Molecules that bind to follistatin polypeptides

Abstract

Provided herein are methods and materials involving conjugating binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) to follistatin polypeptides. For example, provided are binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) that bind to follistatin polypeptides, as well as methods and materials for using one or more such binding molecules to treat a mammal (e.g., a human) with cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 63 / 442,943, filed February 2, 2023. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated by reference into the disclosure of this application.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file entitled "48881-0048WO1_SL.xml." This XML file, created on January 29, 2024, is 581,555 bytes in size. The material in the XML file is incorporated herein by reference in its entirety.

[0003] background Technical Field This specification relates to methods and materials involving binding of molecules (e.g., antibodies, antibody fragments, antibody domains, chimeric antigen receptors (CARs), cell engagers, or antibody-drug conjugates (ADCs)) to follistatin polypeptides. For example, this specification provides binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, or ADCs) that bind to follistatin polypeptides, as well as methods and materials for treating cancer using such binding molecules, alone or in combination with one or more chemotherapeutic agents. This specification also provides cells (e.g., host cells) engineered to express one or more binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to follistatin polypeptides, and methods and materials for using such cells to treat cancer. [Background technology]

[0004] Background information Follistatin (FST), also known as activin-binding protein, is an extracellular glycoprotein (approximately 35 kD in its most prevalent form) encoded in humans by the FST gene. FST was identified as an inhibitor of pituitary follicle-stimulating hormone secretion. FST primarily functions in binding and neutralizing members of the TGF-β superfamily, with a particular emphasis on activin, which is involved in the proliferation, differentiation, and apoptosis of numerous cell types. FST is involved in tumorigenesis, metastasis, and angiogenesis of solid tumors through its interaction with activin. Elevated serum levels of follistatin polypeptide have been reported in patients with ovarian cancer, indicating that follistatin polypeptide may be a potential tumor marker for ovarian cancer diagnosis. Therefore, follistatin polypeptide is a potential target for treating various types of cancer. Summary of the Invention

[0005] overview Provided herein are methods and materials involving binding of molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, or ADCs) to follistatin polypeptides. For example, provided herein are binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, or ADCs) that bind to follistatin polypeptides, as well as methods and materials for using one or more such binding molecules to treat a mammal (e.g., a human) with cancer (e.g., ovarian cancer, lung cancer, and / or prostate cancer). In some cases, provided herein are methods and materials for using one or more binding molecules described herein in combination with one or more chemotherapeutic agents to treat a mammal (e.g., a human) with cancer (e.g., ovarian cancer, lung cancer, and / or prostate cancer).

[0006] Also provided herein are cells (e.g., host cells) engineered to express one or more binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to a follistatin polypeptide, as well as methods and materials for using such cells to treat cancer (e.g., ovarian cancer, lung cancer, and / or prostate cancer). In some cases, the present description provides methods and materials for using one or more such cells in combination with one or more chemotherapeutic agents to treat a mammal (e.g., a human) with cancer (e.g., ovarian cancer, lung cancer, and / or prostate cancer).

[0007] As described herein, binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more CARs, one or more cell engagers, and / or one or more ADCs) can be designed to have the ability to bind to a follistatin polypeptide. For example, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, or ADC) provided herein can be capable of binding to a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of the human follistatin polypeptide set forth in SEQ ID NO: 113 and / or SEQ ID NO: 115 (see, e.g., Example 2).

[0008] As described herein, anti-follistatin binding molecules (e.g., clones #1-#14) bind to a follistatin polypeptide (e.g., human follistatin polypeptide). See, e.g., Figures 4-6. Furthermore, exposing cancer cells to an anti-follistatin binding molecule described herein (e.g., any one of clones #1-#14) and a chemotherapeutic agent (e.g., taxol or cisplatin) enhances the ability of the chemotherapeutic agent to kill the cancer cells. See, e.g., Figures 7-9. These results indicate that the anti-follistatin binding molecules provided herein can be used to treat cancer (e.g., ovarian cancer, lung cancer, and / or prostate cancer).

[0009] In some cases, a single set of three CDRs of an antibody domain (e.g., a VH domain) provided herein (e.g., SEQ ID NOs: 1-3; SEQ ID NOs: 9-11; SEQ ID NOs: 17-19; SEQ ID NOs: 25-27; SEQ ID NOs: 33-35; SEQ ID NOs: 41-43; SEQ ID NOs: 49-51; SEQ ID NOs: 57-59; SEQ ID NOs: 65-67; ​​SEQ ID NOs: 73-75; SEQ ID NOs: 81-83; SEQ ID NOs: 89-91; SEQ ID NOs: 97-99; or SEQ ID NOs: 105-107) is genetically engineered into a CAR to encode follistatin. + cells (e.g., follistatin + Tumor cells and / or follistatin + CARs with the ability to target tumor vasculature + Cells (e.g., CAR + T cells, CAR + CARs such as induced pluripotent stem cells + stem cells, or CARs + NK cells); and by genetic engineering, the Fc region can be incorporated into antibody structures containing follistatin. + cells (e.g., follistatin + Tumor cells and / or follistatin + Antibodies with the ability to target tumor vasculature (e.g., tumor vasculature) can be generated to target follistatin. +and / or can be genetically engineered into cell engagers, such as bispecific T cell engagers (e.g., BiTEs), bispecific killer engagers (e.g., BiKEs), and / or trispecific killer engagers (e.g., TriKEs), to induce ADCC against cells. + cells (e.g., follistatin + Tumor cells and / or follistatin + To generate cell engagers with the ability to target tumor vasculature, targeting follistatin + One or more immune responses can be induced against the cells (e.g., a T cell immune response and / or ADCC using cell engagers in the absence of Fc-containing antibodies). Note that BiKE- and TriKE-mediated killing is sometimes referred to as ADCC even though it is not initiated by the Fc domain when the engager lacks an Fc domain.

[0010] Furthermore, as described herein, the binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, and / or one or more antibody domains) can be used to generate conjugates comprising the binding molecule and a drug. For example, ADCs, such as full-length antibody-drug conjugates, Fab-drug conjugates, scFv-drug conjugates, and / or antibody domain-drug conjugates, can be designed to include an appropriate binding molecule provided herein to generate the conjugate. Such conjugates can be used to target cancer cells (e.g., follistatin + cancer cells) or cancer vasculature (e.g., follistatin + It can be used to deliver drug payloads to target cells, such as cancer vasculature.

[0011] Also as described herein, the binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) can be used to treat a mammal (e.g., a human) with cancer. For example, cancer (e.g., follistatin + A composition comprising one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) described herein can be administered to a mammal (e.g., a human) having cancer (e.g., ovarian, lung, and / or prostate cancer) to reduce the number of cancer cells in the mammal, induce ADCC against cancer cells in the mammal, and / or increase survival time from cancer (e.g., ovarian, lung, and / or prostate cancer) in the mammal.

[0012] Also as described herein, cells (e.g., host cells) can be engineered to express one or more binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, or cell engagers) capable of binding to a follistatin polypeptide. For example, cells such as T cells (e.g., CTLs), stem cells (e.g., induced pluripotent stem cells), or NK cells can be engineered to express one or more CARs capable of binding to a follistatin polypeptide. Such cells (e.g., follistatin-specific CARs) can be engineered to express one or more CARs capable of binding to a follistatin polypeptide. + T cells or NK cells) can be used to treat cancer (e.g., ovarian cancer, lung cancer, and / or prostate cancer).

[0013] In some cases, the binding molecules (e.g., antibodies, antigen-binding fragments, and / or antibody domains) provided herein can be used to detect the presence or absence of a follistatin polypeptide. For example, the binding molecules (e.g., antibodies, antigen-binding fragments, and / or antibody domains) provided herein can be used to detect the presence or absence of a follistatin polypeptide when a specimen (e.g., a biological sample such as a tumor biopsy) obtained from a mammal (e.g., a human) detects the presence or absence of a follistatin polypeptide.+ cells (e.g., follistatin + The present invention can be used to determine whether a cancer cell contains a follistatin polypeptide (e.g., a follistatin + Having the ability to detect the presence or absence of follistatin (cancer cells) can enable clinicians, healthcare professionals, and patients to make better decisions about possible treatment options. + The detection of cancer cells provides clinicians, healthcare professionals, and patients with information about follistatin. + This allows for the selection of an appropriate anti-cancer treatment that targets cancer cells. + Therapies that target cancer cells include administering one or more binding molecules described herein capable of binding to a follistatin polypeptide, and / or administering one or more cells engineered to express a binding molecule described herein (e.g., a follistatin-specific CAR). + Examples include administration of T cells or NK cells.

[0014] In general, one aspect herein features an antibody comprising (or consisting essentially of, or consisting of): (i) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions); (ii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: (iii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 10 (or SEQ ID NO: 10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO: 11 (or SEQ ID NO: 11 with one, two or three amino acid additions, deletions or substitutions); (iv) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 (or SEQ ID NO: 17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 19 with one, two or three amino acid additions, deletions or substitutions), (iv) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:25 (or SEQ ID NO:25 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:26 (or SEQ ID NO:26 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:27 (or (v) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 33 (or SEQ ID NO: 33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 34 (or SEQ ID NO: 34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 35 (or SEQ ID NO: 35 with one, two, or three amino acid additions, deletions, or substitutions),(vi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 41 (or SEQ ID NO: 41 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO: 42 (or SEQ ID NO: 42 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO: 43 (or SEQ ID NO: 43 with the addition, deletion, or substitution of one, two, or three amino acids); (vii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 49 (or SEQ ID NO: 49 with the addition, deletion, or substitution of one, two, or three amino acids), (viii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 49 (or SEQ ID NO: 50 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 50 (or SEQ ID NO: 50 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 51 (or SEQ ID NO: 51 with one, two, or three amino acid additions, deletions, or substitutions); (viii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 57 (or SEQ ID NO: 57 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 58 (or SEQ ID NO: 59 with one, two, or three amino acid additions, deletions, or substitutions), (ix) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 65 (or SEQ ID NO: 65 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 66 (or SEQ ID NO: 66 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 67 (or SEQ ID NO: 68 with one, two, or three amino acid additions, deletions, or substitutions). (x) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 73 (or SEQ ID NO: 73 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 74 (or SEQ ID NO: 74 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 75 (or SEQ ID NO: 75 with one, two, or three amino acid additions, deletions, or substitutions); (xi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 81 (or one, two, or three amino acid additions, deletions, or substitutions),(xii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 89 (or SEQ ID NO: 89 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 90 (or SEQ ID NO: 90 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 91 (or SEQ ID NO: 91 with one, two, or three amino acid additions, deletions, or substitutions); (xiii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 97 (or SEQ ID NO: 88 with one, two, or three amino acid additions, deletions, or substitutions). (xiv) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO: 105 (or SEQ ID NO: 105 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 106 (or SEQ ID NO: 106 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 107 (or SEQ ID NO: 107 with one, two, or three amino acid additions, deletions, or substitutions). The antibody may comprise the ability to bind to SEQ ID NO: 113 and / or SEQ ID NO: 115. The antibody may comprise the heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 8. The antibody may comprise a heavy chain variable domain or region of (ii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 16. The antibody may compriseThe antibody may comprise a heavy chain variable domain or region of (iii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:24. The antibody may comprise a heavy chain variable domain or region of (iv). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:32. The antibody may comprise a heavy chain variable domain or region of (v). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:40. The antibody may comprise a heavy chain variable domain or region of (vi). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:48. The antibody may comprise a heavy chain variable domain or region of (vii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:56. The antibody may comprise a heavy chain variable domain or region of (viii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 64. The antibody may comprise a heavy chain variable domain or region of (ix). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 72. The antibody may comprise a heavy chain variable domain or region of (x). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 80. The antibody may comprise a heavy chain variable domain or region of (xi). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 88. The antibody may comprise a heavy chain variable domain or region of (xii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 96. The antibody may compriseThe antibody may comprise a heavy chain variable domain or region of (xiii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 104. The antibody may comprise a heavy chain variable domain or region of (xiv). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 112. The antibody may be a monoclonal antibody. The antibody may be an scFv antibody. This paragraph may be referred to as the first aspect paragraph.

[0015] In another aspect, the present specification features an antigen-binding fragment comprising (or consisting essentially of, or consisting of): (i) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:1 (or SEQ ID NO:1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:2 (or SEQ ID NO:2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:3 (or SEQ ID NO:3 with one, two, or three amino acid additions, deletions, or substitutions); (ii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 9 (or SEQ ID NO: 9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 10 (or SEQ ID NO: 10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 11 (or SEQ ID NO: 11 with one, two, or three amino acid additions, deletions, or substitutions); (iii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 (or SEQ ID NO: 17 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 18 ( (iv) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:25 (or SEQ ID NO:25 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:26 (or SEQ ID NO:26 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:27 (or (v) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 33 (or SEQ ID NO: 33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 34 (or SEQ ID NO: 34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 35 (or SEQ ID NO: 35 with one, two, or three amino acid additions, deletions, or substitutions),(vi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 41 (or SEQ ID NO: 41 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO: 42 (or SEQ ID NO: 42 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO: 43 (or SEQ ID NO: 43 with the addition, deletion, or substitution of one, two, or three amino acids); (vii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 49 (or SEQ ID NO: 49 with the addition, deletion, or substitution of one, two, or three amino acids), (viii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 49 (or SEQ ID NO: 50 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 50 (or SEQ ID NO: 50 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 51 (or SEQ ID NO: 51 with one, two, or three amino acid additions, deletions, or substitutions); (viii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 57 (or SEQ ID NO: 57 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 58 (or SEQ ID NO: 59 with one, two, or three amino acid additions, deletions, or substitutions), (ix) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 65 (or SEQ ID NO: 65 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 66 (or SEQ ID NO: 66 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 67 (or SEQ ID NO: 68 with one, two, or three amino acid additions, deletions, or substitutions). (x) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 73 (or SEQ ID NO: 73 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 74 (or SEQ ID NO: 74 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 75 (or SEQ ID NO: 75 with one, two, or three amino acid additions, deletions, or substitutions); (xi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 81 (or one, two, or three amino acid additions, deletions, or substitutions),(xii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 89 (or SEQ ID NO: 89 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 90 (or SEQ ID NO: 90 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 91 (or SEQ ID NO: 91 with one, two, or three amino acid additions, deletions, or substitutions); (xiii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 97 (or SEQ ID NO: 88 with one, two, or three amino acid additions, deletions, or substitutions). (xiv) a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO: 105 (or SEQ ID NO: 105 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 106 (or SEQ ID NO: 106 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 107 (or SEQ ID NO: 107 with one, two, or three amino acid additions, deletions, or substitutions). The antigen-binding fragment may comprise the ability to bind to SEQ ID NO: 113 and / or SEQ ID NO: 115. The antigen-binding fragment may comprise the heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 8. The antigen-binding fragment may comprise the heavy chain variable domain or region of (ii). The heavy chain variable domain or region may comprise:The antigen-binding fragment may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 16. The antigen-binding fragment may comprise a heavy chain variable domain or region of (iii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 24. The antigen-binding fragment may comprise a heavy chain variable domain or region of (iv). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 32. The antigen-binding fragment may comprise a heavy chain variable domain or region of (v). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 40. The antigen-binding fragment may comprise a heavy chain variable domain or region of (vi). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 48. The antigen-binding fragment may comprise a heavy chain variable domain or region of (vii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 56. The antigen-binding fragment may comprise a heavy chain variable domain or region of (viii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 64. The antigen-binding fragment may comprise a heavy chain variable domain or region of (ix). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 72. The antigen-binding fragment may comprise a heavy chain variable domain or region of (x). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 80. The antigen-binding fragment may comprise a heavy chain variable domain or region of (xi). The heavy chain variable domain or region mayThe antigen-binding fragment may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 88. The antigen-binding fragment may comprise a heavy chain variable domain or region of (xii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 96. The antigen-binding fragment may comprise a heavy chain variable domain or region of (xiii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 104. The antigen-binding fragment may comprise a heavy chain variable domain or region of (xiv). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 112. The antigen-binding fragment may be monoclonal. The antigen-binding fragment may be a Fab. This paragraph may be referred to as the second aspect paragraph.

[0016] In another aspect, the present specification features an antibody domain comprising (or consisting essentially of, or consisting of): (i) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 2 (or SEQ ID NO: 2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 3 (or SEQ ID NO: 3 with one, two, or three amino acid additions, deletions, or substitutions); (ii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 2 (or SEQ ID NO: 2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 3 (or SEQ ID NO: 3 with one, two, or three amino acid additions, deletions, or substitutions); ) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two or three amino acid additions, deletions or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two or three amino acid additions, deletions or substitutions); (iii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:17 (or SEQ ID NO:17 with one, two or three amino acid additions, deletions or substitutions), SEQ ID NO:18 (or (iv) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 25 (or SEQ ID NO: 25 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 26 (or SEQ ID NO: 26 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 27 (or (v) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 33 (or SEQ ID NO: 33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 34 (or SEQ ID NO: 34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 35 (or SEQ ID NO: 35 with one, two, or three amino acid additions, deletions, or substitutions),(vi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 41 (or SEQ ID NO: 41 with the addition, deletion, or substitution of one, two, or three amino acids), SEQ ID NO: 42 (or SEQ ID NO: 42 with the addition, deletion, or substitution of one, two, or three amino acids), and SEQ ID NO: 43 (or SEQ ID NO: 43 with the addition, deletion, or substitution of one, two, or three amino acids); (vii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 49 (or SEQ ID NO: 49 with the addition, deletion, or substitution of one, two, or three amino acids), (viii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 49 (or SEQ ID NO: 50 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 50 (or SEQ ID NO: 50 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 51 (or SEQ ID NO: 51 with one, two, or three amino acid additions, deletions, or substitutions); (viii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 57 (or SEQ ID NO: 57 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 58 (or SEQ ID NO: 59 with one, two, or three amino acid additions, deletions, or substitutions), (ix) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 65 (or SEQ ID NO: 65 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 66 (or SEQ ID NO: 66 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 67 (or SEQ ID NO: 68 with one, two, or three amino acid additions, deletions, or substitutions). (x) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 73 (or SEQ ID NO: 73 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 74 (or SEQ ID NO: 74 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 75 (or SEQ ID NO: 75 with one, two, or three amino acid additions, deletions, or substitutions); (xi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 81 (or one, two, or three amino acid additions, deletions, or substitutions),(xii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 89 (or SEQ ID NO: 89 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 90 (or SEQ ID NO: 90 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 91 (or SEQ ID NO: 91 with one, two, or three amino acid additions, deletions, or substitutions); (xiii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 97 (or SEQ ID NO: 88 with one, two, or three amino acid additions, deletions, or substitutions). (xiv) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 105 (or SEQ ID NO: 105 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 106 (or SEQ ID NO: 106 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 107 (or SEQ ID NO: 107 with one, two, or three amino acid additions, deletions, or substitutions). This antibody domain may comprise the ability to bind to SEQ ID NO: 113 and / or SEQ ID NO: 115. The antibody domain may comprise the heavy chain variable domain or region of (i). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 8. The antibody domain may comprise a heavy chain variable domain or region of (ii). The heavy chain variable domain or region may comprise:The antibody domain may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 16. The antibody domain may comprise a heavy chain variable domain or region of (iii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 24. The antibody domain may comprise a heavy chain variable domain or region of (iv). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 32. The antibody domain may comprise a heavy chain variable domain or region of (v). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 40. The antibody domain may comprise a heavy chain variable domain or region of (vi). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 48. The antibody domain may comprise a heavy chain variable domain or region of (vii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 56. The antibody domain may comprise a heavy chain variable domain or region of (viii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 64. The antibody domain may comprise a heavy chain variable domain or region of (ix). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 72. The antibody domain may comprise a heavy chain variable domain or region of (x). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 80. The antibody domain may comprise a heavy chain variable domain or region of (xi). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 88. The antibody domain may compriseThe antibody domain may comprise a heavy chain variable domain or region of (xii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 96. The antibody domain may comprise a heavy chain variable domain or region of (xiii). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 104. The antibody domain may comprise a heavy chain variable domain or region of (xiv). The heavy chain variable domain or region may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 112. The antibody domain may be monoclonal. The antibody domain may be a VH domain. This paragraph may be referred to as the third aspect paragraph.

[0017] In another aspect, the present specification features a chimeric antigen receptor comprising (or consisting essentially of, or consisting of) an antigen-binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein the antigen-binding domain comprises (or consists essentially of, or consists of) any antibody, any antigen-binding fragment, or any antibody domain of any of the first aspect paragraph, the second aspect paragraph, or the third aspect paragraph. The antigen-binding domain can comprise a VH domain capable of binding to a follistatin polypeptide. The hinge can comprise a hinge described in Example 23. The transmembrane domain can comprise a transmembrane domain described in Example 24. The chimeric antigen receptor can comprise one or more signaling domains described in Example 25. This paragraph may be referred to as a fourth aspect paragraph.

[0018] In another aspect, this document features a cell comprising (or consisting essentially of, or consisting of) the chimeric antigen receptor of any of the preceding paragraphs. The cell can be a T cell, a stem cell, or an NK cell.

[0019] In another aspect, the present specification features a cell engager comprising (or consisting essentially of, or consisting of) a first antigen-binding domain, a linker, and a second antigen-binding domain, wherein the first antigen-binding domain comprises (or consists essentially of, or consists of) any antibody, any antigen-binding fragment, or any antibody domain of any of the first, second, or third embodiment paragraphs. The first antigen-binding domain can comprise a VH domain capable of binding to a follistatin polypeptide. The first antigen-binding domain can be an IgG capable of binding to a follistatin polypeptide. The linker can comprise a linker described in Example 20 or Example 23. The second antigen-binding domain can bind to a polypeptide expressed on the surface of a T cell. The polypeptide expressed on the surface of a T cell can be a CD3 polypeptide. The second antigen-binding domain can be the antigen-binding domain described in Example 28. The second antigen-binding domain can bind to a polypeptide expressed on the surface of a NK cell. The polypeptide expressed on the surface of the NK cell can be a CD16a, NKG2A, NKG2D, NKp30, NKp44, or NKp46 polypeptide. The second antigen-binding domain can be the antigen-binding domain described in Example 29. The cell engager can comprise a third antigen-binding domain. The third antigen-binding domain can bind to a polypeptide expressed on the surface of the NK cell. The polypeptide expressed on the surface of the NK cell can be a CD16a, NKG2A, NKG2D, NKp30, NKp44, or NKp46 polypeptide. The third antigen-binding domain can be the antigen-binding domain described in Example 29. This paragraph may be referred to as the fifth aspect paragraph.

[0020] In another aspect, the present specification features a nucleic acid comprising (or consisting essentially of, or consisting of) a nucleic acid sequence encoding at least a portion of an antibody, antigen-binding fragment, or antibody domain of any of the first, second, or third aspect paragraphs. The nucleic acid sequence can encode any one of the heavy chain variable domains or regions (i) through (xiv) in the first aspect paragraph above. The nucleic acid can be a viral vector. The nucleic acid can be a phagemid.

[0021] In another aspect, this specification features a nucleic acid comprising (or consisting essentially of, or consisting of) a nucleic acid sequence encoding a chimeric antigen receptor of any of the fourth aspect paragraphs or a cell engager of any of the fifth aspect paragraphs. The nucleic acid can be a viral vector. The nucleic acid can also be a phagemid.

[0022] In another aspect, this document features a host cell comprising (or consisting essentially of, or consisting of) the nucleic acid of any of the preceding paragraphs. This paragraph may be referred to as the sixth aspect paragraph.

[0023] In another aspect, this document features a host cell that expresses any of the chimeric antigen receptors of the fourth aspect paragraph or any of the cell engagers of the fifth aspect paragraph. The host cell can be a T cell, a stem cell, or a NK cell. This paragraph may be referred to as a seventh aspect paragraph.

[0024] In another aspect, the description features an antibody-drug conjugate (ADC) comprising (or consisting essentially of, or consisting of) an antigen-binding domain covalently linked to a drug, wherein the antigen-binding domain comprises an antibody, antigen-binding fragment, or antibody domain of any of the first, second, or third aspect paragraphs. The antigen-binding domain can comprise a VH domain capable of binding to a follistatin polypeptide. The drug can be selected from the group consisting of an auristatin, a mertansine, or a pyrrolobenzodiazepine (PBD) dimer. This paragraph may be referred to as an eighth aspect paragraph.

[0025] In another aspect, this document features a composition comprising (or consisting essentially of, or consisting of) an antibody, antigen-binding fragment, or antibody domain of any of the first, second, or third aspect paragraphs. The composition can include an antibody of any of the first aspect paragraphs. The composition can include an antigen-binding fragment of any of the second aspect paragraphs. The composition can include an antibody domain of any of the third aspect paragraphs.

[0026] In another aspect, this document features a composition comprising (or consisting essentially of, or consisting of) a cell engager of any of the fifth aspect paragraphs.

[0027] In another aspect, this document features a composition comprising (or consisting essentially of, or consisting of) the cells of either the sixth or seventh aspect paragraph.

[0028] In another aspect, this description features a composition comprising (or consisting essentially of, or consisting of) any ADC of any one of the eighth aspect paragraphs. The composition can include a checkpoint inhibitor. The checkpoint inhibitor can be selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab.

[0029] In another aspect, this document features a method of treating a mammal having cancer. The method includes (or consists essentially of, or consists of) administering to the mammal any of the compositions of paragraph 4 above. The mammal can be a human. The cancer is characterized by a follistatin-dependent + Follistatin can be used to treat cancer. + Cancer, follistatin + ovarian cancer, follistatin + Lung cancer and follistatin + The cancer may be selected from the group consisting of: prostate cancer, After the administration step, the number of cancer cells in the mammalian body may be reduced.

[0030] In another aspect, this document features a method of treating a mammal having cancer. The method includes (or consists essentially of, or consists of) (a) administering to the mammal any of the compositions of paragraph 4 referenced in the preceding paragraph, and (b) administering to the mammal a composition comprising a checkpoint inhibitor. The mammal can be a human. The cancer can be caused by a checkpoint inhibitor, such as follistatin. + Follistatin can be used to treat cancer. + Cancer, follistatin + ovarian cancer, follistatin + Lung cancer and follistatin +The checkpoint inhibitor may be selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab. After administering steps (a) and (b), the number of cancer cells in the mammalian body can be reduced.

[0031] In another aspect, this document features a method of conjugating a binding molecule to a follistatin polypeptide. The method includes (or consists essentially of, or consists of) contacting a follistatin polypeptide with an antibody, antigen-binding fragment, or antibody domain of any of the first, second, or third aspect paragraphs. The contacting can be in vitro. The contacting can be in vivo. The contacting can be performed in a mammal by administering the antibody, antigen-binding fragment, or antibody domain to the mammal. The mammal can be a human.

[0032] In another aspect, this document features a method of conjugating a binding molecule to a follistatin polypeptide. The method includes (or consists essentially of, or consists of) contacting the follistatin polypeptide with a chimeric antigen receptor of any of the fourth aspect paragraphs, a cell engager of any of the fifth aspect paragraphs, or an ADC of any of the eighth aspect paragraphs. The contacting can be performed in vitro. The contacting can be performed in vivo. The contacting can be performed in a mammal by administering the chimeric antigen receptor, cell engager, or ADC to the mammal. The mammal can be a human.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.Methods and materials are described herein for use in this disclosure, but other suitable methods and materials known in the art can also be used.The materials, methods, and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.

[0034] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0035] [Figure 1] 1 is a schematic diagram of an exemplary CAR construct designed to express a CAR. A promoter sequence (e.g., a CMV immediate early promoter sequence) may be followed by a signal peptide sequence (e.g., a GM-CSF signal peptide sequence), which may be followed by an scFv provided herein, followed by an optional linker (not shown), followed by an optional hinge (e.g., a CD8 hinge sequence; not shown), followed by a transmembrane sequence (e.g., a CD8 transmembrane sequence), followed by one or more intracellular signaling domain sequences (e.g., a 4-1BB (CD137) intracellular signaling domain sequence and a CD3ζ intracellular signaling domain sequence). [Figure 2]2 is a schematic diagram of an exemplary CAR construct designed to express a CAR. A promoter sequence (e.g., a CMV immediate early promoter sequence) is followed by a signal peptide sequence (e.g., a GM-CSF signal peptide sequence), which is then followed by a VH domain provided herein (e.g., a VH domain designed to include a set of three CDRs, such as CDR1, CDR2, and CDR3 of a VH domain provided herein, wherein the three CDRs are, for example, those set forth in SEQ ID NOs: 1-3; SEQ ID NOs: 9-11; SEQ ID NOs: 17-19; SEQ ID NOs: 25-27; SEQ ID NOs: 33-35; SEQ ID NOs: 41-43; SEQ ID NOs: 49-51; SEQ ID NOs: 57-59; SEQ ID NOs: 65-67; ​​SEQ ID NOs: 73-75; SEQ ID NOs: 81-83; SEQ ID NOs: 89-91; SEQ ID NOs: 97-99; or SEQ ID NOs: 105-107), followed by an optional linker (not shown), followed by an optional hinge (e.g., a CD8 hinge sequence; not shown), followed by a transmembrane sequence (e.g., a CD8 transmembrane sequence), followed by one or more intracellular signaling domain sequences (e.g., a 4-1BB (CD137) intracellular signaling domain sequence and a CD3ζ intracellular signaling domain sequence). [Figure 3] 3 is a schematic diagram of an exemplary BiTE designed with heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 in an Ig format (e.g., IgG1 format). A humanized anti-CD3 scFv (e.g., gOKT3-7 scFv described in U.S. Pat. No. 6,750,325) can be linked to the C-terminus of the light chain via a linker (e.g., a (G4S)3 linker; SEQ ID NO: 139). [Figure 4]Figure 4 is a graph plotting binding of the indicated VH domain binding molecules to recombinant human follistatin polypeptide as determined by ELISA. 200 ng of recombinant human follistatin polypeptide was coated onto an immunosorbent plate, and the indicated concentrations of the indicated binding molecules were added. After incubation and washing, VH binding was detected using a horseradish peroxidase (HRP)-conjugated mouse anti-FLAG tag antibody (A8592, Sigma-Aldrich). [Figure 5] Figure 5 is a graph plotting the binding of the indicated binding molecules in Fc format to recombinant human follistatin polypeptide as determined by ELISA. 200 ng of recombinant human follistatin polypeptide was coated onto an immunosorbent plate, and the indicated concentrations of the indicated binding molecules were added. After incubation and washing, HRP-conjugated goat anti-human IgG Fc (Sigma-Aldrich) was used for detection of VH-Fc binding. Because FST1E8, FST2D4, and FST3B3 are very similar in sequence to each other, only FST1E8 was transferred to the VH-Fc format and used for this ELISA. [Figure 6] Figure 6 is a graph plotting the binding of the indicated VH domain binding molecules to BSA as determined by ELISA. 200 ng of BSA was coated onto an immunosorbent plate, and the indicated concentrations of the indicated binding molecules were added. After incubation and washing, VH binding was detected using a horseradish peroxidase (HRP)-conjugated mouse anti-FLAG tag antibody (A8592, Sigma-Aldrich). [Figure 7]Figure 7 includes graphs of propidium iodide (PI) and annexin V flow analysis of primary ovarian cancer cells treated with the indicated antibodies or anti-follistatin VH clones in the presence or absence of cisplatin. Viable cells are in the lower left quadrant. The results show that IgG alone or anti-follistatin VH clones alone do not significantly affect ovarian cancer cell viability. However, neutralization of follistatin by either tested anti-follistatin VH clone (i.e., FST3G9 or FST1E8) significantly enhanced cisplatin cell killing. [Figure 8] Figure 8 contains graphs plotting nuclear DNA content in ovarian cancer cells treated with or without chemotherapy (Taxol) in the presence or absence of the anti-follistatin VH clone (FST3G9). The results show that Taxol increases nuclear content, which leads to mitotic catastrophe. The addition of the anti-follistatin VH clone (FST3G9) significantly enhanced this effect. P3 = 1N nuclear content; P4 = 2N nuclear content; P5 = 3N nuclear content; and P6 > 3N nuclear content. [Figure 9] Figure 9 includes photographs of immunofluorescence imaging of F-actin (shown in red) and DAPI (shown in blue) in ovarian cancer cells with the indicated treatments. FST-Ab = anti-follistatin VH clone FST3G9. The results show that taxol-treated cells increased in size and became multinucleated with mitotic crisis. Addition of anti-follistatin VH clone (FST3G9) to taxol treatment resulted in fewer cells and only large multinucleated cells in sustained mitotic crisis. [Figure 10] Figure 10 includes three graphs plotting the results of a competitive biolayer interferometry BLItz assay used to map different binding regions of binding molecules in follistatin. Based on these results, binding molecules with five different binding regions in follistatin were isolated and identified. [Figure 11]11 includes graphs plotting the results of a chemotherapy synergy study using propidium iodide (PI) and annexin V flow analysis of primary ovarian cancer cells treated with vehicle, cisplatin (Cis), or Cis in combination with various anti-follistatin VH clones. Viable cells are shown in the lower left quadrant of each graph. The final VH concentration in each treatment group was the same. DETAILED DESCRIPTION OF THE INVENTION

[0036] Detailed Description Provided herein are binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and ADCs) that bind (e.g., specifically bind) to a follistatin polypeptide (e.g., a human follistatin polypeptide). For example, provided herein are binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and ADCs) that bind (e.g., specifically bind) to a polypeptide comprising, consisting essentially of, or consisting of the amino acids set forth in SEQ ID NO: 113 and / or SEQ ID NO: 115 (see, e.g., Example 2).

[0037] As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies, recombinant antibodies, humanized antibodies, human antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two antibodies, diabodies, single-chain variable fragment antibodies (e.g., scFv antibodies), and tandem single-chain variable fragment antibodies (e.g., taFv). Diabodies can include two chains, each having a heavy chain variable domain and a light chain variable domain, where the variable domains can be derived from the same or different antibodies (see, e.g., Hornig and Faerber-Schwarz, Methods Mol. Biol., 907:713-27 (2012); and Brinkmann and Kontermann, MAbs., 9(2):182-212 (2017)). The two variable regions can be linked by a polypeptide linker (e.g., a polypeptide linker having a length of 5 to 10 residues or a polypeptide linker such as those described in Example 20). In some cases, interdomain disulfide bonds may exist in one or both of the heavy and light chain variable domain pairs of a diabody. scFvs are single-chain polypeptide antibodies in which the heavy and light chain variable domains are linked directly or via a polypeptide linker (e.g., a polypeptide linker having a length of 8 to 18 residues, or a polypeptide linker such as those described in Example 20). See also Chen et al., Adv. Drug Deliv. Rev., 65(10):1357-1369 (2013). scFvs can be designed with an orientation in which the heavy chain variable domain is followed by the light chain variable domain, or they can be designed with an orientation in which the light chain variable domain is followed by the heavy chain variable domain. In either case, an optional linker can be placed between the two domains. Examples of scFv structures of the scFvs provided herein include, but are not limited to, the structures described in Example 19.

[0038] The antibodies provided herein may comprise a CDR described herein (e.g., a CDR described in Table 43) and may be configured to be a human antibody, a humanized antibody, or a chimeric antibody. In some cases, the antibodies provided herein may comprise a CDR described herein (e.g., a CDR described in Table 43) and may be a monoclonal antibody. In some cases, the antibodies provided herein may comprise a CDR described herein (e.g., a CDR described in Table 43) and may be configured as an scFv antibody.

[0039] As used herein, the term "antigen-binding fragment" refers to a fragment of an antibody (e.g., a fragment of a humanized antibody, a fragment of a human antibody, or a fragment of a chimeric antibody) that has the ability to bind to an antigen. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', or F(ab')2 antigen-binding fragments. The antigen-binding fragments provided herein may include the CDRs described herein (e.g., the CDRs listed in Table 43) and can be configured to be human, humanized, or chimeric antigen-binding fragments. In some cases, the antigen-binding fragments provided herein may include the CDRs described herein (e.g., the CDRs listed in Table 43) and can be monoclonal antigen-binding fragments. In some cases, the antigen-binding fragments provided herein may include the CDRs described herein (e.g., the CDRs listed in Table 43) and can be configured as Fab antibodies. In some cases, Fab antibodies can include a partial hinge sequence (e.g., EPKSCDKT (SEQ ID NO: 228)) for disulfide bonding between the heavy and light chains of the Fab.

[0040] As used herein, the term "antibody domain" refers to an antibody domain, such as a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain), free of one or more other domains of an antibody. In some cases, an antibody domain can be a single antibody domain (e.g., a VH domain or a VL domain) capable of binding to an antigen. The antibody domains provided herein can include CDRs described herein (e.g., CDRs described in Table 43) and can be human antibody domains (e.g., human VH domains), humanized antibody domains (e.g., humanized VH domains), or chimeric antibody domains (e.g., chimeric VH domains). In some cases, the antibody domains provided herein can include CDRs described herein (e.g., CDRs described in Table 43) and can be monoclonal antibody domains. In some cases, the antibody domains provided herein can include CDRs described herein (e.g., CDRs described in Table 43) and can be engineered as a single VH domain or a single VL domain. Examples of VH domains provided herein include, but are not limited to, the structures described in Examples 3-16.

[0041] The anti-follistatin antibodies, anti-follistatin antigen-binding fragments, or anti-follistatin antibody domains provided herein may be of the IgA, IgD, IgE, IgG, or IgM type, including, but not limited to, IgG or IgM types, such as, for example, IgG1, IgG2, IgG3, IgG4, IgM1, and IgM2. In some cases, the antibodies (e.g., anti-follistatin antibodies) provided herein can be scFv antibodies. In some cases, the antigen-binding fragments (e.g., anti-follistatin antibody fragments) provided herein can be Fab. In some cases, the antibodies (e.g., anti-follistatin antibodies) provided herein can be complete, intact antibodies having the structure described in Example 18. In some cases, the antibody domains (e.g., anti-follistatin antibody domains) provided herein can be VH domains.

[0042] In some cases, the anti-follistatin antibodies, anti-follistatin antigen-binding fragments, or anti-follistatin antibody domains provided herein can be fully human. In some cases, the anti-follistatin antibodies, anti-follistatin antigen-binding fragments, or anti-follistatin antibody domains provided herein can have a low risk of inducing immunogenicity in humans.

[0043] As used herein, the term "chimeric antigen receptor" refers to a chimeric polypeptide designed to include an optional signal peptide, an antigen-binding domain, an optional hinge, a transmembrane domain, and one or more intracellular signaling domains. As described herein, the antigen-binding domain of a CAR provided herein can be designed to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). For example, a CAR provided herein can be designed to include an antibody, antigen-binding fragment, and / or antibody domain component (e.g., a combination of CDRs) described herein as the antigen-binding domain, provided that the antigen-binding domain has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). In some examples, a CAR provided herein can be designed to include an antigen-binding domain comprising a single set of three CDRs (e.g., CDR1, CDR2, and CDR3) of an antibody domain (e.g., a VH domain) provided herein (e.g., SEQ ID NOs: 1-3; SEQ ID NOs: 9-11; SEQ ID NOs: 17-19; SEQ ID NOs: 25-27; SEQ ID NOs: 33-35; SEQ ID NOs: 41-43; SEQ ID NOs: 49-51; SEQ ID NOs: 57-59; SEQ ID NOs: 65-67; ​​SEQ ID NOs: 73-75; SEQ ID NOs: 81-83; SEQ ID NOs: 89-91; SEQ ID NOs: 97-99; or SEQ ID NOs: 105-107). In some cases, the antigen-binding domain of a CAR targeting a follistatin polypeptide can be designed to include a VH domain described herein or an scFv antibody described herein.

[0044] Examples of CAR structures that can be used to generate the CARs provided herein include, but are not limited to, those described in Figures 1 and 2 and Examples 21 and 26.

[0045] In some cases, the CARs provided herein can be designed to include a signal peptide. Any suitable signal peptide can be used to design the CARs described herein. Examples of signal peptides that can be used to produce the CARs described herein include, but are not limited to, the signal peptides derived from human IGKV1-39, IGKV1-16, IGKV1-33, IGKV3-11, IGKV4-1, or IGKV6-21. In some cases, the CARs provided herein can be designed to include a signal peptide that includes, consists essentially of, or consists of one of the amino acid sequences described in Example 22. In some cases, the CARs provided herein can be designed to include a signal peptide that includes, consists essentially of, or consists of one of the amino acid sequences described in Example 22 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof. In some cases, the CARs provided herein can be designed to include a signal peptide that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 22 with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or combinations thereof.

[0046] In some cases, the CAR provided herein can be designed to include a hinge. Any suitable hinge can be used to design the CAR described herein. Examples of hinges that can be used to create the CAR described herein include, but are not limited to, Ig-derived hinges (for example, IgG1-derived hinges, IgG2-derived hinges, or IgG4-derived hinges), Ig-derived hinges containing CD2 and CD3 domains, Ig-derived hinges containing CD2 domains and lacking CD3 domains, Ig-derived hinges containing CD3 domains and lacking CD2 domains, Ig-derived hinges lacking CD2 domains and lacking CD3 domains, CD8α-derived hinges, CD28-derived hinges, and CD3ζ-derived hinges. The CAR provided herein can be designed to include any suitable length of hinge. For example, the CARs provided herein can be designed to include a hinge that is about 3 to about 75 amino acid residues in length (e.g., about 3 to about 65, about 3 to about 50, about 5 to about 75, about 10 to about 75, about 5 to about 50, about 10 to about 50, about 10 to about 40, or about 10 to about 30). In some cases, a linker sequence can be used as a hinge to generate the CARs described herein. For example, any one of the linker sequences shown in Example 20 can be used as a hinge for the CARs described herein.

[0047] In some cases, the CARs provided herein can be designed to include a hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 20 or Example 23. In some cases, the CARs provided herein can be designed to include a hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 20 or Example 23, with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof. In some cases, the CARs provided herein can be designed to include a hinge that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 20 or Example 23, with 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, or 10 or fewer amino acid deletions, additions, substitutions, or combinations thereof.

[0048] The CARs provided herein can be designed to include any suitable transmembrane domain. For example, the transmembrane domains of the CARs provided herein can be, but are not limited to, a CD3ζ transmembrane domain, a CD4 transmembrane domain, a CD8α transmembrane domain, a CD28 transmembrane domain, and a 4-1BB transmembrane domain. In some cases, the CARs provided herein can be designed to include a transmembrane domain that includes, consists essentially of, or consists of one of the amino acid sequences described in Example 24. In some cases, the CARs provided herein can be designed to include a transmembrane domain that includes, consists essentially of, or consists of one of the amino acid sequences described in Example 24 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or a combination thereof. In some cases, the CARs provided herein can be designed to include a transmembrane domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 24, with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or combinations thereof.

[0049] The CARs provided herein can be designed to include one or more intracellular signaling domains. For example, the CARs provided herein can be designed to include one, two, three, or four intracellular signaling domains. Any suitable intracellular signaling domain or combination of intracellular signaling domains can be used to produce the CARs described herein. Examples of intracellular signaling domains that can be used to produce the CARs described herein include, but are not limited to, the CD3ζ intracellular signaling domain, the CD27 intracellular signaling domain, the CD28 intracellular signaling domain, the OX40 (CD134) intracellular signaling domain, the 4-1BB (CD137) intracellular signaling domain, the CD278 intracellular signaling domain, the DAP10 intracellular signaling domain, and the DAP12 intracellular signaling domain. In some cases, the CARs described herein can be designed as first-generation CARs with a CD3ζ intracellular signaling domain. In some cases, the CARs described herein can be designed as second-generation CARs with a CD28 intracellular signaling domain followed by a CD3ζ intracellular signaling domain. In some cases, the CARs described herein can be designed as third-generation CARs having (a) a CD28 intracellular signaling domain followed by (b) a CD27 intracellular signaling domain, an OX40 intracellular signaling domain, or a 4-1BB intracellular signaling domain, followed by (c) a CD3ζ intracellular signaling domain. In some cases, the CARs provided herein can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 25.In some cases, CARs provided herein can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 25, with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof, provided that the intracellular signaling domain has at least some activity to activate intracellular signaling. In some cases, CARs provided herein can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 25, with 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, or 10 or less amino acid deletions, additions, substitutions, or combinations thereof, provided that the intracellular signaling domain has at least some activity to activate intracellular signaling.

[0050] In some cases, a CAR that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59, followed by a hinge such as the hinge / linker described in Example 20 or Example 23 (e.g., an IgG4-derived hinge, a CD8α hinge, or a linker+IgG4-derived hinge), followed by a transmembrane domain such as the transmembrane domain described in Example 24 (e.g., a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains such as one or more intracellular signaling domains described in Example 25 (e.g., a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59, followed by SEQ ID NO:178, SEQ ID NO:184, followed by SEQ ID NO:192, followed by SEQ ID NO:191 (see, e.g., Example 26). In another example, a CAR targeting a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59, followed by SEQ ID NO:180, SEQ ID NO:187, then SEQ ID NO:194, followed by SEQ ID NO:191 (see, e.g., Example 26).

[0051] In some cases, a CAR that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO: 64, followed by a hinge such as the hinge / linker described in Example 20 or Example 23 (e.g., an IgG4-derived hinge, a CD8α hinge, or a linker+IgG4-derived hinge), then a transmembrane domain such as the transmembrane domain described in Example 24 (e.g., a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains such as one or more intracellular signaling domains described in Example 25 (e.g., a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO: 64, followed by SEQ ID NO: 178, SEQ ID NO: 184, then SEQ ID NO: 192, followed by SEQ ID NO: 191 (see, e.g., Example 26). In another example, a CAR that targets a follistatin polypeptide can be designed to include a VH domain that includes SEQ ID NO: 64, followed by SEQ ID NO: 180, followed by SEQ ID NO: 187, followed by SEQ ID NO: 194, followed by SEQ ID NO: 191 (see, e.g., Example 26).

[0052] In some cases, a CAR that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67, followed by a hinge such as the hinge / linker described in Example 20 or Example 23 (e.g., an IgG4-derived hinge, a CD8α hinge, or a linker+IgG4-derived hinge), then a transmembrane domain such as the transmembrane domain described in Example 24 (e.g., a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains such as one or more intracellular signaling domains described in Example 25 (e.g., a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67, followed by SEQ ID NO:178, SEQ ID NO:184, then SEQ ID NO:192, followed by SEQ ID NO:191 (see, e.g., Example 26). In another example, a CAR targeting a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67, followed by SEQ ID NO:180, SEQ ID NO:187, then SEQ ID NO:194, followed by SEQ ID NO:191 (see, e.g., Example 26).

[0053] In some cases, a CAR that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO: 72, followed by a hinge such as the hinge / linker described in Example 20 or Example 23 (e.g., an IgG4-derived hinge, a CD8α hinge, or a linker+IgG4-derived hinge), then a transmembrane domain such as the transmembrane domain described in Example 24 (e.g., a human CD28 transmembrane domain or a CD8α transmembrane domain), followed by one or more intracellular signaling domains such as one or more intracellular signaling domains described in Example 25 (e.g., a human 4-1BB intracellular signaling domain followed by a human CD3ζ intracellular signaling domain). For example, a CAR that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO: 72, followed by SEQ ID NO: 178, SEQ ID NO: 184, then SEQ ID NO: 192, followed by SEQ ID NO: 191 (see, e.g., Example 26). In another example, a CAR targeting a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO:72, followed by SEQ ID NO:180, followed by SEQ ID NO:187, followed by SEQ ID NO:194, followed by SEQ ID NO:191 (see, e.g., Example 26).

[0054] As used herein, the term "cell engager" refers to a polypeptide that comprises two or more antigen-binding domains (e.g., two, three, or four antigen-binding domains) and has the ability to bring two cells together. Examples of cell engagers include, but are not limited to, BiTE, BiKE, and TriKE. Generally, the cell engagers provided herein can be designed to include at least one antigen-binding domain capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) and at least one antigen-binding domain capable of binding to an antigen expressed on the surface of a cell (e.g., a T cell or an NK cell). In some cases, the cell engagers described herein can bind to a follistatin polypeptide via the two or more antigen-binding domains of the cell engager. +cells (e.g., follistatin + The cell engager can bind a specific cell (e.g., a cancer cell) to another cell (e.g., a T cell or an NK cell). Examples of cell engager structures of the cell engagers provided herein include, but are not limited to, the structure shown in Figure 3. In some cases, the anti-CD3 scFv depicted in Figure 3 can be replaced with a different antigen-binding domain capable of binding to an antigen expressed on the surface of a cell (e.g., a T cell or an NK cell).

[0055] When a cell engager comprises an antigen-binding domain capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) and two or more other antigen-binding domains (e.g., two, three, or four other antigen-binding domains), each of the other antigen-binding domains can bind to a different antigen expressed on the surface of a different cell type, but can also bind to different antigens expressed on the surface of the same cell type. For example, TriKE can be designed to have a first antigen-binding domain capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), a second antigen-binding domain capable of binding to a first antigen expressed on the surface of an NK cell (e.g., a CD16 polypeptide, such as a CD16a polypeptide), and a third antigen-binding domain capable of binding to a second antigen expressed on the surface of an NK cell (e.g., an NKG2A polypeptide).

[0056] As described herein, at least one antigen-binding domain of a cell engager provided herein can be designed to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). For example, a cell engager provided herein can be designed to include, as an antigen-binding domain, components (e.g., combinations of CDRs) of an antibody, antigen-binding fragment, and / or antibody domain described herein, provided that the antigen-binding domain has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). In some examples, a cell engager provided herein can be designed to include an antigen-binding domain comprising two sets of three CDRs of an antigen-binding fragment (e.g., CDR1, CDR2, and CDR3 of the heavy chain and CDR1, CDR2, and CDR3 of the light chain).

[0057] In some examples, the cell engagers provided herein can be designed to contain an antigen-binding domain comprising a single set of three CDRs (e.g., CDR1, CDR2, and CDR3) of an antibody domain (e.g., a VH domain) provided herein (e.g., SEQ ID NOs: 1-3; SEQ ID NOs: 9-11; SEQ ID NOs: 17-19; SEQ ID NOs: 25-27; SEQ ID NOs: 33-35; SEQ ID NOs: 41-43; SEQ ID NOs: 49-51; SEQ ID NOs: 57-59; SEQ ID NOs: 65-67; ​​SEQ ID NOs: 73-75; SEQ ID NOs: 81-83; SEQ ID NOs: 89-91; SEQ ID NOs: 97-99; or SEQ ID NOs: 105-107).

[0058] In some cases, the antigen binding domain of a cell engager that targets a follistatin polypeptide can be designed to include a VH domain described herein, or an scFv / Fab antibody described herein. In some cases, the antigen binding domain of a CAR described herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can be designed to include a VH domain described herein, or an scFv / Fab antibody described herein. +It can be used as the antigen binding domain of a cell engager to target cells.

[0059] As described herein, a cell engager can be designed to include at least one antigen-binding domain capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) and at least one other antigen-binding domain. The at least one other antigen-binding domain can be capable of binding to any suitable antigen expressed on the cell surface. For example, a cell engager can bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and at least one other antigen-binding domain. + When designing a cell engager such as a BiTE that connects cells to T cells, the cell engager can include an antigen-binding domain capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) and an antigen-binding domain capable of binding to a polypeptide expressed on the surface of a T cell. An example of a polypeptide expressed on the surface of a T cell that can be targeted by the antigen-binding domain of a cell engager provided herein includes, but is not limited to, a CD3 polypeptide. Examples of antigen-binding domains capable of binding to a polypeptide expressed on the surface of a T cell that can be used to generate a cell engager (e.g., a BiTE) provided herein include, but are not limited to, an anti-CD3 scFv and an anti-CD3 VH domain. Additional examples of amino acid sequences that can be used as antigen-binding domains capable of binding to a polypeptide expressed on the surface of a T cell (e.g., CD3) are described in U.S. Patent No. 6,750,325 (see, e.g., the sequence listing of U.S. Patent No. 6,750,325).

[0060] Examples of BiTE structures that can be used to generate the BiTEs provided herein include, but are not limited to, those shown in Examples 30 and 31.

[0061] In some cases, cell engagers provided herein can be designed to comprise an antigen binding domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 28. In some cases, cell engagers provided herein can be designed to comprise an antigen binding domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 28 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof, provided that the antigen binding domain is capable of binding to a polypeptide expressed on the surface of a T cell. In some cases, the cell engagers provided herein can be designed to contain an antigen-binding domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 28, with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or a combination thereof, provided that the antigen-binding domain is capable of binding to a polypeptide expressed on the surface of a T cell.

[0062] Follistatin +When designing a cell engager, such as BiKE or TriKE, to link cells with NK cells, the cell engager can include an antigen-binding domain capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) and one or more (e.g., one, two, or three) antigen-binding domains capable of binding to a polypeptide expressed on the surface of an NK cell. Examples of polypeptides expressed on the surface of NK cells that can be targeted by the antigen-binding domains of the cell engagers provided herein include, but are not limited to, a CD16 polypeptide (e.g., a CD16a polypeptide), an NKG2A polypeptide, an NKG2D polypeptide, an NKp30 polypeptide, an NKp44 polypeptide, and an NKp46 polypeptide. Examples of antigen-binding domains capable of binding to a polypeptide expressed on the surface of an NK cell that can be used to generate a cell engager (e.g., BiKE or TriKE) provided herein include, but are not limited to, anti-CD16a scFv, anti-NKG2A scFv, anti-NKG2D scFv, anti-NKp30 scFv (see, e.g., BioLegend catalog #325207), anti-NKp44 scFvs, anti-NKp46 scFvs, anti-CD16a VH domain, anti-NKG2A VH domain, anti-NKG2D VH domain, anti-NKp30 VH domain, anti-NKp44 VH domain, and anti-NKp46 VH domain.Additional examples of amino acid sequences that can be used as antigen-binding domains capable of binding to polypeptides expressed on the surface of NK cells (e.g., CD16, NKG2A, NKG2D, or NKp46) are described in McCall et al. (Mol. Immunol., 36(7):433-445 (1999); e.g., anti-CD16 scFv sequences); International Patent Application Publication No. PCT / US2017 / 048721 (see, e.g., the CDRs and sequence listing of the anti-CD16a binding domain); U.S. Patent Application Publication No. 2011 / 0052606 (see, e.g., the CDRs and sequence listing of anti-NKG2A antibodies, such as Z199); U.S. Patent Application Publication No. 2011 / 0150870 (see, e.g., the CDRs and sequence listing of anti-NKG2D antibodies); U.S. Patent Application Publication No. 2018 / 0369373 (see, e.g., the CDRs and sequence listing of anti-NKp46 antibodies); and U.S. Patent Application Publication No. 2017 / 0368169 (see, e.g., the CDRs and sequence listing of anti-NKp46 antibodies).

[0063] In some cases, cell engagers provided herein can be designed to comprise an antigen binding domain (e.g., scFv or VH) that comprises, consists essentially of, or consists of one or more amino acid sequences set forth in Example 29. In some cases, cell engagers provided herein can be designed to comprise an antigen binding domain (e.g., scFv or VH) that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 29, with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof, provided that the antigen binding domain is capable of binding to a polypeptide expressed on the surface of an NK cell. In some cases, the cell engagers provided herein can be designed to include an antigen-binding domain (e.g., scFv or VH) that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 29, with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or a combination thereof, provided that the antigen-binding domain is capable of binding to a polypeptide expressed on the surface of an NK cell.

[0064] In some cases, the cell engagers provided herein can be designed to include a linker disposed between each antigen-binding domain. Any suitable linker can be used to design the cell engagers provided herein. Examples of linkers that can be used to generate the cell engagers described herein include, but are not limited to, the linker sequences described in Example 20. The cell engagers provided herein can be designed to include a linker of any suitable length. For example, the cell engagers provided herein can be designed to include a linker having a length of about 3 to about 100 amino acid residues (e.g., about 3 to about 90, about 3 to about 80, about 3 to about 70, about 3 to about 60, about 3 to about 50, about 3 to about 40, about 3 to about 30, about 3 to about 20, about 3 to about 15, about 5 to about 100, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, or about 12 to about 17). In some cases, a cell engager (e.g., BiTE) provided herein can be designed to include a GGGGSGGGSGGGGS (SEQ ID NO: 139) linker. In some cases, the hinge of a CAR described herein can be used as a linker to generate a cell engager described herein. For example, any one of the sequences described in Example 23 can be used as a linker in a cell engager described herein.

[0065] In some cases, cell engagers provided herein can be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 20 or Example 23. In some cases, cell engagers provided herein can be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 20 or Example 23, with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, additions, substitutions, or combinations thereof. In some cases, cell engagers provided herein can be designed to include a linker that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in Example 20 or Example 23, with no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 amino acid deletions, additions, substitutions, or combinations thereof.

[0066] In some cases, a cell engager (e.g., BiTE) that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59, followed by a linker such as the linker / hinge described in Example 20 or Example 23 (e.g., SEQ ID NO:139 or 159), followed by an antigen binding domain (e.g., an anti-human CD3 scFv) capable of binding to a polypeptide expressed on the surface of a T cell (see, e.g., Example 30).

[0067] In some cases, a cell engager (e.g., a BiTE) that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO: 64, followed by a linker such as the linker / hinge described in Example 20 or Example 23 (e.g., SEQ ID NO: 139 or 159), followed by an antigen binding domain (e.g., an anti-human CD3 scFv) capable of binding to a polypeptide expressed on the surface of a T cell (see, e.g., Example 30).

[0068] In some cases, a cell engager (e.g., BiTE) that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67, followed by a linker such as the linker / hinge described in Example 20 or Example 23 (e.g., SEQ ID NO:139 or 159), followed by an antigen binding domain (e.g., an anti-human CD3 scFv) capable of binding to a polypeptide expressed on the surface of a T cell (see, e.g., Example 31).

[0069] In some cases, a cell engager (e.g., a BiTE) that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO: 72, followed by a linker such as the linker / hinge described in Example 20 or Example 23 (e.g., SEQ ID NO: 139 or 159), followed by an antigen binding domain (e.g., an anti-human CD3 scFv) capable of binding to a polypeptide expressed on the surface of a T cell (see, e.g., Example 31).

[0070] In some cases, a cell engager (e.g., BiKE or TriKE) that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, followed by a linker such as the linker / hinge described in Example 20 or Example 23 (e.g., SEQ ID NO: 139 or 159), followed by one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD16a scFv for BiKE, or an anti-human CD16a scFv and an anti-human NKG2A scFv for TriKE).

[0071] In some cases, a cell engager (e.g., BiKE or TriKE) that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO: 64, followed by a linker such as the linker / hinge described in Example 20 or Example 23 (e.g., SEQ ID NO: 139 or 159), followed by one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD16a scFv for BiKE, or an anti-human CD16a scFv and an anti-human NKG2A scFv for TriKE).

[0072] In some cases, a cell engager (e.g., BiKE or TriKE) that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67, followed by a linker such as the linker / hinge described in Example 20 or Example 23 (e.g., SEQ ID NO: 139 or 159), followed by one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD16a scFv for BiKE, or an anti-human CD16a scFv and an anti-human NKG2A scFv for TriKE).

[0073] In some cases, a cell engager (e.g., BiKE or TriKE) that targets a follistatin polypeptide can be designed to include a VH domain comprising SEQ ID NO: 72, followed by a linker such as the linker / hinge described in Example 20 or Example 23 (e.g., SEQ ID NO: 139 or 159), followed by one or more antigen binding domains capable of binding to a polypeptide expressed on the surface of an NK cell (e.g., an anti-human CD16a scFv for BiKE, or an anti-human CD16a scFv and an anti-human NKG2A scFv for TriKE).

[0074] In certain embodiments, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (or a variant of SEQ ID NO: 1 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (or a variant of SEQ ID NO: 2 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 3 (or a variant of SEQ ID NO: 3 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 3.

[0075] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (or a variant of SEQ ID NO: 1 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (or a variant of SEQ ID NO: 2 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 3 (or a variant of SEQ ID NO: 3 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO:4 (or a variant of SEQ ID NO:4 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO:5 (or a variant of SEQ ID NO:5 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO:6 (or a variant of SEQ ID NO:6 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO:7 (or a variant of SEQ ID NO:7 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0076] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 3 can be designed to include framework regions as set forth in Example 3, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 3 and the framework regions described in Example 3, except that framework region 1 having the amino acids set forth in SEQ ID NO:4 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO:12, framework region 1 having the amino acids set forth in SEQ ID NO:20, or framework region 1 having the amino acids set forth in SEQ ID NO:28. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 3, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0077] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 8. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 8. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 8.

[0078] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:8, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs:1, 2, and 3. For example, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:8, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs:1, 2, and 3.

[0079] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 8, or the amino acids set forth in SEQ ID NO: 8 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 8 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 1, 2, and 3.

[0080] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:1; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:2; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:3. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO:1" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO:1, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:1, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:1, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO:1 include, but are not limited to, those listed in Table 1.

[0081] [Table 1] TIFF2026504407000003.tif14156

[0082] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO:2" refers to a CDR2 that has zero, one, or two amino acid substitutions in SEQ ID NO:2, zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:2, and / or zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:2, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s that consist essentially of the amino acid sequence set forth in SEQ ID NO:2 include, but are not limited to, those listed in Table 2.

[0083] [Table 2]

[0084] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO:3" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO:3, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:3, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:3, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO:3 include, but are not limited to, those listed in Table 3.

[0085] [Table 3]

[0086] In another embodiment, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:9 (or a variant of SEQ ID NO:9 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:10 (or a variant of SEQ ID NO:10 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:11 (or a variant of SEQ ID NO:11 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 4.

[0087] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:9 (or a variant of SEQ ID NO:9 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:10 (or a variant of SEQ ID NO:10 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:11 (or a variant of SEQ ID NO:11 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO: 12 (or a variant of SEQ ID NO: 12 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO: 13 (or a variant of SEQ ID NO: 13 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO: 14 (or a variant of SEQ ID NO: 14 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO: 15 (or a variant of SEQ ID NO: 15 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0088] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 4 can be designed to include framework regions as set forth in Example 4, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 4 and the framework regions described in Example 4, except that framework region 1 having the amino acids set forth in SEQ ID NO: 12 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO: 4, framework region 1 having the amino acids set forth in SEQ ID NO: 20, or framework region 1 having the amino acids set forth in SEQ ID NO: 28. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 4, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0089] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 16. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 16. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0090] In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 16, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 9, 10, and 11. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 16, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 9, 10, and 11.

[0091] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 16, or the amino acids set forth in SEQ ID NO: 16 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 16 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 9, 10, and 11.

[0092] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:9; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:10; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:11. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO:9" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO:9, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:9, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:9, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO:9 include, but are not limited to, those listed in Table 4.

[0093] [Table 4]

[0094] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 10" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO: 10, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 10, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 10, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 10 include, but are not limited to, those listed in Table 5.

[0095] [Table 5] TIFF2026504407000008.tif79156

[0096] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 11" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO: 11, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 11, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 11, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 11 include, but are not limited to, those set forth in Table 6.

[0097] [Table 6] TIFF2026504407000010.tif43155

[0098] In another embodiment, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 17 (or a variant of SEQ ID NO: 17 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 18 (or a variant of SEQ ID NO: 18 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 19 (or a variant of SEQ ID NO: 19 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 5.

[0099] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 17 (or a variant of SEQ ID NO: 17 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 18 (or a variant of SEQ ID NO: 18 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 19 (or a variant of SEQ ID NO: 19 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO:20 (or a variant of SEQ ID NO:20 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO:21 (or a variant of SEQ ID NO:21 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO:22 (or a variant of SEQ ID NO:22 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO:23 (or a variant of SEQ ID NO:23 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0100] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 5 can be designed to include framework regions as set forth in Example 5, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 5 and the framework regions set forth in Figure 4, except that framework region 1 having the amino acids set forth in SEQ ID NO:20 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO:4, framework region 1 having the amino acids set forth in SEQ ID NO:12, or framework region 1 having the amino acids set forth in SEQ ID NO:28. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 5, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0101] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 24. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 24. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 24.

[0102] In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 24, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 17, 18, and 19. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 24, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 17, 18, and 19.

[0103] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 24, or the amino acids set forth in SEQ ID NO: 24 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 24 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 17, 18, and 19.

[0104] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 17; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 18; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 19. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 17" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO: 17, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 17, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 17, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 17 include, but are not limited to, those listed in Table 7.

[0105] [Table 7] TIFF2026504407000012.tif14156

[0106] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 18" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO: 18, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 18, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 18, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 18 include, but are not limited to, those listed in Table 8.

[0107] [Table 8]

[0108] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 19" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO: 19, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 19, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 19, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 19 include, but are not limited to, those set forth in Table 9.

[0109] [Table 9]

[0110] In another embodiment, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 25 (or a variant of SEQ ID NO: 25 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 26 (or a variant of SEQ ID NO: 26 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 27 (or a variant of SEQ ID NO: 27 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 6.

[0111] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 25 (or a variant of SEQ ID NO: 25 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 26 (or a variant of SEQ ID NO: 26 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 27 (or a variant of SEQ ID NO: 27 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO:28 (or a variant of SEQ ID NO:28 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO:29 (or a variant of SEQ ID NO:29 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO:30 (or a variant of SEQ ID NO:30 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO:31 (or a variant of SEQ ID NO:31 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0112] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 6 can be designed to include framework regions as set forth in Example 6, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 6 and the framework regions described in Example 6, except that framework region 1 having the amino acids set forth in SEQ ID NO:28 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO:4, framework region 1 having the amino acids set forth in SEQ ID NO:12, or framework region 1 having the amino acids set forth in SEQ ID NO:20. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 6, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0113] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 32. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 32. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 32.

[0114] In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 32, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 25, 26, and 27. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 32, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 25, 26, and 27.

[0115] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 32, or the amino acids set forth in SEQ ID NO: 32 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 32 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 25, 26, and 27.

[0116] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 25; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 26; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 27. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO:25" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO:25, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:25, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:25, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO:25 include, but are not limited to, those listed in Table 10.

[0117] [Table 10]

[0118] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO:26" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO:26, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:26, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:26, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO:26 include, but are not limited to, those listed in Table 11.

[0119] [Table 11] TIFF2026504407000017.tif79155

[0120] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO:27" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO:27, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:27, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:27, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO:27 include, but are not limited to, those listed in Table 12.

[0121] [Table 12] TIFF2026504407000019.tif43155

[0122] In certain embodiments, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 33 (or a variant of SEQ ID NO: 33 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 34 (or a variant of SEQ ID NO: 34 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 35 (or a variant of SEQ ID NO: 35 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 7.

[0123] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 33 (or a variant of SEQ ID NO: 33 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 34 (or a variant of SEQ ID NO: 34 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 35 (or a variant of SEQ ID NO: 35 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO:36 (or a variant of SEQ ID NO:36 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO:37 (or a variant of SEQ ID NO:37 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO:38 (or a variant of SEQ ID NO:38 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO:39 (or a variant of SEQ ID NO:39 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0124] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 7 can be designed to include framework regions as set forth in Example 7, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 7 and the framework regions described in Example 7, except that framework region 1 having the amino acids set forth in SEQ ID NO: 36 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO: 12, framework region 1 having the amino acids set forth in SEQ ID NO: 20, or framework region 1 having the amino acids set forth in SEQ ID NO: 28. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 7, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0125] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 40. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 40. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 40.

[0126] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 40, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 33, 34, and 35. For example, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 40, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 33, 34, and 35.

[0127] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 40, or the amino acids set forth in SEQ ID NO: 40 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 40 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 33, 34, and 35.

[0128] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 33; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 34; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 35. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 33" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO: 33, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 33, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 33, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 33 include, but are not limited to, those listed in Table 13.

[0129] [Table 13] TIFF2026504407000021.tif14155

[0130] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 34" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO: 34, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 34, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 34, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 34 include, but are not limited to, those listed in Table 14.

[0131] [Table 14]

[0132] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 35" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO: 35, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 35, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 35, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 35 include, but are not limited to, those listed in Table 15.

[0133] [Table 15]

[0134] In another embodiment, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 41 (or a variant of SEQ ID NO: 41 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 42 (or a variant of SEQ ID NO: 42 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 43 (or a variant of SEQ ID NO: 43 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 8.

[0135] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 41 (or a variant of SEQ ID NO: 41 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 42 (or a variant of SEQ ID NO: 42 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 43 (or a variant of SEQ ID NO: 43 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO:44 (or a variant of SEQ ID NO:44 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO:45 (or a variant of SEQ ID NO:45 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO:46 (or a variant of SEQ ID NO:46 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO:47 (or a variant of SEQ ID NO:47 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0136] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 8 can be designed to include framework regions as set forth in Example 8, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 8 and the framework regions described in Example 8, except that framework region 1 having the amino acids set forth in SEQ ID NO:44 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO:4, framework region 1 having the amino acids set forth in SEQ ID NO:20, or framework region 1 having the amino acids set forth in SEQ ID NO:28. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 8, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0137] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 48. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 48. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 48.

[0138] In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 48, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 41, 42, and 43. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 48, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 41, 42, and 43.

[0139] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 48, or the amino acids set forth in SEQ ID NO: 48 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 48 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 41, 42, and 43.

[0140] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 41; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 42; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 43. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO:41" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO:41, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:41, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:41, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO:41 include, but are not limited to, those listed in Table 16.

[0141] [Table 16]

[0142] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 42" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO: 42, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 42, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 42, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 42 include, but are not limited to, those listed in Table 17.

[0143] [Table 17]

[0144] TIFF2026504407000026.tif79155

[0145] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 43" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO: 43, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 43, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 43, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 43 include, but are not limited to, those listed in Table 18.

[0146] [Table 18] TIFF2026504407000028.tif43155

[0147] In another embodiment, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 49 (or a variant of SEQ ID NO: 49 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 50 (or a variant of SEQ ID NO: 50 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 51 (or a variant of SEQ ID NO: 51 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 9.

[0148] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 49 (or a variant of SEQ ID NO: 49 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 50 (or a variant of SEQ ID NO: 50 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 51 (or a variant of SEQ ID NO: 51 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO: 52 (or a variant of SEQ ID NO: 52 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO: 53 (or a variant of SEQ ID NO: 53 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO: 54 (or a variant of SEQ ID NO: 54 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO: 55 (or a variant of SEQ ID NO: 55 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0149] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 9 can be designed to include framework regions as set forth in Example 9, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 9 and the framework regions described in Example 9, except that framework region 1 having the amino acids set forth in SEQ ID NO:52 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO:4, framework region 1 having the amino acids set forth in SEQ ID NO:12, or framework region 1 having the amino acids set forth in SEQ ID NO:28. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 9, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0150] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 56. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 56. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 56.

[0151] In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 56, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 49, 50, and 51. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 56, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 49, 50, and 51.

[0152] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 56, or the amino acids set forth in SEQ ID NO: 56 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 56 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 49, 50, and 51.

[0153] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 49; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 50; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 51. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO:49" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO:49, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:49, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:49, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO:49 include, but are not limited to, those listed in Table 19.

[0154] [Table 19] TIFF2026504407000030.tif13156

[0155] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO:50" refers to a CDR2 that has zero, one, or two amino acid substitutions in SEQ ID NO:50, a CDR2 that has zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:50, and / or a CDR2 that has zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:50, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s that consist essentially of the amino acid sequence set forth in SEQ ID NO:50 include, but are not limited to, those listed in Table 20.

[0156] [Table 20]

[0157] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 51" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO: 51, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 51, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 51, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 51 include, but are not limited to, those set forth in Table 21.

[0158] [Table 21]

[0159] In another embodiment, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 57 (or a variant of SEQ ID NO: 57 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 58 (or a variant of SEQ ID NO: 58 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 59 (or a variant of SEQ ID NO: 59 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 10.

[0160] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 57 (or a variant of SEQ ID NO: 57 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 58 (or a variant of SEQ ID NO: 58 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 59 (or a variant of SEQ ID NO: 59 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO: 60 (or a variant of SEQ ID NO: 60 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO: 61 (or a variant of SEQ ID NO: 61 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO: 62 (or a variant of SEQ ID NO: 62 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO: 63 (or a variant of SEQ ID NO: 63 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0161] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 10 can be designed to include framework regions as set forth in Example 10, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 10 and the framework regions described in Example 10, except that framework region 1 having the amino acids set forth in SEQ ID NO:60 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO:4, framework region 1 having the amino acids set forth in SEQ ID NO:12, or framework region 1 having the amino acids set forth in SEQ ID NO:20. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 10, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0162] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 64. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 64. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 64.

[0163] In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 64, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 57, 58, and 59. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 64, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 57, 58, and 59.

[0164] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 64, or the amino acids set forth in SEQ ID NO: 64 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 64 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 57, 58, and 59.

[0165] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 57; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 58; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 59. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 57" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO: 57, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 57, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 57, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 57 include, but are not limited to, those listed in Table 22.

[0166] [Table 22]

[0167] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 58" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO: 58, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 58, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 58, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 58 include, but are not limited to, those set forth in Table 23.

[0168] [Table 23] TIFF2026504407000035.tif80156

[0169] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 59" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO: 59, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 59, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 59, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 59 include, but are not limited to, those set forth in Table 24.

[0170] [Table 24] TIFF2026504407000037.tif43155

[0171] In certain embodiments, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 65 (or a variant of SEQ ID NO: 65 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 66 (or a variant of SEQ ID NO: 66 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 67 (or a variant of SEQ ID NO: 67 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 11.

[0172] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 65 (or a variant of SEQ ID NO: 65 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 66 (or a variant of SEQ ID NO: 66 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 67 (or a variant of SEQ ID NO: 67 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO:68 (or a variant of SEQ ID NO:68 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO:69 (or a variant of SEQ ID NO:69 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO:70 (or a variant of SEQ ID NO:70 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO:71 (or a variant of SEQ ID NO:71 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0173] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 11 can be designed to include framework regions as set forth in Example 11, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 11 and the framework regions described in Example 11, except that framework region 1 having the amino acids set forth in SEQ ID NO:68 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO:4, framework region 1 having the amino acids set forth in SEQ ID NO:12, or framework region 1 having the amino acids set forth in SEQ ID NO:20. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 11, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0174] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 72. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 72. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 72.

[0175] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 72, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 65, 66, and 67. For example, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 72, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 65, 66, and 67.

[0176] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 72, or the amino acids set forth in SEQ ID NO: 72 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 72 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 65, 66, and 67.

[0177] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 65; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 66; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 67. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 65" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO: 65, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 65, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 65, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 65 include, but are not limited to, those listed in Table 25.

[0178] [Table 25] TIFF2026504407000039.tif14155

[0179] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 66" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO: 66, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 66, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 66, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 66 include, but are not limited to, those set forth in Table 26.

[0180] [Table 26]

[0181] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 67" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO: 67, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 67, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 67, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 67 include, but are not limited to, those set forth in Table 27.

[0182] [Table 27]

[0183] In another embodiment, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73 (or a variant of SEQ ID NO: 73 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74 (or a variant of SEQ ID NO: 74 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75 (or a variant of SEQ ID NO: 75 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 12.

[0184] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 73 (or a variant of SEQ ID NO: 73 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 74 (or a variant of SEQ ID NO: 74 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 75 (or a variant of SEQ ID NO: 75 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO: 76 (or a variant of SEQ ID NO: 76 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO: 77 (or a variant of SEQ ID NO: 77 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO: 78 (or a variant of SEQ ID NO: 78 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO: 79 (or a variant of SEQ ID NO: 79 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0185] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 12 can be designed to include framework regions as set forth in Example 12, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 12 and the framework regions described in Example 12, except that framework region 1 having the amino acids set forth in SEQ ID NO: 76 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO: 4, framework region 1 having the amino acids set forth in SEQ ID NO: 20, or framework region 1 having the amino acids set forth in SEQ ID NO: 28. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 12, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0186] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 80. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 80. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 80.

[0187] In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 80, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 73, 74, and 75. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 80, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 73, 74, and 75.

[0188] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 80, or the amino acids set forth in SEQ ID NO: 80 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 80 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 73, 74, and 75.

[0189] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 73; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 74; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 75. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 73" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO: 73, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 73, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 73, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 73 include, but are not limited to, those listed in Table 28.

[0190] [Table 28]

[0191] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 74" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO: 74, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 74, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 74, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 74 include, but are not limited to, those set forth in Table 29.

[0192] [Table 29] TIFF2026504407000044.tif79155

[0193] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 75" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO: 75, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 75, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 75, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 75 include, but are not limited to, those set forth in Table 30.

[0194] [Table 30] TIFF2026504407000046.tif43155

[0195] In another embodiment, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 81 (or a variant of SEQ ID NO: 81 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 82 (or a variant of SEQ ID NO: 82 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 83 (or a variant of SEQ ID NO: 83 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 13.

[0196] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 81 (or a variant of SEQ ID NO: 81 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 82 (or a variant of SEQ ID NO: 82 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 83 (or a variant of SEQ ID NO: 83 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO: 84 (or a variant of SEQ ID NO: 84 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO: 85 (or a variant of SEQ ID NO: 85 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO: 86 (or a variant of SEQ ID NO: 86 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO: 87 (or a variant of SEQ ID NO: 87 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0197] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 13 can be designed to include framework regions as set forth in Example 13, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 13 and the framework regions described in Example 13, except that framework region 1 having the amino acids set forth in SEQ ID NO: 84 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO: 4, framework region 1 having the amino acids set forth in SEQ ID NO: 12, or framework region 1 having the amino acids set forth in SEQ ID NO: 28. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 13, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0198] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 88. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 88. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 88.

[0199] In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 88, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 81, 82, and 83. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 88, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 81, 82, and 83.

[0200] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 88, or the amino acids set forth in SEQ ID NO: 88 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 88 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 81, 82, and 83.

[0201] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 81; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 82; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 83. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 81" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO: 81, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 81, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 81, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 81 include, but are not limited to, those listed in Table 31.

[0202] [Table 31] TIFF2026504407000048.tif14155

[0203] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 82" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO: 82, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 82, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 82, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 82 include, but are not limited to, those set forth in Table 32.

[0204] [Table 32]

[0205] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 83" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO: 83, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 83, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 83, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 83 include, but are not limited to, those set forth in Table 33.

[0206] [Table 33]

[0207] In another embodiment, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can include a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 89 (or a variant of SEQ ID NO: 89 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 90 (or a variant of SEQ ID NO: 90 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 91 (or a variant of SEQ ID NO: 91 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 14.

[0208] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 89 (or a variant of SEQ ID NO: 89 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 90 (or a variant of SEQ ID NO: 90 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 91 (or a variant of SEQ ID NO: 91 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO:92 (or a variant of SEQ ID NO:92 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO:93 (or a variant of SEQ ID NO:93 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO:94 (or a variant of SEQ ID NO:94 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO:95 (or a variant of SEQ ID NO:95 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0209] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 14 can be designed to include framework regions as set forth in Example 14, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 14 and the framework regions described in Example 14, except that framework region 1 having the amino acids set forth in SEQ ID NO:92 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO:4, framework region 1 having the amino acids set forth in SEQ ID NO:12, or framework region 1 having the amino acids set forth in SEQ ID NO:20. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 14, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0210] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 96. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 96. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 96.

[0211] In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 96, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 89, 90, and 91. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 96, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 89, 90, and 91.

[0212] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 96, or the amino acids set forth in SEQ ID NO: 96 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 96 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 89, 90, and 91.

[0213] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 89; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 90; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 91. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 89" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO: 89, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 89, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 89, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 89 include, but are not limited to, those set forth in Table 34.

[0214] [Table 34]

[0215] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO:90" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO:90, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:90, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:90, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO:90 include, but are not limited to, those set forth in Table 35.

[0216] [Table 35] TIFF2026504407000053.tif79156

[0217] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO:91" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO:91, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:91, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:91, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO:91 include, but are not limited to, those set forth in Table 36.

[0218] [Table 36] TIFF2026504407000055.tif43155

[0219] In another embodiment, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 97 (or a variant of SEQ ID NO: 97 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 98 (or a variant of SEQ ID NO: 98 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 99 (or a variant of SEQ ID NO: 99 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 15.

[0220] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 97 (or a variant of SEQ ID NO: 97 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 98 (or a variant of SEQ ID NO: 98 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 99 (or a variant of SEQ ID NO: 99 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO: 100 (or a variant of SEQ ID NO: 100 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO: 101 (or a variant of SEQ ID NO: 101 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO: 102 (or a variant of SEQ ID NO: 102 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO: 103 (or a variant of SEQ ID NO: 103 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0221] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 15 can be designed to include framework regions as set forth in Example 15, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 15 and the framework regions described in Example 15, except that framework region 1 having the amino acids set forth in SEQ ID NO: 100 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO: 4, framework region 1 having the amino acids set forth in SEQ ID NO: 12, or framework region 1 having the amino acids set forth in SEQ ID NO: 28. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 15, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0222] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 104. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 104. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 104.

[0223] In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 104, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 97, 98, and 99. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 104, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 97, 98, and 99.

[0224] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 104, or the amino acids set forth in SEQ ID NO: 104 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 104 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 97, 98, and 99.

[0225] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 97; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 98; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 99. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO:97" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO:97, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:97, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:97, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO:97 include, but are not limited to, those listed in Table 37.

[0226] [Table 37] TIFF2026504407000057.tif14156

[0227] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO:98" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO:98, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:98, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:98, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO:98 include, but are not limited to, those set forth in Table 38.

[0228] [Table 38]

[0229] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO:99" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO:99, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO:99, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO:99, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO:99 include, but are not limited to, those set forth in Table 39.

[0230] [Table 39]

[0231] In another embodiment, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 105 (or a variant of SEQ ID NO: 105 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 106 (or a variant of SEQ ID NO: 106 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 107 (or a variant of SEQ ID NO: 107 with one or two amino acid modifications). Examples of such binding molecules having these CDRs and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) include, but are not limited to, the VH domains shown in Example 16.

[0232] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) provided herein capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide), having a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO: 105 (or a variant of SEQ ID NO: 105 with one or two amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO: 106 (or a variant of SEQ ID NO: 106 with one or two amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 107 (or a variant of SEQ ID NO: 107 with one or two amino acid modifications), can comprise any suitable framework region. For example, such a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising: framework region 1 having the amino acid sequence set forth in SEQ ID NO: 108 (or a variant of SEQ ID NO: 108 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 2 having the amino acid sequence set forth in SEQ ID NO: 109 (or a variant of SEQ ID NO: 109 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); framework region 3 having the amino acid sequence set forth in SEQ ID NO: 110 (or a variant of SEQ ID NO: 110 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications); and framework region 4 having the amino acid sequence set forth in SEQ ID NO: 111 (or a variant of SEQ ID NO: 111 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications).

[0233] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) having any of the CDRs described in Example 16 can be designed to include framework regions as set forth in Example 16, but can also be designed to include one or more framework regions from another antibody or antibody fragment. For example, an antibody domain (e.g., a VH domain) can be designed to include the three CDRs described in Example 16 and the framework regions described in Example 16, except that framework region 1 having the amino acids set forth in SEQ ID NO: 108 is replaced with framework region 1 having the amino acids set forth in SEQ ID NO: 4, framework region 1 having the amino acids set forth in SEQ ID NO: 12, or framework region 1 having the amino acids set forth in SEQ ID NO: 20. In another example, a Fab or scFv can be designed to include (a) the three CDRs described in Example 16, (b) the framework regions set forth in Examples 3-16, and (c) a light chain variable domain.

[0234] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 112. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 112. In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can comprise (a) a heavy chain variable domain comprising an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO: 112.

[0235] In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 112, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 105, 106, and 107. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can comprise a heavy chain variable domain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO: 112, provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 105, 106, and 107.

[0236] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein that has the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 112, or the amino acids set forth in SEQ ID NO: 112 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions). For example, an antibody domain (e.g., a VH domain) provided herein can have the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) and can comprise a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 112 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and / or amino acid additions), provided that the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NOs: 105, 106, and 107.

[0237] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein having the ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide) can comprise a heavy chain variable domain comprising: (i) a CDR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 105; (ii) a CDR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 106; and (iii) a CDR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 107. As used herein, a "CDR1 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 105" refers to a CDR1 having zero, one, or two amino acid substitutions in SEQ ID NO: 105, a CDR1 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 105, and / or a CDR1 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 105, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR1s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 105 include, but are not limited to, those set forth in Table 40.

[0238] [Table 40]

[0239] As used herein, a "CDR2 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 106" refers to a CDR2 having zero, one, or two amino acid substitutions in SEQ ID NO: 106, a CDR2 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 106, and / or a CDR2 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 106, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR2s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 106 include, but are not limited to, those set forth in Table 41.

[0240] [Table 41]

[0241] As used herein, a "CDR3 consisting essentially of the amino acid sequence set forth in SEQ ID NO: 107" refers to a CDR3 having zero, one, or two amino acid substitutions in SEQ ID NO: 107, a CDR3 having zero, one, two, three, four, or five amino acid residues immediately preceding SEQ ID NO: 107, and / or a CDR3 having zero, one, two, three, four, or five amino acid residues immediately following SEQ ID NO: 107, provided that the binding molecule (e.g., antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) maintains its basic ability to bind to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of CDR3s consisting essentially of the amino acid sequence set forth in SEQ ID NO: 107 include, but are not limited to, those set forth in Table 42.

[0242] [Table 42] TIFF2026504407000063.tif49155

[0243] When designing a single-chain antibody (e.g., scFv) having a heavy chain variable domain and a light chain variable domain, the two regions can be linked directly, or they can be linked using any suitable linker sequence. For example, the heavy chain variable domain can be directly linked to the light chain variable domain via a linker sequence. Examples of linker sequences that can be used to link the heavy chain variable domain and the light chain variable domain to create an scFv include, but are not limited to, the linkers described in Example 20.

[0244] As noted herein, the amino acid sequences described herein can include amino acid modifications (e.g., a specified number of amino acid modifications). Such amino acid modifications can include, but are not limited to, amino acid substitutions, amino acid deletions, amino acid additions, and combinations. In some cases, amino acid modifications can be made to improve the binding and / or contact of a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein with an antigen and / or to improve the functional activity of the binding molecule. In some cases, amino acid substitutions in a specified SEQ ID NO can be conservative amino acid substitutions. For example, conservative amino acid substitutions can be made by replacing one amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains can include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0245] In some cases, amino acid substitutions in a specified SEQ ID NO can be non-conservative amino acid substitutions. Non-conservative amino acid substitutions can be made by replacing one amino acid residue with another amino acid residue having a dissimilar side chain. Examples of non-conservative substitutions include, but are not limited to, (a) replacing a hydrophilic residue (e.g., serine or threonine) with a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine); (b) replacing cysteine ​​or proline with any other residue; (c) replacing a residue with a basic side chain (e.g., lysine, arginine, or histidine) with a residue with an acidic side chain (e.g., aspartic acid or glutamic acid); and (d) replacing a residue with a large side chain (e.g., phenylalanine) with glycine or another residue with a small side chain.

[0246] The percent sequence identity between a particular amino acid or nucleic acid sequence and an amino acid or nucleic acid sequence referenced by a particular sequence identification number is determined as follows: First, the amino acid or nucleic acid sequence is compared to the sequence set forth in the particular sequence identification number using the BLAST 2 Sequences (Bl2seq) program from stand-alone versions of BLASTZ, including BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from the Fish & Richardson website (e.g., www.fr.com / blast / ) or the U.S. government's National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov). Instructions on how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. Bl2seq performs comparisons between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, options are set as follows: -i is set to the file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); -j is set to the file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default settings. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\Bl2seq -ic:\seq1.txt -jc:\seq2.txt -p blastn -oc:\output.txt -q -1 -r 2.To compare two amino acid sequences, Bl2seq options are set as follows: -i is set to the file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to the file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default settings. For example, to generate an output file containing a comparison between two amino acid sequences, the following command can be used: C:\Bl2seq -ic:\seq1.txt -jc:\seq2.txt -p blastp -oc:\output.txt. If the two compared sequences share homology, the specified output file will present the regions of homology as aligned sequences. If the two compared sequences do not share homology, the specified output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where the same nucleotide or amino acid residue is present in both sequences. Matched positions refer to positions where the same nucleotide or amino acid residue is present at the same position in the aligned sequences. The percent sequence identity is determined by dividing the number of matches by the length of the sequence shown in the identified sequence (e.g., SEQ ID NO:8, SEQ ID NO:16, or SEQ ID NO:24), and then multiplying the resulting value by 100. For example, an amino acid sequence that has 100 matches when aligned with the sequence shown in SEQ ID NO:8 is 80 percent identical to the sequence shown in SEQ ID NO:8 (i.e., 100÷125×100=80.0). Note that the percent sequence identity value is rounded to two decimal places. For example, 78.11, 78.12, 78.13, and 78.14 will be truncated to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 will be rounded up to 78.2. Note also that length values ​​are always integers.

[0247] Methods for generating amino acid sequence variants (e.g., amino acid sequences containing one or more alterations with respect to a specified SEQ ID NO) can include site-directed or random mutagenesis (e.g., by PCR) of a nucleic acid encoding an antibody or fragment thereof. See, e.g., Zoller, Curr. Opin. Biotechnol. 3: 348-354 (1992). Both naturally occurring and non-naturally occurring amino acids (e.g., artificially derivatized amino acids) can be used to generate the amino acid sequence variants provided herein.

[0248] Further set forth in Table 43 are a number of representative binding molecules (e.g., antibodies, antigen-binding fragments, and / or antibody domains) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide).

[0249] [Table 43]

[0250] The binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can be produced using any suitable method. For example, the binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) can be produced in recombinant host cells. For example, nucleic acids encoding the binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) can be constructed, introduced into an expression vector, and expressed in a suitable host cell. Example 17 is a sequence listing of nucleic acid sequences encoding representative binding molecules (e.g., antibodies, antigen-binding fragments, and / or antibody domains) described herein. In some cases, the binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) can be recombinantly produced in prokaryotic hosts such as E. coli, Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lactobacillus zeae / casei, or Lactobacillus paracasei.The binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) provided herein can also be expressed in yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, or Yarrowia lipolytica), filamentous fungi of the genus Trichoderma (e.g., T. reesei), or in yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, or Yarrowia lipolytica), or in filamentous fungi of the genus Trichoderma (e.g., T. reesei). They can also be recombinantly produced in eukaryotic hosts such as Aspergillus reesei and Aspergillus filamentous fungi (e.g., A. niger and A. oryzae), protozoa such as Leishmania tarentolae, insect cells, or mammalian cells (e.g., mammalian cell lines such as Chinese hamster ovary (CHO) cells, Per.C6 cells, mouse myeloma NS0 cells, baby hamster kidney (BHK) cells, or the human embryonic kidney cell line HEK293). See, e.g., Frenzel et al. (Front Immunol., 4:217 (2013)).

[0251] In some cases, the antigen-binding fragments or antibody domains provided herein can be produced by proteolysis of intact antibodies. For example, antigen-binding fragments can be obtained by treating antibodies with enzymes such as papain or pepsin. Papain digestion of whole antibodies can be used to produce F(ab)2 or Fab fragments, while pepsin digestion of whole antibodies can produce F(ab')2 or Fab' fragments.

[0252] In some cases, the binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) provided herein can be substantially pure. The term "substantially pure," as used herein with respect to a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC), refers to the binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) being substantially free of other polypeptides, lipids, carbohydrates, and nucleic acids to which it is naturally associated. Thus, a substantially pure binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) provided herein is any binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) that has been removed from its natural environment and is at least 60% pure. The substantially pure binding molecules (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) provided herein can be at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent pure.

[0253] Also provided herein are bispecific binding molecules (e.g., bispecific antibodies, bispecific antigen-binding fragments, and / or bispecific antibody domains) that bind to two different epitopes, at least one of which is an epitope of a follistatin polypeptide (e.g., a human follistatin polypeptide). In some cases, bispecific binding molecules provided herein can be designed to bind to two different epitopes of the same follistatin polypeptide (e.g., a human follistatin polypeptide). In some cases, bispecific binding molecules provided herein can bind to an epitope on a follistatin polypeptide (e.g., a human follistatin polypeptide) and a different polypeptide (e.g., a CD3 polypeptide). Bispecific binding molecules can be produced by chemically linking two different binding molecules (e.g., antibodies, antigen-binding fragments, and / or antibody domains). Bispecific binding molecules can also be produced by fusing two antibody-producing cells (e.g., hybridomas) to create a hybrid cell line that produces two different heavy chains and two different light chains within the same cell, resulting in, for example, a bispecific IgG molecule. See Brinkmann and Kontermann, MAbs., 9(2):182-212(2017).

[0254] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) can be fused or conjugated (e.g., covalently or non-covalently linked) to another polypeptide or other moiety to provide a fusion protein or conjugate. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) can be conjugated to a polymer (e.g., polyethylene glycol (PEG), PEG-modified polyethyleneimine (PEI) (PEI-PEG), and / or polyglutamic acid (PGA) (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer), hyaluronic acid, a fluorescent substance, a luminescent substance, a hapten, an enzyme, a metal chelate, a drug, a radioisotope, and / or a cytotoxic agent. Any suitable method can be used to conjugate (e.g., covalently or non-covalently attach) another polypeptide or other moiety to a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager). For example, another polypeptide or other moiety can be conjugated to a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) using the methods described in U.S. Pat. No. 8,021,661.

[0255] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can be modified with a moiety that improves its stabilization and / or retention in the circulation, e.g., in blood, serum, or other tissues, by, e.g., at least 1.5-fold, 2-fold, 5-fold, 10-fold, or 50-fold. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can be conjugated (e.g., covalently or non-covalently attached) to a polymer, such as a substantially non-antigenic polymer. Examples of substantially non-antigenic polymers that can be used as described herein include, but are not limited to, polyalkylene oxide and polyethylene oxide. In some cases, the polymers used herein can have any suitable molecular weight. For example, polymers having an average molecular weight of about 200 daltons to about 35,000 daltons (e.g., about 1,000 daltons to about 15,000 daltons, or about 2,000 daltons to about 12,500 daltons) can be used. In some cases, the binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, cell engagers, and / or ADCs) can be conjugated (e.g., covalently or non-covalently attached) to a water-soluble polymer. Examples of water-soluble polymers that can be used as described herein include, but are not limited to, hydrophilic polyvinyl polymers, polyvinyl alcohol, polyvinylpyrrolidone, polyalkylene oxide homopolymers, polyethylene glycol (PEG), polypropylene glycol, polyoxyethylenated polyols, and copolymers and / or block copolymers thereof, provided that the water solubility of the copolymer or block copolymer is maintained.

[0256] In some cases, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can be conjugated (e.g., covalently or non-covalently linked) to one or more polyoxyalkylenes (e.g., polyoxyethylene, polyoxypropylene, or block copolymers of polyoxyethylene and polyoxypropylene), polymethacrylate, carbomer, branched or unbranched polysaccharides, or combinations thereof. For example, a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) can be covalently linked to polyoxyethylene.

[0257] The present specification also provides ADCs. As used herein, the term "ADC" refers to a conjugate comprising (a) an antigen-binding domain and (b) at least one drug covalently linked, directly or indirectly, to the antigen-binding domain. In some cases, the ADCs described herein can comprise (a) an antigen-binding domain capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) and (b) at least one drug covalently linked, directly or indirectly, to the antigen-binding domain. Any suitable binding molecule (e.g., an antibody, antigen-binding fragment, and / or antibody domain) provided herein and capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can be used as the antigen-binding domain to generate the ADCs described herein. For example, any of the binding molecules listed in Table 43 can be used to generate ADCs capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide). Examples of drugs that can be used to generate the ADCs described herein include, but are not limited to, auristatins (e.g., monomethylauristatin E (MMAE)), mertansine (DM-1), and pyrrolobenzodiazepine (PBD) dimers. Any suitable ADC linker can be used to covalently link one or more drugs to an antigen-binding domain capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) to generate the ADCs provided herein. For example, a cleavable or non-cleavable ADC linker can be used to covalently link one or more drugs to an antigen-binding domain capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) to generate the ADCs provided herein.Examples of ADC linkers that can be used to covalently attach one or more drugs to an antigen-binding domain capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) for purposes of making the ADCs provided herein include, but are not limited to, ADC disulfide linkers, ADC hydrazone linkers, ADC peptide linkers, ADC thioether linkers, and ADC PEG-containing linkers.

[0258] The present specification also provides nucleic acid molecules (e.g., isolated nucleic acid molecules) having a nucleic acid sequence encoding at least a portion of a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein. For example, the isolated nucleic acid molecules provided herein can include a nucleic acid sequence encoding a VH domain, such as a VH domain set forth in any one of Examples 3-16. In another example, the isolated nucleic acid molecules provided herein can include a nucleic acid sequence encoding a CAR or cell engager described herein (e.g., BiTE, BiKE, or TriKE). The nucleic acids (e.g., isolated nucleic acid molecules) provided herein can be single-stranded or double-stranded nucleic acids of any suitable type (e.g., DNA, RNA, or DNA / RNA hybrid).

[0259] The present specification also provides vectors (e.g., plasmid vectors or viral vectors) containing one or more nucleic acids provided herein. Examples of plasmid vectors that can be designed to contain one or more nucleic acids having a nucleic acid sequence encoding at least a portion of the binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) include, but are not limited to, phagemids. Examples of viral vectors that can be designed to contain one or more nucleic acids having a nucleic acid sequence encoding at least a portion of the binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers) include, but are not limited to, retroviral vectors, parvovirus-based vectors (e.g., adenovirus-based vectors and adeno-associated virus (AAV)-based vectors), lentiviral vectors (e.g., herpes simplex (HSV)-based vectors), poxvirus vectors (e.g., vaccinia virus-based vectors and fowlpox virus-based vectors), and hybrid or chimeric viral vectors. For example, viral vectors having an adenoviral backbone and lentiviral components as described elsewhere (Zheng et al., Nat. Biotech., 18(2): 176-80 (2000); WO 98 / 22143; WO 98 / 46778; and WO 00 / 17376), or viral vectors having an adenoviral backbone and AAV components as described elsewhere (Fisher et al., Hum. Gene Ther., 7:2079-2087 (1996)), can be designed to contain one or more nucleic acids having a nucleic acid sequence encoding at least a portion of a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein.

[0260] In some cases, a vector provided herein (e.g., a plasmid vector or a viral vector) can comprise a nucleic acid sequence encoding an scFv or antibody domain (e.g., a VH domain) provided herein. In some cases, a vector provided herein (e.g., a plasmid vector or a viral vector) can comprise a nucleic acid sequence encoding a CAR provided herein. In some cases, a vector provided herein (e.g., a plasmid vector or a viral vector) can comprise a nucleic acid sequence encoding a cell engager provided herein.

[0261] The vectors provided herein (e.g., plasmid vectors or viral vectors provided herein) can include any suitable promoter and other control sequences (e.g., transcription and translation initiation and termination codons) operably linked to a nucleic acid sequence encoding at least a portion of a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager). In some cases, the promoter used to drive expression can be a constitutive promoter or a regulatable promoter. Examples of regulatable promoters that can be used as described herein include, but are not limited to, inducible promoters, repressible promoters, and tissue-specific promoters. Examples of viral promoters that can be used as described herein include, but are not limited to, adenovirus promoters, vaccinia virus promoters, CMV promoters (e.g., immediate-early CMV promoters), and AAV promoters.

[0262] Any suitable method can be used to generate a nucleic acid molecule (or vector, such as a plasmid vector or viral vector) having a nucleic acid sequence encoding at least a portion of a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein. For example, molecular cloning techniques can be used to generate a nucleic acid molecule (or vector, such as a plasmid vector or viral vector) having a nucleic acid sequence encoding at least a portion of a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein, as described elsewhere (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0263] Also provided herein are host cells comprising a nucleic acid provided herein (e.g., a nucleic acid having a nucleic acid sequence encoding at least a portion of a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager)). Host cells that can be designed to contain one or more nucleic acids provided herein can be prokaryotic or eukaryotic. Examples of prokaryotic cells that can be designed to contain a nucleic acid provided herein include, but are not limited to, Escherichia coli (E. coli) (e.g., Tb-1, TG-1, DH5α, XL-Blue MRF (Stratagene), SA2821, or Y1090 cells), Bacillus subtilis, Salmonella typhimurium, Serratia marcescens, or Pseudomonas (e.g., P. aerugenosa) cells. Examples of eukaryotic cells that can be designed to contain a nucleic acid provided herein include, but are not limited to, insect cells (e.g., Sf9 cells or Ea4 cells), yeast cells (e.g., S. cerevisiae cells), and mammalian cells (e.g., mouse, rat, hamster, monkey, or human cells). For example, VERO cells, HeLa cells, 3T3 cells, Chinese hamster ovary (CHO) cells, W138 BHK cells, COS-7 cells, and MDCK cells can be designed to contain a nucleic acid provided herein. Any suitable method can be used to introduce one or more nucleic acids provided herein (e.g., a vector such as a plasmid or viral vector carrying a nucleic acid sequence encoding at least a portion of a binding molecule provided herein) into a host cell.For example, the nucleic acids provided herein can be introduced into host cells using transformation by calcium chloride treatment, transduction, conjugation, triparental mating, DEAE-dextran transfection, infection, membrane fusion with liposomes, high-velocity DNA-coated particle bombardment, direct microinjection into single cells, electroporation, or a combination thereof (see, e.g., Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Davis et al., Basic Methods in Molecular Biology (1986); and Neumann et al., EMBO J., 1:841 (1982)).

[0264] In some cases, cells such as T cells, stem cells (e.g., induced pluripotent stem cells or mesenchymal stem cells), or NK cells can be designed to express one or more nucleic acids encoding a CAR described herein. For example, a population of T cells can be infected with a viral vector designed to express a nucleic acid encoding a CAR described herein (e.g., a CAR capable of binding to a follistatin polypeptide).

[0265] In some cases, cells such as T cells, stem cells (e.g., induced pluripotent stem cells or mesenchymal stem cells), or NK cells can be designed to express one or more nucleic acids encoding a cell engager described herein. For example, a population of T cells can be infected with a viral vector designed to express a nucleic acid encoding a cell engager described herein (e.g., a cell engager capable of binding to a follistatin polypeptide).

[0266] In some cases, a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein can be produced using a method that includes: (a) introducing nucleic acid encoding a polypeptide into a host cell; (b) culturing the host cell in a culture medium under conditions sufficient to express the polypeptide; (c) recovering the polypeptide from the cell or culture medium; and (d) purifying the polypeptide (e.g., to at least 50, 60, 70, 80, 90, 95, 97, 98, or 99 percent pure).

[0267] In some cases, the binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, cell engagers, and / or ADCs), nucleic acids provided herein (e.g., nucleic acids encoding antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), vectors provided herein (e.g., viral vectors designed to express antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), and / or host cells provided herein (e.g., host cells designed to express antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein) can be formulated as pharmaceutical compositions for administration to a mammal (e.g., a human) with cancer to treat the mammal. In some cases, the binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, cell engagers, and / or ADCs), nucleic acids provided herein (e.g., nucleic acids encoding antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), vectors provided herein (e.g., viral vectors designed to express antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein), and / or host cells provided herein (e.g., host cells designed to express antibodies, antigen-binding fragments, antibody domains, CARs, and / or cell engagers provided herein) can be formulated as pharmaceutical compositions for administration to a mammal (e.g., a human) to reduce the number of cancer cells in the mammal and / or increase the survival time of a mammal afflicted with cancer. For example, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, antibody domains, cell engagers, and / or ADCs) capable of binding to a follistatin polypeptide (e.g., a human follistatin polypeptide) can be formulated as pharmaceutical compositions for administration to a mammal (e.g., a human).In some cases, the pharmaceutical compositions provided herein may include a pharmaceutically acceptable carrier, such as a buffer, salt, surfactant, sugar, osmotic agent, or a combination thereof, as described elsewhere (Gervasi, et al., Eur. J. Pharmaceutics and Biopharmaceutics, 131:8-24 (2018)). Examples of pharmaceutically acceptable carriers that can be used to prepare the pharmaceutical compositions provided herein include, but are not limited to, water, lactic acid, citric acid, sodium chloride, sodium citrate, sodium succinate, sodium phosphate, surfactants (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), dextran 40, or sugars (e.g., sorbitol, mannitol, sucrose, dextrose, or trehalose), or a combination thereof. For example, pharmaceutical compositions designed to include a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, cell engager, and / or ADC) (or a nucleic acid, vector, or host cell provided herein) can be formulated to include a buffer (e.g., acetate buffer, citrate buffer, histidine buffer, succinate buffer, phosphate buffer, or hydroxymethylaminomethane (Tris) buffer), a surfactant (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), and a sugar such as sucrose.Other ingredients that can be included in the pharmaceutical compositions provided herein include, but are not limited to, amino acids such as glycine or arginine, antioxidants such as ascorbic acid, methionine or ethylenediaminetetraacetic acid (EDTA), anticancer agents such as enzalutamide, imatinib, gefitinib, erlotinib, sunitinib, lapatinib, nilotinib, sorafenib, temsirolimus, everolimus, pazopanib, crizotinib, ruxolitinib, axitinib, bosutinib, cabozantinib, ponatinib, regorafinib, ibrutinib, trametinib, perifosine, bortezomib, carfilzomib, batimastat, ganetespib, obatoclax, navitoclax, taxol, paclitaxel, or bevacizumab, or combinations thereof. For example, the pharmaceutical compositions provided herein may comprise one or more binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cells designed to express a CAR capable of binding to a follistatin polypeptide, one or more cell engagers, and / or one or more ADCs) in combination with an anti-PD-1 antibody or a PD-1 inhibitor (e.g., cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrilamide, cefotaxime ... In some embodiments, the therapeutic agent may be formulated to contain a combination of one or more checkpoint inhibitors, such as pembrolizumab, ibuprofen, ribozyme, ribozyme inhibitors (e.g., pembrolizumab, ribozyme inhibitors ...

[0268] In some cases, pharmaceutical compositions provided herein can be formulated to include one or more binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cells designed to express a CAR capable of binding to a follistatin polypeptide, one or more cell engagers, and / or one or more ADCs) in combination with one or more anti-cancer agents. In some cases, one or more binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cells designed to express a CAR capable of binding to a follistatin polypeptide, one or more cell engagers, and / or one or more ADCs) can be used in combination with one or more anti-cancer agents to treat a mammal (e.g., a human) with cancer. Examples of anti-cancer agents that can be used as described herein include, but are not limited to, taxol compounds, cisplatin, monomethyl auristatin E (MMAE), the tubulin inhibitor DM4, the active metabolite SN-38, and pyrrolobenzodiazepine (PDB) dimers.

[0269] In some cases, when a pharmaceutical composition is formulated to include one or more binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cells designed to express a CAR capable of binding to a follistatin polypeptide, one or more cell engagers, and / or one or more ADCs), any suitable concentration of the binding molecule can be used. For example, the pharmaceutical compositions provided herein can be formulated to include one or more binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cells designed to express a CAR capable of binding to a follistatin polypeptide, one or more cell engagers, and / or one or more ADCs). +The composition can be formulated into a liquid containing about 1 mg to about 500 mg of the compound (cell population, cell engager, and / or ADC) per mL (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 2 mg to about 200 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg). In another example, pharmaceutical compositions provided herein can be formulated to be solid or semi-solid containing about 0.5 mg to about 500 mg (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg) of a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, antibody domain, cell engager, and / or ADC). In some cases, pharmaceutical compositions containing a binding molecule provided herein (e.g., an antibody, antigen-binding fragment, and / or antibody domain) can be formulated to be about 1 x 10 5 ~Approx. 1×10 12 (For example, about 1 × 10 5 ~Approx. 1×10 10 , about 1×10 5 ~Approx. 1×10 8 , about 1×10 6 ~Approx. 1×10 12 , about 1×10 6 ~Approx. 1×10 12 , about 1×10 8 ~Approx. 1×10 12 , about 1×10 9 ~Approx. 1×10 12 , about 1×10 6 ~Approx. 1×10 11 , or approximately 1 × 10 7 ~Approx. 1×10 10) can be formulated as a dosage form having a potency of the binding molecule.

[0270] In some cases, when pharmaceutical compositions are formulated to include one or more nucleic acids (e.g., vectors such as viral vectors) encoding at least a portion of a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, CAR, and / or cell engager) provided herein, any suitable concentration of nucleic acid can be used. For example, the pharmaceutical compositions provided herein can be formulated to be liquids containing about 0.5 mg to about 500 mg of the nucleic acids provided herein per mL (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 2 mg to about 200 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg). In another example, the pharmaceutical compositions provided herein can be formulated to be solid or semi-solid containing about 0.5 mg to about 500 mg of the nucleic acid provided herein (e.g., about 1 mg to about 500 mg, about 10 mg to about 500 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 250 mg, about 0.5 mg to about 150 mg, about 0.5 mg to about 100 mg, about 0.5 mg to about 50 mg, about 1 mg to about 300 mg, about 10 mg to about 300 mg, about 25 mg to about 300 mg, about 50 mg to about 150 mg, or about 150 mg to about 300 mg).

[0271] In some cases, pharmaceutical compositions designed to include a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, cell engager, and / or ADC) provided herein can be formulated to include one or more agents capable of reducing aggregation of the binding molecule when formulated. Examples of such agents that can be used as described herein include, but are not limited to, methionine, arginine, lysine, aspartic acid, glycine, glutamic acid, and combinations thereof. In some cases, one or more of these amino acids can be included in the formulation at a concentration of about 0.5 mM to about 145 mM (e.g., about 1 mM to about 145 mM, about 10 mM to about 145 mM, about 100 mM to about 145 mM, about 0.5 mM to about 125 mM, about 0.5 mM to about 100 mM, about 0.5 mM to about 75 mM, or about 10 mM to about 100 mM).

[0272] The pharmaceutical compositions provided herein can take any suitable form. For example, the pharmaceutical compositions provided herein can be designed to be liquid, semi-solid, or solid. In some cases, the pharmaceutical compositions provided herein can be a solution (e.g., an injectable and / or infusible solution), dispersion, suspension, tablet, pill, powder, microemulsion, liposome, or suppository. In some cases, the pharmaceutical compositions provided herein can be lyophilized. In some cases, the pharmaceutical compositions provided herein (e.g., a pharmaceutical composition comprising one or more binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs)) can be formulated with a carrier or coating designed to protect against rapid release. For example, the pharmaceutical compositions provided herein can be formulated as controlled-release or modified-release formulations as described elsewhere (U.S. Patent Application Publication Nos. 2019 / 0241667; 2019 / 0233522; and 2019 / 0233498).

[0273] The present specification also provides a method for producing one or more binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or a nucleic acid, vector, or host cell provided herein (e.g., a CAR + For example, methods are provided for administering to a mammal (e.g., a human) a composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, and / or host cell (e.g., CAR) provided herein). + In some cases, a composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, and / or host cell (e.g., CAR) provided herein) can be administered to a mammal (e.g., a human) with cancer to treat the mammal. + A composition comprising the cancer cells (e.g., a pharmaceutical composition provided herein) can be administered to a mammal (e.g., a human) to reduce the number of cancer cells in the mammal and / or increase the survival time of a mammal afflicted with cancer.

[0274] One or more binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or a nucleic acid, vector, or host cell provided herein (e.g., a CAR +A composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein can be used to treat any suitable cancer. For example, a mammal (e.g., a human) with cancer can be treated by administering to the mammal a composition (e.g., a pharmaceutical composition) comprising one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein. Examples of cancers that can be treated as described herein include, but are not limited to, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, kidney cancer, head and neck cancer, liver cancer, cervical cancer, urothelial cancer, thymic epithelial tumor, and breast cancer. In some cases, follistatin + Cancer (e.g., follistatin + ovarian cancer, follistatin + Lung cancer, or follistatin + A composition (e.g., a pharmaceutical composition) comprising one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein can be administered to a mammal (e.g., a human) with cancer (e.g., prostate cancer) to treat the mammal (e.g., reduce the number of cancer cells in the mammal).

[0275] Any suitable method can be used to administer the compositions (e.g., pharmaceutical compositions) provided herein to a mammal (e.g., a human). For example, compositions provided herein (e.g., pharmaceutical compositions comprising one or more binding molecules provided herein, such as one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs provided herein) can be administered to a mammal (e.g., a human) intravenously (e.g., via intravenous injection or infusion), intratumorally (e.g., via intratumoral injection), subcutaneously (e.g., via subcutaneous injection), intraperitoneally (e.g., via intraperitoneal injection), orally, via inhalation, or intramuscularly (e.g., via intramuscular injection). In some cases, the route and / or method of administration of a composition (e.g., a pharmaceutical composition provided herein) can be adjusted depending on the mammal receiving treatment.

[0276] In some cases, one or more binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or nucleic acids, vectors, or host cells provided herein (e.g., CAR + An effective amount of a composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be an amount that reduces the number of cancer cells in a mammal having cancer without causing significant toxicity to the mammal. In some cases, the effective amount of a composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be an amount that reduces the number of cancer cells in a mammal having cancer without causing significant toxicity to the mammal. +An effective amount of a composition (e.g., a pharmaceutical composition provided herein) comprising a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, cell engager, and / or ADC) provided herein can be an amount that increases survival time in a mammal with cancer compared to a control mammal with a comparable cancer that is not treated with the composition. For example, an effective amount of a binding molecule (e.g., an antibody, antigen-binding fragment, antibody domain, cell engager, and / or ADC) provided herein can be from about 0.001 mg / kg to about 100 mg / kg (e.g., from about 0.001 mg / kg to about 90 mg / kg, from about 0.001 mg / kg to about 80 mg / kg, from about 0.001 mg / kg to about 70 mg / kg, from about 0.001 mg / kg to about 60 mg / kg). / kg, about 0.001mg / kg to about 50mg / kg, about 0.001mg / kg to about 40mg / kg, about 0.001mg / kg to about 30mg / kg, about 0.005mg / kg to about 100mg / kg, about 0.01mg / kg~about 100mg / kg, about 0.05mg / kg~about 100mg / kg, about 0.1mg / kg~about 100mg / kg, about 0.5mg / kg~about 100mg / kg, about 1mg / kg~about 100mg / kg, about 5mg / kg to about 100mg / kg, about 0.01mg / kg to about 25mg / kg, about 0.1mg / kg to about 30mg / kg, about 0.15mg / kg to about 25mg / kg, about 0.2 mg / kg~about 20mg / kg, about 0.5mg / kg~about 20mg / kg, about 1mg / kg~about 30mg / kg, about 1mg / kg~about 25mg / kg, about 1mg / kg~about 20mg / kg, about 2mg / kg The effective dose may be about 20 mg / kg to about 20 mg / kg, about 5 mg / kg to about 30 mg / kg, about 10 mg / kg to about 30 mg / kg, about 15 mg / kg to about 30 mg / kg, about 20 mg / kg to about 30 mg / kg, about 3 mg / kg to about 30 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 1 mg / kg to about 3 mg / kg. The effective dose may remain constant or may be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors may affect the actual effective amount used for a particular application.For example, when treating a mammal with cancer, the severity of the cancer, the route of administration, the age and general health of the mammal, the use of excipients, the possibility of co-administration with other therapeutic or prophylactic treatments, such as the use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may necessitate a greater or lesser effective amount of the compositions provided herein (e.g., pharmaceutical compositions comprising one or more binding molecules provided herein) administered.

[0277] In some cases, one or more binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or nucleic acids, vectors, or host cells provided herein (e.g., CAR + An effective number of administrations of a composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a number that reduces the number of cancer cells in a mammal having cancer without causing significant toxicity to the mammal. In some cases, administration of one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a number that reduces the number of cancer cells in a mammal having cancer without causing significant toxicity to the mammal. +An effective number of administrations of a composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding molecules (e.g., a medicament for treating cancer) can be a number that increases the survival time of a mammal having cancer compared to a control mammal having a comparable cancer but not treated with the composition. For example, an effective number of administrations of a pharmaceutical composition provided herein, such as a pharmaceutical composition comprising one or more binding molecules provided herein, can be about twice daily to once per year (e.g., about twice daily to once per month, about twice daily to once per week, about once daily to once per month, or about once daily to once per week). In some cases, the number of administrations of a pharmaceutical composition provided herein, such as a pharmaceutical composition comprising one or more binding molecules provided herein, can be daily. The number of administrations of a pharmaceutical composition provided herein, such as a pharmaceutical composition comprising one or more binding molecules provided herein, can remain constant or can vary over the course of treatment. Various factors can affect the actual effective number used for a particular application. For example, the severity of the cancer, the route of administration, the age and general health of the mammal, the use of excipients, the possibility of co-administration with other therapeutic or prophylactic treatments, such as the use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may necessitate a greater or lesser actual effective dose of the compositions provided herein (e.g., pharmaceutical compositions comprising one or more binding molecules provided herein).

[0278] In some cases, one or more binding molecules provided herein (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) (or nucleic acids, vectors, or host cells provided herein (e.g., CAR +An effective administration period for a composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a period that reduces the number of cancer cells in the body of a mammal without causing significant toxicity to the mammal. In some cases, the effective administration period for a composition (e.g., a pharmaceutical composition provided herein) comprising one or more binding molecules (e.g., one or more antibodies, one or more antigen-binding fragments, one or more antibody domains, one or more cell engagers, and / or one or more ADCs) provided herein (or a nucleic acid, vector, or host cell (e.g., CAR) provided herein) can be a period that reduces the number of cancer cells in the body of a mammal without causing significant toxicity to the mammal. + The effective administration period of a composition comprising a binding molecule (e.g., a pharmaceutical composition provided herein) can be a period that increases the survival time of a mammal having cancer compared to a control mammal having a comparable cancer that is not treated with the composition. For example, the effective administration period of a pharmaceutical composition provided herein, such as a pharmaceutical composition comprising one or more binding molecules provided herein, can vary from the time of a single administration to several weeks to several months (e.g., 4-12 weeks). Several factors can affect the actual effective administration period used for a particular application. For example, the severity of the cancer, the route of administration, the age and general health of the mammal, the use of excipients, the possibility of coadministration with other therapeutic or prophylactic treatments, such as the use of other agents (e.g., checkpoint inhibitors), and the judgment of the treating physician may require an increase or decrease in the actual effective administration period of a composition provided herein (e.g., a pharmaceutical composition comprising one or more binding molecules provided herein).

[0279] In some cases, the binding molecules (e.g., antibodies, antigen-binding fragments, and / or antibody domains) provided herein can be used to detect the presence or absence of a follistatin polypeptide (e.g., a human follistatin polypeptide) in vitro, in situ, or in vivo (e.g., in vivo imaging in a mammal, such as a human). For example, the binding molecules (e.g., antibodies, antigen-binding fragments, and / or antibody domains) provided herein can be designed to include a label (e.g., a covalently attached radioactive, enzymatic, chromogenic, or fluorescent label). The labeled binding molecule can be used to detect the presence or absence of a follistatin polypeptide (e.g., a human follistatin polypeptide) in a biological sample in vitro. Examples of biological samples that can be evaluated using the binding molecules (e.g., antibodies, antigen-binding fragments, and / or antibody domains) provided herein include, but are not limited to, serum samples, plasma samples, tissue samples, biopsy samples, cell line samples, and tissue culture samples. In some cases, biological samples that can be evaluated as described herein can include mammalian somatic tissues and / or somatic cells that can express a follistatin polypeptide (e.g., a human follistatin polypeptide), such as leukocytes, ovarian tissue or cells, prostate tissue or cells, cardiac tissue or cells, placental tissue or cells, pancreatic tissue or cells, liver tissue or cells, spleen tissue or cells, lung tissue or cells, breast tissue or cells, head and neck tissue or cells, endometrial tissue or cells, colon tissue or cells, colorectal tissue or cells, cervical tissue or cells, stomach tissue or cells, or umbilical cord tissue or cells, etc. In some cases, binding molecules provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) can be immobilized, e.g., on a support, and retention of a follistatin polypeptide (e.g., a human follistatin polypeptide) from a biological sample can be detected on the support, and / or vice versa.In some cases, the binding molecules provided herein (e.g., antibodies, antigen-binding fragments, and / or antibody domains) can be used in applications such as fluorescence polarization, microscopy, ELISA, centrifugation, chromatography, and / or cell sorting (e.g., fluorescence-activated cell sorting).

[0280] In some cases, binding molecules (e.g., antibodies, antigen-binding fragments, and / or antibody domains) provided herein containing a label (e.g., a covalently attached radioactive label) can be used to detect the presence or absence of a follistatin polypeptide (e.g., a human follistatin polypeptide) in a mammal (e.g., a human). For example, binding molecules (e.g., antibodies, antigen-binding fragments, and / or antibody domains) provided herein labeled (e.g., covalently labeled) with a radioactive or MRI-detectable label can be administered to a mammal (e.g., a human), and the mammal can be evaluated using a method for detecting the detectable label. In some cases, the mammal can be scanned to evaluate the location of a labeled binding molecule provided herein within the mammal. For example, the mammal can be imaged using NMR or other tomography techniques.

[0281] Examples of labels that can be attached (e.g., covalently or non-covalently) to the binding molecules (e.g., antibodies, antigen-binding fragments, and / or antibody domains) provided herein include: 131 I, 111 In, 123 I, 99m Tc, 32 P, 33 P, 125 I, 3 H, 14 C, and 188These include, but are not limited to, radioactive labels such as Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron-emitting isotopes detectable with a positron emission tomography ("PET") scanner, chemiluminescent agents such as luciferin, and enzymatic markers such as peroxidase or phosphatase. In some cases, short-range emitters can also be used, such as isotopes detectable with short-range detector probes.

[0282] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0283] Example 1: Obtaining a binding molecule capable of binding to a human follistatin polypeptide To identify binding molecules that bind to the human follistatin polypeptide, a large-scale phage display antibody domain library was panned and screened using the amino acid sequence shown in SEQ ID NO: 113 (Example 2). To identify such binding molecules, a polypeptide containing amino acid residues 1 to 334 of the human follistatin polypeptide shown in Example 2 was fused to an AviTag / His tag / Fc tag sequence at the C-terminus of the follistatin sequence, and the resulting follistatin-AviTag polypeptide was used to pan a human VH domain phage display library. Fourteen VH domains (clones #1 to #14; Examples 3 to 16) were identified.

[0284] Fourteen VH domains (clones #1 to #14) were evaluated for binding affinity and specificity to the human follistatin polypeptide using ELISA (Figure 4). Clones #1 to #14 had EC values ​​of 17, 19, 2.5, 18, 3, 2.5, 3.5, 3.2, 12.3, 0.5, 21, 21, 68, and 23, respectively. 50The VH domains had affinity values ​​(nM) indicating high affinity binding to human follistatin polypeptide. Twelve of the 14 VH domains were used to generate Fc versions of the binding molecules and evaluated for binding to human follistatin polypeptide using ELISA (FIG. 5). Twelve of the 14 VH domains were evaluated for binding to BSA (FIG. 6).

[0285] To investigate whether anti-follistatin binding molecules could enhance cancer cell killing by chemotherapeutic agents such as cisplatin, FST3G9 and FST1E8 were used. Both FST3G9 and FST1E8 enhanced the ability of cisplatin to kill cancer cells (Figure 7). See also Figures 8-9.

[0286] Example 2 Exemplary Human Follistatin Sequences [ka] [ka]

[0287] Example 3: Sequence of Exemplary Anti-Follistatin Clone #1 [ka] [ka]

[0288] Example 4: Sequence of Exemplary Anti-Follistatin Clone #2 [ka] [ka]

[0289] Example 5: Sequence of Exemplary Anti-Follistatin Clone #3 [ka]

[0290] Example 6: Sequence of Exemplary Anti-Follistatin Clone #4 [ka] [ka]

[0291] Example 7: Sequence of Exemplary Anti-Follistatin Clone #5 [ka] [ka]

[0292] Example 8: Sequence of Exemplary Anti-Follistatin Clone #6 [ka]

[0293] Example 9: Sequence of Exemplary Anti-Follistatin Clone #7 [ka]

[0294] Example 10: Sequence of Exemplary Anti-Follistatin Clone #8 [ka] [ka]

[0295] Example 11 Sequence of Exemplary Anti-Follistatin Clone #9 [ka]

[0296] Example 12 Sequence of Exemplary Anti-Follistatin Clone #10 [ka]

[0297] Example 13: Sequence of Exemplary Anti-Follistatin Clone #11 [ka] [ka]

[0298] Example 14: Sequence of Exemplary Anti-Follistatin Clone #12 [ka] [ka]

[0299] Example 15: Sequence of Exemplary Anti-Follistatin Clone #13 [ka]

[0300] Example 16: Sequence of Exemplary Anti-Follistatin Clone #14 [ka] [ka]

[0301] Example 17 Exemplary Nucleic Acid Sequences Encoding Anti-Follistatin Clones [ka] [ka] [ka] [ka]

[0302] Example 18 Exemplary Ig (e.g., IgG1) Structures Heavy chain: heavy chain variable domain + CH1 + hinge + CH2 + CH3 Light chain: light chain variable domain + constant light chain (κ or λ) [ka]

[0303] Example 19 Exemplary scFv Structures Exemplary scFv structures: Heavy chain variable domain / region + linker + light chain variable domain / region Exemplary scFv structures: Light chain variable domain / region + linker + heavy chain variable domain / region

[0304] Example 20 Exemplary Linker Sequences Exemplary linker sequences for scFv, CAR, and / or cell engager [ka]

[0305] Example 21 Exemplary CAR Structures Signal peptide + scFv + (linker / hinge) n1 +Transmembrane domain+(intracellular signaling domain) n2 n1=0, 1, 2, or 3 n2 = 1, 2, 3, 4, or 5 with or without a linker located between consecutive intracellular signaling domains Signal peptide + scFv + linker + hinge + transmembrane domain + intracellular signaling domain + intracellular signaling domain + linker + intracellular signaling domain Signal peptide + scFv + linker + hinge + transmembrane domain + intracellular signaling domain + linker + intracellular signaling domain Signal peptide + scFv + hinge + transmembrane domain + intracellular signaling domain + intracellular signaling domain + linker + intracellular signaling domain Signal peptide + scFv + hinge + transmembrane domain + intracellular signaling domain + intracellular signaling domain + intracellular signaling domain Signal peptide + scFv + linker + transmembrane domain + intracellular signaling domain + intracellular signaling domain + intracellular signaling domain Signal peptide + VH domain + (linker / hinge) n1 +Transmembrane domain+(intracellular signaling domain) n2 n1=0, 1, 2, or 3 n2 = 1, 2, 3, 4, or 5 with or without a linker located between consecutive intracellular signaling domains Signal peptide + VH domain + linker + hinge + transmembrane domain + intracellular signaling domain + intracellular signaling domain + linker + intracellular signaling domain Signal peptide + VH domain + linker + hinge + transmembrane domain + intracellular signaling domain + linker + intracellular signaling domain Signal peptide + VH domain + hinge + transmembrane domain + intracellular signaling domain + intracellular signaling domain + linker + intracellular signaling domain Signal peptide + VH domain + hinge + transmembrane domain + intracellular signaling domain + intracellular signaling domain + intracellular signaling domain Signal peptide + VH domain + linker + transmembrane domain + intracellular signaling domain + intracellular signaling domain + intracellular signaling domain

[0306] Example 22 Exemplary signal peptides for CAR [ka]

[0307] Example 23 Exemplary Hinge for CAR In some cases, these hinges can be used as linkers. [ka] [ka]

[0308] Example 24 Exemplary Transmembrane Domains for CAR [ka]

[0309] Example 25 Exemplary Intracellular Signaling Domains for CARs [ka]

[0310] Example 26 Exemplary CARs and Sequences [ka] [ka]

[0311] Example 27 Exemplary Linker and gOKT3-7 scFv Sequences [ka] [ka]

[0312] Example 28 Exemplary scFv Sequences Exemplary Antigen Binding Domains that Bind to T Cells [ka]

[0313] Example 29 Exemplary scFv Sequences Exemplary antigen-binding domains that can be used to design cell engagers that bind to NK cells [ka] [ka] [ka]

[0314] Example 30 Exemplary BiTEs that Bind Follistatin and T Cells [ka]

[0315] Example 31 Exemplary BiTEs that Bind Follistatin and T Cells [ka]

[0316] Example 32 Combinations of Anti-Follistatin VH Compounds Different VH compounds targeting follistatin (identified as demonstrated in Figure 5) were tested in combination to determine whether they possess dual-targeting activity. To map the distinct binding regions of the binding molecules in follistatin, competitive binding assays with FST3G9, FST19, and FST1 were used. The competitive binding assays indicated that FST3 and FST4 share the same binding epitope as FST3G9, FST19 shares the same binding epitope as FST2, and FST1 shares the same binding epitope as FST6. Other binding molecules exhibited distinct binding epitopes. These competitive BLItz assays allowed the binding molecules to be divided into five groups based on their binding regions in follistatin (Figure 10). Subsequently, chemotherapy synergy studies were performed using propidium iodide (PI) and Annexin V flow analysis of primary ovarian cancer cells treated with vehicle, Cis, or Cis in combination with various anti-follistatin VH clones. These studies demonstrated that while 3G9 increased Cis-mediated killing, combinations of 3G9 with 1E8 and 3G9 with 6 exhibited the highest levels of cell killing (FIG. 11).

[0317] Example 33 Design of a CAR derived from a binding molecule capable of binding to a human follistatin polypeptide Clones #1 to #14 are used to generate CAR clones with the structures shown in Examples 21 or 26. Nucleic acids encoding these CARs under the control of a CMV promoter are introduced into human T cells. CAR-expressing T cells related to the CAR clones express follistatin in the human body. + They are used as effector cells to kill target cells that contain follistatin on their cell surface, such as cancer cells.

[0318] Example 34 Design of BiKE derived binding molecules capable of binding to human follistatin polypeptide Clones #1 to #14 are used to generate BiKEs with the following configuration: VH domain of any one of clones #1 to #14 + (G4S) linker (SEQ ID NO: 158) + anti-NKG2A VH + linker + anti-NKG2A VL. Such BiKEs are synthesized using follistatin + It is used to direct NK cells to kill cells that contain human follistatin on their cell surface, such as cancer cells.

[0319] Example 35 Design of BiTEs derived from binding molecules capable of binding to human follistatin polypeptide Clones #1 to #14 were used to generate BiTEs having the following configuration: the VH domain of any one of clones #1 to #14 + (GSGSS)3 linker (SEQ ID NO: 650) + anti-CD3 scFv + GSGSSG linker (SEQ ID NO: 649) + hIgG1 Fc.

[0320] Such BiTEs include follistatin + It is used to direct T cells to kill cells that contain human follistatin on their cell surface, such as cancer cells.

[0321] Other embodiments While the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to illustrate, but not limit, the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Antibodies, including: (i) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 2 (or SEQ ID NO: 2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 3 (or SEQ ID NO: 3 with one, two, or three amino acid additions, deletions, or substitutions); (ii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions); (iii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 (or SEQ ID NO: 17 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two, or three amino acid additions, deletions, or substitutions): (iv) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:25 (or SEQ ID NO:25 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:26 (or SEQ ID NO:26 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:27 (or SEQ ID NO:27 with one, two, or three amino acid additions, deletions, or substitutions); (v) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 33 (or SEQ ID NO: 33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 34 (or SEQ ID NO: 34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 35 (or SEQ ID NO: 35 with one, two, or three amino acid additions, deletions, or substitutions); (vi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 41 (or SEQ ID NO: 41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 42 (or SEQ ID NO: 42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 43 (or SEQ ID NO: 43 with one, two, or three amino acid additions, deletions, or substitutions); (vii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 49 (or SEQ ID NO: 49 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 50 (or SEQ ID NO: 50 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 51 (or SEQ ID NO: 51 with one, two, or three amino acid additions, deletions, or substitutions); (viii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 57 (or SEQ ID NO: 57 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 58 (or SEQ ID NO: 58 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 59 (or SEQ ID NO: 59 with one, two, or three amino acid additions, deletions, or substitutions); (ix) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 65 (or SEQ ID NO: 65 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 66 (or SEQ ID NO: 66 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 67 (or SEQ ID NO: 67 with one, two, or three amino acid additions, deletions, or substitutions); (x) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 73 (or SEQ ID NO: 73 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 74 (or SEQ ID NO: 74 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 75 (or SEQ ID NO: 75 with one, two, or three amino acid additions, deletions, or substitutions); (xi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 81 (or SEQ ID NO: 81 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 82 (or SEQ ID NO: 82 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 83 (or SEQ ID NO: 83 with one, two, or three amino acid additions, deletions, or substitutions); (xii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 89 (or SEQ ID NO: 89 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 90 (or SEQ ID NO: 90 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 91 (or SEQ ID NO: 91 with one, two, or three amino acid additions, deletions, or substitutions); (xiii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 97 (or SEQ ID NO: 97 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 98 (or SEQ ID NO: 98 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 99 (or SEQ ID NO: 99 with one, two, or three amino acid additions, deletions, or substitutions); or (xiv) A heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 105 (or SEQ ID NO: 105 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 106 (or SEQ ID NO: 106 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 107 (or SEQ ID NO: 107 with one, two, or three amino acid additions, deletions, or substitutions).

2. 2. The antibody of claim 1, comprising the ability to bind to SEQ ID NO: 113 or SEQ ID NO:

115.

3. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (i).

4. The antibody of claim 3, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

8.

5. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (ii).

6. The antibody of claim 5, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

16.

7. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (iii).

8. 8. The antibody of claim 7, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

24.

9. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (iv).

10. 10. The antibody of claim 9, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

32.

11. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (v).

12. 12. The antibody of claim 11, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

40.

13. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (vi).

14. 14. The antibody of claim 13, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

48.

15. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (vii).

16. 16. The antibody of claim 15, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

56.

17. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (viii).

18. 18. The antibody of claim 17, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

64.

19. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (ix).

20. 20. The antibody of claim 19, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

72.

21. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (x).

22. 22. The antibody of claim 21, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

80.

23. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (xi).

24. 24. The antibody of claim 23, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

88.

25. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (xii).

26. 26. The antibody of claim 25, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

96.

27. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (xiii).

28. 28. The antibody of claim 27, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

104.

29. The antibody of claim 1 or 2, comprising the heavy chain variable domain or region of (xiv).

30. 30. The antibody of claim 29, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

112.

31. The antibody of any one of claims 1 to 30, which is a monoclonal antibody.

32. The antibody of any one of claims 1 to 31, which is an scFv antibody.

33. An antigen-binding fragment comprising: (i) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 2 (or SEQ ID NO: 2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 3 (or SEQ ID NO: 3 with one, two, or three amino acid additions, deletions, or substitutions); (ii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions); (iii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 (or SEQ ID NO: 17 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two, or three amino acid additions, deletions, or substitutions): (iv) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:25 (or SEQ ID NO:25 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:26 (or SEQ ID NO:26 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:27 (or SEQ ID NO:27 with one, two, or three amino acid additions, deletions, or substitutions); (v) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 33 (or SEQ ID NO: 33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 34 (or SEQ ID NO: 34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 35 (or SEQ ID NO: 35 with one, two, or three amino acid additions, deletions, or substitutions); (vi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 41 (or SEQ ID NO: 41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 42 (or SEQ ID NO: 42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 43 (or SEQ ID NO: 43 with one, two, or three amino acid additions, deletions, or substitutions); (vii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 49 (or SEQ ID NO: 49 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 50 (or SEQ ID NO: 50 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 51 (or SEQ ID NO: 51 with one, two, or three amino acid additions, deletions, or substitutions); (viii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 57 (or SEQ ID NO: 57 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 58 (or SEQ ID NO: 58 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 59 (or SEQ ID NO: 59 with one, two, or three amino acid additions, deletions, or substitutions); (ix) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 65 (or SEQ ID NO: 65 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 66 (or SEQ ID NO: 66 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 67 (or SEQ ID NO: 67 with one, two, or three amino acid additions, deletions, or substitutions); (x) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 73 (or SEQ ID NO: 73 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 74 (or SEQ ID NO: 74 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 75 (or SEQ ID NO: 75 with one, two, or three amino acid additions, deletions, or substitutions); (xi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 81 (or SEQ ID NO: 81 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 82 (or SEQ ID NO: 82 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 83 (or SEQ ID NO: 83 with one, two, or three amino acid additions, deletions, or substitutions); (xii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 89 (or SEQ ID NO: 89 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 90 (or SEQ ID NO: 90 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 91 (or SEQ ID NO: 91 with one, two, or three amino acid additions, deletions, or substitutions); (xiii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 97 (or SEQ ID NO: 97 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 98 (or SEQ ID NO: 98 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 99 (or SEQ ID NO: 99 with one, two, or three amino acid additions, deletions, or substitutions); or (xiv) A heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 105 (or SEQ ID NO: 105 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 106 (or SEQ ID NO: 106 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 107 (or SEQ ID NO: 107 with one, two, or three amino acid additions, deletions, or substitutions).

34. 34. The antigen-binding fragment of claim 33, comprising the ability to bind to SEQ ID NO: 113 or SEQ ID NO:

115.

35. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (i).

36. 36. The antigen-binding fragment of claim 35, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

8.

37. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (ii).

38. 38. The antigen-binding fragment of claim 37, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

16.

39. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (iii).

40. 40. The antigen-binding fragment of claim 39, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

24.

41. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (iv).

42. 42. The antigen-binding fragment of claim 41, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

32.

43. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (v).

44. 44. The antigen-binding fragment of claim 43, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

40.

45. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (vi).

46. 46. ​​The antigen-binding fragment of claim 45, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

48.

47. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (vii).

48. 48. The antigen-binding fragment of claim 47, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

56.

48. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (viii).

49. 49. The antigen-binding fragment of claim 48, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

64.

50. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (ix).

51. 51. The antigen-binding fragment of claim 50, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

72.

52. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (x).

53. 53. The antigen-binding fragment of claim 52, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

80.

54. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (xi).

55. 55. The antigen-binding fragment of claim 54, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

88.

56. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (xii).

57. 57. The antigen-binding fragment of claim 56, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

96.

58. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (xiii).

59. 59. The antigen-binding fragment of claim 58, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

104.

60. 35. The antigen-binding fragment of claim 33 or 34, comprising the heavy chain variable domain or region of (xiv).

61. 61. The antigen-binding fragment of claim 60, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

112.

62. 62. The antigen-binding fragment of any one of claims 33 to 61, which is monoclonal.

63. 63. The antigen-binding fragment of any one of claims 33 to 62, which is a Fab.

64. An antibody domain comprising: (i) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 1 (or SEQ ID NO: 1 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 2 (or SEQ ID NO: 2 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 3 (or SEQ ID NO: 3 with one, two, or three amino acid additions, deletions, or substitutions); (ii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:11 (or SEQ ID NO:11 with one, two, or three amino acid additions, deletions, or substitutions); (iii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 17 (or SEQ ID NO: 17 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 18 (or SEQ ID NO: 18 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 19 (or SEQ ID NO: 19 with one, two, or three amino acid additions, deletions, or substitutions): (iv) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO:25 (or SEQ ID NO:25 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:26 (or SEQ ID NO:26 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:27 (or SEQ ID NO:27 with one, two, or three amino acid additions, deletions, or substitutions); (v) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 33 (or SEQ ID NO: 33 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 34 (or SEQ ID NO: 34 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 35 (or SEQ ID NO: 35 with one, two, or three amino acid additions, deletions, or substitutions); (vi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 41 (or SEQ ID NO: 41 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 42 (or SEQ ID NO: 42 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 43 (or SEQ ID NO: 43 with one, two, or three amino acid additions, deletions, or substitutions); (vii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 49 (or SEQ ID NO: 49 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 50 (or SEQ ID NO: 50 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 51 (or SEQ ID NO: 51 with one, two, or three amino acid additions, deletions, or substitutions); (viii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 57 (or SEQ ID NO: 57 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 58 (or SEQ ID NO: 58 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 59 (or SEQ ID NO: 59 with one, two, or three amino acid additions, deletions, or substitutions); (ix) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 65 (or SEQ ID NO: 65 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 66 (or SEQ ID NO: 66 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 67 (or SEQ ID NO: 67 with one, two, or three amino acid additions, deletions, or substitutions); (x) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 73 (or SEQ ID NO: 73 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 74 (or SEQ ID NO: 74 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 75 (or SEQ ID NO: 75 with one, two, or three amino acid additions, deletions, or substitutions); (xi) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 81 (or SEQ ID NO: 81 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 82 (or SEQ ID NO: 82 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 83 (or SEQ ID NO: 83 with one, two, or three amino acid additions, deletions, or substitutions); (xii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 89 (or SEQ ID NO: 89 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 90 (or SEQ ID NO: 90 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 91 (or SEQ ID NO: 91 with one, two, or three amino acid additions, deletions, or substitutions); (xiii) a heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 97 (or SEQ ID NO: 97 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 98 (or SEQ ID NO: 98 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 99 (or SEQ ID NO: 99 with one, two, or three amino acid additions, deletions, or substitutions); or (xiv) A heavy chain variable domain or region comprising the amino acid sequence set forth in SEQ ID NO: 105 (or SEQ ID NO: 105 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO: 106 (or SEQ ID NO: 106 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO: 107 (or SEQ ID NO: 107 with one, two, or three amino acid additions, deletions, or substitutions).

65. 65. The antibody domain of claim 64, comprising the ability to bind to SEQ ID NO: 113 or SEQ ID NO:

115.

66. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (i).

67. 67. The antibody domain of claim 66, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

8.

68. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (ii).

69. 69. The antibody domain of claim 68, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

16.

70. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (iii).

71. 71. The antibody domain of claim 70, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

24.

72. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (iv).

73. 73. The antibody domain of claim 72, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

32.

74. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (v).

75. 75. The antibody domain of claim 74, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

40.

76. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (vi).

77. 77. The antibody domain of claim 76, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

48.

78. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (vii).

79. 79. The antibody domain of claim 78, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

56.

80. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (viii).

81. 81. The antibody domain of claim 80, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

64.

82. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (ix).

83. 83. The antibody domain of claim 82, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

72.

84. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (x).

85. 85. The antibody domain of claim 84, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

80.

86. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (xi).

87. 87. The antibody domain of claim 86, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

88.

88. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (xii).

89. 89. The antibody domain of claim 88, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

96.

90. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (xiii).

91. 91. The antibody domain of claim 90, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

104.

92. 66. The antibody domain of claim 64 or 65, comprising the heavy chain variable domain or region of (xiv).

93. 93. The antibody domain of claim 92, wherein the heavy chain variable domain or region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

112.

94. 94. The antibody domain of any one of claims 64 to 93, which is monoclonal.

95. 95. An antibody domain according to any one of claims 64 to 94, which is a VH domain.

96. 96. A chimeric antigen receptor comprising an antigen-binding domain, a hinge, a transmembrane domain, and one or more signaling domains, wherein the antigen-binding domain comprises an antibody, antigen-binding fragment, or antibody domain of any one of claims 1 to 95.

97. 97. The chimeric antigen receptor of Claim 96, wherein the antigen binding domain comprises a VH domain having the ability to bind to a follistatin polypeptide.

98. 98. The chimeric antigen receptor of claim 96 or 97, wherein the hinge comprises the hinge described in Example 23.

99. 99. The chimeric antigen receptor of any one of claims 96 to 98, wherein the transmembrane domain comprises the transmembrane domain described in Example 24.

100. 100. The chimeric antigen receptor of any one of claims 96 to 99, comprising one or more signalling domains described in Example 25.

101. A cell comprising the chimeric antigen receptor of any one of claims 96 to 100.

102. 102. The cell of claim 101, which is a T cell, a stem cell, or a NK cell.

103. 96. A cell engager comprising a first antigen-binding domain, a linker, and a second antigen-binding domain, wherein the first antigen-binding domain comprises the antibody, antigen-binding fragment, or antibody domain of any one of claims 1 to 95.

104. 104. The cell engager of Claim 103, wherein said first antigen binding domain comprises a VH domain capable of binding to a follistatin polypeptide.

105. 104. The cell engager of Claim 103, wherein said first antigen-binding domain is an IgG having the ability to bind to a follistatin polypeptide.

106. 106. The cell engager of any one of claims 103-105, wherein the linker comprises the linker of Example 20 or Example 23.

107. 107. The cell engager of any one of claims 103 to 106, wherein the second antigen-binding domain binds to a polypeptide expressed on the surface of a T cell.

108. 108. The cell engager of Claim 107, wherein the polypeptide expressed on the surface of a T cell is a CD3 polypeptide.

109. 108. The cell engager of claim 107, wherein said second antigen binding domain is an antigen binding domain described in Example 28.

110. The cell engager of any one of claims 103 to 106, wherein the second antigen-binding domain binds to a polypeptide expressed on the surface of a NK cell.

111. 111. The cell engager of claim 110, wherein the polypeptide expressed on the surface of the NK cell is a CD16a, NKG2A, NKG2D, NKp30, NKp44, or NKp46 polypeptide.

112. 111. The cell engager of claim 110, wherein said second antigen binding domain is an antigen binding domain described in Example 29.

113. 113. The cell engager of any one of claims 103 to 112, comprising a third antigen-binding domain.

114. 114. The cell engager of Claim 113, wherein said third antigen-binding domain binds to a polypeptide expressed on the surface of an NK cell.

115. 115. The cell engager of Claim 114, wherein the polypeptide expressed on the surface of the NK cell is a CD16a, NKG2A, NKG2D, NKp30, NKp44, or NKp46 polypeptide.

116. 115. The cell engager of claim 114, wherein said third antigen-binding domain is an antigen-binding domain described in Example 29.

117. 96. A nucleic acid comprising a nucleic acid sequence encoding at least a portion of the antibody, antigen-binding fragment, or antibody domain of any one of claims 1 to 95.

118. 118. The nucleic acid of Claim 117, wherein the nucleic acid sequence encodes the heavy chain variable domain or region of any one of (i) to (xiv) of Claim 1.

119. 119. The nucleic acid of claim 117 or 118, which is a viral vector.

120. 119. The nucleic acid of claim 117 or 118, which is a phagemid.

121. 117. A nucleic acid comprising a nucleic acid sequence encoding the chimeric antigen receptor of any one of claims 96 to 100 or the cell engager of any one of claims 103 to 116.

122. 122. The nucleic acid of claim 121, which is a viral vector.

123. 122. The nucleic acid of claim 121, which is a phagemid.

124. A host cell comprising the nucleic acid of any one of claims 121 to 123.

125. 117. A host cell expressing the chimeric antigen receptor of any one of claims 96 to 100 or the cell engager of any one of claims 103 to 116.

126. 126. The host cell of claim 124 or 125, which is a T cell, a stem cell, or a NK cell.

127. 96. An antibody-drug conjugate (ADC) comprising an antigen-binding domain covalently linked to a drug, wherein the antigen-binding domain comprises the antibody, antigen-binding fragment, or antibody domain of any one of claims 1 to 95.

128. 128. The ADC of claim 127, wherein said antigen-binding domain comprises a VH domain having the ability to bind to a follistatin polypeptide.

129. 129. The ADC of claim 127 or 128, wherein the drug is selected from the group consisting of an auristatin, a mertansine, or a pyrrolobenzodiazepine (PBD) dimer.

130. A composition comprising an antibody, antigen-binding fragment, or antibody domain according to any one of claims 1 to 95.

131. 131. The composition of claim 130, comprising the antibody of any one of claims 1 to 32.

132. 131. The composition of claim 130, comprising the antigen-binding fragment of any one of claims 33 to 63.

133. 131. The composition of claim 130, comprising the antibody domain of any one of claims 64 to 95.

134. 117. A composition comprising the cell engager of any one of claims 103-116.

135. A composition comprising a cell according to any one of claims 101 to 102 and 124 to 126.

136. 130. A composition comprising the ADC of any one of claims 127-129.

137. 137. The composition of any one of claims 130 to 136, comprising a checkpoint inhibitor.

138. 138. The composition of claim 137, wherein the checkpoint inhibitor is selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab.

139. 139. A method of treating a mammal having cancer, comprising administering to said mammal a composition according to any one of claims 130 to 138.

140. 140. The method of claim 139, wherein the mammal is a human.

141. The cancer is treated with follistatin + 141. The method of claim 139 or 140, wherein the cancer is cancer.

142. The follistatin + Cancer, follistatin + ovarian cancer, follistatin + Lung cancer and follistatin + 142. The method of claim 141, wherein the cancer is selected from the group consisting of prostate cancer.

143. 143. The method of any one of Claims 139-142, wherein after said administering step, the number of cancer cells in said mammal is reduced.

144. 1. A method of treating a mammal having cancer, comprising: (a) administering to said mammal the composition of any one of claims 130 to 138; and (b) administering to said mammal a composition comprising a checkpoint inhibitor. The above method, comprising:

145. 145. The method of claim 144, wherein the mammal is a human.

146. The cancer is treated with follistatin + 146. The method of claim 144 or 145, wherein the cancer is cancer.

147. The follistatin + Cancer, follistatin + ovarian cancer, follistatin + Lung cancer and follistatin + 147. The method of claim 146, wherein the cancer is selected from the group consisting of prostate cancer.

148. 148. The method of any one of claims 144-147, wherein the checkpoint inhibitor is selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, AMP-514, avelumab, durvalumab, atezolizumab, KN035, CK-301, AUNP12, CA-170, BMS-986189, and ipilimumab.

149. 149. The method of any one of Claims 144-148, wherein after said administering steps (a) and (b), the number of cancer cells in said mammal is reduced.

150. 100. A method of conjugating a binding molecule to a follistatin polypeptide, comprising contacting the follistatin polypeptide with the antibody, antigen-binding fragment, or antibody domain of any one of claims 1-95.

151. 151. The method of claim 150, wherein said contacting is performed in vitro.

152. 151. The method of claim 150, wherein said contacting is performed in vivo.

153. 153. The method of claim 152, wherein said contacting is effected in a mammal by administering said antibody, antigen-binding fragment, or antibody domain to said mammal.

154. 154. The method of claim 153, wherein the mammal is a human.

155. 130. A method of conjugating a binding molecule to a follistatin polypeptide, comprising contacting the follistatin polypeptide with a chimeric antigen receptor of any one of claims 96-100, a cell engager of any one of claims 103-116, or an ADC of any one of claims 127-129.

156. 156. The method of claim 155, wherein said contacting is performed in vitro.

157. 156. The method of claim 155, wherein said contacting is performed in vivo.

158. 158. The method of Claim 157, wherein said contacting is effected in a mammal by administering said chimeric antigen receptor, said cell engager, or said ADC to said mammal.

159. 159. The method of claim 158, wherein the mammal is a human.

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