Antibodies to MSLN and methods of use thereof

Abstract

The present invention includes human antibodies that bind to the mesothelin (MSLN) protein, which can be used to treat cancer. TIFF2025528835000026.tif81146

Description

[Technical Field]

[0001] This application is an international application claiming priority to U.S. Provisional Patent Application No. 63 / 398,082, filed August 15, 2022, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. The disclosures of these publications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art known to those skilled in the art as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights.

[0004] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing which is incorporated herein by reference in its entirety. The ASCII copy made in [ ] has the name [ ] and is [ ] bytes in size.

[0005] FIELD OF THE INVENTION The present invention includes antibodies to mesothelin (MSLN) and methods of use thereof. [Background technology]

[0006] background Mesothelin (MSLN) is a protein normally expressed in mesothelial cells. MSLN can exist in two forms: a membrane-bound form via a GPI linker and a soluble / shedded form. Summary of the Invention

[0007] An aspect of the present invention is directed to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to a mesothelin protein or fragment thereof, including a heavy chain, a light chain, or a combination thereof.In embodiments, the heavy chain comprises a CDR1 comprising GYTLTTNG (SEQ ID NO: 31), GGTFSSDT (SEQ ID NO: 35), GFTFDDYA (SEQ ID NO: 38), GFKFTDYY (SEQ ID NO: 46), or GYTFTHYA (SEQ ID NO: 49), VSPYNGHI (SEQ ID NO: 32), VNPYNGHI (SEQ ID NO: 34), INPNSGGT (SEQ ID NO: 36), ISWNSGSI (SEQ ID NO: 39), ISWNNGSI (SEQ ID NO: 41), ISWNSGNI (SEQ ID NO: 43), INTSSNHI (SEQ ID NO: 47), or IHAGNGNS (SEQ ID NO: 48). and a CDR2 comprising ARVNRANYYGMDV (SEQ ID NO: 33), ARESALGGSYPLSF (SEQ ID NO: 37), AKDPSSSWLAGAFDI (SEQ ID NO: 40), AKDPSTSWLAGAFDI (SEQ ID NO: 42), AKDAGSSGYFNAFDI (SEQ ID NO: 44), AKSPSSNWYPDAFDI (SEQ ID NO: 45), ARGASWGPL (SEQ ID NO: 48), or AREVGHGMDV (SEQ ID NO: 51), or a combination of these CDRs; , QSVSSSY (SEQ ID NO: 59), SLRRFY (SEQ ID NO: 62), SLIRFY (SEQ ID NO: 65), SLRAYY (SEQ ID NO: 67), SLRNYY (SEQ ID NO: 70), NIGSKS (SEQ ID NO: 73), or SSNIGAGYD (SEQ ID NO: 76), CDR1 including LGS (SEQ ID NO: 53), GAS (SEQ ID NO: 60), GKN (SEQ ID NO: 63), AKN (SEQ ID NO: 71), DDS (SEQ ID NO: 74), or GNT (SEQ ID NO: 77), CDR2 including MQSSTNSAH (SEQ ID NO: 54), MQALQTPLT (SEQ ID NO: 55), MQ and a CDR3 comprising ALQTPL (SEQ ID NO: 56), MQALQTPLT (SEQ ID NO: 57), HASSTNSAH (SEQ ID NO: 58), LQDDSYPLT (SEQ ID NO: 61), NSRDSDGNHVF (SEQ ID NO: 64), NSQDRDGNHVF (SEQ ID NO: 66), NSRDSSGNHLGV (SEQ ID NO: 68), NSRDSSGNHLGS (SEQ ID NO: 69), SSRDSSSDNHVV (SEQ ID NO: 72), QSADYNWLCD (SEQ ID NO: 7475), or QSYDSSLSGYV (SEQ ID NO: 78), or a combination of these CDRs.In an embodiment, the sequence is determined using the IMGT numbering scheme.

[0008] In embodiments, the mesothelin protein is a human mesothelin protein.

[0009] In embodiments, the antibodies are fully human or humanized.

[0010] In embodiments, the antibodies are monospecific, bispecific, or multispecific.

[0011] In embodiments, the antibody is an IgG, e.g., the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0012] In embodiments, the antibody is a single chain antibody, a bispecific antibody, or a bispecific T cell engager.

[0013] In embodiments, the antibody has a binding affinity of at least 1.0×10 −9 M.

[0014] In embodiments, the antibody further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0015] In embodiments, the antibody comprises Gly3-1-H10, TEA2-E10, TEA1-D3, Gly3-2-C1, TEA1-D2, Gly2-2-F7, Gly1-2-H4, Gly1-2-D4, TEA1-E5, Gly3-2-C10, Gly2-1-F2, Gly2-2-B6, Gly1-2-E10, TEA2-C9, or TEA1-E8.

[0016] In embodiments, the antibody competes with binding of Gly3-1-H10, TEA2-E10, TEA1-D3, Gly3-2-C1, TEA1-D2, Gly2-2-F7, Gly1-2-H4, Gly1-2-D4, TEA1-E5, Gly3-2-C10, Gly2-1-F2, Gly2-2-B6, Gly1-2-E10, TEA2-C9, or TEA1-E8.

[0017] In embodiments, the antibody competes with binding of Gly3-1-H10, TEA2-E10, TEA1-D3, Gly3-2-C1, TEA1-D2, Gly2-2-F7, Gly1-2-H4, Gly1-2-D4, TEA1-E5, Gly3-2-C10, Gly2-1-F2, Gly2-2-B6, Gly1-2-E10, TEA2-C9, or TEA1-E8.

[0018] In embodiments, the antibody or fragment is linked to a therapeutic agent.

[0019] In embodiments, the antibody is a single chain fragment, a bispecific antibody, or a bispecific T cell engager (BiTE).

[0020] Aspects of the present invention are also directed to isolated antibodies or fragments thereof that bind to human mesothelin protein. In embodiments, the antibody comprises: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 54; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 54; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 53. or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 56; or (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57; or (f) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 52 CDR1, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 58;or (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 36, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 37, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 59, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 61; or (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 40, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 62, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 64; or (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 40, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 62, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 64. CDR3; (j) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 65, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; (k) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 43, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 44, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 68; (l) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 43, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 44, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69 CDR3;(m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78. CDR3. ;

[0021] Embodiments of the present invention are also directed to isolated scFv antibodies that bind to human mesothelin protein. In embodiments, the antibody comprises: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 54; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 54; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 53. or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 56; or (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57; or (f) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 52 CDR1, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 58;or (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 36, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 37, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 59, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 61; or (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 40, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 62, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 64; or (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 40, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 62, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 64. CDR3; (j) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 65, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; (k) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 43, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 44, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 68; (l) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 43, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 44, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69 CDR3;(m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78. CDR3. In embodiments, the sequence is determined using the IMGT numbering scheme.

[0022] In embodiments, the invention includes an isolated antibody or fragment thereof that binds to human mesothelin protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence at least 90% identical thereto.

[0023] In embodiments, the invention includes an isolated antibody or fragment thereof that binds to human mesothelin protein, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence at least 90% identical thereto.

[0024] In embodiments, the invention includes an isolated antibody or fragment thereof that binds to human mesothelin protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence at least 90% identical thereto.

[0025] In embodiments, the invention includes an isolated scFv that binds to human mesothelin protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence at least 90% identical thereto.

[0026] In embodiments, the invention includes an isolated scFv that binds to a human mesothelin protein, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence at least 90% identical thereto.

[0027] In embodiments, the invention includes an isolated scFv that binds to human mesothelin protein, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence at least 90% identical thereto.

[0028] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:1 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:2.

[0029] In embodiments, the invention includes an isolated scFv that binds to a human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:1 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:2.

[0030] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:3 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:4.

[0031] In embodiments, the invention includes an isolated scFv antibody that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:3 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:4.

[0032] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:5 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:6.

[0033] In embodiments, the invention includes an isolated scFv that binds to a human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:5 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:6.

[0034] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:7 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:8.

[0035] In embodiments, the invention includes an isolated scFv antibody that binds to a human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:7 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:8.

[0036] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:9 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:10.

[0037] In embodiments, the invention includes an isolated scFv antibody that binds to a human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:9 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:10.

[0038] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:11 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:12.

[0039] In embodiments, the invention includes an isolated scFv antibody that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:11 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:12.

[0040] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 13 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 14.

[0041] In embodiments, the invention includes an isolated scFv antibody that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 13 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 14.

[0042] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 15 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 16.

[0043] In embodiments, the invention includes an isolated scFv that binds to a human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 15 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 16.

[0044] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 17 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 18.

[0045] In embodiments, the invention includes an isolated scFv antibody that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 17 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 18.

[0046] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 19 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 20.

[0047] In embodiments, the invention includes an isolated scFv antibody that binds to a human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 19 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 20.

[0048] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:21 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:22.

[0049] In embodiments, the invention includes an isolated scFv antibody that binds to a human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:21 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:22.

[0050] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:23 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:24.

[0051] In embodiments, the invention includes an isolated scFv antibody that binds to a human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:23 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:24.

[0052] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:25 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:26.

[0053] In embodiments, the invention includes an isolated scFv antibody that binds to a human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:25 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:26.

[0054] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:27 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:28.

[0055] In embodiments, the invention includes an isolated scFv that binds to a human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:27 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:28.

[0056] In embodiments, the invention includes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:29 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:30.

[0057] In embodiments, the invention includes an isolated scFv antibody that binds to a human mesothelin protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO:29 and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:30.

[0058] Embodiments of the present invention are also directed to isolated bispecific antibodies comprising an antibody fragment described herein and a second antigen-binding fragment having specificity for a molecule on an immune cell, e.g., the molecule is selected from the group consisting of CCR4, B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, and KIR.

[0059] In embodiments, the fragment and the second fragment are each independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody.

[0060] In embodiments, the bispecific antibody further comprises an Fc fragment.

[0061] Further embodiments of the present invention are directed to bispecific T cell engagers (BiTEs) that bind to the human mesothelin protein. In embodiments, a BiTE comprises: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 54; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 54; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 53. or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 56; or (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57; or (f) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 52 CDR1, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 58;or (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 36, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 37, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 59, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 61; or (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 40, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 62, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 64; or (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 40, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 62, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 64. CDR3; (j) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 65, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; (k) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 43, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 44, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 68; (l) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 43, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 44, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69 CDR3;(m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78. CDR3. In embodiments, the sequence is determined using the IMGT numbering scheme.

[0062] Another aspect of the present invention is directed to a bispecific T cell engager (BiTE) that binds to human mesothelin protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence at least 90% identical thereto.

[0063] Further aspects of the present invention are directed to bispecific T cell engagers (BiTEs) that bind to human mesothelin protein, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence at least 90% identical thereto.

[0064] Yet a further aspect of the present invention is directed to a bispecific T cell engager (BiTE) that binds to human mesothelin protein, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence at least 90% identical thereto.

[0065] Aspects of the invention are also directed to nucleic acids encoding the antibodies or fragments described herein.

[0066] Aspects of the present invention are also directed to pharmaceutical compositions comprising an antibody or fragment thereof described herein and a pharmaceutically acceptable carrier or excipient.

[0067] In embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent, for example, the therapeutic agent is a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine.

[0068] Aspects of the present invention are also directed to isolated cells comprising one or more polynucleotides encoding the antibodies or fragments described herein.

[0069] In embodiments, a vector may comprise a nucleic acid described herein.

[0070] In embodiments, the cells may comprise a vector described herein.

[0071] Aspects of the invention also relate to engineered cells containing chimeric antigen receptors. In embodiments, the chimeric antigen receptor comprises an extracellular ligand-binding domain specific for an antigen on the surface of a cancer cell, the antigen comprising mesothelin, and the extracellular ligand-binding domain comprises an antibody or fragment thereof, the antibody or fragment thereof comprising: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 54; (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 54; (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 54 (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 55; (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 56; (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57;(f) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 52, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 58; (g) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 35, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 36, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 37, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 59, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 61; (h) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 40, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 62, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 64. CDR3; (i) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 40, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 62, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 64; (j) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 65, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; (k) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 43, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 44, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 68 CDR3;(l) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 43, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 44, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69; (m) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 38, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 39, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; (n) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75. or (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78. In embodiments, the sequences are determined using the IMGT numbering scheme;

[0072] In embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29, or a sequence at least 90% identical thereto.

[0073] In embodiments, the antibody or fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30, or a sequence at least 90% identical thereto.

[0074] In embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence at least 90% identical thereto.

[0075] In embodiments, the cells include T cells, NK cells, NKT cells, iPS cells, iPS-derived cells, cell lines, or B cells. For example, the cells include CD4+, CD8+, CD3+ pan T cells, or any combination thereof.

[0076] Aspects of the present invention are also directed to kits. In embodiments, the kits include at least one pharmaceutical composition described herein; a syringe, needle, or applicator for administering at least one antibody to a subject; and instructions for use.

[0077] Further aspects of the invention are directed to methods for detecting the presence of mesothelin in a sample. In embodiments, the methods include contacting the sample with a monoclonal antibody described herein and detecting the presence or absence of an antibody-antigen complex, thereby detecting the presence of mesothelin in the sample.

[0078] In embodiments, the contacting comprises immunohistochemistry, for example, immunohistochemistry comprises precipitation, immunofluorescence, Western blot, or ELISA.

[0079] In embodiments, the sample comprises whole blood, a blood component, a bodily fluid, a biopsy, a tissue, serum, or one or more cells. For example, the one or more cells comprise an in vitro culture. For example, the one or more cells comprise a mesothelin-expressing cell.

[0080] In embodiments, the sample includes a normal sample or a cancerous sample, for example, the sample is an in vitro sample.

[0081] Embodiments may further include obtaining a sample from a subject.

[0082] In embodiments, the cancer expresses mesothelin. In embodiments, the cancer comprises an epithelial cell cancer. For example, the epithelial cell cancer comprises lung cancer, mesothelioma, or ovarian cancer.

[0083] Aspects of the present invention are also directed to methods for treating cancer in a subject. In embodiments, the method comprises administering to the subject a pharmaceutical composition described herein.

[0084] In embodiments, the cancer comprises an epithelial cell cancer, for example, the cancer comprises lung cancer, mesothelioma, or ovarian cancer.

[0085] Other objects and advantages of the present invention will become readily apparent from the description which follows. [Brief explanation of the drawings]

[0086] [Figure 1] Characterization of a set of antibodies for CASS B cell development. Figure 1 provides cell binding curves for anti-MSLN (top) and anti-Muc1 (bottom) scFv-Fc, demonstrating that we previously identified high affinity antibodies.

[0087] [Figure 2] CASS B cell construct testing and B cell transduction. Panel A shows that engineered cells expressing an IgG-BCR construct can bind soluble HA. Panel B shows that lentivirus can be used to achieve high transduction efficiency with multiple DNA constructs and donors. Panel C shows an engineered reporter system in Jurkat cells using an NFAT / NFκB-inducible response element showing increasing levels of GFP expression under various stimulation conditions. Embodiments described herein can be used with the CASS B cell platform.

[0088] [Figure 3] Figure 2 shows that 293T cells transfected with engineered membrane-bound IgG exhibit high levels of membrane IgG binding (Panel A) and soluble HA capture (Panel B).

[0089] [Figure 4-1] Figure 4 is a schematic diagram showing the structure of mesothelin (MSLN). Sources: Mesothelin, Hassan, Raffit et al. Clin Cancer Res, 2004 and New High Affinity Monoclonal Antibodies Recognize Non-Overlapping Epitopes On Mesothelin For Monitoring And Treating Mesothelioma, Zhang, Yi-Fan et al. Scientific Reports, 2015. [Figure 4-2] See description of Figure 4-1.

[0090] [Figure 5] FIG. 1 is a schematic diagram showing the protocol for whole cell panning.

[0091] [Figure 6] 1 is a table showing the results of mesothelioma panning.

[0092] [Figure 7-1] Figure 7 shows representative FACS data and a summary of the FACS screening (14 plates). 14 x 96-well plate colony picking; 3 x Gly wash 1; 3 x Gly wash 2; 4 x Gly wash 3; 4 x TEA elution; FACS of 100K 293T-MSLN+ cells (blue) or 100K 293-MSLN- cells (red). [Figure 7-2] See description of Figure 7-1.

[0093] [Figure 8-1]Figure 8 shows MSLN serum levels. Source: Detection and Quantitation of Serum Mesothelin, a Tumor Marker for Patients with Mesothelioma and Ovarian Cancer, Hassan, Raffit, et al., Clin Cancer Res, 2006 and A prospective trial evaluating the role of mesothelin in undiagnosed pleural effusions, Hooper, Clare E. et al., European Respiratory Journal, 2013. [Figure 8-2] See description of Figure 8-1.

[0094] [Figure 9] Binding curves (MSLN) of purified phages are shown.

[0095] [Figure 10] Kinetic data (untreated supernatant) are shown. Expi293 cells were used to express scFv-Fc in 6-well plates. Two days after transfection, scFv-Fc concentrations in the supernatants were quantified using a Bethyl IgG quantitation kit. These concentrations were used to normalize samples to 10 μg / ml for Octet kinetic analysis. Diluted scFv-Fc from the supernatants was loaded onto an anti-human Fc sensor and allowed to bind soluble recombinant mesothelin (Biolegend), followed by a dissociation step in PBST. Rate constants are shown above. Samples were run using untreated supernatant and were singlets.

[0096] [Figure 11-1] FIG. 11 shows the purified scFv-Fc binding curves. [Figure 11-2] See description of Figure 11-1.

[0097] [Figure 12-1]FIG. 12 shows a killing assay in which 293T-MSLN-BFP cells and 293T-CLDN4-mCard cells were mixed. [Figure 12-2] See description of Figure 12-1.

[0098] [Figure 13-1] FIG. 13 shows a killing assay in which 293T-MSLN-BFP cells and 293T-CLDN4-mCard cells were mixed. [Figure 13-2] See description of Figure 13-1.

[0099] [Figure 14] Killing assay of anti-MSLN monoCAR T cells against Ovcar8 and Ovcar5 cells.

[0100] [Figure 15] Figures 15-16 show that anti-MSLN CAR T cells were incubated at a 5:1 ratio with a 1:1 mixture of ovarian cancer cell lines expressing high (Ov8) or low (Ov5) levels of MSLN for 48 hours. The numbers of Ov8 and Ov5 cells were counted at T0 and T48 using a Celigo image cytometer. Cytotoxic CAR T cells eliminate Ov8 cells (solid line) more efficiently than low MSLN + Ov5 cells (dashed line). These two figures are from the same experiment, differing only in the plates testing different antibodies / CAR T constructs. [Figure 16A] See legend to Figure 15. [Figure 16B] See legend to Figure 15.

[0101] [Figure 17-1] Figure 17 shows a killing assay using sorted cells. Prior to the killing assay, anti-MSLN CART cells were sorted and expanded. Positive numbers indicate killing, and negative numbers indicate growth / proliferation of target cells. As demonstrated herein, anti-MSLN CAR T cells preferentially kill MSLN+OVCAR8 cells, but not MSLN-Skrc59 cells. [Figure 17-2]See description of Figure 17-1. [Figure 17-3] See description of Figure 17-1.

[0102] [Figure 18-1] Figure 18 shows CRISPR knockout MSLN in Ovcar8. [Figure 18-2] See description of Figure 18-1.

[0103] [Figure 19] Figure 1 shows the protocol for the dual CAR T E8-T4E3 killing assay.

[0104] [Figure 20-1] Figure 20 shows a dual CAR T E8-T4E3 killing assay. The CART of donor 2020030401 was sorted and the CART of donor 2020030402 was not sorted. [Figure 20-2] See description of Figure 20-1.

[0105] [Figure 21] Figure 1 shows that non-specific killing of F10 is cell line dependent.

[0106] [Figure 22] This shows that non-specific killing of F10 is donor dependent.

[0107] [Figure 23] Killing assay of Ovcar8 MSLN-negative cell line.

[0108] [Figure 24] Killing assay of Ovcar8 MSLN-negative cell line.

[0109] [Figure 25-1] Figure 25 shows the COV362 binding curve. [Figure 25-2] See description of Figure 25-1.

[0110] [Figure 26-1] FIG. 26 shows the Ovcar8 binding curve. [Figure 26-2] See description of Figure 26-1.

[0111] [Figure 27] This shows that MSLN surface expression on Ovcar8 is higher than that on Cov362.

[0112] [Figure 28-1] Figure 28 shows that anti-MSLN CAR T cells exhibit preferential killing of MSLN+OVCAR8 cells but not MSLN-Skrc59 cells. Prior to the killing assay, anti-MSLN CAR T cells were sorted and expanded. Positive numbers indicate killing, and negative numbers indicate growth / proliferation of target cells. [Figure 28-2] See description of Figure 28-1. [Figure 28-3] See description of Figure 28-1.

[0113] [Figure 29A] Figure 29 shows antibody competition. An anti-His HIS1K sensor was used for competition. The sensor was loaded with 5 μg / ml of MSLN-His (Biolegend) for 60 seconds. YP158 or YP218 was then loaded onto the sensor until receptor saturation was achieved. After a short baseline reading, the sensor was immersed in a well containing eight aMSLN antibodies. [Figure 29B] See legend to Figure 29A.

[0114] [Figure 30] Shows killing of additional MSLN+OvCA cell lines by anti-MSLN E8 CAR T.

[0115] [Figure 31]Killing assay of anti-muc1 and anti-msln dual CAR T is shown. CAR Ts tested: E8-T4E3: anti-mesothelin and anti-muc1 dual CAR T; E8: anti-mesothelin mono CAR T; T4E3: anti-MUC1 mono CAR T; F10: anti-HA mono CAR T as a negative control. In vitro killing protocol using Celigo: 1500 BFP-labeled 293msln+ cells were mixed with 1500 mCardinal-labeled Cov362msln+ / MUC1+ cells. Individual CAR Ts were added at an E:T ratio of 2:1. Data were collected 42 hours after adding CAR T to tumor cells. Killing rates are based on: Panel A; 293msln+; Panel B: Cov362 (msln+ / MUC1+).

[0116] [Figure 32] Schematic diagram of anti-mesothelin CASS B-cell therapy for NSCLC.

[0117] [Figure 33] Non-limiting exemplary antibody properties for CASS B cell development are shown. Cell binding curves (left) and BLI-based kinetic measurements (right) show that one clone (Gly1-2-H4) binds to a conformational epitope present only in the GPI-linked form.

[0118] [Figure 34] Figure 1 shows a schematic diagram of the engineering strategy for dual targeting and affinity fine-tuning of the anti-MUC1 / anti-MSLN scFv targeting moiety to improve tumor killing efficacy and patient safety. Balanced affinity and orientation of the scFv against both targets provides an optimized DFIR CAR targeting design.

[0119] [Figure 35]Anti-MSLN CAR killing. Panel A provides a schematic of the 71 kDa surface glycosylphosphatidylinositol (GPI)-linked glycoprotein that is processed into the 31 kDa megakaryocyte potentiating factor (MPF) and the 40 kDa MSLN, which remains membrane-bound; Panels B and C) Killing of MSLN+OVCAR8 cells (Panel B) but not SK59 RCC cells (Panel C) by three (H4, F2, and E8) anti-MSLN CAR-T cells; Panel D) Killing of MUC1+, MSLN+ CoV362 cells by each anti-MUC1 (T4E3) and anti-MSLN (E8) CAR-T cell and dual-targeting E8-TAE3 CAR-T cell.

[0120] [Figure 36]

[0023] Figure 1 shows the anti-tumor effect of anti-CCR4 mAb 2-3 in vivo. Panel A shows that mAb 2-3 treatment blocks human Treg migration toward CCL22-secreting OvCA cells. Panel B shows that in vivo mAb 2-3 treatment restores IGROV-1 killing by Treg depletion.

[0121] [Figure 37] A schematic diagram of the DFIR CAR is shown. The anti-MUC1 and anti-MLSN targeting moieties are tested at different positions and lengths to optimize orientation-based access to their respective targets. An internal ribosome entry site (IRES) allows CAP-independent translation of the Ab payload.

[0122] [Figure 38]Evaluation of CAR-T cell killing in ccRCC 3D cultures is shown. Panel A provides IF staining of ccRCC patient-derived organotypic spheroids (PDOTS) for Hoechst (blue), Calcein and EpCAM (green), PI and CD8 (red), and CD45, EpCAM, and CAIX (purple). Panel B provides the workflow for generating ccRCC PDOTS. Panel C provides T cell migration and cytokine release. Anti-CAIX G36 CAR-T cells (expressing ZsGreen) were evaluated with ccRCC PDOTS and anti-BCMA A716 (control) CAR-T cells; untransduced T cells and untreated controls were used as negative controls. T cell migration from the side channel to the center channel was monitored and quantified using ZsGreen. CXCL10 release was assessed by ELISA.

[0123] [Figure 39] Antibody binding data are shown. FACS binding represents cell-expressed MSLN, and ELISA binding represents soluble MSLN. This distinction may be important because MSLN can be shed from tumor cells, acting as a decoy for CAR T / Ab therapy. However, there is a slight conformational shift during shedding, which may allow only membrane-bound MSLN to be targeted.

[0124] [Figure 40] The amino acid sequences of anti-mesothelin antibodies are shown. DETAILED DESCRIPTION OF THE INVENTION

[0125] Detailed Description Abbreviations and Definitions

[0126] A detailed description of one or more embodiments is provided herein. However, it should be understood that the present invention can be embodied in various forms. Accordingly, the specific details disclosed herein should not be construed as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art how to use the present invention in any suitable manner.

[0127] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The use of the words "a" or "an" when used in conjunction with the word "comprising" in the claims and / or specification may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0128] Whenever any of the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, it is understood that the phrase "without limitation" is also involved. Similarly, "one example," "exemplary," etc. are understood to be non-limiting.

[0129] The term "substantially" permits deviations from the descriptor that do not adversely affect the intended purpose. It is understood that the descriptor is modified by the term "substantially" even if the word "substantially" is not explicitly recited.

[0130] Terms such as "comprising," "including," "having," and "involving" (and similarly, "comprises," "includes," "has," and "involves") are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprising" and, therefore, should be interpreted as an open term meaning "at least the following" and not excluding additional features, limitations, aspects, etc. Thus, for example, "a process comprising steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the terms "a" or "an" are used, they should be understood to mean "one or more," unless such interpretation is undue to the context.

[0131] As used herein, the term "about" is used to mean approximately, roughly, around, or within a range thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 20 percent above or below (higher or lower).

[0132] Described herein are unique recombinant monoclonal mesothelin (MSLN) antibodies. "Recombinant," as it relates to a polypeptide (such as an antibody) or polynucleotide, can refer to a form of the polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which is one that can be made by combining polynucleotides or polypeptides that do not normally occur together.

[0133] Provided herein are the amino acid sequences of monoclonal MSLN antibodies, in addition to exemplary wild-type IgG constant regions useful in combination with the VH and VL sequences provided herein (see Tables 1-15). The amino acid sequences of the heavy and light chain complementarity determining regions (CDRs) of the MSLN antibodies are shown below in underlined ( CDR1 ), Underlined and bold ( CDR2 ), or Underlined, italic and bold ( CDR3 ) is shown. Table 1. MSLN-WC-R3-Gly3-1-H10 Ab variable region amino acid sequence TIFF2025528835000002.tif46148 Table 2: MSLN-WC-R3-TEA2-E10 Ab variable region amino acid sequence TIFF2025528835000003.tif38148 Table 3. MSLN-WC-R3-TEA1-D3 Ab variable region amino acid sequence TIFF2025528835000004.tif42148 Table 4. MSLN-WC-R3-Gly3-2-C1 Ab variable region amino acid sequence TIFF2025528835000005.tif42148 Table 5. MSLN-WC-R3-TEA1-D2 Ab variable region amino acid sequence TIFF2025528835000006.tif42148 Table 6. MSLN-WC-R3-Gly2-2-F7 Ab variable region amino acid sequence TIFF2025528835000007.tif42148 Table 7. MSLN-WC-R3-Gly1-2-H4 Ab variable region amino acid sequence TIFF2025528835000008.tif42148 Table 8. MSLN-WC-R3-Gly1-2-D4 Ab variable region amino acid sequence TIFF2025528835000009.tif42148 Table 9: MSLN-WC-R3-TEA1-E5 Ab variable region amino acid sequences TIFF2025528835000010.tif42148 Table 10: MSLN-WC-R3-Gly3-2-C10 Ab variable region amino acid sequence TIFF2025528835000011.tif43148 Table 11. MSLN-WC-R3-Gly2-1-F2 Ab variable region amino acid sequence TIFF2025528835000012.tif42148 Table 12: MSLN-WC-R3-Gly2-2-B6 Ab variable region amino acid sequence TIFF2025528835000013.tif42148 Table 13: MSLN-WC-R3-Gly1-2-E10 Ab variable region amino acid sequence TIFF2025528835000014.tif42148 Table 14. MSLN-WC-R3-TEA2-C9 Ab variable region amino acid sequences TIFF2025528835000015.tif38148 Table 15. MSLN-WC-R3-TEA1-E8 Ab variable region amino acid sequence TIFF2025528835000016.tif38148

[0134] The amino acid sequences of the complementarity determining regions of the heavy and light chains of the MSLN antibody are shown in Tables 16A-B below. Table 16A: Heavy chain (V) of MSLN antibody H ) Complementarity-Determining Region (CDR) TIFF2025528835000017.tif150147 Table 16B. Light chain (V) of MSLN antibody L ) Complementarity-Determining Region (CDR) TIFF2025528835000018.tif150147

[0135] The amino acid sequences of the framework regions of the heavy and light chains of the MSLN antibody are shown in Tables 17A-B below. Table 17A: Heavy chain (V) of MSLN antibody H ) Framework region (FR) TIFF2025528835000019.tif168147TIFF2025528835000020.tif153147 Table 17B. Light chain (V) of MSLN antibody L ) Framework region (FR) TIFF2025528835000021.tif239147TIFF2025528835000022.tif72147

[0136] The MSLN antibodies described herein bind to MSLN. In one embodiment, the MSLN antibodies have high affinity and specificity for MSLN. Some embodiments also feature antibodies that share a certain percentage of identity or similarity with the amino acid or nucleotide sequence of the anti-MSLN antibodies described herein. For example, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity when compared to a specific region or the full length of any one of the anti-MSLN antibodies described herein. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid identity when compared to a specific region or the full length of any one of the anti-MSLN antibodies described herein. Sequence identity or similarity for the nucleic acids and proteins of the invention can be determined by sequence comparison and / or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Current Protocols in Molecular Biology, eds. Ausubel et al. (2007). For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence identity or similarity for the nucleic acids and proteins of the invention.

[0137] As used herein, "polypeptide" can encompass a single "polypeptide" as well as multiple "polypeptides" and can refer to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" can refer to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term that can be used to refer to a chain of two or more amino acids can refer to a "polypeptide" herein, and the term "polypeptide" can be used in place of or interchangeably with any of these terms. "Polypeptide" can also refer to post-expression modified products of a polypeptide, such as, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology and is not necessarily translated from a specific nucleic acid sequence. It can be generated by any method, including chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that alter, add, delete, or substitute a single amino acid or a small percentage of amino acids in the encoded sequence are collectively referred to herein as "conservatively modified variants." In some embodiments, the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants of the anti-MSLN antibodies disclosed herein may exhibit increased cross-reactivity to MSLN compared to unmodified MSLN antibodies.

[0138] For example, a "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art as follows: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is replaced with another amino acid residue from the same side chain family. In another embodiment, the amino acid chain can be replaced with a structurally similar chain that differs in the order and / or composition of the side chain family members.

[0139] antibody

[0140] As used herein, "antibody" or "antigen-binding polypeptide" can refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a full-length antibody or any antigen-binding fragment, or single chain thereof. For example, "antibody" can include any protein- or peptide-containing molecule containing at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Non-limiting examples include the complementarity-determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, the variable region of a heavy or light chain, the constant region of a heavy or light chain, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term "antibody" can refer to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" means that the antibody reacts with one or more antigenic determinants of the antigen and not with other polypeptides.

[0141] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to an antibody fragment of F (ab’)2 , F (ab)2 , F ab ', F ab, Fv, scFv, etc. Antibody fragments, regardless of structure, bind to the same antigen recognized by the intact antibody. The term "antibody fragment" can encompass aptamers (such as spiegelmers), minibodies, and diabodies. The term "antibody fragment" can also encompass any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', F(ab'), Fd, Fv, single-chain Fv (scFv), single-chain antibodies, dAb (domain antibodies), minibodies, disulfide-linked Fv (sdFv), fragments containing VL or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.

[0142] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L (scFv) refers to a fusion protein of the variable regions of a single-chain Fv ("scFv") polypeptide molecule. A single-chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed from a gene fusion comprising VH- and VL-encoding genes linked by a peptide-encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883.) In some embodiments, the regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, and the V H N-terminus of V Lor vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. Many methods have been described for identifying chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513, 5,892,019, 5,132,405, and 4,946,778 (each of which is incorporated herein by reference in its entirety).

[0143] Very large naive human scFv libraries have been and can be generated to provide a large source of rearranged antibody genes against a large number of target molecules. Smaller libraries can be constructed from individuals with infectious diseases to isolate disease-specific antibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43 (1992); Zebedee et al., Proc. Natl. Acad. Sci. USA 89:3 175-79 (1992)).

[0144] Antibody molecules obtained from humans are classified into five classes of immunoglobulins: IgG, IgM, IgA, IgE, and IgD, which differ from each other in the nature of the heavy chains present in the molecule. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within each (e.g., γ1-γ4). Certain classes also have subclasses, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, and IgG5, are well characterized and are known to confer functional differentiation. With regard to IgG, a typical immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are joined by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains, which begin at the mouth of the "Y" and continue through the variable region. The immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

[0145] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus at the forked end of the Y-shape to the C-terminus at the bottom of each chain.

[0146] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. The variable domains (VL and VH) of both the light and heavy chains determine antigen recognition and specificity. Conversely, the constant domains (CL and CH1, CH2, or CH3) of the light and heavy chains confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, and complement fixation. The term "antigen-binding site" or "binding portion" may refer to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent sections within the V regions of the heavy and light chains, called "hypervariable regions," are interspersed between more conserved adjacent sections known as "framework regions" or "FRs." Thus, the term "FR" may refer to the amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions" or "CDRs." The VH and VL regions containing the CDRs and frameworks (FRs) of the MSLN antibody are shown in Tables 1 to 17.

[0147] The six CDRs present in each antigen-binding domain are short, noncontiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, the FR regions, show little inter-molecular variability. The framework regions primarily adopt a beta-sheet conformation, and the CDRs form loops that connect them and, in some cases, form part of the beta-sheet structure. The framework regions function to form a scaffold that orients the CDRs through non-covalent interactions between the chains. The antigen-binding domain formed by the arranged CDRs provides a surface complementary to the epitope on the antigen in an immune response, promoting non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and framework regions, respectively, have been previously identified for heavy or light chain variable regions and can be readily identified by one of skill in the art (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDapartment of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0148] Where there are more than one definition for a term used and / or accepted in the art, the definition of the term used herein is intended to encompass such meanings unless specifically and explicitly stated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. This particular region is described in Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The Kabat and Chothia definitions of CDRs include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, the application of either definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs defined by each of the above cited references are set forth in the following table for comparison: The exact residue numbers which comprise a particular CDR will vary depending on the sequence and size of the CDR, and one of skill in the art can routinely determine which residues make up a particular CDR, given the variable region amino acid sequence of an antibody. TIFF2025528835000023.tif47128

[0149] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence, without reliance on other experimental data on the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0150] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine ​​residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2; includes 3-25 amino acids and ends with the sequence WGXG (X is any amino acid). CDR-L1 begins at approximately residue 24 (i.e., after the cysteine ​​residue), includes approximately 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e., after the cysteine ​​residue), includes about 7-11 residues, and ends with the sequence F or WGXG (where X is any amino acid).

[0151] In certain embodiments, the CDRs of an antibody can be determined according to the IMGT numbering system, which is unique to IMGT and is designed to compare variable domains regardless of antigen receptor, chain type, or species [Lefranc M.-P., Immunology Today 18, 509 (1997) / Lefranc M.-P., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V., and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT-specific numbering, conserved amino acids always have the same positions, e.g., cysteine ​​23 (1st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine ​​104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT-specific numbering provides standardized boundaries for the framework regions (FR1-IMGT: positions 1-26, FR2-IMGT: positions 39-55, FR3-IMGT: positions 66-104, and FR4-IMGT: positions 118-128) and the complementarity-determining regions: CDR1-IMGT: positions 27-38, CDR2-IMGT: positions 56-65, and CDR3-IMGT: positions 105-117. Since gaps represent unoccupied positions, the length of the CDR-IMGT (shown between brackets and separated by dots, e.g., [8.8.13]) is important information.The IMGT-specific numbering is used in the 2D graphic representations called IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)] and in the 3D structures of the IMGT / 3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. 32, D208-D210 (2004)].

[0152] As used herein, the term "epitope" may include any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, the VL and VH domains of an antibody, or a subset of complementarity-determining regions (CDRs), combine to form variable regions that define a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of a Y. Epitope determinants may consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and may have specific three-dimensional structural and charge characteristics. For example, antibodies may be raised against N- or C-terminal peptides of a polypeptide. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VL chains. In one embodiment, the antibody is encoded by NCBI reference number: NM_005823 and has the amino acid sequence: It may be for mesothelin (MSLN) with TIFF2025528835000024.tif51135.

[0153] See, for example, the product data sheet for SC110135 (Mesothelin (MSLN) (NM_005823) Human Untagged Clone).

[0154] As used herein, the terms "immunological binding" and "immunological binding properties" can refer to the type of non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ) and K d A smaller K represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, both "on rate constants" (K on ) and "off rate constant" (K off ) can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). K off / K on The ratio of α to β allows for the elimination of parameters not related to affinity, and the equilibrium binding constant K D (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies of the present invention may have an equilibrium binding constant (K) as measured by a kinetic assay, e.g., a radioligand binding assay or similar assay known to those skilled in the art, e.g., BIAcore or Octet (BLI). D ) is ≦1 μM, ≦10 μM, ≦10 nM, ≦10 pM, or ≦100 pM to about 1 pM, it can specifically bind to the MSLN epitope. For example, in some embodiments, D is about 1E-12M and about 1E-11M. D In some embodiments, K Dis about 1E-11M and about 1E-10M. D In some embodiments, K D is about 1E-10M and about 1E-9M. D In some embodiments, K D is about 1E-9M and about 1E-8M D In some embodiments, K D is about 1E-8M and about 1E-7M D In some embodiments, K D is about 1E-7M and about 1E-6M. D For example, in some embodiments, K D is about 1E-12M, and in other embodiments, K D is about 1E-11M. In some embodiments, K D is about 1E-10M, and in other embodiments, K D is about 1E-9M. In some embodiments, K D is about 1E-8M, and in other embodiments, K D is about 1E-7M. In some embodiments, K D is about 1E-6M, and in other embodiments, K D is about 1E-5M. In some embodiments, K D is about 3E-11M, and in other embodiments, K D is about 3E-12M. In some embodiments, K D is approximately 6E-11M. "Specifically binds" or "having specificity" can refer to an antibody that binds to an epitope via the antibody's antigen-binding domain, where the binding involves some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope if it binds to that epitope via its antigen-binding domain more readily than it binds to a random, unrelated epitope.

[0155] For example, MSLN antibodies can be monovalent or bivalent and can comprise a single chain or two chains. Functionally, the binding affinity of MSLN antibodies is 10 -5 M~10-12 For example, the binding affinity of the MSLN antibody is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M or 10 -5 M~10 -6 I am M.

[0156] MSLN proteins, or their derivatives, fragments, analogs, homologs, or orthologs, can be used as immunogens in the generation of antibodies that immunologically specifically bind to these protein components. MSLN proteins, or their derivatives, fragments, analogs, homologs, or orthologs, coupled to proteoliposomes, can be used as immunogens in the generation of antibodies that immunologically specifically bind to these protein components.

[0157] Those skilled in the art will recognize that, without undue experimentation, one can determine whether a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by determining whether the former prevents the latter from binding to MSLN. For example, if the human monoclonal antibody being tested exhibits reduced binding by and competes with a human monoclonal antibody of the invention, then these two monoclonal antibodies bind to the same or closely related epitopes.

[0158] Another method for determining whether a human monoclonal antibody has the specificity of the human monoclonal antibody of the present invention is to preincubate the human monoclonal antibody of the present invention with the MSLN protein with which it normally reacts, then add the human monoclonal antibody to be tested and determine whether the human monoclonal antibody to be tested is inhibited in its ability to bind to MSLN. If the human monoclonal antibody to be tested is inhibited, it may have the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention. Screening of human monoclonal antibodies of the present invention can also be performed using MSLN to determine whether the monoclonal antibody to be tested can neutralize MSLN.

[0159] Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies directed against the proteins of the invention or against their derivatives, fragments, analogs, homologs, or orthologs (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0160] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provide the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen that is the target of the desired immunoglobulin, or an epitope thereof, can be immobilized on a column, and immune-specific antibodies can be purified by immunoaffinity chromatography. Immunoglobulin purification is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia, PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0161] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" can refer to a population of antibody molecules containing only one antibody molecule species consisting of a unique light chain gene product and a unique heavy chain gene product. The complementarity-determining regions (CDRs) of a monoclonal antibody are identical among the molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with an epitope of an antigen characterized by a unique binding affinity for it.

[0162] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is immunized with an immunizing agent to induce lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0163] The immunizing agent may comprise the protein antigen, a fragment thereof, or a fusion protein thereof. For example, peripheral blood lymphocytes are used if cells of human origin are preferred, or spleen cells or lymph node cells are used if a non-human mammalian source is preferred. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line may be a transformed mammalian cell, such as a myeloma cell of rodent, bovine, or human origin. For example, a rat or mouse myeloma cell line is used. The hybridoma cells may be cultured in a suitable medium containing one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma may contain hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances will prevent growth of HGPRT-deficient cells.

[0164] Useful immortalized cell lines are those that fuse efficiently, maintain stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Immortalized cell lines include, for example, mouse myeloma lines available from the Salk Institute Cell Distribution Center (San Diego, California) and the American Type Culture Collection (Manassas, Virginia). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63.)

[0165] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies against the antigen. For example, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis as described in Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies that have a high degree of specificity and high binding affinity for the target antigen.

[0166] After hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103.) Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0167] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0168] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567, which is incorporated herein by reference in its entirety. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention and / or for the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0169] A fully human antibody is an antibody molecule in which the entire sequence of both the light and heavy chains, including, for example, the CDRs, arises from human genes. Such antibodies are referred to herein as "humanized antibodies" or "fully human antibodies." Human monoclonal antibodies, such as fully human antibodies and humanized antibodies, can be prepared by using trioma technology, human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), and EBV hybridoma technology to produce human monoclonal antibodies (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies are available and can be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see Cole, et al., 1985 "MONOCLONAL ANTIBODIES AND CANCER THERAPY", Alan R. Liss, Inc., pp. 77-96).

[0170] A "humanized antibody" may be an antibody derived from a non-human species (e.g., mouse) whose amino acid sequence (e.g., in the CDR regions) has been modified to enhance similarity to antibody variants produced in humans. Antibodies can be humanized by methods known in the art, such as CDR grafting. See also Safdari et al., (2013) Biotechnol Genet Eng Rev.; 29:175-86. Furthermore, humanized antibodies can be produced in transgenic plants as an inexpensive alternative to existing mammalian systems. For example, transgenic plants can be tobacco plants, i.e., Nicotiana benthamiana and Nicotiana tabacum. Antibodies are purified from plant leaves. Stable transformation of plants can be achieved using Agrobacterium tumefaciens or biolistic bombardment. For example, a nucleic acid expression vector containing at least the heavy and light chain sequences can be expressed via transformation in a bacterial culture, i.e., A. tumefaciens strain BLA4404. Infiltration of plants can be achieved by injection. Soluble leaf extracts can be prepared by crushing leaf tissue in a mortar and centrifugation. Isolation and purification of antibodies can be carried out by many methods known to those skilled in the art. Other methods for antibody production in plants are described, for example, in Fischer et al., Vaccine, 2003, 21:820-5, and Ko et al., Current Topics in Microbiology and Immunology, Vol. 332, 2009, pp. 55-78. Thus, the present invention further provides any cell or plant containing a vector encoding or producing an antibody of the present invention.

[0171] Antibodies can be modified by, for example, CDR grafting (EP 239,400; WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering, or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., Proc. Natl. Sci. USA 91:969-973 (1994)) and chain shuffling (U.S. Pat. No. 5,565,332, the entirety of which is incorporated by reference). "Humanization" (also called reshaping or CDR grafting) is a well-established technique understood by those skilled in the art to reduce the immunogenicity of monoclonal antibodies (mAbs) derived from heterologous sources (e.g., rodents) and improve activation of the human immune system (see, e.g., Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008). "Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling," J. Biochem. 144(1):115-20).

[0172] In addition, antibodies (such as human antibodies) can also be produced using other techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Following challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as Marks et al., Bio / Technology 10, 779-783 (1992), Lonberg et al., Nature 368, 856-859 (1994), Morrison, Nature 368, 812-13 (1994), Fishwild et al., Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0173] Human antibodies can also be produced using transgenic non-human animals that are modified to produce fully human antibodies in response to antigen challenge rather than the animal's endogenous antibodies (see WO 94 / 02602 and U.S. Pat. No. 6,673,986). Endogenous genes encoding heavy and light immunoglobulin chains in the non-human host are disabled, and active loci encoding human heavy and light immunoglobulin chains are inserted into the host's genome. Human genes are incorporated, for example, using yeast artificial chromosomes containing the necessary human DNA segments. Animals providing the desired modifications are then obtained as progeny by breeding intermediate transgenic animals containing less than the full complement of modifications. A non-realistic example of such a non-human animal is a mouse, referred to as the Xenomouse™, as disclosed in WO 96 / 33735 and WO 96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained, for example, as polyclonal antibody preparations, directly from animals after immunization with an immunogen of interest, or alternatively, from immortalized B cells derived from animals, such as hybridomas, that produce monoclonal antibodies. Additionally, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly or further modified to obtain antibody analogs, such as, for example, single-chain Fv (scFv) molecules.

[0174] Thus, such a technique can be used to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., WO 98 / 24893, WO 96 / 34096, WO 96 / 33735, U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated by reference in their entireties. Additionally, companies such as Creative BioLabs (Shirley, NY) offer services to provide human antibodies directed against a selected antigen using technology similar to that described above.

[0175] An example of a method for producing a non-human host, exemplified as a mouse, lacking expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. This can be obtained by a method comprising deleting a J segment gene from at least one endogenous heavy chain locus in embryonic stem cells to prevent rearrangement of the locus and the formation of transcripts of the rearranged immunoglobulin heavy chain locus, the deletion being carried out by a targeting vector containing a gene encoding a selectable marker, and producing a transgenic mouse from the embryonic stem cells, the somatic and germ cells of which contain the gene encoding the selectable marker.

[0176] One method for producing a desired antibody, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method involves introducing an expression vector containing a nucleotide sequence encoding the heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding the light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy and light chains.

[0177] In a further improvement of this procedure, methods for identifying clinically relevant epitopes on immunogens and correlative methods for selecting antibodies that immunospecifically bind with high affinity to the relevant epitopes are disclosed in WO 99 / 53049.

[0178] The antibody of interest can also be expressed by a vector containing a DNA segment encoding the single-chain antibody described herein. Vectors include, but are not limited to, chemical conjugates such as those described in International Publication No. WO 93 / 64701, which have a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid-binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in International Application No. PCT / US95 / 02140 (WO 95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid-binding moiety (e.g., protamine), plasmids, phages, viral vectors, and the like. Vectors can be chromosomal, non-chromosomal, or synthetic. Retroviral vectors can also be used, including Moloney murine leukemia virus. DNA viral vectors can also be used, including pox vectors such as orthopox or avipox vectors, herpes virus vectors such as herpes simplex virus (HSV) vectors (see Geller, AI et al, J. Neurochem, 64:487 (1995); Lim, F., et al, DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al, Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI, et al, Proc Natl. Acad. Sci USA 87:1149 (1990)), and adenovirus vectors (LeGal LaSalle et al, Science, 259:988 (1993); Davidson, et al, Nat. Genet. 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995)) and adeno-associated virus vectors (Kaplitt, MG, et al., Nat. Genet. 8:148 (1994)).

[0179] Poxvirus vectors transfer genes into the cytoplasm of cells. Avipoxvirus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be used to transfer nucleic acids into neural cells. Adenovirus vectors result in shorter-term expression (approximately 2 months) than adeno-associated virus (approximately 4 months), which in turn is shorter than HSV vectors. The vector selected will depend on the target cell and the condition being treated. Introduction can be by standard techniques (e.g., infection, transfection, transduction, or transformation). Examples of modes of gene transfer include naked DNA, CaP04 precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0180] Vectors can be used to target essentially any target cell. For example, stereotactic injection can be used to direct vectors (e.g., adenovirus, HSV) to a desired location. In addition, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. A method based on bulk flow, called convection, has also proven effective in delivering large molecules to extended areas of the brain and may be useful for delivering vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injection, as well as oral or other known administration routes.

[0181] These vectors can be used to express large amounts of antibodies that can be used in a variety of ways, for example, to detect the presence of MSLN in a sample. Antibodies can also be used to attempt to bind and destroy MSLN activity.

[0182] In one embodiment, the antibodies described herein may be full-length antibodies, including those that contain an Fc region similar to a wild-type Fc region that binds to an Fc receptor.

[0183] Techniques can be adapted for the production of single chain antibodies specific to the antigenic proteins of the present invention (see, e.g., U.S. Pat. No. 4,946,778). In addition, methods can be used to generate monoclonal antibodies with the desired specificity for the protein or its derivatives, fragments, analogs, or homologs. ab To allow for rapid and effective identification of fragments, F ab Methods can be adapted for the construction of expression libraries (see, e.g., Huse, et al., 1989 Science 246:1275-1281). Antibody fragments containing the idiotype to a protein antigen can be produced by techniques known in the art, including, but not limited to: (i) F produced by pepsin digestion of antibody molecules. (ab’)2 Fragment, (ii)F (ab’)2 F generated by reducing the disulfide bridges of the fragment ab (iii) F fragments produced by treating antibody molecules with papain and a reducing agent. ab fragments, and (iv) F v piece.

[0184] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies can, for example, target immune system cells to unwanted cells (see U.S. Pat. No. 4,676,980) or be used to treat HIV infection (see WO 91 / 00360 and WO 92 / 20373). Antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, as well as those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0185] The antibodies of the present invention can be modified with respect to effector function, for example, to enhance the effectiveness of the antibody in treating cancer. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, antibodies can be engineered with dual Fc regions, thereby potentially possessing enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)). In one embodiment, an antibody of the present invention has a modified Fc region such that the Fc region does not bind to Fc receptors. For example, the Fc receptor is an Fcγ receptor. Antibodies with modified Fc regions such that the Fc region does not bind Fcγ but still binds to neonatal Fc receptors are useful as described herein.

[0186] In embodiments, antibodies of the invention may comprise Fc variants. See, for example, WO 2018 / 145075 and WO 2019 / 183362, which provide Fc variant compositions for enhancing antibody-mediated receptor signaling. In embodiments, Fc variants may comprise amino acid substitutions that alter the antigen-independent effector function of the antibody, such as the circulating half-life of the antibody. Such antibodies exhibit increased or decreased binding to FcRn and therefore have increased or decreased serum half-lives, respectively, when compared to antibodies lacking these substitutions. Fc variants with improved affinity for FcRn may have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where a longer half-life of the administered antibody is preferred, for example, to treat chronic diseases or disorders. In contrast, Fc variants with reduced FcRn-binding affinity may have a shorter resting period, and such molecules may also be useful, for example, for administration to mammals where a shortened circulation time may be advantageous, such as in in vivo imaging, or in situations where the starting antibody has toxic side effects if present in the circulation for an extended period of time. Fc variants with reduced FcRn-binding affinity are also less likely to cross the placenta and are therefore useful in treating diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn-binding affinity may be preferable include applications in which localization to the brain, kidney, and / or liver is preferred. In one embodiment, Fc variant-containing antibodies may exhibit reduced transport from the vasculature across the epithelium of renal glomeruli. In another embodiment, Fc variant-containing antibodies may exhibit reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, an antibody with altered FcRn binding comprises an Fc domain with one or more amino acid substitutions within the "FcRn-binding loop" of the Fc domain. The FcRn-binding loop is composed of amino acid residues 280 to 299 (EU numbering). Exemplary amino acid substitutions that alter FcRn-binding activity are disclosed in WO 05 / 047327, which is incorporated herein by reference.In certain exemplary embodiments, an antibody of the invention, or a fragment thereof, comprises an Fc domain with one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0187] In some embodiments, mutations are introduced into the constant region of the mAb to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the mAb. For example, the mutation is a LALA mutation in the CH2 domain. In one embodiment, the antibody (e.g., a human mAb or a bispecific Ab) contains a mutation on one scFv unit of the heterodimeric mAb that reduces ADCC activity. In another embodiment, the mAb contains mutations on both chains of the heterodimeric mAb that completely eliminate ADCC activity. For example, the mutation introduced into one or both scFv units of the mAb is a LALA mutation in the CH2 domain. These mAbs with variable ADCC activity can be optimized so that the mAb exhibits maximal selective killing toward cells expressing one antigen recognized by the mAb, but minimal killing toward a second antigen recognized by the mAb.

[0188] In other embodiments, antibodies of the invention for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgG1 or IgG4 heavy chain constant region, which may be altered to reduce or eliminate glycosylation. For example, antibodies of the invention may also include Fc variants containing amino acid substitutions that alter the glycosylation of the antibody. For example, Fc variants may have reduced glycosylation (e.g., N-linked or O-linked glycosylation). In some embodiments, Fc variants have reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif, e.g., an N-linked glycosylation motif comprising the amino acid sequence NXT or NXS. In embodiments, the antibody comprises an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU numbering). In embodiments, the antibody comprises an IgG1 or IgG4 constant region comprising S228P and T299A mutations (EU numbering).

[0189] Exemplary amino acid substitutions that reduce or alter glycosylation are disclosed in WO 05 / 018572, which is incorporated herein by reference. In some embodiments, antibodies of the invention or fragments thereof are modified to eliminate glycosylation. Such antibodies or fragments thereof may be referred to as "agly" antibodies or fragments thereof (e.g., "agly" antibodies). Without wishing to be bound by theory, "agly" antibodies or fragments thereof may have an improved safety and stability profile in vivo. An exemplary agly antibody or fragment thereof comprises a deglycosylated Fc region of an IgG4 antibody that lacks Fc effector function, thereby eliminating the potential for Fc-mediated toxicity to normal living tissues and cells that express MSLN. In yet other embodiments, antibodies of the invention or fragments thereof comprise an altered glycan. For example, the antibody can have a reduced number of fucose residues on the N-glycan at Asn297 of the Fc region, i.e., is defucosylated. In another embodiment, the antibody may have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.

[0190] The present invention is also directed to immunoconjugates comprising antibodies conjugated to cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or to radioisotopes (the latter being radioconjugates).

[0191] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Non-limiting examples include: 212 B.I., 131 I, 131 In, 90 Y, and 186 Re is an example.

[0192] Conjugates of antibodies and cytotoxic agents have been prepared using a variety of bifunctional protein-linking agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (see WO 94 / 11026 and U.S. Pat. No. 5,736,137).

[0193] Those skilled in the art will recognize that a wide variety of possible moieties may be attached to a given antibody or other molecule of the invention. (See, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), which is incorporated herein by reference in its entirety.

[0194] Conjugation can be achieved by any chemical reaction that will link two molecules, so long as the antibody and other moiety retain their respective activities. This conjugation can involve many chemical mechanisms, such as covalent bonding, affinity binding, intercalation, coordinate bonding, and complex formation. In one embodiment, the conjugation is a covalent bond. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporation of an external crosslinking molecule. Many bivalent or polyvalent linking agents are useful for linking protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative linking agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of linking agents known in the art, but rather is illustrative of more common linking agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)). Non-limiting examples of linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester).) See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives attached to antibodies via oligopeptide linkers.Non-limiting examples of useful linkers that can be used with the antibodies of the invention include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene) (Pierce Chem. Co., catalog (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate) (Pierce Chem. Co., catalog number 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propianamido]hexanoate (Pierce Chem. Co. catalog number 2165-G); and (v) sulfo-NHS (-hydroxysulfo-succinimide) conjugated to EDC (Pierce Chem. Co., catalog number 2165-G). Chem. Co., Cat. No. 24510).

[0195] The linkers described herein contain components with different attributes, thereby resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond, allowing for the formation of conjugates with improved stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer available conjugates. Sulfo-NHS can enhance the stability of carbodiimide bonds. Carbodiimide bonds (such as EDC) when used in combination with sulfo-NHS form esters that are more resistant to hydrolysis than the carbodiimide bond reaction alone.

[0196] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0197] Non-limiting examples of useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257:286-288 (1982).

[0198] Multispecific antibodies (bispecific and trispecific)

[0199] The embodiments described herein may include monospecific or multispecific antibodies.

[0200] Monospecific antibodies are antibodies that have one or more binding sites that specifically bind to a single antigen.

[0201] A multispecific antibody is an antibody that can recognize two or more different antigens. For example, a bispecific antibody (bsAb) is an antibody that contains two variable domains or scFv units such that the resulting antibody recognizes two different antigens. For example, a trispecific antibody (tsAb) is an antibody that contains two variable domains or scFv units such that the resulting antibody recognizes three different antigens. The present invention provides multispecific antibodies, such as bispecific and trispecific antibodies, that recognize MSLN and a second and / or third antigen. In one embodiment, multispecific antibodies (e.g., bispecific and trispecific antibodies) can comprise an MSLN-specific fusion protein comprising an antibody described herein. Exemplary second and / or third antigens include tumor-associated antigens (e.g., LINGO1), cytokines (e.g., IL-12 (IL-12A (p35 subunit) protein sequence having NCBI Accession No. NP_000873.2; IL-12B (p40 subunit) protein sequence having NCBI Accession No. NP_002178.2; IL-18 (protein sequence having NCBI Accession No. NP_001553.1); IL-15 (protein sequence having NCBI Accession No. NP_000576.1); IL-7 (protein sequence having NCBI Accession No. NP_000871.1); IL-2 (protein sequence having NCBI Accession No. NP_0005 and IL-21 (protein sequence having NCBI Accession No. NP_068575.1), cytokine cognate receptors (e.g., IL-12R), and cell surface receptors. Non-limiting examples of second and / or third antigens include CTLA-4, CXCR4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD47, and CD73. In one embodiment, the bispecific and trispecific antibodies comprise an MSLN fusion protein.For example, the fusion protein can include an antibody comprising a variable domain or scFv unit described herein and a ligand or antigen and / or a third ligand or antigen, such that the resulting antibody recognizes the antigen and binds to a ligand-specific receptor. Exemplary antibody compositions (e.g., VH and / or VL sequences or fragments thereof) useful in designing the MSLN fusion proteins described herein include the anti-CAIX antibodies described in PCT / US2006 / 046350 and PCT / US2015 / 067178; the anti-CXCR4 antibodies described in PCT / US20006 / 005691 and PCT / US2019 / 022272; the anti-CCR4 antibodies described in PCT / US2008 / 088435, PCT / US2013 / 039744, and PCT / US2015 / 054202; Examples of suitable antibodies include, but are not limited to, the anti-PD-L1 antibodies described in PCT Publication Nos. 008 / 088435 and PCT / US2020 / 062815; the anti-PD-1 antibodies described in PCT Publication Nos. 008 / 088435 and 062815; the anti-PD-1 antibodies described in PCT Publication Nos. 037791 and 037781; the anti-GITR antibodies described in PCT Publication No. 043504; the anti-claudin-4 antibodies described in PCT Publication No. 022272; and the anti-MUC1 antibodies described in PCT Publication No. 037783 (each of which is incorporated by reference in its entirety). In one embodiment, the fusion protein further comprises a constant region and / or a linker as described herein. Different formats of multispecific antibodies (e.g., bispecific and trispecific antibodies, such as fusion proteins comprising an antibody and a ligand that recognize MSLN) are described herein.Ligands can be tumor-associated antigens (e.g., LINGO1, ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), MUC1, MSLN, CD19, CD20, CD30, CD40, CD22, RAGE-1, MN-CA IX, RET1, RET2 (AS), prostate-specific antigen (PSA), TAG-72, PAP, p53, Ras, prostein, PSMA, survivin, 9D7, prostate cancer tumor antigen-1 (PCTA-1), GAGE, MAGE, mesothelin, β-catenin, TGF-βRII, BRCA1 / 2, SAP-1, HPV-E6, HPV-E7 (and for additional tumor-associated surface antigens, see International Application No. PCT / US2015 / 067225 and International Application No. PCT / US2015 / 067225, which are incorporated by reference in their entireties). see also PCT / US2019 / 022272); cytokines (e.g., IL-12 (IL-12A (p35 subunit) protein sequence having NCBI accession number NP_000873.2; IL-12B (p40 subunit) protein sequence having NCBI accession number NP_002178.2; IL-18 (protein sequence having NCBI accession number NP_001553.1); IL-15 (protein sequence having NCBI accession number IL-7 (protein sequence with NCBI accession number NP_000871.1); IL-2 (protein sequence with NCBI accession number NP_000577.2); and IL-21 (protein sequence with NCBI accession number NP_068575.1)); CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LI The antigen-specific fragment may be GHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD47, or CD73. Bispecific or trispecific antibodies in various formats are also provided herein. In some embodiments, the anti-MSLN fragment and the second and / or third antigen-specific fragment are each independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.In some embodiments, the bispecific or trispecific antibody further comprises an Fc fragment (e.g., as described in PCT / US2015 / 021529 and PCT / US2019 / 023382, each of which is incorporated by reference in its entirety). The bispecific or trispecific antibody of the invention may comprise a heavy and light chain combination or scFv of the MSLN antibody described herein.

[0202] Multispecific antibodies (e.g., bispecific and trispecific antibodies) of the present invention (e.g., anti-MSLN-scFv fusion proteins) can be constructed using methods known in the art. In some embodiments, a bispecific antibody is a single polypeptide in which two scFv fragments are joined by a long linker polypeptide of sufficient length to allow intramolecular interactions between the two scFv units to form the antibody. In other embodiments, a bispecific antibody is two or more polypeptides linked by covalent or non-covalent bonds. In some embodiments, the amino acid linker shown herein (GGGGSGGGGS; "(G4S)2") can be generated using a longer G4S linker to improve flexibility. For example, the linker can also be "(G4S)3" (e.g., GGGSGGGGSGGGGS); "(G4S)4" (e.g., GGGGSGGGGSGGGGSGGGGS); "(G4S)5" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGS); "(G4S)6" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS); For example, the use of a (G4S)5 linker can provide additional flexibility to the ligands described herein and can improve expression. In some embodiments, the linker can also be a (GS) n , (GGS) n , (GGGS) n , (GGSG) n, (GGSGG) n , or (GGGGS) n where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Non-limiting examples of linkers known to those of skill in the art that can be used to construct the fusions described herein can be found in U.S. Pat. No. 9,708,412, U.S. Patent Application Publication Nos. 20180134789 and 20200148771, and WO 2019051122 (each of which is incorporated by reference in its entirety).

[0203] In another embodiment, multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-MSLN-scFv fusions) can be constructed using the "knob-into-hole" method (Ridgway et al., Protein Eng 7:617-621 (1996)). In this method, Ig heavy chains of two different variable domains are reduced to selectively cleave heavy chain pairing while preserving the heavy-light chain pairing. Two heavy-light chain heterodimers that recognize two or three different antigens / ligands are mixed to promote heteroligand binding pairing mediated through the engineered "knob-in-hole" of the CH3 domain.

[0204] In another embodiment, multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-MSLN-scFv fusions) can be constructed by exchanging heavy-light chain dimers from two or more different antibodies to generate hybrid antibodies, where the first heavy-light chain dimer recognizes MSLN and the second heavy-light chain dimer recognizes a second and / or third antigen. The mechanism of heavy-light chain dimerization is similar to the formation of human IgG4, which also functions as a bispecific molecule. Dimerization of IgG heavy chains is promoted by intramolecular forces such as the pairing of the CH3 domains of each heavy chain with disulfide bridges. The presence of a specific amino acid (R409) in the CH3 domain has been shown to promote dimer exchange and the assembly of IgG4 molecules. Heavy chain pairing is further stabilized by inter-heavy chain disulfide bridges in the hinge region of the antibody. Specifically, in IgG4, the hinge region contains the amino acid sequence Cys-Pro-Ser-Cys (compared to the stable IgG1 hinge region, which contains the sequence Cys-Pro-Pro-Cys) at amino acids 226 to 230. This difference in the sequence of serine at position 229 has been linked to the propensity of IgG4 to form intrachain disulfides in the hinge region (Van der Neut Kolfschoten, M. et al., 2007, Science 317:1554-1557 and Labrijn, A. F. et al., 2011, Journal of Immunol 187:3238-3246).

[0205] Multispecific antibodies of the present invention (e.g., bispecific and trispecific antibodies such as anti-MSLN-scFv fusions) can be generated by introducing the R409 residue in the CH3 domain and a Cys-Pro-Ser-Cys sequence in the hinge region of an antibody that recognizes MSLN or a second and / or third antigen, such that the heavy-light chain dimers are swapped to produce an antibody molecule with one heavy-light chain dimer that recognizes MSLN and a second heavy-light chain dimer that recognizes a second and / or third antigen, where the second and / or third antigen (or ligand) is any antigen (or ligand) disclosed herein. Known IgG4 molecules can also be engineered so that the heavy and light chains recognize MSLN or a second and / or third antigen, as disclosed herein. The use of this method to construct multispecific antibodies of the present invention (e.g., bispecific and trispecific antibodies such as anti-MSLN-scFv fusions) can be advantageous due to the unique characteristics of the IgG4 molecule, whose Fc region differs from other IgG subtypes in that it interacts poorly with effector systems of the immune response, such as complement and Fc receptors expressed by certain leukocytes. This particular property makes these IgG4-based multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-MSLN-scFv fusions) attractive for therapeutic applications, where the antibody must bind to a target and functionally modify a signaling pathway associated with the target, but does not induce effector activity.

[0206] The multispecific antibodies described herein (e.g., bispecific and trispecific antibodies such as anti-MSLN-scFv fusions) can be engineered with a non-depleting heavy chain isotype, such as IgG1-LALA or stabilized IgG4 or one of the other non-depleting variants. In some embodiments, mutations are introduced into the constant region of the bsAb such that the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the bsAb is altered. For example, the mutation is a LALA mutation in the CH2 domain. In one aspect, multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-MSLN-scFv fusions) contain a mutation on one scFv unit of the heterodimeric multispecific antibody that reduces ADCC activity. In another aspect, multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-MSLN-scFv fusions) contain mutations on both chains of the heterodimeric multispecific antibody that completely eliminate ADCC activity. For example, a mutation introduced into one or both scFv units of a multispecific antibody (e.g., bispecific and trispecific antibodies such as anti-MSLN-scFv fusions) is a LALA mutation in the CH2 domain. These multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-MSLN-scFv fusions) with variable ADCC activity can be optimized so that the multispecific antibody exhibits maximal selective killing toward cells expressing one antigen recognized by the multispecific antibody, but minimal killing toward the second antigen recognized by the multispecific antibody.

[0207] The multispecific antibodies (e.g., bispecific antibodies) described herein can be engineered as modular tetrameric bispecific antibodies (tBsAbs). See, e.g., WO 2018 / 071913, incorporated herein by reference in its entirety. For example, a tetravalent antibody can be a dimer of bispecific scFv fragments comprising a first binding site for a first antigen and a second binding site for a second antigen. In embodiments, an anti-MSLN antibody can be the first binding site for a first antigen. In embodiments, an anti-MSLN antibody can be the second binding site for a second antigen. The two binding sites can be linked together via a linker domain. In embodiments, the scFv fragment is a tandem scFv, and the linker domain comprises an immunoglobulin hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region) amino acid sequence. In embodiments, the immunoglobulin hinge region amino acid sequence can be, for example, the linker amino acid sequence (GGGS). x1-6 , (GGGGS) x1-6 , or GSAGSAAGSGEF. In embodiments, the linker domain comprises at least a portion of an immunoglobulin Fc domain, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc domain. In embodiments, at least a portion of the immunoglobulin Fc domain does not comprise a CH2 domain. In embodiments, at least a portion of the immunoglobulin Fc domain may be a CH2 domain. An exemplary CH2 domain amino acid sequence comprises APELLGGPDVFLF (SEQ ID NO: [ ]). The Fc domain can be linked to the C-terminus of an immunoglobulin hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region) amino acid sequence. The linker domain may be flanked at one or both ends by a flexible linker amino acid sequence (e.g., (GGGS) x1-6 , (GGGGS) x1-6 , or GSAGSAAGSGEF).

[0208] In embodiments, the tBsAb may be specific for MSLN and also specific for a target selected from the group consisting of B7H3, CXCR4, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CCR4, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-L1, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, and KIR.

[0209] In embodiments, a multispecific antibody may be a bispecific T cell engager (BiTE). The term "BiTE" (bispecific T cell engager) may refer to a single polypeptide chain molecule having two antigen-binding domains, one of which binds to a T cell antigen. For example, a BiTE may comprise an MSLN antibody or functional fragment thereof disclosed herein and an antibody or fragment thereof that binds to a T cell antigen. For example, the antibody or fragment thereof that binds to a T cell antigen may be specific for CD3.

[0210] In embodiments, the multispecific antibody may be a trispecific T cell engager (TriTE). The term "TriTE" (trispecific T cell engager) may refer to a single polypeptide chain molecule having three antigen-binding domains, one or more of which bind to a T cell antigen. For example, a TriTE may comprise an MSLN antibody or functional fragment thereof disclosed herein and an antibody or fragment thereof that binds to a T cell antigen. For example, the antibody or fragment thereof that binds to a T cell antigen may be specific for CD3, CD28, or both.

[0211] The multispecific antibodies disclosed herein (e.g., bispecific and trispecific antibodies such as anti-MSLN-scFv fusions) may be useful in the treatment of chronic infections, diseases, or medical conditions, such as cancer.

[0212] fusion proteins

[0213] The present invention provides fusion proteins containing the MSLN antibody or functional fragment thereof disclosed herein operably linked to a second protein. The second protein can be, for example, a cytokine or growth factor. In embodiments, the cytokine is IL-2 or TGF-beta and its variants. In some other embodiments, the second protein can be a therapeutic agent such as a toxin, a detectable moiety such as a fluorescent protein for detection, or a biological agent such as an agent that stimulates T cells (i.e., CD3). In some embodiments, the MSLN antibody of the present invention can be operably linked to multiple additional proteins or peptides, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional protein or peptide sequences.

[0214] In some embodiments, the MSLN antibody or functional fragment thereof disclosed herein is directly linked to the second protein. In other embodiments, the MSLN antibody or functional fragment thereof is linked to the second protein via a linker, such as a flexible polypeptide chain. The linker can be any suitable linker of any length, but may be at least 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acids in length. In one embodiment, the linker is an amino acid sequence naturally occurring in a host immunoglobulin molecule, and the presence of the linker is incapable of eliciting an immune response against the linker sequence by the mammal. Fusion proteins of the present invention containing multiple additional proteins directed against an MSLN antibody may have multiple linker sequences linking each additional protein or peptide sequence.

[0215] Fusion proteins of the present invention can be constructed by recombinant methods known to those skilled in the art. For example, an expression vector containing a nucleic acid sequence encoding an MSLN antibody of the present invention can be operably linked to a nucleic acid sequence encoding a second protein and introduced into an expression system for translation and production of the fusion protein. Alternatively, those skilled in the art can readily utilize de novo protein synthesis techniques to produce the fusion proteins described herein.

[0216] Use of antibodies against MSLN

[0217] Antibodies of the present invention that specifically bind to the MSLN protein or a fragment thereof can be administered to treat MSLN-related diseases or disorders. "MSLN-related diseases or disorders" include disease states and / or symptoms associated with disease states in which elevated levels of MSLN and / or activated cell signaling pathways involving MSLN are observed. Exemplary MSLN-related diseases or disorders include, but are not limited to, T cell-suppressed diseases, such as cancer and infectious diseases. In some embodiments, the cancer may be lung cancer, kidney cancer, ovarian cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, uterine cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or stomach cancer. In some embodiments, the infectious disease is a viral disease, such as influenza.

[0218] The antibodies of the present invention, including bispecific, polyclonal, monoclonal, humanized, and fully human antibodies, can be used as therapeutic agents. Such agents can be used to treat cancer in a subject, improve vaccine efficacy, or enhance natural immune responses. Antibody preparations, e.g., those with high specificity and high affinity for their target antigen, can be administered to a subject to exert effects resulting from binding to the target. Administration of the antibody can neutralize, inhibit, or interfere with the activity of the MSLN protein.

[0219] Pharmaceutical Composition

[0220] The antibodies of the present invention that specifically bind to the MSLN protein or a fragment thereof can be administered in the form of a pharmaceutical composition for the treatment of cancer. Principles and considerations for preparing therapeutic pharmaceutical compositions containing antibodies, as well as guidance in selecting components, are provided, for example, in Remington: The Science and Practice of Pharmacy, 20th ed. (Alfonso R. Gennaro, et al., editors), Mack Pub. Co., Easton, Pa., 2000; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide and Protein Drug Delivery (Advances in Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0221] The specific dosage and treatment regimen for a patient will depend on various factors, such as the antibody, variant, or derivative thereof used, the patient's age, weight, general health, sex, and diet, as well as the time of administration, frequency of excretion, concomitant use of drugs, and the severity of the disease being treated. The assessment of such factors by a medical professional is within the skill of one of ordinary skill in the art. The amount will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmaceutical and pharmacokinetic principles well known in the art.

[0222] A therapeutically effective amount of an antibody of the present invention can be the amount necessary to achieve a therapeutic goal. As noted above, this can be a binding interaction between the antibody and its target antigen, which in certain cases interferes with the function of the target. The amount that needs to be administered further depends on the binding affinity of the antibody for its specific antigen and also on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. The dosage of an antigen-binding polypeptide described herein administered to a subject (e.g., a patient) is approximately 0.1 mg / kg to 100 mg / kg of patient body weight, 0.1 mg / kg to 20 mg / kg of patient body weight, or 1 mg / kg to 10 mg / kg of patient body weight. Human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to foreign polypeptides. Therefore, lower dosages and less frequent administration of human antibodies are often possible. Furthermore, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing antibody uptake and tissue (e.g., brain) penetration through modifications such as lipidation. A typical range for therapeutically effective administration of an antibody or antibody fragment of the invention can be, by way of non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight, and a typical administration frequency can range, for example, from twice daily to once weekly.

[0223] When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation may also contain two or more active compounds necessary for the indication being treated, e.g., those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may contain an agent that enhances its function (e.g., a cytotoxic agent, a cytokine (e.g., IL-15), a chemotherapeutic agent, or a growth-inhibitory agent, etc.). Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0224] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively.

[0225] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0226] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time.

[0227] The antibodies or agents of the present invention (also referred to herein as "active compounds"), as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or agent and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" may include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as liposomes and fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0228] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and an agent for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0229] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In embodiments, the composition is sterile and fluid to the extent that easy syringability exists. It may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, can be included in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0230] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the ingredients listed above as needed, and then sterilize by filtration.For example, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other necessary ingredients listed above.For the preparation of sterile injectable solution, the method of preparation is vacuum drying and freeze-drying, which produces powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile filtered.

[0231] Oral compositions include inert diluents or edible carriers. They can be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using fluid carriers for use as mouthwashes, in which the compound in the fluid carrier is orally applied, swirled in the mouth, and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0232] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0233] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams known in the art.

[0234] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0235] In one embodiment, the active compound is prepared with a carrier that will protect the compound against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0236] For the sake of ease of administration and uniformity of dosage, oral or parenteral compositions can be formulated in dosage unit form.As used herein, dosage unit refers to a physically separate unit suitable as a unit dose for the subject to be treated, and each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by and directly depends on the specific characteristics of active compound and the therapeutic effect to be achieved, and the inherent limitations of the technology of compounding such active compound for individual treatment.

[0237] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0238] diagnosis

[0239] The antibody according to the present invention can be used as an agent for detecting the presence of MSLN (or a protein fragment thereof) in a sample. For example, the sample can be a cancer sample or a sample from a subject at risk of having cancer. For example, the cancer can be lung cancer, kidney cancer, ovarian cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, uterine cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or stomach cancer. For example, the antibody can comprise a detectable label. The antibody can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., F ab , scFv, or F (ab)2 ) can be used. With respect to probes or antibodies, the term "labeled" can encompass direct labeling of the probe or antibody by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin for detection with fluorescently labeled streptavidin. The term "biological sample" can include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids present within a subject. Thus, the use of the term "biological sample" includes blood and fractions or components of blood, including serum, plasma, or lymph. That is, the detection methods of the present invention can be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detection of analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations.

[0240] Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology," Vol. 42, J.R.Crowther (Ed.), Human Press, Totowa, NJ, 1995; "Immunoassay," E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays," P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Additionally, in vivo techniques for detecting an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0241] Antibodies to the MSLN protein (or fragments thereof) can be used in methods known in the art related to localizing and / or quantitating the MSLN protein (e.g., for use in measuring levels of the MSLN protein in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging). In certain embodiments, antibodies specific for the MSLN protein, or derivatives, fragments, analogs, or homologs thereof, that contain an antigen-binding domain derived from the antibody, are utilized as pharmaceutically active compounds (hereinafter referred to as "therapeutic agents").

[0242] Antibodies specific for the MSLN protein of the invention can be used to isolate MSLN polypeptides by standard techniques, such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies to the MSLN protein (or fragments thereof) can be used diagnostically to monitor protein levels in tissues as part of a clinical testing procedure, e.g., to determine the effectiveness of a given treatment regimen.

[0243] Detection can be facilitated by conjugating (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125 I, 131 I, 35 S, 32 P, or 3 H is one example.

[0244] Screening Method

[0245] The present invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules, or other drugs), that regulate or otherwise interfere with MSLN activity. Methods for identifying compounds useful for treating cancer are also provided. The present invention also encompasses compounds identified using the screening assays described herein.

[0246] For example, the present invention provides assays for screening candidate or test compounds that modulate the expression and / or activity of MSLN. Test compounds of the present invention can be obtained using any of a number of approaches in combinatorial library methods known in the art, including, but not limited to, biological libraries; spatially addressable parallel solid-phase or liquid-phase libraries; synthetic library methods requiring deconvolution; "one bead, one compound" library methods; and synthetic library methods using affinity chromatography selection. While the biological library approach is limited to peptide libraries, the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds. (See, e.g., Lam, 1997, Anticancer Drug Design 12:145.)

[0247] As used herein, "small molecule" may refer to a composition having a molecular weight of less than about 5 kD, most preferably less than about 4 kD. Small molecules may be, for example, nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the present invention.

[0248] Examples of methods for the synthesis of molecular libraries can be found in the art, for example, DeWitt, et al., 1993. Proc. Natl. Acad. Sci. USA 90:6909; Erb, et al., 1994. Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann, et al., 1994. J. Med. Chem. 37:2678; Cho, et al., 1993. Science 261:1303; Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33:2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop, et al., 1994. J. Med. Chem. 37:1233.

[0249] Libraries of compounds can be stored in solution (see, e.g., Houghten, 1992, Biotechniques 13:412-421), on beads (see, e.g., Lam, 1991, Nature 354:82-84), on chips (see, e.g., Fodor, 1993, Nature 364:555-556), bacteria (see, e.g., U.S. Pat. No. 5,223,409), spores (see, e.g., U.S. Pat. No. 5,233,409), plasmids (Cull, et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869), or on phages (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla, et al., 1990, Science 249:406-406). al., 1990. Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409).

[0250] In one embodiment, a candidate compound is introduced to the antibody-antigen complex to determine whether the candidate compound disrupts the antibody-antigen complex, where disruption of the complex indicates that the candidate compound modulates MSLN activity.

[0251] In another embodiment, at least one MSLN protein is exposed to at least one monoclonal antibody. The formation of an antibody-antigen complex is detected, and one or more candidate compounds are introduced into the complex. If the antibody-antigen complex is disrupted after the introduction of the one or more candidate compounds, the candidate compounds are useful for treating cancer or a proliferative disease or disorder.

[0252] Determining the ability of a test compound to interfere with or disrupt the antibody-antigen complex can be accomplished, for example, by conjugating the test compound with a radioisotope or enzyme label, so that binding of the test compound to the antigen or a biologically active portion thereof can be determined by detecting the labeled compound in the complex. For example, the test compound can be directly or indirectly labeled with 125r, 35S, 14C, or 3H, and the radioisotope can be detected by direct counting of radioemission or by scintillation counting. Alternatively, the test compound can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and detected by measuring the conversion of the enzyme label and an appropriate substrate to product.

[0253] In one embodiment, the assay comprises contacting the antibody-antigen complex with a test compound and determining the ability of the test compound to interact with the antigen or disrupt a pre-existing antibody-antigen complex. In this embodiment, determining the ability of the test compound to interact with the antigen and / or disrupt the antibody-antigen complex comprises determining the ability of the test compound to preferentially bind to the antigen or a biologically active portion thereof compared to the antibody.

[0254] In another embodiment, the assay comprises contacting the antibody-antigen complex with a test compound and determining the ability of the test compound to modulate the antibody-antigen complex. Determining the ability of the test compound to modulate the antibody-antigen complex can be accomplished, for example, by determining the ability of the antigen to bind to or interact with the antibody in the presence of the test compound.

[0255] Those skilled in the art will recognize that in any of the screening methods disclosed herein, the antibody can be an MSLN antibody. Additionally, the antigen can be an MSLN protein or a portion thereof.

[0256] The screening methods disclosed herein can be performed as cell-based or cell-free assays. For cell-free assays involving membrane-bound MSLN protein, it may be desirable to utilize a solubilizing agent to maintain the membrane-bound protein in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, isotridecyl poly(ethylene glycol ether) n, N-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonic acid, 3-(3-cholamidopropyl)dimethylammonium-1-propanesulfonic acid (CHAPS), or 3-(3-cholamidopropyl)dimethylammonium-2-hydroxy-1-propanesulfonic acid (CHAPSO).

[0257] In some embodiments, it may be desirable to immobilize the antibody or antigen to facilitate separation of the complexed form from the uncomplexed form of either or both after introduction of the candidate compound and to adapt the assay to automation. Observation of the antibody-antigen complex in the presence and absence of the candidate compound can be achieved in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, fusion proteins can be provided that add a domain that allows one or both of the proteins to be bound to a matrix. For example, a GST-antibody fusion protein or a GST-antigen fusion protein can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis, MO) or a glutathione-derivatized microtiter plate, then combined with a test compound, and the mixture can be incubated under conditions that promote complex formation (e.g., physiological conditions of salt and pH). After incubation, the beads or microtiter plate wells are washed to remove unbound components, the matrix is ​​immobilized in the case of beads, and the complex is determined directly or indirectly. Alternatively, the complexes can be dissociated from the matrix, and the level of antibody-antigen complex formation can be determined using standard techniques.

[0258] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the present invention. For example, antibodies or antigens (e.g., MSLN proteins) can be immobilized using conjugation of biotin and streptavidin. Biotinylated antibody or antigen molecules can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.) and immobilized to wells of a streptavidin-coated 96-well plate (Pierce Chemical). Alternatively, other antibodies that react with the antibody or antigen of interest but do not interfere with the formation of the antibody-antigen complex of interest can be derivatized to the wells of the plate, and unbound antibody or antigen can be captured in the wells by antibody conjugation. Methods for detecting such complexes include immunodetection of complexes using such other antibodies reactive with the antibody or antigen, in addition to those described herein for GST-immobilized complexes.

[0259] This invention further pertains to novel agents identified by any of the screening assays described herein and uses thereof for treatments described herein.

[0260] Chimeric antigen receptor (CAR) cell therapy

[0261] Also provided herein are cell therapies, such as chimeric antigen receptor (CAR) cell therapies. For example, the cells can be CAR T cells or CAR NK cells.

[0262] CAR cell therapy redirects a patient's T cells and / or NK cells to kill tumor cells, for example, by exogenous expression of a CAR on the T cells or NK cells. CARs can be transmembrane fusion proteins that link the antigen recognition domain of an antibody to the intracellular signaling domain of a T cell receptor and co-receptor or NK cell receptor.

[0263] In one embodiment, a monospecific CAR cell is provided. For example, the anti-MSLN antibody described herein can be used as a targeting moiety of the CAR cell. For example, an MSLN antibody can have low affinity to its antigen but high avidity. In another example, an MSLN antibody can have high affinity to its antigen but low avidity. An antibody with fewer binding sites can have high affinity and low avidity, while an antibody with a larger binding site can have low affinity and high avidity.

[0264] In another embodiment, a bispecific (or dual-target) CAR cell is provided. In another embodiment, the CAR cell is an engineered cell comprising a chimeric antigen receptor, where the chimeric antigen receptor comprises an extracellular ligand-binding domain specific for a first antigen and a second antigen on the surface of a cancer cell, wherein the first antigen comprises an antigen that is not MSLN and the second antigen comprises MSLN.

[0265] In embodiments, the anti-MSLN antibodies or MSLN fusion proteins described herein can be used as a payload for armored CAR cell therapy. For example, suitable cells capable of secreting the anti-MSLN antibodies of the present invention (or alternatively, engineered to express the secreted anti-MSLN antibodies described herein) can be used. The secreted anti-MSLN "payload" can be, for example, a minibody, scFv, IgG molecule, bispecific fusion molecule, and other antibody fragments described herein. After contact or engineering, the cells described herein can be introduced into a patient in need of treatment by infusion therapy, as known to those skilled in the art.

[0266] In embodiments, the patient may have an MSLN-associated disease or disorder, such as a cancer, as described herein. The cells (e.g., T cells) may be, for example, but not limited to, T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof. Exemplary CARs and CAR factories useful in aspects of the present invention include those disclosed in, for example, PCT / US2015 / 067225 and PCT / US2019 / 022272, each of which is incorporated herein by reference in its entirety. In one embodiment, the MSLN antibodies discussed herein can be used to construct multispecific antibodies or as payloads for CAR-T cells or CAR NK cells. For example, in one embodiment, the anti-MSLN antibodies discussed herein can be used for targeting CARs (i.e., as targeting moieties). In another embodiment, the anti-MSLN antibodies discussed herein can be used as targeting moieties, and different MSLN antibodies targeting different epitopes can be used as payloads. In another embodiment, the payload can be an immunomodulatory antibody payload.

[0267] Solid tumors present unique challenges for CAR-T therapy. Some barriers to CAR-T efficacy in solid tumors include heterogeneous antigen expression, poor tissue homing, activation, persistence, and an immunosuppressive tumor microenvironment. Unlike hematological cancers, tumor-associated target proteins are overexpressed between tumors and healthy tissues, resulting in on-target / off-tumor T cell killing of healthy tissues. Furthermore, immunosuppression in the tumor microenvironment (TME) limits CAR-T cell activation toward tumor killing. After such contact or manipulation, the cells can be introduced into a cancer patient in need of treatment by infusion therapy known to those skilled in the art. The cancer patient may have any of the types of cancer disclosed herein. The cells (e.g., T cells) may be, for example, but not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

[0268] In embodiments, CAR cells (i.e., CAR T cells or CAR NK cells) can be generated according to methods known in the art using lentiviral systems (via transduction), retroviral systems (via transfection (electroporation)), and transposon systems (via PiggyBac). Useful promoters for payloads that can be used in the generation of CAR-Ts include, for example, constitutive promoters (where the promoter is the same as that of the CAR-T, e.g., EF1a followed by IRES or 2A); inducible promoters (where the promoter is different from that of the CAR-T, e.g., NFAT, IL-2 promoter); and engineered promoters (such as cytokine MSLN locus "knock-ins" and / or promoters under the control of endogenous promoters). In one embodiment, the MSLN antibody or MSLN fusion protein discussed herein can be used to construct a multispecific antibody or as a payload for a CAR cell. For example, in one embodiment, the anti-MSLN antibody or MSLN fusion protein discussed herein can be used for targeting a CAR (i.e., as a targeting moiety). In one embodiment, the anti-MSLN antibody or MSLN fusion protein discussed herein can be used as a payload secreted by CAR cells. In another embodiment, the anti-MSLN antibody or MSLN fusion protein discussed herein can be used as a targeting moiety, and a different MSLN antibody targeting a different epitope can be used as the payload. In another embodiment, the payload can be an immunomodulatory antibody payload. In some embodiments, the MSLN antibody or MSLN fusion protein described herein for use in CAR-T compositions is not a high-affinity MSLN antibody (e.g., so that the antibody does not bind strongly to its MSLN target). For example, the MSLN antibody or MSLN fusion protein described herein can be used as a payload secreted by CAR cells, and two targeting moieties (e.g., tumor-associated surface antigens) are selected for a particular cancer.Non-limiting examples of tumor-associated surface antigens include ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), MUC1, MSLN, CD19, CD20, CD30, CD40, CD22, RAGE-1, and MN-CA. IX, RET1, RET2 (AS), prostate-specific antigen (PSA), TAG-72, PAP, p53, Ras, prostein, PSMA, survivin, 9D7, prostate cancer tumor antigen-1 (PCTA-1), GAGE, MAGE, mesothelin, β-catenin, TGF-βRII, BRCA1 / 2, SAP-1, HPV-E6, HPV-E7 (also see PCT / US2015 / 067225 and PCT / US2019 / 022272, which are incorporated by reference in their entireties, for additional tumor-associated surface antigens). Exemplary armored CAR-T cells are listed in the table below. TIFF2025528835000025.tif130150

[0269] Those skilled in the art will recognize that CAR cells can be generated from cell sources known to those skilled in the art. Non-limiting examples include T cells, NK cells, iPSC-derived cells (e.g., iPSC-derived T cells and / or iPSC-derived NK cells), peripheral blood cells (e.g., peripheral blood mononuclear cells), umbilical cord blood cells, cell lines (e.g., the NK92 cell line), human embryonic stem cells (hESCs), and CD34+ hematopoietic progenitor cells (HPCs). See, for example, Lu, Hui, et al., "From CAR-T cells to CAR-NK cells: a developing immunotherapy method for hematological malignancies." Frontiers in Oncology (2021): 3151.

[0270] Chimeric B cell receptor

[0271] Engineered B cell receptors, called chimeric B cell receptors, including B cell receptors containing antibodies or antibody fragments preselected for high affinity to specific disease-associated antigens, represent a powerful new approach to disease. B cells function as professional antigen-presenting cells, processing and presenting antigens on MHC class II molecules, enhancing immune cell recognition of tumors and aiding in the spread of neoantigens. Chimeric antibody signaling and secreting (CASS) B cells, a key component of immune memory, simultaneously recruit a broad range of immune cells, providing a robust, lifelong surveillance program that reverses tumor-infiltrating lymphocyte depletion and protects against tumor metastasis and recurrence. In embodiments, B cells may contain chimeric, non-natural, and at least partially artificially engineered receptors. In some cases, engineered chimeric B cell receptors have one, two, three, four, or more components, and in some embodiments, one or more components facilitate B cell targeting or binding to one or more antigen-bearing cells.

[0272] Aspects of the present invention include genetically engineered B cells that have been modified to express and carry a chimeric B cell receptor on their surface. In embodiments, the genetically engineered B cells may comprise a single chimeric B cell receptor that targets one antigen, such as MSLN, or a single chimeric B cell receptor that targets two or more antigens (e.g., a bispecific or multispecific chimeric B cell receptor). In some embodiments, the cells comprise a split chimeric B cell receptor, such as two different scFvs expressed on the B cell surface with different costimulatory domains. Additionally, some embodiments comprise fine-tuned chimeric B cell receptors.

[0273] In embodiments, the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, and the polypeptides assemble together to form the chimeric B cell receptor.

[0274] For example, the extracellular ligand-binding domain can be selected to recognize a ligand, such as MSLN, that acts as a cell surface marker on target cells associated with a disease state. For example, the disease state can be cancer and the target ligand can be a cancer-associated antigen, such as MSLN.

[0275] In embodiments, the extracellular ligand-binding domain may comprise an antigen-binding or antigen-recognition domain derived from an antibody directed against a target antigen, such as an anti-MSLN antibody described herein, hi embodiments, the extracellular ligand-binding domain may comprise an antibody or fragment thereof described herein.

[0276] In one embodiment, the transmembrane domain comprises a stalk region. The stalk region can be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4, or CD28, or all or a portion of an antibody constant region (e.g., CH1, CH2, CH3, or both CH2 and CH3 for an IgG antibody, or CH1, CH2, CH3, CH4, or any combination thereof for an IgM antibody). In other embodiments, the stalk region can be a synthetic sequence that corresponds to a naturally occurring stalk sequence, or can be an entirely synthetic stalk sequence. In one embodiment, the stalk region is a portion of the human CD8 alpha chain.

[0277] The signaling domain or intracellular signaling domain of the chimeric B cell receptor of the present invention is involved in intracellular signal transduction following binding of the extracellular ligand-binding domain to a target, resulting in activation of an immune cell and an immune response. In other words, the signaling domain is involved in activating at least one of the normal functions of the B cell in which the chimeric B cell receptor is expressed. Thus, the term "signaling domain" can refer to the portion of a protein that induces a cell to perform a specialized function, such as, for example, the early activation of Lyn and Syk and the late activation of NFAT and NFκB.

[0278] Chimeric B cell receptors can contain native transmembrane and intracellular domains. In native B cells, B cell receptor binding leads to rapid tyrosine phosphorylation and calcium ion polarization of the intracellular domain, resulting in downstream activation of NFAT and NF-κB. We have engineered an inducible expression system that uses NFAT / NF-κB response elements to drive the expression of secreted proteins and is activated by antigens associated with pathologies such as cancer.

[0279] Distinguishing features of suitable transmembrane polypeptides include their ability to be expressed on the surface of immune cells, such as B cells, and to interact together to induce a cellular response of the immune cells against a predefined target cell. Different transmembrane polypeptides of a chimeric B cell receptor, comprising an extracellular ligand-binding domain and / or a signaling domain, interact together to participate in signal transduction after binding to a target ligand and induce an immune response. The transmembrane domain may be derived from natural or synthetic sources. The transmembrane domain may be derived from any membrane-bound or transmembrane protein.

[0280] Treatment method

[0281] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or favorable clinical results include, but are not limited to, alleviation of symptoms, reduction in extent of disease, stable (i.e., not worsening) state of disease, slowing or delaying of disease progression, improvement or palliation of disease state, remission (partial or total), whether detectable or not. "Treatment" can also refer to prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to suffer from the condition or disorder or those in whom the condition or disorder is to be prevented.

[0282] The present invention provides both prophylactic and therapeutic methods for treating subjects at risk for (or susceptible to) cancer (e.g., when an early detection cancer biomarker is identified in such a subject) or other cell proliferation-related diseases or disorders. Such diseases or disorders include, but are not limited to, diseases or disorders associated with aberrant expression of MSLN and / or aberrant activation of cell signaling pathways involving MSLN. Such diseases or disorders are included in MSLN-associated diseases or disorders. For example, the method is used to treat, prevent, or alleviate the symptoms of cancer. In one embodiment, the method is used to treat, prevent, or alleviate the symptoms of solid tumors. Non-limiting examples of other tumors that can be treated with the compositions described herein include lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or gastric cancer. Additionally, the method of the present invention can be used to treat blood cancers such as leukemia and lymphoma. Alternatively, the method can be used to treat, prevent, or alleviate the symptoms of metastatic cancer. For example, cancers that can be treated or prevented or whose symptoms can be alleviated include B-cell chronic lymphocytic leukemia (CLL), non-small cell lung cancer, melanoma, ovarian cancer, lymphoma, or renal cell carcinoma. Cancers that can be treated or prevented or whose symptoms can be alleviated also include solid tumors with high mutational burden and WBCs in the filtrate.

[0283] Thus, in one aspect, the invention provides a method for preventing, treating, or alleviating symptomatic cancer or cell proliferative disease or disorder in a patient by administering to the patient a monoclonal antibody, scFv antibody, or bispecific antibody of the invention. For example, an anti-MSLN antibody can be administered in a therapeutically effective amount.

[0284] Subjects at risk for cancer or cell proliferation-related diseases or disorders can include patients with a family history of cancer or subjects who have been exposed to agents known or suspected to cause cancer. Administration of a prophylactic agent can occur prior to the manifestation of cancer, such that the disease is prevented or, alternatively, its progression is delayed.

[0285] In another embodiment, tumor cell growth is inhibited by contacting the cell with an anti-MSLN antibody of the invention. The cell can be any cell that expresses MSLN.

[0286] The present invention further provides both prophylactic and therapeutic methods for treating subjects at risk (or susceptible to) chronic or acute viral, bacterial, or parasitic infections. The present invention also provides therapeutic methods for both prophylactic and therapeutic treatment of subjects at risk for a disease, disorder, or condition associated with T cell depletion, or at risk of developing T cell depletion. The present invention also provides therapeutic methods for both prophylactic and therapeutic treatment of subjects at risk for a disease, disorder, or condition associated with T cell depletion, or at risk of developing T cell depletion. Such diseases or disorders include, but are not limited to, HIV, AIDS, and chronic or acute bacterial, viral, or parasitic infections. Other such chronic infections include, for example, those caused by hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus type 1 (HSV-1), Helicobacter pylori, or Toxoplasma gondii. Other acute infectious diseases included are those caused by microorganisms such as gram-positive bacteria, gram-negative bacteria, protozoa, or fungi, e.g., as described herein. Non-limiting examples of infections include influenza infections.

[0287] The present invention also encompasses methods for increasing or enhancing an immune response to an antigen. The immune response is increased or enhanced by administering a monoclonal antibody, scFv antibody, or bispecific antibody of the present invention to a subject. The immune response is enhanced, for example, by enhancing antigen-specific T effector function. The antigen is a viral (e.g., HIV), bacterial, parasitic, or tumor antigen. The immune response is a natural immune response. By natural immune response, we mean an immune response that is the result of an infectious disease. The infectious disease can be an influenza infection. The infectious disease is a chronic infectious disease. An increase or enhancement of the immune response to an antigen can be measured by many methods known in the art. For example, the immune response can be measured by measuring one of the following: T cell activity, T cell proliferation, T cell activation, effector cytokine production, and T cell transcriptional profile. Alternatively, the immune response is a response induced by vaccination.

[0288] Thus, in another aspect, the invention provides a method of increasing vaccine efficacy by administering to a subject a monoclonal or scFv antibody of the invention and a vaccine, wherein the antibody and vaccine are administered sequentially or simultaneously, and the vaccine is a tumor vaccine, a bacterial vaccine, or a viral vaccine.

[0289] Combined use

[0290] The compositions of the present invention described herein can be administered in combination with chemotherapeutic agents. Chemotherapeutic agents that can be administered with the compositions of the present disclosure include antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon α-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); hormones (e.g., medroxycycline, cyclosporine, cycloheximide ... These include, but are not limited to, progesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone; nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard), and thiotepa); steroids and combinations (e.g., betamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0291] In additional embodiments, the compositions of the invention described herein can be administered in combination with cytokines, including but not limited to IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0292] In additional embodiments, the compositions described herein can be administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

[0293] In some embodiments, the compositions described herein can be administered in combination with other immunotherapeutic agents, including, but not limited to, simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, sitatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, dutuzumab, and tuzumab. Rigotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, fizitumumab, framvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab Mab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minletumomab, mitumomab, moxetumomab, narnatumumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratuzumab, onartuzumab, oportuzumab, oregovomab, panitumumab, palsatuzumab, patritumumab, pemtumomab and 3F8.

[0294] The present invention provides a method for treating cancer in a patient by administering two antibodies that bind to the same epitope on the MSLN protein, or alternatively, two different epitopes on the MSLN protein. Alternatively, cancer can be treated by administering a first antibody that binds to MSLN and a second antibody that binds to a protein other than MSLN. In other embodiments, cancer can be treated by administering a bispecific antibody that binds to both MSLN and a protein other than MSLN. For example, the protein other than MSLN can include, but is not limited to, IL-12, IL-12R, IL-2, IL-2R, IL-15, IL-15R, IL-7, IL-7R, IL-21, or IL-21R. For example, the protein other than MSLN can be a tumor-associated antigen, or the protein other than MSLN can be a cytokine. Non-limiting examples of proteins other than MSLN include CTLA-4, CXCR4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD47, and CD73.

[0295] In some embodiments, the invention provides for the administration of anti-PD-1 antibodies, alone or in combination with an additional antibody that recognizes another protein other than MSLN, along with cells capable of generating or enhancing an immune response. For example, these cells can be peripheral blood mononuclear cells (PBMCs), or any cell type found in PBMCs, such as cytotoxic T cells, macrophages, and natural killer (NK) cells.

[0296] Additionally, the present invention provides for the administration of antibodies that bind to MSLN protein with other therapeutic agents, including anti-neoplastic agents such as small molecules, growth factors, cytokines, or biomolecules such as peptides, peptidomimetics, peptoids, polynucleotides, lipid-derived mediators, small biogenic amines, hormones, neuropeptides, and proteases. Small molecules include, but are not limited to, inorganic molecules and small organic molecules. Suitable growth factors or cytokines include IL-2, GM-CSF, IL-12, and TNF-alpha. Small molecule libraries are known in the art. (See Lam, Anticancer Drug Des., 12:145, 1997.)

[0297] Diagnostic Assays

[0298] Anti-MSLN antibodies can be used diagnostically, e.g., to monitor the development or progression of cancer, e.g., as part of a clinical trial procedure to determine the effectiveness of a given treatment and / or prophylactic regimen.

[0299] In some embodiments, for diagnostic purposes, the anti-MSLN antibodies of the invention are linked to a detectable moiety, e.g., to provide a method for detecting cancer cells in a subject at risk for or afflicted with cancer.

[0300] The detectable moiety can be conjugated directly to the antibody or fragment, or indirectly, for example, by using a fluorescent secondary antibody. Direct conjugation can be achieved, for example, by standard chemical coupling of a fluorophore to the antibody or antibody fragment, or through genetic engineering. Chimeric or fusion proteins containing an antibody or antibody fragment linked to a fluorescent or bioluminescent protein can be constructed. For example, Casadei, et al. (Proc Natl Acad Sci USA. 1990 Mar;87(6):2047-51) describe a method for creating a vector construct capable of expressing a fusion protein gene between aequorin and an antibody gene in mammalian cells.

[0301] As used herein, the term "labeled" with respect to a probe or antibody can encompass both direct labeling of the probe or antibody by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin for detection with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject (e.g., biopsy material), as well as tissues, cells, and biological fluids present within a subject. That is, the detection methods of the present invention can be used in vitro and in vivo to detect cells expressing MSLN in biological samples. For example, in vitro techniques for detecting MSLN include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. Additionally, in vivo techniques for detecting MSLN involve introducing a labeled anti-MSLN antibody into a subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0302] In the case of "targeted" conjugates, i.e., conjugates that include a targeting moiety, which is a molecule or feature designed to localize the conjugate to a specific site within a subject or animal, localization can refer to a state in which equilibrium between the bound "localized" entity and the unbound "free" entity within the subject is essentially achieved. The rate at which such equilibrium is achieved depends on the route of administration. For example, a conjugate administered by intravenous injection can achieve localization within minutes of injection, whereas an orally administered conjugate may take several hours to achieve localization. Alternatively, localization can simply refer to the location of an entity within a subject or animal at a selected period of time after the entity is administered. As another example, localization is achieved when the moiety becomes distributed after administration.

[0303] A reasonable estimate of the time required to achieve localization can be made by one skilled in the art. Furthermore, the state of localization as a function of time can be tracked by imaging a detectable moiety (e.g., a luminescent conjugate) according to the method of the present invention, such as with a photodetector device. The "photodetector device" used can be sensitive enough to allow imaging of weak light from within a mammal in a reasonable time and to use the signal from such a device to construct an image.

[0304] If it is possible to use extremely bright light-generating moieties and / or detect light-generating fusion proteins localized near the surface of the object or animal being imaged, then "night vision" goggles or standard sensitive video cameras such as Silicon Intensified Tube (SIT) cameras (e.g., manufactured by Hammamatsu Photonic Systems, Bridgewater, NJ) can be used. However, more sensitive light detection methods are needed.

[0305] At extremely low light levels, the photon flux per unit area becomes so low that the scene being imaged no longer appears continuous. Instead, it is represented by individual photons that differ from each other both temporally and spatially. When viewed on a monitor, such an image appears as sparkling points of light, each representing a single detected photon. By accumulating these detected photons over time in a digital image processor, an image can be acquired and constructed. In contrast to conventional cameras, where the signal at each image point is assigned an intensity value, in photon-counting imaging, the amplitude of the signal is immaterial. The goal is simply to detect the presence of a signal (photon) and count its occurrence relative to its location over time.

[0306] At least two types of photodetector devices described herein can detect individual photons and generate a signal that can be analyzed by an image processor. Noise-reducing photodetector devices achieve sensitivity by reducing the background noise of the photon detector rather than amplifying the photon signal. Noise is primarily reduced by cooling the detector array. Devices include charge-coupled device (CCD) cameras called "back-thinned" cooled CCD cameras. In more sensitive devices, cooling is achieved using, for example, liquid nitrogen, to bring the temperature of the CCD array to approximately -120°C. "Back-thinned" refers to an ultra-thin backplate that shortens the path length photons must travel before being detected, thereby increasing quantum efficiency. A highly sensitive back-thinned cryogenic CCD camera is the "TECH 512" Series 200 camera available from Photometries, Ltd. (Tucson, Arizona).

[0307] "Photon amplification devices" amplify photons before they hit the detection screen. This class includes CCD cameras equipped with intensifier tubes, such as microchannel intensifier tubes. Microchannel intensifier tubes contain a metal array of channels perpendicular to and coextensive with the camera's detection screen. The microchannel array is positioned between the sample, subject, or animal being imaged and the camera. Most of the photons that enter the channels of the array contact the sides of the channels before exiting. A voltage applied across the array results in the emission of many electrons from each photon collision. Electrons from such collisions exit their channels of origin in a "shotgun" pattern and are detected by the camera.

[0308] Even greater sensitivity can be achieved by arranging intensifying microchannel arrays in series, so that electrons generated in the first stage in turn result in an amplified signal of electrons in the second stage. However, the increased sensitivity is achieved at the expense of spatial resolution, which decreases with each additional stage of amplification. An exemplary microchannel intensifier-tube-based single-photon detection device is the C2400 series available from Hamamatsu.

[0309] Image processors process signals generated by photon-counting photodetector devices to construct images that can be displayed on a monitor or printed on a video printer, for example. Such image processors are sold as part of systems that include the highly sensitive photon-counting cameras described herein and are therefore available from the same sources. Image processors can be connected to personal computers, such as an IBM-compatible PC or an Apple Macintosh (Apple Computer, Cupertino, Calif.), which may or may not be included as part of a purchased imaging system. Once images are in the form of digital files, they can be manipulated and printed using a variety of image processing programs (e.g., "ADOBE PHOTOSHOP," Adobe Systems, Adobe Systems, Mt. View, Calif.).

[0310] In one embodiment, the biological sample contains protein molecules from the test subject. One exemplary biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.

[0311] The present invention also encompasses kits for detecting the presence of MSLN- or TIGIT-expressing cells in a biological sample. For example, the kit may include: a labeled compound or agent (e.g., an anti-MSLN scFv or monoclonal antibody) capable of detecting cancer or tumor cells in a biological sample, a means for determining the amount of MSLN in the sample, and a means for comparing the amount of MSLN in the sample with a standard. In some embodiments, the standard is a non-cancerous cell or a cellular extract thereof. The compound or agent may be packaged in a suitable container. The kit may further include instructions for using the kit to detect cancer in a sample.

[0312] Other embodiments

[0313] While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0314] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0315] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, as alternative methods may be used to obtain similar results.

[0316] Example 1 We identified a series of anti-mesothelin (MSLN) antibodies by whole-cell panning. Panning rounds were completed using transduced cells (293T-MSLN or Cf2Th-MSLN) or a tumor cell line naturally enriched in MSLN (OVCAR8). A total of 118 unique antibodies were identified, and lead clones were identified based on binding to OvCar8 and / or Cf2Th cells (see Figure 39). The antibodies were extensively epitope mapped to target a distal region of MSLN, which is also the binding site for MUC16. Furthermore, one of our antibodies (Gly1-2-H4) recognized only cell surface-expressed MSLN and did not bind to plates coated with soluble MSLN, indicating a conformational epitope. A subset of these scFvs was used as the targeting moiety of CAR T cells and showed potent killing of MSLN+ cells.

[0317] MSLN has been identified as a tumor-associated antigen in a wide variety of cancers, including lung and ovarian cancer. MSLN can exist in two forms: a form bound to the cell membrane via a GPI linker and a soluble / shedding form. The binding of the GPI linker causes a slight conformational shift in the protein, which is recognized by one of the inventors' antibodies. By developing a CAR T that can only recognize the membrane-bound form of the protein, the inventors can avoid soluble MSLN acting as a decoy and protect the CAR from premature activation.

[0318] These scFvs can be developed as monoclonal antibodies for therapeutic or diagnostic purposes. Furthermore, they have potential as targeting moieties for CAR T cells against cancers that overexpress MSLN.

[0319] Example 2 Chimeric antibody signaling and secreting (CASS) B cells - a novel combined cellular immunotherapy to achieve cancer treatment

[0320] B cells are a key component of a patient's innate immunity. While other immune cells, including T cells, NK cells, and myeloid cells, have been engineered with chimeric antigen receptors (CARs), B cells have largely been ignored. Their primary role is to recognize foreign invaders via membrane-bound antibodies (B cell receptors, BCRs) and eliminate them by producing antibodies that bind to and eliminate the threatening targets. We propose developing a chimeric antibody signaling and secretion (CASS) B-cell platform by engineering both target-binding domains and secreted antibodies to develop potent anti-cancer therapeutics. The target-binding domain is designed to recognize tumor-associated antigens, and upon binding, leads to the secretion of trispecific T-cell-binding antibodies that effectively stimulate recruited T cells and bring them into contact with tumor cells, further promoting tumor cell lysis. The second major function of B cells is to present foreign proteins to T cells as MHC-II fragments, forming immune cell clusters, leading to their activation and further recruitment of various anti-tumor immune cells. Finally, B cells play a key role in immunological memory, with CASS B cells providing a lifelong anti-tumor surveillance network that protects patients from metastasis and recurrence after the initial tumor has been removed.

[0321] While the CASS B-cell platform is applicable to many cancers, we use high-grade serous ovarian cancer (HGSOC) as an experimental model. HGSOC is the most common and deadly form of ovarian cancer, but it was chosen because few new treatments have been developed and overall survival rates remain poor.

[0322] Aim 1 will first explore the engineering of CASS B cells by constructing a synthetic B cell receptor that recognizes Muc1. Upon binding to MUC1-expressing tumor cells, the CASS B cells inducibly secrete a trispecific T cell-binding antibody (CD3xCD28xMSLN) that recognizes a second tumor-associated mesothelin (MSLN), further activating the T cells and participating in tumor cell recognition.

[0323] Aim 2 will use 2D and 3D tissue culture experiments to validate the CASS B cell engineering and demonstrate both the efficacy of the CASS B cells and the ability of the trispecific antibody to stimulate and bind to T cells.

[0324] Goal 3 is to validate the efficacy of the CASS B cell platform in vivo using humanized mice, enabling detailed molecular characterization of antitumor immune responses. In an embodiment, HGSOC can be tested. Without wishing to be bound by theory, CASS B cells are a pioneering platform with the potential to provide cures for a variety of cancers.

[0325] Immunomodulatory antibodies (Abs) and adoptive cell therapy have had a transformative effect on cancer treatment, shifting the focus from killing tumor cells to activating patients' natural antitumor immunity and reversing the immunosuppressive tumor microenvironment (TME). These are some of the most promising anticancer therapeutics to date, but often show limited efficacy in patients with high-grade serous ovarian cancer (HGSOC). 1-3 This, in part, results in immunologically "cold" tumors with minimal tumor-infiltrating lymphocytes (TILs). 4,5 This is due to the low mutational burden and limited neoantigen production observed in HGSOC tumors. Immune cells arriving at the tumor site encounter numerous immunosuppressive cells, including myeloid-derived suppressor cells, regulatory T cells, and tumor-associated M2 macrophages. 6,7 To address this, embodiments herein include a novel and innovative cellular immunotherapy platform: chimeric antibody-secreting and signaling (CASS) B cells.

[0326] As natural antigen-presenting cells (APCs), CASS B cells utilize their ability to present antigens on MHC class II molecules to facilitate tumor cell recognition and neoantigen dissemination, while also relying on their role in the formation and function of tertiary lymphoid structures (TLS) to secrete chemokines that recruit various immune cell subsets to HGSOC tumor sites. 8,9To further improve tumor recognition and engagement by recruited T cells, we engineered CASS B cells to express modified tumor-associated antigen (TAA)-targeting B cell receptors (BCRs), which, upon activation, induce the expression of anti-CD3xCD28xTAA trispecific T cell-binding antibodies locally at the tumor site. In this study, mucin-1 (Muc1) and mesothelin (MSLN) were chosen for targeting of CASS B cells and T cell engagers, respectively. 10-15 The combination of anti-CD3xCD28 on a trispecific antibody allows efficient stimulation of both CD4 and CD8 T cells, and the anti-MSLN domain targets activated T cells to tumors and promotes potent tumor cell lysis. 16 The trispecific antibody developed here is designed to enhance T cell-mediated cytotoxicity but does not directly inhibit immunosuppressive signals generated in the TME. To block immunosuppression and further enhance the efficacy of both CASS B cells and recruited T cells, we combined CASS B cells with systemic delivery of an anti-PD1 antibody. 17,18 The CASS B cell platform is unique in the field of cell therapy because CASS B cells have no intrinsic cytotoxicity of their own and have the ability to both mount a robust anti-tumor response and enhance the activity of recruited immune cells.

[0327] Described herein are anti-Muc1-targeting CASS B cells that secrete anti-CD3xCD28xMSLN T cell-binding Abs against HGSOC. The modular design of CASS B cells allows them to be easily targeted with other TAAs, and the secreted payload can be tailored to target relevant immune axes, making CASS B cells adaptable to a wide variety of cancers. Because B cells are long-lived and an important part of immune memory, CASS B cells continuously deliver therapeutic payloads in tumors and provide a lifelong immune surveillance system against metastasis and recurrence after the primary tumor is eradicated.

[0328] Without wishing to be bound by theory, local tumor activation of CASS B cells remodels the HGSOC tumor microenvironment through their natural antigen-presenting capacity, while the formation of tertiary lymphoid structures (TLS) leads to the recruitment and activation of a broad subset of immune cells. Furthermore, CASS B cells engineered to secrete anti-CD3xCD28xMSLN trispecific T cell-binding antibodies further restore antitumor immunity, enhancing T cell activation, tumor recognition, and cytotoxic activity.

[0329] The inventors have developed a human phage library consisting of 27 billion species. 19,20,22,23,25 was used to identify a set of anti-MSLN scFvs (Fig. 1, top) and Mucl scFvs (Fig. 1, bottom).

[0330] Our engineered IgG-BCR is constructed using an scFv fused to a membrane-bound IgG1 hinge-Fc that is expressed at high levels and binds to the target antigen (Figure 2, panel A). 26 Transduction experiments using primary B cells demonstrate high-titer transduction for multiple donors and DNA constructs (Figure 2, panel B). We developed an inducible T cell activation assay utilizing the NFAT / NFκB response element (RE) (Figure 2, panel C). A fluorescent protein is used instead of a secreted trispecific T cell engager antibody.

[0331] non-restrictive goals

[0332] Non-limiting Objective 1: Engineering and optimization of CASS B cell constructs and trispecific Ab payloads - Select appropriate Abs (anti-MSLN, Mucl, CD3, CD28) for development of CASS B cells, followed by optimization of the signaling domain and inducible response element (RE).

[0333] Non-limiting objective 2: In vitro evaluation of CASS B and trispecific T cell engagers - In vitro characterization will be performed to identify activation thresholds and quantify Ab secretion levels. 3D organoid cultures will be used for detailed molecular and functional characterization of both CASS B cells and T cell-binding payloads.

[0334] Non-limiting objective 3: In vivo efficacy using an HLA-matched humanized HGSOC mouse model - Final evaluation of the optimized CASS B cell constructs, with or without systemic anti-PD1 combination therapy, will be tested in vivo using a cell line-derived xenograft (CDX) model in humanized mice.

[0335] Non-limiting Objective 1: Engineering and optimization of CASS B cell constructs and trispecific Ab payloads:

[0336] Non-limiting Objective 1A: Antibody discovery and engineering of a trispecific T cell engager - In this sub-objective, we will develop four antibodies: anti-Muc1 for IgG-BCR targeting, and anti-CD3, CD28, and MSLN for a trispecific T cell engager. Affinity tuning and epitope mapping will be performed to further characterize these antibodies.

[0337] Non-limiting Objective 1B: Design and Engineering of CASS B Cell Vectors - Using engineered IgG-BCR targeting constructs and NFAT / NFκB inducible reporter systems, we will design CASS B cell lentiviral vectors. We will validate the ability of IgG-BCR to induce fluorescent protein expression via engineered transcription factor response elements (REs), including various NFAT and NFκB formats with minimal promoters. 27-30 .

[0338] Non-limiting Objective 1C: Optimization of primary B cell transduction and exponential expansion - Because primary B cells require a range of cytokines and stimulatory signals, we further engineered the 3T3-msCD40L feeder cell line to express IL-2, 15, and 21 to support maximal B cell expansion. 31,32 We validate an optimized protocol for transduction and exponential expansion of primary B cells. Lentiviral transduction of primary B cells is optimized by testing different envelope proteins (VSVG, BaEV, GaLV) for lentiviral packaging and promoters driving IgG-BCR expression (Ef1α, SFFV). 33-36 .

[0339] Non-limiting objective 2: In vitro validation of CASS B cell and trispecific T cell engagers

[0340] Non-limiting Objective 2A: In vitro functional validation of anti-Muc1 CASS B cells - CASS B cell constructs will be assembled using the trispecific T cell engager developed in Non-limiting Objective 1. Characterization will be performed using soluble cross-linked Muc1 to determine the activation threshold of CASS B cells and quantify the level of trispecific Ab expression.

[0341] Non-limiting objective 2B: In vitro efficacy of CASS B cells in 3D culture utilizing HGSOC tumor spheroids - Muc1 / MSLN-positive HGSOC cell lines will be implanted into HLA-matched humanized mice. Tumors harvested from these mice will be used with microfluidic chips to generate 3D organoid models of HGSOC in vitro. 37-40 This allows us to validate CASS B cell homing to tumor spheroids, and cytokine analysis of the TME allows detailed validation of the efficacy of the T cell-binding payload. For detailed molecular and mechanistic investigation of CASS B cell and T cell engager function and efficacy, we use single-cell RNA sequencing and multiparameter FACS.

[0342] Non-limiting objective 3: In vivo efficacy using an HLA-matched humanized HGSOC mouse model

[0343] Non-limiting Objective 3A: Validation of anti-tumor efficacy and persistence of anti-Muc1 CASS B cells in humanized NSG-SGM3 mice bearing CDX tumors - HLA-matched humanized NSG-SGM3 mice will be used to generate HGSOC models using Muc1 / MSLN-positive HGSOC cell lines. These models will then be used to validate the efficacy of both the trispecific T cell engager and whole CASS B cells, and various advanced analytical techniques (IHC, multiparameter FACS, scRNAseq) will be used to further investigate the effects of CASS B cell therapy not only on tumors but also on the entire immune system.

[0344] Non-limiting Objective 3B: Validate the efficacy of CASS B cells in combination with systemic anti-PD1 therapy - Using the HGSOC CDX model described in Non-limiting Objective 3A, test the efficacy of CASS B cells in combination with anti-PD1 therapy. Molecular signatures and phenotypic analysis of immune cell depletion markers will be assessed and compared to those identified in Non-limiting Objective 3A.

[0345] Statistical Considerations: With five animals per group for individual pairwise comparisons between conditions of interest and different outcome measures, the power to detect a mean difference equal to 2.5 SD using a two-sample, two-tailed t-test at the 0.05 level is 0.93. In practice, a linear mixed-effects model approach is applied. The Bioconductor R package flowWorkspace processes flow data, and the R package flowFlowJo is used for visualization. Relationships between expression are examined using Spearman correlation and mutual information. The Moran index is used to quantify spatial autocorrelation of proteins on RNAscope ISH images, and Gabriel graphs are used to evaluate spatial patterns correlated with response. Multivariate spatial associations between proteins are assessed using simple Moran eigenvector maps and spatial principal component analysis. Additional statistical analyses are provided in consultation with the Dana-Farber Biostatistics Core.

[0346] In embodiments, further manipulation can be performed by modifying or substituting the IgG-BCR signaling domain. Given our experience with humanized mice, we generate cohorts assuming a 20% dropout rate due to animal death or lack of CD34 engraftment. If adequate humanization by CD34 engraftment cannot be achieved, a potential alternative is humanization by hPBMC injection. The CDX model allows for easy construction of our models for both in vitro and in vivo testing.

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[0380] 33.Girard-Gagnepain,A.et al.Baboon envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early-cytokine-stimulated and resting HSCs.Blood 124,1221-1231(2014).

[0381] 34.Tomas,H.A.et al.Improved GaLV-TR Glycoproteins to Pseudotype Lentiviral Vectors:Impact of Viral Protease Activity in the Production of LV Pseudotypes.Mol.Ther.- Methods Clin.Dev.15,1-8(2019).

[0382] 35.Bernadin,O.et al.Baboon envelope LVs efficiently transduced human adult,fetal,and progenitor T cells and corrected SCID-X1 T-cell deficiency.Blood Adv.3,461-475(2019).

[0383] 36.Winiarska,M.et al.Selection of an optimal promoter for gene transfer in normal B cells.Mol.Med.Rep.16,3041-3048(2017).

[0384] 37.Jenkins,R.W.et al.Ex vivo profiling of PD-1 blockade using organotypic tumor spheroids.Cancer Discov.8,196-215(2018).

[0385] 38.Aref,A.R.et al.3D microfluidic:Ex vivo culture of organotypic tumor spheroids to model immune checkpoint blockade.Lab Chip 18,3129-3143(2018).

[0386] 39. Wan, C. et al. Enhanced Efficacy of Simultaneous PD-1 and PD-L1 Immune Checkpoint Blockade in High-Grade Serious Ovarian Cancer. Cancer Res. 81, 158-173 (2021).

[0387] 40.Odunsi,A.et al.Fidelity of human ovarian cancer patient-derived xenografts in a partially humanized mouse model for preclinical testing of immunotherapies.J.Immunother.Cancer 8,1-13(2020).

[0388] Example 3 Engineering chimeric antibody signaling and secreting (CASS) B cells to achieve cancer cures

[0389] Cancer cells are organisms that have learned to hijack our immune system to favor their own growth. They do this through the impairment of our cellular DNA repair mechanisms, resulting in the upregulation of growth factors and their receptors, leading to uncontrolled tumor growth. This molecular hijacking also leads to the surface expression and secretion of molecules involved in immune checkpoint blockade (ICB). Immunotherapy via anti-PD1 / PDL1 blockade represents a significant advance in the cancer field and is a frontline or standard treatment option for various cancers, including non-small cell lung cancer, melanoma, colorectal cancer, and renal cell carcinoma (1-3). However, while treatment success is well documented, it rarely leads to a cure. Better methods are needed.

[0390] Cell therapy is a method of harnessing the immune system to kill cancer cells. For example, T cell receptor (TCR) and chimeric antigen receptor (CAR) T cells can be directed to and targeted to cancer cells.

[0391] Additionally, embodiments herein include a new form of cell therapy called chimeric antibody signaling and secreting (CASS) B cells, which harness the B cells of the humoral immune system to secrete high levels of their most specialized antibodies. These CASS B cells are engineered to recognize tumor-associated antigens (TAAs) via engineered B cell receptors (BCRs). Upon TAA binding, these cells activate and induce the production of high levels of immunomodulatory bispecific antibodies (BsAbs) at the tumor site. This allows for the reversal of the immunosuppressive tumor microenvironment, similar to the systemic delivery of immune checkpoint-blocking monoclonal antibodies (mAbs). BsAb secretion can be conditionally expressed only upon TAA binding by CASS B cells and is primarily localized at the tumor site. Without wishing to be bound by theory, this represents a step toward a new cell therapy in which locally secreted monoclonal antibodies are dedicated to altering the tumor microenvironment and restoring local anti-tumor immunity.

[0392] Monoclonal antibody (mAb) drugs, which directly kill cancer cells, act as immune checkpoint blockade inhibitors, or disrupt tumor vasculature, are among the most promising anti-cancer therapeutics in development. However, the idea of ​​engineering human B cells to seek out cancer cells and secrete these mAbs at tumor sites in vivo is novel and could offer a powerful new approach to treating both primary and metastatic tumors. It could also provide a lifelong anti-tumor immune surveillance system to prevent cancer recurrence and achieve a "cure." The role of B cells in the immune system is to recognize foreign invaders, whether microorganisms or cancer cells, and eliminate them by producing antibodies that bind to and eliminate the threat. They achieve this by expressing membrane-bound antibodies that act as B cell receptors (BCRs) that bind to tumor antigens, switching B cells from BCR-expressing to antibody-secreting cells. After BCR binding, rapid tyrosine phosphorylation occurs, mediated by two primary tyrosine kinases, Lyn and Syn, and calcium ion polarization. These biochemical events lead to the activation of downstream signaling pathways, resulting in further downstream activation of NF-κB and NFAT signaling, and ultimately B cell proliferation and potent mAb secretion. (5) We exploit these signaling pathways to engineer chimeric antibody signaling and secreting (CASS) B cells that induce clonal CASS B cell proliferation and secretion of immunomodulatory BsAbs at tumor sites.

[0393] Without wishing to be bound by theory, the development of the CASS B cell platform could benefit numerous clinical indications that are sensitive to checkpoint blockade inhibitors or have an immunosuppressive microenvironment. Anti-PD1 / PDL1 therapy has had a transformative effect on the treatment of NSCLC, becoming the frontline therapy for many patients. However, this treatment has limitations in efficacy, durability, and scope of use. (6-7) We use NSCLC as an experimental model utilizing an anti-PD1 / anti-TIGIT bispecific antibody under development in our laboratory.

[0394] An engineered membrane-bound single-chain IgG BCR against mesothelin (MSLN) is used to target CASS B cells to NSCLC tumor sites. This plasmid also contains a second cassette driven by an NFAT or NF-κB response element that drives secretion of an anti-TIGIT / anti-PD1 BsAb. Upon tumor localization and BCR activation, BsAb expression is induced by the native signaling pathway upon BCR engagement of MSLN in NSCLC. Because this is an inducible expression system, there are localized areas of high antibody concentration at the tumor site, significantly reducing on-target, off-tumor effects.

[0395] CASS B cells are engineered in two parts. We successfully transduce B cells with an engineered BCR. To efficiently transduce B cells, lentiviral particles are pseudotyped with gibbon leukemia virus (GALV) or engineered baboon envelope glycoproteins, and in the transfer vector, BCR expression is driven from the spleen focus-forming virus (SFFV) or human elongation factor-1 alpha (EF1α) promoter (8). Next, we validate NFAT and / or NF-κB-inducible expression cassettes for BsAb secretion (9). To determine which response element to use, we first engineered a plasmid to express GFP. Raji cells and primary B cells were transduced with the engineered anti-MSLN BCR and the NFAT / NF-κB-inducible GFP lentiviral vector, and soluble biotinylated MSLN was added to the culture medium containing streptavidin to crosslink the BCR. GFP expression was then measured to determine the optimal response element. We construct membrane-bound IgG (memIgG) engineered with anti-influenza antibodies. Figure 3 shows that our memIgG construct is expressed at high levels and is functionally active, as it can bind soluble HA.

[0396] Next, CASS B cells were constructed by lentiviral transduction with a vector encoding a tumor-specific anti-MSLN BCR and an inducible BsAb replacing GFP. CASS B cell secretion of anti-TIGIT / PD1 BsAb was quantified by first inducing expression using soluble biotinylated MSLN streptavidin and then co-incubating with MSLN A549 NSCLC cells. In a secondary experiment, CASS B cells were tested for in vitro inhibition of depletion by co-culturing with CD3 T cells and A549 cells expressing various combinations of PDL1 and CD155 (the TIGIT ligand). In addition to measuring soluble antibody concentrations, cell activation / depletion was measured by FACS staining. These tumor cells were also stained for binding of anti-MSLN, which inhibits CASS B cell shedding. Next, in vivo experiments were performed in humanized PBL NSG-SGM3 mice bearing A549 tumors to verify the efficacy and persistence of CASS B cells. Efficacy will be measured by local BsAb secretion by immunohistochemical staining, an increase in the B cell population around the tumor site, and changes in tumor size. We will also measure BsAb leakage into the periphery by examining serum secreted BsAb concentrations. To measure persistence, CASS B cells will be detected in both the peripheral blood and the tumor microenvironment (TME). Finally, scRNA sequencing will be used to analyze the effect of BsAb on modulating the TME. Compared to systemic delivery, there will be localized regions of high BsAb concentrations centered around the tumor, leading to improved outcomes and tumor elimination.

[0397] The goal of developing new cancer treatments can be to achieve a "cure." Restoring host anti-tumor immunity at the tumor site can be achieved by understanding the immune elements common or "public" in an individual. Once this is achieved, it is necessary to develop a permanent internal immune surveillance system to prevent cancer recurrence. The inventors have developed a new immune surveillance system using B cells from the immune system that has not been developed for this purpose before.

[0398] We demonstrate the feasibility of engineering chimeric antibody signaling and secreting (CASS) B cells to target mesothelin (MSLN)-expressing NSCLC cells. These anti-MSLN CASS B cells are introduced into the tumor site, where they secrete high levels of a bispecific anti-TIGIT / anti-PDL1 antibody that acts as a dual checkpoint blockade inhibitor. This results in a dynamic shift in the tumor microenvironment that helps reverse T cell exhaustion and restore antitumor immunity. Importantly and practically, this combination cellular immunotherapy requires a one-time administration for the patient's lifetime.

[0399] References cited in this example:

[0400] 1.Rolfo C,Caglevic C,Santarpia M,Araujo A,Giovannetti E,Gallardo CD,Pauwels P,Mahave M.Immunotherapy in NSCLC:A Promising and Revolutionary Weapon.Adv Exp Med Biol.2017;995:97-125.doi:10.1007 / 978-3-319-53156-4_5.PMID:28321814 Review.

[0401] 2. M.-O. Grimm, K. Leucht, V. Gruenwald, S. Foller, New First Line Treatment Options of Clear Cell Renal Cell Cancer Patients with PD-1 or PD-L1 Immune-Checkpoint Inhibitor-Based Combination Therapies. J. Clin. Med. 9, 565 (2020).

[0402] 3.X.Wu,et al.,Application of PD-1 Blockade in Cancer Immunotherapy.Comput.Struct.Biotechnol.J.17,661-674(2019).

[0403] 4.Ryeong Lee B,Sehyun C,Moon J,Joon Kim M,Lee H,Wan Ko H,Chul Cho B,Sup Shim H,Hwang D,Ryun Kim H,and Ha S-J.Combination of PD-L1 and PVR determines sensitivity to PD-1 blockade.JCIinsite 2020;5(14):e128633.https: / / doi.org / 10.1172 / jci.insight.128633.

[0404] 5.Tolar P,Hanna J,Krueger PD,Pierce SK.The constant region of the membrane immunoglobulin mediates B cell-receptor clustering and signaling in response to membrane antigens.Immunity.2009;30(1):44-55.Epub 2009 / 01 / 13.doi:10.1016 / j.immuni.2008.11.007.PubMed PMID:19135393;PMCID:PMC2656684.45.

[0405] 6.Bendell JC,Bedard P,Bang YJ,et al:Phase Ia / Ib dose-escalation study of the anti-TIGIT antibody tiragolumab as a single agent and in combination with atezolizumab in patients with advanced solid tumors.2020 AACR Virtual Annual Meeting II.Abstract CT302.

[0406] 7.M.-J.Ahn,et al.,1400P Vibostolimab,an anti-TIGIT antibody,as monotherapy and in combination with pembrolizumab in anti-PD-1 / PD-L1-refractory NSCLC.Ann.Oncol.31,S887(2020).

[0407] 8.Levy C,Fusil F,Amirache F,Costa C,Girard-Gagnepain A,Negre D,Bernadin O,Garaulet G,Rodriguez A,Nair N,Vandendriessche T,Chuah M,Cosset FL,Verhoeyen E.Baboon envelope pseudotyped lentiviral vectors efficiently transduce human B cells and allow active factor IX B cell secretion in vivo in NOD / SCIDgammac(- / -) mice.J Thromb Haemost.2016;14(12):2478-92.Epub 2016 / 09 / 30.doi:10.1111 / jth.13520.PubMed PMID:27685947.

[0408] 9.Uchibori R,Teruya T,Ido H,Ohmine K,Sehara Y,Urabe M,Mizukami H,Mineno J,Ozawa K.Functional Analysis of an Inducible Promoter Driven by Activation Signals from a Chimeric Antigen Receptor.Mol Ther Oncolytics.2019;12:16-25.Epub 2019 / 01 / 22.doi:10.1016 / j.omto.2018.11.003.PubMed PMID:30662937;PMCID:PMC6325072.

[0409] Example 4 Engineering chimeric antibody signaling and secreting (CASS) B cells to achieve cancer cures

[0410] Immune checkpoint blockade inhibitors (CBIs) and CAR T cells have revolutionized the way cancer is treated. While both of these therapies engage the patient's immune system, neither is able to actively mount an anti-tumor immune response, significantly limiting their scope and efficacy. To address this, we developed chimeric antibody-secreting and signaling (CASS) B cells that express an engineered tumor-targeting B cell receptor and, upon binding, secrete high levels of dual-targeting, bispecific CBI antibodies locally at the tumor site. Because B cells function as professional antigen-presenting cells, they can process and present antigens on MHC class II molecules, further enhancing immune cell recognition of tumors and aiding in the spread of neoantigens. CASS B cells, a key component of immune memory, simultaneously recruit a broad range of immune cells, providing a surveillance program that reverses tumor-infiltrating lymphocyte depletion and protects against tumor metastasis and recurrence.

[0411] Non-small cell lung cancer (NSCLC) was selected as a model for developing anti-MSLN-directed CASS B cells secreting an immunomodulatory anti-PD1 / TIGIT bispecific antibody (bsAb). Objective 1 focuses on the development of the CASS B cell platform. At the end of this phase, we will identify three antibodies and optimize the signaling domains that comprise the CASS B cells. Objective 2 involves in vitro characterization and efficacy testing, providing a clear understanding of the relationship between CASS B cell activation and bsAb secretion while providing a critical analysis of the efficacy of CASS B cells compared with CAR T cells at both the functional and molecular levels. Objective 3 involves in vivo experiments using cell line-derived and patient-derived NSCLC models in humanized mice. Multiparameter flow cytometry, single-cell RNA sequencing, and immunohistochemistry will provide a detailed assessment of the molecular and mechanistic efficacy of the immunomodulatory bsAb and CASS B cell platform.

[0412] We have developed a novel type of combination cellular immunotherapy, chimeric antibody-secreting signaling (CASS) B cells. These B cells express an engineered tumor-associated antigen (TAA) that targets the B cell receptor (BCR), and upon binding, secrete high levels of checkpoint blockade inhibitor (CBI) bispecific antibodies (bsAb) locally at the tumor site. This allows for the reversal of the immunosuppressive tumor microenvironment (TME), restoration of the patient's natural innate and adaptive immune responses, and elimination of cancerous cells. In addition, unlike T cells, B cells can act as professional antigen-presenting (APC) cells, enhancing tumor cell recognition and assisting in the dissemination of neoantigens, thereby both reversing tumor-infiltrating lymphocyte (TIL) depletion and inducing broader and more robust antitumor immune responses. We engineered anti-PD1 / TIGIT bsAb-secreting CASS B cells targeting mesothelin (MSLN) for the treatment of NSCLC. 1-3 .

[0413] The first objective involves the construction and optimization of an engineered anti-MSLN IgG-BCR, an anti-PD1 / TIGIT bsAb delivered at the tumor site, and an inducible response element (RE) to drive bsAb expression. A panel of anti-MSLN, anti-PD1, and anti-TIGIT antibodies will be identified in our laboratory, and lead candidates will be identified through functional assays. Parallel efforts will focus on the development of inducible response elements and optimization of B cell transduction conditions.

[0414] The secondary objective is to functionally validate the anti-MSLN CASS B cells in vitro. Activation assays will be used to quantify bsAb and cytokine secretion levels. Patient-derived organotypic tumor spheroids (PDOTS) will be used to validate the efficacy of CASS B cells and perform molecular / mechanistic comparisons with CAR T cells by cytokine profiling, IHC, and single-cell RNA sequencing (scRNAseq).

[0415] Another goal is to generate cell line-derived (CDX) and patient-derived xenograft (PDX) models of NSCLC using HLA-matched humanized mice to test the efficacy of CASS B cells. These models will be combined with various analytical techniques (IHC, flow cytometry, scRNAseq) to further investigate the effects of CASS B cell therapy on the surrounding TME.

[0416] BACKGROUND: Checkpoint blockade inhibitors (CBIs), monoclonal antibodies (mAbs) and adoptive cell therapy have had a transformative effect on cancer treatment, shifting the focus from killing tumor cells to activating patients' natural antitumor immunity and reversing the immunosuppressive tumor microenvironment (TME). These are some of the most promising anticancer therapeutics to date, but only a small subset of patients experience a complete or durable response, and many patients experience immune-related adverse events (irAEs) of varying severity. 4-7 To combat this, combination CBI therapies such as anti-PD(L)1 / anti-TIGIT are being tested and have demonstrated significant promise in clinical trials. 8-11 An alternative approach is the development of armored or immune-restorative CAR T cells engineered to secrete immunomodulatory payloads directly at the tumor site, increasing efficacy while reducing the on-target / off-tumor side effects seen with systemic delivery. 12,13 .

[0417] In addition to T cells, various immune cells, including natural killer cells (NK-CARs) and macrophages (CAR-Ms), have been utilized to generate novel CARs, and what unites these cells is that they provide direct antitumor activity. 14,15 B cells, a key component of humoral immunity, produce the antibodies upon which the field of immunotherapy first developed. However, they lack intrinsic cytotoxic capacity and have therefore been largely excluded from these advances.

[0418] Without wishing to be bound by theory, the chimeric antibody-secreting signaling (CASS) B-cell platform is a unique B-cell-based cellular therapy that does not rely on direct cytotoxicity but instead utilizes two inherent capabilities of B cells: the ability to secrete high levels of CBI antibodies to reverse the immunosuppressive TME, and the ability to process and present antigens on MHC class II molecules, thereby recruiting CD4+ T cells and enabling enhanced tumor cell recognition and neoantigen dissemination. While inducible, targeted delivery of CBIs reduces irAEs, CASS B cells have the ability to mount robust antitumor responses, making their ability to function as professional APCs unique in the field of cell therapy. This impact is further exemplified by MHC class II neoantigens, which play a key role in innate antitumor responses. 16,17 .

[0419] Described herein are MSLN+-targeted CASS B cells secreting anti-PD1 / TIGIT bsAbs against NSCLC. The modular design of CASS B cells allows for easy targeting of other TAAs, and the secreted payload can be tailored to target relevant immune axes, making CASS B cells adaptable to a wide variety of cancers. Because B cells are long-lived and an important part of immune memory, CASS B cells continuously deliver therapeutic payloads in tumors, providing a lifelong immune surveillance system against metastasis and recurrence after the primary tumor is eradicated. Without wishing to be bound by theory, the long-term persistence and inducibility of the CASS B cell platform could be adapted for other diseases treated with biologic therapies, such as cardiovascular and autoimmune diseases and neurological disorders, which may require long-term disease maintenance and rapid therapeutic administration to treat acute symptoms.

[0420] The lab has been working to develop armored and immune-restorative CAR T cells, including those secreting cytokines, CBI mAbs, and bsAbs (BiTEs), with the goal of enhancing CAR T efficacy by remodeling the local TME. 12,13 Studies using inducible or constitutively expressed payloads have demonstrated that a) binding of bystander immune cells and recruitment of other anti-cancer cell types significantly enhances therapeutic efficacy, and b) proteins secreted at the tumor site remain at high concentrations in the peritumoral area with minimal transfer to serum. 18~20 .

[0421] B cell engineering is a more recent achievement and has primarily focused on generating B cells that secrete neutralizing anti-pathogenic antibodies against RSV and HIV. 21-23 Unlike the cancer therapeutics described herein, these described B cells focus on the systemic production of antibodies that neutralize viral infections and utilize CRISPR / Cas9 to insert recombinant mAbs into the Ig locus of B cells. This has the added benefit of allowing the antibody to continue affinity maturation, a requirement for combating infectious diseases but not for targeting immune markers. Without wishing to be bound by theory, the inventors engineered B cells to express engineered antibodies that are inducibly selected, and upon activation, these modified B cells differentiate not only into Ab-secreting cells but also into memory B cells that provide long-term protection. 24 .

[0422] Non-limiting illustrative research designs

[0423] Goal 1: Engineering and optimization of CASS B cell constructs - Select appropriate scFvs (anti-MSLN, PD1, TIGIT) for development of CASS B cells, followed by optimization of signaling domains and inducible response elements 25-28 Protocols for highly efficient transduction and exponential expansion of primary B cells have also been optimized utilizing various lentiviral envelope proteins and culture conditions. 29-32 .

[0424] Experimental design and procedures

[0425] Antibody discovery, engineering, and optimization: We constructed a human phage library consisting of 27 billion species that has been used to isolate numerous therapeutic antibodies. 33-37 A preliminary set of anti-PD1, TIGIT, and MSLN antibodies (Figure 33) were identified for further manipulation and optimization.

[0426] Designing and engineering bispecific antibodies: PD1 / TIGIT are expressed on immune cells, so immune depletion is undesirable 38,39 The current design utilizes a previously developed and characterized tandem scFv construct, although other designs are also considered.

[0427] B cell isolation, expansion, and transduction: Isolate B cells and test various expansion media formulations 23,40,41 For transduction, B cells are activated, transduced, and sorted 72 hours after transduction.

[0428] Generation of IgG-BCR constructs: Our engineered IgG-BCRs are constructed using scFvs fused to membrane-bound IgG1 hinge-Fc that express at high levels and bind to target antigens. 42 Initial transduction experiments using primary B cells demonstrate high-titer transduction for multiple donors and DNA constructs. We previously developed an inducible T cell activation assay utilizing the NFAT / NFκB RE. In optimization experiments, a fluorescent protein will be used in place of the secreted bsAb.

[0429] Without wishing to be bound by theory, there is no problem with mAb discovery or CASS B cell engineering. The mAb engineering technology employed in our laboratory allows us to develop a panel of previously discovered mAbs to find lead candidates for each target. Without wishing to be bound by theory, we are able to achieve high transduction efficiency and exponential growth of transduced cells for in vivo experiments.

[0430] A lead antibody can be identified for each target, and then vectors can be constructed to successfully transduce / expand CASS B cells.

[0431] Goal 2: In vivo testing and efficacy of CASS B cells - Detailed in vitro characterization will be performed to identify activation thresholds and quantify bsAb secretion levels. Final in vitro assays will be performed using patient-derived organotypic tumor spheroids (PDOTS) to observe CASS B cell homing and perform detailed analysis of bsAb payload.

[0432] Non-limiting exemplary experimental designs and procedures

[0433] CASS B cell activation assay: Supernatants from activated CASS B cells are screened by ELISA to measure bsAb and cytokine concentrations.

[0434] Generation and evaluation of NSCLC PDOTs: Generation of NSCLC PDOTs is performed according to the protocols outlined in Jenkins et al. and Aref et al. 43,44In addition to testing the efficacy of the bsAb and CASS B cells, comparative experiments will be performed to validate therapeutic differences between CASS B cell and CAR T cell treatment. Immune profiling by IHC, scRNAseq, and cytokine profiling will be performed. Due to the limited availability of NSCLC tumor tissue, a backup plan has been devised to use mesothelioma tumors for PDOT generation. This is an ideal alternative to in vitro assays, as mesothelioma has a highly suppressive TME and immune infiltrating cells exhibit high levels of exhaustion markers. 45-48 .

[0435] Expected Results: Objective 2 will provide a deeper understanding of CASS B cell activation and validate optimal anti-MSLN scFvs for selective targeting of MSLN+ tumors. PDOTS will provide a comprehensive dataset of CASS B cell efficacy via cytokine secretion and transcriptional profiles, demonstrating that anti-MSLN CASS B cells propagate to tumors and that secreted bsAb reverses immune cell suppression. Furthermore, greater epitope spreading and bystander immune cell activation will be observed with CASS B cells compared to CAR T cell therapy.

[0436] Milestones: The first milestone in Objective 2 is the generation of CASS B cell activation curves and quantification of bsAb secretion. The next milestone is the establishment of PDOTS and efficacy testing of bsAb systemically and as a CASS B cell payload. Another milestone is the comparison of CASS B cell therapy with CAR T cell therapy and the generation of immune profiles of each therapy via IHC and scRNAseq.

[0437] Objective 3: In vivo efficacy using HLA-matched humanized NSCLC mouse models - Conduct in vivo experiments on cell line-derived xenograft (CDX) models in humanized mice. Use of validated patient-derived xenograft (PDX) models from public repositories for final testing will improve tumor integrity and phenotypic characterization while accurately mimicking the TME of in vivo tumors.

[0438] Experimental design and procedures

[0439] Establishment of NSCLC CDX and PDX models in humanized mice: NSCLC cell lines and PDX models are screened for PDL1 and CD155 expression levels before luciferase conversion 49 Reconstitution of the human immune system is performed as described by the present inventors and others. 50-52 To generate growth curves, various concentrations of NSCLC cell line / PDX tumors are implanted into the hind flanks of humanized mice. 53 .

[0440] Assessment of CASS B cell efficacy in humanized NSCLC mouse models: We will validate the efficacy of bsAb and CASS B cells in vivo using CDX and PDX models. Samples will be analyzed by multiparameter FACS, IHC / ISH, and scRNASeq according to established protocols routinely used in our laboratory. 54-63 .

[0441] Without wishing to be bound by theory, successful NSCLC models will be generated. These models demonstrate that CASS B cells cluster around tumors, secrete high levels of bsAb, reduce the immunosuppressive properties of the TME, and recruit additional antitumor immune cells. IHC / ISH and scRNAseq provide molecular evidence of bsAb efficacy, CASS B cell homing, and APC pathway activation in both CASS B cells and CD4 T cells, and 5' scRNAseq of TCR / BCR will be used to directly monitor epitope spreading among TILs.

[0442] Statistical Considerations: With five animals per group for individual pairwise comparisons between conditions of interest and different outcome measures, the power to detect a difference in means equal to 2.5 SD using a two-sample two-tailed t-test at the 0.05 level is 0.93.

[0443] Milestones: The first milestone for Goal 3 is the generation of CDX and PDX models of NSCLC. Subsequent milestones could be the initiation and completion of planned animal studies testing bsAbs in CDX models and CASS B cells in CDX and PDX models. With the wealth of data generated by these studies, the third milestone could be the completion of data analysis from each animal study.

[0444] References cited in this example:

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[0450] 6.Ott,P.A.& Wu,C.J.Cancer vaccines:Steering t cells down the right path to eradicate tumors.Cancer Discov.9,476-481(2019).

[0451] 7.Rogado,J.et al.Immune-related adverse events predict the therapeutic efficacy of anti-PD-1 antibodies in cancer patients.Eur.J.Cancer 109,21-27(2019).

[0452] 8.Lee,D.H.Update of early phase clinical trials in cancer immunotherapy.BMB Rep.54,70-88(2021).

[0453] 9.Ahn,M.-J.et al.1400P Vibostolimab,an anti-TIGIT antibody,as monotherapy and in combination with pembrolizumab in anti-PD-1 / PD-L1-refractory NSCLC.Ann.Oncol.31,S887(2020).

[0454] 10.Niu,J.et al.1410P Safety and efficacy of vibostolimab,an anti-TIGIT antibody,plus pembrolizumab in patients with anti-PD-1 / PD-L1-naive NSCLC.Ann.Oncol.31,S891-S892(2020).

[0455] 11.Rodriguez-Abreu,D.et al.Primary analysis of a randomized,double-blind,phase II study of the anti-TIGIT antibody tiragolumab(tira) plus atezolizumab(atezo) versus placebo plus atezo as first-line(1L) treatment in patients with PD-L1-selected NSCLC(CITYSCAPE).J.Clin.Oncol.38,9503(2020).

[0456] 12.Suarez,E.R.et al.Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model.Oncotarget 7,(2016).

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[0462] 18.Chmielewski,M.,Kopecky,C.,Hombach,A.A.& Abken,H.IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression.Cancer Res.71,5697-5706(2011).

[0463] 19.Rafiq,S.et al.Targeted delivery of a PD-1-blocking scFV by CAR-T cells enhances anti-tumor efficacy in vivo.Nat.Biotechnol.36,847-858(2018).

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[0465] 21.Voss,J.E.et al.Reprogramming the antigen specificity of b cells using genome-editing technologies.Elife 8,1-22(2019).

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[0468] 24.Nahmad,A.D.et al.Engineered B cells expressing an anti-HIV antibody enable memory retention,isotype switching and clonal expansion.Nat.Commun.11,1-10(2020).

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[0508] Example 5 Designing and testing a dual-targeted fine-tuned immune recovery (DFIR) CAR T-cell therapy to achieve a cure for high-grade serous ovarian cancer

[0509] Ovarian cancer (OvCA) is the fifth leading cause of cancer death among women in the United States. Globally, 200,000 new cases are diagnosed each year, and 120,000–130,000 women die from the disease. 1 Due to the lack of specific symptoms or signs and reliable screening for early detection, the majority of women (60–65%) with OvCA are diagnosed at a late stage, when the cancer has spread beyond the borders of the ovaries. Currently, the standard treatment for OvCA is surgical intervention followed by platinum-containing chemotherapy, such as carboplatin plus paclitaxel. 2,3 Treatment for stage III or IV disease is rarely curative, with a 5-year survival rate of less than 20%. No effective treatments are available for recurrent or metastatic disease that fails first-line therapy. 4 Furthermore, several recent clinical trials of anti-PD(L)1 immune checkpoint inhibitors (ICIs), with and without chemotherapy, have shown low response rates. 5 More effective therapies are urgently needed.

[0510] Chimeric antigen receptor (CAR) T-cell therapy is a new type of "living drug." CARs contain a single-chain variable antibody fragment (scFv) linked to an intracellular signaling blocker containing a CD3-ζ(z) activation domain (first generation), a CD28 or 41BB costimulatory domain (second generation), or both a CD3-ζ and costimulatory domain (third generation). With FDA approval of second-generation CAR T-cell therapy, this new type of cellular immunotherapy has become a mainstream cancer treatment for hematological malignancies. However, these results have not been readily applicable to solid tumors. Indeed, several major hurdles have been recognized that limit the ability to achieve a "cure" for solid tumors using CAR T cells. These include on-target and off-tumor toxicity due to shared tumor-associated antigen (TAA) epitopes on normal tissues, tumor heterogeneity, and an immunosuppressive tumor microenvironment (TME). To address these issues, we constructed dual-targeted fine-tuned immune recovery (DFIR) CAR-T cells for the treatment of solid tumors with an emphasis on improving antitumor efficacy and patient safety.

[0511] This DoD Ovarian Cancer (OvCA) Research Program pilot award will test DFIR CAR-T therapy for high-grade serous ovarian cancer (HGSOC). Specifically, we engineer an anti-MUC1 / anti-MSLN dual-targeting CAR, which increases efficacy by enabling CAR-T activation in the presence of mucin 1 (MUC1) or mesothelin (MSLN), helping to account for solid tumor heterogeneity and mitigate single-antigen escape. 12,13 Because many antigens used to target epithelial tumors are also present at low density on healthy tissue, establishing an improved safety profile for CAR-T cells is important. Improved safety is addressed by fine-tuning the affinity of the scFv targeting moiety to recognize only high-density tumor TAAs and not the same antigens expressed at physiological levels on normal tissue (Figure 34). Immune recovery occurs through local secretion of a bispecific antibody (BsAb) at the tumor site, which functions as a potent checkpoint blockade inhibitor (ICI) and inhibitor of regulatory T cell (Treg) suppression and recruitment. 14 In this project, the first arm of the BsAb is directed against TIGIT, which has been shown to be overexpressed on tumor antigen-specific (TA-specific) CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs) from individuals with HGSOC. The second arm of the BsAb targets CCR4, which is overexpressed on regulatory T cells (Tregs) and is increased within the tumor-infiltrating lymphocyte population in OvCA. 15-18

[0512] Here, we demonstrate that the antitumor efficacy and safety of CAR-T cell therapy for HGSOC can be significantly improved by dual targeting and fine-tuning the binding affinity of the anti-MUC1 and anti-MSLN CAR targeting moieties. We further demonstrate that CAR-T cells can be engineered to secrete bispecific antibodies (BsAbs) that target two major immunosuppressive pathways operative in HGSOC. The locally secreted BsAbs are directed against TIGIT, present on exhausted T cells, Tregs, and NK cells, and CCR4, which is upregulated on Tregs and plays an important role in chemotaxis. Without wishing to be bound by theory, simultaneous blockade of these two pathways reverses immunosuppression and restores local antitumor immunity.

[0513] Specific goals:

[0514] Objective 1 - Validate the efficacy of OvCA cell killing by anti-MUC1 / MSLN DFIR CAR-T cells constitutively secreting anti-CCR4 / TIGIT BsAb locally at the tumor site using in vitro studies. Subtasks include validation of reversal of T cell depletion and restoration of cytokine secretion.

[0515] DFIR CAR-T cells are designed to have high killing potency of single-antigen-positive OvCA cells (MUC1 or MSLN) and double-positive (MUC1 and MSLN) (we refer to this as "and / or" gating). Fine-tuning of binding affinity is achieved by validating anti-MUC1 and anti-MSLN scFvs of different affinities as a single CAR for their ability to kill only tumor cells that overexpress either of these two antigens, but not healthy cells that may express physiological (lower) levels of these antigens.

[0516] The DFIR CAR-T cell antibody payload has been determined by mining scRNAseq data from publicly available HGSOC patient samples to identify "public" signatures of two non-overlapping dysfunctional pathways in tumor-infiltrating immune cells that can be used for therapeutic intervention.

[0517] Patient-derived organotypic tumor spheroids (PDOTS) are used to evaluate both killing by CAR-T cells alone and in comparison with CAR-T cells secreting antibodies that function as ICIs. The results of these ex vivo 3D cultures using the TME guide the in vivo DFIR CAR-T mouse therapeutic model.

[0518] Antibody engineering techniques allow us to fine-tune the affinity of the MUC1 and MSLN scFv targeting moieties to achieve highly effective tumor cell killing.

[0519] The novel anti-CCR4 / TIGIT BsAb was designed to function as a checkpoint control inhibitor of the TIGIT / CD155 axis on Tregs, cytotoxic T cells, and NK cells, and as an antagonist of Treg chemotaxis toward tumor cells secreting the chemokines CCL17 / CCL22.

[0520] Improved efficacy and safety of CARs: Solid tumors can exhibit phenotypic and genotypic heterogeneity. This is due to inherent characteristics that affect critical pathways such as DNA repair and replication, resulting in uncontrolled tumor growth. Furthermore, immunoediting is observed, resulting in evasion of neutralization, which can lead to the proliferation of single-positive tumor cells. This can result in treatment failure when CAR-T cells are directed against a single target. Without wishing to be bound by theory, dual targeting can provide a gating strategy whereby CAR-T cells can target and kill single-positive tumor cells and / or double-positives, the latter of which exhibit synergistic binding and killing due to increased avidity. 19-21 .

[0521] We addressed the dual targeting problem by isolating a human anti-MUC1 scFv against the membrane-proximal SEA domain. We panned a 27 × 109 human scFv phage display library against recombinant MUC1-SEA polypeptide and identified a lead scFv (T4E3). We also obtained a second anti-MUC1-SEA scFv (3D1) that targets a similar domain on MUC1. The binding affinity of T4E3 scFvFc (5.26) is approximately 10-fold higher than that of 3D1 scFvFc (57.02 nM), providing a good therapeutic range for assessing the effect of binding affinity and MUC1 target density on tumor cell killing. Off-target killing of healthy cells expressing low densities of MUC-1 is demonstrated, as even a low E:T (0.8:1) ratio efficiently killed MUC1+ COV263 cells (bottom) (% survival at 48 hours), while control MUC1- HCT116v cells (top) were not killed.

[0522] A similar discovery campaign was conducted to identify anti-MSLN scFvs. To address the conformational changes MSLN undergoes upon release from the GPI linker, we isolated new anti-MSLN antibodies from our phage library using a series of MSLN+ cells in a whole-cell panning approach (Figure 35, panel A). This campaign led to the identification of several potent anti-MSLN scFvs (Figure 35, panel B) and specific killing of MSLN+ OVCAR8 cells but not MSLN-SK59 renal cell carcinoma cells (Figure 35, panel C). Importantly, each MUC1 (T4E3) and MSLN (E8) CAR was able to kill dual MUC1+, MSLN+ CoV362 cells, similar to the dual-targeting T4E3-E8 CAR (Figure 35, panel D).

[0523] IR CAR-T cells alter the TME: Altering the TME is crucial for CAR-T cell therapy to both reverse immune cell depletion at the tumor site and prevent newly arrived CAR-T cells from being depleted at the tumor site. We have invested significant effort in analyzing published scRNAseq data to identify targets for which we can develop BsAbs that can act as potent ICIs and reverse immunosuppression at the tumor site. We have completed these discovery efforts and selected two targets: TIGIT and CCR4. We performed binding data of anti-TIGIT mAb and competition with its immunosuppressive ligand, CD155. This Ab-mediated blockade of CD155 binding to TIGIT can block immunosuppression through this axis.

[0524] CCR4 is a chemokine receptor present on immunosuppressive Tregs that migrate to OvCA tumor sites. Our lead anti-CCR4 antibody is mAb 2-322-24. This antibody blocks in vivo migration of human Tregs to OvCA cells that secrete the chemokines CCL17 and CCL22 (Figure 36, panel A) and restores in vivo tumor cell killing (Figure 36, panel B).

[0525] Study Plan: We will test individual anti-MUC1 and anti-MSLN CARs for killing against OVCAR8, CoV362, and MUC1- or MSLN-transduced 293T cells. We will conduct additional in vitro CAR-T cell killing tests on OVCAR8 and CoV362 cells expressing different densities of MUC1 and MSLN, as well as negative control MUC1-MSLN-SK59 cells and other low-density MSLN+ or MUC1+ cell lines. We will verify killing activity using our published Celigo assay. Results from these tests will be analyzed for the ability to kill tumor cells with different MUC1 and MSLN densities, and safety will be analyzed for lack of killing of control cell lines. These studies will enable us to validate the anti-MUC1 and anti-MSLN scFvs used in the construction of dual-targeting CARs. Next, DF CAR-T cells are constructed with lead anti-MUC1 and anti-MSLN scFvs in both orientations (proximal vs. distal to the T cell membrane) and with different inter-scFv linker lengths (GGGGS) of 1 to 3 (Figures 34 and 37). This ensures that the orientation and accessibility of each scFv to its target is optimized. From these studies, one lead dual-targeting CAR is selected for further testing.

[0526] Delivery of antibody payloads involves a different series of studies using primary OvCA PDOTS. We successfully established a ccRCC ex vivo 3D model that recapitulates the TME and provides a facile means for screening the efficacy of CAR-T variants (Figure 38, panel A). The workflow is shown in Figure 38, panel B. We demonstrated that ccRCC PDOTS serve as a powerful tool for evaluating both CAR-T cell efficacy and migration. Anti-CAIX G36 CAR-T cells significantly migrated from the side channel to the middle channel with ccRCC spheroids and secreted CXCL10 after 6 days of co-incubation (Figure 38, panel C). In our OvCA studies, CAR lentivirus was transduced into healthy donor T cells at a CD4:CD8 ratio of 2:1. After ex vivo purification and expansion, we add DFIR CAR-T cells to the outer wells of the device and test the migration and cytokine secretion of lead DFIR CAR-T cells secreting anti-TIGIT, anti-CCR4, or anti-CCR4 / TIGIT BsAbs. CAR-T migration is measured by co-transducing CAR-T cells with lentivirus expressing ZsGreen, and viability is measured by PI staining. Cytokine release profiles are measured by Luminex.

[0527] We will validate these monoCAR-T cells and DFIR CAR-T cells with different payloads by recovering DFIR CAR-T / TILs and supernatants from the microfluidic device as reported. 26 We will examine whether there are phenotypic and functional characteristics that distinguish anti-MUC1 / MSLN DFIR CAR-T cells secreting anti-CCR4 / TIGIT BsAb from other treatments, and whether there are differences between HGSOC cell donors.

[0528] Results: Without wishing to be bound by theory, anti-MUC1 / MSLN DF CAR-T cells can be engineered to selectively kill OvCA cells that overexpress MUC1 and / or MSLN, but not healthy cells that express these TAAs at physiological (low) levels or are negative for them. Furthermore, anti-MUC1 / MSLN DFIR CAR-T cells secreting anti-CCR4 / TIGIT BsAb exhibit enhanced killing compared to anti-MUC1 / MSLN DF CAR-T cells that do not secrete an ICI payload. Furthermore, DFIR CAR-T cells secreting anti-CCR4 / TIGIT BsAb exhibit greater reversal / prevention of TIL / CAR-T depletion than DFIR CAR-T cells secreting only anti-TIGIT or anti-CCR4 Abs, due to dual ICI pathway blockade. The mechanism of action of our BsAb secreting DFIR CAR-T cells is through both the prevention of CAR-T cell depletion and the restoration of anti-tumor immunity to TILs. Prevention / restoration of CAR-T / TIL depletion is quantified by FACS staining of PD1, TIM3, and LAG3, and determining their cytokine secretion levels (e.g., IFNγ, IL-2).

[0529] Data analysis and statistical considerations: Each CAR-T assay was performed three times, replicated three times across three PBMC donors, and killing percentages were tabulated across these conditions by sample average. We first compared anti-MUC1 and anti-MSLN monoCAR-T cells for specific (efficacy) and background (safety) killing by examining two to three different CARs with different affinities for each target and two to three OvCA cell lines with different MUC1 and MSLN target densities. From these studies, we identified the anti-MUC1 and anti-MSLN CARs to be used and selected the CAR with the greatest efficacy across cell lines while maintaining acceptable safety, e.g., a maximum of 10% background killing, to create our dual-target anti-MUC1 / MSLN DFIR CAR. The effect of antibody payload secretion on DFIR CAR-T cell activation was monitored by functional results in PDOT studies. Outcomes monitored by FACS include reversal of upregulated exhaustion markers (PD1, TIM3, LAG3) in TIL / CAR-T cells and increased TIL / CAR-T survival, as well as increased pro-inflammatory cytokine secretion by Luminex.

[0530] In vitro CAR-T killing: Data are analyzed in GraphPad Prism and a two-way ANOVA for multiple comparisons is used for statistical analysis.

[0531] Cytokine ELISA: Data are analyzed with GraphPad Prism and a two-way ANOVA for multiple comparisons is used for statistical analysis.

[0532] We will conduct in vivo studies to validate the efficacy of CAR-T cells in humanized mice (NSG-SGM3) bearing target 2-HLA-matched ovarian tumors. The studies will be designed with appropriate control groups to separately evaluate the efficacy of anti-MUC1 / anti-MSLN DFIR CAR-T cells and their anti-CCR4 / TIGIT BsAb payloads. Subtasks will include analysis of TILs by scRNASeq, multiparameter FACS analysis, and cytokine secretion profiling from plasma.

[0533] A humanized mouse model will be developed to examine OvCA TME and DFIR CAR-T intervention.

[0534] Using scRNAseq to catalog and quantify TME changes resulting from DFIR CAR-T therapy.

[0535] Rationale and Data: Without wishing to be bound by theory, alteration of the TME in HGSOC can be achieved by combined checkpoint blockade and by blocking Treg recruitment to the tumor site. We demonstrated significant inhibition of Treg migration to OvCA tumor sites in vivo by our anti-CCR4 antibody, which also resulted in significant inhibition of tumor cell proliferation. We demonstrated antitumor efficacy for an IR CAR developed for the treatment of ccRCC. The CAR was constructed using an affinity-tuned anti-CAIX scFv, and the secreted Ab payload was either an anti-PDL1 or an unrelated anti-SARS antibody. As can be seen, the antitumor efficacy of an anti-CAIX CAR secreting an anti-PDL1 Ab, which acts as an ICI for this cancer, is significantly improved.

[0536] Research Plan:

[0537] Primary OvCA PDX models have been established from tumor cells derived from HGSOC ascites. 27 Fourteen of these cell lines had growth kinetics suitable for robust in vivo experiments. Two of these will be characterized by measuring luciferase activity for use in our DFIR CAR-T cell study. These cell lines will be HLA-typed, and we will identify 5 / 6 or 6 / 6 HLA-matched donors from commercial blood companies for the study. We will use G-CSF-mobilized peripheral blood (PB) and purify both CD34+ hematopoietic stem cells (HSCs) and T cells using commercially available immune reconstitution and CAR T cell generation kits. Approximately 3–4 months after reconstitution, when PB hCD45 cells exceed 25%, mice will be intraperitoneally implanted with in vivo passaged tumors. At a later time point, CAR-T cells from the same donor will be injected via the tail vein. Mice are monitored weekly by BLI imaging and sacrificed at appropriate time points based on tumor growth. Ascites fluid is collected and processed for tumor cell count determination. CAR-T cells and TILs are processed for FACS staining of T cell subsets and myeloid lineage and exhaustion markers (PD1, TIM3, and LAG3) and for scRNASeq to characterize changes in the TME.

[0538] Each animal study included five groups: anti-MUC1 / anti-MSLN DFIR CAR-T secreting anti-TIGIT / anti-CCR4 BsAb, anti-MUC1 / anti-MSLN DFIR CAR-T secreting irrelevant BsAb, anti-TIGIT / anti-CCR4 BsAb secreting irrelevant DFIR CAR-T secreting irrelevant BsAb and untransduced T cells (5 groups, n = 6 mice / group). Animals were imaged weekly for luciferase activity, starting from baseline immediately after tumor implantation. Tumor growth was quantified by luciferase activity. Two experiments were performed for each of the two different tumor cell lines. (Total mice = 144, with 36 mice available for each study (accounting for 6 mice per reconstitution due to death, poor immune reconstitution, and infection).)

[0539] For scRNASeq analysis, the bioinformatics pipeline for this project involves a combination of tools, including alAkazam28; Shazam29; Seurat30; and Cumulus31. We examine differences in transcriptional, metabolic, and evolutionary patterns between different CAR-T treatments. Some overlap is observed between subsets. We compare these different treatments to examine whether there are phenotypic and functional characteristics that distinguish anti-MUC1 / MSLN DFIR CAR-T cells secreting anti-CCR4 / TIGIT BsAb from other treatments, as well as differences between T cell donors.

[0540] Results: Without wishing to be bound by theory, anti-MUC1 / anti-MSLN DFIR CAR-Ts secreting anti-TIGIT / anti-CCR4 BsAbs showed the greatest antitumor activity compared to the other groups, followed by anti-MUC1 / anti-MSLN DFIR CAR-Ts secreting irrelevant BsAbs. Furthermore, irrelevant DFIR CAR-Ts secreting anti-TIGIT / anti-CCR4 BsAbs had limited clinical efficacy due to the lack of targeting and proliferation of DFIR CAR-T cells. The other two animal groups showed uncontrolled tumor cell proliferation. Without wishing to be bound by theory, the T cell depletion profiles from both FACS staining and scRNA-Seq correlated with antitumor activity, supporting the contribution of secreted anti-CCR4 / TIGIT BsAbs to the recovery of antitumor molecular and functional properties.

[0541] Data analysis and statistical considerations:

[0542] Tumor Growth: Data are analyzed in GraphPad Prism. BLI is analyzed using Living Image software.

[0543] Flow cytometry: Flow cytometry data are analyzed using FlowJo. PBMCs and peripheral blood mononuclear cells (PMN) cells are stained with their respective markers for appropriate color compensation.

[0544] Single-cell RNA sequencing: 10X Genomics offers a comprehensive analysis suite for data processing (Cell Ranger) and visualization (Loupe Cell Browser). Additionally, R programs such as Seurat can further analyze the data output from Cell Ranger.

[0545] Tumor growth trajectories are summarized by averaging tumor growth at each time point across replicates for each of the four CAR-T therapies and the control therapy (untransduced T cells) in each of the two PDX models. The most promising CAR-T therapy produces the greatest reduction in tumor growth in both PDX models. FACS analysis of T cell subsets and exhaustion markers is performed on weekly blood draws and compared across the five groups over time. This data allows us to investigate and quantify the effects of DFIR CAR-T therapy on T cell differentiation and function over time.

[0546] With six samples per group, the power to detect a difference between means equal to 1.8 standard deviations using a two-sample, two-tailed t-test at the 0.05 level for the most extreme pair of comparisons (control group: irrelevant payload or untransduced irrelevant DFIR CAR-T) is 0.8. However, the statistical method ultimately used will be determined by the size of the cohort. For example, with four mice per group for each pairwise comparison, a much larger effect size (difference between groups) would be required to achieve the same power. Similarly, with four samples per group, the power to detect a difference between group means equal to 2.3 standard deviations using a two-sample, two-tailed t-test at the 0.05 level is 0.8.

[0547] The selected dose of CAR-T cells is set at 1 x 10e6, a level that has resulted in potent tumor cell killing in other CAR-T / mouse cancer models. This dose can be adjusted.

[0548] References cited in this example: 1.Webb PM,Jordan SJ.Epidemiology of epithelial ovarian cancer.Best Pract Res Clin Obstet Gynaecol.2017;41:3-14.Epub 2016 / 10 / 17.doi:10.1016 / j.bpobgyn.2016.08.006.PubMed PMID:27743768.PMCID:NA 2.Agarwal R,Kaye SB.Ovarian cancer:strategies for overcoming resistance to chemotherapy.Nat Rev Cancer.2003;3(7):502-16.Epub 2003 / 07 / 02.doi:10.1038 / nrc1123.PubMed PMID:12835670.PMCID:NA 3.DiSaia PJ,Bloss JD.Treatment of ovarian cancer:new strategies.Gynecol Oncol.2003;90(2 Pt 2):S24-32.Epub 2003 / 08 / 21.doi:10.1016 / s0090-8258(03)00341-x.PubMed PMID:12928003.PMCIDPMC747367 4.Disis ML,Rivkin S.Future directions in the management of ovarian cancer.Hematol Oncol Clin North Am.2003;17(4):1075-85.Epub 2003 / 09 / 10.doi:10.1016 / s0889-8588(03)00054-6.PubMed PMID:12959192.PMCID:NA 5.Borella F,Ghisoni E,Giannone G,Cosma S,Benedetto C,Valabrega G,Katsaros D.Immune Checkpoint Inhibitors in Epithelial Ovarian Cancer:An Overview on Efficacy and Future Perspectives.Diagnostics(Basel).2020;10(3).Epub 2020 / 03 / 12.doi:10.3390 / diagnostics10030146.PubMed PMID:32156035;PMCID:PMC7151145. 6.Lee DW,Barrett DM,Mackall C,Orentas R,Grupp SA.The future is now:chimeric antigen receptors as new targeted therapies for childhood cancer.Clin Cancer Res.2012;18(10):2780-90.Epub 2012 / 05 / 17.doi:10.1158 / 1078-0432.Ccr-11-1920.PubMed PMID:22589486;PMCID:PMC4119811. 7.Yip A,Webster RM.The market for chimeric antigen receptor T cell therapies.Nature Reviews Drug Discovery.2018;17:161-2.PMCID:NA 8.Srivastava S,Riddell SR.Engineering CAR-T cells:Design concepts.Trends Immunol.2015;36(8):494-502.Epub 2015 / 07 / 15.doi:10.1016 / j.it.2015.06.004.PubMed PMID:26169254;PMCID:PMC4746114. 9.Maude SL,Frey N,Shaw PA,Aplenc R,Barrett DM,Bunin NJ,Chew A,Gonzalez VE,Zheng Z,Lacey SF,Mahnke YD,Melenhorst JJ,Rheingold SR,Shen A,Teachey DT,Levine BL,June CH,Porter DL,Grupp SA.Chimeric antigen receptor T cells for sustained remissions in leukemia.N Engl J Med.2014;371(16):1507-17.Epub 2014 / 10 / 16.doi:10.1056 / NEJMoa1407222.PubMed PMID:25317870;PMCID:PMC4267531. 10.Zhu Y,Tan Y,Ou R,Zhong Q,Zheng L,Du Y,Zhang Q,Huang J.Anti-CD19 chimeric antigen receptormodified T cells for B-cell malignancies:a systematic review of efficacy and safety in clinical trials.Eur J Haematol.2016;96(4):389-96.Epub 2015 / 06 / 27.doi:10.1111 / ejh.12602.PubMed PMID:26115358.PMCID:NA 11.Kakarla S,Gottschalk S.CAR T cells for solid tumors:armed and ready to go? 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[0549] Example 6 Tec...

Claims

1. An antibody or antigen-binding fragment thereof that binds to a mesothelin protein or a fragment thereof, comprising a heavy chain, a light chain, or both a heavy chain and a light chain, The aforementioned heavy chain, HCDR1 containing GYTLTTNG (SEQ ID NO: 31), GGTFSSDT (SEQ ID NO: 35), GFTFDDYA (SEQ ID NO: 38), GFKFTDYY (SEQ ID NO: 46), or GYTFTHYA (SEQ ID NO: 49), HCDR2 containing VSPYNGHI (SEQ ID NO: 32), VNPYNGHI (SEQ ID NO: 34), INPNSGGT (SEQ ID NO: 36), ISWNSGSI (SEQ ID NO: 39), ISWNNGSI (SEQ ID NO: 41), ISWNSGNI (SEQ ID NO: 43), INTSSNHI (SEQ ID NO: 47), or IHAGNGNS (SEQ ID NO: 50), HCDR3 containing ARVNRANYYGMDV (SEQ ID NO: 33), ARESALGGSYPLSF (SEQ ID NO: 37), AKDPSSSWLAGAFDI (SEQ ID NO: 40), AKDPSTSWLAGAFDI (SEQ ID NO: 42), AKDAGSSGYFNAFDI (SEQ ID NO: 44), AKSPSSNWYPDAFDI (SEQ ID NO: 45), ARGASWGPL (SEQ ID NO: 48), or AREVGHGMDV (SEQ ID NO: 51), or including combinations thereof; The aforementioned light chain, LCDR1 including QSLLRSNGYNY (SEQ ID NO: 52), QSVSSSY (SEQ ID NO: 59), SLRRFY (SEQ ID NO: 62), SLIRFY (SEQ ID NO: 65), SLRAYY (SEQ ID NO: 67), SLRNYY (SEQ ID NO: 70), NIGSKS (SEQ ID NO: 73), or SSNIGAGYD (SEQ ID NO: 76), LCDR2 including LGS, GAS, GKN, AKN, DDS, or GNT, LCDR3 including MQALQTPL (SEQ ID NO: 56), MQSSTSTNSAH (SEQ ID NO: 54), MQALQTPLT (SEQ ID NO: 55), MQALQTPLT (SEQ ID NO: 57), HASSTNSAH (SEQ ID NO: 58), LQDDSYPLT (SEQ ID NO: 61), NSRRDSDGNHVF (SEQ ID NO: 64), NSQDRDGNHVF (SEQ ID NO: 66), NSRRDSSGNHLGV (SEQ ID NO: 68), NSRRDSSGNHLGS (SEQ ID NO: 69), SSRRDSSSDNHVV (SEQ ID NO: 72), QSADYNWLLCD (SEQ ID NO: 75), or QSYDSSLSGYV (SEQ ID NO: 78), or a combination of those CD-Rs, An antibody or its antigen-binding fragment.

2. The antibody according to claim 1, comprising a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence that is at least 90% identical thereto, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence that is at least 90% identical thereto.

3. The antibodies described herein.

4. A therapeutic antibody that binds to mesothelin, comprising a variable domain and a constant domain, wherein the constant domain is IgG, and the variable domain comprises a framework region and a complementarity determination means for binding to mesothelin.

5. The antibody according to claim 4, wherein the constant region is IgG1 or IgG4.

6. An isolated antibody or fragment thereof that binds to human mesothelin protein, (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 56; or (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (c) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (d) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 34, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 55; or (e) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 57; or (f) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 58; or (g) VH CDR1 containing the amino acid sequence of SEQ ID NO: 35, VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, VL CDR1 containing the amino acid sequence of SEQ ID NO: 59, VL CDR2 containing the amino acid sequence of GAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 61; or (h) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; or (i) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; (j) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 41, VH CDR3 containing the amino acid sequence of SEQ ID NO: 42, VL CDR1 containing the amino acid sequence of SEQ ID NO: 65, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 66; (k) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 68; (l) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 69; (m) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 45, VL CDR1 containing the amino acid sequence of SEQ ID NO: 70, VL CDR2 containing the amino acid sequence of AKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 72; (n) VH CDR1 containing the amino acid sequence of SEQ ID NO: 46, VH CDR2 containing the amino acid sequence of SEQ ID NO: 47, VH CDR3 containing the amino acid sequence of SEQ ID NO: 48, VL CDR1 containing the amino acid sequence of SEQ ID NO: 73, VL CDR2 containing the amino acid sequence of DDS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 75; or (o) VH CDR1 containing the amino acid sequence of SEQ ID NO: 49, VH CDR2 containing the amino acid sequence of SEQ ID NO: 50, VH CDR3 containing the amino acid sequence of SEQ ID NO: 51, VL CDR1 containing the amino acid sequence of SEQ ID NO: 76, VL CDR2 containing the amino acid sequence of GNT, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 78 An antibody or fragment thereof, containing an antibody.

7. An isolated scFv antibody that binds to human mesothelin protein, (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 56; or (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (c) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (d) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 34, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 55; or (e) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 57; or (f) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 58; or (g) VH CDR1 containing the amino acid sequence of SEQ ID NO: 35, VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, VL CDR1 containing the amino acid sequence of SEQ ID NO: 59, VL CDR2 containing the amino acid sequence of GAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 61; or (h) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; or (i) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; (j) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 41, VH CDR3 containing the amino acid sequence of SEQ ID NO: 42, VL CDR1 containing the amino acid sequence of SEQ ID NO: 65, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 66; (k) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 68; (l) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 69; (m) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 45, VL CDR1 containing the amino acid sequence of SEQ ID NO: 70, VL CDR2 containing the amino acid sequence of AKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 72; (n) VH CDR1 containing the amino acid sequence of SEQ ID NO: 46, VH CDR2 containing the amino acid sequence of SEQ ID NO: 47, VH CDR3 containing the amino acid sequence of SEQ ID NO: 48, VL CDR1 containing the amino acid sequence of SEQ ID NO: 73, VL CDR2 containing the amino acid sequence of DDS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 75; or (o) VH CDR1 containing the amino acid sequence of SEQ ID NO: 49, VH CDR2 containing the amino acid sequence of SEQ ID NO: 50, VH CDR3 containing the amino acid sequence of SEQ ID NO: 51, VL CDR1 containing the amino acid sequence of SEQ ID NO: 76, VL CDR2 containing the amino acid sequence of GNT, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 78 scFv antibody, which includes this antibody.

8. An isolated antibody or fragment thereof that binds to human mesothelin protein, comprising a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence at least 90% identical thereto, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence at least 90% identical thereto.

9. An isolated scFv that binds to human mesothelin protein, comprising a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence at least 90% identical thereto, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence at least 90% identical thereto.

10. F(ab) that binds to mesothelin, (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 56; or (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (c) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (d) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 34, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 55; or (e) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 57; or (f) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 58; or (g) VH CDR1 containing the amino acid sequence of SEQ ID NO: 35, VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, VL CDR1 containing the amino acid sequence of SEQ ID NO: 59, VL CDR2 containing the amino acid sequence of GAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 61; or (h) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; or (i) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; (j) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 41, VH CDR3 containing the amino acid sequence of SEQ ID NO: 42, VL CDR1 containing the amino acid sequence of SEQ ID NO: 65, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 66; (k) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 68; (l) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 69; (m) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 45, VL CDR1 containing the amino acid sequence of SEQ ID NO: 70, VL CDR2 containing the amino acid sequence of AKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 72; (n) VH CDR1 containing the amino acid sequence of SEQ ID NO: 46, VH CDR2 containing the amino acid sequence of SEQ ID NO: 47, VH CDR3 containing the amino acid sequence of SEQ ID NO: 48, VL CDR1 containing the amino acid sequence of SEQ ID NO: 73, VL CDR2 containing the amino acid sequence of DDS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 75; or (o) VH CDR1 containing the amino acid sequence of SEQ ID NO: 49, VH CDR2 containing the amino acid sequence of SEQ ID NO: 50, VH CDR3 containing the amino acid sequence of SEQ ID NO: 51, VL CDR1 containing the amino acid sequence of SEQ ID NO: 76, VL CDR2 containing the amino acid sequence of GNT, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 78 F(ab), including F(ab).

11. VhH that binds to mesothelin, (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33; or (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33; or (c) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 34, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33; or (d) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33; or (e) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33; or (f) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33; or (g) VH CDR1 containing the amino acid sequence of SEQ ID NO: 35, VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, VH CDR3 containing the amino acid sequence of SEQ ID NO: 37; or (h) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40; or (i) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 40; (j) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 41, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 42; (k) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 44; (l) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 44; (m) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 45; (n) VH CDR1 containing the amino acid sequence of SEQ ID NO: 46, VH CDR2 containing the amino acid sequence of SEQ ID NO: 47, VH CDR3 containing the amino acid sequence of SEQ ID NO: 48; or (o) VH CDR1 containing the amino acid sequence of SEQ ID NO: 49, VH CDR2 containing the amino acid sequence of SEQ ID NO: 50, and VH CDR3 containing the amino acid sequence of SEQ ID NO: 51 VhH, including.

12. An isolated bispecific antibody comprising a fragment according to any one of claims 1 to 11 and a second antigen-binding fragment having specificity for molecules on immune cells.

13. The bispecific antibody according to claim 12, wherein the molecule is selected from the group consisting of CCR4, B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, and KIR.

14. The bispecific antibody according to claim 12, wherein the aforementioned fragment and the second fragment are each independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.

15. The bispecific antibody according to claim 12, further comprising an Fc fragment.

16. A bispecific T cell engager (BiTE) that binds to human mesothelin protein, (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 56; or (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (c) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (d) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 34, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 55; or (e) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 57; or (f) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 58; or (g) VH CDR1 containing the amino acid sequence of SEQ ID NO: 35, VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, VL CDR1 containing the amino acid sequence of SEQ ID NO: 59, VL CDR2 containing the amino acid sequence of GAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 61; or (h) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; or (i) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; (j) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 41, VH CDR3 containing the amino acid sequence of SEQ ID NO: 42, VL CDR1 containing the amino acid sequence of SEQ ID NO: 65, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 66; (k) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 68; (l) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 69; (m) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 45, VL CDR1 containing the amino acid sequence of SEQ ID NO: 70, VL CDR2 containing the amino acid sequence of AKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 72; (n) VH CDR1 containing the amino acid sequence of SEQ ID NO: 46, VH CDR2 containing the amino acid sequence of SEQ ID NO: 47, VH CDR3 containing the amino acid sequence of SEQ ID NO: 48, VL CDR1 containing the amino acid sequence of SEQ ID NO: 73, VL CDR2 containing the amino acid sequence of DDS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 75; or (o) VH CDR1 containing the amino acid sequence of SEQ ID NO: 49, VH CDR2 containing the amino acid sequence of SEQ ID NO: 50, VH CDR3 containing the amino acid sequence of SEQ ID NO: 51, VL CDR1 containing the amino acid sequence of SEQ ID NO: 76, VL CDR2 containing the amino acid sequence of GNT, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 78 This includes bispecific T cell engagers (BiTEs).

17. A bispecific T cell engager (BiTE) that binds to human mesothelin protein, comprising a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence at least 90% identical thereto, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence at least 90% identical thereto.

18. An antibody according to any one of claims 1 to 11, having at least 90% sequence identity.

19. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 11 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

20. (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 56; or (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (c) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (d) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 34, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 55; or (e) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 57; or (f) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 58; or (g) VH CDR1 containing the amino acid sequence of SEQ ID NO: 35, VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, VL CDR1 containing the amino acid sequence of SEQ ID NO: 59, VL CDR2 containing the amino acid sequence of GAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 61; or (h) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; or (i) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; (j) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 41, VH CDR3 containing the amino acid sequence of SEQ ID NO: 42, VL CDR1 containing the amino acid sequence of SEQ ID NO: 65, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 66; (k) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 68; (l) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 69; (m) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 45, VL CDR1 containing the amino acid sequence of SEQ ID NO: 70, VL CDR2 containing the amino acid sequence of AKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 72; (n) VH CDR1 containing the amino acid sequence of SEQ ID NO: 46, VH CDR2 containing the amino acid sequence of SEQ ID NO: 47, VH CDR3 containing the amino acid sequence of SEQ ID NO: 48, VL CDR1 containing the amino acid sequence of SEQ ID NO: 73, VL CDR2 containing the amino acid sequence of DDS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 75; or (o) VH CDR1 containing the amino acid sequence of SEQ ID NO: 49, VH CDR2 containing the amino acid sequence of SEQ ID NO: 50, VH CDR3 containing the amino acid sequence of SEQ ID NO: 51, VL CDR1 containing the amino acid sequence of SEQ ID NO: 76, VL CDR2 containing the amino acid sequence of GNT, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 78 The pharmaceutical composition according to claim 19, comprising:

21. The pharmaceutical composition according to claim 19, wherein the pharmaceutical composition comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), the amino acid sequence of the HCVR being SEQ ID NO: 7, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 29, and the amino acid sequence of the LCVR being SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO:

30.

22. A nucleic acid encoding an antibody or fragment according to any one of claims 1 to 11.

23. A nucleic acid encoding the bispecific antibody according to claim 12.

24. A nucleic acid encoding BiTE according to any one of claims 16 to 17.

25. A modified cell comprising a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular ligand-binding domain specific to an antigen on the surface of a cancer cell, the antigen comprises mesothelin, the extracellular ligand-binding domain comprises an antibody or a fragment thereof, and the antibody or fragment thereof (a) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 56; or (b) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (c) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 54; or (d) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 34, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 55; or (e) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 57; or (f) VH CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH CDR2 containing the amino acid sequence of SEQ ID NO: 32, VH CDR3 containing the amino acid sequence of SEQ ID NO: 33, VL CDR1 containing the amino acid sequence of SEQ ID NO: 52, VL CDR2 containing the amino acid sequence of LGS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 58; or (g) VH CDR1 containing the amino acid sequence of SEQ ID NO: 35, VH CDR2 containing the amino acid sequence of SEQ ID NO: 36, VH CDR3 containing the amino acid sequence of SEQ ID NO: 37, VL CDR1 containing the amino acid sequence of SEQ ID NO: 59, VL CDR2 containing the amino acid sequence of GAS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 61; or (h) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; or (i) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 40, VL CDR1 containing the amino acid sequence of SEQ ID NO: 62, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 64; (j) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 41, VH CDR3 containing the amino acid sequence of SEQ ID NO: 42, VL CDR1 containing the amino acid sequence of SEQ ID NO: 65, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 66; (k) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 68; (l) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 43, VH CDR3 containing the amino acid sequence of SEQ ID NO: 44, VL CDR1 containing the amino acid sequence of SEQ ID NO: 67, VL CDR2 containing the amino acid sequence of GKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 69; (m) VH CDR1 containing the amino acid sequence of SEQ ID NO: 38, VH CDR2 containing the amino acid sequence of SEQ ID NO: 39, VH CDR3 containing the amino acid sequence of SEQ ID NO: 45, VL CDR1 containing the amino acid sequence of SEQ ID NO: 70, VL CDR2 containing the amino acid sequence of AKN, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 72; (n) VH CDR1 containing the amino acid sequence of SEQ ID NO: 46, VH CDR2 containing the amino acid sequence of SEQ ID NO: 47, VH CDR3 containing the amino acid sequence of SEQ ID NO: 48, VL CDR1 containing the amino acid sequence of SEQ ID NO: 73, VL CDR2 containing the amino acid sequence of DDS, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 75; or (o) VH CDR1 containing the amino acid sequence of SEQ ID NO: 49, VH CDR2 containing the amino acid sequence of SEQ ID NO: 50, VH CDR3 containing the amino acid sequence of SEQ ID NO: 51, VL CDR1 containing the amino acid sequence of SEQ ID NO: 76, VL CDR2 containing the amino acid sequence of GNT, and VL CDR3 containing the amino acid sequence of SEQ ID NO: 78 Manipulated cells, including those containing artificial cells.

26. The manipulated cell according to claim 25, wherein the antibody or fragment thereof comprises a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29 or a sequence that is at least 90% identical thereto, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 or a sequence that is at least 90% identical thereto.

27. The manipulated cells according to claim 25, comprising T cells, NK cells, NKT cells, iPS cells, iPS-derived cells, cell lines, or B cells.

28. The manipulated cells according to claim 25, comprising CD4+, CD8+, CD3+ pan-T cells, or any combination thereof.

29. A pharmaceutical composition for treating cancer, comprising an effective amount of the antibody according to any one of claims 1 to 11.

30. A pharmaceutical composition for inhibiting mesothelin in a target, comprising an effective amount of the antibody described in any one of claims 1 to 11.

31. The pharmaceutical composition according to claim 30, comprising an antibody comprising a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, or a sequence that is at least 90% identical thereto, and a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 2, 4, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30, or a sequence that is at least 90% identical thereto.

32. Use of an antibody according to any one of claims 1 to 11 in the manufacture of a pharmaceutical product for treating cancer.

33. An antibody according to any one of claims 1 to 11, for use in the treatment of therapies.

34. An antibody according to any one of claims 1 to 11, for use in the treatment of cancer.

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