PD-1 (9B) on CD4+ T cells in cynomolgus monkeys treated with CI106 or CI107. [Figure 9B] 9A-9C show T cell activation as measured by expression of CD69 (9A), Ki67 (9B), and PD-1 (9B) on CD4+ T cells in cynomolgus monkeys treated with CI106 or CI107. [Figure 9C] 9A-9C show T cell activation as measured by expression of CD69 (9A), Ki67 (9B), and PD-1 (9B) on CD4+ T cells in cynomolgus monkeys treated with CI106 or CI107. [Figure 10A] Figures 10A-E plot the dose-dependent increase in AST at 48 hours post-dose (10A), ALT at 48 hours post-dose (10B), IL-6 at 8 hours post-dose (10C), IFNg at 8 hours post-dose (10D), and Ki67 at 72 hours post-dose (10E) in cynomolgus monkeys treated with act-104, CI106, or CI107. The dose-response curves for all parameters were shifted for the doubly masked antibody, indicating improved tolerability and reduced pharmacodynamic effects compared to the protease-activated bispecific antibody. [Figure 10B] Figures 10A-E plot the dose-dependent increase in AST at 48 hours post-dose (10A), ALT at 48 hours post-dose (10B), IL-6 at 8 hours post-dose (10C), IFNg at 8 hours post-dose (10D), and Ki67 at 72 hours post-dose (10E) in cynomolgus monkeys treated with act-104, CI106, or CI107. The dose-response curves for all parameters were shifted for the doubly masked antibody, indicating improved tolerability and reduced pharmacodynamic effects compared to the protease-activated bispecific antibody. [Figure 10C] Figures 10A-E plot the dose-dependent increase in AST at 48 hours post-dose (10A), ALT at 48 hours post-dose (10B), IL-6 at 8 hours post-dose (10C), IFNg at 8 hours post-dose (10D), and Ki67 at 72 hours post-dose (10E) in cynomolgus monkeys treated with act-104, CI106, or CI107. The dose-response curves for all parameters were shifted for the doubly masked antibody, indicating improved tolerability and reduced pharmacodynamic effects compared to the protease-activated bispecific antibody. [Figure 10D] Figures 10A-E plot the dose-dependent increase in AST at 48 hours post-dose (10A), ALT at 48 hours post-dose (10B), IL-6 at 8 hours post-dose (10C), IFNg at 8 hours post-dose (10D), and Ki67 at 72 hours post-dose (10E) in cynomolgus monkeys treated with act-104, CI106, or CI107. The dose-response curves for all parameters were shifted for the doubly masked antibody, indicating improved tolerability and reduced pharmacodynamic effects compared to the protease-activated bispecific antibody. [Figure 10E] Figures 10A-E plot the dose-dependent increase in AST at 48 hours post-dose (10A), ALT at 48 hours post-dose (10B), IL-6 at 8 hours post-dose (10C), IFNg at 8 hours post-dose (10D), and Ki67 at 72 hours post-dose (10E) in cynomolgus monkeys treated with act-104, CI106, or CI107. The dose-response curves for all parameters were shifted for the doubly masked antibody, indicating improved tolerability and reduced pharmacodynamic effects compared to the protease-activated bispecific antibody. [Figure 11A] Figures 11A-11C compare the effects of EGFR-binding CI107 and non-EGFR-binding CI128 (RSVxCD3) on the increase in total bilirubin (11A), IL-6 (11B), and PD-1-expressing CD4+ T cells (11C) in cynomolgus monkeys treated with CI107 or CI128. [Figure 11B]Figures 11A-11C compare the effects of EGFR-binding CI107 and non-EGFR-binding CI128 (RSVxCD3) on the increase in total bilirubin (11A), IL-6 (11B), and PD-1-expressing CD4+ T cells (11C) in cynomolgus monkeys treated with CI107 or CI128. [Figure 11C] Figures 11A-11C compare the effects of EGFR-binding CI107 and non-EGFR-binding CI128 (RSVxCD3) on the increase in total bilirubin (11A), IL-6 (11B), and PD-1-expressing CD4+ T cells (11C) in cynomolgus monkeys treated with CI107 or CI128. [Figure 12A] FIG. 12A shows affinity measurements of the v12, v16, and v19 CD3 antibodies compared to hSP34. [Figure 12B] FIG. 12B shows the cytotoxicity of activated or double-masked bispecific antibodies on HT29-luc2 cells. [Figure 13] FIG. 13 shows the PK expansion of the doubly masked antibody CI107 compared to the protease-activated bispecific antibody act-104. [Figure 14A] Figure 14A shows efficacy in the HT29-luc2 tumor intervention model in PBMC-engrafted NSG mice. Antitumor efficacy in this example correlates with the protease sensitivity and substrate cleavability of the test agent, with the most effective test agent being fully protease-activated CI048. [Figure 14B] Figure 14B shows staining of tumor sections for CD3 (dark staining) as a measure of T cell infiltration into the tumor. Tumor T cell infiltration correlates with the protease sensitivity and substrate cleavage of the test article. [Figure 15] Figures 15 and 16 are plots of binding isotherms for the activatable anti-EGFR C225v5 antibody of the disclosure, the activatable anti-EGFR antibody 3954-2001-C225v5 described herein, and the anti-EGFR antibody C225v5. [Figure 16]Figures 15 and 16 are plots of binding isotherms for the activatable anti-EGFR C225v5 antibody of the disclosure, the activatable anti-EGFR antibody 3954-2001-C225v5 described herein, and the anti-EGFR antibody C225v5. [Figure 17] 17-19 illustrate exemplary BAAs provided herein. [Figure 18] 17-19 illustrate exemplary BAAs provided herein. [Figure 19] 17-19 illustrate exemplary BAAs provided herein. [Figure 20] Figure 20 shows the PK of the doubly masked BAA CI107 after administration of a single dose of 600, 2000, or 4000 ug / kg. [Figure 21] FIG. 21 demonstrates that the cytotoxicity of CI090 and CI091 in HT29-luc2 cells was attenuated compared to CI011. [Figure 22] FIG. 22 demonstrates that activation of primary CD8+ T cells by CI090 and CI091 was attenuated compared to CI011. [Figure 23] Figure 23 shows the efficacy of the HT29-luc2 tumor intervention model in PBMC-implanted NSG mice. Anti-tumor efficacy of CI091, CI090, and CI011 is shown. [Figure 24] FIG. 24 plots IL-6 levels in a cynomolgus monkey in vivo study at 8 hours post-dose. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description of the Invention Provided herein are antibodies, activatable antibodies (AA), bispecific antibodies, and bispecific activatable antibodies (BAA).

[0036] In some embodiments, provided herein are humanized antibodies that specifically bind to the epsilon chain of CD3 (CD3ε; interchangeably referred to herein as CD3).

[0037] In some embodiments, provided herein is an IgG1 antibody that specifically binds to epidermal growth factor receptor (EGFR), wherein said antibody comprises a point mutation in the Fc region such that said antibody has reduced effector function.

[0038] In some embodiments, AAs are provided herein, for example, AAs that specifically bind to EGFR or CD3. These AAs are optimized for affinity, effector function, masking, and cleavage.

[0039] In some embodiments, provided herein are BAAs that bind to a target antigen (e.g., a tumor antigen such as a target shown in Table 9) and a second antigen (e.g., an immune effector antigen on an immune effector cell). In some embodiments, the immune effector cell is a white blood cell. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a natural killer (NK) cell. In some embodiments, the immune effector cell is a macrophage. In some embodiments, the immune effector cell is a mononuclear cell, such as a myelomononuclear cell. In some embodiments, the BAA is an immune effector cell-inducing BAA. In some embodiments, the BAA is a white blood cell-inducing BAA. In some embodiments, the BAA is a T cell-inducing bispecific (TCB) AA, also referred to herein as a TCBAA. In some embodiments, the BAA is an NK cell-inducing BAA. In some embodiments, the BAA is a macrophage cell-inducing BAA. In some embodiments, the BAA is a mononuclear cell-inducing BAA, such as a myelomonocytic cell-inducing BAA. In some embodiments, the bispecific antibody binds EGFR and CD3. These BAAs are optimized for affinity, effector function, masking, and cleavage.

[0040] These antibodies, AAs, and BAAs are also provided herein with methods for making and using them.AAs (including their general production) and the identification of masking moieties (MM) and cleavable moieties (CM) are described in International Publication No. WO2009 / 025846 by Daugherty et al., published on February 26, 2009, and WO2010 / 081173 by Stagliano et al., published on July 15, 2010 (both of which are incorporated herein by reference in their entirety).BAAs (including their general production) and the identification of masking moieties (MM) and cleavable moieties (CM) are described in International Publication No. WO2015 / 013671 by Lowman et al., published on January 29, 2015, and WO2016 / 014974 by Irving et al., published on January 28, 2016 (both of which are incorporated herein by reference in their entirety). International Publication No. WO2016 / 014974 to Irving et al., published January 28, 2016, and International Publication No. WO2016 / 118629 to Moore et al., published July 28, 2016, which provide AA, general production, MM, and CM, are also incorporated by reference.

[0041] As used herein, unless otherwise specified, the term "antibody" includes antibodies or antigen-binding fragments thereof that specifically bind their target, including monoclonal antibodies, domain antibodies, single-chain antibodies, Fab fragments, F(ab')2 fragments, scFvs, scAbs, dAbs, single-domain heavy chain antibodies, and single-domain light chain antibodies. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is an scFv antibody. In some embodiments, such antibodies or immunologically active fragments thereof that bind their target are murine, chimeric, humanized, or fully human monoclonal antibodies.

[0042] 1.CD3 antibody Provided herein are antibodies or antigen-binding fragments (AB) that specifically bind to the epsilon chain of CD3 (CD3ε, referred to throughout this specification as CD3).

[0043] Exemplary amino acid sequences of CD3 binding antibodies (variable domains) of the present disclosure are provided in Table 1 (predicted CDR sequences are underlined). As provided below, L3 is a linker, connecting the light and heavy chain variable domains in the exemplary CD3 binding antibodies.

[0044] [Table 1] TIFF2025175286000003.tif181167

[0045] An exemplary scFv linker (referred to herein as "L3" and which links VH and VL) is provided in Table 1-1.

[0046] [Table 2]

[0047] Exemplary CDR sequences for CD3 binding antibodies are provided in Table 2.

[0048] [Table 3]

[0049] As provided herein, a CD3 antibody comprises at least one of the CDR sequences provided in Table 2.

[0050] In some embodiments, the CD3 antibody comprises a heavy chain variable domain set forth in SEQ ID NO:2.

[0051] In some embodiments, the CD3 antibody comprises a heavy chain variable domain set forth in SEQ ID NO:3.

[0052] In some embodiments, the CD3 antibody comprises a light chain variable domain set forth in SEQ ID NO:1.

[0053] In some embodiments, the CD3 antibody comprises a light chain variable domain set forth in SEQ ID NO:4.

[0054] In some embodiments, the CD3 antibody comprises a heavy chain variable domain set forth in SEQ ID NO:2 and a light chain variable domain set forth in SEQ ID NO:1.

[0055] In some embodiments, the CD3 antibody comprises a heavy chain variable domain set forth in SEQ ID NO:3 and a light chain variable domain set forth in SEQ ID NO:1.

[0056] In some embodiments, the CD3 antibody comprises a heavy chain variable domain set forth in SEQ ID NO:3 and a light chain variable domain set forth in SEQ ID NO:4.

[0057] In some embodiments, the CD3 antibody comprises a heavy chain variable domain set forth in SEQ ID NO:2 and a light chain variable domain set forth in SEQ ID NO:4.

[0058] In some embodiments, the CD3 antibody comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3, or comprises a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4.

[0059] In some embodiments, the CD3 antibody comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3, and a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4.

[0060] In some embodiments, the CD3 antibody is an scFv antibody. In some embodiments, the variable domain comprises, from N- to C-terminus, the following structure: LV-HV. In some embodiments, the variable domain comprises, from N- to C-terminus, the following structure: HV-LV.

[0061] In some embodiments, the CD3 antibody is an scFv antibody comprising a heavy chain variable region (VH) linked to a light chain variable region (VL), wherein said VH is linked to the VL by a linker comprising the amino acid sequence of SEQ ID NO: 98. Exemplary sequences with such linkers are provided in Table 1.

[0062] In exemplary embodiments, provided herein is an antibody (AB) that specifically binds to CD3, wherein the antibody is an IgG1 antibody or an scFv linked to an Fc domain, and wherein the antibody comprises an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, and P331, numbered according to the EU index as set forth in Kabat, such that the antibody has reduced effector function. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, the antibody comprises amino acid substitutions at at least two of amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises amino acid substitutions at amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises amino acid substitutions at L234F, L235E, and P331S. In some embodiments, the antibody comprises amino acid substitutions at L234F, L235E, and P331S. In some embodiments, the antibody comprises an Fc region comprising an amino acid substitution at N297. In some embodiments, the Fc region comprises a N297Q mutation. In some embodiments, the antibody comprises the following amino acid substitutions: L234F, L235E, P331S, and N297Q. In some embodiments, the heavy chain variable domain of the antibody comprises any one of SEQ ID NO:2 or SEQ ID NO:3, or the light chain variable domain of AB comprises any one of SEQ ID NO:1 or SEQ ID NO:4.

[0063] 2. Activatable CD3 antibody In some embodiments, any one of the CD3 antibodies provided herein is in the activatable antibody (AA) format.

[0064] As generally provided herein, the AAs of the present invention include MM-CM constructs, also referred to herein as prodomains. Thus, as used herein, the term "prodomain" refers to a polypeptide comprising a masking moiety (MM) and a cleavable moiety (CM). In some embodiments, the MM and CM are separated by a linker, referred to herein as L1. In some embodiments, the prodomain comprises a linker at the carboxyl terminus of the CM; this linker, referred to herein as L2, connects the CM of the prodomain to the AB. In some embodiments, the prodomain comprises a linker between the MM and CM and a linker after the CM. In some embodiments, the MM and CM are not separated by a linker. In certain embodiments, the prodomain comprises one of the following formulas (the following formulas represent the amino acid sequence in either the N-terminal to C-terminal direction or the C-terminal to N-terminal direction): (MM)-L1-(CM), (MM)-(CM)-L2, (MM)-L1-(CM)-L2, or (MM)-(CM). In exemplary embodiments, the prodomain comprises EGFR MM and a CM cleavable by matriptase or an MMP; or CD3ε MM and a CM cleavable by matriptase or an MMP. In some embodiments, the prodomain comprises EGFR MM and a CM cleavable by matriptase and an MMP. In some embodiments, the prodomain comprises CD3ε MM and a CM cleavable by matriptase and an MMP. Activatable antibodies (AA) comprising the prodomain are provided herein. Nucleotides encoding the prodomains of the invention are also provided herein.

[0065] Thus, provided herein is a CD3AA comprising: (a) an antibody or antigen-binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3 (CD3ε), wherein the antibody comprises a heavy chain domain set forth in SEQ ID NO:2 or SEQ ID NO:3, or a light chain domain set forth in SEQ ID NO:1 or SEQ ID NO:4; (b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits binding of the AB to the CD3ε when the AA is in an uncleaved state; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. As described above, both (b) and (c) are part of the prodomain.

[0066] In some embodiments, the AB of CD3AA is any one of the CD3 antibodies described in the preceding section.

[0067] In some embodiments, AB of CD3AA comprises a heavy chain variable domain set forth in SEQ ID NO:2.

[0068] In some embodiments, AB of CD3AA comprises a heavy chain variable domain set forth in SEQ ID NO:3.

[0069] In some embodiments, AB of CD3AA comprises the light chain variable domain set forth in SEQ ID NO:1.

[0070] In some embodiments, AB of CD3AA comprises the light chain variable domain set forth in SEQ ID NO:4.

[0071] In some embodiments, the AB of CD3AA comprises a heavy chain variable domain set forth in SEQ ID NO:2 and a light chain domain set forth in SEQ ID NO:1.

[0072] In some embodiments, the AB of CD3AA comprises a heavy chain variable domain set forth in SEQ ID NO:3 and a light chain domain set forth in SEQ ID NO:1.

[0073] In some embodiments, the AB of CD3AA comprises a heavy chain variable domain set forth in SEQ ID NO:3 and a light chain domain set forth in SEQ ID NO:4.

[0074] In some embodiments, AB is an scFv comprising a heavy chain variable region (VH) linked to a light chain variable region (VL), where said VH is linked to the VL by a linker L3 comprising the amino acid sequence of SEQ ID NO: 98. Exemplary sequences with such linkers are provided in Table 1.

[0075] In some embodiments, the MM of CD3AA comprises any one of the sequences listed in Table 3.

[0076] Exemplary CD3 masking moieties (MMs) of the present invention are provided in Table 3.

[0077] In some embodiments, the MM of CD3AA comprises the sequence set forth in SEQ ID NO: 12. In some embodiments, the MM of CD3AA is the sequence set forth in SEQ ID NO: 10. In some embodiments, the MM of CD3AA is the sequence set forth in SEQ ID NO: 11.

[0078] [Table 4]

[0079] In some embodiments, the CM of CD3AA comprises any one of the sequences set forth in Table 4. Exemplary cleavable moieties (CM) of the present invention are shown in Table 4.

[0080] In some embodiments, the CM of an AA of the present disclosure comprises any one of the sequences listed in Table 4-1.

[0081] [Table 5]

[0082] [Table 6] TIFF2025175286000009.tif16167

[0083] 3. Antibodies with Fc mutations Provided herein are IgG1 antibodies or antibody fragments (e.g., scFv, Fab, F(ab')2) comprising an antigen-binding domain linked to an Fc domain with an Fc mutation, wherein the Fc has reduced effector function (referred to herein as Fc variants). Any of the BAA, AA, and antibodies described herein can comprise any of the Fc variants disclosed herein.

[0084] Antibodies containing these Fc mutations maintain target binding affinity while reducing effector function. Thus, provided herein are antibodies that bind to a target of interest, wherein the antibody is an IgG1 antibody or an Fc-linked antibody fragment, and wherein the Fc region comprises an amino acid substitution at at least one of amino acid positions L234, L235, and P331, as numbered according to the EU index as set forth in Kabat, such that the antibody has reduced effector function. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, there is an additional mutation at N297. In some embodiments, the amino acid substitution is N297Q or N297A.

[0085] In some embodiments, the Fc is selected from the Fc sequences set forth in Table 4-2. In some embodiments, the Fc is selected from SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, and SEQ ID NO: 160, wherein X is any naturally occurring amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, valine) or any non-naturally occurring amino acid. (e.g., trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine).

[0086] [Table 7] TIFF2025175286000011.tif184167

[0087] Antibodies, AAs, bispecific antibodies, and BAAs containing these Fc mutations are provided herein.

[0088] In some embodiments, such Fc variant-containing AAs and BAAs are capable of binding immune effector cells. In some embodiments, they are capable of selectively binding targets present on immune effector cells. In some embodiments, they are capable of binding CD3. In some embodiments, they are capable of binding any of the targets listed in Table 9. In some embodiments, they are capable of binding EGFR.

[0089] Thus, in some embodiments, an activatable antibody (AA) comprising: a) an antibody (AB) that specifically binds a target, wherein said antibody is an IgG1 antibody, and wherein the Fc region of said antibody comprises an amino acid substitution at at least one of amino acid positions L234, L235, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said antibody is reduced; b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits binding of the AB to a target when the AA is in an uncleaved state; and c) a cleavable moiety (CM) coupled to AB, wherein said CM is a polypeptide that functions as a substrate for a protease. Provided herein is an activatable antibody (AA) comprising:

[0090] In some embodiments, the Fc region comprises amino acid substitutions at least at amino acid positions L234, L235, N297, and P331, as numbered by the EU index as set forth in Kabat, to reduce the effector function of the aforementioned AA. In some embodiments, the target is selected from the group consisting of the targets set forth in Table 9.

[0091] In some embodiments, a bispecific activatable antibody (BAA) comprising: a) an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 is i. two heavy chains (AB1 HC) and two light chains (AB1 LC); and ii. a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of said MM1-CM1 construct is linked to each amino terminus of each light chain of said AB1; where: said MM1 inhibits the binding of said AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, an IgG antibody (AB1), b) two scFvs (AB2) each specifically binding to a second target, wherein each AB2 is i. a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each of said AB2 heavy chains is linked to the amino terminus of each of said AB1 heavy chains; and ii. a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of said MM2-CM2 construct is linked to the amino terminus of each said AB2; where: said MM2 inhibits the binding of said AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for a second protease, two scFvs (AB2) Including,

[0092] Provided herein is a bispecific activatable antibody (BAA), wherein said AB1 comprises an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced.

[0093] In some embodiments provided herein, the BAAs provided herein comprise: a) a bispecific activatable antibody (BAA), i) an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein said AB1 is linked to a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl termini of said MM1-CM1 construct are linked to each amino terminus of each light chain of said AB1, wherein said MM1 inhibits the binding of said AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, an IgG antibody (AB1), ii) two scFvs (each AB2) each specifically binding to a second target, wherein each AB2 comprises a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each of said AB2 is linked to the amino terminus of each of said AB1 heavy chains; wherein each AB2 is linked to a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each of said MM2-CM2 constructs is linked to the amino terminus of each of said AB2, wherein said MM2 inhibits the binding of said AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for a second protease, two scFvs (each AB2) Bispecific activatable antibodies (BAA) comprising: Including, wherein said AB1 comprises an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced.

[0094] 4.EGFR antibody Antibodies or antigen-binding fragments (AB) thereof that specifically bind to EGFR are provided herein. Exemplary CDR sequences of EGFR-binding antibodies are provided in Table 5.

[0095] Provided herein are EGFR antibodies, bispecific antibodies having one arm that targets EGFR, an AAs that can bind EGFR upon activation, and BAAs that can bind EGFR upon activation. In some embodiments, the EGFR antibodies comprise the CDRs of Table 5.

[0096] In some embodiments, provided herein is an IgG1 antibody that specifically binds to epidermal growth factor receptor (EGFR) and has reduced effector function, e.g., in a BAA format. The antibody comprises an Fc mutation that reduces effector function while maintaining EGFR binding affinity. Thus, provided herein is an antibody that binds to EGFR, wherein the antibody is an IgG1 antibody and comprises an Fc region containing an amino acid substitution at at least one of amino acid positions L234, L235, and P331, numbered according to the EU index as set forth in Kabat, such that the antibody has reduced effector function. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S.

[0097] In some embodiments, the antibody comprises amino acid substitutions at at least two of the following amino acid positions: L234, L235, and P331.

[0098] In some embodiments, the antibody comprises amino acid substitutions at amino acid positions L234, L235, and P331.

[0099] In some embodiments, the antibody comprises the following amino acid substitutions: L234F, L235E, and P331S.

[0100] In some embodiments, the antibody comprises an Fc region comprising an amino acid substitution at N297 along with at least one amino acid substitution at amino acid positions L234, L235, and / or P331. In some embodiments, the Fc region comprises an N297Q mutation. In some embodiments, the Fc region comprises an N297A mutation.

[0101] In some embodiments, the antibody comprises the following substitutions: L234F, L235E, P331S, and N297Q. In some embodiments, the antibody comprises the following substitutions: L234F, L235E, P331S, and N297A.

[0102] Exemplary CDR sequences for EGFR-binding antibodies described in Kabat are provided in Table 5.

[0103] [Table 8]

[0104] Exemplary amino acid sequences of EGFR-binding antibodies are provided in Table 6 (VL and VH indicate the variable light and variable heavy chains, respectively; LC and HC indicate the light and heavy chains, respectively).

[0105] In some embodiments, the EGFR antibody comprises any one of the sequences provided in Table 6.

[0106] In some embodiments, the heavy chain of the EGFR antibody comprises any one of the sequences set forth in SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76 in Table 6. In some embodiments, the heavy chain EGFR antibody comprises any one of the sequences set forth in SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, and SEQ ID NO:73, where X is any naturally occurring amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, valine) or ...). The Fcmt3 amino acid sequence is selected from non-existent amino acids (e.g., trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine). The designation Fcmt3 comprises three point mutations, wherein the Fc region of the heavy chain of the EGFR antibody comprises the following three point mutations: L234F, L235E, and P331S. Thus, in some embodiments, the EGFR antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: C225v5Fcmt3 HC. In some embodiments, the Fc region of the heavy chain of the EGFR antibody comprises a fourth point mutation, N297Q. The designation Fcmt4 comprises the three Fcmt3 point mutations and the fourth point mutation N297Q. Thus, in such embodiments, the EGFR antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:76.

[0107] [Table 9] TIFF2025175286000014.tif250167TIFF2025175286000015.tif213167

[0108] 5.Activatable EGFR antibody In some embodiments, any one of the EGFR antibodies provided herein is in the AA format (EGFR AA). As described above for CD3AA, the EGFR AA also includes a prodomain.

[0109] Thus, provided herein are AA containing antibodies or antigen-binding fragments (AB) that specifically bind to EGFR. Exemplary CDR sequences of EGFR-binding antibodies are provided in Table 5.

[0110] In some embodiments, the AA comprises: (a) any antibody or antigen-binding fragment thereof (AB) that specifically binds to epidermal growth factor receptor (EGFR); (b) and a prodomain, wherein the prodomain comprises: (i) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits binding of the AB to EGFR when the AA is in an uncleaved state, and wherein the MM comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7; and (ii) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease.

[0111] Exemplary EGFR masking moieties (MMs) of the present invention are provided in Tables 7 and 8.

[0112] [Table 10]

[0113] [Table 11]

[0114] In some embodiments, the MM of EGFR AA comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the MM of EGFR AA comprises the amino acid sequence of SEQ ID NO:85.

[0115] In some embodiments, the CM of an EGFR AA comprises an amino acid sequence selected from the group consisting of the sequences shown in Table 4. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 16.

[0116] In some embodiments, provided herein is an activatable antibody (AA) comprising: (a) an antibody that specifically binds to epidermal growth factor receptor (EGFR), wherein the antibody is an IgG1 antibody, and wherein the Fc region of the antibody comprises an amino acid substitution at at least one of amino acid positions L234, L235, and P331, as numbered according to the EU index as set forth in Kabat, such that the effector function of the AA is reduced; (b) a masking moiety (MM) coupled to AB, wherein the MM reduces or inhibits binding of the AB to EGFR when the AA is in an uncleaved state; and (c) a cleavable moiety (CM) coupled to AB, wherein the CM is a polypeptide that functions as a substrate for a protease. The EGFR IgG1 antibody may be any of the IgG1 antibodies described in the immediately preceding section. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7.

[0117] In one exemplary embodiment, provided herein is an activatable antibody (AA), comprising: (a) an antibody (AB) that specifically binds to epidermal growth factor receptor (EGFR), wherein the AB is an IgG1 antibody, and wherein the Fc region of the AB comprises an amino acid substitution at at least one of amino acid positions L234, L235, and P331, as numbered according to the EU index as set forth in Kabat, such that the effector function of the AA is reduced; (b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits binding of the AB to EGFR when the AA is in an uncleaved state; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, the AB comprises amino acid substitutions at at least two of amino acid positions L234, L235, and P331. In some embodiments, the AB comprises amino acid substitutions at amino acid positions L234, L235, and P331. In some embodiments, the AB comprises amino acid substitutions at L234F, L235E, and P331S. In some embodiments, the AB comprises an Fc region comprising an amino acid substitution at N297. In some embodiments, the Fc region comprises an N297Q mutation. In some embodiments, the AB comprises amino acid substitutions at L234F, L235E, P331S, and N297Q. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7 or Table 8. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 4. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, AA is a portion of BAA. 6. Bispecific activatable antibodies (BAA)

[0118] Provided herein are BAAs (bispecific AAs), which when activated specifically bind to two targets (e.g., bind two different targets or bind two different epitopes on the same target), and may include one of the exemplary structures provided in Figures 17-19.

[0119] In some embodiments, the first target is selected from the group consisting of the targets shown in Table 9 and the second target is selected from the group consisting of the targets shown in Table 9.

[0120] As generally provided herein and as described in the section describing AA above, the BAA of the present invention comprise an MM-CM construct, also referred to herein as a prodomain. Thus, as used herein, the term "prodomain" refers to a polypeptide comprising a masking moiety (MM) and a cleavable moiety (CM). In some embodiments, the MM and CM are separated by a linker, referred to herein as L1. In some embodiments, the prodomain comprises a linker at the carboxyl terminus of the CM; this linker, referred to herein as L2, connects the CM of the prodomain to the AB. In some embodiments, the prodomain comprises a linker between the MM and CM and a linker after the CM. In some embodiments, the MM and CM are not separated by a linker. In certain embodiments, the prodomain comprises one of the following formulas (the following formulas represent the amino acid sequence in either the N-terminal to C-terminal direction or the C-terminal to N-terminal direction): (MM)-L1-(CM), (MM)-(CM)-L2, (MM)-L1-(CM)-L2, or (MM)-(CM). In exemplary embodiments, the prodomain comprises EGFR MM and a CM cleavable by matriptase or an MMP; or CD3ε MM and a CM cleavable by matriptase or an MMP. In some embodiments, the prodomain comprises EGFR MM and a CM cleavable by matriptase and an MMP. In some embodiments, the prodomain comprises CD3ε MM and a CM cleavable by matriptase and an MMP. Provided herein are bispecific activatable antibodies (BAAs) comprising the prodomains. Nucleotides encoding the prodomains of the invention are also provided herein.

[0121] In some embodiments, provided herein are BAAs, wherein said BAA, when activated, specifically binds to two targets (e.g., two different targets; or two different epitopes on the same target), and wherein said BAA, when not activated, has the following structure: a) an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 is i. two heavy chains (AB1 HC) and two light chains (AB1 LC); and ii. a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of said MM1-CM1 construct is linked to each amino terminus of each light chain of said AB1, wherein: 1. said MM1 inhibits the binding of said AB1 to its target; 2. A first masking moiety (MM1), which is a polypeptide that functions as a substrate for a first protease, as described above. IgG antibody (AB1), b) two scFvs (AB2) each specifically binding to a second target, wherein each AB2 is i. a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each of said AB2 heavy chains is linked to the amino terminus of each of said AB1 heavy chains; and ii. a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of said MM2-CM2 construct is linked to the amino terminus of each said AB2, wherein: said MM2 inhibits the binding of said AB2 to its target; The aforementioned CM2 is a second masking moiety (MM2), which is a polypeptide that serves as a substrate for a second protease. Two scFvs (AB2) containing Including, Here, the aforementioned BAA has the following characteristics: i. MM2 comprises the amino acid sequence of SEQ ID NO: 12; ii. MM1 comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7; iii. AB2 comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3 or a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4; and iv. AB1 comprises an Fc region containing an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, or L234, L235, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced. It has at least one of the following.

[0122] In some embodiments, the BAAs provided herein are 1. A bispecific activatable antibody (BAA), wherein said BAA, when activated, specifically binds to two targets and has the following structure: a. an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein said AB1 is linked to a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl termini of said MM1-CM1 construct are linked to each amino terminus of each light chain of said AB1, wherein said MM1 inhibits the binding of said AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, an IgG antibody (AB1), b. two scFvs (each AB2) each specifically binding to a second target, wherein each AB2 comprises a light chain variable region linked to a heavy chain variable region, wherein the carboxyl terminus of each of said AB2 is linked to the amino terminus of each of said AB1 heavy chains; wherein each AB2 is linked to a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each of said MM2-CM2 constructs is linked to the amino terminus of each of said AB2, wherein said MM2 inhibits the binding of said AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for a second protease, two scFvs (each AB2) Including, Here, the aforementioned BAA has the following characteristics: i. MM2 comprises the amino acid sequence of SEQ ID NO: 12; ii. MM1 comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7; iii. AB2 comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3 or a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4; and iv. AB1 comprises an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced. The present invention also includes a bispecific activatable antibody (BAA) having at least one of the following:

[0123] In some embodiments, the BAAs provided herein are a) an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 is a. two heavy chains (AB1 HC) and two light chains (AB1 LC); and b. a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of said MM1-CM1 construct is linked to each amino terminus of each light chain of said AB1, wherein: 1. said MM1 inhibits the binding of said AB1 to its target; 2. A first masking moiety (MM1), which is a polypeptide that functions as a substrate for a first protease, as described above. IgG antibody (AB1), b) two scFvs (AB2) each specifically binding to a second target, wherein each AB2 is a. a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each of said AB2 heavy chains is linked to the amino terminus of each of said AB1 heavy chains; and b. a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of said MM2-CM2 construct is linked to the amino terminus of each said AB2, wherein: 1. said MM2 inhibits the binding of said AB2 to its target; 2. A second masking moiety (MM2), which is a polypeptide that serves as a substrate for a second protease, wherein CM2 is a Two scFvs (AB2) containing Including, wherein said AB1 comprises an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced. In some embodiments, the Fc region comprises amino acid substitutions at at least amino acid positions L234, L235, N297, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced.

[0124] In some embodiments, the BAAs provided herein are (1) A bispecific activatable antibody (BAA), (a) an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein said AB1 is linked to a first masking moiety (MM1) that is linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein a carboxyl terminus of said MM1-CM1 construct is linked to each amino terminus of each light chain of said AB1, wherein said MM1 inhibits the binding of said AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, an IgG antibody (AB1), (b) two scFvs (each AB2) that each specifically bind to a second target, wherein each AB2 comprises a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each of said AB2 is linked to the amino terminus of each of said AB1 heavy chains; wherein each AB2 is linked to a second masking moiety (MM2) that is linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each of said MM2-CM2 constructs is linked to the amino terminus of each of said AB2, wherein said MM2 inhibits the binding of said AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for a second protease, two scFvs (each AB2) Bispecific activatable antibodies (BAA) comprising: Including, wherein said AB1 comprises an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced.

[0125] As provided above, the BAA of the present invention comprises two scFvs (AB2), each of which specifically binds to a second target. The VL and VH of the scFvs can be in any order, either VL-VH or VH-VL.

[0126] In some embodiments, the Fc region of AB1 comprises amino acid substitutions at least at amino acid positions L234, L235, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced. In some embodiments, the first target is selected from the group consisting of the targets set forth in Table 9, and the second target is selected from the group consisting of the targets set forth in Table 9.

[0127] In some embodiments, AB1 binds a target antigen (eg, a tumor antigen) and AB2 binds an immune effector target.

[0128] In some embodiments, AB2 binds a target antigen (eg, a tumor antigen) and AB1 binds an immune effector target.

[0129] In some embodiments, AB1 binds EGFR and AB2 binds CD3.

[0130] In some embodiments, MM1 comprises SEQ ID NO:78.

[0131] In some embodiments, MM2 comprises the amino acid sequence of SEQ ID NO:12.

[0132] In some embodiments, the bispecific AA is CI106, provided in Table 11 of Example 1.

[0133] In some embodiments, the BAA is CI107, provided in Table 11 of Example 1.

[0134] In some embodiments, the BAA is CI011, provided in Table 11 of Example 1.

[0135] In some embodiments, the BAA is CI020, provided in Table 11 of Example 1.

[0136] In some embodiments, the BAA is CI040, provided in Table 11 of Example 1.

[0137] In some embodiments, the BAA is CI079, provided in Table 11 of Example 1.

[0138] In some embodiments, the BAA is CI090, provided in Table 11 of Example 1.

[0139] In one exemplary embodiment, AB1 comprises the amino acid sequence of C225v5Fcmt3 HC or C225v5Fcmt4 HC.

[0140] In some embodiments, the first and second proteases are the same protease. In some embodiments, the first and second proteases are different proteases. 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 and CM2 comprise different amino acid sequences that are cleavable by the same protease or multiple proteases. In some embodiments, CM1 and CM2 are cleavable by more than one protease. In some embodiments, CM1 and / or CM2 are cleavable by a serine protease. In some embodiments, CM1 and / or CM2 are cleavable by a matrix metalloproteinase (MMP). In some embodiments, CM1 and / or CM2 are cleavable by a serine protease and an MMP.

[0141] Exemplary BAAs of the present disclosure include, for example, the BAAs and variants thereof set forth in the Examples provided herein.

[0142] In some non-limiting embodiments, at least one of the ABs in the BAA is specific for CD3 and at least one other AB is a binding partner for any of the targets listed in Table 9.

[0143] In one exemplary embodiment, AB2 of the BAA is specific for CD3 and AB1 is a binding partner for any of the targets listed in Table 9.

[0144] [Table 12] TIFF2025175286000019.tif174167

[0145] In some embodiments, unmasked EGFR-CD3 bispecific antibodies exhibit EGFR-dependent tumor cell killing, while dual-masked EGFR-CD3 BAAs reduce target-dependent cytotoxicity by more than 100,000-fold. In established tumor models where tumor-resident proteases are expected to be active, BAAs have been shown to potently induce tumor regression. In non-human primates, the maximum tolerated dose (MTD) of EGFR-CD3 BAAs is more than 60-fold higher than the MTD of unmasked bispecific antibodies, with tolerated exposure (AUC) being more than 10,000-fold higher. Despite the 60-fold difference in MTD, transient increases in serum cytokines and AST / ALT observed in BAA-treated non-human primates remain lower than those induced by bispecific antibodies. By localizing activity to the tumor microenvironment, BAAs may broaden clinical opportunities for T cell-directed bispecific therapies, which are limited by on-target toxicity, particularly in solid tumors. Furthermore, EGFR-CD3 BAAs have the potential to address EGFR-expressing tumors that respond poorly to existing EGFR-directed therapies.

[0146] 7. Cuttable part (CM) Both the monospecific AAs and BAAs of the present disclosure contain at least one CM when masked and unactivated.

[0147] In some embodiments, the cleavable portion (CM) of an AA or BAA comprises an amino acid sequence that can serve as a substrate for at least one protease, typically an extracellular protease. In the case of a BAA, the CM can be selected based on the protease that coexists in the tissue with the desired target of at least one AB of the BAA or AA. The CM can serve as a substrate for multiple proteases, for example, a serine protease and a second, different protease (e.g., an MMP). In some embodiments, the CM can serve as a substrate for more than one serine protease, for example, matriptase and uPA. In some embodiments, the CM can serve as a substrate for more than one MMP, for example, MMP9 and MMP14.

[0148] Various conditions are known in which a target of interest coexists with a protease, and the substrate of that protease is known in the art. For example, in the case of cancer, the target tissue can be cancer tissue, particularly cancer tissue of solid tumor. There are reports in the literature that protease levels increase in many cancers (e.g., liquid tumors or solid tumors). For example, see La Rocca et al., (2004) British J. of Cancer 90(7):1414-1421. Non-limiting examples of diseases include all types of cancer (including, but not limited to, breast cancer, lung cancer, colorectal cancer, gastric cancer, glioblastoma, ovarian cancer, endometrial cancer, renal cancer, sarcoma, skin cancer, cervical cancer, liver cancer, bladder cancer, intrahepatic cholangiocarcinoma, prostate cancer, melanoma, head and neck cancer (e.g., head and neck squamous cell carcinoma, pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular injury, ischemia, etc.). For example, indications would include leukemia (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 include bone disease or metastasis of cancer unrelated to the origin of the primary tumor; 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; sarcoma (such as osteosarcoma); renal 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). In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is cutaneous squamous cell carcinoma. In some embodiments, the cancer is esophageal squamous cell carcinoma. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is lung squamous cell carcinoma.

[0149] CM ranges from approximately 0.001 to 1500 × 10 depending on the enzyme. 4 M -1 S -1or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 × 10 4 M -1 S -1 It is specifically cleaved at a rate of

[0150] For specific enzymatic cleavage, the enzyme is contacted with the CM. When the AA or BAA comprises at least one AB coupled to the MM and the CM, for example, when the AA comprises an AB coupled to the MM via the CM, and the target and sufficient enzymatic activity are present, the CM can be cleaved. Sufficient enzymatic activity can refer to the ability of the enzyme to contact and cleave the CM. It is easy to imagine that the enzyme may be present in the vicinity of the CM, but cannot cleave it due to protein modifications by other cellular factors or enzymes.

[0151] Exemplary CMs of the present disclosure are provided above in Table 4. In some embodiments, the CM is at most 15 amino acids in length, at most 20 amino acids in length, at most 25 amino acids in length, at most 30 amino acids in length, at most 35 amino acids in length, at most 40 amino acids in length, at most 45 amino acids in length, at most 50 amino acids in length, at most 60 amino acids in length, in the range of 10-60 amino acids in length, in the range of 15-60 amino acids in length, in the range of 20-60 amino acids in length, in the range of 25-60 amino acids in length, in the range of 30-60 amino acids in length, in the range of 35-60 amino acids in length, in the range of 40-50 amino acids in length, The amino acid length ranges from 45 to 60, from 10 to 40, from 15 to 40, from 20 to 40, from 25 to 40, from 30 to 40, from 35 to 40, from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, from 10 to 20, or from 10 to 15.

[0152] 8.Masking part (MM) In both the activatable monospecific CD3 and EGFR AAs and BAAs described above, the AA / BAA comprises a MM. As described herein, the AAs and BAAs of the present invention comprise a prodomain that includes a MM.

[0153] In some embodiments, the MM is selected for use with a specific antibody or antibody fragment.

[0154] In certain embodiments, the MM is not a natural binding partner of AB. In some embodiments, the MM shows no or substantially no homology to any natural binding partner of AB. In other embodiments, the MM is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of AB. In some embodiments, the MM is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of AB. In some embodiments, the MM is about 50% identical to any natural binding partner of AB. In some embodiments, the MM is about 25% identical to any natural binding partner of AB. In some embodiments, the MM is about 20% identical to any natural binding partner of AB. In some embodiments, the MM is about 10% identical to any natural binding partner of AB.

[0155] Exemplary MMs of the present disclosure include those up to 15 amino acids in length, up to 20 amino acids in length, up to 25 amino acids in length, up to 30 amino acids in length, up to 35 amino acids in length, up to 40 amino acids in length, up to 45 amino acids in length, up to 50 amino acids in length, up to 60 amino acids in length, in the range of 10-60 amino acids in length, in the range of 15-60 amino acids in length, in the range of 20-60 amino acids in length, in the range of 25-60 amino acids in length, in the range of 30-60 amino acids in length, in the range of 35-60 amino acids in length, in the range of 40-50 amino acids in length, in the range of 45-60 amino acids in length, It may have a length in the range of 10 to 40 amino acids, a length in the range of 15 to 40 amino acids, a length in the range of 20 to 40 amino acids, a length in the range of 25 to 40 amino acids, a length in the range of 30 to 40 amino acids, a length in the range of 35 to 40 amino acids, a length in the range of 10 to 30 amino acids, a length in the range of 15 to 30 amino acids, a length in the range of 20 to 30 amino acids, a length in the range of 25 to 30 amino acids, a length in the range of 10 to 20 amino acids, a length in the range of 10 to 15 amino acids, or a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0156] As provided herein, the MM inhibits the binding of the AB to its target. The MM binds the antigen-binding domain of the AB and inhibits the binding of the AB to its target. The MM can sterically inhibit the binding of the AB to its target. The MM can allosterically inhibit the binding of the AB to its target. In these embodiments, the AB is modified by or coupled to the MM, and when in the presence of the target, the MM inhibits the binding of the AB to its target in an in vivo assay or in vivo assay. There is no or substantially no binding of the AB to the target, or the binding of the AB to the target is on the order of 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% of the binding of the AB to the target compared to the binding of the AB unmodified by or uncoupled to the MM, the parent AB, or the AB uncoupled to the MM for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 80 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer as measured in an in vitro assay.

[0157] When an AB is coupled to or modified with a MM, the MM "masks," reduces, or otherwise inhibits the specific binding of the AB to its target. When an AB is coupled to or modified with a MM, such coupling or modification may result in a conformational change that reduces or inhibits the ability of the AB to specifically bind to its target.

[0158] Exemplary MMs of the present disclosure are provided in Tables 3, 7, and 8 above.

[0159] For any of the AAs and BAAs provided herein, the masked AB has a lower binding affinity than the unmasked AB.

[0160] 9. Linker In many embodiments, it may be desirable to insert one or more linkers (e.g., flexible linkers) into the AA / BAA construct to impart flexibility to one or more of the MM-CM junctions, the CM-AB / CM-scFv junctions, or both. For example, the AB, MM, and / or CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, particularly Gly and Ser) to impart the desired flexibility. As such, the ability of such BAA constructs to remain intact (non-activated) or be activated as disclosed herein would benefit from the introduction of one or more amino acids to provide a flexible linker.

[0161] For example, in certain embodiments, AA comprises one of the following formulas (the following formulas represent the amino acid sequence in either the N-terminal to C-terminal direction or the C-terminal to N-terminal direction): (MM1)-L1-(CM1)-(AB1) (MM1)-(CM1)-L2-(AB1) (MM1)-L1-(CM1)-L2-(AB1) (MM2)-L1-(CM2)-(AB2) (MM2)-(CM2)-L2-(AB2) (MM2)-L1-(CM2)-L2-(AB2) (wherein MM, CM, and AB are as defined above; L1 and L2 are each independently and optionally present or absent, and are the same or different flexible linkers comprising at least one flexible amino acid (e.g., Gly, Ser)).

[0162] In some embodiments, a BAA comprises two heavy chains, each heavy chain comprising, from N- to C-terminus, the structural arrangement MM2-CM2-AB2-AB1 HC, and two light chains, each light chain comprising, from N- to C-terminus, the structural arrangement MM1-CM1-AB1 LC.

[0163] In some embodiments, structures including linkers are provided in FIG.

[0164] In some embodiments, (MM2)-L1-(CM2)-L2-(AB2) is linked to the heavy chain of AB1, and AB2 is an scFv.

[0165] Linkers suitable for use in the compositions described herein are generally those that provide flexibility to the modified AB or AA, facilitating inhibition of AB binding to a target. Such linkers are commonly referred to as flexible linkers. Suitable linkers can be readily selected and can be any suitable length, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids (including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids), and can 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. In some embodiments, suitable linkers can be 4 to 25 amino acids in length. In some embodiments, suitable linkers can be 5 to 25 amino acids in length. In some embodiments, suitable linkers can be 4 to 20 amino acids in length. In some embodiments, a suitable linker can be 5 to 20 amino acids in length.

[0166] Exemplary linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 88), and (GGGS)n (SEQ ID NO: 89), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, n is from about 1 to about 10, or from about 1 to about 9, or from about 1 to about 8, or from about 1 to about 7, or from about 1 to about 6, or from about 1 to about 5, or from about 1 to about 4, or from about 1 to about 3, or from about 1 to about 2. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to serve as neutral tethers between components. Glycine has significantly more access to the φ-ψ space than the equivalent alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary glycers are provided in Table 9-1.

[0167] [Table 13]

[0168] Those skilled in the art will recognize that the design of an AA may include a linker that is fully or partially flexible, whereby the linker may include a flexible linker and one or more moieties that confer a less flexible structure to provide the desired AA structure.

[0169] 10. Conjugation In some embodiments, any antibody (i.e., AA AB and BAA AB disclosed herein) can be conjugated to an agent. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a detectable moiety. In some embodiments, the agent is an anti-neoplastic agent. In some embodiments, the agent is a toxin or fragment thereof. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent (such as a DNA alkylating agent or DNA interfering agent) or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is an agent selected from the group listed in Table 10.

[0170] [Table 14] TIFF2025175286000022.tif245167TIFF2025175286000023.tif102167

[0171] Those skilled in the art will recognize that a wide variety of possible moieties can be coupled to the resulting antibodies, AAs, and BAAs of the present disclosure (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0172] In some embodiments, the antibody, AA, or BAA comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent.

[0173] In some embodiments, the antibody, AA, or BAA comprises one or more disulfide bonds. In some embodiments, the antibody, AA, or BAA comprises one or more lysines. In some embodiments, the antibody, AA, or BAA can be engineered to comprise one or more disulfide bonds or otherwise engineered to allow for site-specific conjugation.

[0174] 11. Production The present disclosure also provides isolated nucleic acid molecules encoding the antibodies, AAs, or BAAs described herein, and vectors comprising these isolated nucleic acid sequences. The present disclosure provides methods of producing antibodies, AAs, or BAAs by culturing cells under conditions that allow expression of the antibodies, AAs, or BAAs, wherein the cells comprise such nucleic acid molecules.

[0175] In some embodiments, the cell comprises such a vector. In some embodiments, the vector is pLW289. In some embodiments, the vector is pLW246. In some embodiments, the vector is pLW307. In some embodiments, the vector is pLW291. In some embodiments, the vector is pLW352. In some embodiments, the vector is pLW353 (descriptions of these vectors and sequences are provided in Example 1 below).

[0176] 12. Use of Antibodies, AAs, Bispecific Antibodies, and BAAs In some embodiments, the antibody / bispecific antibody / AA / BAA thereof can be used as a therapeutic agent. Such agents would generally be used to treat, alleviate, and / or prevent a disease or condition in a subject. A therapeutic regimen is carried out by identifying a subject, e.g., a human patient or other mammal suffering from (or at risk of developing) a disorder, using standard methods.

[0177] Administration of the antibody / bispecific antibody / AA / BAA thereof can suppress, inhibit or interfere with the signaling function of one or more targets.

[0178] It will be appreciated that the therapeutic entities of the present disclosure are administered with appropriate carriers, excipients, and other agents incorporated into the formulation to provide improved entry, delivery, tolerance, and the like. Numerous suitable formulations can be found in the formulary known to all pharmacists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsifiable concentrates (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. Any of the foregoing mixtures may be suitable for treatment and therapy according to the present disclosure, provided that the active ingredients therein are not inactivated by the formulation and the formulation is physiologically compatible with and tolerated by the route of administration. For further information regarding formulations, excipients, and carriers well known to pharmacists, see also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8(2000); Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2):1-60(2000); Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts." J Pharm Sci. 89(8):967-78(2000); Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311(1998), and references therein.

[0179] Generally, alleviation or treatment of a disease or disorder includes the reduction of one or more symptoms or medical problems associated with the disease or disorder. For example, in the case of cancer, a therapeutically effective amount of a drug can achieve one or a combination of the following: reduce the number of cancer cells; reduce tumor size; inhibit (i.e., reduce and / or stop to some extent) the infiltration of cancer cells into surrounding organs; inhibit tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with cancer. In some embodiments, the compositions of the present disclosure can be used to prevent the onset or recurrence of a disease or disorder in a subject (e.g., other mammals, such as humans or non-human primates, companion animals (e.g., cats, dogs, horses), livestock, working animals, or zoo animals). The terms subject and patient are used interchangeably herein.

[0180] A therapeutically effective amount of an antibody / bispecific antibody / AA / BAA thereof of the present disclosure generally refers to the amount needed to achieve a therapeutic objective.

[0181] A typical therapeutically effective dose range for an antibody / bispecific antibody / AA / BAA thereof of the present disclosure can be, by way of non-limiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequencies can range, for example, from twice daily to once weekly.

[0182] The effectiveness of treatment is determined in association with any known method for diagnosing or treating the particular disorder. Methods of screening for antibodies / bispecific antibodies / AA / BAA possessing the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.

[0183] Other intended uses include use in diagnosis, imaging, prognosis, and detection. In some embodiments, the antibodies / bispecific antibodies / AAs / BAAs are used in methods known within the art related to localizing and / or quantitating targets (e.g., for use in measuring the levels of one or more targets in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging, etc.).

[0184] In some embodiments, antibodies / bispecific antibodies / AA / BAAs are used to isolate one or more targets by standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies, AAs, bispecific antibodies, or BAAs can be used diagnostically to monitor protein levels in tissues as part of a clinical testing procedure, for example, to determine the effectiveness of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 Contains H.

[0185] In yet another embodiment, antibodies, bispecific antibodies, AAs, and BAAs directed to two or more targets can be used as agents for detecting the presence of one or more targets (or fragments thereof) in a sample. In some embodiments, the antibody comprises a detectable label. The antibody is a polyclonal antibody or, in some embodiments, a monoclonal antibody. An intact antibody or a fragment thereof (e.g., Fab, scFv, or F(ab')2) is used. The term "labeled" with respect to a probe or antibody is intended to include direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reaction with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of an antibody with biotin such that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and bodily fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. Thus, the term "biological sample" includes within its scope blood and blood fractions or components (including serum, plasma, or lymph). That is, proteins in biological samples can be detected in vitro and in vivo using the detection methods of the present disclosure. For example, in vitro detection techniques for analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology," Vol. 42, J.R.Crowther (Ed.), Human Press, Totowa, NJ, 1995; "Immunoassay," E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays," P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.Furthermore, in vivo techniques for detection of analyte proteins include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0186] The antibodies, bispecific antibodies, AAs, and bispecific antibodies of the present disclosure are also useful in various diagnostic and preventive preparations.In one embodiment, the antibodies, AAs, bispecific antibodies, and BAAs are administered to patients at risk of developing one or more of the aforementioned disorders.The predisposition of a patient or organ to one or more disorders can be determined using genotypic, serotypic, or biochemical markers.

[0187] In another embodiment of the present disclosure, an antibody, AA, bispecific antibody, BAA is administered to a human individual diagnosed with a clinical indication associated with one or more of the aforementioned disorders. Upon diagnosis, the antibody, AA, bispecific antibody, BAA is administered to reduce or reverse the effects of the clinical indication.

[0188] Antibodies, bispecific antibodies, AAs, and bispecific antibodies are also useful in detecting one or more targets in patient samples and are therefore useful as diagnostic agents. For example, the antibodies, bispecific antibodies, AAs, and bispecific antibodies of the present disclosure are used in in vitro assays (e.g., ELISAs) to detect one or more target levels in patient samples.

[0189] In one embodiment, the antibody, AA, bispecific antibody, BAA is immobilized on a solid support (e.g., the well of a microtiter plate). The immobilized antibody and / or AA serves as a capture antibody for any target(s) that may be present in the test sample. Prior to contacting the immobilized antibody / AA with the patient sample, the solid support is rinsed and treated with a blocking agent, such as milk protein or albumin, to prevent nonspecific adsorption of the analyte.

[0190] Subsequently, the well is treated with a test sample suspected of containing the antigen or a solution containing a standard amount of antigen. Such use is, for example, a serum sample from a subject suspected of having a circulating antigen level that is diagnostic of a disease. After washing away the test sample or standard, the solid support is treated with a detectably labeled second antibody. The labeled second antibody serves as a detection antibody. The level of detectable label is measured, and the concentration of target antigen(s) in the test sample is determined by comparing it with a calibration curve prepared from standard samples.

[0191] It will be appreciated that, based on the results obtained using antibodies, AAs, bispecific antibodies, and BAAs in in vitro diagnostic assays, a subject's disease can be staged based on the expression level of the target antigen(s). For a given disease, blood samples are collected from subjects diagnosed at various stages in the progression of the disease and / or at various points in the therapeutic treatment of the disease. Sample populations that provide statistically significant results for each stage of progression or treatment are used to specify concentration ranges of antigens that can be considered characteristic of each stage of the disease.

[0192] Antibodies, bispecific antibodies, AAs, and BAAs can also be used in diagnostic and / or imaging methods. In some embodiments, such methods are in vitro methods. In some embodiments, such methods are in vivo methods. In some embodiments, such methods are in situ methods. In some embodiments, such methods are ex vivo methods. For example, AAs and bispecific antibodies with an enzymatically cleavable CM can be used to detect the presence or absence of an enzyme capable of cleaving the CM. Such AAs and bispecific antibodies can be used in diagnostics, which may include in vivo detection (e.g., qualitative or quantitative) of enzymatic activity (or, in some embodiments, an environment of increased reduction potential, such as that which may result in the reduction of disulfide bonds) by measuring the accumulation of activating antibodies or bispecific activating antibodies (i.e., antibodies or bispecific antibodies resulting from cleavage of the AA or BAA) in given cells or tissues of a given host organism. Such accumulation of activating bispecific antibodies indicates not only that the tissue expresses enzymatic activity (or increased reduction potential depending on the nature of the CM), but also that the tissue expresses at least one target to which the activating bispecific antibody binds.

[0193] For example, the CM can be selected to be a protease substrate for proteases found at tumor sites, viral or bacterial infection sites, and biologically confined sites (e.g., in abscesses and organs). At least one of the ABs can be an AB that binds a target antigen. Detectable labels (e.g., fluorescent labels, radioactive labels, or radiotracers) can be conjugated to the ABs or other regions of the antibodies, AA, bispecific antibodies, or BAAs using methods familiar to those skilled in the art. Suitable detectable labels are discussed in the context of the screening methods described above, with further specific examples provided below. Using at least one AB specific for a diseased protein or peptide along with a protease whose activity is elevated in the diseased tissue of interest, the AA will exhibit an increased binding rate to diseased tissue compared to tissue in which the CM-specific enzyme is not present at detectable levels, is present at lower levels than in the diseased tissue, or is inactive (e.g., in its zymogenic form or in the form of a complex with an inhibitor). Because small proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because enzymes specific for CM are not present at detectable levels (or are present at levels lower than those in non-diseased tissues, or are present in an inactive conformation), accumulation of activated bispecific antibodies in diseased tissues is enhanced compared to non-diseased tissues.

[0194] In another example, the antibodies / bispecific antibodies / AA / BAAs of the present disclosure can be used to detect the presence or absence of a cleavage agent in a sample. For example, if the antibody / bispecific antibody / AA / BAA contains a CM that is sensitive to cleavage by an enzyme, the BAA can be used to detect the presence of the enzyme in a sample (qualitatively or quantitatively). In another example, if the antibody / bispecific antibody / AA / BAA contains a CM that is sensitive to cleavage by a reducing agent, the antibody / bispecific antibody / AA / BAA can be used to detect the presence of reducing conditions in a sample (qualitatively or quantitatively). To facilitate analysis in these methods, the antibody / bispecific antibody / AA / BAA can be detectably labeled and attached to a support (e.g., a solid support such as a slide or bead). The detectable label can be located on a portion of the antibody / bispecific antibody / AA / BAA that is not released after cleavage; for example, the detectable label can be a quenched fluorescent label or other label that is not detectable until cleavage occurs. The assay can be performed, for example, by contacting an immobilized, detectably labeled antibody / bispecific antibody / AA / BAA with a sample suspected of containing an enzyme and / or reducing agent for a time sufficient for cleavage, followed by washing to remove excess sample and contaminants. The presence or absence of a cleaving agent (e.g., an enzyme or reducing agent) in the sample is then assessed by a change in the detectable signal of the antibody / bispecific antibody / AA / BAA prior to contact with the sample (e.g., the presence and / or increase in a detectable signal due to cleavage of the antibody / bispecific antibody / AA / BAA by the cleaving agent in the sample).

[0195] Such detection methods can be adapted to also provide for the detection of the presence or absence of a target capable of binding to at least one AB of an antibody / bispecific antibody / AA / BAA of the present disclosure. Thus, assays can be adapted to assess the presence or absence of a cleaving agent and the presence or absence of a target of interest. The presence or absence of a cleaving agent can be detected by the presence and / or increase of a detectable label of the antibody / bispecific antibody / AA / BAA, as described above, and the presence or absence of a target can be detected by detection of the target-AB complex (e.g., by use of a detectably labeled anti-target antibody).

[0196] The AA / BAA of the present disclosure is also useful in in situ imaging, for example, to confirm AA activation by protease cleavage and binding to specific targets.In situ imaging is a technique that can localize proteolytic activity and targets in biological samples such as cell cultures or tissue sections.This technique can be used to confirm both binding to a given target and proteolytic activity based on the presence of detectable labels (e.g., fluorescent labels).

[0197] These techniques are useful with any frozen cells or tissue from diseased sites (e.g., tumor tissue) or healthy tissue. These techniques are also useful with fresh cell or tissue samples.

[0198] In these techniques, the AA / BAA is labeled with a detectable label, which can be a fluorescent dye (e.g., a fluorophore, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (TRITC), AlexaFluor® label), a near-infrared (NIR) dye (e.g., a Qdot® nanocrystal), a colloidal metal, a hapten, a radioactive marker, biotin and an amplification reagent (such as streptavidin), or an enzyme (e.g., horseradish peroxidase or alkaline phosphatase).

[0199] Detection of label in a sample incubated with labeled AA or BAA indicates that the sample contains the target and a protease specific for the CM of the AA or BAA of the present disclosure. In some embodiments, the presence of the protease can be confirmed using a broad-spectrum protease inhibitor (such as those described herein) and / or a protease-specific agent (e.g., an antibody such as A11 that is specific for the protease matriptase (MT-SP1) and inhibits the proteolytic activity of MT-SP1); see, e.g., International Publication No. WO 2010 / 129609, published November 11, 2010. The same approach, using a broad-spectrum protease inhibitor (such as those described herein) and / or a more selective inhibitor, can be used to identify a protease or class of proteases specific for the CM of the AA or BAA of the present disclosure. In some embodiments, the presence of the target can be confirmed using an agent specific for the target, or a detectable label can be competed with an unlabeled target. In some embodiments, unlabeled AA can be used with detection by a labeled secondary antibody or a more complex detection system.

[0200] Similar techniques are also useful for in vivo imaging, where detection of a fluorescent signal in a subject (e.g., a mammal, including a human) indicates that the diseased site contains the target and contains a protease specific to the CM of an AA or BAA of the present disclosure.

[0201] These techniques are also useful in kits and / or as reagents for detecting, identifying, or characterizing protease activity in various cells, tissues, and organs based on the protease-specific CM in the AAs or BAAs of the present disclosure.

[0202] 13. Therapeutic Administration It will be appreciated that the therapeutic entities of the present disclosure are administered with appropriate carriers, excipients, and other agents incorporated into the formulation to improve penetration, delivery, tolerance, and the like. Numerous suitable formulations can be found in the formulary known to all pharmacists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsifiable concentrates (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. Any of the foregoing mixtures may be suitable for treatment and therapy according to the present disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible with and tolerated by the route of administration. For further information regarding formulations, excipients, and carriers well known to pharmacists, see also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8(2000); Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2):1-60(2000); Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts." J Pharm Sci. 89(8):967-78(2000); Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311(1998), and references therein.

[0203] In some embodiments, the antibody, bispecific antibody, AA, or BAA (or its conjugate composition) is administered in combination with one or more additional agents or a combination of additional agents. Suitable additional agents include existing pharmaceutical and surgical therapeutic agents for the intended application. For example, the additional agent can be used in combination with an additional chemotherapeutic or anti-neoplastic agent.

[0204] In some embodiments, an antibody, bispecific antibody, AA, or BAA (or conjugated composition thereof) of the present disclosure is administered in conjunction with one or more additional agents selected from the group consisting of an antibody, conjugated antibody, AA, conjugated AA, bispecific antibody, conjugated bispecific antibody, BAA, or conjugated BAA. In some embodiments, the antibody portion of any of the foregoing additional agents is directed to a target (such as one or more targets disclosed in Table 9). In some embodiments, it is recognized that the antibody portion of an antibody, bispecific antibody, AA, or BAA (or conjugated composition thereof) of the present disclosure and the antibody portion of the additional agent are directed to the same target (e.g., both can target EGFR). In some embodiments, they are directed to the same target, but to different epitopes. In some embodiments, they are directed to entirely different targets (e.g., an activatable antibody of the present disclosure that targets EGFR can be administered in conjunction with an AA that targets a different target); similarly, for example, a BAA of the present disclosure that targets EGFR and CD3 can be administered in conjunction with AAs that target different targets.

[0205] In some embodiments, an antibody, bispecific antibody, AA, or BAA (or conjugated composition thereof) of the present disclosure is administered in combination with an immunotherapeutic agent. In some embodiments, an antibody, bispecific antibody, AA, or BAA (or conjugated composition thereof) of the present disclosure is administered in combination with a chemotherapeutic agent. In some embodiments, an antibody, bispecific antibody, AA, or BAA (or conjugated composition thereof) of the present disclosure is administered in combination with both an immunotherapeutic agent and a chemotherapeutic agent. In some embodiments, one or more additional agents are administered with any of these combination embodiments.

[0206] In some embodiments, the antibodies are formulated into a single therapeutic composition and the antibody / bispecific antibody / AA / its BAA and additional agent are administered simultaneously. Alternatively, the antibody / bispecific antibody / AA / its BAA are administered separately from one another (e.g., each is formulated into a separate therapeutic composition and the antibody / bispecific antibody / AA / its BAA and additional agent are administered simultaneously), or the antibody / bispecific antibody / AA / its BAA and additional agent are administered at different times during the treatment regimen. The antibody / bispecific antibody / AA / its BAA and additional agent can be administered in multiple doses.

[0207] The antibody / bispecific antibody / AA / its BAA can be incorporated into a pharmaceutical composition suitable for administration. The principles and considerations involved in the preparation of such compositions, as well as guidance in component selection, are provided, for example, in Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0208] Such compositions typically comprise the antibody / bispecific antibody / AA / BAA thereof and a pharmaceutically acceptable carrier.

[0209] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, as well as isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field (incorporated herein by reference). Suitable examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles (such as fixed oils) can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated.

[0210] The formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0211] The disclosed pharmaceutical compositions are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent (such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents); an antibacterial agent (such as benzyl alcohol or methylparaben); an antioxidant (such as ascorbic acid or sodium bisulfite); a chelating agent (such as ethylenediaminetetraacetic acid (EDTA)); a buffer (such as acetate buffer, citrate buffer, or phosphate buffer), and an agent for adjusting tonicity (such as sodium chloride or dextrose). pH can be adjusted with acids or bases (such as hydrochloric acid or sodium hydroxide). Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0212] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thyromesal. In many cases, it will be appropriate to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), and sodium chloride in the composition. Absorption of injectable compositions can be delayed by including in the composition an agent which delays absorption, such as aluminum monostearate and gelatin.

[0213] Sterile injectable solution can be prepared by incorporating the active compound in the amount required with one or combination of the above-listed components in suitable solvent, and then optionally sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into the sterile vehicle that contains the basic dispersion medium and other necessary components from the above-listed components.For the sterile powder that is used to prepare sterile injectable solution, the preparation method is vacuum drying and freeze-drying, which produces the powder of active ingredient and any other desired components from the solution that is previously sterilized by filtration.

[0214] Oral compositions generally contain inert diluents or edible carriers.Oral compositions can be enclosed in gelatin capsules or compressed into tablets.For therapeutic oral administration, active compounds can be incorporated with excipients and used in the form of tablets, troches or capsules.Oral compositions can also be prepared using fluid carriers for use as mouthwash, where the fluid carrier containing the compound is orally applied, gargled and expectorated or swallowed.Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of a similar nature: a binder (such as microcrystalline cellulose, gum tragacanth, or gelatin); an excipient (such as starch or lactose); a disintegrant (such as alginic acid, Primogel, or corn starch); a lubricant (such as magnesium stearate or Sterotes); a glidant (such as colloidal silicon dioxide); a sweetener (such as sucrose or saccharin); or a flavoring substance (such as peppermint, methyl salicylate, or orange flavoring).

[0215] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0216] Systemic administration can also be by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant that is compatible with the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, surfactants, bile salts, and fusidic acid derivatives.Transmucosal administration can be carried out using nasal sprays or suppositories.For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams as is generally known in the art.

[0217] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0218] In one embodiment, the active compound is prepared using a carrier that protects the compound from rapid elimination from the body (such as sustained / controlled release formulations, including implants and microencapsulated delivery systems).Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.Methods for preparing such formulations will be clear to those skilled in the art.

[0219] For example, the active ingredient can be entrapped in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions prepared, for example, by coacervation techniques or interfacial polymerization.

[0220] Sustained-release preparations can be prepared. Examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles (e.g., films) or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, certain hydrogels release proteins for shorter periods.

[0221] Materials can also be purchased from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes that target infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art (e.g., as described in U.S. Pat. No. 4,522,811).

[0222] In order to facilitate administration and uniform dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically separate unit that is suitable as a single dosage for the subject to be treated; each unit contains a predetermined amount of active compound that is calculated to obtain desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present disclosure is determined by and directly depends on the inherent characteristics of active compound and the specific therapeutic effect that should be achieved and the inherent limitation in the field of formulation of this active compound for individual treatment.

[0223] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0224] The formulation may also contain more than one active compound as necessary for the particular indication being treated (e.g., active compounds with complementary activities that do not adversely affect each other). Alternatively, or in addition, the composition may contain an agent that enhances its function (e.g., a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent, etc.). Such molecules are suitably present in the composition in an amount that is effective for the intended purpose.

[0225] In one embodiment, the active compound is administered in combination therapy, i.e., in combination with other drugs (e.g., therapeutic agents) useful for treating pathological conditions or disorders (such as autoimmune disorders and inflammatory diseases). In this context, the term "in combination" means that the drugs are administered simultaneously or sequentially, substantially simultaneously. When administered sequentially at the start of administration of the second compound, the first of the two compounds is still detectable at effective concentrations at the site of treatment.

[0226] For example, a combination therapy can include one or more antibodies / bispecific antibodies / AAs / BAAs thereof of the present disclosure co-formulated and / or co-administered with one or more additional therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxic or cytostatic agents), as described in more detail below. Additionally, one or more antibodies / bispecific antibodies / AAs / BAAs thereof described herein can be used in combination with two or more therapeutic agents described herein (e.g., one BAA administered with another BAA or AA of the present disclosure). Such combination therapies may advantageously utilize lower amounts of administered therapeutic agents, thereby avoiding potential toxicities or complications associated with various monotherapies.

[0227] In other embodiments, one or more antibodies of the present disclosure can be co-formulated and / or co-administered with one or more anti-inflammatory drugs, immunosuppressants, or metabolic or enzyme inhibitors. Non-limiting examples of drugs or inhibitors that can be used in combination with the antibodies described herein include, but are not limited to, one or more of the following: nonsteroidal anti-inflammatory drug(s) (NSAIDs) (e.g., ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin); sulfasalazine; corticosteroids (such as prednisolone); cytokine-suppressive anti-inflammatory drug(s) (CSAIDs); inhibitors of nucleotide biosynthesis (e.g., prilosporin, riboflavin, riboflavin); inhibitors of pyrimidine biosynthesis, folate antagonists (e.g., methotrexate (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid); and inhibitors of pyrimidine biosynthesis (e.g., dihydroorotate dehydrogenase (DHODH) inhibitors). Suitable therapeutic agents for use in combination with the antibodies of the disclosure include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g., leflunomide), and folate antagonists (e.g., methotrexate).

[0228] Examples of additional inhibitors include one or more of the following: corticosteroids (oral, inhaled, and local injection); immunosuppressants (e.g., cyclosporine, tacrolimus (FK-506)); and mTOR inhibitors (e.g., sirolimus (rapamycin - Rapamune™) or rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779)); agents that interfere with signaling by proinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38, or MAP kinase inhibitors); COX2 inhibitors (e.g., celecoxib, rofecoxib, and derivatives thereof); phosphodiesterase inhibitors (e.g., R973401 (phosphodiesterase type IV inhibitor)); phospholipase inhibitors (e.g., For example, inhibitors of cytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketone analogs); inhibitors of vascular endothelial growth factor or growth factor receptors (e.g., VEGF inhibitors and / or VEGF-R inhibitors); and inhibitors of angiogenesis. Suitable therapeutic agents for use in combination with the disclosed antibodies are immunosuppressants (e.g., cyclosporine, tacrolimus (FK-506)); mTOR inhibitors (e.g., sirolimus (rapamycin) or rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779)); COX2 inhibitors (e.g., celecoxib and variants thereof); and phospholipase inhibitors (e.g., inhibitors of cytosolic phospholipase 2 (cPLA2), e.g., trifluoromethyl ketone analogs).

[0229] Further examples of therapeutic agents that can be combined with the antibodies of the present disclosure include one or more of the following: 6-mercaptopurine (6-MP); azathioprine sulfasalazine; mesalazine; olsalazine; chloroquine / hydroxychloroquine (Plaquinil®); penicillamine; aurothiornalate (intramuscular and oral); azathioprine; colchicine; beta-2 adrenergic receptor agonists (salbutamol, terbutaline, salmeterol); xanthines (theophylline, aminophylline); cromoglycate; nedocromil; ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and adrenergic agents.

[0230] In some embodiments, an antibody / bispecific antibody / AA / BAA thereof of the present disclosure can be combined with one or more antibodies / bispecific antibodies / AA / BAA thereof.

[0231] 14. Kit and Product Manufacturing Provided herein are kits and articles of manufacture that include any one or more of the antibodies, AAs, bispecific antibodies, and BAAs provided herein.

[0232] Kits and articles of manufacture may include any one or more of the antibodies, AAs, bispecific antibodies, and BAAs provided herein in a format suitable for storage or transport.

[0233] The kits and articles of manufacture may include at least a second component.

[0234] The kits and articles of manufacture may include a container, a diluent, a solvent, a second composition, or any components useful for converting a composition in a storage format into a composition suitable for use in a method disclosed herein when conversion is necessary. The method may be, for example, a therapeutic method disclosed herein. The kit may include instructions for use.

[0235] The kits and articles of manufacture can include, for example, a cytotoxic agent or a detectable label in a format suitable for conjugation to the agents disclosed herein, antibodies, AAs, bispecific antibodies, and BAAs provided herein.

[0236] The following examples are included for illustrative purposes and are not intended to limit the scope of the invention.

[0237] Enumeration of Embodiments The invention can be defined with reference to the following enumerated exemplary embodiments.

[0238] 1. A bispecific activatable antibody (BAA), wherein said BAA, when activated, specifically binds to two targets and has the following structure: a. an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein said AB1 is linked to a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl termini of said MM1-CM1 construct are linked to each amino terminus of each light chain of said AB1, wherein said MM1 inhibits the binding of said AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, an IgG antibody (AB1), b. two scFvs (each AB2) each specifically binding to a second target, wherein each AB2 comprises a light chain variable region linked to a heavy chain variable region, wherein the carboxyl terminus of each of said AB2 is linked to the amino terminus of each of said AB1 heavy chains; wherein each AB2 is linked to a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each of said MM2-CM2 constructs is linked to the amino terminus of each of said AB2, wherein said MM2 inhibits the binding of said AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for a second protease, two scFvs (each AB2) Including, Here, the aforementioned BAA has the following characteristics: i. MM2 comprises the amino acid sequence of SEQ ID NO: 12; ii. MM1 comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7; iii. AB2 comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3 or a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4; and iv. AB1 comprises an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced; A bispecific activatable antibody (BAA) having at least one of:

[0239] 2. The BAA of embodiment 1, wherein AB2 comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3 and a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4.

[0240] 3. The BAA of embodiment 1, wherein said AB1 binds a tumor target and said AB2 binds an immune effector target.

[0241] 4. The BAA according to any one of embodiments 1 to 3, wherein said BAA is a T cell-inducing bispecific (TCB) AA (TCBAA).

[0242] 5. The BAA according to any one of embodiments 1 to 4, wherein said AB1 binds EGFR and said AB2 binds CD3ε.

[0243] 6. The BAA of any one of embodiments 1 to 5, wherein said MM1 comprises an amino acid sequence selected from the group consisting of the sequences set out in Table 7.

[0244] 7. The BAA described in any one of embodiments 1 to 5, wherein said MM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 85 and SEQ ID NO: 78.

[0245] 8. The BAA of any one of embodiments 1 to 5, wherein said MM1 comprises SEQ ID NO: 78.

[0246] 9. The BAA of any one of embodiments 1 to 8, wherein said MM2 comprises the amino acid sequence of SEQ ID NO: 12.

[0247] 10. The BAA described in any one of embodiments 1 to 9, wherein said CM comprises the amino acid sequence of SEQ ID NO: 14.

[0248] 11. The BAA of any one of embodiments 1 to 9, wherein said CM comprises the amino acid sequence of SEQ ID NO: 17.

[0249] 12. The BAA of any one of embodiments 1 to 9, wherein said CM comprises the amino acid sequence of SEQ ID NO: 16.

[0250] 13. The BAA of any one of embodiments 1-9, wherein CM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 16.

[0251] 14. The BAA of any one of embodiments 1-9, wherein CM2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 17.

[0252] 15. The BAA according to any one of embodiments 1-14, wherein AB1 comprises an amino acid substitution in at least two of the amino acid positions L234, L235, and P331.

[0253] 16. The BAA of embodiment 15, wherein AB1 comprises amino acid substitutions at amino acid positions L234, L235, and P331.

[0254] 17. The BAA of embodiment 15, wherein AB1 comprises the following amino acid substitutions: L234F, L235E, and P331S.

[0255] 18. The BAA of embodiment 15, wherein said AB1 comprises an Fc region comprising an amino acid substitution at N297.

[0256] 19. The BAA according to any one of embodiments 1-14, wherein AB1 comprises amino acid substitutions at amino acid positions L234F, L235E, P331S, and N297Q.

[0257] 20. The BAA of embodiment 1, wherein the heavy chain of said AB1 comprises any one of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76 listed in Table 6.

[0258] 21. BAA CI106, including the layout and sequence provided in Table 11 and Example 1.

[0259] 22. BAA CI107, including the layout and sequence provided in Table 11 and Example 1.

[0260] 23. BAA CI079, including the layout and sequence provided in Table 11 and Example 1.

[0261] 24. BAA CI090, including the layout and sequence provided in Table 11 and Example 1.

[0262] 25. An activatable antibody (AA), a) an antibody or antigen-binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3 (CD3ε); b) a masking moiety (MM) coupled to AB, wherein said MM reduces or inhibits binding of said AB to said CD3ε when said AA is in an uncleaved state, and wherein said MM comprises the amino acid sequence of SEQ ID NO: 12; and c) a cleavable moiety (CM) coupled to AB, wherein said CM is a polypeptide that functions as a substrate for a protease. activatable antibodies (AA), including:

[0263] 26. The AA of embodiment 25, wherein said CM comprises any one of the sequences listed in Table 4.

[0264] 27. The AA of embodiment 25, wherein said CM comprises a substrate cleavable by a serine protease or MMP.

[0265] 28. The AA of embodiment 25, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 56.

[0266] 29. The AA of embodiment 25, wherein said protease is an MMP.

[0267] 30. The AA of embodiment 25, wherein said protease is a serine protease.

[0268] 31. An AA according to embodiment 25, wherein said AB that specifically binds to CD3 is an antibody according to any one of embodiments 38 to 47.

[0269] 32. An activatable antibody (AA), a. an antibody or antigen-binding fragment thereof (AB) that specifically binds to epidermal growth factor receptor (EGFR); b. a masking moiety (MM) coupled to AB, wherein said MM reduces or inhibits binding of said AB to EGFR when said AA is in an uncleaved state, and wherein said MM comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 7; and c. A cleavable moiety (CM) coupled to AB, wherein said CM is a polypeptide that functions as a substrate for a protease. activatable antibodies (AA), including:

[0270] 33. The AA of embodiment 32, wherein said MM comprises the amino acid sequence of SEQ ID NO: 78.

[0271] 34. An AA according to any one of embodiments 32-33, wherein said CM comprises a substrate cleavable by a serine protease or an MMP.

[0272] 35. An AA according to any one of embodiments 32-33, wherein said CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-56.

[0273] 36. The AA of embodiment 32, wherein the CM comprises the amino acid sequence of SEQ ID NO: 14.

[0274] 37. The AA of embodiment 32, wherein said CM comprises the amino acid sequence of SEQ ID NO: 16.

[0275] 38. An antibody or antigen-binding fragment (AB) that specifically binds to the epsilon chain of CD3 (CD3ε), comprising a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3, or a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4.

[0276] 39. The AB of embodiment 38, wherein said antibody comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3 and a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4.

[0277] 40. The AB of embodiment 38, comprising a heavy chain variable domain as set forth in SEQ ID NO:2.

[0278] 41. The AB of embodiment 38, comprising a heavy chain variable domain as set forth in SEQ ID NO:3.

[0279] 42. The AB of embodiment 38, wherein the antibody comprises a light chain variable domain set forth in SEQ ID NO:1.

[0280] 43. The AB of embodiment 38, comprising a light chain variable domain as set forth in SEQ ID NO:4.

[0281] 44. The AB of embodiment 38, comprising a heavy chain variable domain set forth in SEQ ID NO:2 and a light chain variable domain set forth in SEQ ID NO:1.

[0282] 45. The AB of embodiment 38, comprising a heavy chain variable domain set forth in SEQ ID NO:3 and a light chain variable domain set forth in SEQ ID NO:1.

[0283] 46. ​​The AB of embodiment 38, comprising a heavy chain variable domain set forth in SEQ ID NO:3 and a light chain variable domain set forth in SEQ ID NO:4.

[0284] 47. The AB of embodiment 38, comprising a heavy chain variable domain set forth in SEQ ID NO:2 and a light chain variable domain set forth in SEQ ID NO:4.

[0285] 48. The AB according to any one of embodiments 32-47, wherein said AB is a bispecific AB.

[0286] 49. An AA according to any one of embodiments 32 to 47, wherein said antibody is an scFv.

[0287] 50. An AA according to any one of embodiments 32 to 47, wherein said antibody is an IgG1 antibody.

[0288] 51. An activatable antibody (AA), a) an antibody or antigen-binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3 (CD3ε), wherein said antibody comprises a heavy chain variable domain set forth in SEQ ID NO:2 or SEQ ID NO:3, or a light chain variable domain set forth in SEQ ID NO:1 or SEQ ID NO:4; b) a masking moiety (MM) coupled to AB, wherein said MM reduces or inhibits binding of said AB to said CD3ε when said AA is in an uncleaved state; and c) a cleavable moiety (CM) coupled to AB, wherein said CM is a polypeptide that functions as a substrate for a protease. activatable antibodies (AA), including:

[0289] 52. The AA of embodiment 51, wherein said AB comprises a heavy chain variable domain set forth in SEQ ID NO:2.

[0290] 53. The AA of embodiment 51, wherein said AB comprises a heavy chain variable domain set forth in SEQ ID NO:3.

[0291] 54. The AA according to embodiment 51, wherein said AB comprises a light chain variable domain set forth in SEQ ID NO:1.

[0292] 55. The AA according to embodiment 51, wherein said AB comprises a light chain variable domain set forth in SEQ ID NO:4.

[0293] 56. The AA according to embodiment 51, wherein said AB comprises a heavy chain variable domain set forth in SEQ ID NO:2 and a light chain variable domain set forth in SEQ ID NO:1.

[0294] 57. The AA according to embodiment 51, wherein said AB comprises a heavy chain variable domain set forth in SEQ ID NO: 3 and a light chain variable domain set forth in SEQ ID NO: 1.

[0295] 58. The AA according to embodiment 51, wherein said AB comprises a heavy chain variable domain set forth in SEQ ID NO:2 and a light chain variable domain set forth in SEQ ID NO:4.

[0296] 59. The AA according to embodiment 51, wherein said AB comprises a heavy chain variable domain set forth in SEQ ID NO:3 and a light chain variable domain set forth in SEQ ID NO:4.

[0297] 60. The AA according to any one of embodiments 51 to 59, wherein said MM comprises any one of the sequences listed in Table 3.

[0298] 61. The AA according to any one of embodiments 51 to 59, wherein said CM comprises any one of the sequences listed in Table 4.

[0299] 62. A bispecific activatable antibody (BAA) comprising any one of the AAs of embodiments 51-61.

[0300] 63. An activatable antibody (AA), a. an antibody (AB) that specifically binds to a target, wherein said antibody is an IgG1 antibody, and wherein the Fc region of said antibody comprises amino acid substitutions at amino acid positions L234, L235, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said antibody is reduced; b. a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits binding of the AB to a target when the AA is in an uncleaved state; and c. A cleavable moiety (CM) coupled to AB, wherein said CM is a polypeptide that functions as a substrate for a protease. activatable antibodies (AA), including:

[0301] 64. The AA of embodiment 63, wherein said Fc region comprises amino acid substitutions at least at amino acid positions L234, L235, N297, and P331, as numbered according to the EU index as set forth in Kabat, such that the effector function of said AA is reduced.

[0302] 65. The AA of embodiment 63 or 64, wherein said target is selected from the group consisting of the targets set out in Table 9.

[0303] 66. A bispecific activatable antibody (BAA), a. an IgG antibody (AB1) that specifically binds to a first target, wherein said AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein said AB1 is linked to a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl termini of said MM1-CM1 construct are linked to each amino terminus of each light chain of said AB1, wherein said MM1 inhibits the binding of said AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, an IgG antibody (AB1), b. two scFvs (each AB2) each specifically binding to a second target, wherein each AB2 comprises a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each of said AB2 is linked to the amino terminus of each of said AB1 heavy chains; wherein each AB2 is linked to a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each of said MM2-CM2 constructs is linked to the amino terminus of each of said AB2, wherein said MM2 inhibits the binding of said AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for a second protease, two scFvs (each AB2) Including, wherein said AB1 is a bispecific activatable antibody (BAA) comprising an Fc region comprising an amino acid substitution at at least one of amino acid positions L234, L235, N297, and P331, numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced.

[0304] 67. The BAA of embodiment 66, wherein the Fc region comprises amino acid substitutions at least at amino acid positions L234, L235, N297, and P331, as numbered according to the EU index as set forth in Kabat, such that the effector function of the BAA is reduced.

[0305] 68. The BAA of embodiment 66, wherein said Fc region comprises amino acid substitutions at least at amino acid positions L234, L235, and P331, as numbered according to the EU index as set forth in Kabat, such that the effector function of said BAA is reduced.

[0306] 69. The BAA of any one of embodiments 66-68, wherein the first target is selected from the group consisting of targets shown in Table 9, and the second target is selected from the group consisting of targets shown in Table 9.

[0307] 70. The AA or BAA of any one of the preceding embodiments, wherein said antigen-binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody.

[0308] 71. The AA or BAA of any one of the preceding embodiments, wherein said antibody is a rodent antibody, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody.

[0309] 72. An AA according to any one of embodiments 32-37 and 51-71, wherein said AA is a BAA.

[0310] 73. A pharmaceutical composition comprising the antibody, AA, or BAA of any one of embodiments 1 to 72 and an optional carrier.

[0311] 74. The pharmaceutical composition of embodiment 73, comprising an additional agent.

[0312] 75. The pharmaceutical composition according to embodiment 74, wherein said further agent is a therapeutic agent.

[0313] 76. An isolated nucleic acid molecule encoding the antibody, AA, or BAA of any one of embodiments 1-72.

[0314] 77. A vector comprising the isolated nucleic acid molecule of embodiment 76.

[0315] 78. A vector containing the nucleic acid sequence of pLW289.

[0316] 79. A vector containing the nucleic acid sequence of pLW246.

[0317] 80. A vector containing the nucleic acid sequence of pLW307.

[0318] 81. A vector containing the nucleic acid sequence of pLW291.

[0319] 82. A cell comprising any one of the vectors of embodiments 77 to 81.

[0320] 83. Cells containing pLW289 and pLW246.

[0321] 84. Cells containing pLW307 and pLW291.

[0322] 85. A method for producing an antibody, AA, or BAA described in any one of embodiments 1 to 72 by culturing cells under conditions for expression of the antibody, AA, or BAA, wherein the cells comprise a nucleic acid molecule described in embodiment 76 or a vector described in any one of embodiments 78 to 81.

[0323] 86. A method for treating, alleviating the symptoms of, or delaying the progression of a disorder or disease, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody, AA, or BAA described in any one of embodiments 1-72, or a pharmaceutical composition described in any one of embodiments 73-75.

[0324] 87. The method of embodiment 86, wherein the disorder or disease comprises diseased cells that express EGFR.

[0325] 88. The method of embodiment 86 or 87, wherein the disorder or disease is cancer.

[0326] 89. The method of embodiment 88, wherein the cancer is anal cancer, basal cell carcinoma, brain cancer, bladder cancer, breast cancer, bone cancer, cervical cancer, intrahepatic bile duct cancer, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, small intestine carcinoma, squamous cell carcinoma, skin cancer testicular cancer, thyroid cancer, or uterine cancer.

[0327] 90. A method for inhibiting angiogenesis in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody, AA, or BAA described in any one of embodiments 1-72, or a pharmaceutical composition described in any one of embodiments 73-75.

[0328] 91. The method of any one of embodiments 86-90, comprising administering an additional agent.

[0329] 92. The method of embodiment 91, wherein said additional agent is a therapeutic agent.

[0330] 93. A method for reducing damage to healthy tissue caused by binding of an antibody to its target on diseased tissue and on healthy tissue, comprising administering to a subject in need thereof an AA or BAA or a pharmaceutical composition comprising an AA or BAA, wherein said AA or BAA is an AA or BAA described in any one of the embodiments provided herein.

[0331] 94. A method for improving the tolerability of antibody treatment, comprising administering to a subject in need thereof an AA or BAA or a pharmaceutical composition comprising an AA or BAA, wherein said AA or BAA is an AA or BAA described in any one of the embodiments provided herein.

[0332] 95. A method for recruiting T cells to tumor tissue, comprising administering an AA or BAA or a pharmaceutical composition comprising an AA or BAA to a subject in need thereof, wherein the AA or BAA is an AA or BAA described in any one of the embodiments provided herein.

[0333] 96. An antibody, AA, or BAA according to any one of embodiments 1 to 72, or a pharmaceutical composition according to any one of embodiments 73 to 75, for use as a medicament.

[0334] 97. The antibody, AA, or BAA of any one of embodiments 1-72, or the pharmaceutical composition of any one of embodiments 73-75, for use in a method for treating, alleviating the symptoms of, or delaying the progression of a disorder or disease, wherein the disorder or disease comprises diseased cells that express EGFR.

[0335] 98. The antibody, AA, or BAA of any one of embodiments 1-72, or the pharmaceutical composition of any one of embodiments 73-75, for use in a method for treating cancer, optionally wherein said cancer is anal cancer, basal cell carcinoma, brain cancer, bladder cancer, breast cancer, bone cancer, cervical cancer, intrahepatic bile duct cancer, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, small intestine carcinoma, squamous cell carcinoma, skin cancer testicular cancer, thyroid cancer, or uterine cancer.

[0336] 99. An antibody, AA, or BAA according to any one of embodiments 1 to 72, or a pharmaceutical composition according to any one of embodiments 73 to 75, for use in a method of treatment, wherein the method comprises inhibiting angiogenesis.

[0337] 100. An antibody, AA, or BAA, or pharmaceutical composition for use according to any of embodiments 96 to 99, wherein said use comprises administering an additional agent, and optionally said additional agent is a therapeutic agent.

[0338] Example Example 1. Antibody, BAA, and Activated BAA Sequences, Vector Construction, and Expression Antibody of interest The molecules provided below in Table 11 were constructed and tested. As indicated, activated molecules were produced that were masked and proteolytically cleaved to generate the activated form.

[0339] [Table 15]

[0340] The sequences of the molecules and vectors are provided below. Brackets indicate some components of the molecules shown. In some sequences, linkers are provided. Underlined amino acids indicate predicted CDR sequences. CI011:3954-0001-C225v5N297Q-JF15865-0001-CD3LvHv-HN pLW023:HC JF15865-0001-CD3LvHv-C225v5N297Q(HN) Nucleotide sequence [Spacer SEQ ID NO: 176] [pLW023 without spacer SEQ ID NO: 177] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW023 without spacer SEQ ID NO: 179] QGQSGQ[MMYCGGNEVLCGPRV][GSSGGSGGSGG][LSGRSDNH][GGGS]QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106) OPP022:LC 3954-0001-C225v5 Nucleotide sequence [Spacer SEQ ID NO: 180] [OPP022 without spacer SEQ ID NO: 181] (SEQ ID NO: 107) Amino acid sequence [Spacer SEQ ID NO: 178] [OPP022 without spacer SEQ ID NO: 182] [SDNH][GSSGT][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 108) CI020:3954-Nsub-C225v5N297Q-JF15865-Nsub-hSP34LvHv-HN pLW073:HC C225v5N297Q-JF15865-Nsub-hSP34LvHv(HN) Nucleotide sequence [Spacer SEQ ID NO: 176] [pLW073 without spacer SEQ ID NO: 183] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW073 without spacer SEQ ID NO: 184] QGQSS LKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNKS QVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 110) pLW071:LC 3954-Nsub-C225v5 Nucleotide sequence [Spacer SEQ ID NO: 180] [pLW071 without spacer SEQ ID NO: 185] CAAGGCCAGTCTGGCCAGTGCATCTCACCTCGTGGTTGTCCGGACGGCCCATACGTCATGTACGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGGTGGAGGCTCGGGCGGTGGGAGCGGCGGTTCTGATATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 111) Amino acid sequence [Spacer SEQ ID NO: 178][pLW071 without Spacer SEQ ID NO: 186] QGQSGQ[CISPRGCPDGPYVMY][GSSGGSGGSGGSGGGSGGGSGGS]DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 112) CI040:3954-2001-C225v5N297Q-JF15865-2001-hSP34LvHv-HN pLW101:HC JF15865-2001-CD3LvHv-C225v5N297Q(HN) Nucleotide sequence [Spacer SEQ ID NO: 176] [pLW101 without spacer SEQ ID NO: 187] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW101 without spacer SEQ ID NO: 188] QGQSGQ[MMYCGGNEVLCGPRV][GSSGGSGGGSGG][ISSGLLSGRSDNH][GGGS]QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGSGGGGS]EVQLVESGGG LVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 114) CTX122:LC 3954-2001-C225v5 Nucleotide sequence [Spacer sequence number 180] [CTX122 without spacer sequence number 189] (SEQ ID NO: 115) Amino acid sequence [Spacer sequence number 178] [CTX122 without spacer sequence number 190] QGQSGQ[CISPRGCPDGPYVMY][GSSGGSGGSGGSG][ISSGLLSGRSDNH][GSSGT]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 116) CI048:Activation CI011:3954-0001-C225v5N297Q-JF15865-0001-CD3LvHv-HN The pLW023 and OPP022 sequences encoding the corresponding masked antibody components are provided herein as "pLW023" and "OPP022," respectively, and are summarized in Table 11. Activated pLW023:HC JF15865-0001-CD3LvHv-C225v5N297Q(HN) Amino acid sequence [SDNH][GGGS]QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKG LEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYY CARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 118) Activated OPP022:LC 3954-0001-C225v5 Nucleotide sequence TCCGATAATCATGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 119) Amino acid sequence [[ID=​​​ pLW225:HC h20GG-0001-v16sc-C225v5Fcmt3(HN) Nucleotide sequence [Spacer SEQ ID NO: 191] [pLW225 without spacer SEQ ID NO: 192] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW225 without spacer SEQ ID NO: 193] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGGSGG][LSGRSDNH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 122) OPP022:LC 3954-0001-C225v5 The sequences provided above CI090:3954-0001-C225v5Fcmt4-h20GG-0001-v16sc-HN pLW233:HC h20GG-0001-v16sc-C225v5Fcmt4(HN) Nucleotide sequence [Spacer SEQ ID NO: 191] [pLW233 without spacer SEQ ID NO: 194] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW233 without spacer SEQ ID NO: 195] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGGSGG][LSGRSDNH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 124) OPP022:LC 3954-0001-C225v5 The sequences provided above Activated CI090:Activated- 3954-0001-C225v5Fcmt4-h20GG--0001-v16sc-HN Activated pLW233:HC C225v5Fcmt4-h20GG-0001-v16sc(HN) Nucleic acid sequence Amino acid sequence [SDNH] (SEQ ID NO: 165) Activation OPP022:3954-0001-C225v5 Nucleic acid sequence TCCGATAATCATGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 166) Amino acid sequence [SDNH]GSSGTQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 167) CI104:3954-0011-C225v5Fcmt4-h20GG-0011-v16sc-HN The pLW289 and pLW291 sequences encoding the corresponding masked antibody components are provided herein as "pLW289" and "pLW291," respectively, and are summarized in Table 11. Activation CI104:3954-0011-C225v5Fcmt4-h20GG-0011-v16sc-HN Activated pLW289:HC h20GG-0011-v16sc-C225v5Fcmt4(HN) Nucleotide sequence Amino acid sequence [SDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL][GGGGSGGGGSGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGK GLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIY YCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 126) Activated pLW291:LC 3954-0011-C225v5 Nucleotide sequence TCCGATGATCATGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 127)<000id=1569><000id=1570>Amino acid sequence<000id=1571>[SDDH][GSSGT]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 128)<000id=1572><000id=1573><000id=1574> It should be noted that in the translation, the "id" in "<000id=1569>" etc. in the original text seems to be an incorrect expression. It is likely that it should be " " etc. for correct sequence number representation. The translation is adjusted based on the original text as much as possible while keeping the incorrect "id" form for the purpose of following the requirements. pLW289:HC h20GG-0011-v16sc-C225v5Fcmt4(HN) Nucleotide sequence [Spacer SEQ ID NO: 191] [pLW289 without spacer SEQ ID NO: 196] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW289 without spacer SEQ ID NO: 197] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGGSGG][LSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 130) pLW246:LC CF41-2008-C225v5 Nucleotide sequence [Spacer SEQ ID NO: 176] [pLW246 without spacer SEQ ID NO: 198] (SEQ ID NO: 131) Amino acid sequence [Spacer SEQ ID NO: 178] [pLW246 without spacer SEQ ID NO: 199] QGQSGQG[LSCEGWAMNREQCRA][GGGSSGGS][ISSGLLSGRSDQH][GGGS]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 132) CI107:3954-0011-C225v5Fcmt4-h20GG-2006-v16sc-HN pLW307:HC h20GG-2006-v16sc-C225v5Fcmt4(HN) Nucleotide sequence [Spacer SEQ ID NO: 191] [pLW307 without spacer SEQ ID NO: 200] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW307 without spacer SEQ ID NO: 201] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][ISSGLLSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGSGGGGS]EVQLVESGGG LVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 134) pLW291:LC 3954-0011-C225v5 Nucleotide sequence [Spacer SEQ ID NO: 180] [pLW291 without spacer SEQ ID NO: 202] (SEQ ID NO: 135) Amino acid sequence [Spacer SEQ ID NO: 178] [pLW291 without spacer SEQ ID NO: 203] QGQSGQ[CISPRGCPDGPYVMY][GSSGGSGGSGGSG][LSGRSDDH][GSSGT]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 136) CI127:SynFcmt4-h20GG-0011-v16sc-HN pLW334:HC h20GG-0011-v16sc-SYNAGIS® Fcmt4(HN) Nucleotide sequence [Spacer SEQ ID NO: 191] [pLW334 without spacer SEQ ID NO: 204] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW334 without spacer SEQ ID NO: 205] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGGSGG][LSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKD TSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 138) pLW139:LC SYNAGIS® Nucleotide sequence GACATCCAGATGACCCAGAGCCCCAGCACACTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACATGCAAGTGCCAGCTGAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACACCAGCAAGCTGGCCTCCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGAGTTCACCCTGACAATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGTTTTCAAGGCTCCGGCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 139) Amino acid sequence DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 140) CI128:SynFcmt4-h20GG-2006-v16sc-HN [[ID=十一]] pLW335:HC h20GG-2006-v16sc-SYNAGIS® Fcmt4(HN) Nucleotide sequence [Spacer SEQ ID NO: 191] [pLW335 without spacer SEQ ID NO: 206] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW335 without spacer SEQ ID NO: 207] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][ISSGLLSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGSGGGGS]EVQLVESGGG LVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTIS KDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 142) pLW139:LC Synagis® The sequences provided above CI135:CF41-2008-C225v5Fcmt4-h20GG-0011-v12sc-HN pLW352:HC h20GG-0011-v12sc-C225v5Fcmt4(HN) Nucleotide sequence [Spacer SEQ ID NO: 208] [pLW352 without spacer SEQ ID NO: 209] Amino acid sequence [Spacer SEQ ID NO: 176] [pLW352 without spacer SEQ ID NO: 210] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGGSGG][LSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 146) pLW246:LC CF41-2008-C225v5 Nucleotide sequence The sequence provided above Amino acid sequence The sequence provided above CI136:CF41-2008-C225v5Fcmt4-h20GG-0011-v19sc-HN pLW353:HC h20GG-0011-v19sc-C225v5Fcmt4(HN) Nucleotide sequence [Spacer SEQ ID NO: 191] [pLW353 without spacer SEQ ID NO: 211] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW353 without spacer SEQ ID NO: 212] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][LSGRSDDH][GGGS]QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQ APRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 148) pLW246:LC CF41-2008-C225v5 The sequence provided above CI091: 3954-1490DQH-C225v5Fcmt4 -h20GG-2008-v16sc-HN pLW242:HC C225v5Fcmt4-h20GG-2008-v16sc(HN) Nucleic acid sequence [Spacer SEQ ID NO: 191] [pLW242 without spacer SEQ ID NO: 213] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW242 without spacer SEQ ID NO: 214] QGQSGS[GYLWGCEWNCGGITT]GSSGGSGGSGG[ISSGLLSGRSDQH] (SEQ ID NO: 169) CX320:3954-C225v5-2008 Nucleic acid sequence [Spacer sequence number 180] [CX320 without spacer sequence number 215] CAAGGCCAGTCTGGCCAGTGCATCTCACCTCGTGGTTGTCCGGACGGCCCATACGTCATGTACGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGATCCGGTATATCGAGTGGATTGCTGTCTGGCAGATCTGACCAACACGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 170) Amino acid sequence [Spacer SEQ ID NO: 178][CX320 without Spacer SEQ ID NO: 216] QGQSGQ[CISPRGCPDGPYVMY]GSSGGSGGSGGSG[ISSGLLSGRSDQH]GSSGTQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 171) CI064:SynN297Q-JF15865-0001-hSP34LvHv-HN pLW138:HC SynN297Q-JF15865-0001-hSP34LvHv-HN Nucleic acid sequence [Spacer SEQ ID NO: 176] [pLW138 without spacer SEQ ID NO: 147] Amino acid sequence [Spacer SEQ ID NO: 178] [pLW138 without spacer SEQ ID NO: 153] QGQSGQ[MMYCGGNEVLCGPRV]GSSGGSGGSGG[LSGRSDNH] (SEQ ID NO: 173) pLW139:LC Syn kappa Nucleic acid sequence GACATCCAGATGACCCAGAGCCCCAGCACACTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACATGCAAGTGCCAGCTGAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACACCAGCAAGCTGGCCTCCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGAGTTCACCCTGACAATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGTTTTCAAGGCTCCGGCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 174) Amino acid sequence DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 175) Anti-CD3 scFv variant v12 Light chain LV12 Heavy chain HV12 The sequence provided above Anti-CD3 scFv variant v16 Light Chain LV12 Heavy chain HV20 The sequence provided above Anti-CD3 scFv variant v19 Light Chain LV19 Heavy chain HV20 The sequence provided above Anti-CD3 scFv variant v26 Light Chain LV19 Heavy chain HV12 The sequence provided above

[0341] Vector construction The heavy and light chains were individually cloned into mammalian expression vectors using standard molecular biology techniques. Briefly, DNA fragments encoding the regions of interest were amplified using primers that bind to the ends. Overlapping fragments were combined and amplified using flanking primers as needed to construct the complete desired region. The DNA fragments were then cloned into the expression vector using a commercially available homologous recombination kit (MCLabs, South San Francisco, CA). The mammalian expression vector was a modified version of Invitrogen's cDNA™ 3.1(+) containing a G418 or hygromycin selection marker. Mutations were introduced using a QuikChange kit (Agilent, Santa Clara, CA).

[0342] Expression of AA and doubly masked BAA (BAA) AAs and BAAs were expressed in mammalian cells using a standard transfection kit (Life Technologies, Grand Island, NY). Briefly, 293 cells were transfected with nucleic acids using a lipid-based system according to the manufacturer's recommended protocol. AAs and doubly masked BAAs were purified from cell-free supernatants using Protein A beads (GE, Piscataway, NJ) and concentrated using a standard buffer exchange column (Millipore, Temecula, CA).

[0343] Example 2. Binding of doubly masked bispecific AA to EGFR+ HT-29 cells and CD3ε+ Jurkat cells To determine whether the described EGFR- and CD3ε-masking peptides and protease substrates could inhibit binding in the dually masked bispecific AA, flow cytometry-based binding assays were performed.

[0344] HT-29-luc2 cells (Caliper) and Jurkat cells (Clone E6-1, ATCC, TIB-152) were cultured in RPMI-1640 + glutamax (Life Technologies, catalog no. 72400-047), 10% heat-inactivated fetal bovine serum (HI-FBS, Life Technologies, catalog no. 10438-026), 100 U / ml penicillin, and 100 μg / ml streptomycin (Life Technologies, catalog no. 15140-122) according to the manufacturer's guidelines. The following bispecific activating antibodies were tested: CI048 and CI104 (act-104) and the doubly masked bispecific AAs, CI011, CI106, and CI107. Two versions of SP34 scFv were utilized (i.e., scFv in CI011 and CI048 vs. scFv in CI104, CI106, and CI107). Two versions of EGFR masks were utilized (i.e., EGFR mask in CI011 and CI107 vs. EGFR mask in CI106). Two versions of CD3 masks were utilized (i.e., CD3 mask in CI011 vs. CD3 mask in CI106 and CI107).

[0345] HT29-luc2 cells were detached with Versene™ (Life Technologies, Catalog No. 15040-066), washed, plated at 150,000 cells / well in a 96-well plate, and resuspended in 50 μL of primary antibody. Titration began at the concentrations shown in Figures 1A-1B, followed by 3-fold serial dilutions in FACS stain buffer + 2% FBS (BD Pharmingen, Catalog No. 554656). Cells were incubated with shaking at 4°C for approximately 1 hour, harvested, and washed with 2 x 200 μL of FACS stain buffer. Cells were resuspended in 50 μL of Alexa Fluor 647-conjugated anti-human IgG Fc (10 μg / ml, Jackson ImmunoResearch, Product No. 109-606-008) and incubated with shaking at 4°C for approximately 1 hour. HT29-luc2 cells were harvested, washed, and resuspended in a final volume of 60 μL of FACS staining buffer containing 2.5 μg / ml 7-AAD (BD Biosciences, catalog number 559925). Cells stained with secondary antibody alone were used as a negative control. Data were acquired using a MACSQuant® Analyzer 10 (Miltenyi), and median fluorescence intensity (MFI) of viable cells was calculated using FlowJo® V10 (Treestar). Background-subtracted MFI data were graphed using curve-fit analysis in GraphPad Prism 6.

[0346] Jurkats grown in suspension were harvested, washed, plated at 150,000 cells / well in 96-well plates, and resuspended in 50 μl of primary antibody. Staining and data acquisition were performed as described above for HT29-luc2 cells.

[0347] Figure 1A demonstrates that incorporation of the h20GG CD3ε masking peptide into EGFR-masked BAAs CI106 and CI107 significantly reduces binding to Jurkat cells compared to CI011. In some embodiments, binding to Jurkat cells was reduced by more than 5,000-fold. In some embodiments, the disclosed scFvs also reduced binding. Reduced binding to EGFR+ HT29-luc2 cells was also evident for CI106 and CI107 compared to CI011 (Figure 1B). In some embodiments, binding to EGFR+ HT29-luc2 cells was reduced by more than 1,000-fold. In Figures 1A and 1B, the doubly masked BAA reduces binding compared to the activating bispecific antibody.

[0348] Example 3. EGFR-dependent cytotoxicity of doubly masked BAAs Cytotoxicity assays were performed to determine whether the CD3ε and EGFR masks and protease substrates in CI106 and CI107 could further attenuate cell killing compared to CI011 and CI040. Human PBMCs were purchased in frozen aliquots (HemaCare) and cocultured with EGFR-expressing HT29-luc2 cells at a 10:1 ratio in RPMI-1640 + glutamax (supplemented with 5% heat-inactivated human serum (Sigma, catalog no. H3667)). The following doses of bispecific activating antibodies and doubly masked BAAs were tested: CI011, CI040, activating CI104, CI106, and CI107. Furthermore, the non-EGFR-binding masked bispecific AAs, CI127 and CI128, were used to demonstrate EGFR-dependence of cytotoxicity. After 48 hours, cytotoxicity was assessed using the ONE-Glo™ Luciferase Assay System (Promega, Cat. No. E6130). Luminescence was measured on an Infinite M200 Pro (Tecan). Cytotoxicity rates were calculated and plotted using curve-fit analysis in GraphPad PRISM. EGFR receptor counts in the cell line panel were quantified by flow cytometry using QIFIKIT (Dako).

[0349] Figure 2A demonstrates that killing of EGFR+ HT29-luc2 cells was further attenuated by CI106 and CI107 compared to CI011 and CI040. Figure 2B shows that no cytotoxicity was observed when cells were treated with CI127 and CI128, demonstrating that cell killing is dependent on EGFR targeting. Furthermore, Figure 2B demonstrates a >300,000-fold EC50 shift for the doubly masked bispecific antibodies CI106 and CI107 compared to the protease-activated bispecific antibody act-104. Figure 2C shows the number of EGFR receptors on a panel of cell lines, including HT29. The approximate number of EGFR receptors on HT29 cells is 75,000, indicating that a high density of antigen is not required for the potent cytotoxicity of the tested antibodies.

[0350] Example 4. Primary T cell activation with doubly masked BAA Flow cytometry assays were performed to determine whether masking of CD3ε and EGFR in CI106 and CI107 could attenuate primary T cell activation compared to CI011 and CI040. Human PBMCs and U266 cells were co-cultured according to the conditions described in Example 3. After 48 hours of incubation, cells were pelleted, the medium was removed, and the cells were resuspended in 50 μl of a cocktail containing anti-CD45 VioBlue® (Miltenyi, catalog number 130-002-880), anti-CD8 APC-Vio770 (Miltenyi, catalog number 130-096-561), and anti-CD69 PE (BD Pharmingen, catalog number 555531) in FACS staining buffer + 2% FBS. Cells were stained for 1 hour at 4°C with shaking, collected, washed, and resuspended in a final volume of 60 μL of FACS buffer. Data were acquired on a MACSQuant® Analyzer 10 (Miltenyi) and activation was quantified in FlowJo® V10 (Treestar) as the percentage of CD8+ T cells expressing CD69 relative to the PE isotype control. Data were plotted using curve-fit analysis in GraphPad PRISM6.

[0351] Figure 3A demonstrates that activation of primary CD8+ T cells was attenuated by CI106 and CI107 compared to CI011 and CI040. Figure 3B shows a shift in the dose-response curve for T cell activation with doubly masked antibodies compared to the protease-activated bispecific antibody act-104, demonstrating that masking weakens T cell activation.

[0352] Example 5. Doubly masked bispecific AA of the embodiment induced regression of established HT29-luc2 tumors in mice In this example, CI106 and CI107, dual-masked BAAs targeting EGFR and CD3ε, were analyzed for their ability to induce regression or reduction in growth of established HT-29-Luc2 xenograft tumors in human T cell-engrafted NSG mice.

[0353] The human colon cancer cell line HT29-luc2 was obtained from Perkin Elmer, Inc., Waltham, MA (formerly Caliper Life Sciences, Inc.) and cultured according to established procedures. Purified frozen human PBMCs were obtained from Hemacare, Inc., Van Nuys, CA. NSG™ (NOD.Cg-Prkdcscid Il2rg) tm1Wjl / SzJ) mice were obtained from The Jackson Laboratories, Bar Harbor, ME.

[0354] On day 0, each mouse received 2 x 10 10 cells in 100 μL RPMI + Glutamax (serum-free medium) in the right flank. 6 HT29-luc2 cells were inoculated subcutaneously. Pre-frozen PBMCs from a single donor were administered (ip) at a CD3+ T cell:tumor cell ratio of 1:1 on day 3. Tumor volumes were 200 mm 3 When tumor volume reached 100 mg / kg (approximately day 12), mice were randomized and assigned to treatment and iv dosing groups according to Table 12. Tumor volumes and body weights were measured twice weekly.

[0355] [Table 16]

[0356] Figure 4 plots tumor volume versus days after first treatment dose and demonstrates the dose-dependent effect of the doubly masked bispecific AAs CI106 and CI107 on HT29-luc2 xenograft tumor growth. The most effective dose tested was 1.5 mg / kg, which resulted in tumor regression. Statistical analysis (RMANOVA with Dunnett's algorithm versus PBS control) was performed in GraphPad PRISM. * =p<0.05, ** =p<0.01, **** =p<0.0001.

[0357] Example 6. Doubly masked bispecific AA and bispecific antibodies of the present invention reduce the growth of established HCT116 tumors in mice In this example, the activating CI104, a bispecific antibody targeting EGFR and CD3ε, and the doubly masked BAAs CI106 and CI107 were analyzed for their ability to induce regression or reduction in the growth of established HCT116 xenograft tumors in human T cell-engrafted NSG mice. The human colon cancer cell line HCT116 was obtained from ATCC and cultured in RPMI + Glutamax + 10% FBS according to established procedures. The tumor model was performed as described in Example 5. Mice were dosed according to Table 13.

[0358] [Table 17]

[0359] Figure 5 plots tumor volume versus days after first treatment dose and demonstrates the dose-dependent effect of the doubly masked bispecific AAs, CI106 and CI107, on HCT116 xenograft tumor growth. The most effective dose tested was 1.0 mg / kg, which resulted in tumor stasis. Act-104, dosed at 0.3 mg / kg, also resulted in tumor stasis, demonstrating a three-fold difference in efficacy between the doubly masked bispecific antibody and the protease-activated bispecific antibody. Statistical analysis (RMANOVA with Dunnett's algorithm versus the PBS control) was performed in GraphPad PRISM. * =p<0.05, ** =p<0.01, **** =p<0.0001.

[0360] Example 7. Cross-reactivity of doubly masked bispecific AA to cynomolgus monkey T cells To confirm that cynomolgus monkeys are a relevant virulent species, protease-activated CI104, CI106, and CI107 were used in a flow cytometry-based cell binding assay and an HT29-luc2 cytotoxicity assay using cynomolgus monkey pan-T cells (Bioreclamation IVT), and efficacy was compared to human PBMCs. Protocols were as described in Examples 2 and 3.

[0361] Figures 6A and 6B demonstrate that the EC50s of the doubly masked and protease-activated bispecific antibodies tested in cytotoxicity assays are similar when using either human (6A) or cynomolgus (6B) effector cells. Figures 6C and 6D demonstrate that the binding of the protease-activated and doubly masked antibodies to human (6C) and cynomolgus (6D) T cells is similar.

[0362] Therefore, cynomolgus monkeys were determined to be a suitable species for tolerability studies.

[0363] Example 8. Mutations in the Fc region affect the tolerability of a doubly masked bispecific AA in cynomolgus monkeys In this example, naive cynomolgus monkeys (n=1) were dosed with 600 μg / kg of CI079 and CI090 to assess tolerability. The starting dose of 600 μg / kg was selected based on the previously established MTD of CI011. The monkeys were native to China and weighed 2.5–4 kg. Each study animal was monitored for a minimum of 7 days. Tolerability was assessed based on clinical signs, body weight, and food consumption. This study was performed according to standard operating procedures at SNBL USA, Ltd. (Everett, WA).

[0364] Table 14 describes the clinical findings after administration of CI079 and CI090 (Table 15), doubly masked bispecific AAs (BAAs) that differ only in the Fc region. CI079 contains the Fc mutations L234F, L235E, and P331S. CI090 contains the Fc mutation and the N297Q mutation. No clinical findings were observed after administration of 600 μg / kg of CI090, whereas vomiting was observed in the first 24 hours after administration of CI079, demonstrating that the mutations in the Fc region contribute to the tolerability of these molecules.

[0365] [Table 18]

[0366] [Table 19]

[0367] Example 9. Tolerability of doubly masked BAAs in cynomolgus monkeys In this example, naive cynomolgus monkeys (n=1) were dosed with CI106 and CI107 at 600, 2000, 4000 μg / kg (CI107 only), or 6000 μg / kg (CI107 only) to establish the maximum tolerated dose (MTD) after a single IV bolus administration. The starting dose of 600 μg / kg was selected based on the previously established MTD for CI011. The monkeys were native to China and weighed 2.5–4 kg. Each study animal was monitored for a minimum of 7 days. Tolerability was assessed based on clinical signs, body weight, food consumption, and laboratory analyses, including serum chemistry, hematology, cytokine analysis, and flow cytometry to assess T cell activation. Blood was collected once during acclimation and at pre-dose, 48 h, 72 h (hematology only), and 7 days post-dose for standard serum chemistry and hematology analyses. Blood was collected for cytokine analysis before dosing and 1, 4, 8, and 24 hours after dosing. Flow cytometry was performed on peripheral blood before dosing, 72 hours after dosing, and 7 days after dosing. This study was performed according to standard operating procedures at SNBL USA, Ltd. (Everett, WA).

[0368] CI107 administered at 6000 μg / kg resulted in lethality within 24 hours after dosing. In other groups, abnormal clinical signs (including vomiting and decreased food intake) were observed in cynomolgus monkeys treated with CI106 and CI107 at doses of 2000 μg / kg and above. These findings, when observed, were transient and generally limited to a period of 48 hours after dosing. Serum chemistry findings at these doses included mild elevations of alanine transaminase (ALT) and aspartate aminotransferase (AST) at 48 hours, but these did not exceed the normal range. In CI107-treated animals at 2000 and 4000 μg / kg, total bilirubin increased above the normal range at 48 hours and completely reversed by day 8. Both CI106- and CI107-treated animals showed transient increases in serum cytokines IL-2, IL-6, and IFNg after dosing, which recovered by 24 hours after dosing. Increases in the percentage of T cells expressing CD69, Ki67, and PD-1 were observed 72 hours after dosing, with the percentage of positive cells generally being higher in CI107-treated animals.

[0369] Figures 7A-7C show serum concentrations of ALT (7A), AST (7B), and total bilirubin (7C) pre-dose, 48 hours post-dose, and 7 days post-dose. With the exception of total bilirubin at 2000 μg / kg and 4000 μg / kg, all values ​​are within the established normal range for cynomolgus monkeys. Because only pre-dose data were available for CI107 at 6000 μg / kg, this data was not included.

[0370] Figures 8A-8C plot the increase in serum cytokine levels for IL-2 (8A), IL-6 (8B), and IFN-g (8C).

[0371] 9A-9C show T cell activation as measured by expression of CD69 (9A), Ki67 (9B), and PD-1 (9B) on CD4+ T cells.

[0372] Example 10. Doubly masked bispecific AA is safer than activated bispecific antibodies in cynomolgus monkeys. In this example, protease-activated CI104 and doubly masked CI106 and CI107 were administered to cynomolgus monkeys (n=1) at 60, 180 (activated CI104 only), 600, 2000, 4000 μg / kg (CI107 only), or 6000 μg / kg (CI107 only) to compare the tolerability of masked and unmasked antibodies after a single IV bolus. Tolerance assessments and blood sampling were as described in Example 9. The doubly masked BAAs, CI106 and CI107, were tolerated at dose levels 30-60 times higher than the protease-activated bispecific antibody.

[0373] Figures 10A-E plot the dose-dependent increase in AST at 48 hours post-dose (10A), ALT at 48 hours post-dose (10B), IL-6 at 8 hours post-dose (10C), IFNg at 8 hours post-dose (10D), and Ki67 at 72 hours post-dose (10E). The dose-response curves for all parameters were shifted for the doubly masked antibody, indicating improved tolerability and reduced pharmacodynamic effects compared to the protease-activated bispecific antibody. In some embodiments, the IL-6 dose-response curve was shifted by more than 60-fold.

[0374] Example 11. Tolerability of doubly masked bispecific AAs that bind EGFR is EGFR dependent. In this example, cynomolgus monkeys (n=1) were dosed at 2000 μg / kg with the doubly masked bispecific antibodies CI107 (targeting EGFR and CD3ε) and CI128 (targeting RSV and CD3ε). Tolerability assessments and blood sampling were as described in Example 9 above. There was no effect of CI128 on measures of acute organ toxicity (total bilirubin) and T cell activation (IL-6, PD-1), demonstrating that the toxicity observed in cynomolgus monkeys is dependent on EGFR binding. These data also demonstrate that CD3ε binding alone is not sufficient to induce toxicity.

[0375] 11A-11C compare the effects of EGFR-binding CI107 and non-EGFR-binding CI128 on the increase in total bilirubin (11A), IL-6 (11B), and CD4+ T cells expressing PD-1 (11C).

[0376] Example 12. Humanization of anti-CD3 variants v12, v16, and v19 with different affinities and potencies. This example describes anti-CD3 antibody variants v12, v16, and v19. These three variants were derived from the parent antibody hSP34.

[0377] Anti-human CD3 single-chain variable fragments (scFvs) were humanized by selective mutation of the framework. Briefly, CDRs were grafted onto a series of light chain (LC) and heavy chain (HC) human IgG frameworks, and several amino acids in the variable region framework were selectively mutated. Immunoglobulins were expressed with all possible combinations of LC and HC, and then evaluated for expression level, monomericity, and CD3 affinity using ELISA and on-cell binding to Jurkat cells. Desired combinations of variable regions were expressed as scFvs in a bispecific antibody (TCB) format and then evaluated for expression level, monomericity, CD3 affinity, and function in cytotoxicity assays.

[0378] The affinity of the v12, v16, and v19 variants was measured using surface plasmon resonance (SPR). The surface was HC200m (a linear synthetic polycarboxylate-based carboxylated hydrogel). The surface channels were activated using a standard EDC / NHS amine coupling protocol. Channels 1 and 2 were blank, and channels 3 and 4 contained various anti-human CD3 antibodies. The surfaces were created by diluting v12, v16, v19, and MM194 antibodies to 5 μg / ml in 1.0 mL of 10 mM sodium acetate (pH 4.5).

[0379] Kinetic analysis was performed in PBST (10 mM sodium phosphate (pH 7.4), 150 mM sodium chloride, 0.05% Tween-20) at 20° C. Regeneration was a series of three injections: a single injection of 5 μL of 20 mM sodium hydroxide followed by two injections of 5 μL of freshly made 10 mM sodium hydroxide.

[0380] The preparation was performed with a buffer blank and alternating reverse 3-fold serial dilutions. Human CD3egFc was obtained from Sino Biological Inc. (Beijing, China, Catalog No. CT041-H0305H) and reconstituted with sterile water from a lyophilized formulation based on PBS and stabilizers. Serial dilutions of the analyte in solution started at a concentration of 300 nM or 100 nM human CD3. Processing was performed using Scrubber software.

[0381] These variants were also engineered into doubly masked bispecific AAs targeting EGFR and CD3 using the methods described and used in in vitro cytotoxicity assays as described in Example 3.

[0382] Figure 12A shows affinity measurements of v12, v16, and v19 compared to hSP34. V12 showed the highest affinity at 12 nM, while v16 showed the lowest affinity at 70 nM. Figure 12B shows the cytotoxicity of activated or doubly masked bispecific antibodies against HT29-luc2 cells. There was little difference in the cell killing potency of the activated molecules, with v16 being the most potent. There was also little difference in protection against cell killing for the doubly masked molecules.

[0383] Example 13. Doubly masked BAAs can extend PK in cynomolgus monkeys.

[0384] In this example, cynomolgus monkeys were dosed with the protease-activated bispecific antibody act-104 and the doubly masked bispecific antibody CI107 at 60 μg / kg, 180 μg / kg (act-104), or 2000 μg / kg (CI107). Plasma samples were collected at 5 min (act-104 only), 30 min, 4 h (act-104 only), 24 h, 48 h (act-104 only), 96 h, and 168 h. Plasma concentrations were measured by ELISA using an anti-idiotypic antibody for capture and horseradish peroxidase (HRP)-conjugated anti-human IgG (Fc) for detection, and visualized using 3,3',5,5'-tetramethylbenzidine (TMB). Plasma concentration values ​​were interpolated from the calibration curve and plotted using GraphPad PRISM. Area under the curve (AUC) analysis was also performed.

[0385] Figure 13 shows the expanded PK of the doubly masked molecule CI107 compared to the protease-activated molecule act-104. The exposure (AUC) of CI107 was 448 days. * nM, and act-104 (60 μg / kg) was 0.04 days * nM, representing a >10,000-fold difference in plasma exposure.

[0386] Example 14. Protease cleavage susceptibility of doubly masked bispecific AA correlates with tumor efficacy and tumor T cell infiltration. This example describes antitumor efficacy and tumor T cell infiltration in an HT29-luc2 xenograft model. This model was performed as described in Example 5. For tumor T cell infiltration studies, mice received a single dose of test article, and tumors were harvested 7 days after dosing. Formalin-fixed, paraffin-embedded (FFPE) blocks were prepared and used for histology. The test articles used were CI011, CI020 (a doubly masked bispecific antibody lacking a cleavable substrate), CI040, and CI048. The protease sensitivity and substrate cleavability of the test articles are as follows: CI040 > CI011 > CI020. Mice were dosed according to Table 16.

[0387] [Table 20]

[0388] Figure 14A shows efficacy in the HT29-luc2 tumor intervention model in PBMC-engrafted NSG mice. Antitumor efficacy in this example correlates with the protease sensitivity and substrate cleavability of the test agent, with the most effective test agent being fully protease-activated CI048.

[0389] Figure 14B shows staining of tumor sections for CD3 (dark staining) as a measure of T cell infiltration into the tumor. Tumor T cell infiltration correlates with the protease sensitivity and substrate cleavage of the test article.

[0390] Example 15. Second generation doubly masked bispecific AA are safer in cynomolgus monkeys than first generation molecules. In this example, cynomolgus monkey tolerability data were compared for CI011, CI040, CI048 (first-generation molecules), act-104, CI106, and CI107 (second-generation molecules). The data presented in this example were compiled from two cynomolgus monkey tolerability studies. Protease-activated CI104 and CI048 were administered to cynomolgus monkeys at 20 (CI048 only), 60, or 180 μg / kg (act-104 only). Doubly masked CI011, CI040, CI106, and CI107 were administered at 600, 2000, 4000 (CI107 only), or 6000 (CI107 only) μg / kg to compare the tolerability of single IV bolus doubly masked and activated bispecific antibodies. Tolerability assessment was as described in Example 8.

[0391] Table 17 summarizes the clinical findings after a single dose of the test article. The second-generation protease-activated bispecific antibody act-104 was tolerated at doses two-fold higher than the first-generation protease-activated bispecific antibody CI048. CI106 and CI107 were tolerated at doses 30-60 times higher than the first-generation antibodies CI011 and CI040.

[0392] [Table 21]

[0393] Example 16. Evaluation of masking efficacy of activatable anti-EGFR antibodies Masking the ability of an antibody to bind to its antigen is an example of inhibition of binding and is listed herein as masking efficiency (ME). Masking efficiency is calculated as the K of AA binding. D K of antibody binding measured under the same conditions DThe extent of inhibition depends on the affinity of the antibody for its antigen, the affinity of the inhibitor (i.e., masking moiety) for the antibody, and the concentration of all reactants. The local concentration of the tethered masking moiety peptide (inhibitor) is very high (approximately 10 mM) in AA, so a peptide of moderate affinity will effectively mask AA antigen binding.

[0394] The assay is outlined as follows: Nunc Maxisorp™ plates are coated overnight at 4° C. with 100 μl / well of a 1 μg / ml solution of human EGFR (R and D Systems) in PBS, pH 7.4. The plates are washed with 3×PBST (PBS, pH 7.4, 0.05% Tween-20) and the wells are blocked with 200 μl / well of 10 mg / mL BSA in PBST for 2 hours at RT. The plates are washed with 3×PBST (PBS, pH 7.4, 0.05% Tween-20). A dilution curve can be generated with 10 mg / mL BSA in PBST, as illustrated in Table 18 below. In this example, the highest concentrations are 10 nM for the parent antibody and 400 nM for the AA, although the higher concentrations can be increased or decreased to give fully saturated binding curves for more or less masked AAs.

[0395] [Table 22]

[0396] The binding solution is added to the plate, which is then incubated at room temperature for 1 hour and then washed with 3x PBST (PBS, pH 7.4, 0.05% Tween-20). 100 μl / well of goat-anti-human IgG (Fab specific, Sigma catalog no. A0293) diluted 1:4000 in 10 mg / mL BSA in PBST is added, and the plate is incubated at room temperature for 1 hour. The plate is developed with TMB and 1N HCl. Shown in Figures 15 and 16 are plots of binding isotherms for the activatable anti-EGFR C225v5 antibody of the present disclosure, the activatable anti-EGFR antibody 3954-2001-C225v5 described above, and the anti-EGFR antibody C225v5. Plots were generated in GraphPad PRISM, and the data was fitted to a single-site saturation model, K D Determine K D The values ​​and ME values ​​are provided in Table 19.

[0397] [Table 23]

[0398] Example 17. Pharmacokinetics of doubly masked BAAs in cynomolgus monkeys. In this example, cynomolgus monkeys were dosed with the doubly masked bispecific antibody CI107 at 600 μg / kg, 2000 μg / kg, or 4000 μg / kg. Plasma samples were collected at 30 minutes, 4 hours (600 μg / kg only), 24 hours, 48 ​​hours (600 and 4000 μg / kg only), 96 hours, and 168 hours. Plasma concentrations were measured by ELISA as in Example 13.

[0399] FIG. 20 shows the PK of the doubly masked BAA CI107 following administration of a single iv dose of either 600, 2000, or 4000 μg / kg.

[0400] Example 18. EGFR-dependent cytotoxicity of doubly masked bispecific activatable antibodies To determine whether the anti-CD3ε, CD3 mask, and protease substrate in CI090 and CI091 could render cell killing more vulnerable compared to CI011, cytotoxicity assays were performed using the methods described in Example 3. The following bispecific activating and doubly masked bispecific activatable antibodies were tested at dose levels: CI011, CI090, CI091, activating CI090, and CI048. Additionally, the non-EGFR-binding bispecific activatable antibody CI064 was used to demonstrate EGFR-dependence of cytotoxicity.

[0401] Figure 21 demonstrates that killing of EGFR+ HT29-luc2 cells was further compromised by CI090 and CI091 compared to CI011, but the potency of activating CI090 was equivalent to CI048. There was an increased shift in the EC50 for CI090 and CI091 compared to the activating bispecific antibody, indicating increased masking efficiency of these molecules compared to CI011. No cytotoxicity was observed when cells were treated with CI064, demonstrating that cell killing is dependent on EGFR targeting.

[0402] Example 19. Primary T cell activation with doubly masked bispecific activatable antibodies To determine whether the anti-CD3ε, CD3 mask, and protease substrate in CI090 and CI091 can compromise primary T cell activation compared to CI011, flow cytometry assays were performed as described in Example 4.

[0403] FIG. 22 demonstrates that activation of primary CD8+ T cells by CI090 and CI091 was further attenuated compared to CI011.

[0404] Example 20. Doubly masked bispecific activatable antibodies of the embodiments induced regression of established HT29-luc2 tumors in mice In this example, the bispecific activatable antibodies CI011, CI090, and CI091 were analyzed for their ability to induce regression or reduction in growth of established HT-29-Luc2 xenograft tumors in human PBMC-engrafted NSG mice. Methods are as described in Example 5.

[0405] [Table 24]

[0406] Figure 23 plots tumor volume versus days after first treatment dose and demonstrates that a weekly dose of 1 mg / kg induced tumor regression for all bispecific activatable antibodies tested.

[0407] Example 21. Doubly masked bispecific activatable antibodies induce less cytokine release in cynomolgus monkeys than activating bispecific antibodies. In this example, protease-activated CI104 and double-masked CI011, CI090, and CI091 were administered to cynomolgus monkeys (n=1) at 0.06, 0.18 (activated CI104), or 600 mg / kg (CI011, CI090, CI091). Blood was collected for cytokine analysis before dosing and 1, 4, 8, and 24 hours after dosing. Samples were analyzed using a Life Technologies Monkey Magnetic 29-Plex Panel Kit (product number LCP0005M). Data were acquired using a BioRad BioPlex 200 instrument. The analysis was performed according to standard operating procedures at SNBL USA, Ltd. (Everett, WA).

[0408] Figure 24 plots IL-6 levels 8 hours after dosing. The doubly masked bispecific activatable antibody CI011 induced significantly lower cytokine release than activating CI104, even when delivered at higher doses, demonstrating a masking effect on T cell activation. IL-6 was even more reduced in CI090- and CI091-treated animals, reflecting increased masking efficiency of these molecules compared to CI011. Other embodiments

[0409] While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is limited by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following.

Claims

1. A purified polypeptide comprising the amino acid sequence of SEQ ID NO:

12.

2. 10. The purified polypeptide of claim 1, further comprising a polypeptide that is a substrate for a protease.

3. The purified polypeptide of claim 2 , wherein the substrate is cleavable by a serine protease or a matrix metalloprotease (MMP).

4. The purified polypeptide of claim 3 , wherein the protease is an MMP.

5. The purified polypeptide of claim 3 , wherein the protease is a serine protease.

6. 6. The purified polypeptide of claim 5, wherein the serine protease is matriptase or urokinase-type plasminogen activator (uPA).

7. 3. The purified polypeptide of claim 2, comprising a linker between SEQ ID NO: 12 and the polypeptide that is a substrate for a protease.

8. 3. The purified polypeptide of claim 2, wherein SEQ ID NO: 12 and the polypeptide that is a substrate for a protease are not separated by a linker.

9. 3. The purified polypeptide of claim 2, wherein the polypeptide comprises a linker at the carboxyl terminus of the polypeptide that is a substrate for a protease.

10. The polypeptide is, in either an N-terminal to C-terminal direction or a C-terminal to N-terminal direction, i) (SEQ ID NO: 12)-L1-(CM); ii) (SEQ ID NO: 12)-(polypeptide that is a substrate for a protease)-L2; iii) (SEQ ID NO: 12)-L1-(polypeptide that is a substrate for a protease)-L2; and iv) (SEQ ID NO: 12) - (a polypeptide that is a substrate for a protease); and comprising a formula selected from the group consisting of: The purified polypeptide of claim 2, wherein L1 and L2 are linkers.

11. 11. The purified polypeptide of claim 10, wherein L1 and L2 are identical.

12. 11. The purified polypeptide of claim 10, wherein L1 and L2 are different.

Images