Human mesothelin binding substance

Abstract

Antibodies and antigen-binding fragments thereof that bind to human mesothelin have been described.

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Application No. 63 / 288,162, filed December 10, 2021, the entirety of which is incorporated herein by reference.

[0002] Sequence Listing This application contains a computer readable sequence listing submitted herewith in XML file format, the entire contents of which are incorporated herein by reference. The XML file sequence listing submitted herewith is named "14463-047-228_SEQ_LISTING.xml", was created on December 9, 2022, and is 52,002 bytes in size.

[0003] Field The present invention relates to anti-mesothelin antibodies and antigen-binding fragments thereof capable of binding to human mesothelin. Summary of the Invention

[0004] Mesothelin is a differentiation antigen whose expression is restricted in normal human tissues to mesothelial cells lining the pleura, pericardium, and peritoneum (Chang & Pastan, Proc. Natl. Acad. Sci. USA 93:136-140 (1996); Chang et al., Int. J. Cancer 50:373-381 (1992)). However, mesothelin is highly expressed in several human cancers (e.g., nearly all mesotheliomas and pancreatic adenocarcinomas, as well as approximately 70% of ovarian and 50% of lung adenocarcinomas) (Ordonez, Mod Pathol. 16:192-197 (2003); Argani et al., Clin. Cancer Res. 7:3862-3868 (2001); Hassan et al., Appl. Immunohistochem Mol. Morphol. 13:243-247 (2005); Ordonez, Am. J. Surg. Pathol. 27:1418-1428 (2003)).

[0005] The mesothelin gene encodes a 71 kDa precursor protein that is processed into a 31 kDa shed protein called megakaryocyte potentiating factor (MPF) and a 40 kDa fragment (mesothelin) that is attached to the cell membrane with a glycosylphosphatidylinositol (GPI) anchor. MPF was isolated from the culture supernatant of a pancreatic cancer cell line and was named after its ability to stimulate the megakaryocyte colony-forming activity of interleukin-3 in mouse bone marrow cultures. The biological function of mesothelin is unclear, but recent findings suggest that mesothelin may be involved in ovarian cancer metastasis by binding to MUC16 / CA-125 (Rump et al., J. Biol. Chem. 279:9190-9198 (2004)). Small amounts of cell-bound mesothelin are released into the serum and have been shown to be elevated in patients with mesothelioma and ovarian cancer (Hassan et al., Clin. Cancer Res. 12:447-453 (2006)).

[0006] Mesothelin has limited expression in normal tissues and high expression in some cancers, making it a promising candidate for tumor-specific therapies. These therapies include agents that target mesothelin on the cell surface or induce an immune response against mesothelin. Agents in the clinic or about to enter clinical trials include CAT-5001, MORAb-009, and CRS-207 (Hassan & Ho, Eur. J. Cancer 44:46-53 (2008)).

[0007] Therapeutic antibodies that bind to mesothelin may be effective in the treatment of cancer. Monoclonal antibodies (mAbs), both as monotherapy and in combination, have emerged as one of the fastest growing and most effective therapeutic strategies for the treatment of solid tumors and hematological disorders. Between 2015 and 2017, the U.S. Food and Drug Administration approved 27 therapeutic mAbs (Tsumoto et al., Immunotherapy 11:119-127 (2019)), and in 2017, the total number of clinically used mAbs and biosimilars increased to 57 and 11, respectively (Grilo & Mantalaris, Trends Biotechnol. 37:9-16 (2019)). As of the end of 2019, numerous companies were supporting early-phase clinical trials of more than 550 novel antibody therapeutics, of which approximately half were directed against oncology targets (Kaplon et al., MAbs 12:1-24 (2020)).

[0008] The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to human mesothelin (human MSLN) comprising the amino acid sequence set forth in SEQ ID NO: 1. These human MSLN binding agents have weak or no detectable binding to non-human primate mesothelin and may be useful in the treatment of cancer and proliferative diseases.

[0009] The human MSLN binding agent of the present invention comprises: (a) a heavy chain (HC) variable domain (V) comprising the amino acid sequence set forth in SEQ ID NO: 2, 26, 27, or 28; H ) and a light chain (LC) variable domain (V L ), (b) V comprising the amino acid sequence set forth in SEQ ID NO: 10 H and V comprising the amino acid sequence set forth in SEQ ID NO:11 L or (c) a V comprising the amino acid sequence set forth in SEQ ID NO: 18. H and V comprising the amino acid sequence set forth in SEQ ID NO:19 LThe antibody comprises six complementarity determining regions (CDRs) including: The CDR sequences of the human MSLN binders can be defined according to any numbering scheme useful for defining CDR sequences, including, but not limited to, the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering schemes. In certain embodiments, the CDRs are defined by Kabat or IMGT. In certain embodiments, the CDRs are defined by Kabat.

[0010] In a further embodiment of the human MSLN binding agent, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9.

[0011] In a further embodiment of the human MSLN binding agent, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14, L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:15, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:17.

[0012] In a further embodiment of the human MSLN binding agent, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22, L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:24, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25.

[0013] In a further embodiment, the human MSLN binding agent is selected from the group consisting of (i) an amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence ranging from the second amino acid residue to the 116th amino acid residue set forth in SEQ ID NO:2, in which the first amino acid residue is pyroglutamic acid; (ii) an amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence ranging from the second amino acid residue to the 116th amino acid residue set forth in SEQ ID NO:26, in which the first amino acid residue is pyroglutamic acid. (iii) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:27 or an amino acid sequence ranging from the second amino acid residue to the 116th amino acid residue of the amino acid sequence set forth in SEQ ID NO:27, in which the first amino acid residue is pyroglutamic acid; or (iv) an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:28 or an amino acid sequence ranging from the second amino acid residue to the 116th amino acid residue of the amino acid sequence set forth in SEQ ID NO:28, in which the first amino acid residue is pyroglutamic acid. H and V comprising the amino acid sequence set forth in SEQ ID NO:3. L In further embodiments, the human MSLN binding agent comprises a VV comprising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. H and V comprising the amino acid sequence set forth in SEQ ID NO:3. L , including.

[0014] In a further embodiment, the human MSLN binding agent comprises an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence ranging from the second amino acid residue to the 116th amino acid residue of SEQ ID NO: 10, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. Hand an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 11 or the amino acid sequence ranging from the second amino acid residue to the 107th amino acid residue set forth in SEQ ID NO: 11, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. L In a further embodiment, the human MSLN binding agent comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO:11. L , including.

[0015] In a further embodiment, the human MSLN binding agent comprises an amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence ranging from the second amino acid residue to the 118th amino acid residue of SEQ ID NO: 18, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. H and an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence having the amino acid residues from the second amino acid residue to the 108th amino acid residue of the amino acid sequence set forth in SEQ ID NO: 19, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. L In a further embodiment, the human MSLN binding agent comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO:19. L , including.

[0016] The present invention further relates to a VVLK vector comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. H and V comprising an amino acid sequence having at least 90% identity with the amino acid sequence set forth in SEQ ID NO:3. L The present invention provides a human MSLN binding agent comprising: Hcomprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9.

[0017] The present invention further relates to a VVLK vector comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:10. H and V comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:11. L The present invention provides a human MSLN binding substance comprising:

[0018] The present invention further relates to a VVLK vector comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:10. H and V comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:11. L The present invention provides a human MSLN binding agent comprising: H comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0019] The present invention further relates to a VVLK vector comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:18. H and V comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:19. L The present invention provides a human MSLN binding substance comprising:

[0020] The present invention further relates to a VVLK vector comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:18.H and V comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:19. L The present invention provides a human MSLN binding agent comprising: H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:24, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25.

[0021] In further embodiments, the human MSLN binding agent is an antibody comprising a heavy chain constant domain of the IgG1 isotype. In certain embodiments, the heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:29. In certain embodiments, the heavy chain constant domain comprises an amino acid sequence having an amino acid residue 1 to an amino acid residue 329 of the amino acid sequence set forth in SEQ ID NO:29. In further embodiments, the heavy chain constant domain of the IgG1 isotype comprises an Fc domain comprising one or more mutations that render the constant domain effector silent. In certain embodiments, the effector silent constant domain comprises an amino acid sequence set forth in SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In certain embodiments, the effector silent constant domain comprises an amino acid sequence set forth in SEQ ID NO:30 or an amino acid sequence having an amino acid residue 1 to an amino acid residue 329 of the amino acid sequence set forth in SEQ ID NO:30. In certain embodiments, the effector-silent constant domain comprises an amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence ranging from amino acid residue 1 to amino acid residue 329 of SEQ ID NO: 31. In certain embodiments, the effector-silent constant domain comprises an amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence ranging from amino acid residue 1 to amino acid residue 329 of SEQ ID NO: 32. In certain embodiments, the effector-silent constant domain comprises an amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence ranging from amino acid residue 1 to amino acid residue 329 of SEQ ID NO: 33.In certain embodiments, the effector silent constant domain comprises an amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence ranging from the first amino acid residue to the 329th amino acid residue of SEQ ID NO: 34. In certain embodiments, the effector silent constant domain comprises an amino acid sequence set forth in SEQ ID NO: 35, or an amino acid sequence ranging from the first amino acid residue to the 329th amino acid residue of SEQ ID NO: 35. In certain embodiments, the effector silent constant domain comprises an amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence ranging from the first amino acid residue to the 329th amino acid residue of SEQ ID NO: 36. In certain embodiments, the effector silent constant domain comprises an amino acid sequence set forth in SEQ ID NO: 37, or an amino acid sequence ranging from the first amino acid residue to the 329th amino acid residue of SEQ ID NO: 37. In certain embodiments, the constant domain comprises an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:31, or the amino acid sequence ranging from amino acid residue 1 to amino acid residue 329 set forth in SEQ ID NO:31.

[0022] In further embodiments, the light chain may comprise a human kappa or lambda light chain constant domain. In further embodiments disclosed herein, the light chain constant domain may comprise a human kappa light chain constant domain comprising the amino acid sequence set forth in SEQ ID NO:38.

[0023] The present invention further provides a human MSLN binding agent comprising a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv region, or an ScFv. In certain embodiments, a bispecific antibody is provided, in which one arm of the bispecific antibody comprises a human MSLN binding agent selected from the group consisting of a Fab' fragment or an ScFv, and the other arm of the bispecific antibody comprises a Fab' or ScFv specific for an antigen other than human MSLN.

[0024] The present disclosure further provides a human MSLN binding substance, which includes one or two of the modifications to the human MSLN binding substance provided herein selected from the following: (i) the C-terminal amino acid residue K of the heavy chain is removed; and (ii) the N-terminal amino acid residue E or Q of the VH, VL, heavy chain or light chain is replaced with pyroglutamic acid.

[0025] The present invention further includes compositions comprising a human MSLN binding agent disclosed herein and a pharma- ceutically acceptable carrier or diluent.

[0026] The present invention further provides a method for treating cancer or a proliferative disease in an individual in need of treatment, the method comprising administering to the individual a therapeutically effective amount of a human MSLN binding agent disclosed herein or a composition disclosed herein to treat the cancer or proliferative disease.

[0027] The present invention further provides a human MSLN binding agent or composition disclosed herein for the treatment of cancer or a proliferative disease.

[0028] The invention further provides for the use of a human MSLN binding agent disclosed herein in the manufacture of a medicament for treating cancer or a proliferative disease.

[0029] The present invention further provides combination therapies for treating cancer or proliferative diseases comprising a human MSLN binding agent or composition disclosed herein and a therapeutic agent. In a further embodiment, the therapeutic agent is a chemotherapeutic agent or a therapeutic antibody. In yet another embodiment, the antibody is an anti-PD1 antibody or an anti-PD-L1 antibody.

[0030] The present invention further provides the V H and / or the V of the human MSLN binding agent disclosed herein L Also provided are nucleic acid molecules encoding the nucleic acid molecules disclosed herein. Also provided are expression vectors comprising one or more of the nucleic acid molecules disclosed herein. Also provided are host cells comprising expression vectors comprising one or more of the nucleic acid molecules disclosed herein.

[0031] The present invention further provides a method for producing a human MSLN binding agent, the method comprising: (a) providing a host cell comprising an expression vector comprising one or more of the nucleic acid molecules disclosed herein; (b) culturing the host cell in a medium under conditions suitable for expressing the human MSLN binding agent, and (c) isolating the human MSLN binding agent from the medium.

[0032] The present disclosure further provides human MSLN binding agents obtained by expressing the nucleic acids or expression vectors provided herein in a host cell.

[0033] The present invention further provides a human MSLN binding agent as disclosed herein that is conjugated to a detectable moiety.

[0034] The present invention further provides a method for detecting human MSLN on the cell surface of a patient, the method comprising administering to the patient a human MSLN-binding agent disclosed herein conjugated to a detectable moiety, and detecting cells in the patient that are bound to the human MSLN-binding agent conjugated to the detectable moiety. In certain embodiments, the detectable moiety is detectable by magnetic resonance imaging (MRI) or X-ray imaging.

[0035] The present invention further provides a cell that expresses a human MSLN-binding substance on the cell surface.The expressed human MSLN-binding substance can be a whole antibody, a bispecific antibody, a Fab' fragment, a ScFv, or a tandem ScFv.In certain embodiments, the cell that expresses a human MSLN-binding substance is an immune cell, for example, a T cell or a NK cell.

[0036] The present invention further provides a human MSLN binding agent conjugated to a cytotoxin. The human MSLN binding agent can be a whole antibody, a bispecific antibody, a Fab' fragment, or an ScFv. [Brief description of the drawings]

[0037] [Figure 1] ELISA binding of SV017.47D7.1C2 antibody and positive control MORAB-009 to soluble and membrane-bound human MSLN. Like MORAB-009, SV017.47D7.1C2 binds to both soluble and membrane-bound human MSLN. HuMSLN refers to human MSLN and CHOK1 refers to Chinese hamster ovary K1 cells. [Diagram 2] ELISA binding of SV018.20B12.1D1 antibody and positive control MORAB-009 to soluble and membrane-bound human MSLN. SV018.20B12.1D1 binds to soluble human MSLN less potently than to membrane-bound human MSLN. HuMSLN refers to human MSLN and CHOK1 refers to Chinese hamster ovary K1 cells. [Diagram 3] ELISA binding of SV018.45B6.1G1 antibody and positive control MORAB-009 to soluble and membrane-bound human MSLN. SV018.45B6.1G1 binds to soluble human MSLN less potently than membrane-bound human MSLN. HuMSLN refers to human MSLN and CHOK1 refers to Chinese hamster ovary K1 cells. [Figure 4]1 shows human MSLN generated from a precursor protein of approximately 70 kDa by endoprotease furin cleavage releasing the N-terminal domain MPF ​​(megakaryocyte potentiating factor) and the 40 kDa membrane-bound mesothelin (GPI linked to the cell surface). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] definition So that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0039] As used in this specification, including the appended claims, singular words such as "a," "an," and "the" include the corresponding plural referents unless the context clearly indicates otherwise.

[0040] As used herein, the term "mesothelin" or "MSLN" refers to a differentiation antigen whose expression in normal human tissues is restricted to mesothelial cells lining the pleura, pericardium, and peritoneum. MSLN is generated from a ~70 kDa precursor protein that is cleaved with the endoprotease furin to release MPF (megakaryocyte potentiating factor) at the N-terminal region and 40 kDa membrane-bound mesothelin (GPI linked to the cell surface) (see FIG. 4). The amino acid sequence of the human mesothelin precursor is shown in SEQ ID NO:1. The mature form contains amino acids 296-622.

[0041] As used herein, the term "human MSLN" refers to the mature form of human mesothelin and the term "rhesus MSLN" refers to the mature form of rhesus monkey (Macaca mulatta) mesothelin. The amino acid sequences of the precursor and mature forms of rhesus MSLN are set forth in SEQ ID NO:39.

[0042] As used herein, the term "human MSLN binding agent" refers to an antibody or antigen-binding fragment thereof that binds to soluble and / or membrane-bound human MSLN. Human MSLN binding agents include, but are not limited to, bivalent antibody tetramers (2H+2L), monovalent antibodies (H+L), bispecific antibodies targeting human MSLN and another target, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv regions, and ScFvs.

[0043] As used herein, the term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (KD). Affinity can be measured by common methods known in the art, including KinExA and surface plasmon resonance (SPR; Biacore™). Specific exemplary and illustrative embodiments for measuring binding affinity are described below.

[0044] As used herein, the terms "administration" and "treatment", when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contacting an exogenous pharmaceutical, therapeutic, diagnostic, or composition, including a human MSLN binding agent disclosed herein, with the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell includes contact of a reagent with the cell and contact of a reagent with a body fluid, which is in contact with the cell. "Administration" and "treatment" also refer to in vitro and ex vivo treatment of a cell, for example, with a reagent, diagnostic, binding compound, or another cell. The term "subject" includes any organism, preferably an animal, and more preferably a mammal (e.g., a human, rat, mouse, dog, cat, rabbit). In a preferred embodiment, the term "subject" refers to a human.

[0045] The term "amino acid" as used herein refers to a simple organic compound that contains both a carboxyl group (-COOH) and an amino group (-NH2). Amino acids are the building blocks of proteins, polypeptides, and peptides. Amino acids occur in the L- and D-forms, and in naturally occurring proteins, polypeptides, and peptides, they are in the L-form. The amino acids and their code names are shown in the chart below. [Table 1]

[0046] As used herein, the term "antibody" or "immunoglobulin" refers to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each HC contains a heavy chain variable region or domain (V H ), and a heavy chain constant region or domain. Each light chain is composed of a LC variable region or domain (V L ), and LC constant domains. In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region is composed of three domains (CH1, CH2, and CH3). In general, the basic antibody structural unit of an antibody is a Y-shaped tetramer containing two HC / LC pairs (2H). Each tetramer contains two identical pairs of polypeptide chains, each pair having one LC (about 25 kDa) chain and one HC chain (about 50-70 kDa) (H+L). Each HC:LC pair is composed of one V H : 1 V L Includes a pair of 1 V H : 1 V L The pair of Fabs may be referred to by the term "Fab". Thus, each antibody tetramer comprises two Fabs, one for each arm of the Y-shaped antibody. In certain embodiments, the antibody may include its post-translational modifications (e.g., clipping of the C-terminal lysine of the heavy chain, conversion of glutamine or glutamic acid to pyroglutamic acid) that may occur when recombinantly expressed in a host cell (e.g., CHO cell) or during purification / storage.

[0047] The LC constant domain is composed of one domain, CL. H V H 1. V H 2. V H 3. V H 4. V H 5. V H 6, and V H 7 family members; human V L V κ 1. V κ 2. V κ 3. V κ 4. V κ 5. V κ 6. V λ 1. V λ 2. V λ 3. V λ 4. V λ 5. V λ 6. V λ 7. V λ 8. V λ 9, and V λ 10 contains 16 family members. Each of these family members can be further divided into specific subtypes. H and V L can be further subdivided into regions of hypervariability (called the complementarity determining region (CDR) space) interspersed with more conserved regions (called the framework regions (FR)). H and V L V is composed of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. HThe numbering of amino acids within the IMGT sequence may be determined using the Kabat numbering scheme. See Beranger, et al., Ed. Ginetoux, Correspondence between the IMGT unique numbering for C-DOMAIN, the IMGT exon numbering, the Eu and Kabat numberings: Human IGHG, Created: 17 / 05 / 2001, Version: 08 / 06 / 2016 (accessible at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html).

[0048] The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Usually, the numbering of the amino acids of the heavy chain constant domain starts at 118 according to the Eu numbering scheme. The Eu numbering scheme is based on the amino acid sequence of human IgG1 (Eu), which has a constant domain starting at amino acid position 118 of the amino acid sequence of IgG1 as set forth in Edelman et al., Proc. Natl. Acad. Sci. USA. 63:78-85 (1969), and for the IgG1, IgG2, IgG3, and IgG4 constant domains as set forth in Beranger et al., op.cit.

[0049] The variable regions of the heavy and light chains contain binding domains that include CDRs that interact with an antigen. Numerous methods for defining the CDR sequences of antibody variable domains are available in the art (see Dondelinger et al., Frontiers in Immunol. 9:Article 2278 (2018)). Common numbering schemes include: The Kabat numbering scheme is based on sequence variability and is the most commonly used (see Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (sequences define the CDR regions of antibodies)). The Chothia numbering scheme is based on the location of structural loop regions (see Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997)). The AbM numbering scheme is a compromise between the two used in Oxford Molecular's AbM antibody modeling software (see Karu et al., ILAR Journal 37:132-141 (1995)). The Contact numbering scheme is based on the analysis of available complex crystal structures (see: www.bioinf.org.uk: Prof. Andrew C Martin's Group; Abhinandan & Martin, Mol. Immunol. 45:3832-3839 (2008)). The IMGT (ImMunoGeneTics) numbering scheme is a standardized numbering system for all protein sequences of the immunoglobulin superfamily, including the variable domains of the light and heavy chains of antibodies and the T-cell receptor chains of various species, which counts residues consecutively from 1 to 128 based on germline V sequence alignments (see Giudicelli et al., Nucleic Acids Res. 25:206-11 (1997); Lefranc, Immunol Today 18:509 (1997); Lefranc et al., Dev Comp Immunol. 27:55-77 (2003)).

[0050] The following general rules, disclosed at www.bioinf.org.uk: Prof. Andrew C. Cartin's Group and reproduced below in Table 1, may be used to define the CDRs of an antibody sequence that contain amino acids that interact specifically with the amino acids that comprise the epitope of the antigen to which the antibody binds. Cys residues are the most conserved features, although there are rare instances where these generally constant features do not occur. [Table 2]

[0051] The entire nucleotide sequence of the heavy and light chain variable regions is usually numbered according to Kabat, but the three CDRs within the variable regions may be defined according to any one of the numbering schemes described above.

[0052] In general, the state of the art recognizes that in many cases the CDR3 region of the heavy chain is the primary determinant of antibody specificity, and examples of generating specific antibodies based solely on the CDR3 of the heavy chain are known in the art (e.g., Beiboer et al., J. Mol. Biol. 296:833-849 (2000); Klimka et al., British J. Cancer 83:252-260 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95:8910-8915 (1998); Xu et al., Immunity 13:37-45 (2000)). As used herein, the term "Fc domain" or "Fc" refers to a crystallizable fragment domain or region obtained from an antibody that contains the CH2 and CH3 domains of the antibody. In antibodies, the two Fc domains are linked by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. The Fc domain can be obtained by digesting an antibody with the protease papain. Usually, the amino acids in the Fc domain are numbered according to the Eu numbering convention (see Edelmann et al., Biochem. 63:78-85 (1969)).

[0053] As used herein, the term "antigen" refers to any foreign substance that elicits an immune response in the body.

[0054] As used herein, the term "antigen-binding fragment" refers to a polypeptide(s) comprising a fragment of a full-length antibody that retains the ability to specifically bind to an antigen bound to the full-length antibody and / or compete with the full-length antibody for specific binding to an antigen. Examples of antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv regions, and scFvs.

[0055] As used herein, the term "Fab fragment" refers to a fragment that contains one antibody light chain and the CH1 and V of one antibody heavy chain. H The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. A "Fab fragment" can be the product of papain cleavage of an antibody.

[0056] As used herein, the term "Fab' fragment" refers to a fragment that contains one antibody light chain and one V H and an antigen-binding substance comprising a portion or fragment of one antibody heavy chain containing the CH1 domain up to the region between the CH1 and CH2 domains (so that an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule).

[0057] As used herein, the term "F(ab')2 fragment" refers to a fragment that contains two antibody light chains and a V Hand two heavy chains containing a CH1 domain up to the region between the CH1 and CH2 domains (so that an interchain disulfide bond is formed between the two heavy chains). Thus, F(ab')2 refers to an antigen-binding substance that contains two Fab' fragments that are linked by disulfide bonds between the two heavy chains. "F(ab')2" fragments" can be the pepsin cleavage product of an antibody.

[0058] As used herein, the term "Fv region" refers to an antigen-binding agent that comprises the variable regions from both the heavy and light chains of an antibody, but lacks the constant regions.

[0059] As used herein, the term "ScFv" or "single chain variable fragment" refers to VFvs that are fused or linked together by a short linker peptide of 10 to about 25 amino acids. H and V L The linker is usually rich in glycine for flexibility, rich in serine or threonine for solubility, and rich in V H N-terminus of V L The protein can be linked to the C-terminus of the original immunoglobulin despite the removal of the constant region and the introduction of the linker.

[0060] As used herein, the term "diabody" refers to an antigen-binding agent that comprises a small antibody fragment having two antigen-binding regions, the fragment comprising a light chain variable domain (V L ) linked to a heavy chain variable domain (V H )(V H -V L or V L -V H). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding regions. Diabodies are described in further detail, for example, in EP 404,097; WO 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants, see generally, Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0061] These and other potential constructs are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0062] As used herein, the term "isolated" antibodies or antigen-binding fragments thereof are at least partially free of other biological molecules from the cell or cell culture in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other substances, such as cell debris and growth medium. Isolated antibodies or antigen-binding fragments may further be at least partially free of expression system components, such as biological molecules from the host cell or its growth medium. In general, the term "isolated" does not intend the complete absence of such biological molecules, or the absence of water, buffers, or salts, or the absence of components of a pharmaceutical formulation that includes the antibody or fragment.

[0063] As used herein, the term "monoclonal antibody" refers to a population of substantially homogeneous antibodies. That is, the antibody molecules comprising the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations usually contain a large number of different antibodies with different amino acid sequences in the variable domains, often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352:624-628 (1991), and Marks et al., J. Mol. Biol. 222:581-597 (1991). See also, Presta, J. Allergy Clin. Immunol. 116:731 (2005).

[0064] As used herein, the term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, a gene includes coding sequences and / or regulatory sequences necessary for its expression. For example, "gene" refers to a nucleic acid fragment that expresses an mRNA, a functional RNA, or a specific protein (including regulatory sequences). "Gene" also includes non-expressed DNA segments that form, for example, recognition sequences for other proteins. "Gene" can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and can include sequences designed to have desired parameters. Genes include both naturally occurring nucleotide sequences that code for a molecule of interest and synthetically derived nucleotide sequences that code for a molecule of interest (e.g., complementary DNA (cDNA) derived from a messenger RNA (mRNA) nucleotide sequence).

[0065] As used herein, the term "germline" or "germline sequence" refers to a sequence of an unrearranged immunoglobulin DNA sequence. Any suitable source of unrearranged immunoglobulin sequence may be used. Human germline sequences may be obtained, for example, from the JOINSOLVER® Germline Database on the website of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health. Mouse germline sequences may be obtained, for example, as described in Giudicelli et al., Nucleic Acids Res. 33:D256-D261 (2005).

[0066] As used herein, the term "library" generally refers to a collection of related but diverse polynucleotides, typically on a common vector backbone. For example, a light or heavy chain immunoglobulin library may contain polynucleotides encoding light and / or heavy chain immunoglobulins that are diverse in nucleotide sequence but related on a common vector backbone, e.g., these immunoglobulins are functionally diverse in their ability to form complexes with other immunoglobulins and bind to specific antigens, e.g., in the antibody display system of the invention.

[0067] As used herein, the term "polynucleotide" discussed herein forms part of the present invention. "Polynucleotide", "polynucleic acid", "nucleic acid", or "nucleic acid molecule" includes DNA and RNA (single-stranded or double-stranded). In embodiments of the present invention, polynucleotides (e.g., those encoding immunoglobulin chains or components of the antibody display system of the present invention) may be in association with natural regulatory (expression control) sequences or with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'-non-coding regions, and the like.

[0068] A polynucleotide (e.g., one encoding an immunoglobulin chain or component of the antibody display system of the invention) may be operably associated with a promoter. A "promoter" or "promoter sequence" is, in one embodiment of the invention, a DNA regulatory region capable of binding (e.g., directly or via other promoter-binding proteins or agents) RNA polymerase in a cell and initiating transcription of a coding sequence. A promoter sequence is generally bounded at the 3' end by a transcription initiation site and extends upstream (in the 5' direction) to include the minimum number of bases or elements required to initiate transcription at any level. Within the promoter sequence, a transcription initiation site (conveniently defined, e.g., by mapping with nuclease S1) and protein binding domains (consensus sequences) involved in the binding of RNA polymerase may be found. A promoter may be operably associated with other expression control sequences, including enhancer and repressor sequences, or with a nucleic acid of the invention.Promoters that can be used to control gene expression include the cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist, et al., Nature 290:304-310 (1981)), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), regulatory sequences of metallothionein genes (Brinster et al., Nature 296:39-42 (1982)), prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff et al., Nature 296:39-42 (1982)), and promoters of other vectors, such as the β-lactamase promoter (Villa-Komaroff et al., Nature 296:39-42 (1982)). al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978)), or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)) (see also "Useful proteins from recombinant bacteria" in Scientific American 242:74-94 (1980)), as well as promoter elements derived from yeast or other fungi, such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, or the alkaline phosphatase promoter.

[0069] As used herein, the terms "vector," "cloning vector," and "expression vector" include vehicles (e.g., plasmids) by which DNA or RNA sequences can be introduced into a host cell in order to transform the host and, optionally, to facilitate the expression and / or replication of the introduced sequence. Polynucleotides encoding immunoglobulin chains or components of the antibody display system of the invention may, in one embodiment of the invention, be present within a vector.

[0070] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transplants. It is also understood that not all progeny have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where clear designations are intended, they will be clear from the context.

[0071] As used herein, the term "control sequence" or "regulatory sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for expression in eukaryotes include, for example, promoters, operator or enhancer sequences for the expression of messenger RNA encoding a protein, transcription termination and polyadenylation sequences, and ribosome binding sites to facilitate translation of the messenger RNA.

[0072] As used herein, a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence, e.g., a regulatory sequence. For example, a presequence or secretory leader DNA is operably linked to a polypeptide DNA if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If no such sites exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0073] As used herein, the term "encoding" refers to the inherent property of a particular nucleotide sequence within a polynucleotide (e.g., gene, cDNA, or mRNA) to serve as a template for the synthesis of either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or other polymers and macromolecules in biological processes having a defined amino acid sequence and biological properties resulting therefrom. Thus, a gene codes for a protein if the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to the gene. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence, usually provided in a sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) can be referred to as encoding the protein or other product of the gene or cDNA. Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate versions of each other and code for the same amino acid sequence. Protein- and RNA-encoding nucleotide sequences may contain introns.

[0074] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence.

[0075] As used herein, the term "treat" or "treating" refers to administering a therapeutic agent, such as a composition containing any of the human MSLN binding agents of the present invention, to an individual in need thereof, locally, subcutaneously, intramuscularly, intradermally, or systemically. The amount of therapeutic agent effective for treating an individual's cancer or proliferative disease may vary depending on factors such as the individual's injury or condition, age, and / or weight, as well as the ability of the therapeutic agent to induce a desired response in the individual. Achievement of a therapeutic goal may be assessed by individual and / or any clinical measurement commonly used by a physician or other skilled medical provider to assess the severity or progress of a treatment. Thus, these terms refer to a beneficial result being or will be provided to a human or animal individual in need thereof.

[0076] As used herein, the term "treatment" refers to therapeutic treatments and diagnostic applications as applied to a human or animal individual. "Treatment" as applied to a human or animal individual includes contacting the human or animal subject with an antibody or antigen-binding fragment of the invention.

[0077] As used herein, the term "therapeutically effective amount" refers to an amount of a particular substance sufficient to achieve a desired effect in the individual being treated. For example, this may be the amount necessary to prevent or reduce the severity of a disease or disorder in the individual.

[0078] As used herein, the term "combination therapy" refers to the treatment of a human or animal individual that includes administering a first therapeutic agent and a second therapeutic agent to the individual, either sequentially or simultaneously. Generally, the first and second therapeutic agents are administered to the individual separately, rather than as a mixture; however, there may be embodiments in which the first and second therapeutic agents are mixed prior to administration.

[0079] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced (including the progeny of such cells). Host cells include "genetically modified cells," "transformants," and "transformed cells," which include the primary modified (e.g., transformed) cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. If desired, the host cells can be stably or transiently transfected with a polynucleotide encoding a fusion protein, as described herein.

[0080] Human MSLN binding substance The human MSLN binding agents of the present invention are chimeric or fully human antibodies or antigen-binding fragments thereof that specifically bind to human MSLN. H Domain and V L domains, each domain comprising three CDRs and four frameworks (FRs) in the following arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. These human MSLN binding substances were H 3 CDRs from, and V H and its counterpart, V L The CDR sequences may be defined according to any numbering scheme useful for defining CDR sequences, including, but not limited to, the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering schemes. Guidance for defining CDR sequences may be found in the general rules disclosed at www.bioinf.org.uk: Prof. Andrew C. Martin's Group and reproduced in Table 1.

[0081] In certain embodiments, the CDRs are defined as per Kabat or IMGT. The CDR amino acid sequences shown in Tables 2-4 are shown according to the Kabat numbering scheme for identifying CDR amino acid sequences. [Table 3] [Table 4] [Table 5]

[0082] A particular CDR amino acid sequence determined using any one of the methods for identifying CDR amino acid sequences (see Table 1) may have more or fewer amino acids than a CDR amino acid sequence identified according to any other numbering scheme, but the CDR amino acid sequences will overlap to some extent. Thus, the CDR amino acid sequences defined according to Kabat should not be construed as limiting, and any human MSLN binder whose CDR amino acid sequence is identified with another numbering scheme will fall within the scope of the human MSLN binder of the present invention, provided that the amino acid sequence of such a human MSLN binder includes the six CDR amino acid sequences identified by Kabat. For all human MSLN binders disclosed herein, unless otherwise indicated, the amino acids comprising the entire variable domain are numbered according to the Kabat numbering scheme, regardless of the manner in which the amino acids comprising the CDRs are defined. The heavy chain constant domain is numbered according to the Eu numbering scheme.

[0083] In some embodiments, a heavy chain variable domain (V) comprising the amino acid sequence set forth in SEQ ID NO: 18. H ) and a light chain variable domain (V LProvided herein are human mesothelin binding agents that comprise six complementarity determining regions (CDRs) of an antibody, including: (A) 1, A) 2, A) 3, A) 4, A) 5, A) 6, A) 7, A) 8, A) 9, A) 10, A) 11, A) 12, A) 13, A) 14, A) 15, A) 16, A) 17, A) 18, A) 19, A) 20, A) 21, A) 22, A) 23, A) 24, A) 25, A) 26, A) 27, A) 28, A) 29, A) 30, A) 31, A) 32, A) 33, A) 34, A) 35, A) 36, A) 37, A) 38, A) 39 ...

[0084] In some embodiments, the V comprises the amino acid sequence set forth in SEQ ID NO:10. H and V comprising the amino acid sequence set forth in SEQ ID NO:11 L Provided herein are human mesothelin binding agents comprising six CDRs of an antibody comprising: CDRs: (A) 1 , (B) 2 , (C) 3 , (D) 4 , (E) 5 , (F) 6 , (G) 7 , (H) 8 , (I) 9 , (I) 10 , (I) 11 , (I) 12 , (I) 13 , (I) 14 , (I) 15 , (I) 16 , (I) 17 , (I) 18 , (I) 19 , (I) 20 , (I) 21 , (I) 22 , (I) 23 , (I) 24 , (I) 25 , (I) 26 , (I) 27 , (I) 28 , (I) 29 , (I) 30 , (I) 31 , (I) 32 , (I) 33 , (I) 34 , (I) 35 , (I) 36 , (I) 37 , (I) 38 , (I) 39 , (I) 40 , (I) 41 , (I) 42 , (I) 43 , (I) 44 , (I) 45 , (I) 46 , (I) 47 , (I) 48 , (I) 49 , (I) 50 , (I) 51 , (I) 52 , (I) 53 , (I) 54 , (I) 55 , (I) 56 , (I) 57 , (I) 58 , (I) 59 , (I) 60 , (I) 61 , (I) 62

[0085] In some embodiments, the V comprises an amino acid sequence set forth in SEQ ID NO: 2, 26, 27, or 28. H and V comprising the amino acid sequence set forth in SEQ ID NO:3. L Provided herein are human mesothelin binding agents comprising six CDRs of an antibody comprising:

[0086] In certain embodiments of the invention, a human MSLN binding agent comprises (a) a VDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6. H domain, and (b) a V domain including a CDR1 having the amino acid sequence set forth in SEQ ID NO:7, a CDR2 having the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 having the amino acid sequence set forth in SEQ ID NO:9. L The domains, including the CDR sequences, are defined by the Kabat numbering scheme.

[0087] In certain embodiments of the invention, a human MSLN binding agent comprises (a) a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14. H (b) a V domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 15, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 17. L The domains, including the CDR sequences, are defined by the Kabat numbering scheme.

[0088] In certain embodiments of the invention, a human MSLN binding agent comprises (a) a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:20, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:21, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22. H domain, and (b) a V domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 23, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 24, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 25. L The domains, including the CDR sequences, are defined by the Kabat numbering scheme.

[0089] In further embodiments, the mesothelin binding agent comprises a VV comprising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. H domain and the amino acid sequence set forth in SEQ ID NO:3L Domain, including.

[0090] In some embodiments, the mesothelin binding agent comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO:3. L , including.

[0091] In some embodiments, the mesothelin binding agent comprises an amino acid sequence ranging from the second amino acid residue to the 116th amino acid residue of SEQ ID NO:2, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. H and V comprising the amino acid sequence set forth in SEQ ID NO:3. L , including.

[0092] In some embodiments, the mesothelin binding agent comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO:3. L , including.

[0093] In some embodiments, the mesothelin binding agent comprises an amino acid sequence ranging from the second amino acid residue to the 116th amino acid residue of SEQ ID NO:26, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. H and V comprising the amino acid sequence set forth in SEQ ID NO:3. L , including.

[0094] In some embodiments, the mesothelin binding agent comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO:3. L , including.

[0095] In some embodiments, the mesothelin binding agent comprises an amino acid sequence ranging from the second amino acid residue to the 116th amino acid residue of SEQ ID NO:27, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. H and V comprising the amino acid sequence set forth in SEQ ID NO:3. L , including.

[0096] In some embodiments, the mesothelin binding agent comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO:3. L , including.

[0097] In some embodiments, the mesothelin binding agent comprises an amino acid sequence ranging from the second amino acid residue to the 116th amino acid residue of SEQ ID NO:28, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. H and V comprising the amino acid sequence set forth in SEQ ID NO:3. L , including.

[0098] In a further embodiment, the mesothelin binding agent comprises the amino acid sequence set forth in SEQ ID NO:10. H domain, and V comprising the amino acid sequence set forth in SEQ ID NO:11 L Domain, including.

[0099] In a further embodiment, the mesothelin binding agent comprises the amino acid sequence set forth in SEQ ID NO:10. H and V comprising the amino acid sequence set forth in SEQ ID NO:11. L , including.

[0100] In a further embodiment, the mesothelin binding agent comprises an amino acid sequence ranging from the second amino acid residue to the 116th amino acid residue of SEQ ID NO: 10, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. H and V comprising the amino acid sequence set forth in SEQ ID NO:11. L , including.

[0101] In a further embodiment, the mesothelin binding agent comprises the amino acid sequence set forth in SEQ ID NO:10. H and an amino acid sequence having the second to the 107th amino acid residues of the amino acid sequence set forth in SEQ ID NO: 11, in which the first amino acid residue is pyroglutamic acid; L , including.

[0102] In a further embodiment, the mesothelin binding agent comprises an amino acid sequence having the amino acid sequence ranging from the second amino acid residue to the 116th amino acid residue of SEQ ID NO: 10, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. H and an amino acid sequence having the second to 107th amino acid residues of the amino acid sequence ranging from the second to 107th amino acid residues of SEQ ID NO: 11, in which the first amino acid residue is pyroglutamic acid. L , including.

[0103] In a further embodiment, the mesothelin binding agent comprises the amino acid sequence set forth in SEQ ID NO:18. H and V comprising the amino acid sequence set forth in SEQ ID NO:19. L , including.

[0104] In a further embodiment, the mesothelin binding agent comprises an amino acid sequence ranging from the second amino acid residue to the 118th amino acid residue of SEQ ID NO: 18, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. H and V comprising the amino acid sequence set forth in SEQ ID NO:19. L , including.

[0105] In a further embodiment, the mesothelin binding agent comprises the amino acid sequence set forth in SEQ ID NO:18. H and an amino acid sequence having the second to the 108th amino acid residues of the amino acid sequence set forth in SEQ ID NO: 19, the first amino acid residue of which is pyroglutamic acid; L , including.

[0106] In a further embodiment, the mesothelin binding agent comprises an amino acid sequence ranging from the second amino acid residue to the 118th amino acid residue of SEQ ID NO: 18, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. H and an amino acid sequence having the second to the 108th amino acid residues of the amino acid sequence ranging from the second to the 108th amino acid residues of SEQ ID NO: 19, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid. L , including.

[0107] In further embodiments of the invention, the human MSLN binding agent is an antibody comprising a heavy chain (HC) constant domain of the IgG1 isotype. In certain embodiments, the heavy chain constant domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, compared to the amino acid sequence of the native IgG1 isotype.

[0108] An IgG1 heavy chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 17, or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0109] In certain embodiments of the invention, the constant domains disclosed herein may include a C-terminal lysine, or may lack either a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

[0110] In any of the embodiments disclosed herein, the light chain may comprise a human kappa light chain constant domain comprising SEQ ID NO:38.

[0111] Human MSLN-binding substances containing effector-silent Fc domains Effector-silent human MSLN binding agents of the invention, including full-sized antibodies, may include an HC constant domain or an Fc domain thereof that has been modified such that the antibody exhibits no measurable binding to one or more FcRs or exhibits reduced binding to one or more FcRs compared to an unmodified antibody of the same IgG isotype. The effector-silent antibody may, in further embodiments, exhibit no measurable binding to each of FcγRIIIa, FcγRIIa, and FcγRI, or exhibit reduced binding to each of FcγRIIIa, FcγRIIa, and FcγRI, compared to an unmodified antibody of the same IgG isotype. In certain embodiments, the HC constant domain or Fc domain is a human HC constant domain or Fc domain.

[0112] In certain embodiments, the effector-silent antibody comprises an Fc domain of IgG1 isotype that has been modified to lack N-glycosylation of the asparagine (Asn) residue at position 297 (Eu numbering system) of the HC constant domain. The consensus sequence for N-glycosylation is Asn-Xaa-Ser / Thr (Xaa at position 298 is any amino acid other than Pro), and the consensus sequence for N-glycosylation is Asn-Ser-Thr. This modification can be achieved by replacing the codon encoding Asn at position 297 of the nucleic acid molecule encoding the HC constant domain with a codon encoding another amino acid, such as Ala, Asp, Gln, Gly, or Glu, such as N297A, N297Q, N297G, N297E, or N297D. Alternatively, the codon for Ser at position 298 may be replaced by a codon for Pro, while the codon for Thr at position 299 may be replaced by any codon other than the codon for Ser. In yet another alternative, each of the amino acids comprising the N-glycosylation consensus sequence is replaced by another amino acid. Such modified IgG molecules have no measurable effector function. In certain embodiments, these mutant HC molecules may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, which may be conservative or non-conservative mutations. In further embodiments, such IgGs modified to lack N-glycosylation at position 297 may further comprise one or more additional mutations disclosed herein to eliminate measurable effector function.

[0113] An exemplary IgG1 HC constant domain mutated at position 297 to abolish N-glycosylation of the HC constant domain is shown in SEQ ID NO: 36. In certain embodiments, these mutant HC molecules may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, which may be conservative or non-conservative mutations.

[0114] In certain embodiments, the Fc domain of an IgG1 HC constant domain comprising an effector-silent antibody is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (positions are identified according to Eu numbering), and the HC constant domain is effector-silent. In certain embodiments, the modified IgG1 further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, which substitutions may be conservative or non-conservative mutations.

[0115] In certain embodiments, the HC constant domain comprises an L234A, L235A, and D265S substitution (positions are identified according to Eu numbering). In certain embodiments, the HC constant domain comprises an amino acid substitution at position Pro329 and at least one additional amino acid substitution selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (positions are identified according to Eu numbering). These and other substitutions are disclosed in WO9428027; WO2004099249; WO20121300831, U.S. Patent Nos. 9,708,406; 8,969,526; 9,296,815; Sondermann et al. Nature 406,267-273 (2000).

[0116] In certain embodiments of the above, the HC constant domain comprises L234A / L235A / D265A; L234A / L235A / P329G; L235E; D265A; D265A / N297G; or V234A / G237A / P238S / H268A / V309L / A330S / P331S substitutions, positions identified according to Eu numbering. In certain embodiments, the HC molecule further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, which may be conservative or non-conservative mutations.

[0117] In certain embodiments, the effector-silent antibody comprises an IgG1 isotype, and the Fc domain of the HC constant domain has been modified to be effector-silent by replacing amino acids at positions 233 to 236 of IgG1 with the corresponding amino acids of human IgG2 HC, and by replacing amino acids at positions 327, 330, and 331 with the corresponding amino acids of human IgG4 HC, positions identified according to Eu numbering (Armour et al., Eur. J. Immunol. 29(8):2613-24 (1999); Shields et al., J. Biol. Chem. 276(9):6591-604 (2001)). In certain embodiments, the modified IgG1 further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, which may be conservative or non-conservative mutations.

[0118] In certain embodiments, the effector silent antibody is a V that is fused or linked to a hybrid human immunoglobulin H constant domain. H which comprises, from N-terminal to C-terminal direction, a hinge region, a CH2 domain, and a CH3 domain, wherein the hinge region comprises at least a partial amino acid sequence of a human IgD hinge region or a human IgG1 hinge region; and the CH2 domain is that of a human IgG4 CH2 domain, which portion is replaced at the N-terminal region with 4-37 amino acid residues of the N-terminal region of a human IgG2 CH2 or a human IgD CH2 domain. Such hybrid human HC constant domains are disclosed in U.S. Patent No. 7,867,491, which is incorporated herein by reference in its entirety.

[0119] Exemplary IgG1 HC constant domains include an HC constant domain comprising an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37.

[0120] In certain embodiments of the human MSLN binding agent, the human MSLN binding agent is an antibody comprising an IgG1 Fc domain as disclosed herein, which further comprises a C-terminal lysine or which lacks either the C-terminal lysine or the C-terminal glycine-lysine dipeptide.

[0121] In any of the embodiments disclosed herein, the light chain may comprise a human kappa light chain constant domain comprising SEQ ID NO:38.

[0122] Alternatively or additionally, in another embodiment of the present disclosure, the human MSLN binding agents and other peptides provided herein may undergo post-translational modifications as known in the art. Examples of post-translational modifications include, but are not limited to, chemical modifications such as disulfide bonds, oligosaccharides, N-terminal pyroglutamic acid formation, C-terminal lysine processing (resulting in the removal of lysine), deamidation, isomerization, oxidation, glycation, peptide bond cleavage, non-reducible cross-linking, cleavage, and others known in the art. See Liu, et.al., Heterogeneity of Monoclonal Antibodies, J.Pharma.Sci.vol.97,no.7,pp.2426-2447 (July 2008). Other types of modifications include non-covalent interactions, conformational heterogeneity, and aggregation (ibid.).

[0123] In some embodiments, the N-terminal E or Q of the human MSLN binding agents provided herein is replaced with pyroglutamic acid. In some embodiments, the C-terminal K of the human MSLN binding agents provided herein is removed. In other embodiments, the N-terminal E or Q of the human MSLN binding agents provided herein is replaced with pyroglutamic acid and the C-terminal K (e.g., the heavy chain C-terminal amino acid) of the human MSLN binding agent is removed. The present disclosure includes any of the above post-translational modifications of any of the human MSLN binding agents and polypeptides provided herein. For example, in some embodiments, V HProvided herein are human MSLN binding agents that contain the same sequence as 47D7, except that the first N-terminal amino acid of the region is replaced with pyroglutamic acid and / or the C-terminal amino acid of the heavy chain is deleted. H The first N-terminal amino acid of the domain is replaced with pyroglutamic acid, V L Provided herein are human MSLN binding agents that contain the same sequence as 20B12, except that the first N-terminal amino acid of the region is replaced with pyroglutamic acid and / or the C-terminal amino acid of the heavy chain is removed. H The first N-terminal amino acid of the domain is replaced with pyroglutamic acid, V L Provided herein are human MSLN binding agents that contain the same sequence as 45B6, except that the first N-terminal amino acid of the domain is replaced with pyroglutamic acid and / or the C-terminal amino acid of the heavy chain is removed.

[0124] In certain embodiments, provided herein is an antibody or fragment thereof (e.g., scFv) or polyprotein comprising an amino acid sequence set forth in any one of SEQ ID NOs: 2, 10, 11, 18, 19, and 26-28, except that the first amino acid at the N-terminus is replaced with pyroglutamic acid.

[0125] In other specific embodiments, provided herein is an antibody or fragment thereof comprising the amino acid sequence set forth in any one of SEQ ID NOs: 29-37, except that the C-terminal K has been removed.

[0126] ScFv fusion protein that binds to human MSLN In certain embodiments, the V H and V L V L Domain and V H The domains are expressed as ScFv fusion proteins in which the domains are linked together by a peptide linker. H -V LThe carboxyl terminus of one variable region domain is joined to the amino terminus of another variable domain without compromising the fidelity of the pairing and antigen-binding site. Thus, ScFvs are L The C-terminus of H or a fusion protein linked to the N-terminus of V H The C-terminus of L The peptide linker for linking the variable domains may vary in length from 10 to 25 amino acids and usually (but not necessarily) has a G4S structure (e.g., (G4S) n , where n is 1, 2, 3, 4, or 5), such as hydrophilic amino acids, such as glycine (G) and serine (S). Peptide linkers of shorter length (0-4 amino acids) have also been used, but ScFvs with shorter linkers can form multimers. Commonly, (G4S)3 peptides containing three repeating G4S units are used as ScFv peptide linkers (see, for example, Leath et al., Int. J. Oncol. 24:765-771 (2004); Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Iliades et al., FEBS Lett. 409:437-441 (1997)).

[0127] Exemplary ScFv fusion proteins include those of structure V L -(G4S) n -V H or V H -(G4S) n -V L (a) V L comprises the amino acid sequence set forth in SEQ ID NO:3, H (b) V L comprises the amino acid sequence set forth in SEQ ID NO:3, H (c) V L comprises the amino acid sequence set forth in SEQ ID NO:3,H (d) comprises the amino acid sequence set forth in SEQ ID NO: 27; L comprises the amino acid sequence set forth in SEQ ID NO:3, H (e) V L comprises the amino acid sequence set forth in SEQ ID NO: 11, H comprises the amino acid sequence set forth in SEQ ID NO: 10; or (f) V L comprises the amino acid sequence set forth in SEQ ID NO: 19, H comprises the amino acid sequence set forth in SEQ ID NO:18, wherein n is 1, 2, 3, 4, or 5.

[0128] The ScFvs disclosed herein can be provided in a bispecific format that includes a CD3 binding agent (ScFv) linked by a peptide linker to an ScFv that binds to HSLN, as disclosed herein. When these molecules, called bispecific T cell engagers (BiTEs), bind to CD3 on T cells and to HSLN expressed on the cell surface, the T cells migrate to the tumor site.

[0129] The ScFvs disclosed herein may also be fused to cytotoxins, radioisotopes, cytokines, and enzymes for use in the treatment of cancer, autoimmunity, and / or inflammation. In certain embodiments, the peptide linker may comprise 1-10 G4S peptide units.

[0130] In further embodiments, the ScFvs disclosed herein may be linked or inserted at different positions in an intact IgG molecule to confer dual epitope binding. For example, a bispecific antibody may be provided that comprises two heterodimeric heavy chain constant domains, the N-terminus of one heavy chain constant domain fused to the C-terminus of an ScFv disclosed herein and the N-terminus of the other heavy chain constant domain fused to the C-terminus of an ScFv targeting an antigen other than human MSLN or a Fab' targeting an antigen other than human MSLN.

[0131] Nucleic acid molecules encoding human MSLN-binding substances The present disclosure further provides a nucleic acid molecule encoding a human MSLN binding agent of the present invention. In certain embodiments, the human MSLN binding agent is a V H Domains and V encoded by nucleic acid molecules L The nucleic acid sequence encoding the HSLN-binding agent disclosed herein can be obtained by reverse translating the amino acid sequence of the HSLN-binding agent into a nucleic acid sequence encoding the HSLN-binding agent. The codons of the nucleic acid molecule thus obtained can be further modified to correspond to codons commonly or more efficiently used when translated in a particular cell type. Methods and computer programs for reverse translating and / or optimizing nucleic acid molecules to enhance expression in a particular host cell (e.g., IDT Codon Optimization Tool available from Integrated DNA Technologies, Inc., 1710 Coralville, Commercial Park, Iowa, 52241, USA; U.S. Patent No. 8,326,547; WO2020024917A1) are well known in the art.

[0132] In certain embodiments, HC and LC (or V H and V L ) is HC and LC (or V H and V L ) is fused at its N-terminus to a leader peptide and expressed as a fusion protein to facilitate transport of the antibody through the secretory pathway. In certain embodiments, the N-terminus of the ScFv fusion protein is fused at its N-terminus to a leader or signal peptide to facilitate transport of the antibody through the secretory pathway. Examples of leader / signal peptides that may be used include those comprising the amino acid sequence set forth in SEQ ID NO: 39 or SEQ ID NO: 40. Thus, in certain embodiments, the nucleic acid molecule described above may comprise a polynucleotide encoding a leader peptide linked to the 5' end of the nucleic acid molecule encoding the HSLN binding agent.

[0133] The nucleic acid molecules disclosed herein may contain one or more substitutions that optimize one or more codons to enhance expression of the nucleic acid molecule in a particular host cell, such as a yeast or fungal host cell, a non-human mammalian host cell, a human host cell, an insect host cell, or a prokaryotic host cell.

[0134] Methods for producing human MSLN binding agents The disclosure includes a recombinant method for making a human MSLN binding agent, the method comprising introducing into a host cell: (i) the V H and V L or an expression vector encoding the HC and LC of a human MSLN-binding substance; or (ii) two expression vectors, one encoding the V H or the HC of a human MSLN binding substance, and the V of a human MSLN binding substance. L or encoding the LC of the human MSLN binding agent. H , V L The nucleic acid molecule or polynucleotide encoding the Arginase 1-binding agent or component thereof, e.g., V, is operably linked to a promoter and other transcriptional and translational control sequences. The host cell is cultured under conditions and for a period of time suitable for expression of the nucleic acid molecule, after which the mesothelin-binding agent is isolated from the host cell and / or the medium in which the host cell is grown. See, e.g., WO2004041862, WO2006122786, WO2008020079, WO2008142164, or WO2009068627. The expression vector may be a plasmid or a viral vector. The present invention relates to nucleic acid molecules encoding an Arginase 1-binding agent or component thereof, e.g., V H Or HC only, or V L Alternatively, it also relates to a host or host cell that contains only the HC.

[0135] Eukaryotic and prokaryotic host cells, including mammalian cells, as hosts for the expression of human MSLN binding substances are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, but are not limited to, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines. Thus, mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. By determining which cell line has a high expression level, a particularly preferred cell line is selected. Other cell lines that can be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells, and fungal cells. Fungal cells include yeast cells and filamentous fungal cells, such as Pichia pastoris, Saccharomyces cerevisiae, and Trichoderma reesei. The present disclosure includes any host cell that contains a human MSLN-binding agent of the present disclosure, or that contains one or more nucleic acid molecules encoding such a human MSLN-binding agent, or that contains an expression vector that contains one or more nucleic acid molecules encoding such a human MSLN-binding agent.

[0136] Furthermore, expression of human MSLN binding agents from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos. 0216846B1, 0256055B1, 0323997B1, and 0338841B1. Thus, in an embodiment of the present disclosure, the mammalian host cell lacks a glutamine synthetase gene and grows in the absence of glutamine in the medium, but the nucleic acid molecule encoding an immunoglobulin chain includes a glutamine synthetase gene that complements the genetic deletion in the host cell. Such host cells containing the human MSLN binding agents or nucleic acid(s) or expression vector(s) discussed herein, and expression methods as discussed herein for making human MSLN binding agents using such host cells, are part of the present disclosure.

[0137] The present disclosure includes a method for purifying a human MSLN-binding agent, comprising introducing a sample (e.g., culture medium, cell lysate, or cell lysate fraction, e.g., the soluble fraction of the lysate) containing the human MSLN-binding agent into a purification medium (e.g., a cation exchange medium, an anion exchange medium, and / or a hydrophobic exchange medium) and collecting the purified human MSLN-binding agent from the flow-through fraction of the sample that does not bind to the medium; or discarding the flow-through fraction, eluting the bound human MSLN-binding agent from the medium, and collecting the eluate. In one embodiment of the present disclosure, the medium is in a column to which the sample is applied. In an embodiment of the present disclosure, the purification method is performed after recombinant expression of the human MSLN-binding agent in a host cell, e.g., the host cell is first lysed and, optionally, the lysate is purified from insoluble material before purification on the medium, or the human MSLN-binding agent is secreted by the host cell into the culture medium, and the medium or a portion thereof is added to the purification medium.

[0138] In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic of the glycoprotein produced in the cell line or transgenic animal. Thus, the particular glycosylation pattern of a human MSLN binding agent will vary depending on the particular cell line or transgenic animal used to generate the human MSLN binding agent. Human MSLN binding agents that contain only nonfucosylated N-glycans are part of the present disclosure and can be advantageous because nonfucosylated antibodies have been shown to typically exhibit stronger efficacy both in vitro and in vivo than their fucosylated counterparts (e.g., Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775). These human MSLN binding agents with nonfucosylated N-glycans are unlikely to be immunogenic because the carbohydrate structures are normal components of the population present in human serum IgG.

[0139] The disclosure includes human MSLN binding agents that contain N-linked glycans that are normally added to immunoglobulins produced in Chinese hamster ovary cells (CHON-linked glycans) or modified yeast cells (modified yeast cells (modified N-linked glycans), e.g., Pichia pastoris. For example, in an embodiment of the disclosure, the mesothelin binding agent is a "modified yeast N-linked glycan" or "CHO In an embodiment of the disclosure, the human MSLN binding agent comprises one or more of modified yeast N-linked glycans (e.g., G0 and / or G0-F and / or G1 and / or G1-F and / or G2-F and / or Man5). In an embodiment of the disclosure, the human MSLN binding agent comprises modified yeast N-linked glycans, i.e., G0 and / or G1 and / or G2, optionally further comprising Man5. In an embodiment of the disclosure, the human MSLN binding agent comprises CHON-linked glycans, i.e., G0-F, G1-F, and G2-F, optionally further comprising G0 and / or G1 and / or G2 and / or Man5. In an embodiment of the disclosure, about 80% to about 95% (e.g., about 80-90%, about 85%, about 90%, or about 95%) of all N-linked glycans on the human MSLN binding agent are modified yeast N-linked glycans or CHO N-linked glycans. See, e.g., Nett et al. al. Yeast. 28:237-252 (2011); Hamilton et al. Science. 313:1441-1443 (2006); Hamilton et al. Curr Opin Biotechnol. 18(5):387-392 (2007). For example, in embodiments of the present disclosure, the modified yeast cell is GFI5.0 or YGLY8316, or a strain described in U.S. Patent No. 7,795,002 or Zha et al. Methods Mol Biol. 988:31-43 (2013). See also International Patent Application Publication No. WO2013066765.

[0140] Administration / Pharmaceutical Compositions The human MSLN binding agent may be provided in a suitable pharmaceutical composition comprising the human MSLN binding agent and a pharma- ceutically acceptable carrier. The carrier may be a diluent, adjuvant, excipient, or vehicle in which the human MSLN binding agent is administered. Such vehicles may be liquids, such as water and oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharma- ceutical acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, and colorants. The concentration of the molecule or disclosure in such pharmaceutical formulations can vary widely (i.e., less than about 0.5%, usually at least about 1-15% or as much as 20% by weight) and will be selected primarily based on the required dose, liquid volume, viscosity, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, including other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21.sup.st Edition, Troy, DBed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.

[0141] The mode of administration of the human MSLN binding agent can be any suitable route, for example, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous, pulmonary, mucosal (oral, intranasal, intravaginal, rectal)) or other means well known in the art and recognized by the skilled artisan.

[0142] The human MSLN binding agent can be administered to an individual (e.g., a patient) by any suitable route, for example, parenterally by intravenous (iv) infusion or bolus injection, intramuscularly or subcutaneously, or intraperitoneally. An iv infusion can be administered, for example, over 15, 30, 60, 90, 120, 180, or 240 minutes, or over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.

[0143] The dose administered to an individual with cancer or malignant tumor will be sufficient to ameliorate or at least partially inhibit the disease being treated (a "therapeutically effective amount"), and may optionally be from 0.005 mg / kg to about 100 mg / kg, e.g., from about 0.05 mg / kg to about 30 mg / kg, or from about 5 mg to about 25 mg / kg, or about 4 mg / kg, about 8 mg / kg, about 16 mg / kg, or about 24 mg / kg, or for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg, but may be even higher, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg.

[0144] Fixed unit doses (e.g., 50, 100, 200, 500, or 1000 mg) may also be administered, but the dose may be spread over a range of patient surface areas (e.g., 500, 400, 300, 250, 200, or 100 mg / m 2 Typically, one to eight (e.g., one, two, three, four, five, six, seven, or eight) doses may be administered to treat a cancer or malignant tumor, but nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more doses may be administered.

[0145] The administration of the human MSLN binding agent can be repeated after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, or later. Repeated treatment regimens are also possible, as is chronic administration. Repeated administration can be at the same dose or at different doses. For example, the human MSLN binding agent in the disclosed method can be administered at 8 mg / kg or 16 mg / kg every week for 8 weeks, then at 8 mg / kg or 16 mg / kg every 2 weeks for an additional 16 weeks, and then at 8 mg / kg or 16 mg / kg every 4 weeks by intravenous infusion.

[0146] The human MSLN binding agent may be administered, for example, once a week for a period of six months or more, as a maintenance therapy. For example, the human MSLN binding agent in the disclosed method may be administered, for example, once a week for a period of six months or more, as a maintenance therapy, using a single dose or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, on at least one of, or alternatively on, the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, or 40th day from the start of treatment. In some embodiments, the administration of the compound may be provided in one of the following doses: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks, or any combination thereof, as a daily dosage in an amount of about 0.1 to 100 mg / kg per day, e.g., 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg per day.

[0147] Human MSLN binding agents may also be administered prophylactically to reduce the risk of developing cancer, delay the occurrence of events in the progression of cancer, and / or reduce the risk of recurrence when the cancer is in remission, which may be particularly useful in patients in whom other biological factors make it difficult to localize tumors known to exist.

[0148] The human MSLN binding agents can be lyophilized for storage and reconstituted in a suitable carrier prior to use, a technique that has been shown to be effective for conventional protein preparations, and well-known lyophilization and reconstitution techniques can be used.

[0149] Combination Therapy Treatment The combination therapy of the present disclosure includes a human MSLN binding agent and another therapeutic agent (small molecule or antibody) can be used to treat proliferative diseases, particularly cancer.In certain embodiments, the combination therapy of the present disclosure can be used to treat melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, renal cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colon cancer, cervical cancer, thyroid cancer, or salivary gland cancer.

[0150] In another embodiment, the combination therapy of the present disclosure may be used to treat pancreatic cancer, bronchial cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0151] Combination therapy comprising a human MSLN binding agent and a chemotherapeutic agent The combination therapy of the present disclosure may be administered to individuals with cancer in combination with chemotherapy. The individual may receive chemotherapy at the same time as receiving the combination therapy of the present disclosure. The individual may receive the combination therapy of the present disclosure after completing chemotherapy. The individual may be administered chemotherapy after completing the combination therapy. The combination therapy of the present disclosure may also be administered to individuals with recurrent or metastatic cancer who have disease progression, or who have recurrent cancer and are receiving or have completed chemotherapy.

[0152] The chemotherapy may include a chemotherapeutic agent selected from the following group: (i) alkylating agents (including but not limited to bifunctional alkylating agents, cyclophosphamide, mechlorethamine, chlorambucil, and melphalan); (ii) monofunctional alkylating agents (including, but not limited to, dacarbazine, nitrosoureas, and temozolomide (oral dacarbazine)); (iii) anthracyclines (including, but not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin); (iv) cytoskeletal disrupting agents (taxanes) (including but not limited to paclitaxel, docetaxel, abraxane, and taxotere); (v) Epothilones (including but not limited to ixabepilone and utiderone); (vi) histone deacetylase inhibitors (including but not limited to vorinostat and romidepsin); (vii) inhibitors of topoisomerase I (including but not limited to irinotecan and topotecan); (viii) topoisomerase II inhibitors (including but not limited to etoposide, teniposide, and tafluposide); (ix) Kinase inhibitors (including but not limited to, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib). (x) nucleotide analogs and precursor analogs (including, but not limited to, azacitidine, azathioprine, fluoropyrimidines (e.g., capecitabine, carmofur, doxifluridine, fluorouracil, and tegafur), cytarabine, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine (formerly thioguanine)); (xi) peptide antibiotics (including but not limited to bleomycin and actinomycin), platinum-based drugs (including but not limited to carboplatin, cisplatin, and oxaliplatin); (xii) retinoids (including but not limited to tretinoin, alitretinoin, and bexarotene); and (xiii) Vinca alkaloids and derivatives (including, but not limited to, vinblastine, vincristine, vindesine, and vinorelbine).

[0153] The choice of the dosage of the chemotherapeutic agent in chemotherapy depends on several factors, such as the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissues, or organs of the individual being treated. The administration of the additional therapeutic agent should be in an amount that produces an acceptable level of side effects. Thus, the dosage and frequency of administration of each additional therapeutic agent will depend in part on the particular therapeutic agent, the severity of the cancer being treated, and the characteristics of the patient. Guidance is available for selecting the appropriate doses of antibodies, cytokines, and small molecules. See, for example: Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune. Diseases,Marcel Dekker,New York,NY;Baert et al.(2003)New Engl.J.Med.348:601-608;Milgrom et al.(1999)New Engl.J.Med.341:1966-1973;Slamon et al.(2001)New Engl.J.Med.344:783-792;Beniaminovitz et al. (2000) New Engl. J. Med.342:613-619; Ghosh et al. (2003) New Engl. J. Med.348:24-32; Lipsky et al. (2000) New Engl. J. Med.343:1594-1602; Physicians' Desk Reference 2003 Reference,57th Ed);Medical Economics Company;ISBN:1563634457;57th edition(November 2002).Determination of an appropriate dosing regimen can be made by the clinician using, for example, parameters or factors known or suspected in the art to affect or predicted to affect treatment, and will depend, for example, on the individual's clinical history (e.g., previous treatments), the type and stage of the cancer being treated, and biomarkers of response to one or more therapeutic agents in the combination therapy.

[0154] Thus, the present disclosure further contemplates combination therapy embodiments of the present disclosure that include a chemotherapy step that includes either a platinum-containing chemotherapy, pemetrexed and platinum chemotherapy, or carboplatin and paclitaxel or nab-paclitaxel. In certain embodiments, the combination therapy with the chemotherapy step may be used to treat at least NSCLC and HNSCC.

[0155] The combination therapy may further be used in combination with a chemotherapy step to treat any proliferative disease, particularly cancer. In certain embodiments, the combination therapy of the present disclosure may be used to treat melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, non-Hodgkin's lymphoma, renal cancer, Hodgkin's lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colon cancer, cervical cancer, thyroid cancer, or salivary gland cancer.

[0156] In another embodiment, the combination therapy in combination with the chemotherapy step may further be used to treat pancreatic cancer, bronchial cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0157] In certain embodiments, the combination therapy with chemotherapy step may be used to treat one or more cancers selected from melanoma (metastatic or unresectable), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, MSIHC, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (including advanced cancer), and cutaneous squamous cell carcinoma.

[0158] Combination Therapies Comprising Human MSLN Binding Agents and Therapeutic Antibodies - Patent application The human MSLN binding agents of the present disclosure may be administered in combination with one or more therapeutic agents that are antibodies for the treatment of cancer or proliferative diseases. An individual may receive treatment with a therapeutic antibody at the same time that they are receiving the combination therapy of the present disclosure. An individual may receive the combination therapy of the present disclosure after completing treatment with a therapeutic antibody. An individual may receive treatment with a therapeutic antibody after completing the combination therapy. The combination therapy of the present disclosure may also be administered to individuals with recurrent or metastatic cancer who have disease progression, or who have recurrent cancer and are undergoing or have completed chemotherapy. In certain embodiments, the therapeutic agent targets the programmed cell death 1 receptor or ligand (PD-1 and PD-L1, respectively).

[0159] Exemplary anti-PD-1 antibodies that may be used in combination therapy with a human MSLN binder include any antibody that binds to PD-1 and blocks PD-1 from binding to PD-L1. In further embodiments, the exemplary anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and cemiplimab-rwlc. Exemplary antibodies include the following anti-PD-1 antibodies and compositions comprising an anti-PD-1 antibody and a pharmaceutically acceptable salt:

[0160] Pembrolizumab, also known as KEYTRUDA, lambrolizumab, MK-3475, or SCH-900475, is a humanized anti-PD-1 antibody described in U.S. Pat. No. 8,354,509 and WO 2009 / 114335 and disclosed, for example, in Hamid, et al., New England J. Med. 369(2):134-144 (2013).

[0161] Nivolumab, also known as OPDIVO, MDX-1106-04, ONO-4538, or BMS-936558, is a fully human IgG4 anti-PD-1 antibody described in WO2006 / 121168 and U.S. Pat. No. 8,008,449.

[0162] Cemiplimab-rwlc, also known as cemiplimab, LIBTAYO, or REGN2810, is a recombinant human IgG4 monoclonal antibody described in WO2015112800 and US Pat. No. 9,987,500.

[0163] In certain embodiments, the anti-PD-1 antibody comprises (i) a V domain that contains the three HC-CDRs of pembrolizumab fused or linked to an effector-silent HC constant domain. H and (ii) a V comprising the three LC-CDRs of pembrolizumab fused or linked to an LC kappa or lambda constant domain. L , including.

[0164] In certain embodiments, the anti-PD-1 antibody comprises (i) a VH that comprises the three HC-CDRs of nivolumab fused or linked to an effector-silent HC constant domain, and (ii) a VH that comprises the three LC-CDRs of nivolumab fused or linked to an LC kappa or lambda constant domain. L , including.

[0165] In certain embodiments, the anti-PD-1 antibody comprises (i) a V domain comprising the three HC-CDRs of cemiplimab-rwlc fused or linked to an effector-silent HC constant domain. H and (ii) a V comprising the three LC-CDRs of nivolumab fused or linked to an LC kappa or lambda constant domain. L , including.

[0166] In certain embodiments, the VH of the anti-PD-1 antibody is selected from the group consisting of an IgG1, IgG2, IgG3, or IgG4 HC constant domain (currently, certain V HThe anti-PD-1 antibody may be fused or linked to an IgG1, IgG2, IgG3, or IgG4 HC constant domain (not linked to an Fc domain) or may be linked to an IgG1, IgG2, IgG3, or IgG4 HC constant domain that has been modified to contain one or more mutations in the Fc domain that render the resulting anti-PD-1 antibody effector silent.

[0167] Injection device for administering human MSLN binding substances The present disclosure also provides an injection device comprising the human MSLN binding substance or pharmaceutical composition thereof described herein. An injection device is a device that introduces a substance into a patient's body via a parenteral route, for example, intramuscularly, subcutaneously, or intravenously. For example, the injection device may be a syringe (e.g., one pre-filled with a pharmaceutical composition, e.g., an autoinjector), which includes, for example, a cylinder or barrel for holding a liquid to be injected (e.g., one containing a human MSLN binding substance or pharmaceutical composition thereof), a needle for piercing the skin and / or blood vessel to inject the liquid; and a plunger for pushing the liquid out of the cylinder through the hole in the needle. In an embodiment of the present disclosure, the injection device comprising a human MSLN binding substance or pharmaceutical composition thereof is an intravenous (IV) injection device. Such devices contain a human MSLN binding substance or a pharmaceutical composition thereof in a cannula or trocar / needle that may be attached to a tube that may be attached to a bag or reservoir for holding a liquid (e.g., saline or lactated Ringer's solution containing NaCl, sodium lactate, KCl, CaCl2, optionally containing glucose) to be introduced into the subject's body through the cannula or trocar / needle.

[0168] In an embodiment of the present disclosure, the human MSLN binding substance or pharmaceutical composition thereof can be introduced into the device when a trocar and a cannula are inserted into a vein of a subject and the trocar is removed from the inserted cannula. The IV device can be inserted, for example, into a peripheral vein (e.g., in the hand or arm); into the superior or inferior vena cava, or into the right atrium of the heart (e.g., a central IV), or into the subclavian, internal jugular, or femoral vein and advanced toward the heart until it reaches, for example, the superior vena cava or the right atrium (e.g., a central venous line). In an embodiment of the present disclosure, the injection device is an autoinjector, a jet injector, or an external infusion pump. A jet injector introduces the human MSLN binding substance or pharmaceutical composition thereof into the patient's body using a high-pressure narrow liquid jet that penetrates the epidermis. An external infusion pump is a medical device that delivers the human MSLN binding substance or pharmaceutical composition thereof into the patient's body in a controlled amount. An external infusion pump can be electrically or mechanically driven. Different pumps work in different ways: syringe pumps hold the liquid in a syringe reservoir and a moveable piston controls the liquid delivery, elastomeric pumps hold the liquid in an expandable balloon reservoir and pressure from the elastic walls of the balloon drives the liquid delivery, peristaltic pumps use a series of rollers that pinch a long flexible tube and push the liquid forward, and multi-channel pumps can pump liquid from multiple reservoirs at multiple speeds.

[0169] Kits containing human MSLN binding substances Additionally, kits are provided that include one or more components, including but not limited to, a human MSLN binding agent, as discussed herein, in association with one or more additional components, including but not limited to, an additional therapeutic agent, as discussed herein. The human MSLN binding agent and / or therapeutic agent can be formulated into a pharmaceutical composition, either as a pure composition or in combination with a pharma- ceutically acceptable carrier.

[0170] In one embodiment, the kit contains a human MSLN binding agent or a pharmaceutical composition thereof in one container (e.g., a sterile glass or plastic vial) and an additional therapeutic agent in another container (e.g., a sterile glass or plastic vial).

[0171] In another embodiment, the kit comprises a combination of the present disclosure comprising a human MSLN binding agent or a pharmaceutical composition thereof, optionally in combination with one or more therapeutic agents formulated together in a pharmaceutical composition in a single common container.

[0172] Where the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit may include a device for performing such administration. For example, the kit may include one or more hypodermic needles or other injection devices, as discussed above. Thus, the present disclosure includes kits that include an injection device and a human MSLN binding agent, e.g., the injection device includes the human MSLN binding agent or the human MSLN binding agent is in a separate container.

[0173] The kit may include a package insert containing information regarding the pharmaceutical compositions and dosage forms in the kit. Generally, such information assists patients and physicians in effectively and safely using the enclosed pharmaceutical compositions and dosage forms. For example, the following information regarding the disclosed combinations may be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical trials, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, method of supply, suitable storage conditions, references, manufacturer / distributor information, and patent information.

[0174] The following examples are intended to facilitate a further understanding of the present disclosure.

[0175] Common methods Protein ELISA The wells of a 96-well plate are coated with 50 μL of purified human or rhesus MSLN at a concentration of 1–2 μg / mL in carbonate coating buffer (5.3 g NaHCO3 and 3.2 g Na2CO3 in 2 L H20) or HyClone Dulbecco's phosphate-buffered saline (DPBS; Hyclone, catalog number SH30028.02) and incubated overnight at 4 °C. The wells of the plate are then washed with well wash solution (phosphate-buffered saline containing 0.05% polysorbate 20). The wells of the plate are then blocked for 1–2 h at room temperature by adding Super Block blocking buffer T20 (Thermo Scientific, #37536) and then washed with well wash solution. Next, 50 μL / well of hybridoma supernatant fraction (used neat) or purified antibodies serially diluted 4- or 5-fold in ELISA buffer (DPBS + 0.1% BSA + 0.05% polysorbate 20) starting at 10 μg / mL are added to the wells. The plate is then incubated for 1 hour at room temperature, the wells are washed with well wash, and 50 μL / well of horseradish peroxide (HRP)-conjugated anti-species IgG diluted 1:3000-5000 in ELISA buffer is added to the wells. HRP-conjugated anti-species IgG include goat anti-mouse IgG-HRP (Southern Biotech, catalog number 1030-05), goat anti-rat IgG-HRP (Southern Biotech, catalog number 3030-05), and goat anti-human IgG-HRP (Jackson Immunologics, catalog number 109-036-098). The plate is then incubated for 1 hour at room temperature, the wells are washed with well wash solution, and 50 μL / well of ABTS peroxidase substrate (Kirkgaard & Perry Laboratories) is added to the wells. After 10 minutes, the OD at 405 nm is measured using an ELISA plate reader.

[0176] Cell ELISA for CHO cells Seed enough cells into wells of a 96-well plate to obtain 95-100% confluence on day 0 (the day of the assay). On day 0, remove the medium and add 50 μL / well of hybridoma supernatant fractions, or purified antibodies, starting at 10 μg / mL or equimolar concentration, serially diluted 4- or 5-fold in CHO medium (DMEM F12, 10% bovine serum). The plate is then incubated for 1 hour at room temperature or up to 37 °C. Next, add approximately 50 μL of a given concentration of ligand to the wells of the plate and incubate the plate for 30 minutes at room temperature or up to 37 °C. Wash the wells of the plate with ELISA wash solution (PBS containing 0.05% polysorbate 20). Next, add 50 μL / well of HRP-conjugated anti-species IgG, diluted 1:2000 in CHO medium, to the wells and incubate the plate for 1 hour at room temperature or up to 37 °C. HRP-conjugated anti-species IgG includes goat anti-mouse IgG-HRP (Southern Biotech, Catalog No. 1030-05), goat anti-rat IgG-HRP (Southern Biotech, Catalog No. 3030-05), and goat anti-human IgG-HRP (Jackson Immunologics, Catalog No. 109-036-098). Wash the wells of the plate with ELISA wash solution. Then add TMB substrate (1-Step Ultra TMB-ELISA: Thermo Fisher, Catalog No. 34029) to the wells at 50 μL / well. When the blank starts to turn blue, add 50 μL / well of TMB stop solution (KPL, Catalog No. 50-85-06) to the wells and measure A450-A620 in an ELISA reader. EXAMPLES

[0177] Example 1 Immunization and screening of human MSLN binding substances The mRNA encoding full-length human mature MSLN was used to immunize Trianni™ mice (AbCellera, Vancouver, BC). Spleen cells from immunized animals were then isolated and used for fusion with Sp2 / 0 myeloma partners to generate hybridomas. Hybridomas were then plated separately in 96-well plates to generate hybridoma libraries, and antibody supernatant fractions from plated hybridomas were screened for binding to human MSLN, species cross-reactivity, affinity, binding to cells expressing full-length human MSLN, and binding to human MSLN. To ensure monoclonality, positive binding wells were selected for subcloning. The resulting monoclonal hybridomas were then used for small-scale antibody production and purification. The purified antibodies were subjected to another round of screening, including affinity, epitope binning, full-length protein binding, human MSLN binding, protein and cell binding, and competition assays.

[0178] Hybridoma clones SV017.13B12.1E1, SV017.47D7.1C2, SV017.50G3.1B1, SV017.51F4.1A1, SV018.20B12.1D1, and SV018.45B6.1G1 were selected for analysis.

[0179] Example 2 Antibody production ExpiCHO™ cells growing in suspension were transfected with antibody expression plasmids (HC+LC) using a commercial protocol and ExpiFectamine™ CHO reagent (ThermoFisher) (Sivasubramanian et al., MABS 9:29-42 (2017)). Briefly, on day 0, cells were transfected using 1 μg total DNA per mL of cells (LC:HC ratio 3:2) at a density of 6 million cells per mL, and viability >95% was measured using a Vi-Cell (Beckman-Coulter). On day 1, ExpiCHO™ feed and enhancer were added to the cell culture and the cell culture temperature was reduced to 32° C. On day 5, a second EXPI-CHO™ feed was performed and cell viability was measured using a Vi-Cell (Beckman-Coulter). Depending on whether cell viability exceeded 80%, the cell cultures were harvested on days 8-12, and the harvested cell culture fluids were centrifuged to remove cells and debris to obtain clarified supernatant fractions.

[0180] Antibodies were purified from the clarified supernatant fractions using Protein A resin chromatography (mAbSelect Sure™ LX, GE Healthcare) as follows.

[0181] Protein A resin in loading buffer was incubated with the clarified supernatant fraction overnight at 4° C. on a roller mixer. The Protein A resin was then collected from the clarified supernatant fraction, transferred to a chromatography column, and washed with 10 column volumes (CV) of phosphate-buffered saline (PBS). The antibody was then eluted from the chromatography column using an elution buffer containing 20 mM sodium acetate at pH 3.5. Fractions of 1 column volume (CV) were collected and tested by Bradford assay to determine the presence of protein. In some cases, Protein A purification was followed by anion exchange chromatography (Capto™ Q, GE Healthcare). The purified antibody was buffer exchanged into the final formulation buffer containing 20 mM sodium acetate, 9% sucrose (pH 5.5). Purified antibodies were checked for purity by reducing and non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) (PerkinElmer), concentration was measured at A280, and aggregate content was analyzed by SEC-UPLC (size-exclusion ultra-performance liquid chromatography) using a BEH200 UPLC-SEC analytical column (Waters Corporation). Endotoxin was quantified using Endosafe® nexgen-MCS™ (Charles River). Intact mass was confirmed by Synapt® G2S QTOF or Xevo® G2 TOF (Waters).

[0182] The amino acid sequences of antibodies produced by hybridoma clones SV017.13B12.1E1, SV017.47D7.1C2, SV017.50G3.1B1, SV017.51F4.1A1, SV018.20B12.1D1, and SV018.45B6.1G1 are shown in Table 6.

[0183] Example 3 Antibodies produced by hybridoma clones SV017.13B12.1E1, SV017.47D7.1C2, SV017.50G3.1B1, SV017.51F4.1A1, SV018.20B12.1D1, and SV018.45B6.1G1 were evaluated by ELISA (enzyme-linked immunosorbent assay) for binding to human MSLN and non-human primate mesothelin (rhesus MSLN) both as soluble and membrane-bound proteins expressed by Chinese hamster ovary K1 (CHOK1) cells genetically modified to express human MSLN or rhesus MSLN, and the OVCAR3 cell line is a human ovarian cancer cell line that expresses high levels of human MSLN.

[0184] The results are presented in Table 5 and Figures 1-3. As shown in Table 5, all antibodies show strong binding to membrane-bound human MSLN (CHOK1 or OVCAR3). Antibodies identified from clones SV018.20B12.1D1 and SV018.45.1G1 show no detectable binding to rhesus MSLN and weak binding to soluble human MSLN, and antibodies from clones SV017.13B12.1E1, SV017.47D7.1C2, SV017.50G3.1B1, and SV017.51F4.1A1 show weak binding to rhesus MSLN. [Table 6]

[0185] 1-3 show a comparison of antibodies identified from hybridoma clones SV017.47D7.1C2, SV018.20B12.1D1, and SV018.45B6.1G1 with MORAB-009 as a positive control. MORAB-009 has the generic name amatuximab and is an anti-human MSLN antibody comprising heavy and light chains shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively. Data was generated from ELISA.

[0186] Figure 1 shows that antibody SV017.47D7.1C2 binds to membrane-bound human MSLN with a similar potency as MORAB-009 and can bind to soluble human MSLN with a higher potency than when it binds to membrane-bound human MSLN. Figure 2 shows that antibody SV018.20B12.1D1 binds to human MSLN with about half the potency of MORAB-009 and can bind to soluble human MSLN with a lower potency than when it binds to membrane-bound human MSLN. Figure 3 shows that antibody SV018.45B6.1G1 binds to human MSLN with about half the potency of MORAB-009 and can bind to soluble human MSLN with a lower potency than when it binds to membrane-bound human MSLN.

[0187] The ELISA results show that the antibodies bind strongly to membrane-bound human MSLN and weakly or not at all to membrane-bound rhesus MSLN. Antibodies SV018.20B12.1D1 and SV018.45B6.1G1, which showed no detectable binding to membrane-bound rhesus MSLN, also showed low potency against soluble human MSLN, indicating a reduced ability of these antibodies to act as a sink. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8]

[0188] While the present disclosure has been described herein with reference to illustrated embodiments, it should be understood that the disclosure is not limited thereto. Those skilled in the art and with access to the teachings herein will recognize that additional modifications and embodiments are possible within the scope thereof. Accordingly, the present disclosure is limited only by the scope of the claims appended hereto.

Claims

1. A human mesothelin (MSLN) binding agent, comprising: (a) six complementarity-determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 19; (b) six CDRs of an antibody comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 10 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 11; or (c) six CDRs of an antibody comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 2, 26, 27, or 28 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 3; In (a), (b), or (c), the human mesothelin binding agent, wherein the CDRs are defined using the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering scheme.

2. The human MSLN-binding substance (a) the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; and the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; or (b) the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; and the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; or (c) the VH comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; and the VL comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

25. The human MSLN binding substance according to claim 1.

3. The human MSLN-binding substance (a) a VH comprising: (i) an amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having the second to 116th amino acid residues of the amino acid sequence set forth in SEQ ID NO: 2, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid; (ii) an amino acid sequence set forth in SEQ ID NO: 26, or an amino acid sequence having the second to 116th amino acid residues of the amino acid sequence ranging from the second to 116th amino acid residues set forth in SEQ ID NO: 26, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid; (iii) an amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having the second to 116th amino acid residues of the amino acid sequence set forth in SEQ ID NO: 27, wherein the first amino acid residue is pyroglutamic acid; or (iv) an amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having the second to 116th amino acid residues of the amino acid sequence ranging from the second to 116th amino acid residues set forth in SEQ ID NO: 28, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid; and comprising a VL comprising the amino acid sequence set forth in SEQ ID NO:3; (b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence comprising the second to 116th amino acid residues of the amino acid sequence set forth in SEQ ID NO: 10, wherein the first amino acid residue is pyroglutamic acid; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence comprising the second to the 107th amino acid residues of the amino acid sequence set forth in SEQ ID NO: 11, wherein the first amino acid residue of the amino acid sequence is pyroglutamic acid; (c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence comprising the second to 118th amino acid residues of the amino acid sequence set forth in SEQ ID NO: 18, wherein the first amino acid residue is pyroglutamic acid; and a VL comprising the amino acid sequence set forth in SEQ ID NO: 19, or an amino acid sequence comprising the second to the 108th amino acid residues of the amino acid sequence set forth in SEQ ID NO: 19, wherein the first amino acid residue is pyroglutamic acid; (d) a VH comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28, and a VL comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:3, wherein the VH comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; and the VL comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; (e) a VH comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 10, and a VL comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 11, wherein the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; (f) a VH comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 18, and a VL comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 19, wherein the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25; The human MSLN binding substance according to claim 1.

4. The human MSLN-binding substance of claim 1 , wherein the human MSLN-binding substance comprises an antibody comprising a heavy chain constant domain of an IgG1 isotype and a light chain constant domain of a human kappa or human lambda isotype.

5. (a) the heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO:29, or the amino acid sequence ranging from amino acid residue 1 to amino acid residue 329 of SEQ ID NO:29; or (b) the heavy chain constant domain of the IgG1 isotype comprises an Fc domain comprising one or more mutations that render the constant domain effector silent; The human MSLN binding substance according to claim 4.

6. the effector-silent constant domain is (i) an amino acid sequence having the amino acid residues 1 to 329 of the amino acid sequence set forth in SEQ ID NO: 30, or the amino acid sequence ranging from the amino acid residue 1 to the amino acid residue 329 of SEQ ID NO: 30; (ii) an amino acid sequence having the amino acid residues 1 to 329 of the amino acid sequence set forth in SEQ ID NO: 31, or the amino acid sequence ranging from the amino acid residue 1 to the amino acid residue 329 of SEQ ID NO: 31; (iii) an amino acid sequence having the amino acid residues 1 to 329 of the amino acid sequence set forth in SEQ ID NO: 32, or the amino acid sequence ranging from the amino acid residue 1 to the amino acid residue 329 of SEQ ID NO: 32; (iv) an amino acid sequence having the amino acid residues 1 to 329 of the amino acid sequence set forth in SEQ ID NO: 33, or the amino acid sequence ranging from the amino acid residue 1 to the amino acid residue 329 of SEQ ID NO: 33; (v) an amino acid sequence having the amino acid residues 1 to 329 of the amino acid sequence set forth in SEQ ID NO: 34, or the amino acid sequence ranging from the amino acid residue 1 to the amino acid residue 329 of SEQ ID NO: 34; (vi) an amino acid sequence having the amino acid residues 1 to 329 of the amino acid sequence set forth in SEQ ID NO: 35, or the amino acid sequence ranging from the amino acid residue 1 to the amino acid residue 329 of SEQ ID NO: 35; (vii) an amino acid sequence having the amino acid residues 1 to 329 of the amino acid sequence set forth in SEQ ID NO: 36, or the amino acid sequence ranging from the amino acid residue 1 to the amino acid residue 329 of SEQ ID NO: 36; or (viii) an amino acid sequence having the amino acid residues 1 to 329 of the amino acid sequence set forth in SEQ ID NO: 37, or the amino acid sequence ranging from the amino acid residue 1 to the amino acid residue 329 of SEQ ID NO: 37 The human MSLN-binding substance of claim 5, comprising:

7. The human MSLN binding agent of claim 4 , wherein the light chain constant domain comprises the human kappa isotype comprising the amino acid sequence set forth in SEQ ID NO:

38.

8. The human MSLN-binding substance according to claim 1 , wherein the human MSLN-binding substance comprises a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, an Fv region, or an ScFv.

9. A composition comprising the human MSLN-binding substance according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier or diluent.

10. for the treatment of cancer or proliferative diseases, A human MSLN-binding substance according to any one of claims 1 to 8, or a composition comprising the human MSLN-binding substance.

11. A combination therapy for treating cancer or a proliferative disease, comprising the human MSLN-binding substance of any one of claims 1 to 8 or the composition of claim 9, and a therapeutic agent.

12. The combination therapy of claim 11 , wherein the therapeutic agent is a chemotherapeutic agent or a therapeutic antibody.

13. The combination therapy of claim 12, wherein the antibody is an anti-PD1 antibody or an anti-PD-L1 antibody.

14. A nucleic acid molecule encoding the human MSLN-binding substance according to any one of claims 1 to 8.

15. 15. An expression vector comprising one or more of the nucleic acid molecules of claim 14.

16. A host cell comprising the expression vector of claim 15.

17. A method for producing a human MSLN binding substance, comprising: (a) providing a host cell described in claim 16; (b) culturing the host cell in a culture medium under conditions suitable for expressing the human MSLN binding substance; and (c) isolating the human MSLN binding substance from the culture medium.

18. The human MSLN binding agent of any one of claims 1 to 8, conjugated to a detectable moiety.

19. 19. The human MSLN-binding agent of claim 18, wherein the detectable moiety is detectable by magnetic resonance imaging (MRI) or X-ray imaging.

20. A human MSLN binding substance, comprising: (a) (i) the C-terminal amino acid residue K of the heavy chain is removed; and (ii) the N-terminal amino acid residue E or Q of VH, VL, heavy chain, or light chain is replaced with pyroglutamic acid; or (b) the human MSLN-binding substance is obtained by expressing in a host cell a nucleic acid molecule encoding the human MSLN-binding substance according to any one of claims 1 to 8 or an expression vector comprising one or more nucleic acid molecules encoding the human MSLN-binding substance according to any one of claims 1 to 8; The human MSLN-binding substance.

21. A composition for use in a method for detecting human MSLN on the surface of cells in a patient, the composition comprising the human MSLN binding substance described in claim 18, wherein the method comprises administering the human MSLN binding substance described in claim 18 to the patient and detecting the cells in the patient that are bound to the human MSLN binding substance.

22. A composition for use in therapy, comprising the human MSLN conjugate according to any one of claims 1 to 8.

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