Fully human Anti-mesothelin antibody, SCFV and application as car and bispecific t cell engager

Abstract

Human mesothelia (MSLN) binding proteins, antibodies and antibody-based binding agents, derivatives and ligands, including variant antibodies and fragments thereof are provided. The antibodies and antibody-based agents and derivatives, such as single chain F vs (scFvs), chimeric antigen receptors (CARs) and bispectfic T-cell engagers (BiTEs), are useful in the diagnosis and treatment of conditions associated with activated, elevated or highly expressed MSLN, including cancer, and for modulating immune cells and immune response, including immune response to cancer or cancer antigens. Particular scFvs, CARs and BiTEs and constructs are provided. Cells, including T cells, engineered to express the antibodies, scFvs, CARs or BiTEs are also provided.

Description

[0001] Atty. Docket No. 2745-9 PCTT

[0002] FULLY HUMAN ANTI-MESOTHELIN ANTIBODY, SCFV AND APPLICATION AS CAR AND BISPECIFIC T CELL ENGAGER

[0003] INCORPORATION OF SEQUENCE LISTING

[0004]

[0001] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document encoded as XML in UTF-8 text. The electronic document, created on February 17, 2026, is entitled “2745-9_PCT_ST26.xml”, and is 86,693 bytes in size.

[0005] FIELD OF THE INVENTION

[0006]

[0002] The present invention relates to antibodies and antibody-based binding agents, derivatives and ligands, including variant antibodies and fragments thereof, which bind to human mesothelin (MSLN). The antibodies and antibody -based agents and derivatives, such as single chain Fvs (scFvs), chimeric antigen receptors (CARs) and bispecific T-cell engagers (BITEs), are useful in the diagnosis and treatment of conditions associated with activated, elevated or highly expressed MSLN, including cancer, and for modulating immune cells and immune response, including immune response to cancer or cancer antigens. Cells, including T cells, engineered to express the antibodies, scFvs, CARs or BiTEs are also provided.

[0007] BACKGROUND OF THE INVENTION

[0008]

[0003] Mesothelin is a glycosylphosphatidylinositol (GPI)-linked glycoprotein synthesized as a 69 kDa precursor and proteolytic ally processed into a 30 kDa NH2 -terminal secreted form (formerly referred to as megakaryocyte potentiating factor) and a 40 kDa membrane-bound mesothelin (Yamaguchi N et al (1994) J Biol Chem 269:805-08). Mesothelin is present at relatively low levels in mesothelial cells of the pleura, peritoneum and pericardium of healthy individuals, but is highly expressed in several different cancers, including mesotheliomas, stomach cancer, squamous cell carcinomas, prostate cancer, pancreatic cancer, lung cancer, and ovarian cancer (Hassan R, Bera T, Pastan I (2004) Clin Cancer Res Off J Am Assoc Cancer Res 10:3937-42; McGuire WP et al (1996) N Engl J Med 1996; 334:1-6; Argani P et al (2001) Clin Cancer Res Off J Am Assoc Cancer Res 7:3862-68; Hassan R et al (2005) Appl Immunohistochem Mol Morphol 13:243-47; Li M et al (2008) Mol Cancer Ther 7:286-96. In particular, it has been reported that most serous carcinomas of the ovary and adenocarcinomas of the pancreas express high levels of mesothelin (Yen MJ et al (2006) Clin Cancer Res 12:827-31). High levels of mesothelin have been detected in greater than 55% of lung cancers and greater than 70% of ovarian cancers (Hassan R et al (2005) Appl Immunohistochem 13:243-47; Ho M et al (2007) Clin Cancer Res 13:1571-75). Mature human mesothelin (hMSLN, AA residues 296-606) is a therapeutically relevant cell surfaceAtty. Docket No. 2745-9 PCTT

[0009] antigen highly expressed in several aggressive solid tumours including mesothelioma and pancreatic adenocarcinomas (Hassan, R. & Ho, M (2008). Mesothelin targeted cancer immunotherapy. Eur. J.

[0010] Cancer 44, 46-53; Lv, J. & Li, P. (2019) Biomark. Res. 7, 18; Tang, Z., Qian, M. & Ho, M. (2013) Anticancer Agents Med. Chem. 13, 276-280). In addition, Mesothelin has been suggested as a novel cell surface disease marker and potential therapeutic target in acute myeloid leukemia, with high overexpression observed in 36% of an AML cohort (Kaeding A et al (2021) Blood Adv 5(9):2350-2361).

[0011]

[0004] The limited expression of mesothelin on normal cells makes it a viable target for tumor immunotherapy. Administration of antibodies against mesothelin has been proposed as a strategy for mesothelioma as well as lung, ovarian and pancreatic cancer. Although mesothelin is considered a membrane-bound protein, soluble forms of mesothelin have also been reported in patients’ serum and in the stroma of tumors, including malignant mesothelioma, ovarian cancers, pancreatic cancer or highly metastatic cancers. Soluble receptors can be released in the tumoral microenvironment and act as a decoy for therapeutic antibodies. The use of monoclonal antibodies (mAbs) against membrane targets has been shown to increase the survival of patients having several cancers and several antibody -based molecules that target mesothelin have been developed. These include the chimeric anti-mesothelin mAb MORAb-009, immunotoxins SS1P and RG7787, and the antibody-drug conjugates BAY94-9343 and BMS986148 (Bang S et al (2005) Clin Cancer Res 11:1545-50; Hollevoet K et al (2014) Mol Cancer Ther 13:2040-49; Kelly RJ, Sharon E, Pastan I, Hassan R. (2012) Mol Cancer Ther 11:517-25; de Goeij BE, Lambert JM (2016) Curr Opin Immunol 2016; 40:14-23). Recently, a human single-domain antibody (SDl-hFc) has been described and reported to elicit a potent anti-tumor activity by generating a complement-dependent cytotoxicity (CDC) reaction targeting an epitope in mesothelin close to the cancer cell surface (Tang Z et al (2013) Mol Cancer Ther 12:416-26). Although these mesothelin-targeting agents showed a therapeutic effect against some mesothelin expressing tumors, they were not effective in lower doses, possibly due to the presence of soluble mesothelin in the blood as well as in the extracellular space of tumors, which could reduce the efficacy of the therapeutic mAb by competition. Thus, high levels of soluble forms of the antigen represent a barrier to directing therapy to cellular targets. The ability to develop antibodies that can selectively discriminate between membrane -bound and soluble conformations of a specific protein, and thus target only the membrane-associated antigen, is important and necessary.

[0012]

[0005] The GPI-anchored cell surface protein mesothelin (MSLN) shows elevated expression in many malignancies and is an established clinical-stage target for antibody-directed therapeutic strategies. Of these, the harnessing of autologous patient T cells via engineered anti-MSLN chimeric antigen receptors (CAR-T) is an approach garnering interest. Although generally shown to target tumour MSLN safely, CAR-T trials have failed to deliver the impressive curative or response metrics achieved forAtty. Docket No. 2745-9 PCTT

[0013] hematological malignancies using the same technology. A need exists, therefore, for improved anti-MSLN molecules and / or more optimal ways to leverage immune effector cells.

[0014]

[0006] The citation of references herein shall not be construed as an admission that such is prior art to the present invention.

[0015] SUMMARY OF THE INVENTION

[0016]

[0007] In a general aspect, the present invention provides novel mesothelin (MSLN) antibodies and binding agents, derivatives and ligands, directed against human mesothelin, particularly human antibodies and binding agents, derivatives and ligands based thereon. In an aspect, the antibodies, binding agents, derivatives and ligands are specific for human mesothelin. In an aspect, the antibodies, binding agents, derivatives and ligands are specific for human mesothelin and do not recognize or bind native mouse mesothelin. In an aspect the antibodies, binding agents, derivatives and ligands recognize or bind the Dl-D2 region or domain of human mesothelin MSLN. In an aspect, the antibodies, binding agents, derivatives and ligands recognize or bind the DI fragment or region of human mesothelin. In an aspect, recognition and binding to mesothelin MSLN by the antibodies, binding agents, derivatives and ligands requires divalent cations. In an aspect, in the absence of divalent cations, the antibodies, binding agents, derivatives and ligands do not effectively bind mesothelin MSLN.

[0017]

[0008] The invention provides antibodies, binding agents, derivatives and ligands specifically directed against MSLN for diagnostic and therapeutic purposes. In particular, human antibodies, including binding agents and ligands derived therefrom, specific for human MSLN are provided.

[0018]

[0009] The antibodies, binding agents, derivatives and ligands of the present invention have diagnostic and therapeutic use in cancer and in immune modulation, including modulating the immune response to cancer and in cancer vaccines. The antibodies, binding agents, derivatives and ligands of the invention are applicable in characterizing the presence of and in modulating the activity of MSLN, particularly human MSLN, including MSLN expressed on cells, including cancer and tumor cells.

[0019]

[0010] In a further aspect, the present invention provides an antibody or fragment thereof, particularly a human antibody, and binding agents, derivatives and ligands derived thereof which recognizes mesothelin (MSLN), particularly human MSLN (hMSLN), and is selected from antibody (ies), or binding agents, derivatives and ligands derived therefrom, comprising the light chain and heavy chain variable region CDR1, CDR2 and CDR3 sequences as provided herein. In one such aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region CDRs comprising a CDR1 sequence GYTFTGYY (SEQ ID NO:3), a CDR2 sequence INPNSGGT (SEQ ID NO:5), and a CDR3 sequence AREIYSGSHPDDAFDI (SEQ ID NO:7) is provided. In a further such aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region CDRsAtty. Docket No. 2745-9 PCTT

[0020] comprising a CDR1 sequence GYTFTGYY (SEQ ID NO:3) or GYTFTGYYMH (SEQ ID NO:4), a CDR2 sequence INPNSGGT (SEQ ID NO:5) or WINPNSGGTNYAQKFQD (SEQ ID NO:6), and a CDR3 sequence AREIYSGSHPDDAFDI (SEQ ID NO:7) is provided. In some aspects, the VH CDR2 sequence is WINPNSGGTNYAQKFQG (SEQ ID NO:22)

[0021] [Oil] In one aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising light chain variable region CDRs comprising a CDR1 sequence QGISNS (SEQ ID NO:8), a CDR2 sequence GAS (SEQ ID NO: 10), and a CDR3 sequence QQYYSTPHT (SEQ ID NO: 12) is provided. In a further such aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising light chain variable region CDRs comprising a CDR1 sequence QGISNS (SEQ ID NO:8) or RASQGISNSLA (SEQ ID NO:9), a CDR2 sequence GAS (SEQ ID NO: 10) or GASILES (SEQ ID NO: 11), and a CDR3 sequence QQYYSTPHT (SEQ ID NO: 12) is provided.

[0022]

[0012] In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region (VH) corresponding to amino acid sequence SEQ ID NO:1 is provided. In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region (VH) corresponding to amino acid sequence SEQ ID NO:20 is provided. In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region (VH) corresponding to amino acid sequence SEQ ID NO:1 is provided.

[0023]

[0013] In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising light chain variable region (VL or VK) sequence corresponding to amino acid sequence SEQ ID NO: 2 is provided. In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising light chain variable region (VL or VK) sequence corresponding to amino acid sequence SEQ ID NO: 21 is provided.

[0024]

[0014] In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region (VH) corresponding to amino acid sequence SEQ ID NO:1 or SEQ ID NO:20 and light chain variable region (VL or VK) SEQ ID NO:2 or SEQ ID NO:21 is provided. In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region (VH) corresponding to amino acid sequence SEQ ID NO:20 and light chain variable region (VL or VK) SEQ ID NO:21 is provided. In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region (VH) corresponding to amino acid sequence SEQ ID NO:1 and light chain variable region (VL or VK) SEQ ID NO:2 is provided.

[0025]

[0015] In a further aspect, the invention provides an antibody, or binding agents, derivatives and ligands derived thereof, comprising a heavy chain variable region sequence SEQ ID NO: 1 or SEQ ID NO: 20 or a variant thereof having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity to the heavy chain variable region sequence SEQ ID NO: 1 or SEQ ID NO:20, or comprising 1 to 3 aminoAtty. Docket No. 2745-9 PCTT

[0026] acid substitutions in one or more heavy chain CDR region of SEQ ID NO: 3, 4, 5, 6, 7 or 22, wherein said variant retains human MSLN binding and lack of mouse MSLN reactivity. In a further aspect, the invention provides an antibody, or binding agents, derivatives and ligands derived thereof, comprising a heavy chain variable region sequence SEQ ID NO: 1 or a variant thereof having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity to the heavy chain variable region sequence SEQ ID NO: 1. In a further aspect, the invention provides an antibody, or binding agents, derivatives and ligands derived thereof, comprising a heavy chain variable region sequence SEQ ID NO: 20 or a variant thereof having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity to the heavy chain variable region sequence SEQ ID NO:20. In an aspect, such variants retain binding to the D1-D2 region or domain of human mesothelin MSLN, particularly the DI fragment or region of human mesothelin MSLN. In an aspect, such variants retain the requirement for divalent cations to bind MSLN.

[0027]

[0016] In another aspect, the invention provides an antibody, or binding agents, derivatives and ligands derived thereof, comprising a light chain variable region sequence SEQ ID NO: 2 or SEQ ID NO: 21 or a variant thereof having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity to the light chain variable region sequence SEQ ID NO:2 or SEQ ID NO:21, or comprising 1 to 3 amino acid substitutions in one or more light chain CDR region of SEQ ID NO: 8, 9, 10, 11 or 12, wherein said variant retains human MSLN binding and lack of mouse MSLN reactivity. In an aspect, such variants retain binding to the D1-D2 region or domain of human mesothelin MSLN, particularly the DI fragment or region of human mesothelin MSLN. In an aspect, such variants retain the requirement for divalent cations to bind MSLN.

[0028]

[0017] In aspects of the invention variable heavy and light chain VH and VK sequences of antibody 13F08 and variant antibody 13F08g are provided. Specific exemplary VH and VL / VK variable region sequences are provided in parental 13F08 antibody and variant 13F08g antibody.

[0029]

[0018] The parental 13F08 antibody variable region VH is as follows: QVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQDWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSS

[0030] (SEQ ID NO:1)

[0031]

[0019] The parental 13F08 antibody variable region VK is as follows: DIVLTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQNPRKAPRLLVYGASILESGVPSRFSGSGS GTDYTLTITSLQPEDFATYYCQQYYSTPHTFGQGTKVDIK (SEQ ID NO:2)

[0032]

[0020] The variant 13F08g antibody variable region VH is as follows: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSS

[0033] (SEQ ID NO:20)

[0034]

[0021] The variant 13F08g antibody variable region VK is as follows: DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPRLLVYGASILESGVPSRFSGSG SGTDYTLTISSLQPEDFATYYCQQYYSTPHTFGQGTKVEIK (SEQ ID NO:21)Atty. Docket No. 2745-9 PCTT

[0035]

[0022] In additional aspects, antibody derivatives such as single-chain variable fragments (scFv) are provided that bind human MSLN. The scFvs comprise the heavy chain variable region and the light variable region joined by a peptide linker. The scFv may be a VL / VH construct with the light chain (VL or VK) first followed by the heavy chain (VH) variable region, with a linker between them. The scFv may be a VH / VL construct with the heavy chain (VH) first followed by the light chain (VL or VK) variable region, with a linker between them. Suitable linkers are known and available. Exemplary intra-scFv linkers include for example RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO: 13), GQPKARQEGGSGEGGSGESNAAA (SEQ ID NO: 19) and GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25). Exemplary MSLN scFvs are provided herein and include SEQ ID NO:s 14, 23, 26, 27, 15, 28, 29 and 30.

[0036]

[0023] The scFv may be a VL / VH construct with the light chain (VL or VK) first followed by the heavy chain (VH) variable regions. Exemplary such scFv constructs include

[0037] ScFvs utilizing the RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO: 13) intrascFv linker such as: DIVLTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQNPRKAPRLLVYGASILESGVPSRFSGSGS GTDYTLTITSLOPEDFATYYCOQYYSTPHTFGOGTKVDIKRTVAAOARQEGGSGEGGSGESNAA AQVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNY AQKFQDWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSS

[0038] (SEQ ID NO: 14);

[0039] DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPRLLVYGASILESGVPSRFSGSG SGTDYTLTISSLOPEDFATYYCOOYYSTPHTFGOGTKVEIKRTVAAOAROEGGSGEGGSGESNAA AQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNY AQKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSS

[0040] (SEQ ID NO:23).

[0041] ScFvs utilizing the GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25) such as:

[0042] DIVLTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQNPRKAPRLLVYGASILESGVPSRFSGSGS GTDYTLTITSLOPEDFATYYCOOYYSTPHTFGOGTKVDIKGGGSGGGGSGGGGSGGGGSGOVOL VQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ DWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSS (SEQ ID NO:26);

[0043] DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPRLLVYGASILESGVPSRFSGSG SGTDYTLTISSLOPEDFATYYCOOYYSTPHTFGOGTKVEIKGGGSGGGGSGGGGSGGGGSGOVO LVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKF QGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSS (SEQ ID NO:27).

[0044]

[0024] In another embodiment, an alternative scFv construct provided is a VH / VL (also denoted VH / VK) construct with the heavy chain variable region first, a linker, then the light chain variable region. Exemplary such constructs are:

[0045] ScFvs utilizing the RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO: 13) intra-scFv linker such as: QVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQDWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSRT VAAOAROEGGSGEGGSGESNAAADIVLTOSPSSLSASVGDRVTITCRASOGISNSLAWYOQNPRAtty. Docket No. 2745-9 PCTT

[0046] KAPRLLVYGASILESGVPSRFSGSGSGTDYTLTITSLQPEDFATYYCQQYYSTPHTFGQGTKVDIK

[0047] (SEQ ID NO: 15);

[0048] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSRT VAAOAROEGGSGEGGSGESNAAADIOMTOSPSSLSASVGDRVTITCRASOGISNSLAWYOQKPG KAPRLLVYGASILESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYSTPHTFGQGTKVEIK

[0049] (SEQ ID NO:28)

[0050] ScFvs utilizing the GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25) such as:

[0051] QVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQDWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSS GGGSGGGGSGGGGSGGGGSGDIVLTOSPSSLSASVGDRVTITCRASOGISNSLAWYOQNPRKAP RLLVYGASILESGVPSRFSGSGSGTDYTLTITSLQPEDFATYYCQQYYSTPHTFGQGTKVDIK

[0052] (SEQ ID NO:29); and QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSG GGSGGGGSGGGGSGGGGSGDIOMTOSPSSLSASVGDRVTITCRASOGISNSLAWYOQKPGKAPR LLVYGASILESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYSTPHTFGQGTKVEIK (SEQ ID NO:30).

[0053]

[0025] In relevant aspects of the invention, the MSLN antibody sequences, particularly antibodies incorporating the CDR sequences set out or the variable region sequences provided herein, can be incorporated into chimeric antigen receptors (CARs) to produce CAR expressing cells for targeting the ligand human MSLN or MSLN-expressing cells, particularly MSLN-expressing tumor cells or cancer cells. Chimeric antigen receptors (CARs) are hybrid molecules comprising an antigen-targeting moiety, followed by a linker, transmembrane (TM) domain, and an intracellular domain comprising various endodomains (EDs) involved in T-cell activation. In accordance with the invention, CARs or other antigen targeting molecules contemplated herein target MSLN, and particularly wherein the antigen-targeting moiety is the antibody or derivatives and binding agents recognizing MSLN.

[0054]

[0026] Thus, in another aspect, the present invention provides a CAR comprising particularly the MSLN antibody sequence(s) or binding domains of the invention, a transmembrane domain, and an intracellular domain.

[0055]

[0027] In some embodiments, the intracellular domain comprises a primary signalling domain and a costimulatory domain. In an embodiment, the intracellular domain comprises two or more costimulatory domains. In an alternative embodiment, the CAR does not comprise a costimulatory domain.

[0056]

[0028] In some embodiments, the primary signalling domain comprises a CD3 zeta (CD3Q, CD3 gamma (CD3y), CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, or DAP12 primary signalling domain. In some embodiments, the primary signalling domain comprises a CD3 zeta (CD3Q signalling domain.

[0057]

[0029] In some embodiments, the costimulatory domain comprises a CD28, 4-1BB (CD137), MyD88, 0X40, CD27, CD30, CD40, CD134, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR,Atty. Docket No. 2745-9 PCTT

[0058] BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or TNFR2 costimulatory domain. In some embodiments, the costimulatory domain is a CD28 costimulatory domain. In some embodiments, the costimulatory domain is a 41BB costimulatory domain.

[0059]

[0030] In some embodiments, the transmembrane domain comprises a CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, NKG2C, or TNFR2 transmembrane domain, or the alpha, beta or zeta chain of the T-cell receptor. In some embodiments, the transmembrane domain is a CD27 transmembrane domain.

[0060]

[0031] In some embodiments, the antigen-binding domain, particularly the CD27 extracellular domain, is connected to the transmembrane domain by a hinge region. In some embodiments, the hinge region comprises a CD8 hinge, an IgG hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, or a glycine-serine linker. In certain embodiments, the IgG hinge is from IgG 1, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof. In certain embodiments, the hinge domain comprises the CHI, CH2, CH3 and / or hinge domain of the immunoglobulin. In certain embodiments, the hinge domain is a fragment of the immunoglobulin hinge domain.

[0061]

[0032] In some embodiments, the CAR further comprises a leader sequence or a signal sequence. In some embodiments, the leader sequence is a human leader sequence. In some embodiments, the leader sequence is a signal sequence and is a human signal sequence. In some embodiments, the leader sequence is an N-terminal leader sequence. In some embodiments, the leader sequence or signal sequence is an immunoglobulin signal sequence. Other suitable leader sequences or signal sequences will be known by those skilled in the art. In some embodiments, the leader sequence is cleaved from the CAR during cellular processing and localization of the CAR to the cellular membrane when expressed in a cell.Atty. Docket No. 2745-9 PCTT

[0062]

[0033] In some embodiments, the CAR comprises more than one antigen-binding domain. In some embodiments, the CAR comprises the MSLN binding domain or antibody or variable region sequences as provided herein and another distinct antigen-binding domain. Thus, in some embodiments, the CAR is a multi-specific CAR comprising two or more antigen-binding domains, wherein one of the antigen-binding domains comprises a MSLN antibody or binding domain as provided herein. The two or more antigenbinding domains may bind to the same or different targets.

[0063]

[0034] In addition to the CAR construct, the CAR may further comprise an accessory gene that encodes an accessory peptide. Examples of accessory genes can include a transduced host cell selection marker, an in vivo tracking marker, a cytokine, a suicide gene, or some other functional gene. In some embodiments, the accessory gene is a fluorescent protein, such as, but not limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP). Non-limiting examples of classes of accessory genes that can be used to increase the effector function of CAR containing host cells, include i) secretable cytokines (e.g., but not limited to, IE-7, IE-12, IL-15, IL-18), ii) membrane bound cytokines (e.g., but not limited to, IL-15), iii) chimeric cytokine receptors ( e.g., but not limited to, IL-2 / IL-7, IL-4 / IL-7), iv) constitutive active cytokine recep-tors (e.g., but not limited to, C7R), v) dominant negative receptors (DNR; e.g., but not limited to TGFRII DNR), vi) ligands of co stimulatory molecules (e.g., but not limited to, CD80, 4-1BBL), vii) antibodies, including fragments thereof and bispecific antibodies ( e.g., but not limited to, bispecific T-cell engagers (BiTEs)), or vii) a second CAR.

[0064]

[0035] The invention provides bispecific T Cell engagers (BiTEOs) which are particularly directed against MSLN positive and / or MSLN expressing cells, particularly including against cancer cells or tumor cells. The data provided herein represents the first demonstration that a MSLN-targeting BiTE-T reagent can efficiently control endogenous MSLN+tumor cells both in vitro and in vivo. In an aspect, the invention provides BiTEs directed against MSLN positive and / or MSLN expressing cells wherein the BiTEs include or comprise MSLN binding sequences. In an aspect, the invention provides bispecific T Cell engagers (BiTEs) which are based on and include or comprise the MSLN binding sequences, antibodies described herein. A BiTE refers to a single polypeptide chain molecule that has two antigen binding sites, one of which binds to an immune effector cell antigen (such as for example, CD3) and the second of which binds to an antigen present on the surface of a target cell, such as MSLN in this instance. Thus in an embodiment hereof an MSLN BiTE is provided, wherein the MSLN binding portion comprises an antibody, variable region sequence, having CDR binding sequences, which are particularly directed against and specific for MSLN. In an embodiment hereof an MSLN BiTE is provided, wherein the MSLN binding portion comprises the antibody, variable region sequence, and / or CDR binding domain sequences set out herein.Atty. Docket No. 2745-9 PCTT

[0065]

[0036] MSLN BiTEs can be constructed by one skilled in the art using MSLN binding sequences, including scFvs, provided herein, or based on MSLN binding sequences, antibodies, active fragments thereof, domains thereof, that are otherwise available or known in the art.

[0066]

[0037] Exemplary MSLN BiTEs based on the new and unique MSLN binding sequences and scFvs provided herein, particularly 13F08 and variants thereof, include SEQ ID NO:s 17, 24, 32, 33, 34, 35, 36 and 37. Exemplary MSLN BiTEs using exemplary anti-CD3 scFv sequence are provided. One skilled in the art can construct alternative MSLN BiTEs using different scFvs to replace the anti-CD3 scFv sequences.

[0067]

[0038] An exemplary BiTE provided herein is the BiTE construct, denoted 13F08_UCHT1, where UCHT1 is anti CD3 scFv. This BiTE construct is based on the 13F08 scFv (SEQ ID NO: 14), with the intra-scFv linker RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO: 13), an inter-scFv linker GSASASTGGDAS (SEQ ID NO:16), followed by the anti-CD3 scFv UCHT1. The linkers are underlined. The anti-CD3 scFv UCHT1 is shown in bold:

[0068] >13F08_UCHT1[Parental BiTE], where UCHT1 is anti CD3 scFv DIVLTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQNPRKAPRLLVYGASILESGVPSRFSGSGS GTDYTLTITSLOPEDFATYYCOOYYSTPHTFGOGTKVDIKRTVAAOARQEGGSGEGGSGESNAA AQVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNY AQKFQDWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSS GSASASTGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYY TSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGQPKA ROEGGSGEGGSGESNAAAEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYTMNWVROAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGY YGDSDWYFDVWGQGTLVTVSS (SEQ ID NO: 17)

[0069]

[0039] Another exemplary BiTE in an aspect of the invention is provided below and is based on a variant heavy and light chain VH and VK sequence. This BiTE construct is based on the variant 13F08 scFv, with the intra-scFv linker RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO: 13), an inter-scFv linker GSASASTGGDAS (SEQ ID NO:16), followed by the anti-CD3 scFv UCHT1 (SEQ ID NO:18), including the intra-scFv linker GQPKARQEGGSGEGGSGESNAAA (SEQ ID NO: 19). The linkers are underlined. The anti-CD3 scFv UCHT1 is shown in bold.

[0070] >13F08g[germ_line]_UCHTl[BiTE] DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPRLLVYGASILESGVPSRFSGSG SGTDYTLTISSLOPEDFATYYCOOYYSTPHTFGOGTKVEIKRTVAAOARQEGGSGEGGSGESNAA AQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNY AQKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSS GSASASTGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYY TSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGQPKA RQEGGSGEGGSGESNAAAEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYTMNWVROAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGY YGDSDWYFDVWGQGTLVTVSS (SEQ ID NO 24)

[0071]

[0040] Other exemplary BiTEs include those with the intra-scFV linker GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25):

[0072] >13F08_UCHT1[Parental] where UCHT1 is anti CD3 scFvAtty. Docket No. 2745-9 PCTT

[0073] DIVLTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQNPRKAPRLLVYGASILESGVPSRFSGSGS GTDYTLTITSLOPEDFATYYCOOYYSTPHTFGOGTKVDIKGGGSGGGGSGGGGSGGGGSGOVOL VQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ DWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSGSASAS TGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLES GVPSRFSGSGSGTDYTLTISSLOPEDFATYYCOOGNTLPWTFGOGTKVEIKGOPKARQEGG SGEGGSGESNAAAEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYTMNWVROAPGKGLE WVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSD WYFDVWGQGTLVTVSS (SEQ ID NO 32)

[0074] >13F08g[variant]_UCHTl[BiTE] where UCHT1 is anti CD3 scFv DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPRLLVYGASILESGVPSRFSGSG SGTDYTLTISSLOPEDFATYYCOOYYSTPHTFGOGTKVEIKGGGSGGGGSGGGGSGGGGSGOVO LVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKF QGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSGSASA STGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLE SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCOOGNTLPWTFGOGTKVEIKGQPKARQEG GSGEGGSGESNAAAEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYTMNWVROAPGKGLE WVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSD WYFDVWGQGTLVTVSS (SEQ ID NO 33)

[0075]

[0041] Alternative exemplary MSLN BiTEs based on a VH / VK MSLN scFv sequence, with exemplary anti-CD3 sequence include:

[0076] >13F08 parental VH / VK scFv and intra-scFv linker RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO:13) QVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQDWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSRT VAAOAROEGGSGEGGSGESNAAADIVLTOSPSSLSASVGDRVTITCRASOGISNSLAWYOQNPR KAPRLLVYGASILESGVPSRFSGSGSGTDYTLTITSLQPEDFATYYCQQYYSTPHTFGQGTKVDIK GSASASTGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYY TSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGQPKA RQEGGSGEGGSGESNAAAEVOLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVROAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGY YGDSDWYFDVWGQGTLVTVSS (SEQ ID NO 34)

[0077] >13F08 variant VH / VK scFv and intra-scFv linker RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO:13) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSRT VAAQARQEGGSGEGGSGESNAAADIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPG KAPRLLVYGASILESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYSTPHTFGQGTKVEIK GSASASTGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYY TSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGQPKA RQEGGSGEGGSGESNAAAEVOLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVROAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGY YGDSDWYFDVWGQGTLVTVSS (SEQ ID NO 35)

[0078] >13F08 parental VH / VK scFv and intra-scFV linker GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25):Atty. Docket No. 2745-9 PCTT

[0079] QVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQDWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSG GGSGGGGSGGGGSGGGGSGDIVLTOSPSSLSASVGDRVTITCRASOGISNSLAWYOQNPRKAPRL LVYGASILESGVPSRFSGSGSGTDYTLTITSLQPEDFATYYCQQYYSTPHTFGQGTKVDIKGSASA STGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLE SGVPSRFSGSGSGTDYTLTISSLOPEDFATYYCOOGNTLPWTFGOGTKVEIKGOPKARQEG GSGEGGSGESNAAAEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYTMNWVROAPGKGLE WVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSD WYFDVWGQGTLVTVSS (SEQ ID NO 36)

[0080] >13F08 variant VH / VK scFv and intra-scFV linker GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25): QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSG GGSGGGGSGGGGSGGGGSGDIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPR LLVYGASILESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYSTPHTFGQGTKVEIKGSAS ASTGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRL ESGVPSRFSGSGSGTDYTLTISSLOPEDFATYYCOOGNTLPWTFGOGTKVEIKGOPKARQE GGSGEGGSGESNAAAEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYTMNWVROAPGKG LEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGD SDWYFDVWGQGTLVTVSS (SEQ ID NO 37)

[0081]

[0042] In an aspect, the invention provides cells, particularly T cells, engineered to secrete or express an anti-MSLN+ BiTE. In one aspect, the cells, particularly T cells, are engineered to secrete or express an anti-MSLN+ BiTE comprising the MSLN binding sequences described herein, including the exemplified MSLN antibodies or derivatives provided.

[0082]

[0043] In another aspect, the invention provides cells expressing a BiTE of the invention. In an aspect, the invention provides cells expressing an MSLN-directed BiTE. The cells can particularly be T cells. In an aspect the BiTE expressing cells, particularly T cells, engage, bind and / or target MSLN expressing cells, including tumor, cancer etc cells. In an aspect of the invention, MSLN-directed BiTE expressing cells stimulate or otherwise direct the killing bystander cells, including those of a tumor or cancer.

[0083]

[0044] In another aspect, the present invention provides a nucleic acid encoding an MSLN antibody, binding agent or antibody sequence, or derivative or ligand, of the invention.

[0084]

[0045] In an embodiment, the nucleic acid comprises a nucleotide sequence which is capable of encoding a MSLN binding polypeptide or antibody as set out in any of SEQ ID NOs:l, 2, 20 or 21 or a polypeptide at least about 90%, or at least about 95% identical to any one of SEQ ID NOs:l, 2, 20 or 21. In an embodiment, the nucleic acid comprises a nucleotide sequence which is capable of encoding a MSLN binding polypeptide or antibody comprising variable region sequences comprising heavy and light chain CDR sequences as set out herein, including for example heavy chain CDR sequences SEQ ID NO:s 3 or 4, 5 or 6 or 22 and 7, including for example light chain CDR sequences SEQ ID NO:s 8 or 9, 10 or 11, and 12. Nucleic acid encoding the ScFvs, the CARs or the BiTEs as set out and disclosed herein is also provided.Atty. Docket No. 2745-9 PCTT

[0085]

[0046] In another aspect, the present invention provides a vector comprising the nucleic acid of the invention. In an embodiment, the vector is a DNA vector, an RNA vector, a plasmid, a lentivirus vector, adenovirus or adeno-associated virus vector, or a retrovirus vector. In some embodiments, the nucleic acid of the invention is operably linked to a promoter in the vector.

[0086]

[0047] In another aspect, the present invention provides a cell comprising the antibody, binding protein, binding agent, derivative or ligand of the invention, the nucleic acid of the invention, or the vector of the invention. In a similar aspect, the present invention provides a cell expressing the antibody, binding protein, binding agent, derivative or ligand, the scFv, the CAR, or the BiTE of the invention.

[0087]

[0048] In some embodiments, the cell is an immune effector cell. In some embodiments, the immune effector cell is a T cell or an NK cell. In some embodiments, the immune effector cell is a CD8+ T cell. In some embodiments, the immune effector cell is a CD4+ T cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0088]

[0049] In one embodiment, the cell described herein can further comprise a second (or more) CAR or BiTE, e.g., a second CAR or BiTE that includes a different antigen-binding domain, e.g., to the same target (i.e., MSLN) or a different target, or an additional secreted or membrane-anchored protein. In one embodiment, the second CAR or BiTE includes an antigen-binding domain which binds to a target expressed on the same cancer cell type as the target of the first CAR or BiTE. In one embodiment, the additional secreted protein is a chemokine, cytokine, T cell proliferative factor, or natural ligand. In a further embodiment, the additional protein is a membrane-anchored, truncated receptor or ligand. In one such embodiment, the additional protein is EGFR, and may be a truncated EGFR.

[0089]

[0050] In another aspect, the present invention provides a composition comprising the nucleic acid of the invention, the vector of the invention, or the cell of the invention and a pharmaceutically acceptable carrier.

[0090]

[0051] In another aspect, the present invention provides a method of making an scFv-expressing cell, comprising introducing the nucleic acid of the invention or the vector of the invention, into a cell, under conditions such that the scFv is expressed. In certain embodiments, the method further comprises expanding the population of cells after the nucleic acid molecule encoding a scFv has been introduced. In another aspect, the present invention provides a method of making a CAR-expressing cell, comprising introducing the nucleic acid of the invention or the vector of the invention, into a cell, under conditions such that the CAR is expressed. In certain embodiments, the method further comprises expanding the population of cells after the nucleic acid molecule encoding a CAR has been introduced. In another aspect, the present invention provides a method of making a BiTE-expressing cell, comprising introducing the nucleic acid of the invention or the vector of the invention, into a cell, under conditions such that the BiTE is expressed. In certain embodiments, the method further comprises expanding the population of cells after the nucleic acid molecule encoding a BiTE has been introduced.Atty. Docket No. 2745-9 PCTT

[0091]

[0052] In another aspect, the present invention provides a method of treating a subject having a cancer associated with expression of MSLN, the method comprising administering to the subject the binding protein, antibody, binding agent, derivative or ligand of the invention, the composition of the invention, the scFv of the invention, the CAR of the invention, the BiTE of the invention, or the cell of the invention.

[0092]

[0053] In a related aspect, the present invention provides the binding protein of the invention, particularly the MSLN antibody, binding protein or derivative, the composition of the invention, the scFv of the invention, the CAR of the invention, the BiTE of the invention, or the cell of the invention, for use in the treatment of a cancer associated with expression of MSLN. In a related aspect, the present invention provides the binding protein of the invention, particularly the MSLN antibody, binding protein or derivative, the composition of the invention, the scFv of the invention, the CAR of the invention, the BiTE of the invention, or the cell of the invention, for use in the treatment of a disease associated with altered expression or overexpression of MSLN.

[0093]

[0054] In another related aspect, the present invention provides use of the binding protein of the invention, particularly the MSLN antibody, binding protein or derivative, the composition of the invention, the scFv of the invention, the CAR of the invention, the BiTE of the invention, or the cell of the invention in the manufacture of a medicament for the treatment of a cancer associated with expression of MSLN. In another related aspect, the present invention provides use of the binding protein of the invention, particularly the MSLN antibody, binding protein or derivative, the composition of the invention, the scFv of the invention, the CAR of the invention, the BiTE of the invention, or the cell of the invention in the manufacture of a medicament for the treatment of a cancer associated with altered expression or overexpression of MSLN

[0055] In another aspect, the present invention provides use of the binding protein of the invention, particularly the MSLN antibody, binding protein or derivative, the composition of the invention, the scFv of the invention, the CAR of the invention, the BiTE of the invention, or the cell of the invention in the manufacture of a medicament for the treatment of cancer. In another aspect, the present invention provides use of the binding protein of the invention, particularly the MSLN antibody, binding protein or derivative, the composition of the invention, the scFv of the invention, the CAR of the invention, the BiTE of the invention, or the cell of the invention in the manufacture of a medicament for the treatment of solid tumors.

[0094]

[0056] Examples of cancers that express MSLN include, without limitation, mesotheliomas, stomach cancer, squamous cell carcinomas, prostate cancer, pancreatic cancer, lung cancer, breast cancer (particularly triple-negative cancer), ovarian cancer, and cervical cancer. In an embodiment, the cancer is mesothelioma. In an embodiment, the cancer is ovarian cancer. In an embodiment, the cancer is pancreatic cancer. In an embodiment, the cancer is leukemia. In an embodiment, the cancer is acute myeloid leukemia (AML). In an embodiment, the cancer is pediatric AML. In an embodiment, the subject is a child with AML.Atty. Docket No. 2745-9 PCTT

[0095]

[0057] In some embodiments, a population of cells of the invention are administered. In some embodiments, the cells administered to the subject are allogenic cells or autologous cells. In some embodiments, the cells lack or have low expression of a functional T cell receptor (TCR) or a functional human leukocyte antigen (HLA).

[0096]

[0058] In some embodiments, the treatment comprises administering an agent that increases the efficacy of the cells. In some embodiments, the agent is a protein phosphatase inhibitor, a kinase inhibitor, a cytokine, an inhibitor of an immune inhibitory molecule, or an agent that decreases the level or activity of T regulatory cells.

[0097]

[0059] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In other embodiments, the subject is a non-human mammal.

[0098]

[0060] In another aspect, the present invention provides a method of detecting a cell expressing MSLN, the method comprising contacting the binding protein mutant, antibody, scFv, CAR or BiTE of the invention with a sample comprising the cell and detecting binding between the binding protein and MSLN.

[0099]

[0061] The diagnostic utility of the present invention extends to the use of the MSLN binding peptides, antibodies and / or derivatives of the present invention in assays to characterize tumors or cellular samples or to screen for tumors or cancer, including in vitro and in vivo diagnostic assays. Peptides or polypeptides of the invention may carry a detectable or functional label. They may carry a radioactive label, such as the isotopes3H,14C,32P,35S,36C1,51Cr,57Co,58Co,59Fe,90Y,121I,124I,125I,131I,111In,117Lu,211At,198Au,67Cu,225Ac,213Bi,99Tc and186Re. In an aspect, the label may be an enzyme, including wherein detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.

[0100]

[0062] Conjugates or fusion proteins of the present invention, wherein MSLN binding peptides, antibodies and / or derivatives of the present invention are conjugated or attached to other molecules or agents further include, but are not limited to binding members conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.

[0101]

[0063] The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of, for instance, MSLN. The system or test kit may comprise a labeled component prepared by one of the radioactive and / or enzymatic techniques discussed herein, coupling a label to the MSLN binding peptides, antibodies and / or derivatives of the present invention, and one or more additional immunochemical reagents, at least one of which is a free or immobilized component to be determined or their binding partner(s).

[0102]

[0064] In an aspect of the invention MSLN binding peptides, antibodies and / or derivatives of the present invention as provided herein, may be administered in conjunction with or in a composition of cancer antigen(s) and adjuvant(s), including to patients to promote a more robust priming and activation of theAtty. Docket No. 2745-9 PCTT

[0103] adaptive anti-tumor response to enhance immune therapies directed at cancers. Additional inhibitors to MSLN activity, such as small molecules, antisense or aptamers can also be used to inhibit MSLN activity.

[0104]

[0065] Thus, in an aspect of the invention the MSLN binding peptides, antibodies and / or derivatives of the present invention may be administered alone or in combination with other treatments, therapeutics or agents or cell therapies, either simultaneously or sequentially dependent upon the condition to be treated. Immune modulators may be included in a composition with or administered with MSLN binding peptides, antibodies and / or derivatives of the present invention and / or administered at a different time to enhance immune modulation and / or cancer therapy, including immune therapies or cell therapies directed against cancer. An immune modulator may be an adjuvant. In a further aspect, the MSLN binding peptides, antibodies and / or derivatives of the present invention can also be used as immunostimulant(s) or adjuvant(s) in combined use with antigenic materials such as, without limitation, proteins, peptides, or nucleic acids and so forth in order to produce a protective immune response, such as a T-cell or CTL response to the administered antigen.

[0105]

[0066] The invention provides a method for improving, facilitating or enhancing chimeric antigen receptor (CAR) T cell therapy or BiTE therapy comprising administering one or more MSLN binding peptides, antibodies and / or derivatives of the present invention either simultaneously or sequentially with the CAR T cell(s). In another method of the invention, MSLN binding peptides, antibodies and / or derivatives of the present invention are administered in combination with activated T cells or T cells directed against a cancer antigen or cell cycle regulator.

[0106]

[0067] Pharmaceutical compositions or immunogenic compositions of the invention may further comprise additional antibodies or therapeutic agents. In an aspect, such other agents or therapeutics may be selected from anti-cancer agents or therapeutics, anti-mitotic agents, apoptotic agents or antibodies, or immune modulators, or small molecule inhibitors to immune modulators. More generally these anti-cancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti -mitotic s), inhibitors or signal transduction inhibitors. The composition may be administered with an immune modulator such as an adjuvant. The composition may also be administered with, or may include combinations along with other anti-TGF[3 antibodies, other immunomodulatory antibodies or other anti-tumor antigen antibodies.

[0107]

[0068] The radiolabelled specific binding members, particularly antibodies and fragments and / or derivatives thereof, are useful in in vitro diagnostics techniques and in in vivo radioimaging techniques and in radioimmunotherapy (RIT or RAIT). In the instance of in vivo imaging, the antibodies, fragments, and / or derivatives of the present invention may be conjugated to a radioisotope or to an imaging agent rather than a radioisotope(s), including but not limited to a magnetic resonance image enhancing agent, wherein for instance an antibody molecule is loaded with a large number of paramagnetic ions throughAtty. Docket No. 2745-9 PCTT

[0108] chelating groups. Examples of chelating groups include EDTA, porphyrins, polyamines crown ethers and polyoximes. Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanium, holmium and ferbium. In a further aspect of the invention, radiolabelled antibodies, fragments and / or derivatives targeting MSLN, particularly radioimmunoconjugates, are useful in radioimmunotherapy, particularly as radiolabelled antibodies for cancer therapy, including to kill cancer cells. In a still further aspect, the radiolabelled specific binding members, particularly antibodies fragments and / or derivatives thereof, are useful in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and / or location of cancer cells, precancerous cells, tumor cells, and hyperproliferative cells, prior to, during or following surgery to remove such cells. The combination of radioimaging (such as via PET imaging or immunoPET — where an antibody (Ab), fragment or derivative is labeled with an isotope for radioimaging — and radioimmunotherapy, using the same antibody or antibody derivative with a therapeutic isotope, for example can provide significant advantages in cancer management.

[0109]

[0069] The diagnostic utility of the present invention extends to the use of the antibodies of the present invention in assays to characterize tumors or cellular samples or to screen for tumors or cancer, including in vitro and in vivo diagnostic assays. Further, the antibodies of the invention may be used in radioimmunotherapy, by labelling the antibodies, or fragments thereof, with radionuclides intended for therapeutic use. Labelled antibodies or immunoconjugates or antibody fusion proteins are provided wherein the specific binding members, particularly antibody / ies fragments and / or derivatives thereof, of the present invention are conjugated or attached to one or more label or tag. Examples of detectable and / or therapeutic radiolabels are the isotopes3H,14C,32P,35S,36C1,51Cr,57Co,58Co,59Fe,86Y,90Y,121I,124I,125I,131I,111In,117Lu,177Lu,211At,198Au,64Cu,67Cu,225Ac,213Bi,99Tc,186Re,188Re,89Zr,68Ga and18F (Garaulet et al (2024) Cancers 16(6):2896). In an aspect, the label or tag may be an enzyme, including wherein detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art. The label or tag may be a detectable label bound for example by another specific antibody or may be a label which is a fluroresent molecule or protein, such as a GFP label or a His tag or a myc tag.

[0110]

[0070] Immunoconjugates or antibody fusion proteins of the present invention are aspects provided herein, wherein the specific binding members, particularly antibody / ies fragments and / or derivatives thereof, of the present invention are conjugated or attached to other molecules or agents further include, but are not limited to binding members conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.Atty. Docket No. 2745-9 PCTT

[0111]

[0071] Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

[0112] BRIEF DESCRIPTION OF THE DRAWINGS

[0113]

[0072] Figure 1: Evaluation of 13F08 binding to MSLN under typical assay conditions, (a-d) Kinetic biolayer interferometry sensorgrams for purified 13F08-BiTE and SSl-BiTE binding to immobilized biotinylated hMSLN extracellular region (aa: 296-580). Buffer system: PBS / 0.01% Tween-20 / 0.05 % BSA. (e) Titration ELISA showing the binding of bivalent 13F08-Fc to biotinylated human (e) MSLN relative to representative CAR-active anti-MSLN scFv-Fc comparators, (f) 13F08-Fc does not recognize murine MSLN. Two separate batches of 13F08-Fc are shown in (e) and (f). A murine-MSLN positive mAb (a kind gift from Acrobiosystems) was included as an assay control in (f). Buffer system: PBS / 0.1% Tween-20 / 2% BSA. Data (n=3 technical triplicates) are plotted as mean + / - SEM.

[0114]

[0073] Figure 2: (A) Logarithmic FACS staining of MSLN+and negative endogenous cells lines. Staining was performed using 2 µg / mL for both mAb K1 (upper panels; isotype Ctrl at matched concentration) and the individual Fc-fusions middle panels; Ctrl-Fc was anti-TEMl which stains A673 TEMl+ / MSLN-neg control cells). CD19 staining {lower panels) was performed to assess the expression of CD19 on the indicated cell lines; Raji were used as a positive control for CD19 staining. All data normalized to mode. (B) Median Fluorescence Intensity (MFI) FACS profiling of K1 mAb (left), and 13F08-Fc and comparators (right; all at 2 µg / mL) versus MSLN+and negative control tumor cell lines. Data derived using FACS buffer comprising PBS / 2% FBS. (C) Correlation plot of MSLN expression detected by K1 mAb versus 13F08-Fc binding (fold MFI increase over Ctrl) in different cell lines. Data plotted are the means + / - SEM from n=2-4 independent experiments.

[0115]

[0074] Figure 3: [Relates to Fig. 2]. Western blot detection of FL native hMSLN from various MSLN+endogenous, transfected, and negative cell line extracts.

[0116]

[0075] Figure 4: Western blot detection by 13F08-Fc of specific truncated hMSLN fragments and domains from transiently transfected HEK cell extracts, (a) Schematic of bi-directional expression cassette and MSEN truncation variants expressed in HEK293-6E cells, (b) Western blot performed on whole cell extracts. MSLN#2, major transcript (ISO#2); MSLN#1, minor transcript (ISO#1) with aa insertion (QAPRRPEP) in D2. JM / GPI, juxta-membrane GPI-anchor region recognized by 15B6-Fc (employed as an expression control); the transfection control was provided by GFP on the vector.

[0117]

[0076] Figure 5: Immunoprecipitation and immunoblotting of FE-hMSLN captured from transfected HEK293-6E whole cell extracts by 13F08-Fc and comparators. NT, non-transfected HEKs. The previouslyAtty. Docket No. 2745-9 PCTT

[0118] described anti-MSLN P4 scFv-Fc (Lanitis et. Mol Ther. 2012;20(3):633-43) was arbitrarily chosen as the detection reagent.

[0119]

[0077] Figure 6: FACS titration profiling of MSLN-transfected HEK cells stained with 13F08-Fc and anti-MSLN comparators in the presence of buffer containing 2.5 mM EDTA. (a) Staining of HEK cells transfected with full length (FL)-MSLN (ISO#2, major transcript variant) as a function of scFv-Fc concentration, (b) FACS staining summary of 13F08-Fc and an extended panel of comparator Fc-fusions versus HEK293-6E transfected with various truncated and chimeric MSLN fragments (huD2, huDl-D2, and huD2-D3 contain the ISO#1 D2 insertion as per Suppl. Fig. S3). All Fc-fusions at 0.1 pg / mL; 15B6 recognizes a juxta-membrane (JM) linear epitope and serves as an expression control; MFI: ++, 103-104; +++, 104-105; ++++, 105-106.

[0120]

[0078] Figure 7: Impact of divalent cations on the recognition of MSLN by 13F08. (A) Titration ELISA demonstrating the behavior of bivalent 13F08- and SSl-Fc against biotinylated recombinant hMSLN in the presence of exogenously added metals or FBS. (B) Titration ELISA data showing the binding of purified monovalent 13F08-BiTE to biotinylated human MSLN relative to the representative SSl-BiTE comparator when excess metal cations are available (1mM CaCl2and 2% FBS) or sequestered (2% FBS + 2.5mM EDTA). Data are shown as the mean of n=3 technical replicates + / - SEM. (C) Kinetic biolayer interferometry sensorgrams for purified 13F08-BiTE and SSl-BiTE binding to immobilized biotinylated human MSLN in the presence or absence of CaCF.

[0121]

[0079] Figure 8: Evaluation of 13F08 binding to human MSLN in the presence of metal cations, (a) Titration ELIS As showing the binding of 13F08-Fc and representative anti-MSLN comparators to biotinylated human MSLN when excess metal cations are available (1 mM CaCl2or 2% FBS) or sequestered (2% FBS + 2.5 mM EDTA). Data presented as n=3 technical triplicates + / - SEM. (b) Titration FACS profiling of HEK293-6E cells transiently transfected with FL-MSLN (upper panels) and MSLN+OVCAR8 tumor cells (lower panels) and stained with 13F08-Fc and comparators + / - EDTA included in the FACS buffer (PBS / 2% FBS).

[0122]

[0080] Figure 9: Summary data for the impact of EDTA inclusion or omission on the FACS staining of various transfected MSLN constructs (h, human; m, murine). 13F08-Fc and SSl-Fc at 0.1 ug / mL; MFI: ++, 103-104; +++, 104-105; ++++, 105-106. NT, non-transfected.

[0123]

[0081] Figure 10: Evaluation of purified soluble BiTEs for activity in co-cultures of primary human T cells and target tumor cell lines. (A) Representative IncuCyte screening assays showing kinetic killing profiles for three endogenous MSLN+tumor lines obtained with a panel of purified BiTEs, including 13F08 (upper panels), with associated secretion of IFNy (lower panels). (B) Evaluation of 13F08-BiTE alongside other anti-MSLN scFvs derived from established CARs and formatted as BiTEs (data shown are from donor-matched T cells and are representative of n=4 T cell donors). Data curves and shading indicate meanAtty. Docket No. 2745-9 PCTT

[0124] + / - SEM respectively (n=3 technical replicates); BiTEs were used at a final concentration of 4 nM with an E: T ratio of 2:1 for both (A) and (B); A375 was included as a MSLN-negative control line.

[0125]

[0082] Figure 11: Jurkat NFAT-luciferase reporter cells activated in the presence of purified 13F08 BiTE and MSLN-transfected HEK293-6E cells (E: T ratio 1:1; left), or endogenous MSLN+OVCAR8 cells right. Data presented as n=3 technical triplicates + / - SEM.

[0126]

[0083] Figure 12: In vitro activity of anti-MSLN 13F084-lBBz CAR-transduced T cells towards various target cell lines, (a) Schematic of the 2ndgeneration CAR construct (left) together with relative CAR expression levels determined by FACS following transduction of primary T cells (right), (b) Killing of MSLN+endogenous cell lines by CAR-transduced T cells as determined by IncuCyte assay (upper panels) with associated IFNy secretion (lower panels). A375 cells were included as a MSLN-negative control; effector-to-target (E: T) ratio of 2:1. Representative data are shown (donor-matched T cells). For IFN|3 data points represent triplicates (n=3 biologically independent wells; mean + / - SEM is depicted). Experiments were repeated independently with T cells from at least n=3 different donors with similar results.

[0127]

[0084] Figure 13: Target cell killing mediated by 13F08 BiTE-T secretor effector cells. (A) Schematic illustrating secretory retroviral BiTE constructs (left) with transduction efficiencies determined by FACS (right). (B) IncuCyte kinetic cytotoxicity co-culture assays showing specific and potent killing by 13F08 BiTE-T cells of MSLN+tumor lines whilst sparing the MSLN-negative control lines (A673 and OVCAR5). (C) Heat maps depicting profiles and relative magnitudes of upregulated activation markers and effector cytokines for the co-cultures in (B). Data depicted as fold-difference relative to the CD19 BiTE-T controls after 24 h of co-culture. Assays conducted at an effector-to-target (E: T) ratio of 2: 1. (D) Correlation plot of CD69 expression versus KI mAb FACS staining in different cell lines. Data in (B-D) obtained with donor-matched T cells. Representative data shown. Experiments were repeated with T cells from at least n=3 donors with similar results.

[0128]

[0085] Figure 14: Confirmation of bi-specific target recognition by 13F08 BiTE secreted by primary T cells, (a) Co-localization of secreted FLAG-tagged 13F08 BiTE with T cell CD3s as determined by dual immunofluorescence ImageStream analysis, (b) Staining of MSLN+OVCAR8 tumor cells by 13F08 secreted BiTE.

[0129]

[0086] Figure 15: Engineering with 13F08 BiTE does not lead to an altered cell state or phenotype of the producer BiTE-T cells, (a) Representative Tcmphenotypic marker FACS plots (left). Phenotype illustrated with regard to CD45RO and CCR7 (routinely used to assess T cell state) for n=5 donors (right), (b) Cytokine secretion (assay detection thresholds are indicated as dotted horizontal lines), (c) Tonic activation markers post overnight culture (16 h) of the indicated T cells (40,000 cells seeded in 200 mL of fresh media in a 96-well plate) in the absence of target cells.Atty. Docket No. 2745-9 PCTT

[0130]

[0087] Figure 16: [Relates to Fig. 13]. Correlation of MSLN expression on target cell lines with the induction of 13F08 BiTE-T cell surface activation markers (pink) and secreted cytokines (blue) following 24 h of co-culture. MSLN levels were determined by staining with the KI reference mAb (data from Main, Fig. 1); CD25 and 4-1BB levels were assessed by commercially available FACS antibodies; GM-CSF, IFNy, IL-2, GrzB and TNF secreted to the medium were assessed by CBA (See Methods).

[0131]

[0088] Figure 17: 13F08 BiTE-T can potently prime functional killing of cognate MSLN+target cells by non-transduced (NT) bystander T cells. (A) Schematic illustration of potential mechanisms of bystander T cell recruitment by secreted BiTE. (B) Secreted 13F08- and CD19-BiTEs in BiTE-T cell media can stain CD3s on NT T cells. BiTE constructs as illustrated in Fig. 13A. (C) NT T cell redirected killing of cocultured MSLN+tumor lines by a single addition of 13F08 BiTE-containing culture media supernatant from engineered T cells. A673 cells were used as a MSLN-negative control. (D) Heat map depicting effector cytokine upregulation (post-24 h of culture) of 13F08 BiTE-activated NT cells in the presence of MSLN+tumor lines versus the A673 MSLN-negative control. (E) IncuCyte cytotoxicity co-culture titration assay. Engineered BiTE-T (>75 % transduction efficiency) are combined at the indicated percentage with NT donor-matched T cells (to give a fixed cell count of 100 % total cells). Total T cell:target (OVCAR8) ratio in (C-E) maintained at 2:1 for all conditions. Data curves and shading indicate mean + / - SEM respectively (n=3 technical triplicates). Experiments in (B-E) were repeated independently at least twice with T cells from at least n=2 donors. Representative data are shown.

[0132]

[0089] Figure 18: In vitro functional comparison of 13F08 BiTE-T and CAR-transduced primary human T cells, (a) T cell transduction efficiencies of the 13F08 and CD19 (control) CARs. (b) Kinetic IncuCyte co-culture killing assays for MSLN-negative A673 and MSLN+OVCAR8, H226, and AsPC-1 tumor cell lines, (c) cytokine release heatmap profiles measured at 24 h post co-culture. Data curves and shading indicate mean + / - SEM respectively (n=3). Effector-to-target (E: T) ratio of 1:1. Representative data shown (with donor-matched T cells). Experiments repeated independently at least three times with T cells from at least n=3 donors with similar results.

[0133]

[0090] Figure 19: 13F08 BiTE-T bystander cell killing. (A) Schematic illustration of triple co-culture assay set up. (B) Bystander impact on MSLN-negative cells (A673, A375, HEK293T) by 13F08 BiTE-T cells activated specifically in the presence of cognate MSLN+OVCAR8. OVCAR8 and MSLN-negative tumor cells were differentially labelled for individual fate tracking as shown in (A) and combined according to the proportions indicated in parentheses to correct for their relative in vitro proliferation rates. Data curves and shading indicate mean + / - SEM respectively (n=3 technical triplicates). Experiments were repeated independently at least twice with T cells from at least n=2 donors. Representative data are shown. Assays conducted at an effector-to-target (E: T) ratio of 2:1.Atty. Docket No. 2745-9 PCTT

[0134]

[0091] Figure 20: Resistance of 13F08 BiTE-T to shed and exogenous MSLN decoy fragments, (a) IncuCyte co-culture assay showing A673 insensitivity to 13F08 BiTE-T effector cells in the presence of conditioned OVCAR8 supernatant or exogenous rhMSLN. (b) 13F08 BiTE-T mediated IncuCyte killing of MSLN+OVCAR8 target cells is not inhibited by either shed MSLN from concentrated OVCAR8 spent culture media, or exogenous recombinant purified human MSLN. Concentrated A673 medium included as a negative control. Data curves and shading indicate mean + / - SEM respectively (n=3 technical triplicates). Experiments were repeated independently twice with T cells from n=2 donors with similar results; plots shown with donor-matched T cells. Assays conducted at an effector-to-target (E: T) ratio of 2:1.

[0135]

[0092] Figure 21: In vivo anti-tumor efficacy of adoptively transferred 13F08 BiTE-T cells in NSG mice. (A) Schematic of experimental format for the OVCAR8 xenograft model left) with comparative tumor progression curves in mice treated with two high doses (27x106per dose) of ACT product (right). Significance test: 2-way ANOVA with Sidak correction, curve analysis post ACT1 until d43 (n=6 per group); p=0.0002:***. (B) Ex vivo profiling of pre-clearance residual OVCAR8 tumors (sampled when ~22mm3) from (A) in a replicate experiment (n=7 per group). Tumor Infiltrating ACT T cell count in tumors from mice with the indicated treatment and their expression of Ki67 and 4- IBB proliferation and activation markers, respectively, determined by FACS analysis (data point drop-outs were due to complete tumor clearance at the time of sampling, hence no material available) ACT T cell count in peripheral blood of mice treated with the indicated treatment (per 100 µL; data point drop-outs were due to sample coagulation). Significance test: 2-tailed Mann Whitney; ns, not significant; p<0.05:*; p<0.01:**; p<0.001:***. (C) Progression curves for H226 (n=5 per group), and SKOV3 (n=6 per group) tumor xenografts. Significance tests: 2-way ANOVA with Sidak correction; p<0.01:**; p<0.001:***. (D) Tumor progression curves for an OVCAR8 xenograft model treated with a reduced total ACT cell dose (n=10 per group, left). Significance test: Repeated measures ANOVA with Sidak correction; p<0.0001:****. ACT T cell count in peripheral blood from mice treated as indicated (right). Blood was analysed (per 100 µL) on d29 (~1 week post-tumor clearance in the 13F08 BiTE-T group). Significance test: unpaired, two-tailed t-test; ns, not significant. (E) Tumor progression curves for an AsPC-1 xenograft model (n=13-14 per group) treated with a single reduced ACT dose (left). Significance test: 2-way repeated measures ANOVA with Sidak correction; p<0.0001:****. Ex vivo profiling of T cell-infiltrated tumors at d30 (right). Tumor-infiltrating T cell count and CD25 late activation marker expression on ACT T cells determined by FACS. ACT T cell count in peripheral blood (per 100 µL) from mice that have received the indicated treatment. Significance tests: unpaired, two-tailed t-test. Data are presented as mean + / - SEM; ACT, adoptive T cell transfer; CD19 BiTE-T as irrelevant control. Corresponding animal weight curves are shown in Fig. 22.

[0136]

[0093] Figure 22: BiTE-T ACT-treated mice weights.Atty. Docket No. 2745-9 PCTT

[0137]

[0094] Figure 23: [Relates to Fig. 21] In vivo challenge of MSLN-negative / low OVCAR5 xenograft tumors with 13F08 BiTE-T cells (NSG setting). Significance testing: mixed effects model (REML) with Sidak correction (n=10); P=0.69 (ns, not significant). Data are presented as mean + / - SEM; ACT, adoptive T cell transfer; CD19 BiTE-T as irrelevant control.

[0138]

[0095] Figure 24: 13F08 BiTE-T cells can be rapidly cleared via expressed tEGFR in NSG mice, (a) Schematic of the tEGFR / 13F08 retroviral co-expression construct, (b) Transduction efficiency determined by FACS detection of EGFR and intracellular detection of FLAG; gated on live T cells, (c) IncuCyte coculture killing profiles upper panels) and corresponding effector cytokine levels lower panels) for T cells transduced with the indicated constructs. Significance test: Kruskall-Wallis with Dunn correction (n=3); ns, not significant, (d) OVCAR8 xenografted mice treated by ACT with the indicated engineered T cells followed by intraperitoneal delivery of Cetuximab or Rituximab control Ab treatments (as depicted in schematic, left)', triangles indicate Ab treatments. Tumor progression curves were analyzed by 2-way ANOVA with Tukey correction (n=8 per group); ns, not significant; p<0.0001:****. (e) Animal weights during tEGFR / BiTE-T + Ritux / Cetux treatment. Data are presented as mean + / - SEM; ACT, adoptive T cell transfer; CD19 BiTE-T as irrelevant control.

[0139]

[0096] Figure 25: (A) SDS-PAGE analysis on crude expression culture supernatants (day 5) from HEK cells transfected with various 13F08 BiTE variants and controls. NR, non-reducing; R, reducing; arrows indicate relative migration of NR and R BiTEs. (B) 13F08 myc-tagged BiTE variants secreted from transfected HEK cells bind to immobilized human bio-MSLN (AcroBiosystems) in the presence of 1mM CaCl2. Crude expression supernatants titrated as indicated (not controlled for relative expression levels). VH-VK, reversed orientation of 13F08 scFv; GS, substitution of regular intra- scFv 13F08 linker (SEQ ID NO:13) with GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25); Irr. BiTE, irrelevant negative control BiTE; NT, non-transfected HEK supernatant.

[0140]

[0097] Figure 26: The epitope of 13F08(g) resides principally within the DI domain of human MSLN in transfected HEK cells. HEK293-6E cells were transiently transfected with either the FL MSLN or truncated domain fragments fused to the membrane proximal region containing the epitope of 15B6 (employed as an expression control) and stained by FACS with the indicated Fc-fusion proteins. Staining was carried out with EDTA omitted from the FACS staining and wash buffers (allowing metal retention in the FACS staining system).

[0141]

[0098] Corresponding reference characters indicate corresponding parts throughout the several views. The examples set out herein illustrate several embodiments of the invention but should not be construed as limiting the scope of the invention in any manner.Atty. Docket No. 2745-9 PCTT

[0142] DETAILED DESCRIPTION

[0143] Definitions:

[0144]

[0099] As used herein the term “about” refers to ± 10 %.

[0145]

[0100] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”. It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of’ are also provided.

[0146]

[0101] The term “consisting of’ means “including and limited to”.

[0147]

[0102] The term “consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0148]

[0103] As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

[0149]

[0104] Throughout this application, various embodiments of this disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0150]

[0105] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0151]

[0106] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0152]

[0107] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein areAtty. Docket No. 2745-9 PCTT

[0153] not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0154]

[0108] The term “antibody” describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. CDR grafted antibodies are also contemplated by this term. An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U. S. Patent Nos. 4,816,397 and 4,816,567. The term “antibody(ies)” includes a wild type immunoglobulin (Ig) molecule, generally comprising four full length polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain); including full length functional mutants, variants, or derivatives thereof, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain antibodies; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2). Also included within the meaning of the term “antibody” are any “antibody fragment”.

[0155]

[0109] An “antibody fragment” means a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHI) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of an Fab (Fd) fragment, which consists of the VH and CHI domains; (iv) a variable fragment (Fv), which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E. S. et al., Nature 341, 544-546 (1989)); (vi) a camelid antibody; (vii) an isolated complementarity determining region (CDR); (viii) a Single Chain Fv Fragment wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (ix) a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites (WO94 / 13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)); and (x) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; (xi) multivalent antibody fragments (scFv dimers, trimers and / or tetramers (Power and Hudson, J Immunol. Methods 242: 193-204Atty. Docket No. 2745-9 PCTT

[0156] 9 (2000)); (xii) a minibody, which is a bivalent molecule comprised of scFv fused to constant immunoglobulin domains, CH3 or CH4, wherein the constant CH3 or CH4 domains serve as dimerization domains (Olafsen T et al (2004) Prot Eng Des Sel 17(4):315-323; Hollinger P and Hudson PJ (2005) Nature Biotech 23(9): 1126-1136); and (xiii) other non-full length portions of heavy and / or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.

[0157] [HO] Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are included.

[0158] [Hl] As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and U. S. Patent Nos. 4,816,397 and 4,816,567.

[0159]

[0112] An "antibody combining site" is that structural portion of an antibody molecule comprised of light chain or heavy and light chain variable and hypervariable regions that specifically binds antigen.

[0160]

[0113] The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

[0161]

[0114] Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab')2 and F(v), which portions are preferred for use in the therapeutic methods described herein.

[0162]

[0115] Antibodies may also be bispecific, wherein one binding domain of the antibody is a specific binding member of the invention, and the other binding domain has a different specificity, e.g. to recruit an effector function or the like. Bispecific antibodies of the present invention include wherein one binding domain of the antibody is a specific binding member of the present invention, including a fragment thereof, and the other binding domain is a distinct antibody or fragment thereof, including that of a distinct anti-cancer or anti-tumor specific antibody. The other binding domain may be an antibody that recognizes or targets a particular cell type, as in a neural or glial cell-specific antibody. In the bispecific antibodies of the present invention the one binding domain of the antibody of the invention may be combined with other binding domains or molecules which recognize particular cell receptors and / or modulate cells in a particular fashion, as for instance an immune modulator (e.g., interleukin(s)), a growth modulator or cytokine or a toxin e.g.,Atty. Docket No. 2745-9 PCTT

[0163] ricin) or anti-mitotic or apoptotic agent or factor. Thus, the MSLN antibodies of the invention may be utilized to direct or target agents, labels, other molecules or compounds or antibodies in indications such as wound healing, inflammation, cancer or tumors.

[0164]

[0116] The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may also contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.

[0165]

[0117] The term “antigen binding domain” describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may bind to a particular part of the antigen only, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains. Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0166]

[0118] Immunoconjugates or antibody fusion proteins of the present invention, wherein the antibodies, antibody molecules, or fragments thereof, of use in the present invention are conjugated or attached to other molecules or agents further include, but are not limited to such antibodies, molecules, or fragments conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, antimicrobial agent or peptide, cell wall and / or cell membrane disrupter, or drug.

[0167]

[0119] The term “adjuvant(s)” describes a substance, compound, agent or material useful for improving an immune response or immune cell or component stimulation, and may in some instances be combined with any particular antigen in an immunological, pharmaceutical or vaccine composition. Adjuvants can be used to increase the amount of antibody and effector T cells produced and to reduce the quantity of antigen or immune stimulant or modulator and the frequency of injection. Although some antigens are administered without an adjuvant, there are many antigens that lack sufficient immunogenicity to stimulate a useful immune response in the absence of an effective adjuvant. Adjuvants also improve the immune response from "self-sufficient" antigens, in that the immune response obtained may be increased or the amount of antigen administered may be reduced. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al., Immunology, Second Ed., 1984, Benjamin / Cummings: Menlo Park, California, p. 384). In a preferred aspect an adjuvant is physiologically and / or pharmaceutically acceptable in a mammal, particularly a human. The standard adjuvant for use in laboratory animals is Freund's adjuvant. Freund's Complete adjuvant (FCA) is an emulsion containing mineral oil and killed mycobacteria in saline. Freund'sAtty. Docket No. 2745-9 PCTT

[0168] incomplete adjuvant (FIA) omits the mycobacteria. Both FIA and FCA induce good humoral (antibody) immunity, and FCA additionally induces high levels of cell-mediated immunity. However, neither FCA nor FIA are acceptable for clinical use due to the side effects. In particular, mineral oil is known to cause granulomas and abscesses, and Mycobacterium tuberculosis is the agent responsible for tuberculosis. Previously known and utilized adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvant such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Mineral salt adjuvants include but are not limited to: aluminum hydroxide, aluminum phosphate, calcium phosphate, zinc hydroxide and calcium hydroxide. Preferably, the adjuvant composition further comprises a lipid of fat emulsion comprising about 10% (by weight) vegetable oil and about 1-2% (by weight) phospholipids. Preferably, the adjuvant composition further optionally comprises an emulsion form having oily particles dispersed in a continuous aqueous phase, having an emulsion forming polyol in an amount of from about 0.2% (by weight) to about 49% (by weight), optionally a metabolizable oil in an emulsion-forming amount of up to 15% (by weight), and optionally a glycol ether-based surfactant in an emulsion-stabilizing amount of up to about 5% (by weight). There have been many substances that have been tried to be used as adjuvants, such as the lipid- A portion of gram negative bacterial endotoxin, and trehalose dimycolate of mycobacteria. The phospholipid lysolecithin exhibited adjuvant activity (Arnold et al., Eur. J Immunol. 9:363-366, 1979). Some synthetic surfactants exhibited adjuvant activity, including dimethyldioctadecyl ammonium bromide (DDA) and certain linear polyoxypropylenepolyoxyethylene (POP-POE) block polymers (Snippe et al., Int. Arch. Allergy Appl. Immunol. 65:390-398, 1981; and Hunter et al., J. Immunol. 127:1244-1250, 1981).

[0169]

[0120] The term “specific” may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.

[0170]

[0121] The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

[0171]

[0122] The term “consisting essentially of’ refers to a product, particularly a peptide sequence, of a defined number of residues which is not covalently attached to a larger product. In the case of the peptide of the invention referred to above, those of skill in the art will appreciate that minor modifications to the N- or C-terminal of the peptide may however be contemplated, such as the chemical modification of the terminal to add a protecting group or the like, e.g. the amidation of the C-terminus.Atty. Docket No. 2745-9 PCTT

[0172]

[0123] The term “isolated” refers to the state in which specific binding members of the invention, or nucleic acid encoding such binding members will be, in accordance with the present invention. Members and nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.

[0173]

[0124] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "pg" mean microgram, "mg" means milligram, "ul" or "µl" mean microliter, "ml" means milliliter, "1" means liter.

[0174]

[0125] The terms "antibody", “anti-MSLN antibody”, “MSLN antibody”, “hMSLN antibody”, "13F08 antibody" and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and the profile of activities set forth herein and in the Claims. Exemplary such MSLN antibodies provided herein include antibodies 13F08 and the 13F08 variant as provided and characterized herein. Antibodies provided herein extend to antibodies or proteins, including antibody fragments, having the amino acid sequence data described herein including certain CDR sequences among 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 22, or such as variable region heavy chain sequences and variable region light chain sequences SEQ ID NOs: 1, 2, 20, 21 and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms "antibody", “anti-MSEN antibody”, “MSEN antibody”, “hMSLN antibody”, "13F08 antibody" and the exemplary antibodies are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.

[0175]

[0126] The amino acid residues described herein are preferred to be in the " L" isomeric form. However, residues in the " D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin-binding is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.

[0176]

[0127] It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus.Atty. Docket No. 2745-9 PCTT

[0177] Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.

[0178]

[0128] A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo i.e., capable of replication under its own control.

[0179]

[0129] A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

[0180]

[0130] A " DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

[0181]

[0131] An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.

[0182]

[0132] A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0183]

[0133] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

[0184]

[0134] A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain " TATA" boxes and " CAT" boxes. Prokaryotic promoters contain Shine -Dalgarno sequences in addition to the -10 and -35 consensus sequences.Atty. Docket No. 2745-9 PCTT

[0185]

[0135] An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

[0186]

[0136] A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

[0187]

[0137] The term "oligonucleotide," as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

[0188]

[0138] The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either singlestranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

[0189]

[0139] The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

[0190]

[0140] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.Atty. Docket No. 2745-9 PCTT

[0191]

[0141] A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0192]

[0142] Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.

[0193]

[0143] It should be appreciated that also within the scope of the present invention are DNA sequences encoding specific binding members (antibodies) of the invention which code for e.g. an antibody having amino acid sequence as provided herein or comprising the CDR domain region sequences set out herein, but which are degenerate thereto. By "degenerate to" is meant that a different three -letter codon is used to specify a particular amino acid.

[0194]

[0144] Mutations can be made in the sequences encoding the amino acids, antibody fragments, CDR region sequences, such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention includes sequences containing amino acid changes and substitutions, including conservative changes, which do not significantly alter the activity or binding characteristics of the resulting protein.Atty. Docket No. 2745-9 PCTT

[0195]

[0145] The following is one example of various groupings of amino acids:

[0196] Amino acids with nonpolar R groups

[0197] Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine

[0198] Amino acids with uncharged polar R groups

[0199] Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

[0200] Amino acids with charged polar R groups (negatively charged at Ph 6.0)

[0201] Aspartic acid, Glutamic acid

[0202] Basic amino acids (positively charged at pH 6.0)

[0203] Lysine, Arginine, Histidine (at pH 6.0)

[0204]

[0146] Another grouping may be those amino acids with phenyl groups:

[0205] Phenylalanine, Tryptophan, Tyrosine

[0206]

[0147] Another grouping may be according to molecular weight (z.e., size of R groups).

[0207]

[0148] Particularly preferred substitutions are:

[0208] - Lys for Arg and vice versa such that a positive charge may be maintained;

[0209] - Glu for Asp and vice versa such that a negative charge may be maintained;

[0210] - Ser for Thr such that a free -OH can be maintained; and

[0211] - Gin for Asn such that a free NH2can be maintained.

[0212]

[0149] Exemplary and preferred conservative amino acid substitutions include any of: glutamine (Q) for glutamic acid (E) and vice versa; leucine (L) for valine (V) and vice versa; serine (S) for threonine (T) and vice versa; isoleucine (I) for valine (V) and vice versa; lysine (K) for glutamine (Q) and vice versa; isoleucine (I) for methionine (M) and vice versa; serine (S) for asparagine (N) and vice versa; leucine (L) for methionine (M) and vice versa; lysine (L) for glutamic acid (E) and vice versa; alanine (A) for serine (S) and vice versa; tyrosine (Y) for phenylalanine (F) and vice versa; glutamic acid (E) for aspartic acid (D) and vice versa; leucine (L) for isoleucine (I) and vice versa; lysine (K) for arginine (R) and vice versa.

[0213]

[0150] Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (z.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces -turns in the protein's structure.

[0214]

[0151] Two amino acid sequences are "highly homologous" or "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90% or 95% or 98% or 99%) are identical, or represent conservative substitutions. The CDR regions of two antibodies are substantially homologous when one or more amino acids, or one or a few, or one to three, or one or two are substituted with a similar or conservative amino acid substitution, and wherein theAtty. Docket No. 2745-9 PCTT

[0215] antibody / antibodies have the profile of binding and activities of one or more of the antibodies, particularly one or more of antibody 13F08, 13F08 variant, or derivative scFv, CAR or BiTE disclosed herein.

[0216]

[0152] A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

[0217]

[0153] A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

[0218]

[0154] The term "agent" means any molecule, including polypeptides, antibodies, polynucleotides, chemical compounds and small molecules. In particular the term agent includes compounds such as test compounds or drug candidate compounds.

[0219]

[0155] The term "agonist" refers to a ligand that stimulates the receptor the ligand binds to in the broadest sense.

[0220]

[0156] The term "assay" means any process used to measure a specific property of a compound. A "screening assay" means a process used to characterize or select compounds based upon their activity from a collection of compounds.

[0221]

[0157] The term "preventing" or "prevention" refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop) in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.

[0222]

[0158] The term "prophylaxis" is related to and encompassed in the term ‘prevention’, and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization;Atty. Docket No. 2745-9 PCTT

[0223] and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.

[0224]

[0159] " Therapeutically effective amount" means that amount of a drug, compound, antimicrobial, antibody, or pharmaceutical agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician. In particular, with regard to gram-positive bacterial infections and growth of gram-positive bacteria, the term “effective amount” is intended to include an effective amount of a compound or agent that will bring about a biologically meaningful decrease in the amount of or extent of tumor regression and or increase in length of a subject’s survival or period disease-free or in remission. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the growth or amount of tumor size, or enhanced survival or disease-free period by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent.

[0225]

[0160] The term "treating" or "treatment" of any disease or infection refers, in one embodiment, to ameliorating the disease or infection (i.e., arresting the disease or growth of the infectious agent or bacteria or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or infection, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, "treating" or "treatment" relates to slowing the progression of a disease or reducing an infection.

[0226]

[0161] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

[0227]

[0162] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "pg" mean microgram, "mg" means milligram, "ul" or "µl" mean microliter, "ml" means milliliter, "1" means liter.

[0228]

[0163] “Treatment” or “treat” or “treating” as used herein refers to an approach for obtaining beneficial or desired results. For purposes of the present application, beneficial or desired results include, but are not limited to, alleviation of a symptom and / or diminishment of the extent of a symptom and / or preventing a worsening of a symptom associated with a disease or condition. In one aspect, “treatment” or “treating” includes one or more of the following: (a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and / or diminishing the extent of the disease or condition); (b) slowing or arresting the development of one or more symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, delaying the worsening or progression of theAtty. Docket No. 2745-9 PCTT

[0229] disease or condition); and (c) relieving the disease or condition, e.g., causing the regression of clinical symptoms, ameliorating the disease state, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival.

[0230]

[0164] “Therapeutically effective amount” or “effective amount” as used herein refers to an amount that is effective to elicit the desired biological or medical response, including the amount of a compound that, when administered to a subject for treating a disease, is sufficient to affect such treatment for the disease. The effective amount will vary depending on the compound, the disease, and its severity and the age, weight, etc., of the subject to be treated. The effective amount can include a range of amounts. As is understood in the art, an effective amount may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved. Suitable doses of any co-administered compounds may optionally be lowered due to the combined action (e.g., additive or synergistic effects) of the compounds.

[0231]

[0165] The term “subject” is meant any subject, particularly a mammalian subject, in need of treatment with a peptide or polypeptide provided herein. Mammalian subjects include, but are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows, apes, monkeys, orangutans, and chimpanzees, and so on. In one aspect, the subject is a human subject. In an aspect, the subject is a domesticated animal. In an aspect the subject is a cat or a dog.

[0232] Detailed Disclosure:

[0233]

[0166] The present invention provides an antibody or fragment thereof, particularly a human antibody, and binding agents, derivatives and ligands derived thereof which recognizes mesothelin (MSLN), particularly human MSLN (hMSLN), and is selected from antibody(ies), or binding agents, derivatives and ligands derived therefrom, comprising the light chain and heavy chain variable region CDR1, CDR2 and CDR3 sequences as provided herein. In one such aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region CDRs comprising a CDR1 sequence GYTFTGYY (SEQ ID NO:3), a CDR2 sequence INPNSGGT (SEQ ID NO:5), and a CDR3 sequence AREIYSGSHPDDAFDI (SEQ ID NO:7) is provided. In a further such aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region CDRs comprising a CDR1 sequence GYTFTGYY (SEQ ID NO:3) or GYTFTGYYMH (SEQ ID NO:4), a CDR2 sequence INPNSGGT (SEQ ID NO:5) or WINPNSGGTNYAQKFQD (SEQ ID NO:6), and a CDR3 sequence AREIYSGSHPDDAFDI (SEQ ID NO:7) is provided. In some aspects, the VH CDR2 sequence is WINPNSGGTNYAQKFQG (SEQ ID NO:22)Atty. Docket No. 2745-9 PCTT

[0234]

[0167] In one aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising light chain variable region CDRs comprising a CDR1 sequence QGISNS (SEQ ID NO:8), a CDR2 sequence GAS (SEQ ID NO: 10), and a CDR3 sequence QQYYSTPHT (SEQ ID NO: 12) is provided. In a further such aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising light chain variable region CDRs comprising a CDR1 sequence QGISNS (SEQ ID NO:8) or RASQGISNSLA (SEQ ID NO:9), a CDR2 sequence GAS (SEQ ID NO: 10) or GASILES (SEQ ID NO: 11), and a CDR3 sequence QQYYSTPHT (SEQ ID NO: 12) is provided.

[0235]

[0168] Antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region (VH) corresponding to amino acid sequence SEQ ID NO:1 is provided. In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region (VH) corresponding to amino acid sequence SEQ ID NO:20 is provided. In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region (VH) corresponding to amino acid sequence SEQ ID NO:1 is provided. Antibody, or binding agents, derivatives and ligands derived thereof, comprising light chain variable region (VL or VK) sequence corresponding to amino acid sequence SEQ ID NO: 2 is provided. In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising light chain variable region (VL or VK) sequence corresponding to amino acid sequence SEQ ID NO: 21 is provided.

[0236]

[0169] In an aspect, antibody, or binding agents, derivatives and ligands derived thereof, comprising heavy chain variable region (VH) corresponding to amino acid sequence SEQ ID NO:1 or SEQ ID NO:20 and light chain variable region SEQ ID NO:2 or SEQ ID NO:21 is provided.

[0237]

[0170] In a further aspect, the invention provides an antibody, or binding agents, derivatives and ligands derived thereof, comprising a heavy chain variable region sequence SEQ ID NO: 1 or SEQ ID NO: 20 or a variant thereof having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity to the heavy chain variable region sequence SEQ ID NO: 1 or SEQ ID NO:20, or comprising 1 to 3 amino acid substitutions in one or more heavy chain CDR region of SEQ ID NO: 3, 4, 5, 6, 7 or 22, wherein said variant retains human MSLN binding and lack of mouse MSLN reactivity. In an aspect, such variants retain binding to the D1-D2 region or domain of human mesothelin MSLN, particularly the DI fragment or region of human mesothelin MSLN. In an aspect, such variants retain the requirement for divalent cations to bind MSLN.

[0238]

[0171] In another aspect, the invention provides an antibody, or binding agents, derivatives and ligands derived thereof, comprising a light chain variable region sequence SEQ ID NO: 2 or SEQ ID NO: 21 or a variant thereof having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% amino acid identity to the light chain variable region sequence SEQ ID NO:2 or SEQ ID NO:21, or comprising 1 to 3 amino acid substitutions in one or more light chain CDR region of SEQ ID NO: 8, 9, 10, 11 or 12, wherein saidAtty. Docket No. 2745-9 PCTT

[0239] variant retains human MSLN binding and lack of mouse MSLN reactivity. In an aspect, such variants retain binding to the D1-D2 region or domain of human mesothelin MSLN, particularly the DI fragment or region of human mesothelin MSLN. In an aspect, such variants retain the requirement for divalent cations to bind MSLN.

[0240]

[0172] Human mesothelin proteins (two isoforms) and their DI and D2 regions or domains are as follows:

[0241]

[0173] Human mesothelin Isoform 1 (NCBI Reference Sequence: NP_005814.2)

[0242] 1 malptarpll gscgtpalgs llfllfslgw vqpsrtlage tgqeaapldg vlanppniss

[0243] 61 Isprqllgfp caevsglste rvrelavala qknvklsteq Irclahrlse ppedldalpl

[0244] 121 dlllflnpda fsgpqactrf fsritkanvd llprgaperq rllpaalacw gvrgsllsea

[0245] 181 dvralgglac dlpgrfvaes aevllprlvs cpgpldqdqq eaaraalqgg gppygppstw

[0246] 241 svstmdalrg llpvlgqpii rsipqgivaa wrqrssrdps wrqpertilr prfrrevekt

[0247] 301 acpsgkkare ideslifykk weleacvdaa llatqmdrvn aipftyeqld vlkhkldely

[0248] 361 pqgypesviq hlgylflkms pedirkwnvt sletlkalle vnkghemspq vatlidrfvk

[0249] 421 grgqldkdtl dtltafypgy Icslspeels svppssiwav rpqdldtcdp rqldvlyp

[0250] 481 rlafqnmngs eyfvkiqsfl ggaptedlka Isqqnvsmdl atfmklrtda vlpltvaevq

[0251] 541 kllgphvegl kaeerhrpvr dwilrqrqdd Idtlglglqg gipngylvld Ismqealsgt

[0252] 601 pcllgpgpvl tvlalllast la

[0253] DI region aa’s 296-390 (bold)

[0254] D2 region aa’s 391-478 (italics)

[0255]

[0174] Human mesothelin Isoform 2 (NCBI Reference Sequence: NP_037536.2

[0256] 1 malptarpll gscgtpalgs llfllfslgw vqpsrtlage tgqeaapldg vlanppniss

[0257] 61 Isprqllgfp caevsglste rvrelavala qknvklsteq Irclahrlse ppedldalpl

[0258] 121 dlllflnpda fsgpqactrf fsritkanvd llprgaperq rllpaalacw gvrgsllsea

[0259] 181 dvralgglac dlpgrfvaes aevllprlvs cpgpldqdqq eaaraalqgg gppygppstw

[0260] 241 svstmdalrg llpvlgqpii rsipqgivaa wrqrssrdps wrqpertilr prfrrevekt

[0261] 301 acpsgkkare ideslifykk weleacvdaa llatqmdrvn aipftyeqld vlkhkldely

[0262] 361 pqgypesviq hlgylflkms pedirkwnvt sletlkalle vnkghemspq aprrplpqva

[0263] 421 tlidrfvkgr gqldkdtldt Itafypgylc slspeelssv ppssiwavrp qdldtcdprq

[0264] 481 Idvlyp aA afqnmngsey fvkiqsflgg aptedlkals qqnvsmdlat fmklrtdavl

[0265] 541 pltvaevqkl Igphveglka eerhrpvrdw ilrqrqddld tlglglqggi pngylvldls

[0266] 601 mqealsgtpc llgpgpvltv lalllastla

[0267] DI region aa’s 296-390 (bold)

[0268] D2 region aa ’s 391 -486 ( italics )

[0269]

[0175] Thus, the DI epitope region amino acid sequence is evektacpsgkkareideslifykkweleacvdaallatqmdrvnaipftyeqldvlkhkldelypqgypesviqhlgylflkmspedirkwnvt (SEQ ID NO:31)

[0270]

[0176] In aspects of the invention variable heavy and light chain VH and VK sequences of antibody 13F08 and variant antibody 13F09g are provided. Specific exemplary VH and VE / VK variable region sequences are set out in those characteristic of parental 13F08 antibody and variant 13F08g antibody.

[0271]

[0177] In additional aspects, antibody derivatives such as single-chain variable fragments (scFv) are provided that bind human MSEN. The scFvs comprise the heavy chain variable region and the light variable region joined by a peptide linker. The scFv may be a VL / VH construct with the light chain (VL or VK) firstAtty. Docket No. 2745-9 PCTT

[0272] followed by the heavy chain (VH) variable region, with a linker between them. Suitable linkers are known and available. Exemplary intra-scFv linkers include for example RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO:13), GQPKARQEGGSGEGGSGESNAAA (SEQ ID NO:19) and GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25).

[0273]

[0178] The unique specificity and affinity of the antibodies and fragments, binding agents, derivatives and ligands of the invention provides diagnostic and therapeutic uses to identify, characterize and target conditions associated with MSLN expression, activity or activation. In particular, antibodies, binding agents, derivatives and ligands of the invention targeting MSLN are useful in modulating immune response. In an aspect thereof, antibodies binding agents, derivatives and ligands of the invention targeting MSLN are useful in modulating immune response against cancer, cancer or tumor cells, and cancer or tumor antigens. The antibodies, binding agents, derivatives and ligands have applicability in therapeutic treatment or management of cancer. The antibodies, binding agents, derivatives and ligands have applicability in enhancing the anti-cancer immune response and in enhancing cancer vaccines. The antibodies binding agents, derivatives and ligands have applicability in enhancing the therapeutic effect including the anticancer and / or anti-cellular effect of radiation therapy(ies). In a particular aspect the antibodies, binding agents, derivatives and ligands of the invention are applicable in treatment, management and / or prevention of cancers, including in cancer recurrence and metastasis. In aspects, cancers that express MSLN include, without limitation, mesotheliomas, stomach cancer, squamous cell carcinomas, prostate cancer, pancreatic cancer, lung cancer, breast cancer (particularly triple-negative cancer), ovarian cancer, and cervical cancer. In an embodiment, the cancer is mesothelioma. In an embodiment, the cancer is ovarian cancer. In an embodiment, the cancer is pancreatic cancer. In an embodiment, the cancer is leukemia. In an embodiment, the cancer is acute myeloid leukemia (AML). In an embodiment, the cancer is pediatric AML. In an embodiment, the subject is a child with AML.

[0274]

[0179] In an aspect of the invention the antibodies, binding agents, derivatives and ligands specifically directed against MSLN may be administered in conjunction with or in a composition of cancer antigen(s) and adjuvant(s), including to patients to promote a more robust priming and activation of the adaptive antitumor response to enhance immune therapies directed at cancers. Additional inhibitors to MSLN activity, such as small molecules, antisense or aptamers can also be used to inhibit MSLN activity.

[0275]

[0180] Potent anti-tumor immunity requires modulating multiple arms of host immune response and targeting pathways that contributes to tumor cell growth and survival. Combining agents that modulate immune response and arrest tumor growth and progression can generate anticancer immunity and arrest tumor growth to improve clinical outcomes (Vanneman, M (2012) Nature Reviews Cancer (12):237-251). Thus, in an aspect of the invention the antibodies, binding agents, derivatives and ligands specifically directed against MSLN may be administered alone or in combination with other treatments, therapeutics orAtty. Docket No. 2745-9 PCTT

[0276] agents, either simultaneously or sequentially dependent upon the condition to be treated. Immune modulators may be included in a composition with or administered with antibodies, binding agents, derivatives and ligands specifically directed against MSLN and / or administered at a different time to enhance immune modulation and / or cancer therapy, including immune therapies directed against cancer. An immune modulator may be an adjuvant. Applicable immune modulators include IDO, TDO (Flatten M (2012) Cancer Research 72(21):5435-40), a-galactosyl ceramide and analogs thereof such as threitolceramide (ThrCer) and ThrCer 6, TLR ligands such as poly I: C (TLR3), MPL (TLR4), imiquimod (TLR7), R848 (TLR8) or CpG (TLR9), iCOS, CTLA-4, PD1, PD1 ligand, TIGIT, 0X40 and 0X40 ligand, Lag3, GITR, GITR ligand interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, T cell modulators including modulators of CD8+T cells, cytokines or hormones which stimulate the immune response or reduction or elimination of cancer cells or tumors. Additional immunomodulators are small molecules, antagonist antibodies or agonist antibodies targeting the applicable immune modulators including IDO, TDO, Toll like receptor family or iCOS, CTLA-4, PD1, PD1 ligand, TIGIT, 0X40 and 0X40 ligand, interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, T cell modulators including modulators of CD8+T cells, cytokines which stimulate the immune response or reduction or elimination of cancer cells or tumors.

[0277]

[0181] There are several recognized and known methods and approaches to determine the CDRs in an antibody. The most commonly used CDR identification methods at present are Kabat (Wu TT, Kabat EA (1970) J Exp Med 132:211-250; Kabat EA et al (1983) Sequence of Proteins of Immunological Interest. Bethesda: National Institute of Health), IMGT (Lefranc MP et al (2003) Dev Comp Immunol 27:55-77) and Chothia (Chothia C, Lesk AM (1987) J Mol Biol 196:901-917; Chothia C et al (1989) Nature 342:877-883; Lefranc MP et al (2003) Dev Comp Immunol 27:55-77). Each of these methods has devised a unique residue numbering scheme according to which it numbers the hypervariable region residues and the beginning and ending of each of the six CDRs is then determined according to certain key positions. IMGT was generally utilized in the present studies. While these different approaches may identify slightly offset CDR sequences, they generally provide overlapping sequences and amino acids and can be useful in combination to identify amino acids which should be maintained or conserved and those that may be suitable for variation or alteration while maintaining binding.

[0278]

[0182] Portions or domains of the antibodies of the invention are contemplated and incorporated, including any portion or domain, including those modified or fused to reagents, labels or other domains or fragments, wherein the portions or domains retain the characteristics of the antibodies hereof, including MSLN specific binding, as exemplified in antibodies, binding agents, derivatives and ligands specifically directed against MSLN hereof. Antibodies and antibody fragments of the invention include smaller recombinant antibody fragments (for example, classic monovalent antibody fragments (Fab, scFv) andAtty. Docket No. 2745-9 PCTT

[0279] engineered variants (diabodies, triabodies, minibodies and single-domain antibodies) that retain the targeting specificity of the whole antibodies (mAbs) (for review see Hollinger P and Hudson PJ (2005) Nature Biotech 23(9): 1126-1136). These include for example domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E. S. et al., Nature 341, 544-546 (1989)); camelid antibody; isolated complementarity determining region (CDR); Single Chain Fv Fragments wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites (WO94 / 13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 906444-6448, (1993)); a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; multivalent antibody fragments (scFv dimers, trimers and / or tetramers (Power and Hudson, J Immunol. Methods 242: 193-2049 (2000)); and minibody, which is a bivalent molecule comprised of scFv fused to constant immunoglobulin domains, CH3 or CH4 (for example IgGl (CH3) and IgE (CH4)), wherein the constant CH3 or CH4 domains serve as dimerization domains (Olafsen T et al (2004) Prot Eng Des Sel 17(4):315-323; Hollinger P and Hudson PJ (2005) Nature Biotech 23(9): 1126-1136). These smaller antibodies and engineered variants or fragments can be produced more economically and may possess other unique and superior properties for a range of diagnostic and therapeutic applications. For example, scFV2-Fc can accumulate in higher abundance in tumor or tissue, and a minibody is approximately 80kD and may be ideal for therapy because of higher uptake in tissues, have faster clearance and have better tissue to blood ratios than intact immunoglobulin (150kDa) or Fab’2 (HOkDa). The antibody fragments may be forged into multivalent and multispecific reagents, linked to therapeutic payloads (such as radionuclides, toxins, enzymes, liposomes and viruses) and engineered for enhanced therapeutic efficacy. Recently, single antibody domains have been engineered and selected as targeting reagents against hitherto immunosilent cavities in enzymes, receptors and infectious agents.

[0280]

[0183] Specific binding members of the present invention may further comprise antibody constant regions or parts thereof. For example, specific binding members based on the sequences hereof may be attached at their C-terminal end to antibody light chain constant domains including human CK or C7_ chains, preferably Ck chains. Similarly, specific binding members based on the sequences of Figure(s) 7, 8, 10, 11, 12 or 13 may be attached at their C-terminal end to all or part of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE, IgD and IgM and any of the isotype sub-classes, particularly IgGl, IgG2b, and IgG4. IgGl is preferred.Atty. Docket No. 2745-9 PCTT

[0281]

[0184] The antibodies, or any fragments thereof, may be conjugated or recombinantly fused to any cellular toxin, bacterial or other, e.g. pseudomonas exotoxin, ricin, or diphtheria toxin. The part of the toxin used can be the whole toxin, or any particular domain of the toxin. Such antibody-toxin molecules have successfully been used for targeting and therapy of different kinds of cancers, see e.g. Pastan, Biochim Biophys Acta. 1997 Oct 24;1333(2): Cl-6; Kreitman et al., N Engl J Med. 2001 Jul 26;345(4):241-7; Schnell et al., Leukemia. 2000 Jan;14(l):129-35; Ghetie et al., Mol Biotechnol. 2001 Jul;18(3):251-68.

[0282]

[0185] Bi- and tri-specific multimers can be formed by association of different scFv molecules and have been designed as cross-linking reagents for T-cell recruitment into tumors (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics), see e.g. Todorovska et al., J Immunol Methods. 2001 Feb l;248(l-2):47-66; Tomlinson et al., Methods Enzymol.

[0283] 2000;326:461-79; McCall et al., J Immunol. 2001 May 15; 166(10):6112-7.

[0284]

[0186] Antibodies, fragments and / or derivatives thereof of the invention may be labelled with a detectable or functional label. Such labeled antibodies, fragments and / or derivatives have applicability in radioimaging and radioimmunotherapy. Radiolabelled antibodies, fragments and / or derivatives targeting MSEN, particularly radioimmunoconjugates, are useful in radioimmunotherapy, particularly as radiolabelled antibodies for cancer therapy, including to kill cancer cells. Radiolabelled antibodies fragments and / or derivatives thereof, are useful in radioimaging and radioimmuno-guided surgery techniques, to identify and indicate the presence and / or location of cancer cells, precancerous cells, tumor cells, and hyperproliferative cells, prior to, during or following surgery. The combination of radioimaging and radioimmunotherapy, using the same antibody, fragment or derivative with a therapeutic isotope, for example can provide significant advantages in cancer management.

[0285]

[0187] Eabels, including detectable labels and therapeutic labels, include, but are not limited to, radiolabels such as the isotopes3H,14C,32P,35S,36C1,51Cr,57Co,58Co,59Fe,86Y,90Y,1211,124I,1251,131I,111In,117Lu,177Lu,211At,198Au,64Cu,67Cu,225Ac,213Bi,99Tc,186Re,188Re,89Zr,68Ga and18F, which may be attached to antibodies, fragments and / or derivatives of the invention using conventional chemistry known in the art of antibody imaging. Eabels also include fluorescent labels (for example fluorescein, rhodamine, Texas Red) and labels used conventionally in the art for MRI-CT imaging. They also include enzyme labels such as horseradish peroxidase, β-glucoronidase, β-galactosidase, urease. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin. Functional labels include substances which are designed to be targeted to the site of a tumor to cause destruction of tumor tissue. Such functional labels include cytotoxic drugs such as 5-fluorouracil or ricin and enzymes such as bacterial carboxypeptidase or nitroreductase, which are capable of converting prodrugs into active drugs at the site of a tumor.Atty. Docket No. 2745-9 PCTT

[0286]

[0188] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., MALDI mass spectrometry), arraybased oligo-peptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping).

[0287]

[0189] In certain aspects, competition binding assays can be used to determine whether an antibody is competitively blocked, e.g., in a dose dependent manner, by another antibody for example, an antibody binds essentially the same epitope, or overlapping epitopes, as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes in competition binding assays such as competition ELISA assays, which can be configured in all number of different formats, using either labeled antigen or labeled antibody. In a particular embodiment, an antibody can be tested in competition binding assays with an antibody described herein.

[0288]

[0190] In addition, antibodies that recognize and bind to the same or overlapping epitopes can be identified using routine techniques such as an immunoassay, for example, by showing the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competition binding assays also can be used to determine whether two antibodies have similar binding specificity for an an antigen or an epitope, including a particular epitope on an antigen or protein target. Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits specific binding of another antibody to a common antigen or target antigen.

[0289]

[0191] The antibodies, binding agents, derivatives and ligands specifically directed against MSLN can be used in isolation, for example to characterize, identify, locate or tag MSLN, including MSLN expressed on cells. The antibodies, binding agents, derivatives and ligands specifically directed against MSLN can be combined with other proteins or protein domains, or can be fused with other proteins or protein domains or covalently attached with other proteins or protein domains. In aspects, the antibodies, binding agents, derivatives and ligands specifically directed against MSLN are conjugated with other proteins or domains.

[0290]

[0192] In some embodiments, the antibodies, binding agents, derivatives and ligands specifically directed against MSLN is conjugated to another compound, for example being conjugated to a half-life extending moiety. In some embodiments, the binding protein is conjugated to a polymer (e.g., PEG). In some embodiments, the binding protein is conjugated to a cytotoxic agent. In some embodiments, theAtty. Docket No. 2745-9 PCTT

[0291] antibodies, binding agents, derivatives and ligands specifically directed against MSLN is conjugated to an immunomodulatory compound, protein or domain. In some embodiments, antibodies, binding agents, derivatives and ligands specifically directed against MSLNis conjugated to or combined with an immune stimulator or immune activator, such as an interleukin, such as IL-2, or an interferon, such as IFN-y.

[0292]

[0193] In aspects of the invention, antibodies, binding agents, derivatives and ligands specifically directed against MSLN are included or incorporated in immunotherapeutic constructs or components or compounds, which are effective and active by virtue of the MSLN binding, interaction.

[0293]

[0194] Such immunotherapeutic constructs or components or compounds include but are not limited to entities which are designed and utilized specifically to alter immunological responses and / or immune cell responses. These include stimulating or inhibit an immune or immune cell response, activating immune cells against an antigen or other cell, such as a cancer antigen or cancer or tumor cell.

[0294]

[0195] Immunotherapeutic approaches utilizing antibodies, binding agents, derivatives and ligands specifically directed against MSLN provided include but are not limited to protein or cell therapies such as chimeric antigen receptors (CARs), bispecific T cell engagers (BiTEs), which utilize protein constructs or cells expressing the protein constructs to modulate the immune system, immune response, anti-cancer response, etc. Other approaches utilize antibodies, binding agents, derivatives and ligands specifically directed against MSLN conjugated to another compound, such as a cytotoxic agent, immunomodulatory agent.

[0295]

[0196] Adoptive cell transfer (ACT) is an important new pillar in cancer therapy, based on collecting and using patients’ own immune cells to treat their cancer. There are several types of ACT, including TILs, TCRs, and CARs (Haanen et al. (2018) J Immunother Cancer 474:449-461). One approach uses immune cells that have penetrated the environment in and around the tumor, known as tumor-infiltrating lymphocytes (TILs). Another approach to ACT involves engineering patients' T cells to express a specific T-cell receptor (TCR) to recognize tumor cell antigens (Mackall et ai (2019) Nature Medicine 25:1341-1355). Chimeric antigen receptors (CARs) use portions of synthetic antibodies directed against specific surface cell antigens and CAR T cell therapy has advanced significantly in clinical development. In CAR therapy, T cells are isolated from a patient and genetically engineered to produce CARs so the T cells recognize and attach to a specific antigen on tumor cells.

[0296]

[0197] The MSLN antibody of the invention, or a fragment thereof, may further be used for constructing a chimeric antigen receptor (CAR), wherein the CAR comprises the antigen binding domain of the TGF-P3 antibody, a transmembrane domain, a costimulatory signaling region, and a signaling domain. In these and other embodiments, the antigen binding domain may be a Fab or a scFv of the MSEN antibody, including as disclosed herein. In yet a further embodiment, MSEN is present in a tumor microenvironment or on cells in the tumor microenvironment. In yet other embodiments, the costimulatoryAtty. Docket No. 2745-9 PCTT

[0297] signaling region comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

[0298]

[0198] T-cells modified to express chimeric antigen receptor (CAR) and administered alone have been subject to suppression within the hostile tumor microenvironment. As a way of a non-limiting example, further modification of these cells to express secretable scFvs (eg, PD-1, PDL-1, or CTLA-4) (known as Armored CARs) have improved anti-tumor function due to their ability to modulate the tumor microenvironment and resist suppressive factors (for example as described in US Patent 10,124,023 and Brentjens et al (2018) Nat Biotechnol 36(9): 847-856). In one such embodiment Armored CARs expressing one or more MSLN antibody of the present invention, including one or more scfv thereof, are contemplated, wherein the MSLN antibody enhances the CAR cells activity and blocks immune suppression, including such as suppression by endogenous TGF [3. In another embodiment, the MSLN antibody of the invention could be used in Adoptive Cell Therapy (ACT) where the MSLN antibody, or fragments thereof, would be genetically introduced into T-cells, preferably but not limited to, tumor infiltrating lymphocytes (TILs), isolated from cancer patients, and then such T-cells would be expanded and delivered back into the patients whereby the T-cells would target the tumor and express and secrete the MSLN antibody, or fragment thereof, in the local tumor microenvironment to counter the immunosuppressive environment there. In another embodiment, exogenous MSLN antibody, or a fragments thereof, can be added to the expanded T-cell population when delivering back into the patients for ACT.

[0299]

[0199] The MSLN antibody of the invention could also be used in Adoptive Cell Therapy (ACT) where the MSLN antibody, or fragments or derivatives thereof, would be genetically introduced into T-cells isolated from cancer patients, and then such T-cells would be expanded and delivered back into a patient whereby the T-cells would target the tumor and express the MSLN antibody, or fragment or derivative thereof, in the local tumor microenvironment to counter the immunosuppressive environment there. Preferably the T-cells used would tumor infiltrating lymphocytes (TILs).

[0300]

[0200] In vivo animal models of cancer or animal xenograft studies may be utilized by the skilled artisan to further or additionally screen, assess, and / or verify the specific binding members and antibodies or fragments thereof of the present invention, including further assessing MSLN modulation and inhibition in vivo and inhibiting tumor progression, recurrence, metastasis, or immune response against tumor cells or response to antigens or vaccines, including tumor or cancer antigens or vaccines. Such animal models include, but are not limited to models of immune response, immune modulation, vaccination, cancer, cancer metastasis. Models of cancers whose recurrence or metastasis are associated with elevated levels of MSLN or MSLN surface expression are particularly susceptible to and targeted by the antibodies of the presentAtty. Docket No. 2745-9 PCTT

[0301] invention. Such cancers include melanomas, breast, lung and prostate cancer. Exemplary and suitable models are known and readily available to the skilled artisan and include those referenced and / or described herein and known in the art.

[0302]

[0201] Antibodies of the present invention may be administered to a patient in need of treatment via any suitable route, including by injection, including intraperitoneally, intramuscularly, subcutaneous, intravenous, into the bloodstream or CSF, or directly into the site of the tumor or by intratumoral administration or intratumoral injection. The precise dose will depend upon a number of factors, including whether the antibody is for diagnosis or for treatment, the size and location of the tumor, the precise nature of the antibody (whether whole antibody, fragment, diabody, etc), and the nature of the detectable or functional label attached to the antibody. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician.

[0303] CHIMERIC ANTIGEN RECEPTORS (CARs)

[0304]

[0202] The term "chimeric antigen receptor" or " CAR" refers to a polypeptide or set of polypeptides, which when in an immune effector cell, provides the cell with specificity for a target cell, for example a cancer cell, and with intracellular signal generation.

[0305]

[0203] A CAR cell-surface receptor provided herein comprises an extracellular target-binding domain (e.g., a MSLN binding domain, such as MSLN antibody or derivative provided herein), a transmembrane domain, and an intracellular domain or endodomain, comprising a signalling domain and optionally at least one co-stimulatory signaling domain (referred to also as intracellular (IC) domain herein), all in a combination that is not naturally found together on a single protein. This particularly includes receptors wherein the extracellular domain and the cytoplasmic or intracellular domain are not naturally found together on a single receptor protein. The chimeric antigen receptors of the present invention are intended primarily for use with lymphocytes such as T cells and natural killer (NK) cells.

[0306]

[0204] Chimeric antigen receptors (CARs) are hybrid molecules comprising an antigen-targeting moiety or binding domain, followed by a linker, transmembrane (TM) domain, and various endodomains (EDs) involved in T-cell activation. CARs may include a single endodomain, such as the ED of CD3-zeta only, to signal T cell activation. CARs also have one or more additional EDs or co-stimulatory EDs, as well such as CD28 and 4-1BB, to provide additional T cell signalling.

[0307]

[0205] Linkers connecting the one or more domains may be selected from those described herein and others known in the art. Linkers include and may be selected from, for example RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO:13), GSASASTGGDAS (SEQ ID NO:16), GQPKARQEGGSGEGGSGESNAAA (SEQ ID NO: 19), GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25), ASTGGDAS (SEQ ID NO:38); a GS linker such as GGGGSGGGGS, etc.Atty. Docket No. 2745-9 PCTT

[0308]

[0206] The CARs described and provided herein comprise at least an extracellular binding domain comprising the MSLN binding domain of the invention, as well as a transmembrane domain and an intracellular domain (also referred to as a “cytoplasmic signalling domain” or "an intracellular signalling domain"). In some embodiments, the CAR further comprises one or more additional polypeptide sequences. Exemplary additional polypeptide sequences include, but are not limited to, signal sequences, epitope tags, and polypeptides that produce a detectable signal. In some embodiments, the CAR further comprises a leader sequence, such as a signal sequence. As used herein the term “leader sequence” refers to a sequence of amino acids which, when fused to a CAR sequence, aids localization of the CAR to a cell membrane of a cell expressing the CAR. In some embodiments, the leader sequence is an N-terminal leader sequence. In some embodiments, the leader sequence is an N-terminal signal sequence. In some embodiments, the leader sequence is a signal sequence and is an immunoglobulin signal sequence. In some embodiments, the leader sequence is cleaved from the CAR during cellular processing and localization of the CAR to the cellular membrane when expressed in a cell.

[0309]

[0207] In some embodiments, the CAR domains are in the same polypeptide chain (e.g., comprise a chimeric fusion protein). In some embodiments, the domains are contiguous with each other. In some embodiments, the domains are not contiguous with each other, e.g., are in different polypeptide chains, such as a split CAR. In some embodiments, the different polypeptide chains include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen or ligand-binding domain to an intracellular signalling domain.

[0310] Target Binding Domain

[0311]

[0208] The binding domain comprises a MSLN binding domain, antibody, fragment, derivative as described and provided herein.

[0312] Transmembrane Domain

[0313]

[0209] In various embodiments, a CAR can be designed to comprise a transmembrane domain that is attached to the target binding domain of the CAR, for positioning of the CAR in a cell membrane. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one embodiment, the transmembrane domain is one that is associated with one of the other domains of the CAR e.g., in one embodiment, the transmembrane domain may be from the same protein that the primary signalling domain, costimulatory domain or the hinge region is derived from. In anotherAtty. Docket No. 2745-9 PCTT

[0314] embodiment, the transmembrane domain is not derived from the same protein that any other domain of the CAR is derived from.

[0315]

[0210] In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one embodiment, the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a CAR-expressing cell. In another embodiment, the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR-expressing cell.

[0316]

[0211] The transmembrane domain may be derived either from a natural or from a recombinant source. In one embodiment the transmembrane domain is capable of signalling to the intracellular domain(s) whenever the CAR has bound to its target. A transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, TNFR2, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD Ila, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, NKG2C, or a functional variant thereof.

[0317]

[0212] In some instances, the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge, such as a hinge from a human protein. As used herein, the term “hinge region” or “hinge domain” refers to any suitable amino acid sequence positioned between the transmembrane domain and the antigen binding domain or CD27 extracellular domain. Suitable hinge sequences are available and known in the art. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (such as an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8 alpha hinge. In one embodiment, the hinge or spacer comprises an IgG4 hinge. In one embodiment, the hinge region comprises an IgD hinge. In another embodiment, the hinge comprises a CD8 alpha hinge region.

[0318]

[0213] Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-Atty. Docket No. 2745-9 PCTT

[0319] serine doublet provides a particularly suitable linker. For example, in one embodiment, the linker comprises the amino acid sequence of GGGGSGGGGS. In one embodiment, the hinge or spacer comprises a KIR2DS2 hinge.

[0320] Intracellular Domain

[0321]

[0214] The intracellular domain of the CAR is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced. The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term "intracellular domain" refers to the portion of a CAR which transduces the effector function signal and directs the cell to perform a specialized function. To the extent that a truncated portion of the intracellular domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular domain is meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal. Intracellular domains for use in the CAR of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0322]

[0215] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary and / or costimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signalling sequences: those that initiate antigendependent primary activation through the TCR (referred to herein as a “primary signalling domain”) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (referred to herein as a “costimulatory domain”).

[0323]

[0216] As used herein, the phrase “primary signalling domain” refers to a domain present in the intracellular domain of a CAR, which is capable of inducing, or inhbiting, antigen-dependent primary activation signalling in a cell expressing the CAR. For example, primary activiation signalling such as that initiated by, for instance, binding of a TCR / CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, such as proliferation, activation, differentiation, and the like. In this regard, a primary signalling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.

[0324]

[0217] Primary signalling domains that are of use in the invention include those of CD3 zeta (CD3ζ, common FcR gamma (FCERIG), Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12, or a functional variant thereof. In one embodiment, a CAR of the invention comprises an intracellular signalling domain, e.g., a primary signalling domain of CD3-zeta, or a functional variant thereof. In one embodiment, a primary signalling domain comprises a modifiedAtty. Docket No. 2745-9 PCTT

[0325] primary signalling domain, e.g., a mutated signaling domain which has altered (e.g., increased or decreased) activity as compared to the native domain.

[0326]

[0218] The intracellular domain of the CAR can comprise the primary signalling domain by itself or it can be combined with another intracellular signalling domain(s) useful in the context of a CAR of the invention. For example, in some embodiments, the intracellular domain of the CAR comprises a primary signalling domain and a costimulatory domain.

[0327]

[0219] A “costimulatory domain” refers to a portion of the CAR which is derived from an intracellular domain of a costimulatory molecule and which is capable of providing a costimulatory signal in a cell expressing the CAR upon antigen binding. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include TNFR2, CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CAR-T cells in vitro and augments human T cell persistence and antitumour activity in vivo. Further examples of such costimulatory molecules include MyD88, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAE, CDlla, EFA-1, ITGAM, CD1 lb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, EFA-1, ITGB7, TNFR2, TRANCE / RANKE, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD19a.

[0328]

[0220] The primary signalling domain and the costimulatory domain within the cytoplasmic portion of the CAR of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signalling sequence. In one embodiment, a glycine- serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker.

[0329]

[0221] In one embodiment, the intracellular domain is designed to comprise the signalling domain of CD3-zeta and the signalling domain of CD28. In one embodiment, the intracellular domain is designed to comprise the signalling domain of CD3-zeta, the signalling domain of CD28, and the signalling domain of 4-1BB, or a functional variant thereof. In some embodiments, different linker sequences may be used between the different domains of the CAR, e.g., a (GGGS)n linker, wherein n is 1-20 (e.g., 1, 2, 3, 4, 5, 6,Atty. Docket No. 2745-9 PCTT

[0330] 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the CAR is a multi-specific (e.g., a bispecific or a trispecific) CAR.

[0331]

[0222] A regulatable CAR (RCAR) is a CAR for which activity can be controlled is desirable to optimize the safety and efficacy of a CAR therapy. There are many ways CAR activities can be regulated. For example, inducible apoptosis using, e.g., a caspase fused to a dimerization domain, can be used as a safety switch in the CAR therapy of the instant invention. In some embodiments, the CAR of the invention is a split CAR. The split CAR approach is described in more detail in publications WO2014 / 055442 and WO2014 / 055657.

[0332]

[0223] Once a CAR described herein is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following target binding, sustain T cell expansion in the absence of re- stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of CARs of the present invention are known and available to one skilled in the art and are described herein, including in the examples hereof. In vitro expansion of CAR-T cells following antigen stimulation can be measured by flow cytometry. Animal models can also be used to measure a CAR’s activity. For example, a xenograft model such as that utilized herein can be utilized. Assessment of cell proliferation and cytokine production has been previously described and is shown and utilized in the examples provided herein. Imaging technologies can be used to evaluate specific trafficking and proliferation of CARs in tumor-bearing animal models.

[0333] BISPECIFIC T CELL ENGAGERS (BiTEs)

[0334]

[0224] A BiTE generally refers to a single polypeptide chain molecule that has two antigen binding sites, one of which binds to an immune effector cell antigen (e.g., CD3) and the second of which binds to an antigen present on the surface of a target cell, e.g., MSLN. When both targets are engaged, the BiTE molecule forms a bridge between the cytotoxic T cell and the tumor cell, which enables the T cell to recognize the tumor cell and fight it through an infusion of toxic molecules. The tumor-binding arm of the molecule can be altered to create different BiTE constructs that target different types of cancer. BiTEs are typically produced as recombinant, glycosylated proteins secreted by higher eukaryotic cell lines. Accordingly, in another embodiment of this invention, the protein of the invention is a BiTE.

[0335]

[0225] MSLN BiTEs can be constructed by one skilled in the art using MSLN binding sequences, including scFvs, provided herein, or based on MSLN binding sequences, antibodies, active fragments thereof, domains thereof, that are otherwise available or known in the art. Available or alternative MSLN binding sequences / antibodies include the SSI and M5 sequences. Other MSLN binders include SSI, M5, 15B6, P4 and MH1 (21, 23, 24, 27, 33) Chowdhury PS et al (1998) Proc Natl Acad Sci USA 95(2):669-74; Ho M et al (2011) Int J Cancer 128(9):2020-30; Haas AR, Golden RJ, Litzky LA, et al. Two cases of severe pulmonary toxicity from highly active mesothelin-directed CAR T cells. Mol Ther. 2023;31(8):2309-25.Atty. Docket No. 2745-9 PCTT

[0336] Liu X et al (2022) Proc Natl Acad Sci USA 119(19):e2202439119; Lanitis E et al (2012) Mol Ther 20(3):633-43; Hassan R et al (2023) Nat Med 29(8):2099-109). Cells, particularly T cells, expressing MSLN BiTEs are a particular aspect of the invention. Such cells produce MSLN-directed molecules active against other cells that are MSLN+ or in the vicinity of MSLN+ cells as bystander cells.

[0337]

[0226] Exemplary MSLN BiTEs based on the new and unique MSLN binding sequences and scFvs provided herein, particularly 13F08 and variants thereof, include SEQ ID NO:s 17, 24, 32, 33, 34, 35, 36 and 37. Exemplary MSLN BiTEs using exemplary anti-CD3 scFv sequence are provided. One skilled in the art can construct alternative MSLN BiTEs using different scFvs to replace the anti-CD3 scFv sequences.

[0338]

[0227] An exemplary BiTE provided herein is the BiTE construct, denoted 13F08_UCHT1, where UCHT1 is anti CD3 scFv. This BiTE construct is based on the 13F08 scFv (SEQ ID NO: 14), with the intra-scFv linker RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO: 13), an inter-scFv linker GSASASTGGDAS (SEQ ID NO:16), followed by the anti-CD3 scFv UCHT1. The linkers are underlined. The anti-CD3 scFv UCHT1 is shown in bold:

[0339] >13F08_UCHT1[Parental BiTE], where UCHT1 is anti CD3 scFv DIVLTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQNPRKAPRLLVYGASILESGVPSRFSGSGS GTDYTLTITSLOPEDFATYYCOOYYSTPHTFGOGTKVDIKRTVAAOARQEGGSGEGGSGESNAA AQVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNY AQKFQDWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSS GSASASTGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYY TSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGQPKA RQEGGSGEGGSGESNAAAEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYTMNWVROAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGY YGDSDWYFDVWGQGTLVTVSS (SEQ ID NO: 17)

[0340]

[0228] Another exemplary BiTE in an aspect of the invention is provided below and is based on a variant heavy and light chain VH and VK sequence. This BiTE construct is based on the variant 13F08 scFv, with the intra-scFv linker RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO: 13), an inter-scFv linker GSASASTGGDAS (SEQ ID NO:16), followed by the anti-CD3 scFv UCHT1 (SEQ ID NO:18), including the intra-scFv linker GQPKARQEGGSGEGGSGESNAAA (SEQ ID NO: 19). The linkers are underlined. The anti-CD3 scFv UCHT1 is shown in bold.

[0341] >13F08g[germ_line]_UCHT1[BiTE] DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPRLLVYGASILESGVPSRFSGSG SGTDYTLTISSLOPEDFATYYCOOYYSTPHTFGOGTKVEIKRTVAAOARQEGGSGEGGSGESNAA AQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNY AQKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSS GSASASTGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYY TSRLESGVPSRFSGSGSGTDYTLTISSLOPEDFATYYCOOGNTLPWTFGOGTKVEIKGOPKA ROEGGSGEGGSGESNAAAEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYTMNWVROAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGY YGDSDWYFDVWGQGTLVTVSS (SEQ ID NO 24)

[0342]

[0229] Other exemplary BiTEs include those with the intra-scFV linker GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25):Atty. Docket No. 2745-9 PCTT

[0343] >13F08_UCHT1[Parental] where UCHT1 is anti CD3 scFv DIVLTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQNPRKAPRLLVYGASILESGVPSRFSGSGS GTDYTLTITSLOPEDFATYYCOOYYSTPHTFGOGTKVDIKGGGSGGGGSGGGGSGGGGSGQVOL VQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ DWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSGSASAS TGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLES GVPSRFSGSGSGTDYTLTISSLOPEDFATYYCOOGNTLPWTFGOGTKVEIKGOPKARQEGG SGEGGSGESNAAAEVOLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVROAPGKGLE WVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSD WYFDVWGQGTLVTVSS (SEQ ID NO 32)

[0344] >13F08g[variant]_UCHTl[BiTE] where UCHT1 is anti CD3 scFv DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPRLLVYGASILESGVPSRFSGSG SGTDYTLTISSLOPEDFATYYCOOYYSTPHTFGOGTKVEIKGGGSGGGGSGGGGSGGGGSGOVO LVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKF QGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSGSASA STGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLE SGVPSRFSGSGSGTDYTLTISSLOPEDFATYYCOOGNTLPWTFGOGTKVEIKGOPKARQEG GSGEGGSGESNAAAEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYTMNWVROAPGKGLE WVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSD WYFDVWGQGTLVTVSS (SEQ ID NO 33)

[0345]

[0230] Alternative exemplary MSLN BiTEs based on a VH / VK MSLN scFv sequence, with exemplary anti-CD3 sequence include:

[0346] >13F08 parental VH / VK scFv and intra-scFv linker RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO:13) QVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQDWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSRT VAAQARQEGGSGEGGSGESNAAADIVLTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQNPR KAPRLLVYGASILESGVPSRFSGSGSGTDYTLTITSLQPEDFATYYCQQYYSTPHTFGQGTKVDIK GSASASTGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYY TSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGQPKA RQEGGSGEGGSGESNAAAEVOLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVROAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGY YGDSDWYFDVWGQGTLVTVSS (SEQ ID NO 34)

[0347] >13F08 variant VH / VK scFv and intra-scFv linker RTVAAQARQEGGSGEGGSGESNAAA (SEQ ID NO:13) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSRT VAAQARQEGGSGEGGSGESNAAADIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPG KAPRLLVYGASILESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYSTPHTFGQGTKVEIK GSASASTGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYY TSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGQPKA RQEGGSGEGGSGESNAAAEVOLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVROAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGY YGDSDWYFDVWGQGTLVTVSS (SEQ ID NO 35)

[0348] >13F08 parental VH / VK scFv and intra-scFV linker GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25):Atty. Docket No. 2745-9 PCTT

[0349] QVQLVQSGAEVKKPGASVRVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQDWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSG GGSGGGGSGGGGSGGGGSGDIVLTOSPSSLSASVGDRVTITCRASOGISNSLAWYOQNPRKAPRL LVYGASILESGVPSRFSGSGSGTDYTLTITSLQPEDFATYYCQQYYSTPHTFGQGTKVDIKGSASA STGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLE SGVPSRFSGSGSGTDYTLTISSLOPEDFATYYCOOGNTLPWTFGOGTKVEIKGOPKARQEG GSGEGGSGESNAAAEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYTMNWVROAPGKGLE WVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSD WYFDVWGQGTLVTVSS (SEQ ID NO 36)

[0350] >13F08 variant VH / VK scFv and intra-scFV linker GGGSGGGGSGGGGSGGGGSG (SEQ ID NO:25): QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHPDDAFDIWGQGTMVTVSSG GGSGGGGSGGGGSGGGGSGDIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPR LLVYGASILESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYSTPHTFGQGTKVEIKGSAS ASTGGDASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRL ESGVPSRFSGSGSGTDYTLTISSLOPEDFATYYCOOGNTLPWTFGOGTKVEIKGOPKARQE GGSGEGGSGESNAAAEVOLVESGGGLVOPGGSLRLSCAASGYSFTGYTMNWVROAPGKG LEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGD SDWYFDVWGQGTLVTVSS (SEQ ID NO 37)

[0351]

[0231] Additional BiTE constructs based on alternative anti-MSLN scFvs are provided and contemplated herein, as well as cells, particularly T cells, expressing these BiTE constructs. In particular, M5, SSI and 15B6-based BiTEs are described as follows. The example provided herein describes binding activity and evaluation of scFv-FC constructs based on SSI and M5 and also BITE constructs based on SSI.

[0352]

[0232] M5-based BITE (W02015090230A1; Haas AR et al (2023) Mol Ther31(8):2309-25); M5 scFv is in the VH-VK orientation. Inter-scFv linker (underlined) ASTGGDAS (SEQ ID NO:38) is truncated by 3 AAs relative to the VL-VH 13F08(g) linker (GSASASTGGDAS). UCHTl(anti CD3 scFv) sequence is shown in bold.

[0353] > M5_ BiTE[M5-UCHTl] QVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNSGGTNYA QKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGG GSGGGGSGGGGSDIVMTQSPSSLSASVGDRVTITCRASQSIRYYLSWYQQKPGKAPKLLIYTASIL QNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQTYTTPDFGPGTKVEIKASTGGDASDIQMT QSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSG TDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGQPKARQEGGSGEGGSGESNAA AEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVST YNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLV TVSSAPTKASQASEQKLISEFELNGHHHHHHH* (SEQ ID NO 39)

[0354]

[0233] SS 1 (murine anti-MSLN) (Chowdhury PS et al (1998) Proc Natl Acad Sci USA 95(2):669-74) SSI scFv is in the VH-VK orientation. Inter-scFv linker (underlined) ASTGGDAS (SEQ ID NO:38). UCHTl(anti CD3 scFv) sequence is shown in bold.

[0355] > SS1 _BiTE[SSl-UCHTl]Atty. Docket No. 2745-9 PCTT

[0356] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKF RGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGG GSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGV PGRFSGSGSGNSYSLTISSVEAEDDATYYCOOWSGYPLTFGAGTKLEIKASTGGDASDIOMTOS PSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTD YTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGQPKARQEGGSGEGGSGESNAAAE VQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTY NQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVT VSSAPTKASQASEQKLISEFELNGHHHHHHH* (SEQ ID NO 40)

[0357]

[0234] 15B6 (murine; recognizes the membrane proximal stalk linear epitope; US 11390683-B2; crystal structure sequence 7UBC). 15B06 scFv is in the VL-VH orientation. Inter-scFv linker underlined (GSASASTGGDAS) (SEQ ID NO: 16). UCHT1 (anti CD3 scFv) sequence is shown in bold.

[0358] >15B6_ BiTE[15B6-UCHTl] QAVVTQESALTTSPGETVTLTCRSSTGAVTTGNYPNWVQEKPDHLFTGLIAGTNNRAPGVPARF SGSLIGDKAALTITGAQTEDEAIYFCALWFSSHWVFGGGTKLTVLGQPKSSQGGGGSGGGGSGG GGSEVQLQQSGPVLVKPGASVKISCKASGYSFTGYYMHWVRQSNGKSLEWIGRINPYTGVPSYK HNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCARELGGYWGQGTTLTVSSGSASASTGGD ASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPS RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGQPKARQEGGSGEG GSGESNAAAEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALI NPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDV WGQGTLVTVSSAPTKASQASEQKLISEFELNGHHHHHHH* (SEQ ID NO:41)

[0359]

[0235] An alternative 15B6 BiTE based on a variant VL-VH sequence with a VH framework 2 variant sequence is contemplated. This variant has been used for active CAR-T constructs. Variant amino acids are shown as underlined and bold.

[0360] QAVVTQESALTTSPGETVTLTCRSSTGAVTTGNYPNWVQEKPDHLFTGLIAGTNNRAPGVPARF SGSLIGDKAALTITGAQTEDEAIYFCALWFSSHWVFGGGTKLTVLGQPKSSQGGGGSGGGGSGG GGSEVQLQQSGPVLVKPGASVKISCKASGYSFTGYYMHWVRQSLVKRLEWIGRINPYTGVPSYK HNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCARELGGYWGQGTTLTVSSGSASASTGGD ASDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPS RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGQPKARQEGGSGEG GSGESNAAAEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALI NPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDV WGQGTLVTVSSAPTKASQASEQKLISEFELNGHHHHHHH* (SEQ ID NO 42)

[0361]

[0236] Exemplary BiTEs provided herein utilize the immune effector cell antigen (such as for example, CD3) as the second antigen binding site. One skilled in the art can construct alternative MSLN BiTEs using different scFvs to replace the anti-CD3 scFv sequences.

[0362]

[0237] Certain BiTEs provided herein utilize the UCHT1 anti-CD3. In other embodiments, alternative CD3 binding sequences may be utilized by one skilled in the art. Thus, the VH and VL regions of the CD3 specific domain can be derived from a CD3 specific antibody known and available in the art. In some aspects, the CD3 specific antibody may be selected from the X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, Flll-409, CLB-T3.4.2, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIIL46, XIII-Atty. Docket No. 2745-9 PCTT

[0363] 87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, 0KT3D, M-T301, SMC2 and F101.01. These CD3-specific antibodies are well known in the art and for example described in Tunnacliffe (1989), Int. Immunol. 1, 546-550. In another embodiment, said VH and VL regions of said CD3 specific domain are derived from OKT-3 or TR-66. Other VH and VL regions are or are derived from an antibody or antibody derivative specifically directed against CD3 described by Traunecker (1991), EMBO J. 10, 3655-3659. In accordance with this invention, said VH and VL regions are derived from antibodies or antibody derivatives and the like which are capable of specifically recognizing human CD3 epsilon in the context of other TCR subunits, e.g. in mouse T cells transgenic for human CD3 epsilon. These transgenic mouse cells express human CD3 epsilon in a native or near native conformation.

[0364]

[0238] Additional anti-CD3 scFvs (T cell activation) are available and known that can be used as alternatives to the anti-CD3 sequence UCHT1. These can be assembled as scFvs in either VH-VL(K) or VL(K)-VH orientation, and appended to 13F08 scFv, or to other alternative MSLN-binding scFvs. Some VH and VK / VL sequences of other alternative CD3 binders are provided below:

[0365]

[0239] Blinatumomab_aCD3 (derived from OKT3)

[0366] > VH DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQK FKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (SEQ ID NO:43)

[0367] > VK DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGS GSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO:44)

[0368]

[0240] Mosunetuzumab_aCD3

[0369] > VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWIGWIYPGDGNTKYNE KEKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSS (SEQ ID NO:45)

[0370] > VK DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVP DRESGSGSGTDETLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIK (SEQ ID NO:46)

[0371]

[0241] Glofitamab_aCD3

[0372] > VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYA DSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

[0373] (SEQ ID NO:47)

[0374] > VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARF SGS LLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO:48)

[0375]

[0242] Epcoritamab_aCD3Atty. Docket No. 2745-9 PCTT

[0376] > VH EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYY ADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVS

[0377] S (SEQ ID NO:49)

[0378] > VL QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARF SGSLIGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVL (SEQ ID NO:50)

[0379]

[0243] Odronextamab_aCD3_derived in house from Velocimouse

[0380] > VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADS VKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDVWGQGTTVTVAS

[0381] (SEQ ID NO:51)

[0382] > VK EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSG SGTEFTLTISSLQSEDFAVYYCQHYINWPLTFGGGTKVEIK (SEQ ID NO:52)

[0383]

[0244] Tarlatamab_aCD3_ (I2C)

[0384] > VH EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYY ADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

[0385] (SEQ ID NO:53)

[0386] > VL QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFS GSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL (SEQ ID NO:54)

[0387]

[0245] Talquetamab_aCD3 (variant of I2C)

[0388] > VH EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYY AASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

[0389] (SEQ ID NO:55)

[0390] > VL QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARF SGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO:56)

[0391]

[0246] Xaluritamig_aCD3 (variant of I2C)

[0392] > VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYY ADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGDSYVSWFAYWGQGTLVTVSS

[0393] (SEQ ID NO:57)

[0394] > VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIGGTNKRAPGVPARF SGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFGGGTKLTVL (SEQ ID NO:58)

[0395]

[0247] In an important aspect, the invention provides cells, particularly T cells, engineered to secrete or express an anti-MSLN+ BiTE. In one aspect, the cells, particularly T cells, are engineered to secrete orAtty. Docket No. 2745-9 PCTT

[0396] express an anti-MSLN+ BiTE comprising the MSLN binding sequences described herein, including the exemplified MSLN antibodies or derivatives provided.

[0397]

[0248] In another aspect, the invention provides cells expressing a BiTE of the invention. In an aspect, the invention provides cells expressing an MSLN-directed BiTE. The cells can particularly be T cells. In an aspect the BiTE expressing cells, particularly T cells, engage, bind and / or target MSLN expressing cells, including tumor, cancer etc cells. In an aspect of the invention, MSLN-directed BiTE expressing cells stimulate or otherwise direct the killing bystander cells, including those of a tumor or cancer.

[0398]

[0249] Thus, MSLN-directed BiTE-T cells are an embodiment of the invention. In one such aspect, T cells engineered to express MSLN-directed BiTEs are provided. In an aspect, the BiTE is continuously produced by the engineered T cell and can arm itself and bystander T cells. This production may be enhanced in the TME as the BiTE-T is activated and upregulates its protein synthesis which is beneficial to provide a significant boost of anti-tumor immunity specifically within the tumor, including as provided and demonstrated herein. The live T cell can persist long-term in patients, thus frequent or regular / repeated injection of a purified BiTE product may not be necessary, so long as the engineered T cells persist and are active.

[0399]

[0250] In some embodiments, the binding protein of the invention is conjugated to another compound. Conjugates could help extend half-life or impart other biological activites. Methods for conjugation of the binding protein (MSLN antibody or scFv etc) will be apparent to the skilled person and / or described herein. All forms and methods of conjugation (i.e., binding) are contemplated by the present invention, including, for example, direct conjugation between the binding protein and another compound / moiety as described herein or indirect binding (e.g., by virtue of a linker between the binding protein and the other compound / moiety). In one embodiment, the conjugate is formed by a chemical conjugation (e.g., by an amine bond or disulphide bond) or by genetic fusion.

[0400]

[0251] In one embodiment, the disclosure provides a fusion protein comprising the binding protein of the invention (MSLN antibody or scFv etc) and the other compound. For example, the other compound can be positioned at the N-terminus of the protein, C-terminus of the protein or any combination thereof. In one embodiment, the binding protein is conjugated to the other compound via a linker. For example, the linker can be a peptide linker. In one embodiment, the linker is a flexible linker. A “flexible” linker is an amino acid sequence which does not have a fixed structure (secondary or tertiary structure) in solution. Such a flexible linker is therefore free to adopt a variety of conformations. Flexible linkers suitable for use in the present invention are known in the art. The linker may comprise any amino acid sequence that does not substantially hinder interaction of the binding region with its target. Preferred amino acid residues for flexible linker sequences include, but are not limited to, glycine, alanine, serine, threonine proline, lysine, arginine, glutamine and glutamic acid.Atty. Docket No. 2745-9 PCTT

[0401]

[0252] The linker sequences between the binding regions preferably comprise five or more amino acid residues. The flexible linker sequences according to the present invention consist of 5 or more residues, preferably, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or 25 or 30 or more residues. In an embodiment of the invention, the flexible linker sequences consist of 5, 7, 10, 13 or 16 or 30 residues.

[0402]

[0253] Exemplary compounds that can be conjugated to a binding protein of the invention may be selected from human serum albumin or functional fragment thereof, an immunoglobulin Fc region or functional fragment thereof, afamin, alpha-fetoprotein, vitamin D binding protein, antibody fragments that bind to albumin and polymers.

[0403]

[0254] In particular embodiments, the binding protein, antibody, antibody derivative is conjugated to an engager protein. In one such embodiment, the binding protein, antibody, antibody derivative is conjugated to Fc, either as single peptides or directionally assembled heterominers (eg. “knobs into holes” Fc modules). Such Fc fusions and Fc fusion strategies provide a soluble druggable option, for example versus BiTEs. MSLN-binding domain-Fc fusions are contemplated and were generated and tested as provided herein. Alternative such Fc type fusions and Fc conjugated molecules are thus contemplated and included in the invention.

[0404]

[0255] In an embodiment, the binding protein is conjugated to a cytotoxic agent. Cytotoxic agents include any agent that is detrimental to the growth, viability or propagation of cells. Examples of suitable cytotoxic agents and chemotherapeutic agents that can be conjugated to binding proteins in accordance with this aspect of the invention include, e.g., l-(2chloroethyl)-l,2-dimethanesulfonyl hydrazide, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyne-13-one, 1 -dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-amino camptothecin, actinomycin D, amanitins, aminopterin, anguidine, anthracycline, anthramycin (AMC), auristatins, bleomycin, busulfan, butyric acid, calicheamicins, camptothecin, carminomycins, carmustine, cemadotins, cisplatin, colchicin, combretastatins, cyclophosphamide, cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine, diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracin dione, disorazoles, dolastatin, doxorubicin, duocarmycin, echinomycins, eleutherobins, emetine, epothilones, esperamicin, estramustines, ethidium bromide, etoposide, fluorouracils, geldanamycins, gramicidin D, glucocorticoids, irinotecans, leptomycins, leurosines, lidocaine, lomustine (CCNU), maytansinoids, mechlorethamine, melphalan, mercatopurines, methopterins, methotrexate, mithramycin, mitomycin, mitoxantrone, N8-acetyl spermidine, podophyllotoxins, procaine, propranolol, pteridines, puromycin, pyrrolobenzodiazepines (PDBs), rhizoxins, streptozotocin, tally somycins, taxol, tenoposide, tetracaine, thioepa chlorambucil, tomaymycins, topotecans, tubulysin, vinblastine, vincristine, vindesine, vinorelbines, and derivatives of any of the foregoing. Other cytotoxic agents known in the art are contemplated within the scope of the present invention, including, e.g., protein toxins such ricin, C. difficile toxin, pseudomonas exotoxin, ricin,Atty. Docket No. 2745-9 PCTT

[0405] diphtheria toxin, botulinum toxin, bryodin, saporin, pokeweed toxins (i.e., phytolaccatoxin and phytolaccigenin).

[0406]

[0256] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For intravenous, injection, or injection at the site of affliction, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and / or other additives may be included, as required.

[0407]

[0257] A composition may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the binding protein of the invention and other agents or therapeutics such as anti-cancer agents or therapeutics, hormones, anti-mitotic agents, anti-apoptotic agents, antibodies, or immune modulators. More generally these anti-cancer agents may be but are not limited to tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as nonsteroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. The composition can be administered in combination (either sequentially (i.e. before or after) or simultaneously) with tyrosine kinase inhibitors (including, but not limited to AG1478 andZD1839, STI571, OSI-774, SU-6668), doxorubicin, temozolomide, cisplatin, carboplatin, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5 -fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, lomustine, and / or other chemotherapeutic agents. Thus, these agents may be specific anti -cancer agents, or immune cell response modulators or may be more general anti-cancer and anti-neoplastic agents such as doxorubicin, cisplatin, temozolomide, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5 -fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, or lomustine. In addition, the composition may be administered with hormones such as dexamethasone, immune modulators, such as interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, cytokines, agonist or antagonist antibodies to regulators of immune response which stimulate, enhance, or derepress the immune response and reduction or elimination ofAtty. Docket No. 2745-9 PCTT

[0408] cancer cells or tumors. The composition may also be administered with, or may include combinations along with anti-tumor antigen antibodies.

[0409]

[0258] The present invention further provides an isolated nucleic acid encoding an antibody, derivative, binding agent, ligand, or an scFv, CAR or BiTE, of the present invention. Nucleic acid includes DNA and RNA. In a preferred aspect, the present invention provides a nucleic acid which codes for a polypeptide of the invention as defined above, including any polypeptides as set out herein.

[0410]

[0259] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above. The present invention also provides a recombinant host cell which comprises one or more constructs as above. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a binding protein may be isolated and / or purified using any suitable technique, then used as appropriate.

[0411]

[0260] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate.

[0412]

[0261] Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. The present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a binding member polypeptide. Another feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host. A wide variety of host / expression vector combinations may be employed in expressing the DNA sequences of this invention.

[0413]

[0262] It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention.Atty. Docket No. 2745-9 PCTT

[0414]

[0263] Antibodies and fragments of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member. Thus pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous, or by deposition at a tumor site.

[0415]

[0264] The binding members and antibodies of the present invention, and in a particular embodiment the antibody having sequence represented herein, or active fragments thereof, and single chain, recombinant or synthetic antibodies derived therefrom, and particularly antibody comprising the heavy chain CDR region sequences and the light chain CDR region sequences as provided herein, can be prepared in pharmaceutical compositions, including a suitable vehicle, carrier or diluent, or including an adjuvant and / or immune modulator, for administration in instances wherein therapy is appropriate, such as to treat cancer or stimulate or enhance immune response, including immune response against cancer. Such pharmaceutical compositions may also include means for modulating the half-life of the binding members, antibodies or fragments by methods known in the art such as pegylation. Such pharmaceutical compositions may further comprise additional antibodies or therapeutic agents.

[0416]

[0265] A composition of the present invention may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the binding member, particularly antibody or fragment thereof, herein described and other agents or therapeutics such as anti-cancer agents or therapeutics, anti-mitotic agents, apoptotic agents or antibodies, or immune modulators, or small molecule inhibitors to immune modulators. More generally these anti-cancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti -mitotic s), inhibitors or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. In addition, the composition may be administered with immune modulators, such as a-galactosyl ceramide, interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, cytokines or hormones which stimulate the immune response and reduction or elimination of cancer cells or tumors. The composition may be administered with an immune modulator such as an adjuvant. The composition may also be administered with, or may include combinations along with other anti-TGFp antibodies, other immunomodulatoryAtty. Docket No. 2745-9 PCTT

[0417] antibodies or other anti-tumor antigen antibodies. In an aspect, the composition is administered in combination with another antibody, particularly an anti-tumor antigen antibody.

[0418]

[0266] A composition may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the binding member, particularly antibody or fragment thereof, herein described and other agents or therapeutics such as anticancer agents or therapeutics, hormones, anti-mitotic agents, anti-apoptotic agents, antibodies, or immune modulators. More generally these anti -cancer agents may be but are not limited to tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti -mitotic s), or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. The composition can be administered in combination (either sequentially (i.e. before or after) or simultaneously) with tyrosine kinase inhibitors (including, but not limited to AG1478 and ZD1839, STI571, OSI-774, SU-6668), doxorubicin, temozolomide, cisplatin, carboplatin, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, lomustine, and / or other chemotherapeutic agents. Thus, these agents may be specific anti-cancer agents, or immune cell response modulators or may be more general anti-cancer and anti-neoplastic agents such as doxorubicin, cisplatin, temozolomide, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, or lomustine. In addition, the composition may be administered with hormones such as dexamethasone, immune modulators, such as interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, cytokines, agonist or antagonist antibodies to regulators of immune response which stimulate, enhance, or derepress the immune response and reduction or elimination of cancer cells or tumors. The composition may also be administered with, or may include combinations along with other anti-tumor antigen antibodies.

[0419]

[0267] In addition, the present invention contemplates and includes therapeutic compositions for the use of the antibody(ies) or fragments in combination with conventional radiotherapy.

[0420]

[0268] The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of this invention. A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a specific binding member or antibody, polypeptide analog thereof or fragment thereof, as described herein as an active ingredient. In an embodiment, the composition comprises an antigen capable of modulating the specific binding of the present binding member / antibody with a target cell. In an embodiment the composition comprises an antigen or vaccine formulation, particularly a tumor antigen or cancer vaccine.Atty. Docket No. 2745-9 PCTT

[0421]

[0269] The preparation of therapeutic compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions. However, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

[0422]

[0270] A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0423]

[0271] The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of peptide / MHC or tumor antigen binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. Suitable regimes for initial administration and follow on administration are also variable, and may include an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain appropriate and sufficient concentrations in the blood or at the site of desired therapy is contemplated.

[0424]

[0272] The present invention also relates to a variety of diagnostic applications, including methods for detecting the expression of or elevated presence of MSLN, MSLN-mediated cancer, or cancer more generally, evaluating the presence or amount of MSLN-expressing or -responsive cells, by reference to their ability to be recognized by the present specific binding member(s). Peptide complexes can be identified, targeted, labeled, and / or quantitated on cells, including immune cells and / or tumor cells.

[0425] TABLE OF SEQUENCES SEQ DESCRIPTION Nuclei SPECIES SEQUENCE

[0426] ID c acid

[0427]

[0428] NO orAtty. Docket No. 2745-9 PCTT

[0429] amino

[0430] acid

[0431] 13F08 VH amino Homo OVOLVOSGAEVKKPGASVRVSCKASGYTFTGYY acid sapiens MHWVROAPGOGLEWMGWINPNSGGTNYAOKF QDWVTMTRDTSISTAYMELSRLRSDDTAVYYCA REIYSGSHPDDAFDIWGQGTMVTVSS

[0432] 13F08 VK amino Homo DIVLTOSPSSLSASVGDRVTITCRASQGISNSLAW acid sapiens YOONPRKAPRLLVYGASILESGVPSRFSGSGSGTD YTLTITSLOPEDFATYYCOOYYSTPHTFGOGTKV DIK

[0433] Heavy chain amino Homo GYTFTGYY

[0434] CDR1 acid sapiens

[0435] Heavy chain amino Homo GYTFTGYYMH

[0436] CDR1 acid sapiens

[0437] Heavy chain amino Homo INPNSGGT

[0438] CDR2 acid sapiens

[0439] Heavy chain amino Homo WINPNSGGTNYAQKFQD

[0440] CDR2 acid sapiens

[0441] Heavy chain Amin Homo AREIYSGSHPDDAFDI

[0442] CDR3 o acid sapiens

[0443] Light chain Amin Homo QGISNS

[0444] CDR1 o acid sapiens

[0445] Light chain Amin Homo RASQGISNSLA

[0446] CDR1 o acid sapiens

[0447] Light chain Amin Homo GAS

[0448] CDR2 o acid sapiens

[0449] Light chain Amin Homo GASILES

[0450] CDR2 o acid sapiens

[0451] Light chain Amin Homo QQYYSTPHT

[0452] CDR3 o acid sapiens

[0453] Intra-scFv linker Amin Artificial RTVAAQARQEGGSGEGGSGESNAAA

[0454] o acid sequence

[0455] 13F08 parental Amin Artificial DIVLTQSPSSLSASVGDRVTITCRASQGISNSLAWY scFv o acid sequence QQNPRKAPRLLVYGASILESGVPSRFSGSGSGTDY VK / VH TLTITSLQPEDFATYYCQQYYSTPHTFGQGTKVDI scFv KRTVAAQAROEGGSGEGGSGESNAAAOVOLVOS sequence GAEVKKPGASVRVSCKASGYTFTGYYMHWVRQ APGQGLEWMGWINPNSGGTNYAQKFQDWVTMT RDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHP DDAFDIWGQGTMVTVSS

[0456] 13F08 scFv Amin Artificial QVQLVQSGAEVKKPGASVRVSCKASGYTFTGYY VH / VL o acid sequence MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ DWVTMTRDTSISTAYMELSRLRSDDTAVYYCAR

[0457] scFv EIYSGSHPDDAFDIWGOGTMVTVSSRTVAAOARQ sequence EGGSGEGGSGESNAAADIVLTOSPSSLSASVGDRV TITCRASQGISNSLAWYQQNPRKAPRLLVYGASIL ESGVPSRFSGSGSGTDYTLTITSLQPEDFATYYCQ

[0458]

[0459] QYYSTPHTFGQGTKVDIKAtty. Docket No. 2745-9 PCTT

[0460] Inter-scFv linker Amin Artificial GSASASTGGDAS

[0461] o acid sequence

[0462] Linker

[0463] 13F08_UCHT1 Amin Artificial DIVLTQSPSSLSASVGDRVTITCRASQGISNSLAWY parental BiTE o acid sequence QQNPRKAPRLLVYGASILESGVPSRFSGSGSGTDY TLTITSLQPEDFATYYCQQYYSTPHTFGQGTKVDI KRTVAAQAROEGGSGEGGSGESNAAAOVOLVOS GAEVKKPGASVRVSCKASGYTFTGYYMHWVRQ APGQGLEWMGWINPNSGGTNYAQKFQDWVTMT RDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHP DDAFDIWGOGTMVTVSSGSASASTGGDASDIOM TQSPSSLSASVGDRVTITCRASQDIRNYLNWYQ QKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTD YTLTISSLQPEDFATYYCQQGNTLPWTFGQGT KVEIKGOPKARQEGGSGEGGSGESNAAAEVOL VESGGGLVQPGGSLRLSCAASGYSFTGYTMNW VRQAPGKGLEWVALINPYKGVSTYNQKFKDRF TISVDKSKNTAYLQMNSLRAEDTAVYYCARSG YYGDSDWYFDVWGQGTLVTVSS

[0464] anti-CD3 scFv Amin DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNW UCHT1 o acid YQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTD YTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKV EIKGOPKARQEGGSGEGGSGESNAAAEVOLVESG GGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVDKS KNTAYLQMNSLRAEDTAVYYCARSGYYGDSDW YFDVWGQGTLVTVSS

[0465] Intra-scFv linker Amin Artificial GQPKARQEGGSGEGGSGESNAAA

[0466] o acid sequence

[0467] Linker

[0468] 13F08 VH Amin Homo OVOLVOSGAEVKKPGASVAVSCKASGYTFTGYY variant o acid sapiens MHWVROAPGOGLEWMGWINPNSGGTNYAOKF QGWVTMTRDTSISTAYMELSRLRSDDTAVYYCA REIYSGSHPDDAFDIWGQGTMVTVSS

[0469] 13F08 VK Amin Homo DIGMTOSPSSLSASVGDRVTITCRASOGISNSLAW variant o acid sapiens YOQ / fPGKAPRLLVYGASILESGVPSRFSGSGSGTD YTLTISSLOPEDFATYYCOOYYSTPHTFGOGTKV EIK

[0470] Variant heavy Amin Homo WINPNSGGTNYAQKFQG

[0471] chain CDR2 o acid sapiens

[0472] 13F08g variant Amin Artificial DlgMTQSPSSLSASVGDRVTITCRASQGISNSLAW scFV o acid sequence YQQA'PGKAPRLLVYGASILESGVPSRFSGSGSGTD YTLTLS’SLQPEDFATYYCQQYYSTPHTFGQGTKVE scFv IKRTVAAQAROEGGSGEGGSGESNAAAOVOLVO sequence SGAEVKKPGASVXVSCKASGYTFTGYYMHWVRQ APGQGLEWMGWINPNSGGTNYAQKFQGWVTMT RDTSISTAYMELSRLRSDDTAVYYCAREIYSGSHP

[0473]

[0474] DDAFDIWGQGTMVTVSSAtty. Docket No. 2745-9 PCTT

[0475] 13F08g Amin Artificial DIQMTQSPSSESASVGDRVTITCRASQGISNSEAW variant_UCHT o acid sequence YQQKPGKAPREEVYGASIEESGVPSRFSGSGSGTD BiTE YTETISSEQPEDFATYYCQQYYSTPHTFGQGTKVE BiTE IKRTVAAQAROEGGSGEGGSGESNAAAOVOLVO construct SGAEVKKPGASVKVSCKASGYTFTGYYMHWVR QAPGQGEEWMGWINPNSGGTNYAQKFQGWVTM TRDTSISTAYMEESRERSDDTAVYYCAREIYSGSH PDDAFDIWGOGTMVTVSSGSASASTGGDASDIO MTQSPSSLSASVGDRVTITCRASQDIRNYLNWY QQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQQGNTLPWTFGQG TKVEIKGQPKARQEGGSGEGGSGESNAAAEVO LVESGGGLVQPGGSLRLSCAASGYSFTGYTMN WVRQAPGKGLEWVALINPYKGVSTYNQKFKD RFTISVDKSKNTAYLQMNSLRAEDTAVYYCAR SGYYGDSDWYFDVWGQGTLVTVSS

[0476] Intra-scFv linker Amin Artificial GGGSGGGGSGGGGSGGGGSG

[0477] o acid sequence

[0478] 13F08 parental Amin Artificial DIVETQSPSSESASVGDRVTITCRASQGISNSEAWY scFv o acid sequence QQNPRKAPREEVYGASIEESGVPSRFSGSGSGTDY TETITSEQPEDFATYYCQQYYSTPHTFGQGTKVDI

[0479] scFv KGGGSGGGGSGGGGSGGGGSGOVOEVOSGAEV KKPGASVRVSCKASGYTFTGYYMHWVRQAPGQ GEEWMGWINPNSGGTNYAQKFQDWVTMTRDTSI STAYMEESRERSDDTAVYYCAREIYSGSHPDDAF DIWGQGTMVTVSS

[0480] 13F08g variant Amin Artificial DIQMTQSPSSESASVGDRVTITCRASQGISNSEAW scFV o acid sequence YQQKPGKAPREEVYGASIEESGVPSRFSGSGSGTD YTETISSEQPEDFATYYCQQYYSTPHTFGQGTKVE

[0481] scFv IKGGGSGGGGSGGGGSGGGGSGQVOLVQSGAEV KKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GEEWMGWINPNSGGTNYAQKFQGWVTMTRDTSI STAYMEESRERSDDTAVYYCAREIYSGSHPDDAF DIWGQGTMVTVSS

[0482] 13F08 variant Amin Artificial QVQEVQSGAEVKKPGASVKVSCKASGYTFTGYY scFv o acid sequence MHWVRQAPGQGEEWMGWINPNSGGTNYAQKFQ VH / VK GWVTMTRDTSISTAYMEESRERSDDTAVYYCAR scFv EIYSGSHPDDAFDIWGQGTMVTVSSRTVAAQARQ EGGSGEGGSGESNAAADIQMTQSPSSESASVGDR VTITCRASQGISNSEAWYQQKPGKAPREEVYGASI EESGVPSRFSGSGSGTDYTETISSEQPEDFATYYCQ QYYSTPHTFGQGTKVEIK

[0483] 13F08g parental Amin Artificial QVQEVQSGAEVKKPGASVRVSCKASGYTFTGYY scFV o acid sequence MHWVRQAPGQGEEWMGWINPNSGGTNYAQKFQ VH / VK DWVTMTRDTSISTAYMEESRERSDDTAVYYCAR scFv EIYSGSHPDDAFDIWGOGTMVTVSSGGGSGGGGS GGGGSGGGGSGDIVETOSPSSESASVGDRVTITCR ASQGISNSEAWYQQNPRKAPREEVYGASIEESGVP SRFSGSGSGTDYTETITSEQPEDFATYYCQQYYST

[0484]

[0485] PHTFGQGTKVDIKAtty. Docket No. 2745-9 PCTT

[0486] 13F08g variant Amin Artificial QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYY scFV o acid sequence MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ VH / VK GWVTMTRDTSISTAYMELSRLRSDDTAVYYCAR scFv EIYSGSHPDDAFDIWGQGTMVTVSSGGGSGGGGS GGGGSGGGGSGDIQMTQSPSSLSASVGDRVTITCR ASQGISNSLAWYQQKPGKAPRLLVYGASILESGV PSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYS TPHTFGQGTKVEIK

[0487] Human MSLN Amin human evektacpsgkkareideslifykkweleacvdaallatqmdrvnaipftye DI epitope o acid qldvlkhkldelypqgypesviqhlgylflkmspedirkwnvt region

[0488] 13F08 parental Amin Artificial DIVLTQSPSSLSASVGDRVTITCRASQGISNSLAWY BiTE with scFv o acid sequence QQNPRKAPRLLVYGASILESGVPSRFSGSGSGTDY VK / VH TLTITSLQPEDFATYYCQQYYSTPHTFGQGTKVDI BiTE KGGGSGGGGSGGGGSGGGGSGOVOLVOSGAEV KKPGASVRVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPNSGGTNYAQKFQDWVTMTRDTSI STAYMELSRLRSDDTAVYYCAREIYSGSHPDDAF DIWGOGTMVTVSSGSASASTGGDASDIOMTOSPS SLSASVGDRVTITCRASQDIRNYLNWYQQKPG KAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTI SSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK GOPKAROEGGSGEGGSGESNAAAEVOLVESGG GLVQPGGSLRLSCAASGYSFTGYTMNWVRQAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVD KSKNTAYLQMNSLRAEDTAVYYCARSGYYGDS DWYFDVWGQGTLVTVSS

[0489] 13F08 variant Amin Artificial DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAW BiTE with scFv o acid sequence YQQKPGKAPRLLVYGASILESGVPSRFSGSGSGTD VK / VH YTLTISSLQPEDFATYYCQQYYSTPHTFGQGTKVE BiTE IKGGGSGGGGSGGGGSGGGGSGOVOLVOSGAEV KKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPNSGGTNYAQKFQGWVTMTRDTSI STAYMELSRLRSDDTAVYYCAREIYSGSHPDDAF DIWGOGTMVTVSSGSASASTGGDASDIOMTQSPS SLSASVGDRVTITCRASQDIRNYLNWYQQKPG KAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTI SSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK GOPKAROEGGSGEGGSGESNAAAEVOLVESGG GLVQPGGSLRLSCAASGYSFTGYTMNWVRQAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVD KSKNTAYLQMNSLRAEDTAVYYCARSGYYGDS DWYFDVWGQGTLVTVSS

[0490] 13F08 parental Amin Artificial QVQLVQSGAEVKKPGASVRVSCKASGYTFTGYY BiTE with scFv o acid sequence MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ VH / VK DWVTMTRDTSISTAYMELSRLRSDDTAVYYCAR BiTE EIYSGSHPDDAFDIWGOGTMVTVSSRTVAAOARQ EGGSGEGGSGESNAAADIVLTOSPSSLSASVGDRV TITCRASQGISNSLAWYQQNPRKAPRLLVYGASIL

[0491]

[0492] ESGVPSRFSGSGSGTDYTLTITSLQPEDFATYYCQAtty. Docket No. 2745-9 PCTT

[0493] OYYSTPHTFGOGTKVDIKGSASASTGGDASDIOM TQSPSSLSASVGDRVTITCRASQDIRNYLNWYQ QKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTD YTLTISSLQPEDFATYYCQQGNTLPWTFGQGT KVEIKGOPKARQEGGSGEGGSGESNAAAEVOL VESGGGLVQPGGSLRLSCAASGYSFTGYTMNW VRQAPGKGLEWVALINPYKGVSTYNQKFKDRF TISVDKSKNTAYLQMNSLRAEDTAVYYCARSG YYGDSDWYFDVWGQGTLVTVSS

[0494] 13F08 variant Amin Artificial QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYY BiTE with scFv o acid sequence MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ VH / VK GWVTMTRDTSISTAYMELSRLRSDDTAVYYCAR BiTE EIYSGSHPDDAFDIWGQGTMVTVSSRTVAAQARQ EGGSGEGGSGESNAAADIQMTQSPSSLSASVGDR VTITCRASQGISNSLAWYQQKPGKAPRLLVYGASI LESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQ OYYSTPHTFGOGTKVEIKGSASASTGGDASDIOM TQSPSSLSASVGDRVTITCRASQDIRNYLNWYQ QKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTD YTLTISSLQPEDFATYYCQQGNTLPWTFGQGT KVEIKGQPKARQEGGSGEGGSGESNAAAEVQL VESGGGLVQPGGSLRLSCAASGYSFTGYTMNW VRQAPGKGLEWVALINPYKGVSTYNQKFKDRF TISVDKSKNTAYLQMNSLRAEDTAVYYCARSG YYGDSDWYFDVWGQGTLVTVSS

[0495] 13F08 parental Amin Artificial QVQLVQSGAEVKKPGASVRVSCKASGYTFTGYY BiTE with scFv o acid sequence MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ VH / VK DWVTMTRDTSISTAYMELSRLRSDDTAVYYCAR BiTE EIYSGSHPDDAFDIWGOGTMVTVSSGGGSGGGGS GGGGSGGGGSGDIVLTOSPSSLSASVGDRVTITCR ASQGISNSLAWYQQNPRKAPRLLVYGASILESGVP SRFSGSGSGTDYTLTITSLQPEDFATYYCQQYYST PHTFGOGTKVDIKGSASASTGGDASDIOMTOSPS SLSASVGDRVTITCRASQDIRNYLNWYQQKPG KAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTI SSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK GOPKAROEGGSGEGGSGESNAAAEVOLVESGG GLVQPGGSLRLSCAASGYSFTGYTMNWVRQAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVD KSKNTAYLQMNSLRAEDTAVYYCARSGYYGDS DWYFDVWGQGTLVTVSS

[0496] 13F08 variant Amin Artificial QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYY BiTE with scFv o acid sequence MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ VH / VK GWVTMTRDTSISTAYMELSRLRSDDTAVYYCAR BiTE EIYSGSHPDDAFDIWGQGTMVTVSSGGGSGGGGS GGGGSGGGGSGDIQMTQSPSSLSASVGDRVTITCR ASQGISNSLAWYQQKPGKAPRLLVYGASILESGV PSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYS TPHTFGOGTKVEIKGSASASTGGDASDIOMTQSP SSLSASVGDRVTITCRASQDIRNYLNWYQQKPG

[0497]

[0498] KAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTIAtty. Docket No. 2745-9 PCTT

[0499] SSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK GOPKAROEGGSGEGGSGESNAAAEVOLVESGG GLVQPGGSLRLSCAASGYSFTGYTMNWVRQAP GKGLEWVALINPYKGVSTYNQKFKDRFTISVD KSKNTAYLQMNSLRAEDTAVYYCARSGYYGDS DWYFDVWGQGTLVTVSS

[0500] Inter-ScFv Amin Artificial ASTGGDAS

[0501] linker o acid sequence

[0502] linker

[0503] M5_ BiTE[M5- Amin Artificial QVQLVQSGAEVEKPGASVKVSCKASGYTFTDYY UCHT1] o acid sequence MHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ GRVTMTRDTSISTAYMELSRLRSDDTAVYYCASG

[0504] BiTE WDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSG GGGSDIVMTQSPSSLSASVGDRVTITCRASQSIRY YLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCLQTYTTPDFGPGTK VEIKASTGGDASDIOMTOSPSSLSASVGDRVTIT CRASQDIRNYLNWYQQKPGKAPKLLIYYTSRL ESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQGNTLPWTFGQGTKVEIKGQPKARQEGGSG EGGSGESNAAAEVQLVESGGGLVQPGGSLRLS CAASGYSFTGYTMNWVRQAPGKGLEWVALIN PYKGVSTYNQKFKDRFTISVDKSKNTAYLQMN SLRAEDTAVYYCARSGYYGDSDWYFDVWGQG TLVTVSSAPTKASQASEQKLISEFELNGHHHHH HH SSI Amin Artificial QVQLQQSGPELEKPGASVKISCKASGYSFTGYTM _BiTE[SSl- o acid sequence NWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKA UCHT1] TLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYD BiTE GRGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSD IELTQSPAIMSASPGEKVTMTCSASSSVSYMHWY QQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNS YSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKL EIKASTGGDASDIOMTQSPSSLSASVGDRVTITC RASQDIRNYLNWYQQKPGKAPKLLIYYTSRLE SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQ QGNTLPWTFGQGTKVEIKGQPKARQEGGSGE GGSGESNAAAEVQLVESGGGLVQPGGSLRLSC AASGYSFTGYTMNWVRQAPGKGLEWVALINP YKGVSTYNQKFKDRFTISVDKSKNTAYLQMNS LRAEDTAVYYCARSGYYGDSDWYFDVWGQGT LVTVSSAPTKASQASEQKLISEFELNGHHHHHH

[0505] H

[0506] 15B6_ Amin Artificial QAVVTQESALTTSPGETVTLTCRSSTGAVTTGNYP BiTE[15B6- o acid sequence NWVQEKPDHLFTGLIAGTNNRAPGVPARFSGSLI UCHT1] GDKAALTITGAQTEDEAIYFCALWFSSHWVFGGG BiTE TKLTVLGQPKSSQGGGGSGGGGSGGGGSEVQLQ

[0507] QSGPVLVKPGASVKISCKASGYSFTGYYMHWVR

[0508]

[0509] QSNGKSLEWIGRINPYTGVPSYKHNFKDKASLTVAtty. Docket No. 2745-9 PCTT

[0510] DKSSSTAYMELHSLTSEDSAVYYCARELGGYWG QGTTLTVSSGSASASTGGDASDIQMTQSPSSLSAS VGDRVTITCRASQDIRNYLNWYQQKPGKAPKL LIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGQGTKVEIKGQPKA RQEGGSGEGGSGESNAAAEVQLVESGGGLVQP GGSLRLSCAASGYSFTGYTMNWVRQAPGKGL EWVALINPYKGVSTYNQKFKDRFTISVDKSKNT AYLQMNSLRAEDTAVYYCARSGYYGDSDWYF DVWGQGTLVTVSSAPTKASQASEQKLISEFELN GHHHHHHH

[0511] Variant 15B6 Amin Artificial QAVVTQESALTTSPGETVTLTCRSSTGAVTTGNYP BiTE o acid sequence NWVQEKPDHLFTGLIAGTNNRAPGVPARFSGSLI GDKAALTITGAQTEDEAIYFCALWFSSHWVFGGG

[0512] BiTE TKLTVLGQPKSSQGGGGSGGGGSGGGGSEVQLQ QSGPVLVKPGASVKISCKASGYSFTGYYMHWVR QSLVKRLEWIGRINPYTGVPSYKHNFKDKASLTV DKSSSTAYMELHSLTSEDSAVYYCARELGGYWG QGTTLTVSSGSASASTGGDASDIQMTQSPSSLSAS VGDRVTITCRASQDIRNYLNWYQQKPGKAPKL LIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGQGTKVEIKGQPKA RQEGGSGEGGSGESNAAAEVQLVESGGGLVQP GGSLRLSCAASGYSFTGYTMNWVRQAPGKGL EWVALINPYKGVSTYNQKFKDRFTISVDKSKNT AYLQMNSLRAEDTAVYYCARSGYYGDSDWYF DVWGQGTLVTVSSAPTKASQASEQKLISEFELN GHHHHHHH

[0513] B linatumomab_ Amin Murine DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTM aCD3 o acid HWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDK VH ATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYD DHYCLDYWGQGTTLTVSS

[0514] B linatumomab_ Amin Murine DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNW aCD3 o acid YQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGT VK SYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTK LELK

[0515] Mosunetuzumab Amin EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYI o acid HWVRQAPGQGLEWIGWIYPGDGNTKYNEKEKGR _aCD3 VH

[0516] ATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYS NY YFD YWGQGTL VT VS S

[0517] Mosunetuzumab Amin DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTR o acid KNYLAWYQQKPGQPPKLLIYWASTRESGVPDRES _aCD3 VK

[0518] GSGSGTDETLTISSLQAEDVAVYYCTQSFILRTFG QGTKVEIK

[0519] Glofitamab_aC Amin EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAM o acid NWVRQAPGKGLEWVSRIRSKYNNYATYYADSVK D3 VH

[0520] GRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRH GNFGNSYVSWFAYWGQGTLVTVSS

[0521]

[0522] Atty. Docket No. 2745-9 PCTT

[0523] Glofitamab_aC Amin QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNY o acid ANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGS D3 VK

[0524] LLGGKAALTLSGAQPEDEAEYYCALWYSNLWVF GGGTKLTVL

[0525] Epcoritamab_aC Amin EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAM o acid NWVRQAPGKGLEWVARIRSKYNNYATYYADSV D3 VH

[0526] KDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVR HGNFGNSYVSWFAYWGQGTLVTVSS

[0527] Epcoritamab_aC Amin QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYA o acid NWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSLI D3 VK

[0528] GDKAALTITGAQADDESIYFCALWYSNLWVFGG GTKLTVL

[0529] Odronextamab_ Amin EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYTM aCD3 VH o acid HWVRQAPGKGLEWVSGISWNSGSIGYADSVKGR FTISRDNAKKSLYLQMNSLRAEDTALYYCAKDNS GYGHYYYGMDVWGQGTTVTVAS

[0530] Odronextamab_ Amin EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAW aCD3 VK o acid YQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTE FTLTISSLQSEDFAVYYCQHYINWPLTFGGGTKVE IK

[0531] Tarlatamab_aC Amin EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAM D3 VH o acid NWVRQAPGKGLEWVARIRSKYNNYATYYADSV KDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISYWAYWGQGTLVTVSS

[0532] Tarlatamab_aC Amin QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP D3 VL o acid NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLL GGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGG GTKLTVL

[0533] Talquetamab_a Amin EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAM CD3 VH o acid NWVRQAPGKGLEWVARIRSKYNNYATYYAASV KGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR HGNFGNSYVSWFAYWGQGTLVTVSS

[0534] Talquetamab_a Amin QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYA CD3 VL o acid NWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLL GGKAALTLSGVQPEDEAEYYCALWYSNLWVFGG GTKLTVL

[0535] Xaluritamig_aC Amin EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAM D3 VH o acid NWVRQAPGKGLEWVGRIRSKYNNYATYYADSV KGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR HGNFGDSYVSWFAYWGQGTLVTVSS

[0536] Xaluritamig_aC Amin QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNY D3 VL o acid ANWVQQKPGKSPRGLIGGTNKRAPGVPARFSGSL LGGKAALTISGAQPEDEADYYCALWYSNHWVFG GGTKLTVL

[0537] 13F08g scFv Amin DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAW with C-terminal o acid YQQKPGKAPRLLVYGASILESGVPSRFSGSGSGTD myc / his tags YTLTISSLQPEDFATYYCQQYYSTPHTFGQGTKVE IKRTVAAQARQEGGSGEGGSGESNAAAQVQLVQ SGAEVKKPGASVKVSCKASGYTFTGYYMHWVR

[0538]

[0539] QAPGQGLEWMGWINPNSGGTNYAQKFQGWVTMAtty. Docket No. 2745-9 PCTT

[0540] TRDTSISTAYMELSRLRSDDTAVYYCAREIYSGSH PDDAFDIWGOGTMVTVSSGSASAST. SYO / fC / . SY / W LNGHHHHHHH

[0541] 60 hMSLN Amin EVEKTACPSGKKAREIDESLIFYKKWELEACVDA ectodomain o acid ALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQG [E296-G439] YPESVIQHLGYLFLKMSPEDIRKWQVTSLETLKAL with C-terminal LEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDT his tag LT A FYPGA / / / / / / / / / / / / / /

[0542] (underlined) and

[0543] N388Q

[0544]

[0545] mutation

[0546]

[0273] The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.

[0547]

[0274] While the present disclosure has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to adapt to particular situations without departing from the scope of the present disclosure. Therefore, it is intended that the present disclosure not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out the present disclosure, but that the present disclosure will include all embodiments falling within the scope and spirit of the appended claims.

[0548] EXAMPLE 1

[0549] Introduction

[0550]

[0275] The re-directing of patient autologous effector T cells to attack malignant tumors, independently of any natural cognate recognition mediated through TCR-pMHC interactions, is now an established therapeutic paradigm, with both chimeric antigen receptor (CAR-T) and soluble bi-specific engager (BiTE®, Amgen trademark; hereafter BiTE) technologies having revolutionized the treatment of certain blood cancers. For solid tumors, however, these approaches have met with only limited clinical success. In addition to several “immune escape” mechanisms comprising various physical and immunosuppressive chemical barriers that actively exclude and / or dysregulate tumor- targeting lymphocytes (1), solid tumors typically lack public surface antigens that are completely absent from critical healthy tissues.

[0551] Nevertheless, significant over-expression of certain tumor-specific and tumor-microenvironment associated antigens (TAAs), together with the presence of disease-related neo-epitopes, splice-isoforms and conformers, can provide adequate targeting differentiation and opportunities for therapeutic intervention, as can approaches specifically tailored to tumor physiology (2-5).

[0552]

[0276] One such TAA of active clinical interest, the differentiation antigen Mesothelin (MSLN), is a post-translationally processed, glycosylphosphatidylinositol (GPI)-anchored cell surface-bound protein,Atty. Docket No. 2745-9 PCTT

[0553] whose normal physiological expression is limited and restricted to the mesothelial cells of the pleura, pericardium and peritoneum, with trace amounts reported in a limited set of additional tissues (6, 7). MSLN was originally identified in human ovarian carcinoma cells (6) and has subsequently been found to be overexpressed in many diverse cancers, including malignant mesothelioma, [triple-negative] breast cancers, ovarian carcinomas, pancreatic cancer, and pediatric acute myeloid leukemia, amongst others (7-11). Increased MSLN expression has typically been associated with a poorer prognosis for patients across multiple tumor indications (9, 12-14). As of January 2025, ClinicalTrials.gov reports -130 interventional NCT studies targeting MSLN expressed in tumors, with the majority (-56%) involving engineered CAR-or TCR-based ACT strategies, with or without the incorporation of soluble molecule co-therapy.

[0554] Interestingly, the soluble T cell CD3 engager class is represented by only three Phase 1 / 2 studies (NCT06756035; NCT06255665; NCT03872206). To-date, although demonstrating encouraging safety and tolerance data, clinical trials of anti-MSLN CAR-T products have shown only limited efficacy as monotherapies, with no studies having yet progressed beyond phase 2.

[0555]

[0277] Mature, processed, MSLN is a 40kDa protein with the majority of its structure shown to comprise a predominantly super-helical solenoid arrangement with armadillo-like repeats defining a series of three stacked regions or domains (15). The N-terminal region is suggested to be the most immunogenic, least flexible part of the structure, hosting recognition sites for several key clinical antibodies, in addition to the heavily glycosylated cell adhesion mucin, CA-125, which can co-occur as a prognostic factor in MSLN+tumors (16). The C-terminal region of MSLN contains the GPI-anchor transamidation signal motif together with several exposed labile peptide bonds in the juxta-membrane sequence that are substrates for certain proteases capable of shedding the MSLN into the tumor stroma and serum of patients (17).

[0556] Truncated splice-isoform(s) also contribute to this circulating pool of MSLN which may reach concentrations sufficient to act as a decoy ligand for anti-MSLN therapeutics (18). The function of MSLN under normal physiological conditions remains unclear, although KO models and its GPI-linkage support its involvement in tumor cell adhesion, migration, and metastasis (19). Little is known about how native, GPI-anchored MSLN is organized at the cell surface, or how GPI moieties may directly or indirectly influence its conformation, although studies suggest that the removal of GPI moieties - and the lipid interactions they mediate - from certain membrane proteins can perturb distant epitopes recognised by antibody reagents (20).

[0557]

[0278] The anti-MSLN antibody fragments currently exploited for [pre]clinical CARs and / or immunotoxins have typically emerged via immunization or in vitro affinity-driven display technologies and share the characteristic of strong binding to recombinant MSLN or its soluble fragments, possessing KDs in the sub- to double-digit nM range (21-31). Previously, we used a simple “affinity-blind” phenotypic library selection and screening approach to enrich a fully human scFv clone, LABC_13F08,Atty. Docket No. 2745-9 PCTT

[0558] that demonstrated compelling anti-MSLN CAR effector activity yet, surprisingly, lacked the expected significant binding to recombinant MSLN in our routine in vitro ligand binding assays (32). We thus sought to investigate further the enigmatic nature of this molecule, its apparent contradictory and atypical biochemical behavior, and its potential for T cell redirection toward MSLN-expressing tumor lines and clinical applications involving engineered CAR or TCR-based adoptive cell therapy (ACT) strategies.

[0559]

[0279] We performed ELISA, label-free kinetic binding assays, FACS, Western blotting, and transient recombinant MSLN expression to characterize the recognition properties of a novel CAR-active human scFv clone, LABC_13F08. To investigate T cell redirection, we conducted kinetic IncuCyte co-culture killing assays using transduced primary T cells and MSLN+target cell lines, and assessed levels of activation markers and effector cytokines. The antitumor potential of LABC_13F08 formatted either as a CAR or a bi-specific engager (BiTE®) was evaluated in vivo using transduced human primary T cells and immunocompromised NGS mice xenografted with ovarian, mesothelioma and pancreatic MSLN+tumor cell lines.

[0560]

[0280] The LABC_13F08 scFv is highly unusual and distinct from existing [pre]clinical anti-MSEN antibody fragments, exhibiting an absolute requirement for divalent cations to drive MSEN recognition. As a monovalent BiTE®, LABC_13F08 demonstrates robust in vitro potency. Additionally, primary human T cells engineered for constitutive secretion of the 13F08-BiTE exhibit strong anti -tumour activity towards in vivo ovarian and mesothelioma xenograft models, and show encouraging levels of monotherapy control in a challenging pancreatic model. LABC_13F08 BiTE secreted from engineered T cells (BiTE-T) can both recruit non-engineered bystander T cells and also induce activation-dependent MSLN-independent bystander killing of tumor cells lacking cognate tumor antigen. To address any safety concerns 13F08 BiTE-T cells can be rapidly targeted for clearance via a molecular “off’ switch.

[0561]

[0281] The novel LABC_13F08 scFv exhibits a mode of binding to MSLN which is not observed in typical anti-MSLN antibodies. Efficacious targeting by a T cell secreted engager would represent a clinically differentiated approach for the treatment of MSLN+tumors.

[0562] MATERIALS AND METHODS

[0563]

[0282] KEY RESOURCES TABLE

[0564] REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies

[0565] Cat#

[0566] Mouse Anti-Human C-MYC Alexa Fluor® 647, Clone

[0567] Bio-Rad MCA2200A647, 9E10

[0568] RRID: AB_566936 Cat# 901518, PE anti-HA.ll Epitope Tag, Clone 16B12 BioLegend

[0569] RRID: AB_2629623 DYKDDDDK Epitope Tag Alexa Fluor® 647, Clone #

[0570] R& D Systems Cat# IC8529R

[0571]

[0572] 1042EAtty. Docket No. 2745-9 PCTT

[0573] Direct-Blot™ HRP anti-HA.ll Epitope Tag, Clone

[0574] BioLegend Cat# 901519, 16B12 RRID: AB_2686981

[0575] Cat. 317438; Brilliant Violet 605™ anti-human CD4, Clone: OKT4 BioLegend RRID: AB_1121899

[0576] 5

[0577] Cat# 344704, FITC anti-human CD8, clone: SKI BioLegend

[0578] RRID: AB_1877178 Cat# 304029, Pacific Blue™ anti-human CD45, clone: HI30 BioLegend

[0579] RRID: AB_2174123 Cat# 304210, APC anti-human CD45RO, clone: UCHL1 BioLegend

[0580] RRID: AB_314426 Cat# 302925, PE / Cyanine7 anti-human CD28, clone: CD28.2 BioLegend RRID: AB_1064400

[0581] 4

[0582] Brilliant Violet 711™ anti-human CD197 (CCR7), Cat# 353228, BioLegend

[0583] clone: G043H7 RRID: AB_2563865

[0584] Cat# 356407, PerCP / Cyanine5.5 anti-human CD27, clone: M-T271 BioLegend

[0585] RRID: AB_2561905 Cat# 301042, Brilliant Violet 650™ anti-human CD8a, Clone RPA-T8 BioLegend

[0586] RRID: AB_2563505 Brilliant Violet 605™ anti-mouse TER-119 BioLegend Cat# 116239, RRID: AB_2562447 ThermoFisher

[0587] LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit Cat# L34957

[0588] Scientific

[0589] ThermoFisher

[0590] LIVE / DEAD™ Fixable Near-IR Dead Cell Stain Kit Cat# L10119

[0591] Scientific

[0592] Cat# 350505, Brilliant Violet 421™ anti-human Ki-67, Clone Ki-67 BioLegend RRID: AB_1089691

[0593] 5 PerCP / Cyanine5.5 anti-human CD137 (4-1BB), Clone Cat# 309814, BioLegend

[0594] 4B4-1 RRID: AB_2205686

[0595] Cat# 344604, FITC anti-human CD4, Clone SK3 BioLegend

[0596] RRID: AB_1937227 BD Pharmingen™ PE Mouse Anti-Human CD45, Clone

[0597] BD Biosciences Cat# 555483, HI30 RRID: AB_395875

[0598] Cat# 310914, APC / Cyanine7 anti-human CD69, Clone FN50 BioLegend

[0599] RRID: AB_314849 APC anti-human CD25, Clone BC96 BioLegend Cat# 302610, RRID: AB_314280 BD Pharmingen™ Purified Rat Anti-Mouse Cat# 553142, BD Biosciences

[0600] CD16 / CD32 (Mouse BD Fc Block™), Clone 2.4G2 RRID: AB_394657

[0601] Santa Cruz Cat# sc-33672 Anti-Mesothelin (KI) Alexa Fluor® 647, Clone kl

[0602] Biotechnology AF647

[0603] Santa Cruz Cat# sc-24636, normal mouse IgGl Alexa Fluor® 647

[0604] Biotechnology RRID: AB 737215 Brilliant Violet 421™ anti-human EGFR, Clone AY13 BioLegend Cat# 352911, RRID: AB_2562213 Cat# 344606, PE anti-human CD4, Clone SK3 BioLegend

[0605]

[0606] RRID: AB_1937246Atty. Docket No. 2745-9 PCTT Cat# 304010, PE / Cyanine5 anti -human CD45, clone: HI30 BioLegend

[0607] RRID: AB_314398 Cat# 344722, APC anti-human CD8, Clone: SKI BioLegend

[0608] RRID: AB_2075388 Cat# 410712, APC anti-human IgG Fc, Clone M1310G05 BioLegend

[0609] RRID: AB_2565790 Cat# 317344, Brilliant Violet 421™ anti-human CD3, Clone OKT3 BioLegend

[0610] RRID: AB_2565849 DYKDDDDK Tag Monoclonal Antibody (FG4R), HRP, ThermoFisher Cat# MA1-91878- HRP,

[0611] Clone FG4R Scientific

[0612] RRID: AB_2537626 Cat# 302208, PE anti-human CD19, Clone HIB19 BioLegend

[0613] RRID: AB_314237 Cat# 400113, PE Mouse IgGl, K Isotype Ctrl (FC), Clone MOPC-21 BioLegend

[0614] RRID: AB_326435 Cat# 302208, PE anti-human CD 19, Clone HIB 19 BioLegend

[0615] RRID: AB_314237 Erbitux® (cetuximab) Merck

[0616] MabThera® (Rituximab) Roche

[0617] Alexa Fluor 647 AffiniPure Goat Anti-Human IgG Jackson Cat# 109-605-098 probe Immunoresearch RRID: AB_2337889 horseradish peroxidase (HRP) conjugated goat antiCat# A9917

[0618] Sigma Aldrich

[0619] mouse IgG antibody RRID: AB 258476 recombinant anti-human IgG-Fc HRP antibody Sino Biological Cat# SSA001 RRID: AB_2892579 Santa Cruz Cat# sc-33672 mouse monoclonal antibody clone KI

[0620] Biotechnology RRID: AB 627930 AcroBiosystems (gift;

[0621] Monoclonal anti-human / mouse MSLN human IgGl

[0622] non-commercial)

[0623] Santa Cruz Cat# sc-47778 mouse anti-human-b-actin

[0624] Biotechnology RRID: AB 626632

[0625] Cat# A9917 anti-mouse-Fab-HRP Sigma-Aldrich

[0626] RRID: AB_258476 Biological Samples

[0627] Transfusion

[0628] Human blood (buffy coat - blood bag with + / - 50ml)

[0629] Interregionale CRS Cat# 92020 Chemicals, Peptides, and Recombinant Proteins

[0630] Ficoll-Paque™ PLUS Cytiva Cat# 17-1440-02 T4 DNA ligase New England Biolabs Cat# M0202S RetroNectin® Recombinant Human Fibronectin

[0631] Takara Cat# T100B Fragment

[0632] ThermoFisher

[0633] Dynabeads™ Human T- Activator CD3 / CD28 Cat# 11131D Scientific

[0634] Restriction endonucleases New England Biolabs Various ThermoFisher

[0635] TurboFect Transfection Reagent Cat# R0531

[0636] Scientific

[0637] Opti-MEM™ I Reduced Serum Medium, no phenol red Gibco Cat# 11058-021

[0638]

[0639] Recombinant Human IL-2 PeproTech Cat# 200-02Atty. Docket No. 2745-9 PCTT

[0640] Recombinant Human IL-7 Miltenyi Biotec Cat# 130-095-363 Recombinant Human IL- 15 Miltenyi Biotec Cat# 130-095-765 Calcein-AM BioLegend Cat# 425201

[0641] Santa Cruz

[0642] Polybrene Cat# NC9840454

[0643] Biotechnology

[0644] Liberase™ TL Research Grade Roche Cat# 05401020001 DNase I Roche Cat# 11284932001

[0645] Thermo Fisher

[0646] TRIzol™ Reagent Cat# 15596026 Scientific

[0647] RIPA Lysis and Extraction Buffer Thermo Scientific Cat. 89900 Halt Protease and Phosphatase Inhibitor Cocktail Thermo Scientific Cat. 78441 FectoPRO transfection reagent Polyplus Cat. 101000007 Biotinylated full-length recombinant human mesothelin AcroBiosytems Cat. MSN-H82E9 Biotinylated recombinant murine mesothelin AcroBiosytems Cat. MSN-M82E7 Recombinant murine mesothelin R& D Systems Cat. 8604-MS-050 Recombinant hMSLN-histag R& D Systems Cat. 3265-MS-050 Cell Dissociation Buffer Enzyme Free PBS-based Gibco Cat. 3151014 SuperSignal™ West Pico PLUS Chemiluminescent Thermo Scientific Cat. 34577 Substrate

[0648] eBioscience™ Cell Stimulation Cocktail (PMA / Invitrogen Cat. 00-4970-03 ionomycin

[0649] QUANTI-Luc luciferase substrate Invivogen Cat. rep-qlcl Critical Commercial Kits / Assays

[0650] Thermo Fisher

[0651] PureLink™ HiPure Plasmid Maxiprep Kit Cat# K210006 Scientific

[0652] EasySep™ Human CD4+and CD8+T Cell Isolation Kits Stem Cell Cat# 17952 and Technologies 17953 Human IFN-gamma DuoSet ELISA R& D Systems Cat# DY285B Various as per stated BD™ Cytometric Bead Array (CBA) Human Sets BD Biosciences cytokine or chemokine eBioscience™ Intracellular Fixation & Permeabilization ThermoFisher

[0653] Buffer Set Scientific Cat# 88-8824-00 ThermoFisher

[0654] eBioscience™ Protein Transport Inhibitor Cocktail Cat# 00-4980-03 Scientific

[0655] BD Pharm Lyse™ Lysing Buffer BD Biosciences Cat# 555899 MiniCollect® TUBE K2E K2EDTA Greiner Bio-One Cat# 450532 Microvette® 500 Serum Gel CAT SARSTEDT Cat# 20.1344 Nunc® MaxiSorp™ 384 well plates Sigma-Aldrich Cat. P6491-1CS QuantaBlu™ Fluorogenic Peroxidase Substrate Kit Thermo Scientific Cat. 15169 Streptavidin (SA) probes GatorBio Cat. 160002 Dynabeads Protein A Immunoprecipitation Kit Invitrogen Cat. 10006D 4-12% Bis-Tris NuPAGE Mini Protein Gel Invitrogen Cat. NP0329BOX IBlot 2 PDVF Regular Stacks Invitrogen Cat. IB24001 Pierce Protein Concentrator PES, 10K MWCO Thermo Scientific Cat. 88527 human Mesothelin ELISA detection kit Invitrogen Cat. EH322RB Experimental Models: Cell Lines

[0656] Cat# HTB-77, SKOV-3 ATCC

[0657]

[0658] RRID: CVCE_0532Atty. Docket No. 2745-9 PCTT

[0659] Cat# CRL- 1682, AsPC-1 ATCC

[0660] RRID: CVCL_0152 UPENN Ovarian

[0661] OVCAR-8 Cancer Research RRID: CVCL_1629

[0662] Center (Gift)

[0663] UPENN Ovarian

[0664] OVCAR-5 Cancer Research RRID: CVCL_1628

[0665] Center (Gift)

[0666] Cat# HTB-161 OVCAR-3 ATCC

[0667] RRID: CVCL_0465 Cat# 93021013, HeLa Merck

[0668] RRID: CVCL_0030 Cat# CRL-5826, H226 ATCC

[0669] RRID: CVCL_1544 Cat# CRL -1598, A-673 ATCC

[0670] RRID: CVCL_0080 Cat# CRL- 1619 A-375 ATCC

[0671] RRID: CVCL_0132 Cat# CCL-86, Raji ATCC

[0672] RRID: CVCL_0511 HEK293T Cat# CRL-3216;

[0673] ATCC RRID: CVCL_0063 HEK293-6E NRC, Canada RRID: CVCL_HF20 Jurkat NFAT-Lucia cells Invivogen Cat. jktl-nfat Experimental Models: Organisms / S trains

[0674] The Jackson

[0675] NOD-scid IL2Rγnull(NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ) RRID: BCBC_4142

[0676] Laboratory

[0677] Recombinant DNA

[0678] pSTEVe8_13F08 This study

[0679] pSTEVe8_SS1 This study

[0680] pSTEVe8_M5 This study

[0681] pSTEVe8_15B06 This study

[0682] pSTEVe8_MH1 This study

[0683] pSTEVe8_P4 This study

[0684] pSTEVe41_ 13F08-UCHT 1 This study

[0685] pSTEVe41_SS1-UCHT1 This study

[0686] pSTEVe41_P4-UCHTl This study

[0687] pSTEVe41_1C1m-UCHT1 This study

[0688] pSTEVe41_FMC63-UCHT1 This study pSTEVe159_MSLN(variants) This study

[0689] SFG_3XF_13F08_UCHT1 This study

[0690] SFG_HA_αCD19(FMC63)-UCHT1 This study

[0691] pRRL-aCD19(FMC63)_4-lBBZ This study

[0692] pRRL- aMSLN_13F08_CAR 4-1BBZ This study

[0693] pRRL- aMSLN_06B 11_CAR 4-1BBZ This study

[0694] pRRL- aMSLN_38B7_CAR 4-1BBZ This study

[0695] SFG_tEGFR-2A_3XF_13F08-UCHTl This study

[0696] SFG_tEGFR-2A_3XF_ αCD19(FMC63)-UCHT1 This study

[0697]

[0698] pCMVR8.74 Didier Tronohttps: / / www.addgene.org /

[0699] Atty. Docket No. 2745-9 PCTT

[0700] pVSV-G Akitsu Hotta

[0701] pEQ-PAM (plasmid encoding gag and pol) Elio Vanin

[0702] RDF (plasmid encoding the RD114 envelope protein) M. K. Collins

[0703] Software and Algorithms

[0704] https: / / www.flowjo.c FlowJo Version 10.9.0 Software TreeStar Inc.

[0705] om /

[0706] Prism 10 GraphPad

[0707] software / prism / https: / / www. snapgen SnapGene GSL Biotech

[0708] e.com / https: / / www.r- R version 3.5.1 The R Foundation

[0709] project.org / FCAP Array™ Software Version 3.0 BD Biosciences Cat# 652099 Image Data Exploration and Analysis Software

[0710] Amnis Corporation

[0711] (IDEAS®)

[0712] Other

[0713] CytoFLEX Beckman Coulter

[0714] IncuCyte® S3 Live-Cell Analysis Instrument Sartorius Cat# 4647 IncuCyte® Nuclight Red Lentivirus Sartorius Cat# 4625 IncuCyte® Nuclight Green Lentivirus Sartorius Cat# 4626 Cytek® Amnis® ImageStream®xMk II Imaging Flow

[0715] Cytek Biosciences Part# 100220 Cytometer

[0716] Synergy Hl plate reader BioTek

[0717] 405 automatic plate washer BioTek

[0718] GatorPrime instrument GatorBio

[0719] Fusion FX imaging system Vilber

[0720]

[0721] iBlot 2 Gel Transfer Device Invitrogen

[0722]

[0283] Cell lines and cultures

[0723]

[0284] Cells were sourced as indicated in the Key Resources Table. Human Mesothelioma H226, human Ovarian Adenocarcinoma SKOV3, human high grade ovarian serous adenocarcinoma OVCAR8, OVCAR5, and OVCAR3, human pancreatic ductal adenocarcinoma AsPC-1, human papillomavirus-related cervical adenocarcinoma HeLa, and human melanoma A-375 cell lines were maintained in RPMI with stable glutamine supplemented with 10% FBS (Gibco, Thermo Fisher Scientific), 100 U / mL penicillin, and 100 pg / ml streptomycin sulfate. Human Ewings Sarcoma A673 cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM) with stable glutamine supplemented with 10% FBS, 100 U / mL penicillin, and 100 pg / mL streptomycin sulfate. Jurkat NFAT-Lucia luciferase reporter cells (Invivogen), Raji and human HEK 293T cells were cultured in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U / mL penicillin, and 100 pg / mL streptomycin sulfate.

[0724]

[0285] HEK293-6E cells (licenced from National Research Council of Canada) were grown in Freestyle F17 medium (Life Technologies) containing 4 mM GlutaMAX, 0.1% Pluronic® F-68 (Life Technologies) and 25 pg / mL G418 (Fisher Scientific). All cells were maintained at 37°C, 5% CO2 with agitation in aAtty. Docket No. 2745-9 PCTT

[0725] humidified incubator and passaged 3 times a week to ensure exponential growth conditions. Cells were routinely tested for mycoplasma contamination.

[0726]

[0286] Recombinant construct generation and protein production

[0727]

[0287] Soluble and cell surface protein expression in HEK293-6E. Previously reported anti-MSLN CAR antibody warhead sequences and the anti-CD19 FMC63 control scFv were extracted from public sources (SSI, Pat. US7081518A; M5, Pat. W02015090230Al; P4, Pat. US9272002B2; 15B6, PDB:7U8C; MH1, Pat. US 10543271B2; FMC63, Pat. US7446179A2) and synthesized by GeneArt (Invitrogen). For soluble protein production from HEK293-6E cells, gene fragments were cloned under the control of a CMV promotor into pTT5-based episomal vectors comprising an OriP sequence (1). For Fc-fusions (vector pSTEVe8), scFvs were cloned upstream of a human IgGl Fc (hinge-CH2CH3) to encode in-frame, dimerizing Fc-fusions. For soluble BiTE production (vector pSTEVe41), warhead sequences were fused in-frame to an anti-human CD3 scFv (UCHT1; pat. US6054297A) separated by a flexible linker (12 AA). Transient transfection of HEK293-6E and subsequent protein purification was conducted as previously described (2).

[0728]

[0288] Full length MSLN and truncated / mutated variant ORFs (described in the main text) were synthesized by GeneArt (Invitrogen) and cloned in the sense orientation into a pTT-5 based episomal expression vector containing an introduced bi-directional promoter (rpll3a) expression cassette (pSTEVe159). For a transfection reporter, the vector also encoded GFP in the antisense orientation. Vector DNA was transfected into HEK293-6E using FectoPRO transfection reagent (Polyplus) according to the manufacturer’s guidelines. MSLN expression and scFv-Fc binding were assessed by flow cytometry 48 h post-transfection.

[0729]

[0289] Primary T cell transduction constructs: For T cell secretion, BiTEs were cloned into SFG retroviral vectors (3) such that the 13F08 BiTE sequence was appended to a CD8a signal peptide and an N-terminal 3xFLAG tag, and the control FMC63 anti-CD19 BiTE was placed downstream of a human IFN-beta signal peptide and an N-terminal HA tag. Sequences were codon-optimized for human expression and synthesized by GeneArt (Invitrogen). For truncated EGFR co-expression, tEGFR (4) was cloned upstream of the BiTE-encoding DNA in the SFG constructs, separated by a P2A self-cleaving peptide sequence.

[0730]

[0290] To generate anti-mesothelin CAR constructs, anti-mesothelin scFv sequences were cloned into a pRRL 4-1BB CD3C 2ndgeneration CAR lentiviral vector backbone (5) appended to an a human IFN-beta signal peptide and an N-terminal Myc tag. For control anti-CD19 CAR construct, the anti-CD19 scFv (FMC63) was placed downstream of a human IFN-beta signal peptide and an HA tag.

[0731]

[0291] All subcloning and DNA manipulations were performed according to standard molecular biology techniques.

[0732]

[0292] Enzyme-linked immunosorbent assay (ELISA).Atty. Docket No. 2745-9 PCTT

[0733]

[0293] Nunc® MaxiSorp™ 384 well plates (Sigma-Aldrich) were pre-coated with neutravidin, blocked with PBS-TM (0.1% Tween 20, 5% skimmed milk), and coated with 1 μg / mL biotinylated full-length recombinant human (AcroB iosystems) or murine mesothelin protein (R& D Systems). Dilution series of anti-mesothelin purified scFv-Fc or BiTE were incubated for Ih at RT with gentle agitation. Bound BiTEs were detected using a primary recombinant anti-myc tag antibody (derived from parental mAb clone 9E10, in-house) and a horseradish peroxidase (HRP) conjugated goat anti-mouse IgG antibody (Sigma Aldrich; 1:10000). Bound scFv-Fc were detected using a recombinant anti-human IgG-Fc HRP antibody (SinoBiological). QuantaBlu™ Fluorogenic Peroxidase Substrate Kit (Thermo Scientific) was used to measure fluorescence using a plate reader BioTek Synergy Hl (320nm / 405nm excitation / emission). ELISAs were performed in parallel against both mesothelin antigen and mock biotinylated human TEM1 recombinant ECD protein (produced in-house) to assess non-specific binding. Wells were washed between each step (typically 3x) using 120 pL PBS-T dispensed from a BioTek 405 automatic plate washer.

[0734]

[0294] To assess the influence of metal cations, individual scFv-Fc or BiTE molecules in PBS-TB (0.1% Tween 20, 2% BSA) were supplemented with either 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂ or 1 mM ZnCl₂ and binding to immobilized target in ELISA wells was allowed to proceed at RT for 1 h. In parallel, scFv-Fc or BiTE binding was assessed in PBS / 2% FBS with or without the addition of 2.5 mM EDTA.

[0735]

[0295] Biolayer interferometry (BLI)

[0736]

[0296] Biolayer Interferometer (BLI) analysis was performed on a GatorPrime instrument (GatorBio). Recombinant biotinylated human mesothelin protein (AcroBiosystems) was immobilized on preequilibrated Streptavidin (SA) Biosensors (GatorBio) until 2.2 nm shift was reached. Sensors were submerged for 180 s (or 1800 s for long association conditions) into wells containing from 450 nM to 0 nM purified BiTE analytes (1 / 2 dilution series) (association step). For dissociation, sensors were dipped into PBS / 0.01% Tween20 / 0.05% BSA buffer for 300 s. Biosensor probes were regenerated between experiments with 3 cycles of 10 mM Glycine-HCl pH 1.5 for 5 s. To assess the influence of metal cations on binding, association and dissociation buffers were supplemented with 1 mM CaCl₂ or 2.5 mM EDTA

[0297] All runs were performed in parallel against a mock biotinylated human TEM1 recombinant ECD protein (produced in-house) to control for non-specific binding.

[0737]

[0298] Data were analyzed using the Gator data analysis software (GatorBio). The sensorgrams were fitted to a 1:1 binding model for data analysis.

[0738]

[0299] Western blotting and immunoprecipitation

[0739]

[0300] Preparation of cell extracts from MSLN-transfected HEK293-6E: Cells were harvested 2 days after transfection, washed with PBS and lysed using cold RIPA Lysis and Extraction Buffer (Thermo Scientific) supplemented with lx Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) (without EDTA; 1 mL RIPA for every 107cells). Lysis was carried out for 30 min on ice. DNA was fragmented byAtty. Docket No. 2745-9 PCTT

[0740] mechanical disruption using a 26G needle attached to a 1 mL syringe. Then, cell debris were removed by a 30 min centrifugation step at 16000 g. Extracts of non-transfected HEK293-6E cells were processed the same way.

[0741]

[0301] Preparation of cell extracts from tumor cell lines: Whole cell extracts were prepared by detaching adherent cells using 10 mM EDTA and washed with complete RPMI containing 10% FBS. Cells were then lysed and cell debris removed as described above.

[0742]

[0302] Western blotting procedure: The total protein concentration in cell extracts was determined using a Tryptophan Fluorescence method (6). 20 pg of cell protein was separated by SDS-PAGE on 4-12% BisTris NuPAGE gels (Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen) using an iBlot™ 2 Gel Transfer Device (Invitrogen). Membranes were blocked (PBS / 0.1% Tween 20 / 2% Milk) overnight at 4°C before being challenged for 1 h with various anti-MSLN molecules: mouse monoclonal antibody clone KI (Santa Cruz Biotechnology; 1:500) or in house produced human scFv-Fc (13F08, SSI) at 0.1 μg / mL in PBS-T / 2% skim milk. In parallel, staining of identically loaded lanes was conducted using a mouse anti-human-[3-actin (Santa Cruz Biotechnology; 1: 1000) as a loading control. Following primary antibody incubations, membranes were washed four times in PBS-T and the signals were developed using an anti-mouse-Fab-HRP (Sigma- Aldrich; 1:5000) or an anti-human-Fc-HRP secondary conjugate (Sino Biological; 1:10000) and SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific). Visualization was performed on a Fusion FX imaging system (Vilber). The western blot detection of MSLN-transfected HEK293-6E cell extracts was carried out in a similarl manner, except the in-house produced 13F08-Fc was used at 0,5 μg / mL in PBS-T / 2% skim milk.

[0743]

[0303] Immunoprecipitation procedure: Transfected HEK-MSLN and HEK-NT cell extracts were processed using the Dynabeads Protein A Immunoprecipitation Kit (Invitrogen) following the manufacturer’s instructions. Briefly, 1 pg of purified scFv-Fc were incubated for 1 h at room temperature with 30uL of washed Protein A Dynabeads from the kit. Beads were washed once with PBS, blocked with PBS / 2% BSA for 15 min. Blocked coated beads were washed once again and incubated for 4 h at 4°C with 250 μL of prepared cell extract. Then, magnetic beads were washed once with 1 mL of PBS / 0.05% Tween 20 / 0.5 M NaCl and once more with 1 mL PBS. Elution of immunoprecipitated proteins from the beads was performed for 10 min at RT using 100 pL of 0.5 M NH4OH / O.5 mM EDTA pH 11.5. An equivalent of 11 pL of eluted fraction were separated by SDS-PAGE, under non-reducing condition, on a 4-12% Bis-Tris NuPAGE Protein Gel (Invitrogen) alongside 50 ng and 100 ng recombinant hMSLN-histag (R& D Systems). Resolved proteins were transferred onto a PDVF membrane (Invitrogen) using the iBlot 2 Gel Transfer Device and the membrane was blocked overnight at 4°C (PBS / 2% Milk / 0.1% Tween 20). The following day, immunoprecipitated MSLN protein was detected by using anti-hMSLN P4-Fc at 0.2 pg / mL. Following the primary antibody incubation, the signals were developed using an anti-human IgG-Fc-HRPAtty. Docket No. 2745-9 PCTT

[0744] secondary conjugate (Sino Biological; 1:10000) and SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific). Visualization was performed on a Fusion FX imaging system (Vilber).

[0745]

[0304] FACS-based profiling of MSLN on endogenous tumor cell lines

[0746]

[0305] Reference staining using K1 mAb: Equivalent numbers of the indicated cell lines were stained with LIVE / DEAD™ Fixable Aqua dye (1:500 in PBS, 20 min at RT) followed by staining with anti-Mesothelin (KI) Alexa Fluor® 647 (Santa Cruz Biotechnology) or normal mouse IgGl Alexa Fluor® 647 Isotype control (Santa Cruz Biotechnology) at matched concentrations (2 pg / mL final).

[0747]

[0306] Control staining using anti-CD19'. Following viability staining as stated above, cell lines were stained using PE anti-human CD19 (BioLegend; 1:100 in FACS buffer).

[0748]

[0307] Test staining using Fc-fusions: Following viability staining as stated above, equivalent numbers of the indicated cell lines were stained with the following soluble Fc-fusions that were produced and purified in-house: 13F08-Fc, SSl-Fc, M5-Fc, 15B6-Fc, Ctrl-Fc, or IgG control (all at 2 pg / mL). Cells were then washed and stained with APC anti-human IgG Fc Antibody (Clone M1310G05).

[0749]

[0308] Titration staining of OVCAR8 cells (+ / - EDTA): Cells were lifted from T-75 flasks using Cell Dissociation Buffer (Gibco), blocked with FACS buffer on ice for 30 min and then 1x105blocked cells were resuspended in 100 pL purified, serially diluted, scFv-Fc. After 45 min of incubation on ice, the cells were washed 2x with FACS buffer. Binding of scFv-Fc molecules was detected using an Alexa Fluor 647 AffiniPure Goat Anti -Human IgG probe (Jackson Immunoresearch; 1:1000 dilution) with incubation for 45 min on ice. Samples were then washed 2x with FACS buffer and dead cells were stained with 40,6-Diamidino-2-phenylindole (DAPI; 0.6 pg / mL). The procedure was carried out using two different FACS buffers throughout: PBS / 2% FBS / 0.02% NaN3 either with or without 2.5 mM EDTA.

[0750]

[0309] In all cases, stained cells were analysed by Cytoflex LX (Beckman Coulter) with data analysis performed using FlowJo vlO (FlowJo LLC).

[0751]

[0310] FACS based profiling of recombinant MSLN on HEK293-6E transfected cells

[0752]

[0311] HEK293-6E transfected cells expressing full length MSLN and truncated / mutated ORF variants were collected, 48 h post-transfection. Cells were processed essentially as described above for OVCAR8 titration staining. Incubation was carried out with 0.1 pg / mL in FACS buffer + / - 2.5 mM EDTA.

[0753]

[0312] Jurkat NF AT activation reporter cell assay

[0754]

[0313] HEK293-6E cells transfected with full length GPI-anchored MSLN (105) were seeded in 96-well U-bottom plates. Subsequently, 105Jurkat NF AT -Lucia reporter cells (Invivogen) were added to each well. Purified BiTEs were added to a final concentration of 2 nM. Similarly, OVCAR8 and Raji cells were seeded with Jurkat NF AT -Lucia reporter cells at various effector:target ratios and incubated with 1 nM 13F08 BiTE. Phorbol myristate acetate (PMA)Zionomycin (Invitrogen) was included as a maximum positive response control. After 24 h of co-culture, the supernatants were collected and mixed with an equal volumeAtty. Docket No. 2745-9 PCTT

[0755] of QUANTI-Luc luciferase substrate (Invivogen). Luminescence was measured immediately using a BioTek Synergy Hl plate reader.

[0756]

[0314] Quantitation of shed endogenous MSLN in conditioned OVCAR8 media.

[0757]

[0315] 0VCAR8 cells were grown in T-150 flasks for 4 days without media change. Media supernatant was collected, 0,22 pm filtered, and concentrated 12x using a 10K MWCO PES membrane concentrator (Pierce; Thermo Scientific). Shed mesothelin from concentrated supernatant was quantified using the human Mesothelin ELISA detection kit (Invitrogen) following manufacturer’s instructions.

[0758]

[0316] Primary human T cell isolation

[0759]

[0317] Human primary CD4+and CD8+T cells were isolated from PBMCs from blood derived from anonymous human healthy donors (buffy coats, Transfusion Interregionale CRS) using Ficoll-Paque™ density gradient centrifugation (Ficoll-Paque™ PLUS, Cytiva) and immunomagnetic negative selection (EasySep™ Human CD4+and CD8+T Cell Isolation Kits, Stem Cell Technologies).

[0760]

[0318] Virus production and T cell Transduction

[0761]

[0319] Virus production: Retroviral supernatant was produced by triple transfection of HEK293T. Briefly, 80% confluent HEK293T cells in a T150 flask were co-transfected with 18 pg Gag-Pol (Peq-Pam), 7 pg RD114 and 22 pg of the retroviral vector encoding the gene of interest using TurboFect Transfection Reagent (ThermoFisher Scientific). The three plasmids were mixed with 3 mL Opti-MEM™ I Reduced Serum Medium (Gibco) and 120 μL TurboFect, and this transfection mixture was incubated for 30 minutes at room temperature prior to its addition to HEK293T cells. Cultures were returned to the incubator and the following day medium was replenished. At 48 h and 74 h post transfection, viral -containing supernatant was harvested, passed through a 0.45 pm filter (Sartorius), and concentrated by ultracentrifugation at 24,000 g at 4°C for 2 h (Avanti JXN-30 high-speed centrifuge). Virus was then used immediately, or aliquoted, frozen on dry ice, and stored at -80°C. Lentivirus was produced and concentrated as described above, but in the triple plasmid mixture the following three plasmids were used instead: 18 pg pCMVR8.74, 7 pg VSV-G, and 15 pg lenti viral vector encoding the gene of interest.

[0762]

[0320] T cell transduction: Freshly isolated human CD4+and CD8+T cells, were activated on day 0 using CD3 / CD28 human T-Activator Dynabeads™ (ThermoFisher Scientific) at a 2: 1 bead:cell ratio and cultured in complete RPMI medium supplemented with 50 IU / mL recombinant human IL-2 (PeproTech) at IxlO6cells / mL (1 ml / well) in 24-well plate. Complete RPMI medium comprised RPMI 1640 medium with stable glutamine supplemented with 10% heat-inactivated FBS (Gibco), 100 U / ml penicillin, and 100 pg / mL streptomycin sulfate. T cell transductions were performed on day 1 post activation. Retrovirus transductions were performed using RetroNectin (Takara Bio)-coated non-treated tissue culture plates (Greiner Bio-One) according to the Takara protocol. Virus-binding to RetroNectin was facilitated by centrifuging the plates for 1.5 hours at 32°C at 2000 g (as per manufacturer’s instructions). Following centrifugation, activatedAtty. Docket No. 2745-9 PCTT

[0763] donor-matched CD4+and CD8+T cells were pooled together at 20% CD4+and 80% CD8+, and added to RetroNectin / retrovirus-coated plates. For transductions in 24-well plates IxlO6T cells were added in each well, and for 48-well plates 0.5xl06T cells were added. Plates were then centrifuged at 300 g for 10 minutes to promote contact of T cells with viral particles, and returned in the incubator. Lentiviral transductions were performed by direct addition of viral particles onto activated T cells (pooled donor-matched CD4+and CD8+, 20% and 80%, respectively, as stated above). From day 2 post transduction T cells were expanded in complete RPMI medium supplemented with human IL-7 and IL- 15 cytokines (both at 10 ng / mL final concentration; Miltenyi Biotec) and T cell cultures were maintained at 0.5 xlO6cells / mL as we described previously to ensure optimal T cell growth (7). Dynabeads™ were removed on day 5-6, and transduction efficiency and T cell viability were assessed by flow cytometry on day 7, unless otherwise stated.

[0764]

[0321] Extracellular and Intracellular staining of transduced T cells

[0765]

[0322] The transduction efficiency of anti-CD19 and anti-Mesothelin CARs was assessed by cell-surface staining of epitope tags using anti-HA antibody (BioLegend) and anti-c-Myc antibody (Bio-Rad), respectively. For assessing the transduction efficiency in BiTE-engineered T cells, cells were intracellularly stained using eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (ThermoFisher Scientific) according to the manufacturer’s protocol. Anti-HA.11 Epitope Tag Antibody (BioLegend) was used to detect the HA-tagged CD 19 BiTE, and anti-DYKDDDDK (FLAG) Epitope Tag Alexa Fluor® 647 antibody (R& D Systems) was used to detect the FLAG-tagged 13F08 BiTE. In truncated EGFR and BiTE co-expressing T cells, transduction efficiency was assessed by detecting both the truncated EGFR and BiTE. Truncated EGFR was detected by cell surface staining using BV421 anti-human EGFR (BioLegend). Following cell surface staining, cells were intracellularly stained as described above to detect the expressed BiTEs. Cell viability was assessed using LIVE / DEAD™ Fixable Aqua dye. Cells were analyzed using flow cytometry (CytoFLEX; Beckman Coulter).

[0766]

[0323] ImageStream analysis for co-localization of BiTE with CD3s

[0767]

[0324] BiTE-engineered T cells were stained with Brilliant Violet 421™ anti-human CD3, Clone OKT3 (BioLegend) and anti-DYKDDDDK (FLAG) Epitope Tag Alexa Fluor® 647 antibody (Clone: 1042E; R& D Systems). Antibodies were used at 1:200 and 1:10 in FACS buffer, respectively. Samples were then run on an ImageStreamX to assess co-localization of CD3 staining with FLAG staining. Single-color controls were also acquired to calculate a compensation matrix, which was applied in the analysis to correct for fluorescence spillover. Specifically, samples were acquired on a 2-camera, 12-channel ImageStreamX imaging flow cytometer (Cytek Biosciences) at low speed and high magnification (60X). Cells were excited using a 405 nm laser (30mW), a 488 nm laser (lOOmW), a 642 nm laser (150mW) and a 785nm Side Scatter (SSC) laser (2.34 mW). Only events with bright field area greater than 1 pm2(to exclude cell debris) and non-saturating pixels (Raw max pixel values below 4096) were collected. Data were acquired for at leastAtty. Docket No. 2745-9 PCTT

[0768] 15,000 events / sample. Co-localization of CD3 staining with FLAG staining was quantified by using the Colocalization Wizard embedded in IDEAS® 6.3 software, which calculates the co-localization between any 2 fluorescent channels by using the Bright Detail Similarity R3 Feature (8)

[0769]

[0325] T cell state analysis

[0770]

[0326] Two weeks post transduction and following expansion in IL-7 / IL-15-supplemented media, T cell state was assessed using flow cytometry. Equivalent numbers of the indicated T cells were sampled from cultures and stained using LIVE / DEAD™ Fixable Aqua (1:500 in PBS) followed by cell surface staining with the following anti-human antibodies (1:100 in FACS buffer): CD45 (clone: HI30), CD45RO (clone: UCHL1), CD28 (clone: CD28.2), CD197 (CCR7; clone: G043H7), CD27 (clone: M-T271). Fluorescence minus-one (FMO) controls for each antibody performed to establish gating when analyzing the data.

[0771]

[0327] Co-culture kinetic cytotoxicity assays (IncuCyte)

[0772]

[0328] Adherent target tumor cell lines were transduced using Incucyte® Nuclight Red or Green Lentivirus (Sartorius) to stably express nuclear-restricted mKate2 (red fluorescent protein) or GFP (green fluorescent protein), respectively, according to supplier’s instructions. Cells were subsequently sorted using FACS to ensure homogeneous high expression of the nuclear-restricted fluorescent protein. Fluorescent tumor cells were seeded into flat-bottom 96-well plates (10,000 cells per well in 100 pF of their respective culture medium), and allowed to adhere for approximately 5 h in a normal humidified incubator (37 °C and 5% CO2). In triple co-cultures (with bystander impact experiments with 2 cell lines: OVCAR8 and A673, A375 or HEK293T), the total tumor cell number was 10,000, as stated above, comprised of the indicated percentage of each differentially -labeled cell line. The ratio of OVCAR8: MSLN-negative cell line was optimized so that at 24 h post seeding there is a 1:1 cell line representation in the well. Effector cells were then added in 100 pF of media onto the adherent target cells at the indicated E: T ratio (2:1, unless otherwise stated) and plates were monitored in an IncuCyte® S3 Five-Cell Analysis System, housed in a cell culture incubator at 37°C with 5% CO2. Unless otherwise stated, phase contrast and fluorescence scans (using 4x or 10x objective) were acquired every 2 h for a minimum of 48 h. Analysis of total fluorescent area (for the respective fluorescent protein) in each well was then performed and the resulting values were used as a surrogate quantification of tumor cell growth kinetics over time. Values were normalized to baseline (value at time 0) to calculate fold-change in fluorescence (Normalized cell index; where cell is the tumor cell line indicated in plot). Unless otherwise stated, the line-connected data points are averages from n=3 biologically independent wells (triplicates). Shaded area represents SEM.

[0773]

[0329] Co-cultures with soluble BiTEs'. In co-cultures of T cells with soluble purified BiTEs, isolated nontransduced non-bead-activated resting T cells (20% CD4+with 80% CD8+mix) were co-cultured with the indicated Nuclear Red target cells (which were produced and seeded as stated above) at 2:1 T cell to target ratio in the presence of the indicated soluble BiTE at 4nM final concentration. Supernatant samples wereAtty. Docket No. 2745-9 PCTT

[0774] harvested at 22 h post co-culture and screened for IFN-y cytokine using IFN-y ELISA (R& D Systems) as per the manufacturer’s protocol.

[0775]

[0330] Restimulation assays: For re-stimulation (re-challenge) assays, co-cultures were set up and monitored using the IncuCyte® S3 Live-Cell Analysis System as described above. At the indicated times, plates were removed from the IncuCyte, and 10,000 Nuclear Red fresh target cells were added to the wells. Plates were then returned to the IncuCyte, and monitoring was resumed until further intervention (rechallenge, as indicated), or until assay endpoint.

[0776] Co-cultures with secreted BiTE-containing supernatants: Non-transduced T cells were co-cultured with target cells at a 2:1 E: T ratio. Prior to loading in the IncuCyte®, co-cultures were supplemented with cell-free supernatant from the indicated BiTE-engineered / secreting T cells or non-transduced T cell control. The volume of cell-free supernatant used in co-cultures was 100 pL and this was harvested from lxlO6 / mL cultured T cells. Target cells were seeded in 80 pL, and T cells were added in 20 EX so that the final coculture volume is maintained at 200 pL.

[0777]

[0331] T cell activation marker determination following co-culturing with target cells

[0778]

[0332] T cells were co-cultured with Nuclear Red target cells at the indicated E: T ratios as described above with the difference that 20,000 target cells were seeded to increase the number and duration of cell:cell interaction events. After 24 h, co-cultures were harvested and transferred to V-bottom 96-well plates. Samples were then stained with LIVE / DEAD™ Fixable Aqua dye, followed by staining for human CD45 and the indicated T cell activation markers: human 4- IBB, CD69, and CD25 (1:100 in FACS buffer; BioLegend; antibodies listed in Key Resources Table). Samples were then analyzed by flow cytometry. Cell size, Nuclear Red fluorescence and CD45 staining were used to discriminate target cells from T cells. On-target specific activation index was calculated by dividing the Median Fluorescence Intensity (MFI) of the indicated activation marker in the T cell population of the targeting BiTE (13F08) by the MFI of the irrelevant control anti-CD19 BiTE (utilizing the FMC63 scFv).

[0779]

[0333] CBA assay

[0780]

[0334] Cytometric bead array (CBA; BD Biosciences) was used to quantify GM-CSF, IFNy, IL-2, GrzB and TNF released to the medium upon co-culture of T cells with target cells after 24 h (unless otherwise stated). Supernatant from co-cultures was incubated with the capture bead mix (determined by the panel of cytokines / chemokines to be quantified) for 2 h or O / N, followed by a 1 h incubation with the respective PE detection antibodies. Beads were then washed twice and resuspended in assay wash buffer (BD Biosciences). Beads were analyzed using flow cytometry (CytoFLEX; Beckman Coulter). Prior to sample acquisition compensation was performed using beads supplied with the BD CBA kit to ensure proper bead separation / alignment in the 2D matrix (NIR- A vs Red- A) and maximum resolution of the PE signal. Results were analyzed using FCAP Array Software v3.0 (BD Biosciences) using manual bead clustering.Atty. Docket No. 2745-9 PCTT

[0781]

[0335] Mouse models

[0782]

[0336] NSG (NOD. C -PrkdcsculIl2rg‘mlWilISzJ) mice were obtained from the Jackson Laboratory and were housed and bred at the Epalinges UNIL animal facility in compliance with guidelines. All experiments were conducted according to protocols approved by the Service of Consumer and Veterinary Affairs (SCAV) of the Canton of Vaud.

[0783]

[0337] Adoptive cell transfer studies

[0784]

[0338] Tumor cells were injected subcutaneously in the flank of NSG mice followed by monitoring every 2-3 days to assess tumor establishment and growth. For the 0VCAR8 model, 6xl06cells were injected. For all other models, 2xl06cells were injected. Once tumors reached the required size, mice were randomized into treatment groups (according to the randomization method stated below) and were treated by intravenous transfer of engineered T cells at the indicated times. Mice were carefully monitored, and tumor length (L; greatest longitudinal measurement) and width (W; greatest transverse measurement) measured by caliper every 2-3 days. Tumor volumes (V) were calculated using the formula: V = (L x W2) / 2. Mean tumor volumes for each group were determined ± SEM. Mice were sacrificed once tumors reached 1000 mm3or, according to regulation, if they became distressed or moribund.

[0785]

[0339] Mouse randomization method

[0786]

[0340] Mouse randomization was performed using R (www.r-project.org) using an in-house developed algorithm. Mice were randomly assigned in treatment groups and statistical tests (ANOVA to assess variance of group means; Levene's to assess equality of variances between different groups) were performed on recorded tumor volumes for each randomization. The randomization with the maximum product of p-values (from ANOVA and Levene's tests), following 104-105runs, was selected to ensure minimal tumor size variability between groups at the 1st ACT treatment.

[0787]

[0341] Ex vivo tissue processing and analysis

[0788]

[0342] Tumors and / or blood where indicated were harvested from the treated animals. Tumors were cut into pieces using a scalpel and subsequently dissociated in RPMI supplemented with 200 ug / mL Liberase TL (Roche) and 5 U / mL DNase I (Sigma- Aldrich) for 1 h at 37°C on a rocker, followed by passage through a media-primed 70-μm cell strainer to achieve a homogeneous single cell suspension.

[0789]

[0343] Blood was collected in MiniCollect® K2E K2EDTA tubes (Greiner Bio-One) and processed with RBC lysing buffer (BD Pharm Lyse, BD Biosciences) as per manufacturer’s instructions to remove RBCs prior to further processing / staining

[0790]

[0344] Collection at terminal point was performed by cardiac puncture under deep terminal anesthesia and collected blood was transferred to MiniCollect® K2E K2EDTA tubes prior to processing as indicated above.Atty. Docket No. 2745-9 PCTT

[0791]

[0345] T cell phenotyping by flow cytometry: Following tumor dissociation, samples were initially stained with LIVE / DEAD™ Fixable Aqua dye (1:500 in PBS, 20 minutes at RT). Extracellular staining was then performed to detect the surface markers stated, using the respective antibodies indicated in the Key Resources table. Unless otherwise stated, antibodies were used at 1:100 in FACS buffer (PBS, 2% FBS) and staining was performed on ice for 30 minutes. Samples were then fixed / permeabilized using eBioscience™ Intracellular Fixation & Permeabilization Buffer Set and stained for intracellular markers (anti-human Ki-67) as per the manufacturer’s protocol. Prior to extracellular and intracellular antibody staining, samples were blocked using an Fc-block (rat anti-Mouse CD16 / CD32; BD Biosciences). Following staining, samples were washed twice in FACS buffer, and analyzed using flow cytometry. Blood samples were additionally stained with anti-mouse TER- 119 antibody to exclude mouse RBC.

[0792]

[0346] Staining of tumor cells with mAb K1: Tumor samples were processed as indicated above and stained with LIVE / DEAD™ Fixable Aqua dye (1:500 in PBS, 20 minutes at RT). Samples were subsequently washed and stained with Alexa Fluor® 647-conjugated KI mAb or Isotype control (both used at 2 pg / mL final concentration in 100 pL FACS buffer). Prior to antibody staining, samples were blocked using an Fc-block (rat anti-Mouse CD16 / CD32; BD Biosciences).

[0793]

[0347] Statistical analyses and heatmaps

[0794]

[0348] Statistical analyses were performed using GraphPad Prism 10 software. Analysis of differences between two datasets was performed using unpaired two-tailed Student’s t test. Statistical analyses of three or more groups were performed using a one-way ANOVA test followed by the post-hoc test stated in the figure legend. Statistical analysis of tumor growth curves was performed using a two-way repeated-measures ANOVA with the indicated post hoc correction test. Significance levels are indicated in the figures (p < 0.05:*; p < 0.01:**; p < 0.001:***; p < 0.0001:****). Unless otherwise stated, data presented are means ± SEM.

[0795]

[0349] Heatmaps were generated using R and display the on-target upregulation of the indicated marker (in comparison to control). Fold-difference was calculated by dividing the values in 13F08-BiTE / (CAR) with the matched values in the control CD19-BiTE / (CAR) group (i.e. on-target difference). Values were then log-transformed to fix skewness and scaled for uniformity between the different markers.

[0796]

[0350] In vivo T cell depletion mediated by tEGFR / Cetuximab

[0797]

[0351] Animals were treated intraperitoneally with 1 mg / mouse of the indicated drug (Cetuximab or Rituximab) at the indicated times and intervals. Drugs were administered twice (1 dosing after each ACT). Tumor growth and animal wellbeing were monitored every 2-3 days.

[0798]

[0352] METHODS REFERENCES

[0799] 1. Durocher Y, Perret S, Kamen A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res.

[0800] 2002;30(2): E9.Atty. Docket No. 2745-9 PCTT

[0801] 2. Fierle JK, Abram-Saliba J, Brioschi M, deTiani M, Coukos G, Dunn SM. Integrating SpyCatcher / SpyTag covalent fusion technology into phage display workflows for rapid antibody discovery. Sci Rep. 2019;9(l):12815.

[0802] 3. Lindemann D, Patriquin E, Feng S, Mulligan RC. Versatile retrovirus vector systems for regulated gene expression in vitro and in vivo. Mol Med. 1997;3(7):466-76.

[0803] 4. Wang X, Chang WC, Wong CW, Colcher D, Sherman M, Ostberg JR, et al. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood.

[0804] 2011;118(5): 1255-63.

[0805] 5. Barde I, Laurenti E, Verp S, Wiznerowicz M, Offner S, Viornery A, et al. Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease. Gene Ther.

[0806] 2011; 18(11): 1087-97.

[0807] 6. Wisniewski JR, Gaugaz FZ. Fast and sensitive total protein and Peptide assays for proteomic analysis. Anal Chem. 2015;87(8):4110-6.

[0808] 7. Lanitis E, Rota G, Kosti P, Ronet C, Spill A, Seijo B, et al. Optimized gene engineering of murine CAR-T cells reveals the beneficial effects of IL-15 coexpression. J Exp Med. 2021;218(2).

[0809] 8. George TC, Fanning SL, Fitzgerald-Bocarsly P, Medeiros RB, Highfill S, Shimizu Y, et al. Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow. J Immunol Methods. 2006;311(l-2):117-29.

[0810] RESULTS LABC-13F08 Exhibits an Unusual and Novel Metal-Dependency in Binding to a D1-D2 Fragment ofMSLN

[0811]

[0353] Functional CAR activity requires the physical bridging of a CAR scFv with target antigen on a tumor cell. We thus sought to understand the discordance in our previous LABC-13F08 scFv (hereafter 13F08) data which demonstrated potent CAR activation towards MSLN+cells in the absence of appreciable in vitro binding to soluble recombinant(r) human(h) MSEN. Convenient monovalent (BiTE) and dimeric bivalent forms of 13F08 (Fc-fusion) were thus produced and purified from transiently transfected HEK293-6E cells and their binding properties variously compared with reference to representative clinical and pre-clinical comparator molecules, including SSI, M5, 15B6, P4 and MH1 (21, 23, 24, 27, 33). Initially, we performed kinetic biolayer interferometry (BEI) against immobilized high quality, C-terminally biotinylated rhMSLN. In contrast to SS1, a clinically validated CAR-active scFv included as a positive control, the 13F08-BiTE bound weakly, with extended association-phase data indicating a monomer KD in the μM range (Figure 1 A-D). Similarly, titration ELISA data revealed weak binding of the bivalent 13F08-Fc relative to representative comparator anti-MSLN Fc-fusions, and also no cross-reactivity with biotinylated recombinant murine MSEN (Figure 1E, F).

[0812]

[0354] Despite the apparent poor recognition of bulk rhMSLN by 13F08, we reasoned that it must stain MSLN+cell lines in order to account for the previously observed activity of the 13F08 scFv CAR. We thus used FACS to profile the binding of 13F08-Fc against a panel of endogenous MSLN+tumor and negative control cell lines. Here, we also included both clinical (SSI, M5) and pre-clinical (15B6) CAR-active binders as Fc-fusion comparators, as well as the original anti-MSEN reference mAb, KI (6).Atty. Docket No. 2745-9 PCTT

[0813] Although substantial differences in median MFI staining signals were evident across the different lines, likely reflecting distinct MSLN epitope exposure / accessibility environments (a consequence of differences in PTM profiles and / or possible masking due to cis-acting ligands), 13F08-Fc selectively recognized the six tested MSLN+cells, albeit with smaller MFI shifts for HeLa and 0VCAR3. For SK0V3, heterogeneous staining was evident by logarithmic FACS histogram (although less so by median MFI) consistent with the KI reference staining (Figure 2A, 2B). Further, we confirmed a significant correlation in target cell staining between 13F08 and the KI reference (Figure 2C). Western blot analysis performed on solubilized whole cell extracts of these lines mirrored this FACS data, confirming the ability of 13F08-Fc to specifically recognize endogenous native MSLN (Figure 3). In this study, we also observed specific Western staining of MSLN from extracts of HEK293-6E suspension cells transfected with both the major (ISO#2) and minor (ISO#1) full length (FL) GPI-anchored MSLN transcript variants (mature, processed ORFs) and a truncated fragment comprising the two N-terminal domains (D1-D2), but not of various other domain fragments (Figure 4A, B). Additionally, 13F08-Fc could also efficiently immuno-precipitate FL-MSLN from solubilized cells in this recombinant system (Figure 5). Of note, commercial rhMSLN included as a lane marker in these immune-blotting experiments was also readily stained by 13F08-Fc which was unexpected in light of our ELISA / BLI findings. A further discrepancy was highlighted by FACS profiling of MSLN-transfected HEK293-6E cells. Here, in contrast to the strong staining observed towards certain endogenous MSLN+tumor lines (H226, 0VCAR8, AspC-1), 13F08-Fc stained transfected HEKs only partially and exhibited significantly lower staining sensitivity than seen for the comparator Fc-fusions (Figure 6A). Confirming the above WB observations, only transfected FL-MSLN and the D1-D2 truncated fragment gave convincing MFI-shifts (Figure 6B).

[0814] Interestingly, this partial staining behaviour was retained for a chimeric D1-D2 fragment when human DI was fused to murine D2 but not for the vice versa arrangement. As expected, fully murine D1-D2 expressed in HEKs was not recognized by 13F08 (Figure 6B). All comparators gave the expected fragment recognition profiles in accordance with the published literature.

[0815] The Binding of LABC-13F08 to MSLN has an Absolute Requirement for Divalent Cations

[0355] To elucidate these contextual inconsistencies in MSLN recognition by 13F08, we asked whether an explanation could be found in the different buffer and / or media systems employed in the various assays. Indeed, we established that strong apparent binding of 13F08 correlated only with the inclusion of potentially rich sources of metal cations in the experiment (milk as a blocking reagent in WB and IP; FBS routinely included in the FACS buffer when staining tumor lines). Thus, in our above ELISA / BLI experiments, the free metal availability would be low and restricted to ions passively associated with the purified BSA used as a blocking reagent and diluent carrier. Similarly, the suspension HEK293-6E cellsAtty. Docket No. 2745-9 PCTT

[0816] used for FL-MSLN (and fragment) transfections were cultured in serum-free medium, with FACS analysis conducted in buffers including an excess of the strong chelator, EDTA.

[0817]

[0356] Subsequently, we revisited our binding assays, conducting them in the presence or absence of exogenously added metals or EDTA, and confirmed a striking requirement for divalent cations in the interaction of 13F08 with purified rhMSLN protein, MSLN-transfected HEK293-6E cells, and endogenous MSLN+0VCAR8 cells (Figure 7, Figure 8, and Figure 9). Several metals were shown to promote strong monophasic binding, with this dependency only evident for 13F08 and not the anti-MSLN comparators examined. Kinetic BLI measurements performed in the presence of 1mM CaCl₂ indicated a monomer KD towards rhMSLN in the region of 9 nM, whereas EDTA inclusion in the buffer completely ablated residual binding (Figure 7C). Interestingly, substitution of the native MSLN GPI-anchor motif with the type I TM domain of human HER2 in our transfection FACS assay appeared to ablate the partial bi-phasic staining when EDTA was included in the staining buffer, whereas in the absence of EDTA (and in the presence of 2% FBS), this variant was stained strongly (Figure 9).

[0818]

[0357] Collectively, our MSLN truncation and chimeric fragment data indicates that the epitope recognized by 13F08 resides within the D1-D2 fragment, is insensitive to the presence of the minor transcript (ISO#1) D2 splice insertion, does not involve the N-glycon in the D1-D2 linker, and that binding to both FL-MSLN and D1-D2 is strongly facilitated by divalent metal cations present at high levels in serum and milk (Figure 9) (34,35). Thus, 13F08 represents a novel anti-MSLN antibody which is distinguished from other reported molecules by its unusual recruitment of metal to enable single-digit nM KD target binding.

[0819] LABC-13F08 Efficiently Mobilizes T Cells as a Purified BiTE

[0820]

[0358] As LABC-13F08 was originally isolated from a functional CAR screen, we next asked whether this T cell redirection property also extended to the scFv BiTE format. We expressed and purified a panel of several promising scFv candidates (emerging from both our CAR phenotypic and classical phage display screens) as soluble scFvl(aMSLN)-scFv2(aCD3s) BiTEs, and screened their capacity in vitro to trigger effector activity against MSLN+tumor lines in co-cultures containing primary T cells.

[0821] Unexpectedly, 13F08 appeared to be the most potent of the tested BiTEs in an IncuCyte kinetic cell killing assay, specifically driving killing of 0VCAR8, H226, and SK0V3 cognate target cells, inducing IFN-y secretion, and activating an NF AT reporter Jurkat cell line in the presence of both hMSLN-transfected HEK and endogenously expressing 0VCAR8 cells (Figure 10A and Figure 11). Interestingly, we observed that certain comparator CAR scFvs exhibited similarly compelling in vitro efficacy in the purified BiTE format. Here, SSI, M5, and 15B6 compared favourably with 13F08 in their control of 0VCAR8 and SK0V3 target cells (Figure 11).Atty. Docket No. 2745-9 PCTT

[0822] T Cells Engineered to Constitutively Secrete LABC_13F08 Monovalent BiTE Show Specific and Potent In Vitro Activity Towards MSLN+Tumor Lines.

[0823]

[0359] Intrigued by our findings we next asked whether human T cells could be engineered to secrete active 13F08 BiTE in quantities sufficient to drive efficient T cell redirection in the presence of cognate target cells. To confirm the translation / expression potential of a codon-optimized version of the 13F08 scFv in primary human T cells, we first confirmed its expected functional behavior as a 2ndgeneration 4- I BBc CAR (Figure 12). Subsequently, we engineered T cells to express a secreted FLAG-tagged 13F08-BiTE (Figure 13A). We applied imaging flow cytometry to confirm the presence of a functional CD3s- interacting BiTE (Figure 14), and flow cytometry to confirm binding of T cell-secreted 13F08-BiTE to native tumor cell-expressed MSLN (Figure 14B). Importantly, we also observed that the 13F08-BiTE T cells (hereafter, BiTE-T) predominantly exhibited a Tcmphenotype (induced by our T cell expansion protocol) that was indistinguishable from that of non-transduced (NT) T cells as indicated by positivity for the discriminating CD45RO, CCR7, CD28, and CD27 Tcmmarkers (Figure 15A). Additionally, 13F08 BiTE-T cells do not show any significant cytokine release nor upregulation of tonic signalling markers relative to the NT or CD19 CAR-engineered T cell backgrounds (Figure 15 B, C).

[0824]

[0360] We next performed kinetic IncuCyte assays to assess the extent of any specific target cell killing. Encouragingly, the growth curves of all MSLN+tumor lines, including SKOV3 and HeEa which are stained only modestly with 13F08-Fc, were efficiently suppressed by the 13F08 BiTE-T cells indicating that secreted BiTE concentrations rapidly achieve levels that exceed activation thresholds in these cultures (Figure 13A, B). Notably, the A673 and OVCAR5 MSLN-negative control lines were completely spared, with negligible secretion of effector cytokines or induction of activation markers on the T cells in these cultures (Figure 13C). Additionally, the induction of typical activation markers and certain effector cytokines in 13F08 BiTE-T co-cultures was seen to correlate with the levels of tumor cell surface MSLN as determined by the commercial K1 reference mAb (Figure 13D and Figure 16). We next confirmed the binding of T cell-secreted BiTEs to NT T cells (Figure 17A, B) and asked whether BiTE-T secreted 13F08 BiTE could function in trans to recruit NT bystander T cells to mediate killing of cognate MSLN+target cells. We observed that a single supplementation with 13F08 BiTE-T cell media is sufficient to robustly redirect non-engineered T cells to kill MSLN+targets, and to trigger effector cytokine secretion (Figure 17 C, D). Additionally, we also titrated the percentage composition of 13F08 BiTE-T cells present in the bulk effector cell population at a standard 2: 1 effector to target ratio.

[0825] Strikingly, we observed substantial control of OVCAR8 MSLN+cells with as few as 3% of the effectors arising from the 13F08 BiTE-T preparation (Figure 17E). Notably, the real-time killing kinetics also suggested a lag at low BiTE-T cell numbers likely indicating an accumulation phase whereby secretedAtty. Docket No. 2745-9 PCTT

[0826] BiTE levels gradually attain the necessary threshold concentrations required for subsequent widespread T cell recruitment and effector activation (Figure 17E).

[0827]

[0361] Finally, we asked how 13F08 BiTE-T cell in vitro efficacy would compare with 13F08 CAR-T (4-1BBQ cells against 0VCAR8, H226 and AsPC-1 cells. Here, we used a sub-optimal E: T ratio of 1:1 with high cell transduction efficiencies (-80% for both BiTE and CAR). We observed that 13F08 BiTE-secreting T cells exhibited rapid and robust target cell killing kinetics, particularly towards H226, when compared alongside their 13F08 CAR-T counterparts. The increased apparent potency exhibited by the 13F08 BiTE-T cell format correlated with target-specific cytokine release profiles that were more robust and uniform than those seen for the CAR (Figure 18). We speculate that the profound recruitment of potentially all T cells present in the co-culture (including NT, as highlighted in Figure 17), together with the activation signal proceeding through engagement of the sensitive native CD3 / TCR complex likely accounts for the observed performance of 13F08 BiTE-T.

[0828] 13F08 BiTE-T Cells Demonstrate Both MSLN+Target Cell-Dependent Bystander Killing In Vitro and Exhibit Insensitivity to Soluble MSLN Decoy Fragments

[0829]

[0362] The localized “bystander” killing of adjacent non-cognate target cells by pre-activated CD8+ cytotoxic T cells has been previously described (27, 36), and strategies to induce or stimulate this activity to augment the efficacy of CAR-T therapies towards solid tumors are areas of active investigation (37). Interestingly, significant in vitro killing of MSLN-negative bystander cells was previously reported during the initial characterization of the P4 anti-MSLN CAR (27). In an IncuCyte assay comprising cocultures of cognate MSLN+OVCAR8 cells and A673 or A375 tumor cells lacking MSLN, we observed robust killing of both A673 and A375 cells by 13F08 BiTE-T cells only in the presence of MSLN+OVCAR8 cells which was not due to soluble factors present in the OVCAR8 culture supernatant (Figure 19 A-C and Figure 20A). This would imply the activation of a pathway - or pathways - associated with the engagement of death receptors (for example FAS or DR5) on A673 and A375 cells. Interestingly, in co-cultures containing the non-tumor HEK293T cell line, bystander killing by activated 13F08 BiTE-T was not observed, although the growth of HEKs was significantly slowed (Figure 19D). Future studies will be required to further elucidate these data. We also asked whether 13F08 BiTE-T cell killing efficacy could be impacted by the presence and decoy potential of soluble “decoy” MSLN fragments shed from tumor cells by cell surface protease activity (17). We observed no apparent inhibition of OVCAR8 killing kinetics when native shed MSLN from spent and concentrated OVCAR8 supernatants (160 ng / mL final concentration) was included in the assay. Similarly, 1.3 ug / mL of commercial, purified rhMSLN was also without effect (Figure 20B).Atty. Docket No. 2745-9 PCTT

[0830] LAB_13F08 BiTE-Sectering T Cells Show Potent In Vivo Anti-Tumor Activity in ACT Xenograft Models

[0831]

[0363] We next performed an in vivo study in which we treated established subcutaneous OVCAR8 tumors in immunodeficient NSG mice with two cycles of intravenously infused high dose 13F08 BiTE-T (Figure 21A). Notably, we observed complete tumor eradication by 13F08 BiTE-T monotherapy with no outward signs of toxicity (Figure 22). Additionally, we also analysed tumor tissue from mice cohorts treated in parallel at an advanced stage of tumor control (once tumor volumes had decreased to ~22mm3). Tumors from those mice treated with 13F08 BiTE-T cells showed not only strikingly higher levels of infiltration by the CD45+ACT product than the control CD19 BiTE control tumors, but also elevated Ki-67 expression, suggestive of a higher proliferative capacity of these cells in response to MSLN-driven antigen encounter (Figure 21B). 13F08 BiTE-T ACT cell levels determined at this timepoint for peripheral blood were similarly present at frequencies higher than observed for the CD19 control.

[0832] Extending the study, we challenged 13F08 BiTE-T cells against xenografted H226 and SKOV3 tumors, observing a rapid clearance of the former following ACT (Figure 21C). In our previous profiling experiments, SKOV3 cells exhibited weak and heterogeneous anti-MSLN staining, with low anti-MSLN WB sensitivity (Figure 2 and Figure 3). Nevertheless, 13F08 BiTE-T cells achieved an impressive level of tumor control, with outgrowth only resuming approximately 10 days after ACT. We speculate that the escape of SKOV3 xenograft tumors may be due to the survival of a pre-existing compartment of cells expressing levels of MSLN beneath the threshold for functional 13F08-BiTE control. We cannot, however, exclude inefficient bystander killing as a contributary factor, either due to weaker T cell activation generally (due to lower MSLN antigen density), or to the absence of compatible death receptors on SKOV3.

[0833]

[0364] To better evaluate the in vivo potency of 13F08 BiTE-T cells, we performed two further experiments using lower dose ACT T cell regimens. Encouragingly, two ACT cycles comprising a total of 18xl06BiTE-T cells were sufficient to eradicate OVCAR8 tumors (Figure 21D). Interestingly, ex vivo analysis of peripheral blood T cell counts 1 week post-tumor clearance revealed the presence of only a few surviving cells, suggesting only limited capacity for survival in the absence of ongoing tumor stimulation. In a further experiment, we generated an additional xenograft model using AsPC-1, an aggressive pancreatic tumor line known to be difficult to control in the xenograft setting38. We treated mice bearing established AsPC-1 tumors with a single low dose of 10xl0613F08 BiTE-T cells, and observed a significant tumor growth arrest. Ex vivo analysis confirmed a strong infiltration and activation of T cells relative to the CD19 BiTE-T control treatment and, as for OVCAR8, a low T cell presence in the peripheral blood (Figure 2 IE). The low persistence of the ACT product in the circulation observed for these two distinct NGS models also explains the absence of any obvious GvHD in these experiments.Atty. Docket No. 2745-9 PCTT

[0834] Consistent with this, for all in vivo models, we observed no indications of peripheral toxicity as assessed by the monitoring of animal behavior and weight over the period of the study (Figure 22).

[0835]

[0365] As a negative in vivo control to further confirm the specificity of 13F08 BiTE-T cells, mice were implanted with 0VCAR5, a line that profiles as either negative or very low for MSLN expression by FACs staining (Figure 2 and Figure 3) (27). As expected, 0VCAR5 tumors were not controlled by 13F08 BiTE-T cells (Figure 23).

[0836] LAB_13F08 BiTE-Sectering T Cells Can Be Rapidly Cleared Using a Truncated EGFR Safety Switch

[0837]

[0366] The absence of observable toxicity in our NGS xenograft models following the systemic ACT of constitutively expressing 13F08 BiTE-T cells provides confidence that this novel scFv does not exhibit any obvious off-target or “sticky” behaviour when challenged with a galaxy of murine cell-surface tissue antigens. As 13F08 does not recognize native murine MSLN or, by inference, those healthy somatic mouse tissues that express low endogenous target levels, these models cannot inform on the risk to safety of possible on-target / off-tumor toxicity when translating to the human setting. Hence, we considered it prudent to consider incorporating a mechanism to ablate adoptively transferred 13F08 BiTE-T cells to further mitigate clinical risk. One such reported strategy is the co-expression of a truncated non-signalling EGFR (tEGFR) which retains the depleting epitope recognized by the FDA-approved antibody drug, Cetuximab (39). We therefore re-engineered our BiTE-T vector with an upstream tEGFR sequence appended for co-expression via a P2A (Figure 24A, B). Encouragingly, in vitro co-culture IncuCyte killing experiments revealed no impact of the co-expressed tEGFR on target cell killing kinetics or on the secretion of the effector molecules, IFNy and Granzyme B, relative to the singly-engineered 13F08 BiTE-T cells (Figure 24C). We next asked whether Cetuximab-mediated clearance could operate in vivo using an NSG xenograft model. Although lacking NK cells, immune compartments potentially capable of eliciting ADCC / P (e.g. macrophages, neutrophils) are still present in this strain. We treated 0VCAR8 tumor-bearing mice with two ACT cycles using co-engineered tEGFR / 13F08 or tEGFR / CD19 BiTE-T cells. One day following each ACT, mice were treated with Cetuximab or Rituximab control antibody and monitored for tumor growth (Figure 24D). Importantly, we observed that administration of Cetuximab, but not Rituximab, resulted in a total cessation of tumor control by dual-engineered tEGFR / 13F08 BiTE-T cells. Additionally, these dual-engineered BiTE-T cells and their respective antibody treatments induced no apparent toxicity in the mice (Figure 24E). Hence, our data suggest that rapid depletion of the 13F08 BiTE engineered T cell population post-infusion using the tEGFR system is feasible and efficient, and may merit consideration in a clinical setting.

[0838] DISCUSSIONAtty. Docket No. 2745-9 PCTT

[0839]

[0367] CAR-T or soluble T cell engagers offer great potential as toolkit components for the treatment of solid tumors, not least due to their extraordinary sensitivity and effector-driven amplification potential. For either approach, it can be argued that the requisite targeting scFvs (or other equivalent fragments) should ideally derive from tailored, bottom-up screening programs with these specific applications as the primary focus. In phenotypic reporter-enrichment experiments we previously identified LABC_13F08, a fully-human “fit-for-purpose” anti-MSLN scFv capable of driving productive cell-bridging and strong signal transduction as a generic 2ndgeneration CAR.

[0840]

[0368] In the current study, we report three key findings. Firstly, 13F08 retains potent and selective T cell redirection activity when reformatted, expressed, and purified as a classical tandem BiTE with a humanized anti-CD3s scFv. We have observed previously that molecules emerging from our CAR screen may be predisposed to such dual-functionality, which is likely due to common requirements for a permissive bridging distance and geometry between the epitope and T cell surface, together with an intrinsic minimal monovalent affinity requirement for registering in our CAR reporter assay.

[0841]

[0369] Secondly, we identify an unusual, absolute requirement for divalent metal cations in the interaction of 13F08 with hMSLN. None of the comparator molecules we evaluated exhibited this requirement and, to the best of our knowledge, no studies implicating metal co-ordination in the binding of any other anti-MSLN antibody fragment or known natural ligand have been described. Additionally, no bound metal ion adduct is evident in the few reported MSLN crystal structures. This would argue against a metal-induced conformational shift within MSLN that creates / exposes an epitope for 13F08, and would instead favor a model of an atomic co-ordination shell that is exclusively derived from sequence features within the 13F08 CDRs, or that is shared between MSLN and the scFv. Interfacial metal ion involvement in antibody recognition is rare with only a few examples reported - all of which utilize Ca2+to either form an electrostatic “bridge” to the antigen, or to stabilize a particular paratope conformation (41-43).

[0842] Interestingly, in our BLI kinetic affinity experiments, Ca2+was seen to promote the greatest affinity gain, bringing the effective monovalent KD of 13F08 into the single-digit nM range, which is sufficient to account for its observed soluble BiTE activity. As the concentration of accessible Ca2+in the extracellular milieu of the tumor microenvironment is considered to be in excess of ImM, the level of this co-factor is unlikely to be limiting for optimal 13F08 function in vivo. Ongoing structural and biochemical studies will aim to further elucidate the details of this novel MSLN-Ca2+-antibody binding interaction.

[0843]

[0370] Finally, we were interested to explore whether our 13F08 BiTE could have utility in ACT as a T cell secreted anti-tumor agent. The secreted BiTE-T conceptual paradigm is gaining traction as an alternative, or partner strategy, to CAR-T. To-date, several studies have reported encouraging pre-clinical proof-of-concept data using secreted BiTEs targeting several distinct TAAs including CD123, EGFR, EphA2, IL13Rα2, Frα, FAP and EGFRvIII tumor antigens, either as single agents or paired with a coAtty. Docket No. 2745-9 PCTT

[0844] transduced second BiTE or a CAR (43-48). Recently, a dual EGFRvIII-CAR / EGFR-BiTE approach was the subject of a glioblastoma clinical trial report (49). Collectively, this body of data suggests that leveraging the therapeutic potency of the BiTE drug format in a BiTE-T ACT setting has great potential. To counter a rapid clearance due to their small size and lack of an Fc domain, tandem soluble BiTE drugs are clinically administered by continuous infusion which is associated with narrow therapeutic safety windows and non-specific systemic toxicities (50). By contrast, infused BiTE-T cells, which retain the potential to persist and expand, can traffic to tumor sites where they can deliver locally active concentrations of BiTE. Interestingly, at least two studies using different tumor xenograft mouse models have shown that, following ACT, secreted BiTE levels in the peripheral blood remain below thresholds of detection (44, 45). This raises the intriguing question of whether constitutively expressing BiTE-T cell reagents may possess a clinical safety advantage over soluble BiTE drugs. One may speculate that intrinsically lower levels of mRNA and constitutive protein expression in unstimulated BiTE-T cells could work in concert with renal and pharmacological clearance mechanisms to resist the accumulation of secreted BiTE in the systemic circulation. Following trafficking to the tumor and extravasation, basal levels of secreted BiTE appear sufficient to activate BiTE-T producer cells in the presence of tumor antigen. The resulting upregulation of BiTE-T global protein expression could, in principle, lead to increased BiTE secretion at the tumor site and bystander recruitment within the TME (45).

[0845]

[0371] To the best of our knowledge, our data represents the first demonstration that a MSLN-targeting BiTE-T reagent can efficiently control endogenous MSLN+tumor cells both in vitro and in vivo. Further, our data indicates that 13F08 BiTE-T cells exhibit or promote target cell-dependent bystander killing and show no functional sensitivity to shed or soluble MSLN fragments. Additionally, we observe that 13F08 BiTE-T cells can be rapidly ablated in vivo using a commercial antibody safety “switch”. Previous BiTE-T studies have all enlisted existing “off-the-shelf’ rodent / humanized mAbs which the authors have reverse-engineered into scFvs for BiTE construction. In contrast, 13F08 represents the first reported BiTE-T fully human scFv warhead isolated de novo as a functional CAR / BiTE.

[0846]

[0372] As a stand-alone monotherapy, 13F08 BiTE-T persistence and proliferation in the clinical setting could be a concern due to the absence of adequate co-stimulatory signaling. This has been raised by others who observed significant checkpoint expression and a loss of memory and naive BiTE-T cell phenotypes following prolonged antigen stimulation in a mouse model (48). Currently, it is unknown if chronically stimulated, non-proliferating BiTE-T cells could persist within a malignancy and retain competency as bio-reactors, continuing to secrete active levels of diffusible BiTE. If this is the case, one may speculate that local bystander TILs, phenotypically diverse and lacking the signatures of chronic antigen stimulation, could still be effectively recruited for tumor control. Studies to explore this scenario,Atty. Docket No. 2745-9 PCTT

[0847] together with the further augmentation of 13F08 BiTE-T performance through co-engineering with additional effector and or signaling components for persistence and expansion are ongoing.

[0848]

[0373] The data describe LABC_13F08, a novel metal-dependent human anti-MSLN scFv, and demonstrate for the first time that a BiTE-T cell strategy can be leveraged to control and even eliminate different endogenous MSLN+tumor cells both in vitro and in vivo. Conceptually, this presents a significantly differentiated approach to current anti-MSEN CAR-T ACT, wi...

Claims

Atty. Docket No. 2745-9 PCTTClaims*1. An isolated antibody or active fragment thereof which recognizes and binds human mesothelia (MSLN), wherein the antibody or fragment comprises a heavy chain variable region sequence comprising:(a) a CDR1 sequence GYTFTGYY (SEQ ID NO:3) or GYTFTGYY H (SEQ ID NO:4);(b) a CDR2 sequence 1NPNSGGT (SEQ ID NO:5), W1NPNSGGTNYAQK. FQD (SEQ ID NO:6) or W1NPNSGGTNYA. QKFQG (SEQ ID O:22); and(c) a CDR3 sequence AREIYSGSHTPDDAFDI (SEQ ID NO:7);and further comprising a light chain variable region comprising:(a) a CDR1 sequence QGISNS (SEQ ID NO: 8) or RASQGISNSLA (SEQ ID NO:9);(b) a CDR2 sequence GAS (SEQ ID NO: 10) or G ASILES (SEQ ID NO: 11 ); and(c) a CDR3 sequence QQYYSTPHT (SEQ ID NO: 12).

2. The isolated antibody or active fragment of claim 1 comprising a heavy chain variable region sequence comprising:(a) a CDR1 sequence GYTFTGYY (SEQ ID NO:3);(b) a CDR2 sequence INPNSGGT (SEQ ID NO: 5); and(c) a CDR3 sequence AREIYSGSHPDDAFDI (SEQ ID NO:7);and further comprising a light chain variable region comprising:(a) a CDR1 sequence QGISNS (SEQ ID NO: 8);(b) a CDR2 sequence GAS (SEQ ID NO: 10); and(c) a. CDR3 sequence QQYYSTPHT (SEQ ID NO: 12).

3. The isolated anti body of clai m 1 or 2, wherein the antibody recogn izes or binds the D I -D2 fragment or region of human MSLN.4, The isolated antibody of claim 1 or 2. wherein the antibody particularly recognizes or binds the DI region of human MSLN.

5. The isolated antibody of claim 1 or 2, wherein recognition and binding to MSLN requires divalent cations.Atty. Docket No. 2745-9 PCTT6. The isolated antibody of any one f claims 1-5, wherein the anti body or antigen binding fragment comprises a heavy chain variable region (VH) sequence corresponding to the amino acid sequence SEQ ID NO: 1 or SEQ ID NQ:20 or a sequence having at least 90% identity to the amino acid sequence SEQ ID NO:1 or SEQ ID NO:20;and / or comprises the light chain variable region (VL or VK) sequence corresponding to the amino acid sequence SEQ ID NO:2 or SEQ ID NO:21 or a sequence having at least 90% identity to the amino acid sequence SEQ ID NO:2 or SEQ ID NO:21.

7. A single-chain variable fragment (scFv) of the antibody of any one of claims 1-8,8. The scFv of claim 7, wherein the scFv is selected from SEQ ID NO: 14, 23, 26, 27, 15, 18, 29 and 30,9. A composition comprising the antibody or active fragment or scFv of one any of claims 1-8 and a pharmaceutically acceptable carrier.

10. A cell comprising and / or expressing the MSLN antibody or active fragment or scFv of one any of claims 1-8.

11. A chimeric antigen receptor (CAR) or bispecific T cell engager (BiTE) comprising the antibody or active fragment or scFv of any one of claims 1-8 for targeting MSLN or MSLN-expressing cells.

12. The BiTE of claim 11, wherein the BiTE comprises the scFv selected from SEQ ID NO: 14, 23, 26, 27, 15, 18, 29 and 30.

13. The BiTE of claim 11 or 12, comprising antibody or active fragment or scFv targeting MSLN or MSLN-expressing cells and further comprising CD3 binding sequence or anti-CD3 scFv sequence.

14. The BiTE of any one of claims 11-13, wherein the BiTE is selected from SEQ ID NO: 17, 24, 32, 33, 34. 35, 36 and 37.

15. A T cell expressing an MSLN-specific BITE, wherein the T cell engages with MSLN* or MSLN expressing cells.Atty. Docket No. 2745-9 PCT16. A cell, particularly an immune cell, comprising the MSLN CAR or BiTE of any of claims 11-14.

17. A nucleic acid encoding a MSLN antibody or active fragment or scFv of any of claims 1 -8, or a CAR or BiTE of any of claims 11 -1418. A vector comprising the nucleic acid of claim 17,19. A cell, particularly an immune e ffector cell, comprising the nucleic acid of claim 17 or the vector of claim 18,20. A method of treating a subject having a cancer or disease associated with expression of M’SLN, the method comprising administering to the subject the MSLN antibody or active fragment or scFv of one any of claims 1-8, the composition of claim 9, the cell of claim 10, the CAR or BiTE of any of claims 11-14, the cell of claim 15 or 16, or the cell of claim 19.

21. The method of claim 20, wherein the cancer is selected from mesothelioma, stomach cancer, squamous cell carcinomas, prostate cancer, pancreatic cancer, lung cancer, breast cancer (particularly triple-negative cancer), cervical cancer, leukemia (particularly. AML) and ovarian cancer.

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