Antibodies, activatable antibodies, bispecific antibodies, and bispecific activatable antibodies and methods for using the same.

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 of related applications This application claims the benefits 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 entire contents of each are incorporated herein by reference). Field of Invention

[0002] Antibodies, activatable antibodies (AA), bispecific antibodies, and bispecific activatable antibodies (BAA) are provided herein. Methods for producing and using these antibodies, AA, bispecific antibodies, and BAA are also provided herein.

[0003] Sequence listing reference The "Sequence Listing," submitted electronically via EFS-Web in computer-readable format (CRF) at the same time as this specification, with the filename CYTX-045-US_SEQLIST_10-12-18_ST25.txt in accordance with 37 §1.821 of the Federal Code of Regulations, is incorporated herein by reference. An electronic copy of the Sequence Listing was created on December 10, 2018, and its size on disk is 440 kilobytes. [Background technology]

[0004] background While antibody-based therapies have proven effective for some diseases, their efficacy is limited in some cases due to toxicity resulting from the broad range of targets they express. Furthermore, antibody-based therapies have other limitations, such as being rapidly cleared from the circulation after administration. [Overview of the Initiative] [Means for solving the problem]

[0005] In the field of small molecule therapeutics, strategies are being developed to provide prodrugs of active chemicals. These prodrugs are administered in a relatively inactive (or significantly less active) form. Upon administration, the prodrug is metabolized in vivo to an active compound. Such prodrug strategies can increase the options for drugs aimed at targeting specific effects and reducing adverse effects.

[0006] Therefore, antibodies that mimic the desirable characteristics of small molecule prodrugs are still needed in the field of antibody-based therapeutics. Summary of the Invention

[0007] Antibodies, bispecific antibodies, activatable antibodies, and bispecific activatable antibodies, methods for producing them, and methods for using them are provided herein. These have applications in therapeutic and diagnostic agents. Using the activatable antibodies and bispecific activatable antibodies of this disclosure, it is possible to reduce damage to healthy tissue, which is generally caused by antibodies binding to their targets not only on diseased tissue but also on healthy tissue.

[0008] Therefore, according to one embodiment, a bispecific activatable antibody (BAA) is provided herein, wherein the BAA, when activated, specifically binds to two targets and has the following structure: a) An IgG antibody (AB1) that specifically binds to the first target, wherein the aforementioned AB1 is a. Two heavy chains (AB1 HC) and two light chains (AB1 LC); and b. A first masking portion (MM1) that is connected to the first cleavable portion (CM1) to form an MM1-CM1 construct, wherein the carboxyl terminus of the aforementioned MM1-CM1 construct is connected to the respective amino terminus of each light chain of the aforementioned AB1. Including, here, c. The aforementioned MM1 inhibits the binding of the aforementioned AB1 to its target; d. The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, IgG antibody (AB1), b) Two scFv(AB2) each specifically bind to a second target, where each AB2 is a. A light chain variable region linked to a heavy chain variable region, wherein the carboxyl terminus of each AB2 mentioned above is linked to the respective amino terminus of the AB1 heavy chain; and b. A second masking portion (MM2) connected to a second cleavable portion (CM2) to form an MM2-CM2 structure, wherein the carboxyl terminus of the aforementioned MM2-CM2 structure is connected to the amino terminus of each of the aforementioned AB2 portions of the second masking portion (MM2). Including, here, c. The aforementioned MM2 inhibits the binding of the aforementioned AB2 to its target; d. The aforementioned CM2 is a polypeptide that functions as a substrate for the second protease, consisting of two scFv(AB2) polypeptides. Includes, c) Here, the aforementioned BAA has the following characteristics: a.MM2 contains the amino acid sequence of SEQ ID NO: 12; b.MM1 contains an amino acid sequence selected from the group of sequences shown in Table 7; c.AB2 contains a heavy chain variable domain described in SEQ ID NO: 2 or SEQ ID NO: 3 or a light chain variable domain described in SEQ ID NO: 1 or SEQ ID NO: 4; and d.AB1 includes an Fc region containing an amino acid substitution at at least one of the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, so as to reduce the effector function of the aforementioned BAA. It has at least one of the following.

[0009] In some embodiments, the BAA provided herein is A bispecific activatable antibody (BAA), wherein the 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 the aforementioned AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein the aforementioned AB1 is linked to a first masking portion (MM1) linked to a first cleavable portion (CM1) to form an MM1-CM1 construct, wherein the carboxyl terminus of the aforementioned MM1-CM1 construct is linked to the amino terminus of each light chain of the aforementioned AB1, wherein the aforementioned MM1 inhibits the binding of the aforementioned AB1 to its target; the aforementioned CM1 is a polypeptide that functions as a substrate for a first protease, IgG antibody (AB1), b. Two scFvs (each AB2) that each specifically bind 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 the aforementioned AB2s is linked to the amino terminus of each of the aforementioned AB1 heavy chains; wherein each AB2 is linked to a second masking portion (MM2) linked to a second cleavable portion (CM2) to form an MM2-CM2 construct, wherein the carboxyl terminus of each of the aforementioned MM2-CM2 constructs is linked to the amino terminus of each of the aforementioned AB2s, wherein the aforementioned MM2 inhibits the binding of the aforementioned AB2 to its target; the aforementioned CM2 is a polypeptide that functions as a substrate for a second protease, two scFvs (each AB2) comprising, wherein 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 shown in Table 7; iii. AB2 comprises the heavy chain variable domain described in SEQ ID NO: 2 or SEQ ID NO: 3 or the light chain variable domain described in SEQ ID NO: 1 or SEQ ID NO: 4; and iv. AB1 comprises an Fc region with an amino acid substitution at at least one of positions L234, L235, N297, and P331 of the amino acids numbered according to the EU index described in Kabat such that the effector function of the aforementioned BAA is reduced comprising a bispecific activatable antibody (BAA) having at least one of

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

[0011] In another embodiment, a bispecific activatable antibody (BAA) is, a) An IgG antibody (AB1) that specifically binds to the first target, wherein the aforementioned AB1 is i. Two heavy chains (AB1 HC) and two light chains (AB1 LC); and ii. A first masking portion (MM1) that is linked to the first cleavable portion (CM1) to form an MM1-CM1 construct, wherein the carboxyl terminus of the aforementioned MM1-CM1 construct is linked to the amino terminus of each light chain of the aforementioned AB1, and here, The aforementioned MM1 inhibits the binding of the aforementioned AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, the first masking moiety (MM1). Includes IgG antibody (AB1), b) Two scFv(AB2) each specifically bind to a second target, where each AB2 is i. A heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 mentioned above is linked to the respective amino terminus of the AB1 heavy chain; and ii. A second masking portion (MM2) connected to the second cleavable portion (CM2) to form an MM2-CM2 structure, wherein the carboxyl terminus of the aforementioned MM2-CM2 structure is connected to the amino terminus of each of the aforementioned AB2, The aforementioned MM2 inhibits the binding of the aforementioned AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for the second protease, the second masking moiety (MM2). Two scFv(AB2) including Includes, Herein, AB1 provides herein a bispecific activatable antibody (BAA) comprising an Fc region containing an amino acid substitution at at least one of the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, such that the effector function of the BAA is reduced. In some embodiments, the Fc region contains an amino acid substitution at at least the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, such that the effector function of the BAA is reduced. In some embodiments, the Fc region contains an amino acid substitution at at least the amino acid positions L234, L235, and P331, numbered by the EU index described in Kabat, such that the effector function of the BAA is reduced. In some embodiments, the first target is selected from the group of targets shown in Table 9, and the second target is selected from the group of targets shown in Table 9.

[0012] In some embodiments, the BAA provided herein is a) A bispecific activatable antibody (BAA), i) an IgG antibody (AB1) that specifically binds to a first target, wherein the aforementioned AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein the aforementioned AB1 is linked to a first masking portion (MM1) linked to a first cleavable portion (CM1) to form an MM1-CM1 construct, wherein the carboxyl terminus of the aforementioned MM1-CM1 construct is linked to the amino terminus of each light chain of the aforementioned AB1, The aforementioned MM1 inhibits the binding of the aforementioned AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, IgG antibody (AB1), ii) Two scFv (each AB2) each specifically bind to a second target, wherein each AB2 includes a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 is linked to the respective amino terminus of the aforementioned AB1 heavy chain; wherein each AB2 is linked to a second masking region (MM2) linked to a second cleavable region (CM2) to form an MM2-CM2 construct, wherein the carboxyl terminus of each MM2-CM2 construct is linked to the amino terminus of each AB2, The aforementioned MM2 inhibits the binding of the aforementioned AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for the second protease, consisting of two scFv Includes, Here, AB1 comprises a bispecific activatable antibody (BAA) comprising an Fc region containing an amino acid substitution at at least one of the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, such that the effector function of the BAA is reduced. In some embodiments, the Fc region contains an amino acid substitution at at least the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, such that the effector function of the BAA is reduced. In some embodiments, the Fc region contains an amino acid substitution at at least the amino acid positions L234, L235, and P331, numbered by the EU index described in Kabat, such that the effector function of the BAA is reduced. In some embodiments, the first target is selected from the group of targets shown in Table 9, and the second target is selected from the group of targets shown in Table 9.

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

[0014] In another embodiment, the following activatable antibody (AA) is provided herein, comprising: (a) an antibody or antigen-binding fragment (AB) that specifically binds to the epidermal growth factor receptor (EGFR); (b) a masking moiety (MM) coupled to AB, wherein the MM reduces or inhibits the 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 sequences shown in Table 7; and (c) a cleavable moiety (CM) coupled to 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 that can be cleaved by a serine protease or MMP. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 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, AA is part of BAA.

[0015] In another embodiment, an activatable antibody (AA) is, The following describes an activatable antibody (AA) comprising: (a) an antibody or antigen-binding fragment (AB) that specifically binds to the epsilon chain of CD3, wherein the antibody comprises a heavy chain variable domain described in SEQ ID NO: 2 or SEQ ID NO: 3, or a light chain variable domain described in SEQ ID NO: 1 or SEQ ID NO: 4; (b) a masking moiety (MM) coupled to AB, wherein the MM reduces or inhibits the binding of the AB to the CD3 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. In some embodiments, AB comprises a heavy chain variable domain described in SEQ ID NO: 2. In some embodiments, AB comprises a heavy chain variable domain described in SEQ ID NO: 3. In some embodiments, AB comprises a light chain variable domain described in SEQ ID NO: 1. In some embodiments, AB comprises a light chain variable domain described in SEQ ID NO: 4. In some embodiments, AB comprises a heavy chain variable domain described in SEQ ID NO: 2 and a light chain variable domain described in SEQ ID NO: 1. In some embodiments, AB includes the heavy chain variable domain described in SEQ ID NO: 3 and the light chain variable domain described in SEQ ID NO: 1. In some embodiments, AB includes the heavy chain variable domain described in SEQ ID NO: 2 and the light chain variable domain described in SEQ ID NO: 4. In some embodiments, AB includes the heavy chain variable domain described in SEQ ID NO: 3 and the light chain variable domain described in SEQ ID NO: 4. In some embodiments, MM includes any one of the sequences listed in Table 3. In some embodiments, CM includes any one of the sequences listed in Table 4. In some embodiments, AA is part of BAA.

[0016] In another embodiment, the following activatable antibody (AA) is provided herein, comprising: (a) an antibody or antigen-binding fragment (AB) that specifically binds to the epsilon chain of CD3; (b) a masking moiety (MM) coupled to AB, wherein the MM reduces or inhibits the binding of the AB to the CD3 when the AA is in an uncleaved state, wherein the MM comprises the amino acid sequence of SEQ ID NO: 12; and (b) a cleavable moiety (CM) coupled to AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments, the CM comprises one of the sequences listed in Table 4. In some embodiments, the CM comprises a substrate that can be cleaved by a serine protease or 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 BAA.

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

[0018] In another embodiment, the Specified Antibody or Antigen-Conjugated Fragment (AB) of an antibody or its antigen-conjugated fragment (AB) that specifically binds to the epsilon chain of CD3, wherein the antibody comprises a heavy chain variable domain described in SEQ ID NO: 2 or SEQ ID NO: 3 or a light chain variable domain described in SEQ ID NO: 1 or SEQ ID NO: 4. In some embodiments, the antibody comprises a heavy chain variable domain described in SEQ ID NO: 2 or SEQ ID NO: 3 and a light chain variable domain described in SEQ ID NO: 1 or SEQ ID NO: 4. In some embodiments, AB comprises a heavy chain variable domain described in SEQ ID NO: 2. In some embodiments, AB comprises a heavy chain variable domain described in SEQ ID NO: 3. In some embodiments, AB comprises a light chain variable domain described in SEQ ID NO: 1. In some embodiments, AB comprises a light chain variable domain described in SEQ ID NO: 4. In some embodiments, AB comprises a heavy chain variable domain described in SEQ ID NO: 2 and a light chain variable domain described in SEQ ID NO: 1. In some embodiments, AB comprises a heavy chain variable domain described in SEQ ID NO: 3 and a light chain variable domain described in SEQ ID NO: 1. In some embodiments, AB comprises a heavy chain variable domain described in SEQ ID NO: 3 and a light chain variable domain described in SEQ ID NO: 4. In some embodiments, AB includes the heavy chain variable domain described in SEQ ID NO: 2 and the light chain variable domain described in SEQ ID NO: 4. In some embodiments, the antibody is bispecific AB. In some embodiments, the antibody is scFv. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is part of AA or part of BAA.

[0019] In another embodiment, the present invention provides an antibody (AB) that specifically binds to EGFR or CD3, wherein the aforementioned antibody is an IgG1 antibody or an scFv linked to an Fc domain, wherein the aforementioned antibody comprises an Fc region containing an amino acid substitution at at least one of the amino acid positions L234, L235, and P331, numbered by the EU index described in Kabat, such that the effector function of the aforementioned antibody is reduced. In some embodiments, the amino acid substitution is one or more of L234F, L235E, and P331S. In some embodiments, the antibody contains an amino acid substitution at at least two of the amino acid positions L234, L235, and P331. In some embodiments, the antibody contains an amino acid substitution at the amino acid positions L234, L235, and P331. In some embodiments, the antibody contains an amino acid substitution at L234F, L235E, and P331S. In some embodiments, the antibody contains an Fc region containing an amino acid substitution at N297. In some embodiments, the Fc region contains the N297Q mutation. In some embodiments, the antibody contains the L234F, L235E, P331S, and N297Q amino acid substitutions. In some embodiments, the antibody heavy chain contains one of SEQ ID NOs: 69, 70, 71, 72, 73, 74, 75, and 76 as listed in Table 6. In some embodiments, the antibody heavy chain variable domain contains either SEQ ID NOs: 2 or 3, and the light chain variable domain of AB contains either SEQ ID NOs: 1 or 4. In some embodiments, the antibody is part of AA or part of BAA.

[0020] In another embodiment, pharmaceutical compositions comprising any one of the above-mentioned BAA, AA, and antibody, as well as an optional carrier, are also provided herein. In another embodiment, pharmaceutical compositions comprising any one of the above-mentioned BAA, AA, and antibody, as well as a carrier, are also provided herein. In some embodiments, the composition comprises a further agent, for example, the further agent may be a therapeutic agent.

[0021] In other embodiments, isolated nucleic acid molecules encoding any one of the above-mentioned BAA, AA, and antibody are also provided herein. Vectors containing nucleic acids are also provided. In some embodiments, the vector includes the nucleic acid sequence pLW289. In some embodiments, the vector includes the nucleic acid sequence pLW246. In some embodiments, the vector includes the nucleic acid sequence pLW307. In some embodiments, the vector includes the nucleic acid sequence pLW291. In some embodiments, the vector includes the nucleic acid sequence pLW352. In some embodiments, the vector includes the nucleic acid sequence pLW246. In some embodiments, the vector includes the nucleic acid sequence pLW353.

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

[0023] In another embodiment, a method for producing the antibodies, AA, or BAA provided above by culturing cells under conditions that express the antibodies, AA, or BAA, wherein the aforementioned cells include the relevant nucleic acid molecules or vectors provided herein.

[0024] In another embodiment, methods for treating a disorder or disease, alleviating its symptoms, or slowing its progression are provided herein, comprising administering a therapeutically effective amount of the above-mentioned antibody / AA / BAA / pharmaceutical composition to a subject in need thereof. In some embodiments, the disorder or disease comprises disease cells expressing EGFR. In some embodiments, the disorder or disease is cancer. In some embodiments, cancer is anal cancer, basal cell carcinoma, brain cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, intrahepatic cholangiocarcinoma, 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, such as Epstein-Barr virus-associated lymphoma, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, and non-Hodgkin 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 embodiment, the present invention provides a method for inhibiting angiogenesis in a subject, comprising administering a therapeutically effective amount of the above-mentioned antibody / AA / BAA / pharmaceutical composition to a subject in need thereof. In some embodiments, the method comprises administering a further agent. In some embodiments, the further agent is a therapeutic agent.

[0026] In another embodiment, a method is provided herein for reducing damage to healthy tissue resulting from an antibody binding to a target on diseased tissue (e.g., cancerous tissue) and 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 aforementioned AA or BAA is one of the AA or BAAs of the embodiments provided herein.

[0027] In another embodiment, a method for improving the tolerability of an antibody treatment is provided herein, 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 with cancer), wherein the aforementioned AA or BAA is one of the AA or BAAs of the embodiments provided herein.

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

[0029] In another embodiment, an antibody, AA, BAA, or pharmaceutical composition of any one of the embodiments provided herein is provided herein for use as a pharmaceutical. The pharmaceutical may be used in a manner that reduces damage to healthy tissue resulting from the binding of the antibody to its target on affected tissue and on healthy tissue. The pharmaceutical may be used to improve the tolerability of antibody treatment.

[0030] In another embodiment, the present invention provides an antibody, AA, BAA, or pharmaceutical composition of any one of the embodiments provided herein for use in a manner of treating a disorder or disease, alleviating its symptoms, or slowing its progression, wherein the aforementioned disorder or disease comprises disease cells expressing EGFR.

[0031] In another embodiment, antibodies, AAs, BAAs, or pharmaceutical compositions of any one of the embodiments provided herein for use in a method of treating cancer; wherein, optionally, the aforementioned cancer is anal cancer, basal cell carcinoma, brain cancer, bladder cancer, breast cancer, bone cancer, cervical cancer, intrahepatic cholangiocarcinoma, 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 may include the recruitment of T cells into tumor tissue.

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

[0033] Any one of the embodiments of the antibody, AA, BAA, or pharmaceutical composition provided herein may be used in a treatment method comprising administering a further agent; optionally, the further agent is a therapeutic agent. [Brief explanation of the drawing]

[0034] [Figure 1] Figure 1A demonstrates that incorporation of the h20GG CD3ε masking peptide into CI106 and CI107, which are EGFR-masked BAAs, significantly reduces binding to Jurkat cells compared to CI011. Reduction in binding to EGFR+HT29-luc2 cells was also evident for CI106 and CI107 compared to CI011 (Figure 1B). [Figure 2]Figure 2A demonstrates that EGFR+HT29-luc2 cell death 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 death is dependent on EGFR targeting. Figure 2B also shows a more than 300,000-fold EC50 shift for the bi-masked antibodies (i.e., BAA with both EGFR and CD3 target-binding domains masked) CI106 and CI107 compared to the protease-activated bispecific antibody (i.e., BAA activated by protease treatment) act-104 (referred interchangeably as CI104). Figure 2C shows the number of EGFR receptors in a panel of cell lines including HT29. The approximate number of EGFR receptors on HT29 cells is 75,000, indicating that high-density antigens are not required for potent cytotoxicity of the BAA tested. [Figure 3] Figure 3A demonstrates that primary CD8+ T cell activation 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 double-masked antibodies compared to the protease-activating bispecific antibody act-104, demonstrating that masking weakens T cell activation. [Figure 4] Figure 4 plots tumor volume versus days after initial treatment administration, demonstrating the dose-dependent effect of the double-masked, bispecific AAs CI106 and CI107 on the growth of HT29-luc2 xenograft tumors. [Figure 5] Figure 5 plots tumor volume versus days after initial treatment administration, demonstrating the dose-dependent effect of CI106 and CI107, two bi-masked, bi-specific AAs, on the growth of HCT116 xenograft tumors. [Figure 6A] Figures 6A–6B demonstrate that the EC50 values ​​of the tested double-masked bispecific antibodies 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 EC50 values ​​of the tested double-masked bispecific antibodies and protease-activated bispecific antibodies are similar when using either human (6A) or cynomolgus monkey (6B) effector cells. [Figure 6C] Figures 6C–6D demonstrate the similar binding of protease-activating antibodies and double-masked antibodies to human (6C) and cynomolgus monkey (6D) T cells. [Figure 6D] Figures 6C–6D demonstrate the similar binding of protease-activating antibodies and double-masked antibodies to human (6C) and cynomolgus monkey (6D) T cells. [Figure 7A] Figures 7A–7C show serum concentrations of ALT (7A), AST (7B), and total bilirubin (7C) before administration, 48 hours after administration, and 7 days after supplemental administration in cynomolgus monkeys treated with CI106 or CI107. [Figure 7B] Figures 7A–7C show serum concentrations of ALT (7A), AST (7B), and total bilirubin (7C) before administration, 48 hours after administration, and 7 days after supplemental administration in cynomolgus monkeys treated with CI106 or CI107. [Figure 7C] Figures 7A–7C show serum concentrations of ALT (7A), AST (7B), and total bilirubin (7C) before administration, 48 hours after administration, and 7 days after supplemental administration in cynomolgus monkeys treated with CI106 or CI107. [Figure 8A] Figures 8A–8C plot the increases 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] Figures 8A–8C plot the increases 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]Figures 8A–8C plot the increases 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] Figures 9A–9C show T cell activation as measured by the expression of CD69 (9A), Ki67 (9B), and PD-1 (9B) on CD4+ T cells in cynomolgus monkeys treated with CI106 or CI107. [Figure 9B] Figures 9A–9C show T cell activation as measured by the expression of CD69 (9A), Ki67 (9B), and PD-1 (9B) on CD4+ T cells in cynomolgus monkeys treated with CI106 or CI107. [Figure 9C] Figures 9A–9C show T cell activation as measured by the 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 increases in AST (10A) at 48 hours post-administration, ALT (10B) at 48 hours post-administration, IL-6 (10C) at 8 hours post-administration, IFNg (10D) at 8 hours post-administration, and Ki67 (10E) at 72 hours post-administration in cynomolgus monkeys treated with act-104, CI106, or CI107. The dose-response curves for all parameters are shifted for the double-masked antibody, which indicates improved tolerability and reduced pharmacodynamic effects compared to the protease-activated bispecific antibody. [Figure 10B] Figures 10A–E plot the dose-dependent increases in AST (10A) at 48 hours post-administration, ALT (10B) at 48 hours post-administration, IL-6 (10C) at 8 hours post-administration, IFNg (10D) at 8 hours post-administration, and Ki67 (10E) at 72 hours post-administration in cynomolgus monkeys treated with act-104, CI106, or CI107. The dose-response curves for all parameters are shifted for the double-masked antibody, which indicates improved tolerability and reduced pharmacodynamic effects compared to the protease-activated bispecific antibody. [Figure 10C] Figures 10A–E plot the dose-dependent increases in AST (10A) at 48 hours post-administration, ALT (10B) at 48 hours post-administration, IL-6 (10C) at 8 hours post-administration, IFNg (10D) at 8 hours post-administration, and Ki67 (10E) at 72 hours post-administration in cynomolgus monkeys treated with act-104, CI106, or CI107. The dose-response curves for all parameters are shifted for the double-masked antibody, which indicates improved tolerability and reduced pharmacodynamic effects compared to the protease-activated bispecific antibody. [Figure 10D] Figures 10A–E plot the dose-dependent increases in AST (10A) at 48 hours post-administration, ALT (10B) at 48 hours post-administration, IL-6 (10C) at 8 hours post-administration, IFNg (10D) at 8 hours post-administration, and Ki67 (10E) at 72 hours post-administration in cynomolgus monkeys treated with act-104, CI106, or CI107. The dose-response curves for all parameters are shifted for the double-masked antibody, which indicates improved tolerability and reduced pharmacodynamic effects compared to the protease-activated bispecific antibody. [Figure 10E] Figures 10A–E plot the dose-dependent increases in AST (10A) at 48 hours post-administration, ALT (10B) at 48 hours post-administration, IL-6 (10C) at 8 hours post-administration, IFNg (10D) at 8 hours post-administration, and Ki67 (10E) at 72 hours post-administration in cynomolgus monkeys treated with act-104, CI106, or CI107. The dose-response curves for all parameters are shifted for the double-masked antibody, which indicates 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 of 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 of 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 of total bilirubin (11A), IL-6 (11B), and PD-1-expressing CD4+ T cells (11C) in cynomolgus monkeys treated with CI107 or CI128. [Figure 12A] Figure 12A shows the affinity measurements of CD3 antibodies v12, v16, and v19 compared to hSP34. [Figure 12B] Figure 12B shows the cytotoxic effects of activated or double-masked bispecific antibodies on HT29-luc2 cells. [Figure 13] Figure 13 shows the expanded PK of the double-masked antibody CI107 compared to the protease-activating bispecific antibody act-104. [Figure 14A] Figure 14A shows the efficacy of the HT29-luc2 tumor intervention model in PBMC-transplanted NSG mice. In this example, the antitumor efficacy correlated with the protease sensitivity and substrate cleavage of the test substance, with the most effective test substance being CI048 fully activated by protease. [Figure 14B] Figure 14B shows the staining (dark staining) of tumor sections for CD3 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 substance. [Figure 15] Figures 15 and 16 are plots of conjugation isotherms for the activatable anti-EGFR C225v5 antibody of this 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 conjugation isotherms for the activatable anti-EGFR C225v5 antibody of this disclosure, the activatable anti-EGFR antibody 3954-2001-C225v5 described herein, and the anti-EGFR antibody C225v5. [Figure 17] Figures 17-19 illustrate exemplary BAAs provided herein. [Figure 18] Figures 17-19 illustrate exemplary BAAs provided herein. [Figure 19] Figures 17-19 illustrate exemplary BAAs provided herein. [Figure 20] Figure 20 shows the PK of double-masked BAA CI107 after administration of a single dose of 600, 2000, or 4000 ug / kg. [Figure 21] Figure 21 demonstrates that the cytotoxicity of CI090 and CI091 in HT29-luc2 cells was attenuated compared to CI011. [Figure 22] Figure 22 demonstrates that the 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-transplanted NSG mice. It demonstrates the antitumor efficacy of CI091, CI090, and CI011. [Figure 24] Figure 24 plots IL-6 levels in cynomolgus monkeys 8 hours after drug administration in an in vivo study. [Modes for carrying out the invention]

[0035] Detailed description of the invention Antibodies, activatable antibodies (AA), bispecific antibodies, and bispecific activatable antibodies (BAA) are provided herein.

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

[0037] In some embodiments, IgG1 antibodies that specifically bind to the epidermal growth factor receptor (EGFR) are provided herein, wherein the antibody contains a point mutation in the Fc region such that the effector function of the antibody is reduced.

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

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

[0040] Methods for producing and using these antibodies, AA, and BAA are also provided herein. Identification of AA (including its general production) and the masking moiety (MM) and cleavable moiety (CM) is described in Daugherty et al., International Publication No. WO2009 / 025846, published February 26, 2009, and Stagliano et al., International Publication No. WO2010 / 081173, published July 15, 2010 (both in their entirety are incorporated herein by reference). Identification of BAA (including its general production) and the masking moiety (MM) and cleavable moiety (CM) is described in Lowman et al., International Publication No. WO2015 / 013671, published January 29, 2015, and Irving et al., International Publication No. WO2016 / 014974, published January 28, 2016 (both in their entirety are incorporated herein by reference). International publications WO2016 / 014974 by Irving et al., published on January 28, 2016, and WO2016 / 118629 by Moore et al., published on July 28, 2016, which provide AA, general production, MM, and CM, are also referred to.

[0041] As used herein, unless otherwise specified, the term “antibody” includes an antibody or its antigen-binding fragment that specifically binds its target, and includes monoclonal antibodies, domain antibodies, single-chain antibodies, Fab fragments, F(ab')2 fragments, scFv, scAb, dAb, 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 antibody or its immunologically active fragment that binds its target is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody.

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

[0043] Exemplary amino acid sequences of the CD3-binding antibody (variable domain) of this disclosure are provided in Table 1 (the putative CDR sequence is underlined). As provided below, L3 is a linker that ligates the variable domains of the light and heavy chains in the exemplary CD3-binding antibody.

[0044] [Table 1] TIFF0007860317000002.tif181167

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

[0046] [Table 2]

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

[0048] [Table 3]

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

[0050] In some embodiments, the CD3 antibody contains the heavy chain variable domain described in SEQ ID NO: 2.

[0051] In some embodiments, the CD3 antibody contains the heavy chain variable domain described in SEQ ID NO: 3.

[0052] In some embodiments, the CD3 antibody contains the light chain variable domain described in SEQ ID NO: 1.

[0053] In some embodiments, the CD3 antibody contains the light chain variable domain described in SEQ ID NO: 4.

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

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

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

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

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

[0059] In some embodiments, the CD3 antibody comprises a heavy chain variable domain described in SEQ ID NO: 2 or SEQ ID NO: 3, and a light chain variable domain described 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 has the following structure in the direction from the N-terminus to the C-terminus: LV-HV. In some embodiments, the variable domain has the following structure in the direction from the N-terminus to the C-terminus: 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), where the aforementioned VH is linked to the VL by a linker containing the amino acid sequence of SEQ ID NO: 98. Exemplary sequences having such a linker are provided in Table 1.

[0062] In exemplary embodiments, the present invention provides an antibody (AB) that specifically binds to CD3, wherein the aforementioned antibody is an IgG1 antibody or an scFv linked to an Fc domain, wherein the aforementioned antibody comprises an Fc region containing an amino acid substitution at at least one of the amino acid positions L234, L235, and P331, numbered by the EU index described in Kabat, such that the effector function of the aforementioned antibody is reduced. In some embodiments, the amino acid substitution is one or more of L234F, L235E, and P331S. In some embodiments, the antibody contains an amino acid substitution at at least two of the amino acid positions L234, L235, and P331. In some embodiments, the antibody contains an amino acid substitution at the amino acid positions L234, L235, and P331. In some embodiments, the antibody contains an amino acid substitution at L234F, L235E, and P331S. In some embodiments, the antibody contains an Fc region containing an amino acid substitution at N297. In some embodiments, the Fc region contains the N297Q mutation. In some embodiments, the antibody contains the amino acid substitutions L234F, L235E, P331S, and N297Q. In some embodiments, the heavy chain variable domain of the antibody contains either SEQ ID NO: 2 or SEQ ID NO: 3, or the light chain variable domain of AB contains either SEQ ID NO: 1 or SEQ ID NO: 4.

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

[0064] As is commonly provided herein, the AA of the present invention comprises 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 includes a linker at the carboxyl terminus of the CM; this linker, referred to herein as L2, ligates the CM of the prodomain to AB. In some embodiments, the prodomain includes a linker between the MM and CM and a linker behind 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 an amino acid sequence in either the N-terminus-C-terminus direction or the C-terminus-N-terminus direction): (MM)-L1-(CM), (MM)-(CM)-L2, (MM)-L1-(CM)-L2, or (MM)-(CM). In exemplary embodiments, the prodomain comprises EGFR MM and CM cleavable by matryptase or MMP; or CD3εMM and CM cleavable by matryptase or MMP. In some embodiments, the prodomain comprises EGFR MM and CM cleavable by matryptase and MMP. In some embodiments, the prodomain comprises CD3εMM and CM cleavable by matryptase and MMP. Activatable antibodies (AAs) containing the prodomain are provided herein. Nucleotides encoding the prodomain of the present invention are also provided herein.

[0065] Accordingly, the present invention provides a CD3AA comprising: (a) an antibody or antigen-binding fragment (AB) that specifically binds to the epsilon chain (CD3ε) of CD3, wherein the antibody or antigen-binding fragment (AB) comprises the heavy chain domain described in SEQ ID NO: 2 or SEQ ID NO: 3, or the light chain domain described in SEQ ID NO: 1 or SEQ ID NO: 4; (b) a masking moiety (MM) coupled to AB, wherein the MM reduces or inhibits the binding of the AB to the CD3ε 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. As described above, (b) and (c) are both part of a prodomain.

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

[0067] In some embodiments, the AB of CD3AA includes the heavy chain variable domain described in Sequence ID No. 2.

[0068] In some embodiments, the AB of CD3AA includes the heavy chain variable domain described in SEQ ID NO: 3.

[0069] In some embodiments, AB of CD3AA includes the light chain variable domain described in Sequence ID No. 1.

[0070] In some embodiments, the AB of CD3AA includes the light chain variable domain described in Sequence ID No. 4.

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

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

[0073] In some embodiments, the AB of CD3AA includes the heavy chain variable domain described in SEQ ID NO: 3 and the light chain domain described 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 the aforementioned VH is linked to VL by a linker L3 containing the amino acid sequence of SEQ ID NO: 98. Exemplary sequences having such a linker are provided in Table 1.

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

[0076] An exemplary CD3 masking portion (MM) of the present invention is provided in Table 3.

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

[0078] [Table 4]

[0079] In some embodiments, the CM of CD3AA includes one of the sequences listed in Table 4. Exemplary cleavable portions (CMs) of the present invention are shown in Table 4.

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

[0081] [Table 5]

[0082] [Table 6] TIFF0007860317000008.tif16167

[0083] 3. Antibodies with Fc mutations This specification provides IgG1 antibodies having an Fc mutation or antibody fragments containing an antigen-binding domain linked to an Fc domain (e.g., scFv, Fab, F(ab')2), where Fc has reduced effector function (referred to herein as Fc variants). Any of the BAAs, AAs, and antibodies described herein may contain any of the Fc variants disclosed herein.

[0084] Antibodies containing these Fc mutations maintain target binding affinity while reducing effector function. Therefore, the following antibodies are provided herein that bind to a target of interest, wherein the aforementioned antibody is an IgG1 antibody or an antibody fragment linked to Fc, where the aforementioned Fc region contains an amino acid substitution at at least one of the amino acid positions L234, L235, and P331, numbered by the EU index described in Kabat, such that the effector function of the aforementioned antibody is reduced. In some embodiments, the amino acid substitution is one or more of L234F, L235E, and P331S. In some embodiments, there is a further mutation at N297. In some embodiments, the amino acid substitution is N297Q or N297A.

[0085] In some embodiments, Fc is selected from the Fc sequences shown in Table 4-2. In some embodiments, Fc is selected from SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, and SEQ ID NO: 160, 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, pyrrolidine, selenocysteine, serine, threonine, tryptophan, tyrosine, valine) or any naturally occurring amino acid (For example, selected from the group consisting of 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] TIFF0007860317000010.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 can bind to immune effector cells. In some embodiments, they can selectively bind to targets present on immune effector cells. In some embodiments, they can bind to CD3. In some embodiments, they can bind to any of the targets listed in Table 9. In some embodiments, they can bind to EGFR.

[0089] Therefore, in some embodiments, the activatable antibody (AA) is, a) an antibody (AB) that specifically binds to a target, wherein the antibody is an IgG1 antibody, and the Fc region of the antibody (AB) contains an amino acid substitution at at least one of the amino acid positions L234, L235, and P331, numbered by the EU index described in Kabat, such that the effector function of the AA described above is reduced; b) Masking portions (MM) coupled to AB, wherein the aforementioned MM reduces or inhibits the binding of the aforementioned AB to the target when the aforementioned AA is in an uncleaved state; and c) A cleavable moiety (CM) coupled to AB, wherein the aforementioned CM is a polypeptide that functions as a substrate for a protease. This specification provides for activatable antibodies (AA) containing the following.

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

[0091] In some embodiments, a bispecific activatable antibody (BAA) is used. a) An IgG antibody (AB1) that specifically binds to the first target, wherein the aforementioned AB1 is i. Two heavy chains (AB1 HC) and two light chains (AB1 LC); and ii. A first masking portion (MM1) that is connected to the first cleavable portion (CM1) to form an MM1-CM1 construct, wherein the carboxyl terminus of the aforementioned MM1-CM1 construct is connected to the respective amino terminus of each light chain of the aforementioned AB1. Including, here, The aforementioned MM1 inhibits the binding of the aforementioned AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, IgG antibody (AB1), b) Two scFv(AB2) each specifically bind to a second target, where each AB2 is i. A heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 mentioned above is linked to the respective amino terminus of the AB1 heavy chain; and ii. A second masking portion (MM2) connected to a second cleavable portion (CM2) to form an MM2-CM2 structure, wherein the carboxyl terminus of the aforementioned MM2-CM2 structure is connected to the amino terminus of each of the aforementioned AB2 portions of the second masking portion (MM2). Including, here, The aforementioned MM2 inhibits the binding of the aforementioned AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for the second protease, consisting of two scFv(AB2) polypeptides. Includes,

[0092] Herein, AB1 provides herein a bispecific activatable antibody (BAA) comprising an Fc region containing an amino acid substitution at at least one of the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, such that the effector function of the BAA is reduced.

[0093] In some embodiments provided herein, the BAA provided herein is a) A bispecific activatable antibody (BAA), i) an IgG antibody (AB1) that specifically binds to a first target, wherein the aforementioned AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein the aforementioned AB1 is linked to a first masking portion (MM1) linked to a first cleavable portion (CM1) to form an MM1-CM1 construct, wherein the carboxyl terminus of the aforementioned MM1-CM1 construct is linked to the amino terminus of each light chain of the aforementioned AB1, The aforementioned MM1 inhibits the binding of the aforementioned AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, IgG antibody (AB1), ii) Two scFv (each AB2) each specifically bind to a second target, wherein each AB2 includes a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 is linked to the respective amino terminus of the aforementioned AB1 heavy chain; wherein each AB2 is linked to a second masking region (MM2) linked to a second cleavable region (CM2) to form an MM2-CM2 construct, wherein the carboxyl terminus of each MM2-CM2 construct is linked to the amino terminus of each AB2, The aforementioned MM2 inhibits the binding of the aforementioned AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for the second protease, consisting of two scFv molecules (each AB2). Includes bispecific activatable antibodies (BAA) Includes, Here, the aforementioned AB1 includes an Fc region containing an amino acid substitution at at least one of the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, such that the effector function of the aforementioned BAA is reduced.

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

[0095] This specification provides EGFR antibodies, bispecific antibodies having one arm that targets EGFR, AAs capable of binding to EGFR upon activation, and BAAs capable of binding to EGFR upon activation. In some embodiments, the EGFR antibody includes the CDRs shown in Table 5.

[0096] In some embodiments, for example, IgG1 antibodies in BAA form that specifically bind to the epidermal growth factor receptor (EGFR) and reduce its effector function are provided herein. The antibodies contain Fc mutations that reduce effector function while maintaining EGFR binding affinity. Thus, antibodies that bind to EGFR are provided herein, wherein the aforementioned antibody is an IgG1 antibody, and wherein the aforementioned antibody contains an Fc region having an amino acid substitution at at least one of the amino acid positions L234, L235, and P331, numbered by the EU index described in Kabat, such that the effector function of the aforementioned antibody is reduced. In some embodiments, the amino acid substitution is one or more of L234F, L235E, and P331S.

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

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

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

[0100] In some embodiments, the antibody includes an Fc region containing 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 contains the N297Q mutation. In some embodiments, the Fc region contains the N297A mutation.

[0101] In some embodiments, the antibody includes substitutions of L234F, L235E, P331S, and N297Q. In some embodiments, the antibody includes substitutions of L234F, L235E, P331S, and N297A.

[0102] Table 5 provides exemplary CDR sequences of EGFR-binding antibodies described in Kabat.

[0103] [Table 8]

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

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

[0106] In some embodiments, the heavy chain of the EGFR antibody comprises one of the sequences described in SEQ ID NOs: 67, 69, 70, 71, 72, 73, 74, 75, and 76, as listed in Table 6. In some embodiments, the heavy chain EGFR antibody comprises one of the sequences described in SEQ ID NOs: 67, 69, 71, and 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, pyrrolidine, selenocysteine, serine, threonine, tryptophan, tyrosine, valine) or any naturally occurring amino acid The non-existent amino acids are selected from (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 notation Fcmt3 includes three point mutations, where the Fc region of the heavy chain of the EGFR antibody includes the following three point mutations: L234F, L235E, and P331S. Thus, in some embodiments, the EGFR antibody includes a heavy chain having the amino acid sequence described in SEQ ID NO: C225v5Fcmt3 HC. In some embodiments, the Fc region of the heavy chain of the EGFR antibody includes a fourth point mutation N297Q. The notation Fcmt4 includes the Fcmt3 three-point mutation and the fourth point mutation N297Q. Therefore, in such embodiments, the EGFR antibody comprises a heavy chain having the amino acid sequence described in SEQ ID NO: 76.

[0107] [Table 9] TIFF0007860317000013.tif250167TIFF0007860317000014.tif213167

[0108] 5.Activatable EGFR antibody In some embodiments, one of the EGFR antibodies provided herein is in AA form (EGFR AA). As with CD3AA above, EGFR AA also contains a prodomain.

[0109] Therefore, AAs comprising an antibody or its antigen-binding fragment (AB) that specifically binds to EGFR are provided herein. Exemplary CDR sequences of EGFR-binding antibodies are provided in Table 5.

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

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

[0112] [Table 10]

[0113] [Table 11]

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

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

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

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

[0118] This specification provides a bispecific AA (BAA), where the BAA, when activated, specifically binds to two targets (for example, to two different targets or to 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 of targets shown in Table 9, and the second target is selected from the group of targets shown in Table 9.

[0120] As is generally provided herein and as described in the section describing the above AA, the BAA of the present invention comprises 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 includes a linker at the carboxyl terminus of the CM; this linker, referred to herein as L2, ligates the CM of the prodomain to the AB. In some embodiments, the prodomain includes a linker between the MM and CM and a linker behind 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 an amino acid sequence in either the N-terminus-C-terminus direction or the C-terminus-N-terminus direction): (MM)-L1-(CM), (MM)-(CM)-L2, (MM)-L1-(CM)-L2, or (MM)-(CM). In exemplary embodiments, the prodomain comprises EGFR MM and CM cleavable by matryptase or MMP; or CD3εMM and CM cleavable by matryptase or MMP. In some embodiments, the prodomain comprises EGFR MM and CM cleavable by matryptase and MMP. In some embodiments, the prodomain comprises CD3εMM and CM cleavable by matryptase and MMP. Bispecific activatable antibodies (BAAs) containing the prodomain are provided herein. Nucleotides encoding the prodomain of the present invention are also provided herein.

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

[0122] In some embodiments, the BAA provided herein is 1. A bispecific activatable antibody (BAA) wherein, when activated, the aforementioned BAA specifically binds to two targets and has the following structure: a. An IgG antibody (AB1) that specifically binds to a first target, wherein the aforementioned AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein the aforementioned AB1 is linked to a first masking portion (MM1) linked to a first cleavable portion (CM1) to form an MM1-CM1 construct, wherein the carboxyl terminus of the aforementioned MM1-CM1 construct is linked to the amino terminus of each light chain of the aforementioned AB1, The aforementioned MM1 inhibits the binding of the aforementioned AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, IgG antibody (AB1), b. Two scFv (each AB2) each specifically bind to a second target, wherein each AB2 includes a light chain variable region linked to a heavy chain variable region, wherein the carboxyl terminus of each AB2 is linked to the respective amino terminus of the aforementioned AB1 heavy chain; wherein each AB2 is linked to a second masking region (MM2) linked to a second cleavable region (CM2) to form an MM2-CM2 construct, wherein the carboxyl terminus of each MM2-CM2 construct is linked to the amino terminus of each AB2, The aforementioned MM2 inhibits the binding of the aforementioned AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for the second protease, consisting of two scFv molecules (each AB2). Includes, Here, the aforementioned BAA has the following characteristics: i.MM2 contains the amino acid sequence of SEQ ID NO: 12; ii. MM1 contains an amino acid sequence selected from the group of sequences shown in Table 7; iii.AB2 contains a heavy chain variable domain described in SEQ ID NO: 2 or SEQ ID NO: 3 or a light chain variable domain described in SEQ ID NO: 1 or SEQ ID NO: 4; and iv.AB1 includes an Fc region containing an amino acid substitution at at least one of the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, so as to reduce the effector function of the BAA described above. It comprises a bispecific activatable antibody (BAA) having at least one of the following:

[0123] In some embodiments, the BAA provided herein is a) An IgG antibody (AB1) that specifically binds to the first target, wherein the aforementioned AB1 is a. Two heavy chains (AB1 HC) and two light chains (AB1 LC); and b. A first masking portion (MM1) that is linked to the first cleavable portion (CM1) to form an MM1-CM1 construct, wherein the carboxyl terminus of the aforementioned MM1-CM1 construct is linked to the amino terminus of each light chain of the aforementioned AB1, and here, 1. The aforementioned MM1 inhibits the binding of the aforementioned AB1 to its target; 2. The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, the first masking moiety (MM1) Includes IgG antibody (AB1), b) Two scFv(AB2) each specifically bind to a second target, where each AB2 is a. A heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 mentioned above is linked to the respective amino terminus of the AB1 heavy chain; and b. A second masking portion (MM2) connected to the second cleavable portion (CM2) to form an MM2-CM2 structure, wherein the carboxyl terminus of the aforementioned MM2-CM2 structure is connected to the amino terminus of each of the aforementioned AB2, and here, 1. The aforementioned MM2 inhibits the binding of the aforementioned AB2 to its target; 2. The aforementioned CM2 is a polypeptide that functions as a substrate for the second protease, the second masking moiety (MM2). Two scFv(AB2) including Includes, Here, AB1 includes an Fc region containing an amino acid substitution at at least one of the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, so as to reduce the effector function of the BAA described above. In some embodiments, the Fc region contains an amino acid substitution at at least the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, so as to reduce the effector function of the BAA described above.

[0124] In some embodiments, the BAA provided herein is (1) A bispecific activatable antibody (BAA), (a) an IgG antibody (AB1) that specifically binds to a first target, wherein the aforementioned AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein the aforementioned AB1 is linked to a first masking portion (MM1) linked to a first cleavable portion (CM1) to form an MM1-CM1 construct, wherein the carboxyl terminus of the aforementioned MM1-CM1 construct is linked to the amino terminus of each light chain of the aforementioned AB1, The aforementioned MM1 inhibits the binding of the aforementioned AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, IgG antibody (AB1), (b) Two scFv (each AB2) each specifically bound 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 AB2 is linked to the respective amino terminus of the AB1 heavy chain; wherein each AB2 is linked to a second masking region (MM2) linked to a second cleavable region (CM2) to form an MM2-CM2 construct, wherein the carboxyl terminus of each MM2-CM2 construct is linked to the amino terminus of each AB2; The aforementioned MM2 inhibits the binding of the aforementioned AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for the second protease, consisting of two scFv molecules (each AB2). Includes bispecific activatable antibodies (BAA) Includes, Here, the aforementioned AB1 includes an Fc region containing an amino acid substitution at at least one of the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, such that the effector function of the aforementioned BAA is reduced.

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

[0126] In some embodiments, the Fc region of AB1 includes amino acid substitutions at least at amino acid positions L234, L235, and P331, numbered by the EU index described in Kabat, so as to reduce the effector function of the BAA described above. In some embodiments, the first target is selected from the group of targets shown in Table 9, and the second target is selected from the group of targets shown in Table 9.

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

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

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

[0130] In some embodiments, MM1 includes sequence number 78.

[0131] In some embodiments, MM2 includes 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, BAA is CI107, as provided in Table 11 of Example 1.

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

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

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

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

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

[0139] In one exemplary embodiment, AB1 comprises the amino acid sequence 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 contain the same amino acid sequence. In some embodiments, CM1 and CM2 contain different amino acid sequences. In some embodiments, CM1 and CM2 contain different amino acid sequences that can be cleaved by the same protease or multiple proteases. In some embodiments, CM1 and CM2 can be cleaved by one or more proteases. In some embodiments, CM1 and / or CM2 can be cleaved by a serine protease. In some embodiments, CM1 and / or CM2 can be cleaved by a matrix metalloproteinase (MMP). In some embodiments, CM1 and / or CM2 can be cleaved by a serine protease and an MMP.

[0141] The exemplary BAAs of this disclosure include, for example, the BAAs and their variants shown in the examples provided herein.

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

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

[0144] [Table 12] TIFF0007860317000018.tif174167

[0145] In some embodiments, unmasked EGFR-CD3 bispecific antibodies exhibit EGFR-dependent tumor cell death, while double-masked EGFR-CD3 BAAs reduce target-dependent cytotoxicity to less than 1 / 100,000. 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 tolerable dose (MTD) of EGFR-CD3 BAAs is more than 60 times that of unmasked bispecific antibodies, and the acceptable exposure (AUC) is more than 10,000 times. Despite the 60-fold difference in MTD, the transient increases in serum cytokines and AST / ALT observed in non-human primates treated with BAAs are still lower than those induced by bispecific antibodies. By localizing its activity to the tumor microenvironment, BAAs may expand the opportunity for clinically performing T-cell-inducing bispecific therapies, which are limited by on-target toxicity, particularly in solid tumors. Furthermore, EGFR-CD3 BAAs may be effective in addressing EGFR-expressing tumors that do not respond well to existing EGFR-targeted therapies.

[0146] 7. Cuttable part (CM) Both the single-specific AA and BAA of this disclosure include at least one CM when masked and unactivated.

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

[0148] Various conditions under which the target tissue coexists with a protease are known, and the substrates of that protease are known in that field. Taking cancer as an example, the target tissue can be cancerous tissue, particularly solid tumor tissue. There are reports in various papers that protease levels are increased in many cancers (e.g., humoral or solid tumors). See, for example, La Rocca et al, (2004) British J. of Cancer 90(7):1414-1421. Non-limiting examples of diseases include: all types of cancer (breast cancer, lung cancer, colorectal cancer, gastric cancer, glioblastoma, ovarian cancer, endometrial cancer, kidney cancer, sarcoma, skin cancer, cervical cancer, liver cancer, bladder cancer, intrahepatic cholangiocarcinoma, prostate cancer, melanoma, head and neck cancers (e.g., head and neck squamous cell carcinoma, pancreatic cancer, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular injury, ischemia, etc., but not limited to these). 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 metastatic cancer unrelated to bone disease or primary tumor origin; breast cancer (including, in non-limiting examples, 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, in non-limiting examples, non-small cell lung cancer); multiple myeloma; ovarian cancer; pancreatic cancer; prostate cancer; sarcoma (such as osteosarcoma); renal cancer (including, in non-limiting examples, renal cell carcinoma); and / or skin cancer (including, in non-limiting examples, 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 is produced by enzymes at a rate of approximately 0.001 to 1500 x 10 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 cut at this speed.

[0150] For specific enzymatic cleavage, the enzyme is brought into contact with the CM. When AA or BAA contains at least one AB coupled to MM and CM, for example, when AA contains AB coupled to MM via CM, and a target and sufficient enzymatic activity are present, the CM can be cleaved. Sufficient enzymatic activity may refer to the enzyme's ability to contact and cleave the CM. It is easy to imagine that the enzyme may be present near the CM but unable to cleave it due to other cellular factors or protein modifications of the enzyme.

[0151] Exemplary CMs of this disclosure are provided in Table 4 above. In some embodiments, the CM is up to 15 amino acid length, up to 20 amino acid length, up to 25 amino acid length, up to 30 amino acid length, up to 35 amino acid length, up to 40 amino acid length, up to 45 amino acid length, up to 50 amino acid length, up to 60 amino acid length, amino acid length in the range of 10-60, amino acid length in the range of 15-60, amino acid length in the range of 20-60, amino acid length in the range of 25-60, amino acid length in the range of 30-60, amino acid length in the range of 35-60, and 40-50. It has an amino acid length in the range of 45-60, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 10-30, 15-30, 20-30, 25-30, 10-20, or 10-15.

[0152] 8. Masking area (MM) In both the activatable single-specific CD3 and EGFR AA and BAA described above, the AA / BAA contains MM. As described herein, the AA and BAA of the present invention contain a prodomain containing MM.

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

[0154] In some embodiments, MM is not a natural binding partner of AB. In some embodiments, MM shows no homology or substantially no homology to any natural binding partner of AB. In other embodiments, MM is approximately 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, MM is approximately 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, MM is approximately 50% identical to any natural binding partner of AB. In some embodiments, MM is approximately 25% identical to any natural binding partner of AB. In some embodiments, MM is approximately 20% identical to any natural binding partner of AB. In some embodiments, MM is approximately 10% identical to any natural binding partner of AB.

[0155] The exemplary MMs of this disclosure include those with a maximum amino acid length of 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, 50 amino acids, 60 amino acids, amino acids in the range of 10-60, 15-60, 20-60, 25-60, 30-60, 35-60, 40-50, and 45-60. The amino acid lengths may be in the range of 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 10-30, 15-30, 20-30, 25-30, 10-20, 10-15, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0156] As provided herein, MM inhibits the binding of AB to its target. MM binds to the antigen-binding domain of AB, thereby inhibiting the binding of AB to its target. MM can sterically inhibit the binding of AB to its target. MM can allosterically inhibit the binding of AB to its target. In these embodiments, AB is modified by or coupled to MM and in the presence of a target, and in vivo assays or in When measured by a vitro assay, there is no binding of AB to the target or virtually no binding of AB to the target, or the binding of AB to the target is approximately 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 120%, 150%, or 80 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer, compared to the binding of AB not modified by MM or not coupled to MM, parental AB, or AB not coupled to MM to the target.

[0157] When AB is coupled to or modified by MM, MM “masks,” reduces, or otherwise inhibits the specific binding of AB to its target. When AB is coupled to or modified by MM, such coupling or modification may result in a structural change that reduces or inhibits AB’s ability to specifically bind to its target.

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

[0159] In either AA or BAA 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., mobile linkers) into the AA / BAA construct to provide mobility to one or more of the MM-CM junction, CM-AB / CM-scFv junction, or both. For example, AB, MM, and / or CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser) to provide the desired mobility. As such, the ability of such a BAA construct to be intact (unactivated) or to be activated as disclosed herein would benefit from introducing one or more amino acids to provide mobile linkers.

[0161] For example, in one embodiment, AA includes one of the following formulas (where the following formula represents the amino acid sequence either from the N-terminus to the C-terminus or from the C-terminus to the N-terminus): (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) (In the formula, MM, CM, and AB are as defined above; L1 and L2 are independently and optionally present or absent, and they are the same or different mobile linkers containing at least one mobile amino acid (e.g., Gly, Ser)).

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

[0163] In some embodiments, a structure including a linker is provided in Figure 17.

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

[0165] A linker suitable for use in the compositions described herein is generally a linker that provides mobility to the modified AB or AA, thereby facilitating the inhibition of AB binding to a target. Such a linker is generally called a mobile linker. A suitable linker can be readily selected and may be any suitable length of various lengths (1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids (including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids)), and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid lengths. In some embodiments, a suitable linker may be 4 to 25 amino acid lengths. In some embodiments, a suitable linker may be 5 to 25 amino acid lengths. In some embodiments, a suitable linker may be 4 to 20 amino acid lengths. In some embodiments, a suitable linker may be 5 to 20 amino acids long.

[0166] Exemplary linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO: 88), and (GGGS)n (SEQ ID NO: 89) (wherein n is an integer of at least 1)), glycine-alanine polymers, alanine-serine polymers, and other mobile linkers known in the art. In some embodiments, n is about 1 to about 10, or about 1 to about 9, or about 1 to about 8, or about 1 to about 7, or about 1 to about 6, or about 1 to about 5, or about 1 to about 4, or about 1 to about 3, or about 1 to about 2. Glycine and glycine-serine polymers are relatively unstructured and may therefore be able to function as neutral linkers between components. Glycine is significantly closer to the φ-ψ space than its equivalent alanine and is far less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary liquors are provided in Table 9-1.

[0167] [Table 13]

[0168] Those skilled in the art will recognize that the AA design may include linkers that are all or partially movable, thereby including one or more parts that provide a movable linker and a less movable structure to provide a desired AA structure.

[0169] 10. Conjugation In some embodiments, any antibody (i.e., AB of AA and AB of BAA as disclosed herein) can be conjugated to the drug. In some embodiments, the drug is a therapeutic agent. In some embodiments, the drug is a detectable portion. In some embodiments, the drug is an antineoplastic agent. In some embodiments, the drug is a toxin or a fragment thereof. In some embodiments, the drug is conjugated to AB via a linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the drug is a microtubule inhibitor. In some embodiments, the drug 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 drug is a drug selected from the group listed in Table 10.

[0170] [Table 14] TIFF0007860317000021.tif245167TIFF0007860317000022.tif102167

[0171] Those skilled in the art will recognize that a wide variety of possible parts can be coupled to the antibodies, AAs, and BAAs obtained in this disclosure (see, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, JMCruse and RELewis, 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 includes a detectable portion. In some embodiments, the detectable portion is a diagnostic agent.

[0173] In some embodiments, the antibody, AA, or BAA contains one or more disulfide bonds. In some embodiments, the antibody, AA, or BAA contains one or more lysine bonds. In some embodiments, the antibody, AA, or BAA can be manipulated to contain one or more disulfide bonds, or otherwise manipulated to enable site-specific conjugation.

[0174] 11. Production This disclosure also provides isolated nucleic acid molecules encoding antibodies, AAs, or BAAs as described herein, and vectors comprising these isolated nucleic acid sequences. This disclosure provides a method for producing antibodies, AAs, or BAAs by culturing cells under conditions that express antibodies, AAs, or BAAs, wherein the aforementioned cells contain such nucleic acid molecules.

[0175] In some embodiments, the cells include such vectors. 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 (these vectors and sequences described are provided in Example 1 below).

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

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

[0178] It will be recognized that the therapeutic entities of the Disclosure may be administered with appropriate carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, and tolerance. Numerous suitable formulations can be found in Chapter 87, particularly by Blaug and Seymour, of the prescription collection known to all pharmacists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)). 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, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. Any of the aforementioned mixtures may be suitable in the treatments and therapies of the Disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible with the route of administration and is tolerable. For further information regarding formulations, excipients, and carriers familiar to pharmacists, see 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 their cited references.

[0179] Generally, the alleviation or treatment of a disease or disorder involves reducing one or more symptoms or medical problems associated with that disease or disorder. For example, in the case of cancer, a therapeutically effective dose of a drug may achieve one or a combination of the following: reducing the number of cancer cells; reducing the size of the tumor; inhibiting the invasion of cancer cells into surrounding organs (i.e., reducing and / or stopping it to some extent); inhibiting tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating one or more symptoms associated with cancer to some extent. In some embodiments, compositions of the present disclosure can be used to prevent the onset or recurrence of a disease or disorder in a subject (e.g., a human or a non-human primate, a companion animal (e.g., a cat, a dog, a horse), livestock, a working animal, or other mammals such as zoo animals). The terms subject and patient are used interchangeably herein.

[0180] The therapeutically effective amount of antibody / bispecific antibody / AA / BAA in this disclosure generally refers to the amount required to achieve a therapeutic objective.

[0181] The general range of therapeutically effective doses of the antibody / bispecific antibody / AA / BAA of this disclosure may be, as a non-limiting example, approximately 0.1 mg / kg body weight to approximately 50 mg / kg body weight. The general frequency of administration may be, for example, in the range of twice a day to once a week.

[0182] The effectiveness of the treatment is determined in relation to any known method for diagnosing or treating a particular disorder. Methods for screening antibodies / bispecific antibodies / AA / BAA possessing the desired enantiomerity include, but are not limited to, enzyme-linked immunosorbent assays (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 antibody / bispecific antibody / AA / BAA is used in a manner known in the art related to the localization and / or quantification of a target (e.g., use in measuring the level of one or more targets in a suitable physiological sample, use in diagnostic methods, and use in protein imaging, etc.).

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

[0185] In yet another embodiment, antibodies directed to two or more targets, bispecific antibodies, AAs, BAAs 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 includes a detectable label. The antibody is a polyclonal antibody, or in some embodiments, a monoclonal antibody. An intact antibody or 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 terminal labeling of an antibody with biotin so 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 in the subject. Therefore, the term “biological sample” is used in scope to include blood and fractions or components of blood (including serum, plasma, or lymph). That is, proteins in biological samples can be detected in vitro and in vivo using the detection methods disclosed herein. For example, in vitro detection techniques for analytical proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. Procedures for performing immunoassays are described, for example, in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, JRCrowther (Ed.), Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christophorus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.Furthermore, the in vivo detection technique of the analyte protein involves introducing into the subject an antibody which is a labeled anti-analyte protein. For example, the antibody can be labeled with a radioactive marker whose presence and location in the 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 prophylactic formulations. In one embodiment, an antibody, AA, bispecific antibody, BAA is administered to a patient at risk of developing one or more of the aforementioned disorders. A predisposition to one or more disorders of the patient or organ can be determined using a genotype marker, serotype marker, or biochemical marker.

[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 mitigate or reverse the effects of the clinical indication.

[0188] Antibodies, bispecific antibodies, AAs, and bispecific antibodies are also useful in the detection of one or more targets in patient samples and are thus 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., ELISA) to detect one or more target levels in patient samples.

[0189] In one embodiment, an antibody, AA, bispecific antibody, BAA is immobilized on a solid support (e.g., a well of a microtiter plate). The immobilized antibody and / or AA functions as a capture antibody for any target(s) that may be present in the test sample. Prior to contact of 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 non-specific adsorption of analytes.

[0190] Subsequently, the wells are treated with a test sample suspected to contain the antigen or with a solution containing a standard amount of the antigen. Such use is, for example, a serum sample from a subject suspected to have circulating antigen levels that would lead to a diagnosis of disease. After washing away the test sample or standard, the solid support is treated with a second antibody that has been detected and labeled. The labeled second antibody functions as a detection antibody. The level of detectable labeling is measured, and the concentration of the target antigen(s) in the test sample is determined by comparison with a calibration curve prepared from the standard samples.

[0191] Based on results obtained using antibodies, AA, bispecific antibodies, and BAA in in vitro diagnostic assays, it will be recognized that the disease of a subject can be staged based on the expression level of target antigen(s). For a given disease, blood samples are collected from subjects diagnosed as being at various stages of disease progression and / or various points in the therapeutic treatment of the disease. Using sample populations that provide statistically significant results for each stage of progression or treatment, the concentration range of antigens that may be considered characteristic of each stage is specified.

[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. In some embodiments, such methods are in vivo. In some embodiments, such methods are in situ. In some embodiments, such methods are ex vivo. For example, AAs and bispecific antibodies having enzymatically cleavable CMs can be used to detect the presence or absence of an enzyme capable of cleaving CMs. Such AAs and bispecific antibodies can be used in diagnostics, which may include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential, such as one that can result in the reduction of disulfide bonds) by measuring the accumulation of activated antibodies or bispecific activated antibodies (i.e., antibodies or bispecific antibodies resulting from the cleavage of AAs or BAAs) in a given cell or tissue of a given host organism. Such accumulation of activated bispecific antibodies indicates not only that the tissue expresses enzyme activity (or an increase in reduction potential dependent on the properties of CMs), but also that the tissue expresses at least one target to which the activated bispecific antibody binds.

[0193] For example, CM can be selected to be a protease substrate of a protease found in tumor sites, viral or bacterial infection sites, and biologically closed sites (e.g., in abscesses and organs). At least one AB may be an AB that binds to the target antigen. Using methods familiar to those skilled in the art, a detectable label (e.g., fluorescent label, radioactive label, or radiotrace) can be conjugated to the AB or other region of the antibody, AA, bispecific antibody, BAA. Appropriate detectable labels are considered in the context of the above screening methods, and further specific examples are provided below. Using at least one AB specific to a diseased protein or peptide along with a protease whose activity is elevated in the affected tissue of interest, AA will show an increased binding rate to the affected tissue compared to tissue where the CM-specific enzyme is not present at a detectable level, is present at a lower level than in the affected tissue, or is inactive (e.g., in proto-enzymatic form or in the form of a complex with an inhibitor). Because small proteins and peptides are rapidly removed from the blood by the renal filtration system, and because enzymes specific to CM are not present at detectable levels (or are present at lower levels than in non-affected tissues, or exist in an inactive higher-order structure), the accumulation of activated bispecific antibodies in affected tissue is enhanced compared to non-affected tissue.

[0194] In another example, the antibodies, antibody / bispecific antibody / AA / BAA of this 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 enzymatic cleavage, the BAA can be used to detect the presence of the enzyme in the 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 the sample (qualitatively or quantitatively). To facilitate analysis in these methods, the antibody / bispecific antibody / AA / BAA can be detectably labeled and conjugated to a support (e.g., a solid support such as a slide or beads). The detectable label can be placed on a portion of the antibody / bispecific antibody / AA / BAA that is not released after cleavage, for example, the detectable label may be a quenched fluorescent label or other label that is not detectable until cleavage occurs. The assay can be carried out, for example, by contacting an immobilized and detectably labeled antibody / bispecific antibody / AA / BAA with a sample suspected to contain an enzyme and / or reducing agent for a sufficient amount of time to cleave, and then 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 evaluated by the change in the detectable signal of the antibody / bispecific antibody / AA / BAA before contact with the sample (e.g., the presence and / or increase of a detectable signal resulting from 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 detection of the presence or absence of a target capable of binding to at least one AB of the antibody / bispecific antibody / AA / BAA of the Disclosure. Thus, the assay can be adapted to evaluate the presence or absence of a cleavage agent and the presence or absence of a target of interest. The presence or absence of a cleavage agent can be detected by the presence and / or increase of a detectable label on the antibody / bispecific antibody / AA / BAA as described above, and the presence or absence of a target can be detected by the detection of the target-AB complex (e.g., by the use of a detectably labeled anti-target antibody).

[0196] The AA / BAA of this disclosure is also useful, for example, in in situ imaging 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. Using this technique, it is possible to confirm both binding to a given target and proteolytic activity based on the presence of a detectable label (e.g., a fluorescent label).

[0197] These techniques are useful with any frozen cells or tissues derived from affected areas (e.g., tumor tissue) or healthy tissue. They are also useful with fresh cell or tissue samples.

[0198] These techniques label AA / BAA with a detectable label. The detectable label may be a fluorescent dye (e.g., fluorophores, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (TRITC), AlexaFluor® label), near-infrared (NIR) dye (e.g., Qdot® nanocrystals), colloidal metal, hapten, radioactive marker, biotin and amplification reagent (e.g., streptavidin), or enzyme (e.g., horseradish peroxidase or alkaline phosphatase).

[0199] Detection of the label in a sample incubated with labeled AA or BAA indicates that the sample contains the target and a protease specific to the CM of AA or BAA of this 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 to the protease matriptase (MT-SP1) and inhibits the proteolytic activity of MT-SP1); see, for example, international publication number WO 2010 / 129609, published November 11, 2010. Using the same approach by using a broad-spectrum protease inhibitor (such as those described herein) and / or a more selective inhibitor, a protease or protease class specific to the CM of AA or BAA of this disclosure can be identified. In some embodiments, the presence of the target can be confirmed using a target-specific agent, or the detectable label can be made to compete with an unlabeled target. In some embodiments, unlabeled AA can be used in conjunction with detection by a labeled secondary antibody or a more complex detection system.

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

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

[0202] 13. Therapeutic administration The therapeutic entities of this disclosure will be recognized as being administered with appropriate carriers, excipients, and other agents incorporated into the formulation to improve transfer, delivery, and tolerance, etc. Numerous suitable formulations can be found in Chapter 87, particularly by Blaug and Seymour, of the prescription collection known to all pharmacists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)). 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, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. Any of the aforementioned mixtures may be suitable in the treatments and therapies of this disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible with the route of administration and is tolerable. For further information regarding formulations, excipients, and carriers familiar to pharmacists, see 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 their cited references.

[0203] In some embodiments, an antibody, bispecific antibody, AA, or BAA (or a conjugated composition thereof) is administered in combination with one or more further agents, or a combination of further agents. Suitable further agents include existing pharmaceutical and surgical agents for the intended application. For example, the further agents can be used in combination with further chemotherapeutic or antineoplastic agents.

[0204] In some embodiments, an antibody, bispecific antibody, AA, or BAA (or a conjugated composition thereof) of the Disclosure is administered in combination with one or more further agents selected from the group consisting of antibodies, conjugated antibodies, AA, conjugated AA, bispecific antibodies, conjugated bispecific antibodies, BAA, or conjugated BAA. In some embodiments, the antibody portion of any of the aforementioned further agents is directed to a target (such as one or more targets disclosed in Table 9). In some embodiments, the antibody portion of the antibody, bispecific antibody, AA, or BAA (or a conjugated composition thereof) and the antibody portion of the further agent are recognized as being directed to the same target (for example, both can target EGFR). In some embodiments, they are directed to the same target but target different epitopes. In some embodiments, they are directed to entirely different targets (for example, an activatable antibody of the Disclosure targeting EGFR can be administered in combination with an AA targeting a different target); similarly, for example, a BAA of the Disclosure targeting EGFR and CD3 can be administered in combination with an AA targeting a different target.

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

[0206] In some embodiments, these antibodies are formulated into a single therapeutic composition, and the antibody / bispecific antibody / AA / its BAA and further agents are administered simultaneously. Alternatively, the antibodies / bispecific antibodies / AA / its BAA are administered individually (for example, each is formulated into a separate therapeutic composition, and the antibody / bispecific antibody / AA / its BAA and further agents are administered simultaneously), or the antibodies / bispecific antibodies / AA / its BAA and further agents are administered at different times during the treatment regimen. The antibodies / bispecific antibodies / AA / its BAA and further agents can be administered in multiple doses.

[0207] Antibodies / bispecific antibodies / AA / their BAA can be incorporated into pharmaceutical compositions suitable for administration. 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 include an antibody / bispecific antibody / AA / its BAA 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, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington’s Pharmaceutical Sciences, a standard reference text in the art, which is 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. The use thereof in the compositions is intended, except in cases where any conventional media or agents are incompatible with the active compound.

[0210] Formulations used for in vivo administration must be sterile. This can be 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 administration (e.g., intravenous, intradermal, subcutaneous), oral administration (e.g., inhalation), transdermal administration (i.e., topical), transmucosal administration, 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 oil, 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. The pH can be adjusted with an acid or a base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules made of glass or plastic, disposable syringes, or multi-dose vials.

[0212] Suitable pharmaceutical compositions for use by injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. 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 exhibit fluidity to a degree that allows for easy use in a syringe. The composition must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Carriers may be solvents or dispersion media, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintenance of the required particle size in the case of dispersions, and the use of surfactants. Microbial activity can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, and thiromesal). In many cases, it would be appropriate to include isotonic agents, such as sugars, polyhydric alcohols (e.g., mannitol, sorbitol), and sodium chloride in the composition. The absorption of the injectable composition can be delayed by including absorption-delaying agents (e.g., aluminum monostearate and gelatin) in the composition.

[0213] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound along with one or a combination of the listed components in a suitable solvent, and subsequently sterilizing by filtration, if necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a base dispersion medium and other necessary components derived from the listed components. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which yield a powder of the active ingredient and any further desired components derived from a pre-filtered sterilized solution.

[0214] Oral compositions generally contain an inert diluent or an edible carrier. Oral compositions can be encapsulated in gelatin capsules or compressed into tablets. For therapeutic oral administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a gargle, where the fluid carrier containing the compound is applied orally, gargled with, and spat out, or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, and lozenges may contain any of the following ingredients or compounds of similar properties: binders (such as microcrystalline cellulose, tragacanth gum, or gelatin); excipients (such as starch or lactose); disintegrants (such as alginic acid, Primogel, or corn starch); lubricants (such as magnesium stearate or Sterotes); flow enhancers (such as colloidal silicon dioxide); sweeteners (such as sucrose or saccharin); or flavorings (such as peppermint, methyl salicylate, or orange flavoring).

[0215] For inhalation administration, the compound is delivered in the form of an aerosol spray from a pressurized container, pressurized dispenser, or nebulizer containing a suitable propellant (e.g., a gas such as carbon dioxide).

[0216] Systemic administration may also be by mucosal or dermal means. For mucosal or dermal administration, a penetrating agent that conforms to the barrier to be penetrated is used in the formulation. Such penetrating agents are generally known in the art and, for example, for mucosal administration, include surfactants, bile salts, and fusidic acid derivatives. Mucosal administration can be carried out using nasal sprays or suppositories. For dermal administration, the active compound is formulated into an ointment, plaster, gel, or cream, as is generally known in the art.

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

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

[0219] For example, active ingredients can be captured in microcapsules prepared by coacervation technology or interfacial polymerization (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.

[0220] Sustained-release preparations can be prepared. Examples of sustained-release preparations include a semipermeable matrix of a solid hydrophobic polymer containing an antibody, the matrix being in the form of a fabricated object (e.g., a thin film) or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), L-glutamic acid and γ-ethyl-L-glutamate copolymers, 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. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules over a period of 100 days, while certain hydrogels release proteins over a shorter period.

[0221] Materials can also be purchased from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing 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. Patent No. 4,522,811).

[0222] To facilitate administration and standardize dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit forms. As used herein, a dosage unit form refers to a physically distinct unit suitable as a single dose for a subject to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical carrier. The specifications of the dosage unit forms in this disclosure are determined and directly depend on the inherent characteristics of the active compound, the specific therapeutic effect to be achieved, and the inherent limitations in the field of formulation of such active compound for the treatment of an individual.

[0223] The pharmaceutical composition may be included in a container, pack, or dispenser along with instructions for administration.

[0224] The formulation may also contain more than one active compound necessary for the specific indication being treated (e.g., active compounds having complementary activity that does not adversely affect each other). Alternatively, the composition may further contain agents that enhance its function (e.g., cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors). Such molecules are appropriately present in the composition in amounts effective for the intended purpose.

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

[0226] For example, a combination therapy may include one or more antibodies / bispecific antibodies / AAs / their BAAs of the Disclosure that are co-formulated and / or co-administered with one or more further therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxic agents or cell proliferation inhibitors), as described in more detail below. Furthermore, one or more antibodies / bispecific antibodies / AAs / their BAAs described herein may be used in combination with two or more therapeutic agents described herein (e.g., one BAA is administered with another BAA or AA of the Disclosure). Such combination therapies can take advantage of lower doses of therapeutic agents and thus avoid the potential toxicity or complications associated with various monotherapies.

[0227] In other embodiments, one or more antibodies of the present disclosure may be formulated and / or administered concurrently 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: non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin); sulfasalazine; corticosteroids (e.g., prednisolone); cytokine-suppressing anti-inflammatory drugs (CSAIDs); nucleotide biosynthesis inhibitors (e.g., pre- Inhibitors of ion 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 this disclosure include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g., leflunomide), and folate antagonists (e.g., methotrexate).

[0228] Further examples of 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)); drugs that interfere with signaling by pro-inflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38, or MAP kinase inhibitors); COX2 inhibitors (e.g., celecoxib, rofecoxib, and their derivatives); phosphodiesterase inhibitors (e.g., R973401 (phosphodiesterase type IV inhibitor)); phospholipase inhibitors (e.g., Examples include inhibitors of cytoplasmic 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 antibodies of the disclosure 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 its variants); and phospholipase inhibitors (e.g., inhibitors of cytoplasmic phospholipase 2 (cPLA2), e.g., trifluoromethyl ketone analogs).

[0229] Further examples of therapeutic agents that can be combined with the antibodies of this 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; β-2 adrenergic receptor agonists (salbutamol, terbutaline, salmeterol); xanthines (theophylline, aminophylline); cromoglycates; nedocromil; ketotifen; ipratropium and oxytropium; mycophenolate mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and adrenergic agents.

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

[0231] 14. Manufacturing of kits and products Kits and products containing any one or more of the antibodies, AAs, bispecific antibodies, and BAAs provided herein are provided herein.

[0232] The kits and products may contain any one or more of the antibodies, AAs, bispecific antibodies, and BAAs provided herein in a form suitable for storage or transport.

[0233] The kit and product may contain at least the second component.

[0234] The kit and product may include a container, diluent, solvent, second composition, or any component useful for converting a composition in storage form into a composition suitable for use in the methods disclosed herein, if conversion is required. The methods may, for example, be therapeutic methods disclosed herein. The kit may include instructions for use.

[0235] The kits and products may include, for example, cytotoxic agents or detectable labels in a form suitable for conjugation with the drugs disclosed herein, antibodies provided herein, AAs, bispecific antibodies, and BAAs.

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

[0237] List of embodiments The present invention can be defined by reference to the following exemplary embodiments.

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

[0239] 2. The BAA according to Embodiment 1, wherein AB2 comprises a heavy chain variable domain described in SEQ ID NO: 2 or SEQ ID NO: 3 and a light chain variable domain described in SEQ ID NO: 1 or SEQ ID NO: 4.

[0240] 3. The BAA according to Embodiment 1, wherein AB1 binds to a tumor target and AB2 binds to an immune effector target.

[0241] 4. The BAA described in any one of Embodiments 1 to 3, wherein the BAA is a T cell-induced bispecific (TCB)AA (TCBAA).

[0242] 5. The BAA described in any one of Embodiments 1 to 4, wherein AB1 binds to EGFR and AB2 binds to CD3ε.

[0243] 6. The BAA described in any one of Embodiments 1 to 5, wherein the aforementioned MM1 includes an amino acid sequence selected from the group consisting of sequences shown in Table 7.

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

[0245] 8. The BAA described in any one of Embodiments 1 to 5, wherein the aforementioned MM1 includes Sequence ID No. 78.

[0246] 9. The BAA described in any one of Embodiments 1 to 8, wherein the aforementioned MM2 includes the amino acid sequence of SEQ ID NO: 12.

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

[0248] 11. The BAA described in any one of Embodiments 1 to 9, wherein the aforementioned CM includes the amino acid sequence of SEQ ID NO: 17.

[0249] 12. The BAA described in any one of Embodiments 1 to 9, wherein the aforementioned CM includes the amino acid sequence of SEQ ID NO: 16.

[0250] 13. The BAA described in any one of Embodiments 1 to 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 described in any one of Embodiments 1 to 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 to 14, wherein AB1 includes amino acid substitutions at least two of the amino acid positions L234, L235, and P331.

[0253] 16. The BAA according to Embodiment 15, wherein AB1 includes amino acid substitutions at amino acid positions L234, L235, and P331.

[0254] 17. The BAA according to Embodiment 15, wherein AB1 comprises the amino acid substitutions L234F, L235E, and P331S.

[0255] 18. The BAA according to Embodiment 15, wherein the aforementioned AB1 includes an Fc region containing an amino acid substitution at N297.

[0256] 19. The BAA described in any one of Embodiments 1 to 14, wherein AB1 includes amino acid substitutions at amino acid positions L234F, L235E, P331S, and N297Q.

[0257] 20. The BAA according to Embodiment 1, wherein the heavy chain of AB1 described above includes one of SEQ ID NOs. 69, SEQ ID NOs. 70, SEQ ID NOs. 71, SEQ ID NOs. 72, SEQ ID NOs. 73, SEQ ID NOs. 74, SEQ ID NOs. 75, and SEQ ID NOs. 76 as described in Table 6.

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

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

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

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

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

[0263] 26. The AA according to Embodiment 25, wherein the aforementioned CM includes one of the sequences listed in Table 4.

[0264] 27. The AA according to Embodiment 25, wherein the aforementioned CM comprises a substrate that can be cleaved by a serine protease or MMP.

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

[0266] 29. The AA according to Embodiment 25, wherein the aforementioned protease is an MMP.

[0267] 30. AA according to Embodiment 25, wherein the aforementioned protease is a serine protease.

[0268] 31. The AA according to Embodiment 25, wherein the AB that specifically binds to the aforementioned CD3 is the antibody described in any one of Embodiments 38 to 47.

[0269] 32. Activatable antibodies (AA), a. Antibodies or antigen-binding fragments (AB) that specifically bind to the epidermal growth factor receptor (EGFR); b. A masking moiety (MM) coupled to AB, wherein the aforementioned MM reduces or inhibits the binding of the aforementioned AB to EGFR when the aforementioned AA is in an uncleaved state, and wherein the aforementioned MM includes an amino acid sequence selected from the group consisting of sequences shown in Table 7; and c. A cleavable moiety (CM) coupled to AB, wherein the aforementioned CM is a polypeptide that functions as a substrate for a protease. Activatable antibodies (AA) including [specific antibody].

[0270] 33. The AA according to Embodiment 32, wherein the aforementioned MM includes the amino acid sequence of Sequence ID No. 78.

[0271] 34. The AA according to any one of Embodiments 32 to 33, wherein the aforementioned CM comprises a substrate that can be cleaved by a serine protease or MMP.

[0272] 35. The AA according to any one of embodiments 32 to 33, wherein the aforementioned CM includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 18 to 56.

[0273] 36. The AA according to Embodiment 32, wherein the aforementioned CM includes the amino acid sequence of SEQ ID NO: 14.

[0274] 37. The AA according to Embodiment 32, wherein the aforementioned CM includes the amino acid sequence of SEQ ID NO: 16.

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

[0276] 39. The antibody according to Embodiment 38, wherein the antibody comprises the heavy chain variable domain described in SEQ ID NO: 2 or SEQ ID NO: 3 and the light chain variable domain described in SEQ ID NO: 1 or SEQ ID NO: 4.

[0277] 40. AB according to Embodiment 38, comprising the heavy chain variable domain described in Sequence ID No. 2.

[0278] 41. AB according to Embodiment 38, comprising the heavy chain variable domain described in Sequence ID No. 3.

[0279] 42. The antibody described above comprises the light chain variable domain described in SEQ ID NO: 1, as described in Embodiment 38.

[0280] 43. AB according to Embodiment 38, comprising the light chain variable domain described in Sequence ID No. 4.

[0281] 44. AB according to Embodiment 38, comprising the heavy chain variable domain described in Sequence ID No. 2 and the light chain variable domain described in Sequence ID No. 1.

[0282] 45. AB according to Embodiment 38, comprising the heavy chain variable domain described in Sequence ID No. 3 and the light chain variable domain described in Sequence ID No. 1.

[0283] 46. ​​AB according to Embodiment 38, comprising the heavy chain variable domain described in Sequence ID No. 3 and the light chain variable domain described in Sequence ID No. 4.

[0284] 47. AB according to Embodiment 38, comprising the heavy chain variable domain described in Sequence ID No. 2 and the light chain variable domain described in Sequence ID No. 4.

[0285] 48. The AB described above is a bispecific AB as described in any one of Embodiments 32 to 47.

[0286] 49. The AA according to any one of Embodiments 32 to 47, wherein the antibody described above is scFv.

[0287] 50. AA according to any one of Embodiments 32 to 47, wherein the aforementioned antibody is an IgG1 antibody.

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

[0289] 52. AA according to Embodiment 51, wherein the aforementioned AB includes the heavy chain variable domain described in Sequence ID No. 2.

[0290] 53. The AA according to Embodiment 51, wherein the aforementioned AB includes the heavy chain variable domain described in Sequence ID No. 3.

[0291] 54. AA according to Embodiment 51, wherein the aforementioned AB includes the light chain variable domain described in Sequence ID No. 1.

[0292] 55. AA according to Embodiment 51, wherein the aforementioned AB includes the light chain variable domain described in Sequence ID No. 4.

[0293] 56. AA according to Embodiment 51, wherein the aforementioned AB includes the heavy chain variable domain described in Sequence ID No. 2 and the light chain variable domain described in Sequence ID No. 1.

[0294] 57. AA according to Embodiment 51, wherein the aforementioned AB includes the heavy chain variable domain described in Sequence ID No. 3 and the light chain variable domain described in Sequence ID No. 1.

[0295] 58. AA according to Embodiment 51, wherein AB includes the heavy chain variable domain described in Sequence ID No. 2 and the light chain variable domain described in Sequence ID No. 4.

[0296] 59. AA according to Embodiment 51, wherein AB includes the heavy chain variable domain described in Sequence ID No. 3 and the light chain variable domain described in Sequence ID No. 4.

[0297] 60. The AA described in any one of Embodiments 51 to 59, wherein the aforementioned MM includes one of the sequences listed in Table 3.

[0298] 61. The AA described in any one of Embodiments 51 to 59, wherein the aforementioned CM includes one of the sequences listed in Table 4.

[0299] 62. A bispecific activatable antibody (BAA) comprising any one of the AAs described in Embodiments 51 to 61.

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

[0301] 64. The AA according to Embodiment 63, wherein the Fc region described above includes amino acid substitutions at least at amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, such that the effector function of the AA described above is reduced.

[0302] 65. The AA according to embodiment 63 or 64, wherein the aforementioned target is selected from the group consisting of the targets shown in Table 9.

[0303] 66. Bispecific activatable antibody (BAA), a. An IgG antibody (AB1) that specifically binds to a first target, wherein the aforementioned AB1 comprises two heavy chains (AB1 HC) and two light chains (AB1 LC); wherein the aforementioned AB1 is linked to a first masking portion (MM1) linked to a first cleavable portion (CM1) to form an MM1-CM1 construct, wherein the carboxyl terminus of the aforementioned MM1-CM1 construct is linked to the amino terminus of each light chain of the aforementioned AB1, The aforementioned MM1 inhibits the binding of the aforementioned AB1 to its target; The aforementioned CM1 is a polypeptide that functions as a substrate for the first protease, IgG antibody (AB1), b. Two scFv (each AB2) each specifically bind to a second target, wherein each AB2 includes a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 is linked to the respective amino terminus of the aforementioned AB1 heavy chain; wherein each AB2 is linked to a second masking region (MM2) linked to a second cleavable region (CM2) to form an MM2-CM2 construct, wherein the carboxyl terminus of each MM2-CM2 construct is linked to the amino terminus of each AB2, The aforementioned MM2 inhibits the binding of the aforementioned AB2 to its target; The aforementioned CM2 is a polypeptide that functions as a substrate for the second protease, consisting of two scFv molecules (each AB2). Includes, Here, AB1 is a bispecific activatable antibody (BAA) comprising an Fc region containing an amino acid substitution at at least one of the amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, such that the effector function of the BAA is reduced.

[0304] 67. The BAA according to Embodiment 66, wherein the Fc region described above includes amino acid substitutions at least at amino acid positions L234, L235, N297, and P331, numbered by the EU index described in Kabat, such that the effector function of the BAA described above is reduced.

[0305] 68. The BAA according to Embodiment 66, wherein the aforementioned Fc region includes amino acid substitutions at least at amino acid positions L234, L235, and P331, numbered by the EU index described in Kabat, such that the effector function of the BAA is reduced.

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

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

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

[0309] 72. The AA described in any one of embodiments 32 to 37 and 51 to 71, wherein the aforementioned AA is BAA.

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

[0311] 74. The pharmaceutical composition according to Embodiment 73, comprising further agents.

[0312] 75. The pharmaceutical composition according to Embodiment 74, wherein the further agent described above is a therapeutic agent.

[0313] 76. An isolated nucleic acid molecule encoding an antibody, AA, or BAA as described in any one of Embodiments 1 to 72.

[0314] 77. A vector comprising the isolated nucleic acid molecule described in Embodiment 76.

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

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

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

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

[0319] 82. A cell containing 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 according to any one of Embodiments 1 to 72 by culturing cells under conditions that express an antibody, AA, or BAA, wherein the cells include a nucleic acid molecule according to Embodiment 76 or a vector according to any one of Embodiments 78 to 81.

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

[0324] 87. The method according to Embodiment 86, wherein the aforementioned disorder or disease comprises disease cells expressing EGFR.

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

[0326] 89. The method according to Embodiment 88, wherein the aforementioned cancer is anal cancer, basal cell carcinoma, brain cancer, bladder cancer, breast cancer, bone cancer, cervical cancer, intrahepatic cholangiocarcinoma, 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 a therapeutically effective amount of an antibody, AA, or BAA described in any one of Embodiments 1 to 72, or a pharmaceutical composition described in any one of Embodiments 73 to 75, to a subject in need thereof.

[0328] 91. The method according to any one of embodiments 86 to 90, comprising administering a further drug.

[0329] 92. The method according to Embodiment 91, wherein the further drug described above is a therapeutic agent.

[0330] 93. A method for reducing damage to healthy tissue resulting from the binding of antibodies to its targets on affected tissue and on healthy tissue, comprising administering AA or BAA or a pharmaceutical composition comprising AA or BAA to a subject in need thereof, wherein the aforementioned AA or BAA is the AA or BAA described in any one of the embodiments provided herein.

[0331] 94. A method for improving the tolerability of an antibody treatment, comprising administering AA or BAA or a pharmaceutical composition comprising AA or BAA to a subject in need thereof, wherein the aforementioned AA or BAA is the 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 AA or BAA or a pharmaceutical composition comprising AA or BAA to a subject requiring such recruitment, wherein the aforementioned AA or BAA is the 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 pharmaceutical.

[0334] 97. 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 manner of treating a disorder or disease, alleviating its symptoms, or delaying its progression, wherein the aforementioned disorder or disease comprises disease cells expressing EGFR.

[0335] 98. 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 treating cancer, wherein the cancer is optionally anal cancer, basal cell carcinoma, brain cancer, bladder cancer, breast cancer, bone cancer, cervical cancer, intrahepatic cholangiocarcinoma, 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, wherein the method described above includes inhibiting angiogenesis.

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

[0338] Examples Example 1. Sequence construction, vector construction, and expression of antibody, BAA, and activated BAA. Target antibody The molecules provided in Table 11 below were constructed and tested. As shown, the activated molecules were produced in a manner that they were masked and then cleaved by proteolysis to generate the activated form.

[0339] [Table 15]

[0340] The sequences of the molecules and vectors are provided below. Square brackets indicate some of the components of the molecule shown. Linkers are provided in some sequences. Underlined amino acids indicate the putative CDR sequence. CI011:3954-0001-C225v5N297Q-JF15865-0001-CD3LvHv-HN pLW023:HC JF15865-0001-CD3LvHv-C225v5N297Q(HN) Nucleotide sequence [Spacer SEQ ID NO: 176][pLW023 does not include Spacer SEQ ID NO: 177] amino acid sequence [Spacer SEQ ID NO: 178][pLW023 does not include Spacer SEQ ID NO: 179] QGQSGQ[MMYCGGNEVLCGPRV][GSSGGSGGSGG][LSGRSDNH][GGGS]QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 106) OPP022:LC 3954-0001-C225v5 Nucleotide sequence [Spacer SEQ ID NO: 180][OPP022 (excluding Spacer SEQ ID NO: 181)] (Sequence ID 107) amino acid sequence [Spacer SEQ ID NO: 178][OPP022 does not include Spacer SEQ ID NO: 182] [SDNH][GSSGT][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 108) CI020:3954-Nsub-C225v5N297Q-JF15865-Nsub-hSP34LvHv-HN pLW073:HC C225v5N297Q-JF15865-Nsub-hSP34LvHv(HN) Nucleotide sequence [Spacer SEQ ID NO: 176][pLW073 does not include Spacer SEQ ID NO: 183] amino acid sequence [Spacer SEQ ID NO: 178][pLW073 does not include Spacer SEQ ID NO: 184] QGQSS LKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNKS QVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 110) pLW071:LC 3954-Nsub-C225v5 Nucleotide sequence [Spacer SEQ ID NO: 180][pLW071 does not include 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(Sequence ID 112) CI040:3954-2001-C225v5N297Q-JF15865-2001-hSP34LvHv-HN pLW101:HC JF15865-2001-CD3LvHv-C225v5N297Q(HN) Nucleotide sequence [Spacer SEQ ID NO: 176][pLW101 does not include Spacer SEQ ID NO: 187] amino acid sequence [Spacer SEQ ID NO: 178][pLW101 does not include Spacer SEQ ID NO: 188] QGQSGQ[MMYCGGNEVLCGPRV][GSSGGSGGGSGG][ISSGLLSGRSDNH][GGGS]QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGSGGGGS]EVQLVESGGG LVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 114) CTX122:LC 3954-2001-C225v5 Nucleotide sequence [Spacer SEQ ID NO: 180][CTX122 without Spacer SEQ ID NO: 189] (Sequence ID 115) amino acid sequence [Spacer SEQ ID NO: 178][CTX122 without Spacer SEQ ID NO: 190] QGQSGQ[CISPRGCPDGPYVMY][GSSGGSGGSGGSG][ISSGLLSGRSDNH][GSSGT]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 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 [SDNH][GSSGT]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 120) CI079:3954-0001-C225v5Fcmt3-h20GG-0001-v16sc-HN pLW225:HC h20GG-0001-v16sc-C225v5Fcmt3(HN) Nucleotide sequence [Spacer SEQ ID NO: 191][pLW225 does not include Spacer SEQ ID NO: 192] amino acid sequence [Spacer SEQ ID NO: 178][pLW225 does not include Spacer SEQ ID NO: 193] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGGSGG][LSGRSDNH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 122) OPP022:LC 3954-0001-C225v5 The sequence 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 does not include Spacer SEQ ID NO: 194] amino acid sequence [Spacer SEQ ID NO: 178][pLW233 does not include Spacer SEQ ID NO: 195] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGGSGG][LSGRSDNH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 124) OPP022:LC 3954-0001-C225v5 The sequence 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](Sequence ID 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(Sequence ID 126) Activated pLW291:LC 3954-0011-C225v5 Nucleotide sequence TCCGATGATCATGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 127) Amino acid sequence [SDDH][GSSGT]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 128) CI106:CF41-2008-C225v5Fcmt4-h20GG-0011-v16sc-HN pLW289:HC h20GG-0011-v16sc-C225v5Fcmt4(HN) Nucleotide sequence [Spacer SEQ ID NO: 191][pLW289 does not include Spacer SEQ ID NO: 196] amino acid sequence [Spacer SEQ ID NO: 178][pLW289 does not include Spacer SEQ ID NO: 197] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGGSGG][LSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 130) pLW246:LC CF41-2008-C225v5 Nucleotide sequence [Spacer SEQ ID NO: 176][pLW246 without Spacer SEQ ID NO: 198] (Sequence ID 131) amino acid sequence [Spacer SEQ ID NO: 178][pLW246 does not include Spacer SEQ ID NO: 199] QGQSGQG[LSCEGWAMNREQCRA][GGGSSGGS][ISSGLLSGRSDQH][GGGS]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 132) CI107:3954-0011-C225v5Fcmt4-h20GG-2006-v16sc-HN pLW307:HC h20GG-2006-v16sc-C225v5Fcmt4(HN) Nucleotide sequence [Spacer SEQ ID NO: 191][pLW307 does not include Spacer SEQ ID NO: 200] amino acid sequence [Spacer SEQ ID NO: 178][pLW307 does not include Spacer SEQ ID NO: 201] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][ISSGLLSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGSGGGGS]EVQLVESGGG LVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 134) pLW291:LC 3954-0011-C225v5 Nucleotide sequence [Spacer SEQ ID NO: 180][pLW291 does not include Spacer SEQ ID NO: 202] (Sequence ID 135) amino acid sequence [Spacer SEQ ID NO: 178][pLW291 does not include Spacer SEQ ID NO: 203] QGQSGQ[CISPRGCPDGPYVMY][GSSGGSGGSGGSG][LSGRSDDH][GSSGT]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 136) CI127:SynFcmt4-h20GG-0011-v16sc-HN pLW334:HC h20GG-0011-v16sc-Synagis(registered trademark)Fcmt4(HN) Nucleotide sequence [Spacer SEQ ID NO: 191][pLW334 does not include Spacer SEQ ID NO: 204] amino acid sequence [Spacer SEQ ID NO: 178][pLW334 does not include Spacer SEQ ID NO: 205] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGGSGG][LSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKD TSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 138) pLW139:LC Synagis (registered trademark) Nucleotide sequence GACATCCAGATGACCCAGAGCCCCAGCACACTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACATGCAAGTGCCAGCTGAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACACCAGCAAGCTGGCCTCCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGAGTTCACCCTGACAATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGTTTTCAAGGCTCCGGCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 139) Amino acid sequence DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 140) CI128:SynFcmt4-h20GG-2006-v16sc-HN pLW335:HC h20GG-2006-v16sc-Synagis(registered trademark)Fcmt4(HN) Nucleotide sequence [Spacer SEQ ID NO: 191][pLW335 does not include Spacer SEQ ID NO: 206] amino acid sequence [Spacer SEQ ID NO: 178][pLW335 does not include Spacer SEQ ID NO: 207] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][ISSGLLSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGSGGGGS]EVQLVESGGG LVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTIS KDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 142) pLW139: LC Synagis (registered trademark) The sequence 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 does not include Spacer SEQ ID NO: 209] amino acid sequence [Spacer SEQ ID NO: 176][pLW352 does not include Spacer SEQ ID NO: 210] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGGSGG][LSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 146) pLW246:LC CF41-2008-C225v5 Nucleotide sequence The array provided above amino acid sequence The array 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 does not include Spacer SEQ ID NO: 211] amino acid sequence [Spacer SEQ ID NO: 178][pLW353 does not include Spacer SEQ ID NO: 212] QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][LSGRSDDH][GGGS]QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQ APRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQP GGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS KSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 148) pLW246:LC CF41-2008-C225v5 The array 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 does not include Spacer SEQ ID NO: 213] amino acid sequence [Spacer SEQ ID NO: 178][pLW242 does not include Spacer SEQ ID NO: 214] QGQSGS[GYLWGCEWNCGGITT]GSSGGSGGSGG[ISSGLLSGRSDQH](Sequence ID 169) CX320:3954-C225v5-2008 nucleic acid sequence [Spacer SEQ ID NO: 180] [CX320 does not include Spacer SEQ ID NO: 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(Sequence ID 171) CI064:SynN297Q-JF15865-0001-hSP34LvHv-HN pLW138:HC SynN297Q-JF15865-0001-hSP34LvHv-HN nucleic acid sequence [Spacer SEQ ID NO: 176][pLW138 (excluding Spacer SEQ ID NO: 147)] amino acid sequence [Spacer SEQ ID NO: 178][pLW138 (excluding Spacer SEQ ID NO: 153)] QGQSGQ[MMYCGGNEVLCGPRV]GSSGGSGGSGG[LSGRSDNH](Sequence ID 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 array provided above Anti-CD3 scFv variant v16 Light Chain LV12 Heavy chain HV20 The array provided above Anti-CD3 scFv variant v19 Light Chain LV19 Heavy chain HV20 The array provided above Anti-CD3 scFv variant v26 Light Chain LV19 Heavy chain HV12 The array provided above

[0341] Vector construction The heavy and light chains were cloned separately into mammalian expression vectors using standard molecular biology techniques. Briefly, DNA fragments encoding the region of interest were amplified using end-binding primers. Duplicate fragments were combined and amplified using adjacent primers as needed to construct the complete desired region. Subsequently, the DNA fragments were cloned into expression vectors using a commercially available homologous recombination kit (MCLabs, South San Francisco, CA). The mammalian expression vectors are modified versions of Invitrogen's cDNA® 3.1(+) containing either the G418 or hygromycin selection marker. Mutations were introduced using the QuikChange kit (Agilent, Santa Clara, CA).

[0342] Expression of AA and double-masked BAA (BAA) AA and BAA 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. AA and double-masked BAA were purified from cell-free supernatant using protein A beads (GE, Piscataway, NJ) and concentrated using a standard buffer exchange column (Millipore, Temecula, CA).

[0343] Example 2. Binding of double-masked, bispecific AA to EGFR+HT-29 cells and CD3ε+Jurkat cells. Flow cytometry-based binding assays were performed to determine whether the substrates of the EGFR and CD3ε-masking peptides and proteases described can inhibit binding in double-masked, bispecific AA.

[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 guidance. The following bispecific activating antibodies CI048 and CI104 (act-104), as well as the bispecific AAs CI011, CI106, and CI107, which are bi-masked, were tested. Two versions of SP34 scFv were used (i.e., scFv in CI011 and CI048 versus scFv in CI104, CI106, and CI107). Two versions of EGFR masks were used (i.e., EGFR masks in CI011 and CI107 versus EGFR mask in CI106). Two versions of CD3 masks were used (i.e., CD3 mask in CI011 versus CD3 masks in CI106 and CI107).

[0345] HT29-luc2 cells were detached with Versene® (Life Technologies, catalog number 15040-066), washed, plated in 96-well plates at 150,000 cells / well, and resuspended in 50 μL of primary antibody. The dose was started at the concentrations shown in Figures 1A-1B, followed by 3-fold serial dilutions with FACS staining buffer + 2% FBS (BD Pharmingen, catalog number 554656). The cells were incubated at 4°C for approximately 1 hour with shaking, harvested, and washed with 2 × 200 μL of FACS staining buffer. The cells were resuspended in 50 μL of AlexaFluor 647-conjugated anti-human IgG Fc (10 μg / ml, Jackson ImmunoResearch, product number 109-606-008) and incubated at 4°C for approximately 1 hour with shaking. HT29-luc2 cells were collected, washed, and resuspended in 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 MACSQuant® Analyzer 10 (Miltenyi), and the median fluorescence intensity (MFI) of viable cells was calculated using FlowJo® V10 (Treestar). The MFI data with background subtraction was graphed using curve fitting analysis in GraphPad Prism6.

[0346] Jurkat cells grown in suspension were harvested, washed, plated in 96-well plates at 150,000 cells / well, 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 the incorporation of the h20GG CD3ε masking peptide into CI106 and CI107, which are EGFR-masked BAAs, significantly reduces binding to Jurkat cells compared to CI011. In some embodiments, binding to Jurkat cells was reduced to less than 1 / 5,000. In some embodiments, the disclosed scFv also reduced binding. The reduction in binding to EGFR+HT29-luc2 cells was also evident with CI106 and CI107 compared to CI011 (Figure 1B). In some embodiments, binding to EGFR+HT29-luc2 cells was reduced to less than 1 / 1,000. In Figures 1A and 1B, the double-masked BAAs reduce binding compared to the activated bispecific antibody.

[0348] Example 3. EGFR-dependent cytotoxicity of double-masked BAA Cytotoxicity assays were performed to determine whether masking CD3ε and EGFR and protease substrates in CI106 and CI107 could further attenuate cell death compared to CI011 and CI040. Human PBMCs were purchased in frozen aliquots (HemaCare) and co-cultured in RPMI-1640 + glutamax (supplemented with 5% heat-inactivated human serum (Sigma, catalog no. H3667)) with EGFR-expressing HT29-luc2 cells in a 10:1 ratio. The following bispecific activating antibodies and bi-masked BAAs were tested for dose settings: CI011, CI040, activated CI104, CI106, and CI107. Furthermore, EGFR-dependent cytotoxicity was demonstrated using non-EGFR-binding masked bispecific AAs, CI127 and CI128. After 48 hours, cytotoxicity was assessed using the ONE-Glo® luciferase assay system (Promega, catalog number E6130). Luminescence was measured using Infinite M200 Pro (Tecan). Cytotoxicity rates were calculated and plotted using curve-fit analysis in GraphPad PRISM. The number of EGFR receptors in the cell line panel was quantified by flow cytometry using QIFIKIT (Dako).

[0349] Figure 2A demonstrates that EGFR+HT29-luc2 cell death 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 death is dependent on EGFR targeting. Furthermore, Figure 2B shows an EC50 shift of over 300,000-fold for the bispecific antibodies CI106 and CI107, which were bi-masked compared to the protease-activated bispecific antibody act-104. Figure 2C shows the number of EGFR receptors in a panel of cell lines including HT29. The approximate number of EGFR receptors on HT29 cells is 75,000, indicating that high antigen density is not required for potent cytotoxicity of the tested antibodies.

[0350] Example 4. Primary T cell activation by double-masked BAA Flow cytometry assays were performed to determine whether masking 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, the cells were pelleted, the medium was removed, and the cells were resuspended in a 50 μl cocktail containing anti-CD45 VioBlue® (Miltenyi, catalog no. 130-002-880), anti-CD8 APC-Vio770 (Miltenyi, catalog no. 130-096-561), and anti-CD69 PE (BD Pharmingen, catalog no. 555531) in FACS staining buffer + 2% FBS. The cells were stained at 4°C for 1 hour with shaking, harvested, washed, and resuspended in a final volume of 60 μL of FACS buffer. Data were acquired using MACSQuant® Analyzer 10 (Miltenyi), and activation was quantified as the percentage of CD8+ T cells expressing CD69 relative to PE isotype control using FlowJo® V10 (Treestar). The data were plotted using curve fitting analysis in GraphPad PRISM6.

[0351] Figure 3A demonstrates that primary CD8+ T cell activation 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 double-masked antibodies compared to the protease-activating bispecific antibody act-104, demonstrating that masking weakens T cell activation.

[0352] Example 5. The dual-masked, dual-specific AA of the embodiment induced regression of established HT29-luc2 tumors in mice. In this example, we analyzed the ability of CI106 and CI107, which are dual-masked BAAs targeting EGFR and CD3ε, to induce regression or reduction in the growth of established HT-29-Luc2 xenograft tumors in human T cell-transplanted NSG mice.

[0353] 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(trademark)(NOD.Cg-Prkdcscid Il2rg tm1Wjl I obtained the / SzJ) mouse from The Jackson Laboratories, Bar Harbor, ME.

[0354] On day 0, 2 × 10⁶ units of RPMI + Glutamax (serum-free medium) were placed in the right flank of each mouse. 6 Individual HT29-luc2 cells were subcutaneously inoculated. Pre-frozen PBMCs from a single donor were administered on day 3 in a CD3+ T cell:tumor cell ratio of 1:1 (ip). Tumor volume was 200 mm². 3 Upon reaching this stage (approximately day 12), mice were randomized and assigned to either a treatment group or an IV medication group according to Table 12. Tumor volume and body weight were measured twice weekly.

[0355] [Table 16]

[0356] Figure 4 plots tumor volume versus days after initial treatment, demonstrating the dose-dependent effect of the double-masked, bispecific AAs CI106 and CI107 on the growth of HT29-luc2 xenograft tumors. The most effective test dose was 1.5 mg / kg, which resulted in tumor regression. Statistical analysis (RMANOVA with Dunnett against PBS controls) was performed using GraphPad PRISM. * =p<0.05, ** =p<0.01, **** = p < 0.0001.

[0357] Example 6. The dual-masked, bispecific AA and bispecific antibody of the embodiment reduce the growth of established HCT116 tumors in mice. In this example, we analyzed the ability of bispecific antibodies targeting EGFR and CD3ε, activated CI104, and bimasked BAAs, CI106 and CI107, to induce regression or reduction in the growth of established HCT116 xenograft tumors in human T cell-transplanted NSG mice. 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 run as described in Example 5. Mice were administered the drugs according to Table 13.

[0358] [Table 17]

[0359] Figure 5 plots tumor volume versus days after initial treatment administration, demonstrating the dose-dependent effect of the double-masked, bispecific AAs CI106 and CI107 on the growth of HCT116 xenograft tumors. The most effective test dose was 1.0 mg / kg, which induced tumor quiescence. Act-104 administered at 0.3 mg / kg also resulted in tumor quiescence, demonstrating a 3-fold difference in efficacy between the double-masked, bispecific antibody and the protease-activated bispecific antibody. Statistical analysis (RMANOVA with Dunnett against PBS control) was performed using GraphPad PRISM. * =p<0.05, ** =p<0.01, **** = p < 0.0001.

[0360] Example 7. Cross-reactivity to double-masked, bispecific AA cynomolgus monkey T cells. To confirm that cynomolgus monkeys are the relevant toxic species, protease-activated CI104, CI106, and CI107 were used in flow cytometry-based cell binding assays and HT29-luc2 cytotoxicity assays using cynomolgus monkey pan-T cells (Bioreclamation IVT), and their potency was compared to that of human PBMCs. The protocols were as described in Examples 2 and 3.

[0361] Figures 6A and 6B demonstrate that the EC50 of the double-masked bispecific antibody and the protease-activating bispecific antibody tested in cytotoxic assays is similar when using either human (6A) or cynomolgus monkey (6B) effector cells. Figures 6C and 6D demonstrate that the binding of the protease-activating antibody and the double-masked antibody to human (6C) and cynomolgus monkey (6D) T cells is similar.

[0362] Therefore, the crab-eating macaque was determined to be a suitable species for tolerability studies.

[0363] Example 8. Mutations in the Fc region affect the tolerability of double-masked, bispecific AA in cynomolgus monkeys. In this study, naive cynomolgus monkeys (n=1) were administered 600 μg / kg of CI079 and CI090 to evaluate 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 intake. This study was conducted at SNBL USA, Ltd. (Everett, WA) following standard operating procedures.

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

[0365] [Table 18]

[0366] [Table 19]

[0367] Example 9. Tolerance of double-masked BAA in cynomolgus monkeys In this example, naive cynomolgus monkeys (n=1) were administered CI106 and CI107 at doses of 600, 2000, 4000 μg / kg (CI107 only), or 6000 μg / kg (CI107 only) to establish the maximum tolerable dose (MTD) after a single IV bolus. 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. Tolerance was assessed based on clinical signs, body weight, food intake, and laboratory analysis including serological, hematological, cytokine analysis, and flow cytometry to evaluate T cell activation. Blood was collected once during acclimatization and before, 48 hours, 72 hours (hematology only), and 7 days after administration for standard serological and hematological analysis. Blood samples were collected for cytokine analysis before medication administration and at 1, 4, 8, and 24 hours after administration. Flow cytometry was performed on peripheral blood before medication administration, 72 hours after administration, and 7 days after administration. This study was conducted at SNBL USA, Ltd. (Everett, WA) according to standard operating procedures.

[0368] CI107 administered at 6000 μg / kg was lethal within 24 hours of administration. 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, if present, were transient and generally limited to within 48 hours after administration. Serological 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 animals treated with CI107 at 2000 and 4000 μg / kg, total bilirubin increased above the normal range at 48 hours and completely reversed by day 8. In both CI106-treated and CI107-treated animals, transient increases in serum cytokines IL-2, IL-6, and IFNg were observed after administration and recovered within 24 hours post-administration. Increases in the percentage of T cells expressing CD69, Ki67, and PD-1 were observed at 72 hours post-administration, and generally, the percentage of positive cells was higher in CI107-treated animals.

[0369] Figures 7A–7C show serum concentrations of ALT (7A), AST (7B), and total bilirubin (7C) before, 48 hours after, and 7 days after administration. All values, except for total bilirubin at 2000 μg / kg and 4000 μg / kg, are within the established normal range for cynomolgus monkeys. Only pre-administration data was available for CI107 at 6000 μg / kg, so 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] Figures 9A-9C show T cell activation as measured by the expression of CD69 (9A), Ki67 (9B), and PD-1 (9B) on CD4+ T cells.

[0372] Example 10. Double-masked bispecific AA is safer than activated bispecific antibodies in cynomolgus monkeys. In this example, protease-activated CI104 and double-masked CI106 and CI107 were administered to cynomolgus monkeys (n=1) at doses of 60, 180 (activated CI104 only), 600, 2000, 4000 μg / kg (CI107 only), or 6000 μg / kg (CI107 only), and the tolerability of masked and unmasked antibodies after a single IV bolus was compared. Tolerability evaluation and blood collection were as described in Example 9. The double-masked BAAs, CI106 and CI107, were tolerable at dose levels 30 to 60 times higher than those of the protease-activated bispecific antibodies.

[0373] Figures 10A–E plot the dose-dependent increases of AST (10A), ALT (10B), IL-6 (10C), IFNg (10D), and Ki67 (10E) at 48 hours post-administration, and 8 hours post-administration, respectively. The dose-response curves for all parameters are shifted for the double-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 shifted more than 60-fold.

[0374] Example 11. The tolerability of a double-masked, bispecific AA binding to EGFR is EGFR-dependent. In this example, cynomolgus monkeys (n=1) were administered 2000 μg / kg of the double-masked bispecific antibodies CI107 (targeting EGFR and CD3ε) and CI128 (targeting RSV and CD3ε). Tolerability evaluation and blood sampling were performed as described in Example 9 above. CI128 had no effect on the measured values ​​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 insufficient to induce toxicity.

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

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

[0377] Humanization of anti-human CD3 single-chain variable fragments (scFv) was performed by selective mutation of the framework. Briefly, CDRs were grafted onto a series of light-chain (LC) and heavy-chain (HC) human IgG backbones, and several amino acids in the variable region framework were selectively mutated. Immunoglobulins were expressed in all possible combinations of LC and HC, and then evaluated for expression levels, monomer percentage, and CD3 affinity using ELISA and on-cell binding to Jurkat cells. The variable regions of preferred combinations were expressed as scFv in the form of bispecific antibody (TCB), and then evaluated for expression levels, monomer percentage, CD3 affinity, and function in cytotoxic assays.

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

[0379] The kinetic analysis was performed at 20°C in PBST (10 mM sodium phosphate (pH 7.4), 150 mM sodium chloride, 0.05% Tween-20). Regeneration consisted of 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 prepared 10 mM sodium hydroxide.

[0380] The preparation was carried out using alternating reverse 3-fold serial dilutions with a buffer blank. Human CD3egFc was derived from Sino Biological Inc., (Beijing, China, catalog number CT041-H0305H) and reconstituted from a PBS-based lyophilized preparation using sterile water. Serial dilutions of the analyte in solution were initiated at concentrations of 300 nM or 100 nM human CD3. The analysis was performed using Scrubber software.

[0381] These variants were also manipulated using the described method to create bi-masked, bi-specific AAs targeting EGFR and CD3, which were then used in in vitro cytotoxic 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 bispecific antibodies or bispecific antibodies bispecific antibodies against HT29-luc2 cells. There was only slight difference in the cell-killing efficacy of the activated molecules, with v16 being the most potent. There was also only slight difference in protection against cell death for the bispecific molecules.

[0383] Example 13. Double-masked BAA can enhance PK in cynomolgus monkeys.

[0384] In this example, cynomolgus monkeys were administered 60 μg / kg, 180 μg / kg (act-104), or 2000 μg / kg (CI107) of the protease-activated bispecific antibody act-104 and the bi-masked bispecific antibody CI107. Plasma samples were collected at 5 minutes (act-104 only), 30 minutes, 4 hours (act-104 only), 24 hours, 48 ​​hours (act-104 only), 96 hours, and 168 hours. Plasma concentrations were measured by ELISA using an anti-idiotype antibody for capture and horseradish peroxidase (HRP)-labeled 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-activating molecule act-104. The exposure (AUC) for CI107 is 448 days. * It is nM, and for act-104 (60 μg / kg), it takes 0.04 days. * The value was nM, showing a difference of more than 10,000 times compared to plasma exposure.

[0386] Example 14. Protease cleavage sensitivity of double-masked, bispecific AA correlates with tumor efficacy and tumor T cell infiltration. This example describes the antitumor efficacy and tumor T cell infiltration in an HT29-luc2 xenograft model. The model was performed as described in Example 5. In the tumor T cell infiltration study, mice were administered a single dose of the test substance, and tumors were collected 7 days after administration. Formalin-fixed paraffin-embedded (FFPE) blocks were prepared and used for histology. The test substances used were CI011, CI020 (a double-masked, bispecific antibody lacking a cleavable substrate), CI040, and CI048. The protease sensitivity and substrate cleavage of the test substances were as follows: CI040 > CI011 > CI020. Mice were administered the test substances according to Table 16.

[0387] [Table 20]

[0388] Figure 14A shows the efficacy of the HT29-luc2 tumor intervention model in PBMC-transplanted NSG mice. In this example, the antitumor efficacy correlated with the protease sensitivity and substrate cleavage of the test substance, with the most effective test substance being CI048 fully activated by protease.

[0389] Figure 14B shows the staining (dark staining) of tumor sections for CD3 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 substance.

[0390] Example 15. The second-generation double-masked, double-specific AA is safer in cynomolgus monkeys than the first-generation molecule. 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 doses of 20 (CI048 only), 60, or 180 μg / kg (act-104 only). Double-masked CI011, CI040, CI106, and CI107 were administered at doses of 600, 2000, 4000 (CI107 only), or 6000 (CI107 only) μg / kg to compare the tolerability of single IV bolus doses of double-masked bispecific antibodies and activated bispecific antibodies. The evaluation of tolerability was as described in Example 8.

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

[0392] [Table 21]

[0393] Example 16. Evaluation of the masking effectiveness of an activatable anti-EGFR antibody. Masking of an antibody's ability to bind to its antigen is an example of binding inhibition and is listed herein as masking efficiency (ME). Masking efficiency is defined as the K of AA binding. D The antibody-bound K measured under identical conditions DIt can be calculated by dividing by [the specified factor]. The range of inhibition depends on the antibody's affinity for its antigen, the affinity of the inhibitor (i.e., the masking portion) for the antibody, and the concentration of the total reactant. The local concentration of the anchored masking portion peptide (inhibitor) is very high in AA (approximately 10 mM), and therefore, a peptide with moderate affinity will effectively mask AA antigen binding.

[0394] The assay is outlined below: Nunc Maxisorp® plates are coated overnight at 4°C with 100 μl / well of 1 μg / ml human EGFR (R and D Systems) solution 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 at RT for 2 hours with 200 μl / well of 10 mg / mL BSA in PBST. The plates are washed with 3×PBST (PBS, pH 7.4, 0.05% Tween-20). Dilution curves can be prepared with 10 mg / mL BSA in PBST, as illustrated in Table 18 below. In this example, the maximum concentrations are 10 nM for the parent antibody and 400 nM for AA, but the higher concentration can be increased or decreased to obtain fully saturated binding curves for AA with stronger or weaker masking.

[0395] [Table 22]

[0396] The conjugation solution is added to the plate, which is then incubated at room temperature for 1 hour, and then washed with 3×PBST (PBS, pH 7.4, 0.05% Tween-20). 100 μl / well of goat-anti-human IgG (Fab-specific, Sigma catalog number 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 colored with TMB and 1N HCl. Figures 15 and 16 show conjugation isotherm plots for the activated anti-EGFR C225v5 antibody of this disclosure, the activated anti-EGFR antibody 3954-2001-C225v5 mentioned above, and anti-EGFR antibody C225v5. The plots were created in GraphPad PRISM, and the data were fitted to a single-site saturation model. D Determine. K D The values ​​and ME values ​​are provided in Table 19.

[0397] [Table 23]

[0398] Example 17. Pharmacokinetics of double-masked BAA in cynomolgus monkeys. In this example, cynomolgus monkeys were administered 600 μg / kg, 2000 μg / kg, or 4000 μg / kg of double-masked bispecific antibody CI107. 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] Figure 20 shows the PK of double-masked BAA CI107 after administration of a single IV dose of 600, 2000, or 4000 μg / kg.

[0400] Example 18. EGFR-dependent cytotoxicity of a double-masked, bispecific, activatable antibody. To determine whether anti-CD3ε, CD3 masks, and protease substrates in CI090 and CI091 could further jeopardize cell death compared to CI011, cytotoxicity assays were performed using the method described in Example 3. The following bispecific activating antibodies and bispecific activatable antibodies with bi-masking were tested for dose settings: CI011, CI090, CI091, activated CI090, and CI048. Furthermore, the EGFR-dependent cytotoxicity was demonstrated using the non-EGFR-binding bispecific activatable antibody CI064.

[0401] Figure 21 demonstrates that EGFR+HT29-luc2 cell death was further weakened by CI090 and CI091 compared to CI011, although the potency of activated CI090 was equal to that of CI048. Increased EC50 shifts were observed for CI090 and CI091 compared to activated bispecific antibodies, indicating increased masking efficiency of these molecules compared to CI011. No cytotoxicity was observed when cells were treated with CI064, demonstrating that cell death is dependent on EGFR targeting.

[0402] Example 19. Primary T cell activation by a double-masked, bispecific, activatable antibody. To determine whether anti-CD3ε, CD3 mask, and protease substrates can weaken primary T cell activation in CI090 and CI091 compared to CI011, flow cytometry assays were performed as described in Example 4.

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

[0404] Example 20. The dual-masked, dual-specific activatable antibody of the embodiment 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 the growth of established HT-29-Luc2 xenograft tumors in human PBMC-grafted NSG mice. The method is as described in Example 5.

[0405] [Table 24]

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

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

[0408] Figure 24 plots IL-6 levels 8 hours after administration. The bispecific activatable antibody CI011, double-masked, induced significantly lower cytokine release than activated CI104, even when delivered at higher doses, demonstrating its masking effect on T cell activation. IL-6 levels were even more reduced in animals treated with CI090 and CI091, reflecting increased masking efficiency for these molecules compared to CI011. Other Embodiments

[0409] Although the present invention is described in conjunction with its detailed description, the foregoing description is intended to be illustrative and does not limit the scope of the invention, which is limited by the scope of the appended claims. Other embodiments, advantages, and modifications are within the scope of the following:

Claims

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

12.

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

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

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

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

6. The purified polypeptide according to claim 5, wherein the serine protease is a matryptase or a urokinase-type plasminogen activator (uPA).

7. The purified polypeptide according to claim 2, comprising a linker between SEQ ID NO: 12 and the polypeptide which is a substrate for a protease.

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

9. The purified polypeptide according to claim 2, wherein the purified polypeptide contains a linker at the carboxyl terminus of the polypeptide which is a substrate for a protease.

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

11. The purified polypeptide according to claim 10, wherein L1 and L2 are the same.

12. A purified polypeptide according to claim 10, wherein L1 and L2 are different.

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