Compositions and uses of alternatively formatted anti-mesothelin antibodies for cancer treatment

Abstract

Tumors utilize multiple mechanisms to evade the host's antitumor immune response. Humoral immunosuppression is one such mechanism. Tumors produce circulating factors that can suppress antibody- or complement-mediated immune responses, enhancing their own survival. Mesothelin is a cell surface protein overexpressed by several cancer types associated with tumors exhibiting an immunosuppressive microenvironment. Anti-mesothelin antibodies are often subject to such immunosuppressive microenvironments. However, alternatively formatted anti-mesothelin antibodies are effective in killing mesothelin-expressing cancers regardless of the tumor microenvironment immune status. TIFF2023546791000022.tif102170

Description

[Technical Field]

[0001] Technical field of the invention The present invention relates to the field of therapeutic antibodies effective in specifically targeting mesothelin-expressing cancers in immunocompetent and immunosuppressive tumor microenvironments. In particular, the present invention relates to methods, kits, and compositions comprising antibody-based agents with improved therapeutic efficacy in inhibiting cancer growth, regardless of the microenvironment immune status. [Background technology]

[0002] Background of the Invention The mechanism of tumorigenesis involves a combination of the accumulation of mutated genes that enhance dysregulated cell proliferation and the generation of immune evasion mechanisms that allow survival within affected patients. Cellular (mainly T cell-mediated) and humoral (mainly antibody-mediated) immunity are the primary mechanisms by which vertebrate host organisms defend against infectious pathogens and dysregulated host cells. Over the past few years, the use of immune checkpoint inhibitors that can overcome suppressed cell-mediated immunity has become a key focus in cancer biology, resulting in the activation of CD8+ / CD9 ... +It has shown potent effects in inducing T cell killing (Hodi FS, et al. N Engl J Med 363:711-723, 2010). Recent translational studies have shown that tumors also produce factors that suppress humoral immune pathways, inhibiting antibody-mediated mechanisms of tumor killing, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and opsonization (Vergote I, et al. J Clin Oncol 34:2271-2278; Kline JB, et al. J Clin Oncol 5:15, 2018; Wang W et al. Cytogenet Genome Res 152:169-179, 2017; Kline JB et al. Eur J Immunol. 48:1872-1882, 2018; Dai S, et al. PLos Pathog 9:e1003114, 2013; Melero I, et al. Nat Reviews Cancer 7:95-106, 2007). Factors that suppress humoral immune pathways inhibit the efficacy of clinically used therapeutic antibodies that have been reported to exhibit tumor-killing effects via ADCC and CDC, including, but not limited to, rituximab, obinutuzumab, trastuzumab, pertuzumab, cetuximab, alemtuzumab, and some experimental antibodies (DiLillo DJ, Ravetech JV, Cancer Immunol Res 3:704-713, 2015; Ruck T, et al. Int J Mol Sci 16:16414-16439, 2015; Pelaia C, et al. Biomed Res Int 4839230:1-9, 2018; Zhou X, et al. Oncologist 13:954-966, 2008; Hsu YF, et al. Mol Cancer 9:-8, 2010; Spiridon CI, et al. Clin Cancer Res 8:1720-1730,2002;Kline JB,et al.Eur J Immunol 48:1872-1882,2018;Yamashita-Kashima Y,et al.Clin Cancer Res 17:5060-5070,2011).Antibody-mediated humoral immune responses are controlled by the coordinated association of antibodies with cell surface antigens. When this interaction is optimal, cell surface-bound antibodies engage with activating Fcγ receptors on natural killer (NK) cells or dendritic / myeloid / monocytic cells (all cells involved in ADCC are referred to herein as "immune effector cells"). This engagement not only initiates ADCC but also leads to the killing of antibody-bound cells through the classical complement CDC pathway via association with the C1q complement initiation protein and through opsonization by phagocytes (Reuschenbach M, et al. Cancer Immunol Immunother 58:1535-1544, 2009). Inhibitors of the humoral immune response reduce the ability of therapeutic antibodies to use these mechanisms, reducing their therapeutic efficacy (Wang W, et al. Cytogenet Genome Res 152:169-179, 2017; Kline JB, et al. Eur J Immunol 48:1872-1882, 2018; Felder M, et al. Gyn Oncol 152:618-628, 2019).

[0003] The MUC16 / CA125 protein (referred to herein as CA125) inhibits humoral immune responses by binding to negative immunoregulatory receptors of the SIGLEC family, inhibiting NK cell activation (Belisle JA, et al. Mol Cancer 9:1476-4598, 2010), and by directly binding to a subset of IgG1, IgG3, and IgM antibodies. Binding to antibodies disrupts the Fc region, reducing the effectiveness of IgG1 and IgG3 type antibodies in engaging the activating Fcγ receptors FCGR2A (also called CD32a) and FCGR3A (also called CD16a) on immune effector cells, and / or disrupting the ability of all three antibody classes to associate with complement-mediated proteins, including C1q (Pantankar MS, et. al. Gyncol Oncol 99:704-713, 2005; Kline JB, et al. OncoTarget 8:52045-52060, 2017; Kline JB, et al. J. Clin. Oncol. 5:15, 2018; Wang W, et al. Cytogenet Genome Res 152:169-179, 2017; Kline JB, et al. Eur J Immunol. 48:1872-1882, 2018). These studies include clinical trials of experimental anti-cancer antibodies that rely on immune effector mechanisms for their pharmacological activity. In these clinical studies, CA125 levels have been found to correlate with clinical outcomes (Vergote I, et al. J Clin Oncol 34:2271-2278, 2016; Nicolaides NC, et al. Cancer Biol Ther 13:1-22, 2018). In a study of the clinically used rituximab antibody in patients with follicular lymphoma, a 31.4% improvement in 5-year progression-free survival was observed in patients with CA125 levels within the normal range compared with patients with CA125 levels above the normal range (Prochazka V, et al. Int J Hematol 96:58-64, 2012). There is a continuing need in the art to develop agents that can effectively kill tumors with immunocompetent and immunosuppressive microenvironments.

[0004] Mesothelin is a cell surface protein overexpressed by several tumor types, including mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, bile duct cancer, gastric cancer, and endometrial cancer. Some of these tumor types have been found to exhibit humoral immunosuppression, which potentially reduces the efficacy of antibody-based anti-mesothelin therapy (Rump A, et al. J Biol Chem 279:9190-9198, 2004; Hassan R, et al. Cancer Immunol 7:20-30, 2007; Kaneko O, et al. J Biol Chem 284:3739-3749, 2009; Hassan R. Lung Cancer 68:455-459, 2010; Kelly RJ, et al. J Clin Oncol suppl 32:61, 2014). In particular, lung cancer, ovarian cancer, pancreatic cancer, and mesothelioma have been reported to express the humoral immunosuppressive CA125 protein (Vergote I, et al. J Clin Oncol 34:2271-2278, 2016; Nicolaides NC, et al. Cancer Biol Ther 13:1-22, 2018; Liu L, Oncotarget. 7:5943-5956, 2016; Cedres S, et al. Clin Lung Cancer 12:172-179, 2011; Emoto S, et al. Gastric Cancer 15:154-161, 2012). Nicolaides et al. (2018) reported humoral immunosuppression in findings from a phase 2 clinical trial of the anti-mesothelin antibody amatuximab in first-line mesothelioma treatment in addition to standard therapy. In that study, patients with high CA125 levels treated with amatuximab had worse progression-free survival (PFS) and overall survival (OS) outcomes than patients with low CA125 levels, supporting the idea that anti-mesothelin antibodies, which rely on humoral immune function, are less effective in humoral immunosuppressive cancers such as those listed above. Alternative strategies are needed to overcome this mechanism and develop novel antibody-based drugs that may offer a broader range of treatment options for patients with or without humoral immunosuppressive cancers.There is a need in the art for compositions and methods that are effective for killing mesothelin-expressing tumor types that exhibit a humoral immunosuppressive phenotype (e.g., expression of the immunosuppressive CA125 protein) and immunocompetent mesothelin-expressing tumor types. Summary of the Invention

[0005] One embodiment is an antibody-drug conjugate (ADC) that includes an anti-mesothelin antibody containing complementarity-determining regions (CDRs) having the amino acid sequences set forth in SEQ ID NOS: 7-12. One such anti-mesothelin antibody includes SEQ ID NOS: 1 and 2. The ADC also includes a topoisomerase inhibitor.

[0006] Another embodiment is a method of using an anti-mesothelin antibody that does not bind to CA125 as part of an antibody-drug conjugate (ADC). The anti-mesothelin antibody comprises a CDR having the amino acids set forth in SEQ ID NOS: 7-12. The cellular uptake of this anti-mesothelin antibody is greater than that of anti-mesothelin antibodies that bind to either soluble or membrane-bound CA125. One such anti-mesothelin antibody that does not bind to CA125 that can be used comprises the amino acid sequence of SEQ ID NOS: 1 and 2. The method includes administering the ADC to a human in need of anti-mesothelin therapy. The method may also include using the ADC to detect mesothelin epitopes in target cells.

[0007] Another embodiment is a method of treating a cancer patient having a mesothelin-expressing tumor by administering to the patient an antibody-drug conjugate (ADC) comprising an anti-mesothelin antibody having a CDR comprising the amino acids set forth in SEQ ID NOS: 7-12 and a topoisomerase inhibitor. One such anti-mesothelin antibody that can be used includes SEQ ID NOS: 1 and 2.

[0008] Another embodiment is a bispecific antibody (BSP) comprising a mesothelin-binding portion comprising the amino acid sequences of SEQ ID NOs: 7-12 and a human cell surface antigen CD3-binding portion.

[0009] In another embodiment, a method of treating a mesothelin-expressing cancer in a patient is provided in which a bispecific antibody (BSP) comprising a mesothelin-binding portion comprising an amino acid sequence of SEQ ID NO:7-12 and a human cell surface antigen CD3-binding portion is administered to the patient, thereby treating the mesothelin-expressing cancer.

[0010] One aspect of the present invention is an antibody comprising the amino acid sequences shown in SEQ ID NO: 1 [MES light chain] and SEQ ID NO: 2 [MES heavy chain]. Antibody binding by the immunosuppressive CA125 protein is significantly reduced, thereby enhancing antibody internalization. This is particularly useful for ADC-mediated tumor cell killing when the antibody is part of an antibody-drug conjugate.

[0011] Another aspect of the present invention is an antibody comprising the amino acid sequences set forth in SEQ ID NO:1 [MES light chain] and SEQ ID NO:2 [MES heavy chain], conjugated to a cytotoxic compound selected for its ability to kill immunocompetent and immunosuppressive mesothelin-expressing cancer cells. The cytotoxic compound can be a topoisomerase inhibitor, microtubule inhibitor, alkylating agent, or kinase inhibitor. This conjugate is referred to as an "MES-ADC."

[0012] Another aspect of the present invention is a stable cell line containing a mammalian expression vector having the nucleotide sequences shown in SEQ ID NO: 19 [MES light chain] and SEQ ID NO: 5 [MES heavy chain], which encodes the parent MES-1 antibody produced by the cell line and then optionally chemically conjugated to a cytotoxic agent.

[0013] Another aspect of the present invention is a method of treating a patient who has mesothelin-expressing cancer cells and expresses elevated levels of CA125 compared to healthy human populations. An MES-ADC antibody is administered to the patient. The MES-ADC antibody consists of the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 and is linked to the cytotoxic topoisomerase inhibitor SN38. The antibody-cytotoxin is optionally covalently linked by a cleavable linker.

[0014] Yet another aspect of the present invention is a method for treating a patient who has mesothelin-expressing cancer cells and expresses elevated levels of CA125 compared to healthy human populations. An MES-ADC antibody is administered to the patient. The MES-ADC antibody consists of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 and is conjugated to the cytotoxic topoisomerase inhibitor PNU159682. The antibody-cytotoxin may be covalently attached by a cleavable linker.

[0015] In one embodiment, an MES-ADC comprises an antibody consisting of SEQ ID NO:1 and SEQ ID NO:2 linked to the cytotoxic PNU159682 or SN38 topoisomerase inhibitor. Two or more cytotoxins may be attached to the antibody via chemical ligation by linking to free cysteines on the antibody generated by partial reduction. The cysteines may be endogenous to the immunoglobulin sequence or may be engineered into the parent antibody sequence.

[0016] In another aspect of the invention, MES-ADC chemical ligation is carried out using cleavable linkers, such as, but not limited to, Val-Cit-PAB, MA-PEG4-VC-PAB-DMAE, Fmoc-Val-Cit-PAB, Fmoc-Val-Cit-PAB-PNP, MC-Val-Cit-PAB-PNP, Phe-Lys(Trt)-PAB, Fmoc-Phe-Lys(Trt)-PAB, Fmoc-Phe-Lys(Trt)-PAB-PNP, Ala-Ala-Asn-PAB TFA salts, Fmoc-Ala-Ala-Asn-PAB-PNP, Fmoc-Gly3-Val-Cit-PAB, Fmoc-Gly3-Val-Cit-PAB-PNP, Py-ds-Prp-OSu, Py-ds-dmBut-OSu, Py-ds-dmBut-OPFP, Py-ds-Prp-OPFP, PEG8-triazole-PABC-peptide-mc, etc. are utilized, and these chemical structures are known in the art.

[0017] In another embodiment of the present invention, MES-ADC chemical ligation utilizes enzymatically non-cleavable linkers such as, but not limited to, SMCC, MAL-HA-OSu, MAL-di-EG-OPFP, MAL-tri-EG-OPFP, MAL-tetra-EG-OPFP, N3-di-EG-OPFP, N3-tri-EG-OPFP, N3-tetra-EG-OPFP, ALD-BZ-OSu, ALD-di-EG-OSu, ALD-tetra-EG-OSu, ALD-di-EG-OPFP, ALD-tetra-EG-OPFP, MC-EDA, PHA-di-EG-OPFP, PHA-tetra-EG-OPFP, etc. The chemical structures of these are known in the art.

[0018] In another aspect of the invention, the linker is optimized for (a) antibody conjugation to a cytotoxin; (b) stability in the systemic circulation, organ parenchyma / stroma, and tumor microenvironment; and / or (c) improved killing of immunosuppressive tumors by the MES-ADC.

[0019] In another embodiment of the invention, a cytotoxin is attached to one or more lysine residues in the light and heavy chains of an anti-mesothelin antibody. One such antibody comprises the amino acid sequence set forth in SEQ ID NO:1 and / or SEQ ID NO:2.

[0020] In another embodiment of the invention, the cytotoxin is attached to the C-terminus of the heavy chain contained in SEQ ID NO:2 via transamidation.

[0021] Another aspect of the present invention is a bispecific antibody comprising the amino acid sequences shown in SEQ ID NO:3 and SEQ ID NO:2 (single-chain anti-CD3 and MES-1 heavy chain fused to MES-1 light chain, respectively). The bispecific antibody is capable of killing immunocompetent and immunosuppressive mesothelin-expressing cancer cells. The single-chain anti-CD3 antibody is capable of killing mesothelin-expressing cancer cells. + and / or CD8 + The bispecific antibody recognizes a cell surface antigen expressed on lymphocytes and is referred to herein as MES-BSP, i.e., a bispecific antibody targeting mesothelin.

[0022] Another aspect of the invention is a bispecific antibody that targets mesothelin, where the antibody is not bound by the CA125 immunosuppressive protein. One such antibody comprises the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 2. The bispecific antibody can be used to treat mesothelin-expressing cancers or other diseases associated with mesothelin expression.

[0023] Another aspect of the present invention is a stable cell line containing one or more mammalian expression vectors having nucleotide sequences encoding the antibody shown in SEQ ID NO: 19 [MES light chain cDNA] and SEQ ID NO: 5 [MES heavy chain cDNA] genetically linked to a second antibody.

[0024] Another aspect of the present invention is a method for treating a patient who has mesothelin-expressing cancer cells and expresses elevated levels of CA125 compared to healthy human populations. An MES-BSP antibody is administered to the patient. The MES-BSP antibody consists of the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:2, and the bispecific antibody recognizes mesothelin and the human CD3 protein.

[0025] Another aspect of the present invention is a method for treating a patient who has mesothelin-expressing cancer cells and expresses elevated levels of CA125 compared to healthy human populations. An MES-BSP antibody is administered to the patient. The MES-BSP antibody comprises the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 and is capable of recognizing the human CD8 protein.

[0026] In yet another aspect of the invention, an antibody comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 is fused to a single-chain antibody comprising the amino acid sequence of SEQ ID NO: 6 fused to the N-terminus of a human IgG1 light chain (SEQ ID NO: 1) and / or the N-terminus of an IgG heavy chain (SEQ ID NO: 2), resulting in a bispecific antibody capable of binding to mesothelin (Entrez Gene ID: 10232) and CD3 epsilon (CD3E) protein (Entrez Gene ID: 916), referred to herein as MES-BSP.

[0027] In another embodiment of the invention, the fusion of the amino acid sequence of SEQ ID NO: 1 with the amino acid sequence of SEQ ID NO: 6, joined via a linker, results in a light chain fusion polypeptide consisting of SEQ ID NO: 3, which, in combination with SEQ ID NO: 2, creates a functional MES-BSP capable of binding (a) to mesothelin on target cancer cells in the presence or absence of CA125 and (b) to CD3E on lymphoid cells, resulting in target cell killing. The linker can be an amino acid, polypeptide, or a non-biological chemical compound.

[0028] Yet another aspect of the present invention is a MES-BSP comprising complementarity determining regions (CDRs) having the amino acid sequences of SEQ ID NOs: 7, 8, 9; SEQ ID NOs: 10, 11, 12; SEQ ID NOs: 13, 14, 15; and SEQ ID NOs: 16, 17, 18 (up to three amino acids may be modified within one or more CDRs).

[0029] In another embodiment of the invention, the linker in SEQ ID NO: 3 is optimized for (a) standard antibody formation with an IgG1 heavy chain (SEQ ID NO: 2) and / or (b) improved killing of immunosuppressive tumors by MES-BSP, and the linker comprises any combination of 20 naturally occurring amino acid units to maximize the spatial distance between the anti-mesothelin light chain (SEQ ID NO: 1) and the anti-CD3E single chain (SEQ ID NO: 6).

[0030] In another embodiment of the invention, MES-BSP comprises an anti-CD3E single chain genetically linked to the N-terminus of the heavy chain of SEQ ID NO: 2. The linker is optimized for (a) standard antibody formation with an IgG1 light chain (SEQ ID NO: 1) and / or (b) improved killing of immunosuppressive tumors by MES-BSP, and the linker comprises any combination of 20 naturally occurring amino acid units to maximize the spatial distance between the anti-mesothelin heavy chain (SEQ ID NO: 2) and the anti-CD3E single chain (SEQ ID NO: 6).

[0031] Another aspect of the present invention is a stable cell line containing a mammalian expression vector having the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:4 encoding the MES-1 antibody genetically linked to a second antibody.

[0032] Another aspect of the present invention is an antibody having the CDR amino acid sequences of SEQ ID NOS: 7-12 grafted onto a rabbit IgG backbone (referred to herein as rMES-1), which is not bound by the CA125 protein.

[0033] Another aspect of the present invention is a method for monitoring mesothelin-expressing tumors in patients using an antibody that is not bound by CA125. A body fluid or tissue sample from the patient is contacted with an antibody containing the CDRs of SEQ ID NOS: 7-12, e.g., antibody rMES-1. Mesothelin-positive patients can be treated with MES-ADC or MES-BSP.

[0034] In another aspect of the present invention, kits for treating immunocompetent and immunosuppressive mesothelin-binding cancers are provided. The kits preferably include antibodies containing the CDRs of SEQ ID NOS: 7-12 grafted onto a rodent IgG backbone. The antibodies can be used to monitor tumors for mesothelin expression via immunohistochemistry (IHC) on biopsied tissue or via flow cytometry on circulating tumor cells (CTCs). If a positive signal is detected, the patient can be treated with an MES-ADC comprising the amino acid sequences of SEQ ID NOS: 1 and 2 chemically conjugated to a cytotoxin, which can be a topoisomerase inhibitor. Both diagnostic and therapeutic antibodies can be provided in the kit.

[0035] In another aspect of the present invention, kits for treating immunocompetent and immunosuppressive mesothelin-binding cancers are provided. The kits include antibodies containing the CDRs of SEQ ID NOS: 7-12 preferentially grafted onto a rodent IgG backbone. The antibodies can be used to monitor tumors for mesothelin expression via IHC on biopsied tissue or via flow cytometry on circulating tumor cells (CTCs). If a positive signal is detected, the patient can be treated with MES-BSP consisting of SEQ ID NOS: 2 and 3. Both diagnostic and therapeutic antibodies are provided in the kit.

[0036] Another aspect of the present invention is the use of MES-ADC, which is administered as a single agent or in combination with standard of care chemotherapy to patients, a subset of whom expresses CA 125. CA 125 expression can be determined using serum analysis or biopsy via methods used by those skilled in the art.

[0037] Another aspect of the present invention is the use of MES-BSP, administered as a single agent or in combination with standard of care chemotherapy to patients, a subset of whom expresses CA 125. CA 125 expression can be determined using serum analysis or biopsy via methods known in the art.

[0038] These and other aspects of the present invention, which will be apparent to those skilled in the art upon reading this specification, provide methods, compositions, and kits for use in improving therapeutic response in patients with mesothelin-expressing cancers, regardless of the immune status of the tumor microenvironment. The refractoriness of MES-ADC and / or MES-BSP agents to CA125 inhibition contributes to this improved treatment. [The present invention 1001] An antibody-drug conjugate (ADC) comprising an anti-mesothelin antibody comprising a complementarity-determining region (CDR) having the amino acids set forth in SEQ ID NOS: 7 to 12, and a topoisomerase inhibitor. [The present invention 1002] The ADC of the present invention, wherein the antibody comprises the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2. [The present invention 1003] The ADC of the present invention, wherein the antibody is covalently linked to the topoisomerase inhibitor. [The present invention 1004] 1001. The ADC of the present invention, wherein the antibody is linked to a topoisomerase inhibitor via a cleavable linker. [The present invention 1005] 1004. The ADC of the present invention, which is encapsulated in a liposome. [The present invention 1006] 1003. The ADC of the present invention, wherein the topoisomerase inhibitor is (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxylic acid (PNU159682). [The present invention 1007] 1006. An ADC of the invention, wherein PNU159682 is covalently attached to the anti-mesothelin antibody via the linker maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MA-PEG4-VC-PAB-DMAE). [The present invention 1008] The ADC of the present invention, wherein the linker is attached to a cysteine ​​in the anti-mesothelin antibody. [The present invention 1009] The ADC of the present invention 1007, wherein the antibody comprises the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2. [The present invention 1010] 1009. The ADC of the present invention, wherein the DAR of the ADC is 2 or more and 6 or less. [The present invention 1011] 1007. An ADC of the present invention, wherein the topoisomerase inhibitor is (2S,3S,4S,5R,6S)-6-[[(19S)-10,19-diethyl-19-hydroxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]heneicosa-1(21),2,4(9),5,7,10,15(20)-heptaen-7-yl]oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (SN38). [The present invention 1012] 1011. An ADC of the invention, wherein SN38 is covalently attached to the anti-mesothelin antibody via the linker methyl (2S,3S,4S,5R,6S)-3,4,5-triacetyloxy-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (MAC-glucuronide). [The present invention 1013] 1011. An ADC of the invention, wherein SN38 is covalently attached to the anti-mesothelin antibody via the linker PEG8 triazole-PABC-peptide-MC. [The present invention 1014] 1011. The ADC of the present invention, wherein SN38 is covalently linked to a cysteine ​​in the anti-mesothelin antibody. [The present invention 1015] The ADC of the present invention, wherein the anti-mesothelin antibody comprises the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2. [The present invention 1016] 1015 ADCs of the present invention having a DAR of 2 or more and 6 or less. [The present invention 1017] 1. A method of treating a cancer patient having a mesothelin-expressing tumor, comprising: administering to the patient an antibody-drug conjugate (ADC) comprising an anti-mesothelin antibody comprising the amino acids set forth in SEQ ID NOS: 7 to 12 and a topoisomerase inhibitor. A method comprising: [The present invention 1018] 1017. The method of claim 1017, wherein said cancer is selected from the group consisting of mesothelioma, breast cancer, lung cancer, colorectal cancer, gastrointestinal cancer, endometrial cancer, bile duct cancer, and pancreatic cancer. [The present invention 1019] detecting the presence of a mesothelin epitope in the patient by contacting a body sample from the patient with an antibody comprising an amino acid sequence of SEQ ID NOs: 7-12. The method of the present invention 1017 further comprising: [The present invention 1020] The method of claim 1017, wherein said patient has a high level of CA125 compared to a population of healthy humans. [The present invention 1021] 1017. The method of claim 1017, wherein a plurality of patients are treated by administering the bispecific antibody, said plurality of patients comprising at least one patient having an elevated level of CA125 relative to a population of healthy humans and at least one patient having a normal level of CA125. [The present invention 1022] A bispecific antibody (BSP) comprising a mesothelin-binding portion comprising the amino acid sequences of SEQ ID NOs: 7 to 12, and a cell surface antigen CD3-binding portion. [The present invention 1023] The bispecific antibody of the present invention 1022, wherein the cell surface antigen CD3-binding portion comprises the CDR amino acid sequences of SEQ ID NOs: 13 to 18. [The present invention 1024] The light chain of an anti-mesothelin antibody comprising the amino acid sequence of SEQ ID NO: 1 fused to a single-chain antibody recognizing the human cell surface antigen CD3 comprising the amino acid sequence of SEQ ID NO: 6. 1022. A bispecific antibody of the present invention comprising: [The present invention 1025] 1022. The bispecific antibody of the present invention, wherein the bispecific antibody comprises a light chain of an anti-mesothelin antibody comprising the amino acid sequence of SEQ ID NO: 1, and the light chain is linked to the CD3 single-chain antibody by a spacer unit comprising one or more units of the amino acid GGGGS (SEQ ID NO: 20). [The present invention 1026] A nucleic acid vector encoding the bispecific antibody of the present invention. [The present invention 1027] 1026. A nucleic acid vector of the present invention comprising the nucleic acid sequences of SEQ ID NO:4 and SEQ ID NO:5. [The present invention 1028] A stable cell line comprising one or more nucleic acids encoding the bispecific antibody of the invention. [The present invention 1029] 1028. The stable cell line of the present invention, wherein the one or more nucleic acids comprise the nucleic acid sequences of SEQ ID NO:4 and SEQ ID NO:5. [The present invention 1030] 1. A method for treating a mesothelin-expressing cancer in a patient, comprising: administering to said patient a bispecific antibody of the present invention, thereby treating said mesothelin-expressing cancer. A method comprising: [The present invention 1031] 1030. The method of claim 1030, wherein said mesothelin-expressing cancer is selected from the group consisting of mesothelioma, breast cancer, lung cancer, colorectal cancer, gastrointestinal cancer, endometrial cancer, bile duct cancer, and pancreatic cancer. [The present invention 1032] testing the patient by contacting a body sample from the patient with an antibody comprising a complementarity determining region (CDR) comprising the amino acid sequence of SEQ ID NOs: 7-12, thereby detecting mesothelin epitopes in the cancer of the patient. The method of the present invention 1030 further comprising: [The present invention 1033] 1030. The method of claim 1030, wherein a plurality of patients are treated by administering said bispecific antibody, said plurality of patients comprising at least one patient having an elevated level of CA125 compared to a population of healthy humans and at least one patient having a normal level of CA125. [The present invention 1034] The method of claim 1030, wherein said patient has a high level of CA125 compared to a population of healthy humans. [Brief explanation of the drawings]

[0039] [Figure 1A]Screening for anti-mesothelin antibodies not bound by the immunosuppressive CA125 protein; identification of the MES-1 antibody (SEQ ID NOs: 1 and 2). Briefly, 96-well ELISA plates were coated with 15 KU / mL soluble CA125, 1 μg / mL recombinant human mesothelin protein as a positive control, and 1 μg / mL human serum albumin (HSA) as a negative control, and probed with 2.5 μg / mL of different anti-mesothelin antibodies to determine whether they were bound by CA125. Wells were washed, and binding was detected using a secondary anti-human or anti-rodent Fc-horseradish peroxidase (HRP)-conjugated antibody and 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The reaction was stopped using 0.1 N H2SO4, and wells were quantified at 450 nm using a multiwell plate reader (Varioskan™, ThermoFisher). As shown, antibody MES-1 [Ab-3 (lane 3)] consisting of SEQ ID NOs: 1 and 2 was not bound by CA125, whereas all four other anti-mesothelin antibodies tested were bound by CA125. Values ​​were determined from the average of triplicate wells. Statistical analysis was performed using Student's T-test. [Figure 1B]These results demonstrate enhanced uptake of an anti-mesothelin antibody not bound by CA125 (see Ab-3 in Figure 1A ), in contrast to an anti-mesothelin antibody bound by CA125 (see Ab-4 in Figure 1A ). To measure antibody internalization, a pHrodo™ fluorescent assay (ThermoFisher) was used, conjugating a pH-sensitive fluorescent dye to the CA125-insensitive Ab-3 (also referred to herein as MES-1) and CA125-sensitive Ab-4 antibodies. The antibodies were tested for uptake by incubating each with the human ovarian cancer cell line OVCAR3, which expresses mesothelin and membrane-bound CA125. The parental cell line was used to generate an isogenic CA125 knockdown line (referred to as OVCAR3-KO) using shRNA. Both Ab-3 and Ab-4 uptake were measured in replicates in black 96-well microplates over 24 hours by measuring intracellular fluorescence using a Varioskan™ plate reader. As shown, Ab-3 (MES-1) was internalized similarly in both cell lines, whereas Ab-4 was internalized similarly to Ab-3 in OVCAR-KO cells but not in the parental OVCAR3 cells expressing CA125. This indicates a negative effect of CA125-mediated Ab-4 uptake, in contrast to CA125-refractory Ab-3. Statistical analysis was performed using a two-tailed Student's t-test. [Figure 2A]Cytotoxic payloads and linkers were tested to develop a reformatted MES-1 for optimal killing of immunocompetent and immunosuppressive mesothelin-expressing cancer cells. Figure 2A provides the chemical structures of various types of cytotoxic payloads (i.e., microtubule inhibitors, DNA alkylating agents, topoisomerase inhibitors, and protein kinase inhibitors; see, e.g., Yaghoubi S, et al. J Cell Physiol 235:31-64, 2019; Wang RE, et al. J Am Chem Soc 137:3229-3232, 2015) that were tested against immunosuppressive and immunocompetent mesothelin-expressing target cells. Figure 2B provides the chemical structures of various enzymatically cleavable, enzymatically uncleavable, and non-cleavable linkers that were tested to generate MES-ADCs against immunosuppressive and immunocompetent mesothelin-expressing target cells. Figure 2C provides a summary of the cytotoxicity of potential ADC payloads against mesothelin-expressing cancer cells or control cell lines (Table 1). Briefly, 96-well tissue culture plates were seeded with 5,000 cells / well of mesothelin-positive NCI-N87 (CA125+ gastric cancer), SW1990 (CA125+ pancreatic cancer), HAY (CA125+ mesothelioma), YOU (CA125- mesothelioma), OVCAR3 (CA125+ ovarian cancer), and A549 (mesothelin-negative lung cancer) cells in 100 μL of RPMI supplemented with 7.5% heat-inactivated fetal bovine serum (FBS), along with 0.0001–500 ng / mL of cytotoxin or negative control. Cultures were incubated at 37°C and 5% CO2 for 72 hours and then quantified for viability using crystal violet staining. Dried stained wells were solubilized using 1% SDS in phosphate-buffered saline (PBS) and quantified by colorimetric densitometry at 570 nm in a Varioskan™ multiwell plate reader. Several cytotoxins tested were able to significantly kill mesothelin target cells, regardless of CA125 expression, as well as non-mesothelin-expressing control cells A549 and CHO (not shown).As shown, the microtubule inhibitor MMAE, as well as the topoisomerase inhibitors SN38 and PNU159682, were found to have the highest potency, with EC50 values ​​ranging from 0.008 to 5 ng / mL. Experiments represent at least triplicate wells. Statistical analysis was performed using a two-tailed Student's T-test. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 3A] MES-1 and MES-ADC Composition, Purification, and Structural Analysis. Figure 3A shows the analysis of CHO-GS-produced and Protein A-purified MES-1 antibody. Size-exclusion (SEC) HPLC analysis of purified MES-1 demonstrated highly homogeneous antibody species, a prerequisite for generating homogeneous antibody-drug conjugates with desired drug-to-antibody ratios (DARs). Figure 3B provides a schematic diagram of the SN38-MES-ADC and PNU-MES-ADC compositions using cleavable linkers. Figure 3C shows the DAR homogeneity of SN38-MES-ADC and PNU-MES-ADC as determined by hydrophobic interaction chromatography (HIC-HPLC) and size-exclusion chromatography (SEC-HPLC). Both MES-ADCs were generated by partial reduction and cysteine ​​ligation of MES-1. DARs were calculated from HIC peak areas and retention times. As shown, SN38-MES-ADC and PNU-MES-ADC provide reproducible representative profiles for generating both of these ADCs with desired DARs of 2-6. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4A]MES-ADC Conjugate Format and In Vitro and In Vivo Target Cell Killing. Figure 4A shows target cell killing mediated by MES-ADCs containing the two most potent cytotoxins, SN38 and PNU159682, observed from the free cytotoxicity assay in Figure 2C. MES-ADCs were generated using cleavable linkers and tested for killing of various target and control cell lines listed in Table 1. Assays were performed as described in Figure 2C. Briefly, 96-well tissue culture plates were seeded with 5,000 cells / well of mesothelin-positive NCI-N87, SW1990, HAY, YOU, OVCAR3, and CHO-MES cells, as well as mesothelin-negative A549 and CHO cells as negative controls. Cells were cultured with various concentrations of MES-ADC via limiting dilution (ranging from 0.01 to 100 ng / mL) or with a negative control compound in 100 μL of RPMI supplemented with 7.5% heat-inactivated fetal bovine serum (FBS). Cultures were incubated at 37°C and 5% CO for 72 hours, and then viable cells were quantified by crystal violet staining. Dried stained wells were solubilized using 1% SDS in PBS and quantified by colorimetric densitometry at 570 nm on a Varioskan™ multiwell plate reader. As shown, SN38-MES-ADC and PNU-MES-ADC demonstrated the most significant cell killing across all mesothelin-expressing target cells, regardless of CA125 expression status, while the mesothelin-negative strains A549 or CHO (not shown) were unaffected, demonstrating the selectivity and potency of both ADCs for mesothelin-expressing cells. Next, we tested the lead MES-ADC with different linker formats (Figure 4B). Using NCI-N87 and SW1990 cells as target cells and PNU-MES-ADC as a representative MES-ADC, we performed potency analysis with enzymatically cleavable or non-enzymatically cleavable linker formats. Cells were cultured and grown as described above, with varying concentrations of each PNU-MES-ADC via limiting dilution. As shown, the enzymatically cleavable PNU-MES-ADC was significantly more potent than the non-enzymatically cleavable PNU-MES-ADC (gray and blue lines).A549 mesothelin-negative cells were unaffected by either ADC (not shown), again demonstrating the efficacy and target specificity of the MES-ADC. (Figure 4C) NCI-N87, SW1990 tumor cell lines, and a panel of PDX-derived tumors were screened for mesothelin expression via IHC using the rMES-1 detection antibody and a CA125 commercial antibody to confirm whether the expression profiles of these two important proteins were maintained in vivo. Both NCI-N87 and SW1990 cell lines, removed as tumor fragments from xenografts, maintained expression of both proteins. After screening multiple samples, two PDX tumor fragments were identified as having similar levels of mesothelin expression; one also expressed strong levels of CA125 (mesothelioma #PXF1118), while the other showed undetectable expression (non-small cell lung adenocarcinoma (NSCLC) #LXFA983). Figure 4D: In vivo testing of SN38-MES-ADC and PNU-MES-ADC. Initially, 1 x 10 tumor cells were injected into the flanks of multiple athymic nude mice for SW1990 cells or SCID mice for N87 cells. Once measurable tumor lesions (>100 mm) were established, mice were divided into groups and tested for tumor killing using cleavable and non-cleavable PNU-MES-ADC formats, SN38-MES-ADC, and PBS as a negative control. As shown, both the cleavable SN38-MES-ADC and PNU-MES-ADC formats were found to be significantly more effective in vivo than the enzymatically non-cleavable format (not shown) (upper panel: SN38-MES-ADC P<0.049 at 10 mg / kg, P<0.0003 at 20 mg / kg; lower panel: PNU-MES-ADC P<0.011), reflecting their in vitro target cell killing efficacy. (Figure 4E) The cleavable PNU-MES-ADC was tested against immunocompetent LXFA983 (CA125 low) and immunosuppressive PXF1118 (CA125 high) PDX-derived tumor xenografts to determine efficacy against these different tumor types. Tumor fragments were implanted into the flanks of athymic nude mice and treated with PNU-MES-ADC or PBS control.As shown, PNU-MES-ADC was equally effective in killing and inducing regression of both tumors regardless of microenvironment immune (CA125 expression) status (P<0.012). Experiments represent at least triplicate wells for in vitro assays and at least five subjects for in vivo assays. Statistical analysis was performed using a two-tailed Student's T-test. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 5] Single- and multiple-dose administration of cleavable PNU-MES-ADC and its antitumor effect on CA125-positive, mesothelin-expressing PXF1118 tumors. Tumor fragments were implanted into the flanks of athymic nude mice and allowed to grow to an average size of 100 mm. Mice with similar tumor sizes were divided into four groups of six and treated with PBS, 0.25 mg / kg PNU-MES-ADC, or 30 μg / kg free PNU159682 on days 1, 8, and 15 after randomization. The fourth group, using 0.75 mg / kg PNU-MES-ADC, received a single dose on day 1 after randomization, and mice were monitored for tumor growth for over 50 days. As shown, single-dose PNU-MES-ADC was equally effective in killing tumors, causing regression, and maintaining a durable response for over 50 days in mice treated with multiple doses of PNU-MES-ADC, as opposed to mice treated with PBS or free drug (P≦0.0061). Experiments represent six subjects per group. Statistical analysis was performed using a two-tailed Student's T-test. [Figure 6A]ADCC activity of MES-BSP and control antibodies against immunosuppressive cancer cells. To test the efficacy of MES-BSP immune-mediated targeting of immunosuppressive cancer cells, we employed an ADCC assay using primary peripheral blood mononuclear cells (PBMCs) and a Jurkat-CD16a ADCC reporter assay. In Figure 6A, MES-BSP, MES-1, and anti-mesothelin meso-Ab-4 (see Figure 1A, lane 4) antibodies were tested for PBMC immune-mediated killing of the human OVCAR3 ovarian cancer cell line, which expresses mesothelin and produces high amounts of the immunosuppressive CA125 protein. Briefly, 10,000 OVCAR3 cells per well in a 96-well black plate were cultured overnight in 65 μL of RPMI supplemented with 7.5% fetal bovine serum and 1% L-glutamine (R7.5). The next day, 35 μL of various concentrations of MES-BSP, MES-1, and meso-Ab-4 in R7.5 medium and 2.5 × 10 PBMCs were added to each well, and the plates were incubated at 37°C and 5% CO for 72 hours. The wells were then washed three times with 250 μL of R7.5 medium to remove the PBMCs, and the viability of the attached OVCAR3 target cells was quantified using a Varioskan™ luminescence plate reader via Cell Titer GLO® according to the manufacturer's instructions (Promega). As shown, both the MES-1 and MES-BSP antibodies demonstrated tumor cell killing, in contrast to meso-Ab-4, but MES-BSP demonstrated significantly higher target cell killing. This demonstrates the ability of MES-1 in the BSP format to provide enhanced killing over parental MES-1 alone when utilizing immune-mediated targeting. To determine the effect of the immunosuppressive CA125 protein on ADCC activity, the MES-1 and meso-Ab-4 antibodies were tested on OVCAR3 cells and the OVCAR3-CA125 knockdown (OVCAR-KO) cell line, generated using the shRNA vector TRCN0000262686 (Sigma-Aldrich) as previously described (Kline JB, et. al. OncoTarget 8:52045-52060, 2017), using the Jurkat-CD16a ADCC reporter cell line according to the manufacturer's instructions (Promega Corp).The Jurkat ADCC system employs a luciferase readout, and the luciferase signal intensity represents the ADCC activity of the antibody against target cells. As shown in Figure 6B, MES-1 had significantly higher ADCC activity against OVCAR3 than meso-Ab-4, whereas both MES-1 and meso-Ab-4 had similar ADCC activity against the OVCAR-KO cell line. This demonstrated the utility of the natural CA125-refractory MES-1 parent antibody in an alternative MES-1 format (i.e., ADC or BSP) as a drug capable of effectively killing immunosuppressive tumor cells. MES-BSP was equally effective against both the OVCAR3 and OVCAR-KO lines. All experiments were performed in triplicate. Statistical analysis was performed using a two-tailed Student's t-test. [Figure 6B] See legend to Figure 6A. [Figure 7] MES-BSP was tested in vivo using humanized PBMC nude mice and a mesothelin-positive, CA125-expressing human mesothelioma cell line. On day 0, 8 × 10 mesothelioma cells were implanted into athymic nude mice. On day 11, when the average lesion size was approximately 50 mm, 1 × 10 human peripheral blood mononuclear cells (PBMCs) were administered intraperitoneally. The following day, 3 mg / kg MES-BSP or PBS was administered three times daily for three weeks, and tumor growth was monitored (N = 5). As shown, MES-BSP caused a 70% reduction in CA125-positive mesothelioma tumors compared to control-treated mice (P = 0.033). Statistical analysis was performed using a two-tailed Student's t-test. DETAILED DESCRIPTION OF THE INVENTION

[0040] Detailed Description of the Invention The present inventors have developed novel therapeutic agents that can effectively kill mesothelin-positive cancer cells, regardless of the immune status of the tumor cell microenvironment. Without wishing to be limited to a particular theory or mechanism of action, Applicants believe that the amino acid sequences encoded by SEQ ID NOS: 1 and 2 (referred to herein as MES-1 antibodies) and SEQ ID NOS: 2 and 3 (referred to herein as MES-BSP antibodies) are refractory to binding by the immunosuppressive protein CA125, which negatively impacts both: (a) antibody-mediated immune responses (Kline JB, et. al. OncoTarget 8:52045-52060, 2017; Kline JB, et al. Eur J Immunol 48:1872-1882, 2018; Kline JB, et al. J Clin Oncol 5:15, 2018; Nicolaides NC, et al. Cancer Biol Ther 13:1-22, 2018), and (b) antibody-drug conjugate (ADC) uptake and internalized delivery of cytotoxic agents, which is a prerequisite for potent ADC-mediated target cell killing (Nicolaides NC, et. al. U.S. Patent Application No. 16 / 984,444; Chalouni C and Doll SJ Exp Clin Cancer Res 37:20-32, 2018). This immunosuppressive mechanism is thought to involve direct binding of CA125 to the affected antibody (Nicolaides NC, et al. Cancer Biol Ther 13:1-22, 2018).

[0041] Additionally, the present application provides methods for identifying additional antibody-based therapies that are effective against tumors regardless of the tumor microenvironment immune status. These include testing parent antibodies for binding to tumor-produced immunosuppressive proteins, such as CA125. Such antibodies can be formatted into antibody-drug conjugate or bispecific formats and empirically tested to optimize efficacy in killing tumors with an immunosuppressive microenvironment by using various payloads as well as gene fusions and chemical linkers.

[0042] The methods and kits described herein can be used to monitor and confirm the eligibility of mesothelin-expressing patients for treatment with MES-ADC or MES-BSP by testing the patient's tumor cells with antibodies containing the CDR amino acid sequences of SEQ ID NOS: 7-12 using methods for antigen expression testing known in the art.

[0043] The methods, compositions, and kits can be classified into two categories of antibody formats. In one category, MES-ADCs are developed that contain an immunoglobulin light chain (SEQ ID NO: 1) and heavy chain (SEQ ID NO: 2) chemically conjugated to a cytotoxic agent. Direct binding to the antibody component by CA125 is low or zero. The number of cytotoxic moieties per antibody molecule (referred to as the drug:antibody ratio (DAR)) can vary depending on the method of conjugation, with a minimum DAR of 2 and a maximum DAR of 12, preferably 2, 3, 4, 5, or 6. The cytotoxic agent can be, but is not limited to, the topoisomerase inhibitor SN38 or PNU159682. The cytotoxic moiety is attached to the antibody by a chemical or peptide linker. The linker can be cleavable or non-cleavable, as known to those skilled in the art. The desired combination of linker, cytotoxin, and DAR can be empirically determined to optimize the activity of the MES-ADC against mesothelin-mediated tumor cell killing in vitro and / or in vivo. Tumor cell viability can be determined using methods known in the art.

[0044] Suitable cytotoxins include, but are not limited to, safe and preferably biodegradable cytotoxins, such as monomethyl dolastatin 10, auristatin E, monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F, HTI-286, tubulysin M, maytansinoid AP-3, maytansinol, DM1, DM4, Boc-Val-Dil-Dap-OH, Boc-Val-Dil-Dap-Phe-Ome, Boc-Val-Dil-Dap-Doe, Boc-Val-Dil-Dap- These include, but are not limited to, Nrp, Boc-N-Me-Val-Val-Dil-Dap-OH, Tubulysin IM-1, Tubulysin IM-2, Tubulysin IM-3, Dasatinib, Duocarmycin SA, Duocarmycin TM, Duocarmycin MA, Duocarmycin DM, Nemorubicin, PNU-159682, Calicheamicin γ1, N-acetyl-calicheamicin γ1, α-amanitin, PBD-dimer, etc., the structures of which are known in the art.

[0045] Suitable linkers include, but are not limited to, safe and preferably biodegradable linkers, such as Val-Cit-PAB, Fmoc-Val-Cit-PAB, Fmoc-Val-Cit-PAB-PNP, MC-Val-Cit-PAB-PNP, Phe-Lys(Trt)-PAB, Fmoc-Phe-Lys(Trt)-PAB, Fmoc-Phe-Lys(Trt)-PAB-PNP, Ala-Ala-Asn-PAB TFA salt, Fmoc-Ala-Ala-Asn-PAB-PNP, Fmoc-Gly3-Val-Cit-PAB, Fmoc-Gly3-Val-Cit-PAB-PNP, MAC glucuronide phenol, Py-ds-Prp-OSu, Py-d s-dmBut-OSu, Py-ds-dmBut-OPFP, Py-ds-Prp-OPFP, SMCC, MAL-HA-OSu, MAL-di-EG-OPFP, MAL-tri-EG-OPFP, MAL-tetra-EG-OPFP, MA These include, but are not limited to, PEG-VC-PAB-DMAE, MC-EDA, N3-di-EG-OPFP, N3-tri-EG-OPFP, N3-tetra-EG-OPFP, ALD-BZ-OSu, ALD-di-EG-OSu, ALD-tetra-EG-OSu, ALD-di-EG-OPFP, ALD-tetra-EG-OPFP, PHA-di-EG-OPFP, PHA-tetra-EG-OPFP, PEG-triazole-PABC-peptide-mc, etc. These chemical structures are known in the art.

[0046] One embodiment is an MES-ADC that has low CA125 binding and comprises the MES-1 antibody (SEQ ID NOs: 1 and 2) covalently attached to the PNU159682 topoisomerase inhibitor by a MA-PEG4-VC-PAB-DMAE cleavable linker.

[0047] Another embodiment is an MES-ADC that has low CA125 binding and comprises the MES-1 antibody (SEQ ID NOs: 1 and 2) covalently linked to an SN38 topoisomerase inhibitor by a MAC glucuronide phenol or PEG8-triazole-PABC-peptide-mc cleavable linker.

[0048] In another category of antibody formats, MES-BSP (bispecific antibody) contains the immunoglobulin heavy chain of the MES-1 antibody (SEQ ID NO: 2) and a chimeric light chain (SEQ ID NO: 3), which has low CA125 binding. The chimeric light chain is a genetic fusion (amino to carboxyl order) of an anti-CD3 single-chain antibody (SEQ ID NO: 6) fused to the MES-1 light chain (SEQ ID NO: 1). The anti-CD3 single-chain antibody moieties are genetically linked via a spacer containing any one of 20 known natural or modified amino acids, and the linker may be two or more amino acids separating the light chain from the single chain, as known in the art. The desired linker amino acid composition and length can be determined by the amino acid sequence of the human CD3 + The activity of MES-BSP in tumor cell killing in vitro and / or in vivo in the presence of lymphocytes can be empirically determined to optimize tumor cell viability. Tumor cell viability can be determined using any of a variety of methods known in the art.

[0049] In therapeutic applications, MES-ADC or MES-BSP can be administered as monotherapy or in combination with standard of care.

[0050] Compositions can be formed during the course of practicing the methods. They can be preformed, individually packaged, and provided in entities that have a cytotoxin library for screening, or that, for example, allow for the use of various linkers to link cytotoxins to MES-1 antibodies. Similarly, components of the assays and methods described herein can be packaged together in a container and sold as a kit. Kit components can, but need not, be mixed together. They can, for example, be provided in separate or divided containers. Any selection of detection antibodies and MES-ADCs or MES-BSPs described herein can be assembled into compositions or kits.

[0051] While several known antibodies are highly sensitive to CA125 immunosuppression, the compositions described herein are useful for treating patients with mesothelin-expressing cancers, regardless of the tumor microenvironment immune status, with MES-ADC or MES-BSP, each of which contains one or more of the MES-1 parent sequences (SEQ ID NOS: 1-3) that are refractory to CA125 binding, thereby enabling effective ADC internalization and killing in the MES-ADC format and effective immune-mediated killing in the MES-BSP format.

[0052] In some instances, MES-ADC may need to be formulated in liposomes to enhance its therapeutic window in patients with mesothelin-expressing cancers. Any liposome formulation for delivery of MES-ADC may be used to treat patients. Conventional liposomes consist of a lipid bilayer composed of cationic, anionic, or neutral (phospho)lipids and cholesterol, encapsulating an aqueous volume. Suitable liposome compositions include, but are not limited to, the guanidinium-cholesterol cationic lipid bis(guanidinium)-tren-cholesterol (BGTC) combined with the co-lipid dioleoylphosphatidylethanolamine (DOPE). Another example of a suitable liposome formulation is the aminoglycoside lipid dioleylsuccinylparomomycin (DOSP) with the imidazole-based helper lipid MM27. Liposomes can be sterically stabilized, for example, by coating the liposomes using polyethylene glycol.

[0053] The present inventors provide compositions, kits, and methods for identifying patients with mesothelin-positive cancers and treating them with MES-ADC or MES-BSP. The methods may include diagnosing the patient's tumor for mesothelin expression using a CA125-refractory MES-1 detection antibody containing the CDRs of SEQ ID NOS: 7-12. If the assay is positive, the tumor can be treated with MES-ADC (SEQ ID NOS: 1 and 2) or MES-BSP (SEQ ID NOS: 2 and 3), optionally conjugated to a cytotoxin that is a topoisomerase inhibitor. The diagnostic and therapeutic steps may be independent or may be performed in conjunction. For cancers in which mesothelin is highly overexpressed, the use of such preliminary screening may be optional.

[0054] Another embodiment is a MES-ADC containing CDRs (SEQ ID NOs: 7-12), any of which may be modified by up to three amino acids individually or in combination, so long as they remain CA125 refractory.

[0055] In another embodiment, the cytotoxin of the MES-ADC is SN38 or PNU159682 linked to a cleavable linker. The linker is a PEG8-triazole-PABC-peptide-mc linker (C 50 H 79 N9O 16 ) (the entire construct is referred to as SN38-MES-ADC-1), a MAC glucuronide phenol-linker attached to SN38 (the entire construct is referred to as SN38-MES-ADC-2), or a MA-PEG4-VC-PAB-DMAE linker attached to PNU159682 (the entire construct is referred to as PNU-MES-ADC).

[0056] In another embodiment, the MES-1 light chain (SEQ ID NO: 1) is linked to the CD3 single chain (SEQ ID NO: 6) using an amino acid linker unit composed of the amino acids GGGGS (SEQ ID NO: 20). The linker is composed of one or more linker units. By way of example, and without wishing to be limited by length or amino acid composition, the MES-1 light chain and the anti-CD3 single chain may be linked to one, two, three, or more units. Linker optimization can be performed by measuring the CD3 linker using any method used in the art to measure tumor cell killing as described herein. + Optimal killing of mesothelin target cells can be determined in the presence of lymphocytes.

[0057] In yet another embodiment, the linker unit connects the MES-1 light chain to an immunocompetent or immunosuppressive microenvironment and human CD3 + Any combination of known natural or modified amino acids, and any length, can be genetically linked to an anti-CD3 single chain that can be empirically tested to optimize tumor cell killing in mesothelin-positive tumors with lymphocytes.

[0058] Another embodiment has a MES-BSP containing the amino acid sequences of SEQ ID NOs: 7-18, any of which may be modified by up to three amino acids individually or in combination, so long as they remain refractory to CA125 binding.

[0059] In some embodiments, functional methods are used to generate CD3 using various gene linkers. + Optimizing the effect of MES-BSP on killing mesothelin-positive tumor cells in the presence of lymphocytes. The term "effect" generally refers to the effect of CD3 + It refers to a 10% or greater change in target cell killing compared to lymphocytes when the agent is incubated alone, and depending on the antibody and agent used, it can also refer to at least a 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75% change compared to the control.

[0060] Yet another embodiment includes a method for screening antibody drug conjugates (ADCs) with various cytotoxins and / or linkers for pharmacokinetic (PK), pharmacodynamic (PD), or pharmacological (PL) activity, including cellular internalization. In some embodiments, the ADCs are added to cells in vitro and tested for target cell killing. ADCs with significant killing effects are suitable for therapeutic testing. Similarly, the term effect can refer to a change of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75% compared to a control, depending on the antibody and drug used.

[0061] Various terms and terminology ("terms") relating to the embodiments contained herein are used throughout the specification and claims herein. Such terms should be given their ordinary meaning in the art unless otherwise specified. Other specifically defined terms should be construed in a manner consistent with the definition provided.

[0062] As used herein and in the appended claims, the singular forms "a," "an," and "the" also include plural references unless the context clearly dictates otherwise. By way of example, reference to a "cell" can include a combination of two or more cells, etc. Reference to a "probe" can include a detection antibody, MES-ADC, MES-BSP, or an independent probe for monitoring the tumor microenvironment immune status via any analytical method known in the art.

[0063] The term "about," when used in reference to a quantified value, such as an amount, duration, or the like, is intended to encompass a variation of up to ±9% from the specified value, since such variation is appropriate for carrying out the disclosed method. Unless otherwise indicated, all values ​​expressing quantities of reagents, such as molecular weights, molar concentrations, reaction conditions, percentages, etc., used in the specification and claims should be understood in all instances as being quantified by the term "about." Accordingly, unless indicated to the contrary, the numerical values ​​set forth in the following specification and written claims are approximations that may vary depending on the desired properties of the compositions, agents, and / or methods sought to be obtained by the present invention. At the very least, and not intended to limit the scope of this application, each numerical value should be evaluated at least in accordance with the number of reported significant digits and by ordinary rounding techniques known in the art.

[0064] The term "antibody" as used herein is intended broadly to encompass antibody molecules, including immunoglobulins (also referred to as "Ig") or polyclonal antibodies (also referred to as pAb), monoclonal antibodies (also referred to as mAb), including murine, human, humanized, and chimeric mAbs, bispecific antibodies (also referred to as BSPs), antibody-drug conjugates (also referred to as ADCs), antibody-fusion immunotoxins, and antibody fragments. Generally, antibodies are proteins or polypeptide chains that bind to a specific antigen. The antigen is the structure specifically recognized by a given antibody. A standard antibody is a heterotetrameric glycosylated protein composed of two light chains and two heavy chains lined through a complex of disulfide and hydrogen bonds. The term "disulfide bridges thereof" refers to disulfide bridges contained within the heavy chain hinge region, as known in the art. Each heavy chain contains a variable domain (VH) followed by a number of constant domains (called Fc domains). Each light chain contains a variable domain (VL) and a constant domain, with the constant domain of the light chain aligned with the first constant domain of the heavy chain, and the light chain VL aligned with the variable domain of the heavy chain. The antibody light chains of any species can be assigned to one of two different types, kappa (κ) and lambda (λ), based on the amino acid sequence within the constant domain.

[0065] The term "single chain" refers to a single chain antibody, the structures of which are known in the art.

[0066] An immunoglobulin light chain (LC) or heavy chain (HC) contains a "framework" region flanking three "antigen-binding sites," also called complementarity-determining regions (CDRs) based on reported sequence variability (Wu TT, Kabat EA. J Exp Med 132:211-250, 1970). Generally, an antigen-binding site is composed of six CDRs: three in the VH (CDRH1, CDRH2, CDRH3) and three in the VL (CDRL1, CDRL2, CDRL3) (Kabat EA, et al. 5 thEd. PHS, National Institutes of Health, Bethesda, Md., 1991).

[0067] "Specific binding" or "specifically binds" refers to an antibody or antigen-binding fragment that binds to an antigen (including sequences contained within the antibody itself) with greater affinity than it does to other antigens. Typically, a specific antibody or antigen-binding fragment binds to an antigen with a higher affinity than about 5 x 10 -6 Equilibrium dissociation constant K below M D binds to the target antigen.

[0068] "Antibody derivative" or "alternative format" refers to an antibody, as defined above, that is modified by the covalent attachment of another molecule, such as through peptide chemistry (i.e., amidation), gene fusion, and / or post-translational moieties not typically associated with antibodies (i.e., glycosyl, acetyl, and / or phosphoryl).

[0069] The term "antibody dynamic conformation" refers to any change in structure that may affect antibody function, CA125 binding, or cellular internalization.

[0070] The term "monoclonal antibody (mAb)" refers to an antibody that is derived from a single cell clone, including any eukaryotic or prokaryotic cell clone, or a phage clone, and not the method by which it is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology, but can also include recombinant methods.

[0071] "Fab domain" refers to any antibody sequence from the N-terminus through the antibody hinge disulfide region, as known in the art.

[0072] An "Fc domain" is known in the art and refers to any antibody sequence comprising the C-terminus to the antibody hinge disulfide region.

[0073] "Mesothelin" refers to the whole protein or naturally modified forms of the mesothelin protein (GenBank: AAH03512.1).

[0074] An "antigen" is an entity to which an antibody or antibody fragment specifically binds. This includes binding to an antibody or protein of interest.

[0075] The term "CA125" refers to the gene product of the MUC16 gene (HGNC:15582; OMIM:606154), which exists in soluble and membrane-bound forms. It binds to antibodies within the Fab domain and has been reported to affect the humoral immune function of the bound antibody (Kline JB, et al. Oncotarget 8:52045-52060, 2017) and ADC uptake.

[0076] The term "CA125-refractory" refers to antibodies that exhibit low or no binding to CA125 protein as measured using any method known in the art.

[0077] The terms "CD3" and "CD3E" refer to the CD3-epsilon protein expressed on human lymphocytes.

[0078] The terms "cancer," "malignant," "dysregulated," and "tumor" are well known in the art and refer to the presence of cells, also referred to as dysregulated cells, that have uncontrolled cell growth and morphological characteristics that differ from normal cell types of similar origin. Malignant refers to cancer cells that have the potential to cause morbidity and / or mortality. As used, "cancer and tumor" includes precancerous and malignant types.

[0079] As used, the term "soluble" refers to a protein or non-protein agent that is not attached to the cell membrane of a cell. For example, a soluble agent can be released, secreted, or exported from normal or cancerous cells into biological fluids, including serum, whole blood, plasma, urine, or the microfluids of cells, including tumors.

[0080] As used herein, the "level" of a specified protein or non-protein agent (including CA125) refers to one or more levels of the agent determined using any method known in the art for measuring protein and / or non-protein agent levels in vitro or in vivo. Such methods include gel electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitation, absorption spectroscopy, colorimetry, spectrophotometry, flow cytometry, immunodiffusion (single or duplex), liquid-phase assays, immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, fluorescence resonance energy transfer (FRET), Förster resonance energy transfer, electrochemiluminescence immunoassay, and the like. In one embodiment, the level of CA125 is determined using probe-based technology, as described in more detail below.

[0081] The term "humoral immunosuppression" refers to any antibody, antibody fragment, bispecific antibody (BSP), or antibody-drug conjugate (ADC) that is directly bound by CA125 and whose dynamic structure is altered. CA125 is produced by malignant cells such as ovarian cancer and mesothelioma (Nicolaides NC, et al. Cancer Biol Ther 19:622-630, 2018) and is induced by lymphoma from normal surrounding epithelial cells (Sanusi et al. Perit Dial 21:495-500, 2001). It has been reported that CA125 binds to certain antibodies and alters their dynamic structure, potentially affecting biological activities such as ADCC, CDC, opsonization, and internalization, as well as PK, PD, and PL profiles.

[0082] The term "antibody drug conjugate (ADC)" refers to any antibody conjugated or fused to a chemical, polypeptide, nucleic acid, or radionuclide that has toxic activity against a cell.

[0083] The term "cleavable linker" refers to a chemical or amino acid linker that can be cleaved extracellularly or intracellularly by any common mechanism, including, but not limited to, enzyme or protease digestion, acid degradation, pH, chemical reduction, chemical oxidation, hydrolysis, etc.

[0084] The term "non-cleavable linker" refers to a chemical or amino acid linker that is not generally cleaved extracellularly by common mechanisms such as, but not limited to, enzyme or protease digestion, chemical reduction, chemical oxidation, hydrolysis, etc.

[0085] The terms "enzymatically cleavable linker" and "enzymatically non-cleavable linker" refer to a linker that is cleavable or non-cleavable by an enzyme or protease.

[0086] The term "bispecific antibody (BSP)" refers to any antibody that can bind to two or more different antigens. A BSP can include, but is not limited to, at least two full-length antibodies, one full-length antibody and one single-chain antibody, or two single-chain antibodies, each binding to a different antigen or to different epitopes on the same antigen.

[0087] The term "standard antibody" refers to an immunoglobulin light chain bound to an immunoglobulin heavy chain, which antibody is capable of specifically recognizing the antigen. The standard antibody can be fused to another antibody capable of specifically recognizing a second antigen.

[0088] The terms "immunosuppressed microenvironment," "immune-suppressed microenvironment," "immunosuppressed tumor microenvironment," and "immune-suppressed tumor microenvironment" refer to tumors that produce or express factors that suppress cellular or humoral immune function and activity, such as, but not limited to, PDL1 or CA125, respectively.

[0089] The terms "immunocompetent microenvironment," "immuno-competent microenvironment," "immunocompetent tumor microenvironment," "immuno-competent tumor microenvironment," "immune-proficient," "immunoproficient," and "immune-proficient" refer to tumors that do not produce or express factors that suppress cellular or humoral immune function or activity.

[0090] "Immune or immunomicroenvironment status" or "microenvironment immune status" refers to the determination of whether a tumor is immunoresponsive or immunosuppressive. The term also refers to tumors that produce immunosuppressive proteins, and tumors that produce such proteins are considered to have an immunosuppressive microenvironment.

[0091] The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to an in vitro or in vivo process by which antibodies bind to antigens on the surface of cells and can then associate with immune-effector cells via sequences within the Fc domain of the antibody, resulting in the release of toxins that can kill the bound cells.

[0092] The term "complement-dependent cytotoxicity (CDC)" refers to an in vitro or in vivo process in which an antibody binds to an antigen on the surface of a eukaryotic or prokaryotic cell and can then associate with the C1q protein via a sequence within the Fc domain of the antibody, thereby initiating the classical complement cascade and killing the bound cell.

[0093] The term "internalization" refers to the process by which an antibody, antibody fragment, or ADC can bind to an antigen on the surface of a cell and then be internalized through mechanisms known to those of skill in the art.

[0094] The term "pharmacokinetics (PK)" refers to the time that an antibody maintains its steady-state concentration when administered to a subject.

[0095] The term "pharmacodynamics (PD)" refers to the study of the biochemical and physiological effects of antibody-based drugs and their mechanisms of action, including the correlation of their biochemical structure with their actions and effects when administered to a subject.

[0096] The term "pharmacology (PL)" refers to the known effects of an antibody in managing or killing diseased cells in vitro or in vivo.

[0097] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Fluids can include biological fluids, including liquid solutions that have come into contact with a subject or biological source, including cell and organoid culture media, urine, saliva, lavage fluids, etc.

[0098] As used, the term "control sample" refers to any clinically or non-clinically relevant control sample, including, for example, a sample from a healthy subject not afflicted with a particular cancer type or a cell different from its parent cell.

[0099] The term "control level" refers to an accepted or predetermined level of a protein or non-protein agent used to compare with the level of the same agent in a sample derived from a subject or used in an in vitro assay.

[0100] As used herein, the "difference" between the signal of a therapeutic antibody and a control is generally any difference that can be statistically determined using statistical methods commonly used in the art, and is at least a 10% or greater difference compared to the control. Depending on the antibody and probe used, it may also refer to a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75%.

[0101] The term "inhibit" or "inhibition of" means to reduce by a statistically measurable amount or to prevent entirely.

[0102] The term "functional" in reference to antibodies, antibody-containing moieties (i.e., BSPs, ADCs, etc.) used in accordance with the described methods indicates that the antibody is capable of binding to an antigen or CA125, respectively, and / or is capable of binding to and killing target cells in vitro or in vivo.

[0103] The term "target cell" refers to a eukaryotic or prokaryotic cell or cell population that expresses an antigen for a particular antibody or antibody-containing moiety.

[0104] The term "therapeutic window" refers to the effectiveness of a drug in inhibiting tumor growth within a manageable and tolerably toxic dose.

[0105] The term "pharmaceutically acceptable" refers to a substance that is pharmacologically and toxicologically acceptable for administration to a patient and that is manufactured using techniques known in the art. This includes agents approved by federal or state regulatory agencies or listed in the United States Pharmacopoeia or other generally recognized pharmacopeias for animal and human use. The term "pharmaceutically compatible ingredient" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or matrix vehicle used to administer an anticancer agent. A "pharmaceutically acceptable carrier" refers to a matrix that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is nontoxic to the host.

[0106] The terms "effective amount" and "therapeutically effective" are used interchangeably and in reference to administering a pharmaceutical agent in an amount sufficient to produce an enhanced clinical outcome in a patient. An effective amount of the agent is administered in an "effective regimen" according to the methods described herein. The term "effective regimen" refers to a combination of an amount of the agent and frequency of administration sufficient to achieve an enhanced clinical outcome for a patient with a particular cancer. Enhanced efficacy is an improved clinical outcome when a patient is administered an agent that can overcome a pathological condition better than the parent compound or that can enhance the clinical outcome of an effective regimen.

[0107] The terms "patient" and "subject" are used interchangeably to refer to humans and other non-human animals, including veterinary subjects, receiving therapeutic agent treatment. The term "non-human animal" includes all vertebrate animals. In one embodiment, the subject is a human.

[0108] A "therapeutic agent" is typically substantially free of undesired contaminants, meaning that the agent is typically at least about 50% w / w (weight / weight) pure and substantially free from interfering proteins and contaminants.

[0109] The term "immune effector cell" refers to any cell, including but not limited to, NK, myeloid, monocyte, or dendritic cell, that can effect antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (opsonization) upon binding to an antibody-bound target cell. The cells can be purified or present in admixture in the form of peripheral blood mononuclear cells (PBMC).

[0110] The term "dysregulated cell" refers to any cell that is considered abnormal relative to the parent cell. These include transformed cells, malignant cells, virally infected cells, cells that grow autonomously through autoregulation, or prokaryotic pathogens.

[0111] The term "humoral response" refers to ADCC, CDC, opsonization, or internalization of the antibody into target cells by the test antibody.

[0112] The term "drug" refers to an anti-mesothelin ADC or BSP.

[0113] The term "significantly" refers to a statistical result with a P value of less than 0.05 as determined by any number of programs, including Student's T-test.

[0114] Detection antibodies, therapeutic MES-ADC and MES-BSP compositions; kits and methods for treating patients with mesothelin-expressing cancers Provided herein are compositions, kits, and methods for identifying such agents that can effectively inhibit mesothelin-positive cancers, regardless of their microenvironmental immune status, including MES-ADC and MES-BSP (both referred to as agents). In some embodiments of the methods described herein for identifying optimal MES-ADCs and MES-BSPs, the methods involve identifying antibody components of ADCs and / or BSPs that are CA125-refractory and can circumvent either of their negative tumor cell killing activities (i.e., tumor uptake of ADCs, immune responses of bispecific antibodies, etc.). In other embodiments, CA125-refractory MES-ADCs are composed of two or more cytotoxins linked via cleavable or non-cleavable linkers, and the ability of the MES-ADC to significantly improve cytotoxicity against mesothelin-expressing immunosuppressive target cells is tested for efficacy against immunocompetent target cells. In some embodiments, the MES-ADC cytotoxin is a topoisomerase inhibitor of the SN38 or PNU159682 class. In another embodiment, the MES-ADC has a drug-to-antibody ratio (DAR) of 2-6 and comprises an MES-1 antibody conjugated to SN38 via a MAC glucuronide phenol or PEG8-triazole-PABC-peptide-mc linker. In yet another embodiment, the MES-ADC has a DAR of 2-6 and comprises an MES-1 antibody conjugated to PNU159682 via a MA-PEG4-VC-PAB-DMAE linker. An example is shown schematically in Figure 3B. The kit is comprised of an MES-ADC that can identify optimal ADC formats (cytotoxins and linkers) through screening assays used in the art, including ADC killing assays against immunosuppressive and immunocompetent mesothelin-expressing tumor types. Additionally, the kit consists of an optimal MES-ADC and an rMES-1 detection antibody, both of which bind to mesothelin in the presence or absence of CA125, allowing for the identification of patients with mesothelin-expressing cancers for treatment with MES-ADC, regardless of the tumor microenvironment immune status.

[0115] Another embodiment is a method for identifying an optimal MES-BSP. This method involves generating an optimized MES-BSP, where the MES-BSP light chain comprises the amino acids listed in SEQ ID NO: 1 or the heavy chain comprises the amino acids listed in SEQ ID NO: 2 linked at its N-terminus to an anti-CD3 single-chain antibody (SEQ ID NO: 6). In another embodiment, the MES-BSP comprises an MES-1 light chain fused to an anti-CD3 single-chain via a genetically linked spacer. The spacer can be composed of any combination and length of natural or modified amino acids, but one optional linkage between the MES-1 light chain and the CD3 single chain is via the genetically encoded linker unit(s) "GGGGS (SEQ ID NO: 20)." Linkage can be via one or more units. The optimal spacer unit can be determined using functional killing assays of immunosuppressive and immunocompetent mesothelin-expressing target cells using assays commonly used in the art. An example of a screen is discussed in Example 3, and the results are shown in Figure 6. In another embodiment, a kit is comprised of optimal MES-BSP and rMES-1 detection antibodies to identify patients with mesothelin-expressing cancers for treatment with MES-BSP, regardless of the tumor microenvironment immune status.

[0116] In a method for identifying optimally active MES-ADC or MES-BSP agents, the antibody is added to a culture of mesothelin-expressing target cells that naturally or recombinantly express an immunosuppressive protein. Cultures are monitored for target cell viability using standard killing assays, comparing responses to MES-ADC- or MES-BSP-treated cells and control-treated cells. A change of at least 10% is typically considered a significant effect. Depending on the agent and assay used, a significant effect can also be defined as a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75%.

[0117] In some methods for identifying optimally active MES-ADCs or MES-BSPs, drugs or exogenously added soluble CA125 protein are added to cultures of mesothelin-expressing target cells that naturally express CA125. Cultures that compare the response of MES-ADC or MES-BSP and CA125 treatment with control or no CA125 treatment are monitored for target cell viability using standard killing assays. A change of at least 10% is typically considered a significant effect on target cell killing by ADCC, CDC, and / or ADC. Depending on the drug and assay used, a significant effect can also be defined as a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75%.

[0118] Also provided herein are methods for treating cancer subjects using MES-ADC or MES-BSP agents. For example, patients may have mesothelin-expressing cancers, such as, but not limited to, mesothelioma, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, bile duct cancer, or endometrial cancer. Several anti-mesothelin antibodies have been reported to be bound by CA125 (see Figure 1), which may disrupt their internalization as ADCs or suppress their immunokilling effects as BSPs, making the use of MES-ADCs or MES-BSPs that are not bound or affected by CA125 desirable.

[0119] In some embodiments of methods for treating a subject with an MES-ADC or MES-BSP agent, patients with cancers that express immunosuppressive proteins, such as CA125, rendering their tumor microenvironment immunosuppressive may be treated with the MES-ADC or MES-BSP agent alone or in combination with standard of care. In some embodiments of methods for treating a subject with an immunosuppressive microenvironment, mesothelin-expressing cancers known to express CA125 are described herein. The MES-ADC or MES-BSP agent is administered to the subject if the subject has a baseline CA125 level above the normal range. In some embodiments of methods for treating a subject with a CA125-expressing cancer described herein, the method comprises administering the MES-ADC or MES-BSP agent alone. In yet another embodiment, the MES-ADC or MES-BSP agent is administered in combination with chemotherapy. The chemotherapy can be any chemotherapeutic or biologic agent considered standard of care at the time the subject is treated. In the methods of treatment described herein, CA125 expression levels may be determined by any means known in the art and may be defined in the art as being within or above the normal range.

[0120] In other embodiments, patients are identified as having a mesothelin-expressing cancer using an rMES-1 detection antibody, and patients positively bound by rMES are treated with an MES-ADC or MES-BSP agent without determining the tumor microenvironment immune status, since MES-ADC or MES-BSP is effective in both immunosuppressive and immunocompetent microenvironments. In some embodiments of the methods of treating a subject with a mesothelin-expressing cancer described herein, the method comprises administering an MES-ADC or MES-BSP agent alone. In yet another embodiment, the MES-ADC or MES-BSP agent is administered in combination with chemotherapy. The chemotherapy can be any chemotherapeutic or biologic agent considered standard of care at the time the subject is treated.

[0121] In some embodiments of the methods of treatment described herein, exemplary cancers known to express mesothelin include, but are not limited to, mesothelioma, lung cancer, colorectal cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer, breast cancer, and pancreatic cancer, many of which have been reported to produce CA125.

[0122] The methods of the invention can be combined with other therapeutic modalities, such as surgery (e.g., debulking surgery), radiation, targeted therapy, chemotherapy, immunotherapy, the use of growth factor inhibitors, or anti-angiogenic factors. The MES-ADC or MES-BSP agent can be administered simultaneously to a patient undergoing surgery, chemotherapy, or radiation therapy. Alternatively, the patient can receive surgery, chemotherapy, or radiation therapy before or after administration of the standard of care, at least one hour and up to several months before or after administration of the MES-ADC or MES-BSP agent. Some embodiments of the treatment methods provided herein comprise administering to the subject, in addition to the MES-ADC or MES-BSP agent, a therapeutically effective amount of a platinum-based chemotherapy and / or an antifolate and / or a PARP inhibitor, with or without an antibody against a tumor-specific antigen or an immune checkpoint protein.

[0123] In some embodiments of the methods of treatment described herein, the subject may have received first-line surgical resection of the tumor, first-line platinum-based therapy, first-line antifolate-based therapy, first-line platinum and antifolate-based therapy, a PARP inhibitor and / or an immune checkpoint inhibitor for the treatment of cancer prior to administration of the MES-ADC or MES-BSP agent.

[0124] Administration of therapeutic agents (including MES-ADC or MES-BSP agents, antifolates, platinum-based chemotherapy, PARP inhibitors and / or immune checkpoint inhibitors) according to the methods of treatment described herein can be by any means known in the art.

[0125] In yet another embodiment, MES-ADC or MES-BSP agents containing CDR sequences contained within SEQ ID NOS: 7-12, numbered according to IMGT® (international ImMunoGeneTics information system®), other than standard antibody formats, may be used. Administration of these modified MES-ADC or MES-BSP agents may precede, coincide with, or follow administration of any additional standard of care. Treatment may include surgery and treatment with the current standard of care used at the time of treatment.

[0126] Where appropriate, therapeutic agents (including MES-ADC or MES-BSP agents) can be administered using a variety of delivery systems, including intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Agents can be administered, for example, by infusion or bolus injection, via systemic or local approaches, or by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa, etc.).

[0127] The therapeutic agent can be administered by injection via a syringe, catheter, suppository, or any implantable matrix or device.

[0128] The therapeutic agents for use as described herein and pharmaceutical compositions thereof may be administered orally in any acceptable dosage form, such as capsules, tablets, aqueous suspensions, solutions, and the like.

[0129] Suitable methods of administering the therapeutic agent include, but are not limited to, intravenous injection and intraperitoneal administration at a final concentration suitable for effective treatment.

[0130] MES-ADC or MES-BSP agents in combination with other drugs can be administered as pharmaceutical compositions containing a therapeutically or prophylactically effective amount of the therapeutic agent(s) and one or more pharmaceutically acceptable or compatible ingredients.

[0131] The amount of a therapeutic agent that is effective in treating or preventing cancer can be determined by standard clinical techniques. Additionally, in vitro assays can optionally be used to help identify optimal dosage ranges required for the MES-ADC or MES-BSP agent. Effective amounts can be extrapolated from dose-response curves for the MES-ADC or MES-BSP agent derived from in vitro cell-based assays, animal models, or other non-human test systems.

[0132] For example, the toxicity and therapeutic efficacy of a drug can be determined by the LD 50 (lethal dose for 50% of the population) and ED 50 The LD50 value (the dose therapeutically effective in 50% of the population) can be determined in cell cultures or experimental animals by standard pharmaceutical procedures. The dose ratio between toxic and therapeutic effects is the therapeutic index or therapeutic range, and the LD50 value is the therapeutic index. 50 / ED 50 The therapeutic index can be expressed as a ratio. Drugs that exhibit a large therapeutic index are preferred. If the drug exhibits toxic side effects, a delivery system that targets the drug to the site of the affected tissue can be used to minimize potential damage to non-mesothelin-expressing cells, thereby reducing side effects. Alternatively, if necessary, formulations such as, but not limited to, liposome encapsulation can be used to improve the therapeutic index.

[0133] Dosage and dosing schedules can vary depending on the active drug concentration, which can depend on the needs of the subject.

[0134] Another embodiment labels rMES-1 detection antibodies to detect mesothelin-expressing cells in CA125-expressing or non-expressing tumors for diagnostic applications to detect and / or monitor the status of mesothelin-expressing tumor cells in vitro or in situ during or after treatment with a MES-ADC or MES-BSP agent. Labeling can be by any method known in the art for labeling antibodies for diagnostic monitoring.

[0135] Kit construction for optimizing the activity of MES-ADC and MES-BSP Further provided herein are kits for generating optimized MES-ADC and MES-BSP agents suitable for killing immunosuppressive and immunocompetent mesothelin-expressing tumors.

[0136] The kit may include an MES-ADC agent containing a cytotoxin conjugated to an antibody consisting of SEQ ID NOs: 1 and 2 that is capable of killing two or more types of mesothelin-expressing cells or tumors regardless of the microenvironment immune status, wherein the cytotoxin has topoisomerase inhibitory activity with an IC50 of 100 μM or less.

[0137] The kit may also include a MES-BSP agent containing an antibody or antibody fragment having one of SEQ ID NOs: 7-12 linked to an anti-CD3 single chain (SEQ ID NO: 6) that is capable of killing immunosuppressive and immunocompetent mesothelin-expressing cells or tumors, wherein the linkage between the anti-mesothelin antibody and the anti-CD3 antibody is via an optimized spacer, and the agent kills mesothelin-expressing cells with an IC50 of 100 μg / mL or less.

[0138] The above disclosure broadly describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for illustrative purposes only and are not intended to limit the scope of the present invention. [Example]

[0139] Example 1 - Screening for anti-mesothelin antibodies that are naturally refractory to immunosuppressive oncoproteins Several studies have reported that antibodies bound by CA125 are negatively affected in inducing humoral-mediated immune killing and ADC-mediated killing of target cells (Kline JB, et al. OncoTarget 8:52045-52060, 2017; Kline JB, et al. Eur J Immunol 48:1872-1882, 2018; Kline JB, et al. J Clin Oncol 5:15, 2018; Nicolaides NC, et al. Cancer Biol Ther 13:1-22, 2018; Nicolaides et al. U.S. Patent Application No. 1698444). To identify anti-mesothelin antibodies not naturally bound by CA125, we obtained several anti-mesothelin antibodies from academic and commercial sources (e.g., National Cancer Institute, Creative Biolabs, Novus) and tested them for CA125 binding. Figure 1 shows representative results of an enzyme-linked immunosorbent assay (ELISA) to screen for CA125 binding of different anti-mesothelin antibodies. Recombinant mesothelin was used as a positive control, and human serum albumin (HSA) was used as a negative control. Briefly, 96-well plates were coated overnight at 4°C with 50 μL / well of 15 kJ / mL human CA125 protein, 1 μg / mL mesothelin, or 1 μg / mL HSA in 0.05 M carbonate buffer (pH 9.5). The next day, the plates were washed three times with 125 μL of 0.05 M phosphate buffer (pH 7.2) and then blocked with 5% bovine serum albumin in 0.05 M phosphate buffer for 1 hour at room temperature. The wells were then washed three times with 125 μL of 0.05 M phosphate buffer (pH 7.2) and probed with various anti-mesothelin antibodies (meso-Ab1–meso-Ab5) at 2.5 μg / mL. As shown in Figure 1, several anti-mesothelin antibodies were bound by CA125, but unexpectedly, meso-Ab3 (lane 3), consisting of MES-1 antibodies (SEQ ID NO: 1 and SEQ ID NO: 2), did not bind. All antibodies bound to mesothelin protein at similar levels, whereas none of the antibodies bound to the HSA negative control.These results identify MES-1 as a naturally occurring CA125-refractory binding antibody and suggest that such antibodies could be used to treat cancer cells or tumors that produce such immunosuppressants. To confirm that CA125 binding differentially affected the internalization of MES-1 (Ab-3 in Figure 1A) compared with the anti-mesothelin antibody Ab-4, which is bound by CA125 (Figure 1A), we performed internalization assays using mesothelin-expressing OVCAR3 cells (Kline JB, et al., OncoTarget 8:52045-52060, 2017), which naturally overexpress membrane-bound CA125, and for comparison, an shRNA-mediated OVCAR3 CA125 knockdown line (referred to as OVCAR-KO) generated using a strategy similar to that reported by Kline et al. to generate isogenic cells. Both antibodies were fluorescently labeled using the pH-sensitive pHrodo fluorescent dye system according to the manufacturer's protocol (ThermoFisher, thermofisher.com / adcdiscovery). The antibodies were then incubated with OVCAR3 or OVCAR-KO cells in replicates in 96-well microplates for 24 hours, and cellular uptake was quantified by fluorescence using a Varioskan™ plate reader (ThermoFisher). As shown in Figure 1B, the MES-1 Ab (Ab-3), in contrast to Ab-4, was efficiently internalized in both CA125-expressing OVCAR3 and CA125-knockdown OVCAR-KO cells. This supports the unexpected finding that the MES-1 antibody-encoding sequence is naturally CA125-refractory, qualifying it as an antibody-ADC component for treating CA125-expressing tumor cells using the methods taught herein.

[0140] Example 2 - Creation of an anti-mesothelin antibody drug conjugate (ADC) capable of killing immunosuppressive and immunocompetent mesothelin-expressing cancer cells To determine whether MES-1 is refractory to the immunosuppressive effects of CA125 and potentially other immunosuppressive proteins produced by mesothelin-expressing tumor cells, we formulated MES-1 into an ADC format and tested its efficacy in killing immunosuppressive mesothelin-expressing cancer cells expressing CA125. Here, we provide biochemical analyses of MES-1 and MES-ADC compositions, as well as examples of the efficacy of MES-ADCs in various formats required to effectively kill immunosuppressive and immunocompetent mesothelin-expressing tumor cells. We present the use of CA125-unbinding anti-mesothelin antibodies (SEQ ID NO: 1 and SEQ ID NO: 2) conjugated to specific payloads and specific linker types to maximize killing of immunosuppressive target cells. As mentioned above, Nicolaides et al. (Cancer Biol Ther 19:622-630, 2018; Nicolaides et al., U.S. Patent Application No. 16 / 981,444) reported that CA125 binding to the anti-mesothelin antibody amatuximab suppressed humoral immune function and reduced tumor uptake compared to those not bound by CA125, resulting in reduced ADC killing by ADCs against target antigens bound by CA125. To determine the maximum efficacy that MES-1 antibodies in ADC format could have against tumors with immunocompetent and immunosuppressive microenvironments, the inventors tested several MES-1-based ADCs conjugated to various cytotoxic payloads and various linker combinations in vitro and then in human tumor xenografts. To generate these ADCs, the inventors first needed to generate recombinant MES-1 in a system that produces homogeneous MES-1 protein. Recombinant Chinese hamster ovary (CHO) cells were used for recombinant MES-1 production because large amounts of homogenous antibodies have previously been produced using this system.cDNAs encoding the MES-1 light chain (SEQ ID NO: 4) and MES-1 heavy chain (SEQ ID NO: 5) were synthesized by polymerase chain reaction (PCR), and the PCR fragments were cloned into a pXC vector (referred to herein as pNAV0047) containing two CMV-driven expression cloning cassettes and a glutamine synthetase (GS) gene cassette. To generate stable recombinant fusion protein-producing cell lines, 6 × 10 CHOK1SV-GSKO cells containing a knockout endogenous GS gene were cultured. 5 The cells were cultured overnight at 37°C and 5% CO2 in CD-CHO medium (Irving Scientific) supplemented with 6 mM L-glutamine at a concentration of 2.0 × 10 cells / mL. 7 Cells were resuspended with 20 μg of the expression plasmid pNAV0047 in a total volume of 700 μL in CD-CHO medium, transferred to a 0.4 cm electroporation cuvette, and electroporated at 300 V / 900 μF using a BioRad GenePulser II. The cells were immediately transferred to a flask containing 30 mL of CD-CHO medium supplemented with 6 mM L-glutamine and incubated overnight at 37°C and 5% CO in a shaking platform incubator. The next day, cells were harvested and resuspended in 30 mL of CD-CHO / SP4 medium containing 50 μM MSX. High-titer producing clones were selected over a two-week selection period. Selected pools were then subcloned by limiting dilution, and established clones were tested for recombinant MES-1 antibody production. Productive subclones (expressing >1 mg / mL) were expanded and analyzed for antibody production and quality (target antigen binding by ELISA and protein homogeneity by SEC-HPLC). The best-quality clones were then expanded and dialyzed in PBS buffer, after which the antibodies were purified from the culture medium using Protein A column affinity chromatography. The antibodies were quantified and analyzed for homogeneity by size exclusion chromatography (SEC-HPLC) and antigen binding. As shown in Figure 3A, the MES-1 producer was able to produce high-quality, homogeneous antibodies and was used for ADC production.

[0141] Here, we describe a screen for effective cytotoxin-linker combinations that act equally effectively against immunosuppressive and immunocompetent target cancer cells. To identify the best candidates that meet this criterion, we first tested various classes of cytotoxins, including DNA alkylating agents, microtubule inhibitors, and topoisomerase inhibitors (Figure 2A). In addition, we also used combinations of different linkers (cleavable and non-cleavable) to determine whether they affected efficacy (Figure 2B). To test their efficacy, we used several mesothelin-expressing tumor cell lines (a subset of which also coexpress the immunosuppressive CA125 protein). These cell lines are listed in Table 1.

[0142] Table 1. Cell lines tested for MES-ADC killing TIFF0007759668000001.tif48141

[0143] The most potent cytotoxin identified in our screen was the auristatin microtubule inhibitor MMAE (mean EC 50 1.39ng / mL) (Li C, et. al. mAbs 12:1699768, 2020), and two topoisomerase inhibitors, SN38 (mean EC 50 2.44ng / mL) (Meyer-Losic F, et.al. Clin Cancer Res 14:2145-2155, 2008) and PNU159682 (average EC 500.014 ng / mL) (referred to herein as PNU) (Quintieri L, et. al. Clin Cancer Res 11:1608-1617, 2005; Carlson RH. Oncol Times 38:8-10, 2016) (Figure 2C). Based on these results, we next incorporated lead cytotoxins into ADC formats using standard cleavable linkers and retested them against the tumor cell line panel shown in Table 1. Previous reports have shown that the drug-to-antibody ratio (DAR) is an important characteristic for ADC target cell killing, but it is also a parameter that can be difficult to control during manufacturing when using partial reduction and chemical linkage to free cysteines (Farras M, et. al. Mabs. 12:1702-262, 2020). Schematics of SN38-MES-ADC and PNU-MES-ADC are provided in Figure 3B. In many cases, reproducible control of DAR is inherent in the structure and purity of the starting antibody. As shown in Figure 3A, purification of the MES-1 antibody using our manufacturing system allowed us to generate a homogeneous starting antibody, which, upon partial denaturation, routinely produced ADCs with DARs of 2–4 or 4–6 (Figure 3C). Preliminary cell-killing assays of MES-ADCs using cleavable linkers found that SN38- and PNU-MES-ADCs exhibited the most potent targeted killing of all mesothelin-expressing strains, regardless of CA125 status, likely due to unperturbed cellular uptake, whereas strains not expressing mesothelin were unaffected (Figure 4A). Because PNU-MES-ADC exhibited the most potent killing activity, we next tested PNU-MES-ADC in a cleavable format (MA-PEG4-VC-PAB-DMAE) and an enzymatically non-cleavable format (MC-EDA). Contrary to expectations, we found that the cleavable format was nearly 100-fold more potent than the enzymatically non-cleavable format (Figure 4B).

[0144] To determine the in vivo efficacy of the lead MES-ADC drug, we next tested the therapeutic efficacy of the cleavable and non-enzymatically cleavable formats of SN38-MES-ADC and PNU-MES-ADC in mouse xenograft models using the mesothelin- and CA125-expressing tumor cell lines NCI-N87 and SW1990, as well as in patient-derived tumor xenografts (PDX) bearing mesothelin-expressing and non-CA125-expressing tumors. To confirm mesothelin and CA125 expression in xenograft and PDX tumors, we examined xenograft-derived fragments via immunohistochemistry (IHC) using the rMES-1 detection antibody containing SEQ ID NOs: 7–12 on a rabbit IgG backbone and a commercially available anti-CA125 antibody, respectively (Figure 4C). Briefly, 5 μM sections of paraffin-embedded tumor fragments were cut and then attached to glass slides. Sections were deparaffinized and prepared for antigen retrieval in boiling 10 mM sodium citrate (pH 6.0) for 10 minutes, then equilibrated in phosphate-buffered saline-0.05% Tween-20 (PBS-T). Sections were then quenched for endogenous peroxidase activity using 0.3% peroxidase / methanol for 10 minutes and blocked for 1 hour in 10% goat serum in PBS-T. Slides were then rinsed in PBS-T and probed for mesothelin via rMES-1 or rabbit anti-CA125 (Novus) for 1.5 hours using 3 mg / mL of each primary antibody diluted in blocking buffer, followed by washing, secondary blocking for 1 hour, and probing with 5 μg / mL of anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour. Control slides were incubated without primary antibody. Slides were washed with PBS-T and then exposed using eBioscience™ DAB Advanced Chromogenic Substrate as recommended by the manufacturer (ThermoScientific). Finally, samples were counterstained with hematoxylin, coverslipped, and analyzed for antigen expression under a light microscope.Both NCI-N87 and SW1990 xenograft tumors were found to coexpress mesothelin and CA125 (not shown), whereas non-small cell lung adenocarcinoma PDX #LXFA983 and mesothelioma PDX #PXF1118 expressed equivalent levels of mesothelin, with only PDX #PXF118 showing CA125 positivity (Figure 4C, upper panel). We then used these lines in mouse CDX (cell line-derived xenograft) and PDX (patient-derived xenograft) in vivo assays.

[0145] In the SW1990 pancreatic cancer CDX model, 1 × 10 7 Tumor cells were injected into the flanks of multiple athymic nude mice. Established tumors (136–154 mm) were obtained. 3 Mice bearing the tumor were randomized and intravenously administered SN38-MES-ADC at either 10 mg / kg or 20 mg / kg or PBS on days 8, 9, and 10 after implantation. SN38-MES-ADC treatment reduced tumor growth by 25% or 53% at 10 mg / kg or 20 mg / kg, respectively, relative to the vehicle-treated group on day 39, and the difference was statistically significant (P ≤ 0.049 for 10 mg / kg; P ≤ 0.0003 for 20 mg / kg) (Figure 4D, upper panel).

[0146] In the NCI-N87 gastric cancer CDX model, 1 × 10 7 Tumor cells were injected into the flanks of SCID mice. Established tumors (126 mm 3 Mice bearing PNU-MES-ADC were randomized and administered intravenously as indicated below. The enzymatically cleavable format of PNU-MES-ADC was administered at 0.25 mg / kg or 0.5 mg / kg on days 1, 8, 15, 25, 32, and 44 after randomization. PNU-MES-ADC cleavable format treatment reduced tumor growth by 45% at 0.5 mg / kg, a difference that was statistically significant (p≦0.050) (FIG. 4D, lower panel). In the same NCI-N87 gastric cancer CDX model, established tumors (126 mm 3Mice bearing the PNU-MES-ADC (PNU-MES-ADC) were randomized and administered either SN38-MES-ADC, PNU-MES-ADC non-enzymatically cleavable format (0.625 mg / kg on day 1, 1.25 mg / kg on day 5, 2.5 mg / kg on day 9, and 5 mg / kg on day 13), or PBS intravenously at 20 mg / kg on days 1, 3, 5, 7, 9, 11, 13, and 15 after randomization. SN38-MES-ADC treatment reduced tumor growth by 48%, a difference that was statistically significant (p≦0.011) (not shown). PNU-MES-ADC non-enzymatically cleavable format treatment reduced tumor growth by 36%, a difference that was less effective than PNU-MES-ADC with an enzymatically cleavable format but was still statistically significant (p≦0.033).

[0147] In the LXFA983 NSCLC PDX model, tumor fragments were implanted into the flanks of multiple athymic nude mice. Established tumors (128 mm 3 Mice bearing the PNU-MES-ADC cleavable format were randomized and administered either the PNU-MES-ADC cleavable format or PBS intravenously at 0.625 mg / kg (days 1, 4, and 8) or 0.4 mg / kg (days 12 and 16) on days 1, 4, 8, 12, and 16 after randomization. The PNU-MES-ADC cleavable format induced significant tumor regression that was statistically significant (p≦0.0003) (Figure 4E, upper panel).

[0148] In the PXF1118 mesothelin PDX model, tumor fragments were implanted into the flanks of multiple athymic nude mice. Established tumors (117–124 mm) were 3 Mice bearing the PNU-MES-ADC cleavable format were randomized and administered either the PNU-MES-ADC cleavable format or PBS intravenously at 0.625 mg / kg (days 1, 4, and 8) or 0.4 mg / kg (days 12, 16, and 20) on days 1, 4, 8, 12, 16, and 20 after randomization. The PNU-MES-ADC cleavable format induced significant tumor regression that was statistically significant (p≦0.012) (Figure 4E, lower panel).

[0149] To further evaluate the efficacy of reduced-dose and single-dose administration of PNU-MES-ADC, we performed a repeat in vivo experiment using the CA125-expressing PXF1118 PDX line in the model design described above. Briefly, athymic nude mice were prepared as described above, and once established tumors were confirmed, they were divided into four groups of six mice each. Mice were then treated on days 1, 8, and 15 with PBS, 0.25 mg / kg PNU-MES-ADC, or 30 μg / kg free PNU159682 (equivalent to the toxic dose of 0.25 mg / kg PNU-MES-ADC), or a single treatment with 0.75 mg / kg PNU-MES-ADC on day 1. Mice were followed for tumor response and health status for over 50 days. As shown in Figure 5 at day 49, a single dose of the cleavable format of MES-ADC at 0.75 mg / kg significantly reduced established tumor growth (P < 0.0061) and was sufficient to maintain a similar durable response as three weekly doses of PNU-MES-ADC at 0.25 mg / kg, in contrast to mice treated with PBS or free PNU159682 (PN-free), demonstrating that this format and composition is effective for treating mesothelin-expressing tumors, regardless of whether they express CA125. Drug treatment was well tolerated in all groups.

[0150] These data support the teaching that compositions of matter containing the cleavable formats of PNU-MES-ADC and SN38-MES-ADC can effectively kill tumors with immunosuppressive and immunocompetent tumor microenvironments. The finding that other payloads, such as MMAE microtubule inhibitors, or linkers, such as enzymatically non-cleavable MC-EDA, are less effective suggests that the common use of tumor-targeted ADCs cannot simply overcome tumor immunosuppression; rather, 1) active screening for antibodies that are not bound by immunosuppressants such as CA125, 2) screening for optimal payload cytotoxicity, and 3) screening for optimal linkers are required to develop potent and effective ADC agents capable of treating immunosuppressive and immunocompetent tumors, as described in the invention taught herein.

[0151] Example 3 - Generation of an anti-mesothelin bispecific antibody (MES-BSP) capable of killing immunosuppressive and immunocompetent mesothelin-expressing cancer cells The use of antibodies to target tumor cells has typically involved blocking cytokine binding to cytokine receptors to suppress tumor cell growth and / or humoral-mediated immunokilling via ADCC, CDC, and / or opsonization of immune effector cells. As noted above, tumor-produced CA125 protein, as well as other tumor-produced proteins (the latter are personal observations of NCN and JBKLG), have the ability to inhibit humoral-mediated antibody killing of target cells. The use of antibodies that are naturally refractory to binding of immunosuppressive proteins allows tumor cell killing even in the presence of such proteins. As described in Example 1, the MES-1 antibody was identified by screening anti-mesothelin antibodies for their ability to avoid CA125 binding. To enhance MES-1 immune-mediated killing, we targeted CD3 binding to the proximal surface of target cells, a key feature for BSP antibody tumor cell killing. +MES-1 was engineered to potentially utilize immune-mediated killing via cytotoxic T cell recruitment and subsequent T cell activation (Staerz UD, et al. Nature 314;628-631, 1985). The present inventors teach herein the use of a non-CA125-binding anti-mesothelin antibody genetically fused to a second antibody capable of binding to a cell surface antigen expressed on T lymphocytes, resulting in improved immune-mediated tumor cell killing regardless of the immune status of the tumor microenvironment. As an example, we demonstrate the use of a single-chain antibody fused to the MES-1 antibody capable of binding to the CD3 antigen on T cells. As described in Example 2, Nicolaides et al. (Cancer Biol Ther 19:622-630, 2018) have previously shown that CA125 binding to the anti-mesothelin antibody amatuximab results in suppressed humoral immune function. To determine the maximum efficacy that a BSP-formatted MES-1 antibody could have against tumors with immunocompetent and immunosuppressive microenvironments, we investigated several MES-BSP formats linking an anti-CD3 single-chain antibody (SEQ ID NO: 6) to the MES-1 light chain (SEQ ID NO: 1) or heavy chain (SEQ ID NO: 2), and proceeded with the standard MES-1 heavy chain (SEQ ID NO: 2) and a CD3 single chain fused to the N-terminus of the MES-1 light chain using the amino acid linker GGGS, as shown in SEQ ID NO: 3. We then generated recombinant MES-BSP antibodies as described below and tested their activity against immunocompetent and CA125 protein-immunosuppressed cancer cell lines.

[0152] To generate high-quality MES-BSP, recombinant Chinese hamster ovary (CHO) cells were engineered to express MES-BSP for large-scale production. The CD3 single-chain antibody was cloned upstream of the mature N-terminal domain of the MES-1 light chain via a genetic linker encoding the amino acids GGGGS, as shown in SEQ ID NO: 3. The MES-1 light chain-CD3 single-chain fusion cDNA and the MES-1 heavy chain cDNA were cloned into a pXC vector (referred to herein as pNAV0071) containing two CMV-driven expression cassettes and a glutamine synthase (GS) gene cassette. To generate a stable recombinant fusion protein-producing cell line, 6 × 10 CHOK1SV-GSKO cells containing a knockout endogenous GS gene were cultured at 4 °C for 1 h. 5 The cells were cultured overnight at 37°C and 5% CO2 in CD-CHO (Irving Scientific) supplemented with 6 mM L-glutamine at a concentration of 2.0 × 10 cells / mL. 7Cells were resuspended with 20 μg of the expression plasmid pNAV0071 in a total volume of 700 μL in plain CD-CHO, transferred to a 0.4 cm electroporation cuvette, and electroporated at 300 V / 900 μF using a BioRad GenePulser II. The cells were immediately transferred to a flask containing 30 mL of warm CD-CHO supplemented with 6 mM L-glutamine and incubated overnight at 37°C, 5% CO in a shaking platform incubator. The next day, cells were harvested and resuspended in 30 mL of CD-CHO / SP4 containing 50 μM MSX for selection. Selected pools were then subcloned by limiting dilution, and conditioned medium was tested for recombinant MES-BSP antibody production from established clones via ELISA using anti-human Fc-HRP as a probe. Productive subclones (producing >0.5 mg / mL) were expanded and analyzed for antibody production and quality (target antigen binding and protein homogeneity). The best-quality clones were then expanded, and the MES-BSP antibodies were purified from the culture medium using Protein A column affinity chromatography and dialysis in PBS buffer. The MES-BSP antibodies were then quantified and analyzed for homogeneity via SDS-PAGE analysis and for antigen binding via ELISA. High-quality preparations were then tested for efficacy against various tumor cell lines.

[0153] MES-BSP was first tested for humoral immune killing via ADCC in the presence of the mesothelin-expressing immunosuppressive OVCAR3 tumor cell line, which naturally overexpresses CA125 protein, and human PBMCs, in comparison with the parental MES-1 antibody and the humoral immune suppressive meso-Ab-4 antibody (Ab-4, lane 4, Figure 1). As shown in Figure 6A, MES-BSP exhibited significantly greater killing of the CA125-producing OVCAR-3 cell line than MES-1 or meso-Ab-4, whereas meso-Ab-4 showed no killing activity. This was likely due to CA125, as the ADCC activity of meso-Ab-4 was similar to that of MES-1 against the OVCAR3 CA125 knockdown OVCAR-KO cell line (Figure 6B). In this assay, we used the Jurkat-CD16a ADCC reporter cell line to monitor ADCC activity via a luciferase readout according to the manufacturer's instructions (Promega Corp). Similar lines have previously been published, demonstrating significantly enhanced antibody-mediated humoral responses with CA125-influenced antibodies, in contrast to parental OVCAR cells (Kline JB, et al. OncoTarget 8:52045-52060, 2017; Nicolaides NC, et al. Cancer Biol Ther 13:1-22, 2018). Finally, to demonstrate the in vivo efficacy of MES-BSP against mesothelin-expressing, CA125-positive tumor cells, we used a humanized PBMC mouse model and implanted mesothelioma-derived tumor cells expressing both mesothelin and CA125 into athymic nude mice. Once tumors were established, mice were implanted with human peripheral blood mononuclear cells (PBMCs) followed by MES-BSP or control treatment. As shown in Figure 7, MES-BSP was able to statistically significantly suppress tumor growth, confirming its usefulness for treating mesothelin-expressing cells in the CA125 immunosuppressive tumor microenvironment. Here, we demonstrate that CA125 can significantly affect antibody efficacy by using immune-mediated killing to which CA125 can bind. Furthermore, we provide compositions in which anti-mesothelin BSP antibodies can effectively kill immunosuppressive tumor cells.

[0154] (Table 2) Sequence identification (all sequences are from N to C terminus) The underlines indicate the antibody CDRs in the anti-mesothelin-directed antibody. Underlined italics indicate the CDRs in anti-CD3 directed antibodies. Bold text represents the light or heavy chain constant region. Bold italics represent the spacers between the CD3 single light chain and the heavy chain, and between the CD3 single chain and the anti-mesothelin light chain fusion.

[0155] SEQ ID NO: 1 (MES light chain) TIFF0007759668000002.tif17165

[0156] SEQ ID NO: 2 (MES heavy chain) TIFF0007759668000003.tif35166

[0157] SEQ ID NO: 3 (MES-BSP: single chain anti-CD3 fused to MES light chain) TIFF0007759668000004.tif41166

[0158] Anti-CD3 VH is first GGGGS x The anti-CD3 VL is located before the linker, the anti-CD3 VL is located between the first and second linkers, and the anti-mesothelin VL is located after the second linker.

[0159] SEQ ID NO: 4 (MES-BSP: single chain anti-CD3 fused to MES light chain) with modified leader sequence underlined TIFF0007759668000005.tif85165

[0160] SEQ ID NO: 5 (MES heavy chain) with modified leader sequence underlined TIFF0007759668000006.tif97165

[0161] SEQ ID NO: 6 [anti-CD3 HC and LC single chain] TIFF0007759668000007.tif23165

[0162] SEQ ID NO:7 TIFF0007759668000008.tif6128

[0163] SEQ ID NO:8 TIFF0007759668000009.tif6128

[0164] SEQ ID NO:9 TIFF0007759668000010.tif6128

[0165] SEQ ID NO: 10 TIFF0007759668000011.tif6128

[0166] SEQ ID NO: 11 TIFF0007759668000012.tif6128

[0167] SEQ ID NO: 12 TIFF0007759668000013.tif6128

[0168] SEQ ID NO: 13 TIFF0007759668000014.tif6128

[0169] SEQ ID NO: 14 TIFF0007759668000015.tif6128

[0170] SEQ ID NO: 15 TIFF0007759668000016.tif6128

[0171] SEQ ID NO: 16 TIFF0007759668000017.tif6128

[0172] SEQ ID NO: 17 TIFF0007759668000018.tif6128

[0173] SEQ ID NO: 18 TIFF0007759668000019.tif6128

[0174] SEQ ID NO: 19 [MES light chain cDNA] with modified leader sequence underlined TIFF0007759668000020.tif46165

Claims

1. An antibody-drug conjugate (ADC) comprising an anti-mesothelin antibody comprising the amino acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2 and a topoisomerase inhibitor, wherein the ADC is refractory to CA125 binding.

2. 2. The ADC of claim 1, wherein the antibody is covalently attached to the topoisomerase inhibitor.

3. 3. The ADC of claim 1 or 2, wherein the antibody is attached to a topoisomerase inhibitor via a cleavable linker.

4. 4. The ADC of claim 3, which is encapsulated in a liposome.

5. 5. The ADC of any one of claims 1 to 4, wherein the topoisomerase inhibitor is (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-{[(1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5][1,3]oxazolo[2,3-c][1,4]oxazin-3-yl]oxy}-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxylic acid (PNU159682).

6. 6. The ADC of claim 5, wherein PNU159682 is covalently attached to the anti-mesothelin antibody via the linker maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MA-PEG4-VC-PAB-DMAE).

7. 7. The ADC of claim 6, wherein the linker is attached to a cysteine ​​in the anti-mesothelin antibody.

8. 5. The ADC of any one of claims 1 to 4, wherein the topoisomerase inhibitor is (2S,3S,4S,5R,6S)-6-[[(19S)-10,19-diethyl-19-hydroxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]heneicosa-1(21),2,4(9),5,7,10,15(20)-heptaen-7-yl]oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (SN38).

9. 9. The ADC of claim 8, wherein SN38 is covalently attached to the anti-mesothelin antibody via the linker methyl (2S,3S,4S,5R,6S)-3,4,5-triacetyloxy-6-[2-amino-4-(hydroxymethyl)phenoxy]oxane-2-carboxylate (MAC-glucuronide).

10. 9. The ADC of claim 8, wherein SN38 is covalently attached to the anti-mesothelin antibody via the linker PEG8 triazole-PABC-peptide-MC.

11. 9. The ADC of claim 8, wherein SN38 is covalently linked to a cysteine ​​in the anti-mesothelin antibody.

12. The ADC according to any one of claims 1 to 11, wherein the drug:antibody ratio (DAR) of the ADC is 2 or more and 6 or less.

13. A bispecific antibody (BSP) comprising a mesothelin-binding portion and a cell surface antigen CD3-binding portion, wherein the bispecific antibody comprises the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 2 and is refractory to CA125 binding.

14. A nucleic acid vector encoding the bispecific antibody of claim 13.

15. 15. The nucleic acid vector of claim 14, comprising the nucleic acid sequences of SEQ ID NO: 4 and SEQ ID NO:

5.

16. 14. A stable cell line comprising one or more nucleic acids encoding the bispecific antibody of claim 13.

17. 17. The stable cell line of claim 16, wherein the one or more nucleic acids comprise the nucleic acid sequences of SEQ ID NO: 4 and SEQ ID NO:

5.

18. A pharmaceutical composition for treating a cancer patient having a mesothelin-expressing tumor, comprising the antibody-drug conjugate (ADC) of any one of claims 1 to 12.

19. 14. A pharmaceutical composition for treating a mesothelin-expressing cancer in a patient, comprising the bispecific antibody of claim 13.

20. 20. The pharmaceutical composition of claim 18 or 19, wherein the cancer is selected from the group consisting of mesothelioma, breast cancer, lung cancer, colorectal cancer, gastrointestinal cancer, endometrial cancer, bile duct cancer, and pancreatic cancer.

21. The pharmaceutical composition of any one of claims 18 to 20, wherein the patient has a high level of CA125 compared to a healthy human population.

22. 21. The pharmaceutical composition of any one of claims 18 to 20, wherein the pharmaceutical composition is administered to a plurality of patients, the plurality of patients comprising at least one patient having a high level of CA125 compared to a healthy human population and at least one patient having a normal level of CA125.

23. 21. The pharmaceutical composition of any one of claims 18 to 20, wherein the patient has been identified as having a mesothelin epitope in the cancer of the patient by contacting a body sample from the patient with an antibody comprising the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2.

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