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

Abstract

Tumors use a variety of mechanisms to avoid the host's anti-tumor immune response. Humoral immune suppression is one of the mechanisms. Tumors produce circulating factors that can suppress antibody or complement-mediated immune responses to enhance their own survival. Mesothelin is a cell surface protein that is overexpressed by several cancer types that are associated with a microenvironment that exhibits immune suppression. Anti-mesothelin antibodies often suffer from such immune suppression microenvironments. However, an alternatively formatted anti-mesothelin antibody is effective in killing mesothelin-expressing cancers, independent of the tumor microenvironment immune status.

Description

Technical Field

[0001] This invention relates to the field of therapeutic antibodies that effectively and specifically target mesothelin-expressing cancers in both immune-intact and immunosuppressive tumor microenvironments. Specifically, this invention relates to methods, kits, and compositions containing antibody-based agents that exhibit improved therapeutic efficacy in inhibiting cancer growth, independent of the immune status of the microenvironment. Background Technology

[0002] The mechanisms of tumorigenesis involve the combined accumulation of mutated genes that enhance dysregulated cell growth and the generation of immune evasion mechanisms that enable them to survive in affected patients. Cellular immunity (primarily T-cell-mediated immunity) and humoral immunity (primarily antibody-mediated immunity) are the main mechanisms by which vertebrate host organisms defend against infectious pathogens and dysregulated host cells. In recent years, the use of immune checkpoint inhibitors that can overcome suppressed cell-mediated immunity has been demonstrated in releasing activated CD8+. +The robust role of T cells in killing tumor subsets (Hodi FS et al. N Engl J Med 363:711-723, 2010). Recent translations have confirmed that tumors also produce factors that suppress humoral immune pathways, which in turn inhibit tumor killing through mechanisms such as antibody-dependent cell cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), and opsonization-mediated antibody-mediated mechanisms (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 inhibit the humoral immune pathway suppress the effects of clinically used therapeutic antibodies. These therapeutic antibodies have been reported to exhibit tumor-killing activity via ADCC and CDC, such as, but not limited to, rituximab, obinutuzumab, trastuzumab, pertuzumab, cetuximab, alemtuzumab, and many experimental antibodies (DiLillo DJ, Ravetech JV, Cancer Immunol Res 3:704-713, 2015; Ruck T et al. Int JMol 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 synergistic effect of antibody-cell surface antigen binding.When this interaction is optimal, antibodies bound to the cell surface bind to Fc-γ-activating receptors on natural killer (NK) cells or dendritic / myeloid / monocyte cells (any cells involved in ADCC are referred to as “immune effector cells” in this document). This binding initiates ADCC and binds to C1q complement initiation proteins, leading to antibody-bound cell death via the classical complement-CDC pathway and opsonization via phagocytes (Reuschenbach M et al., Cancer Immunol Immunother 58:1535-1544, 2009). Inhibitors of humoral immune responses reduce the ability of therapeutic antibodies to use these mechanisms, thereby diminishing 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 here as CA125) inhibits NK cell activation by binding to negative immunomodulatory receptors of the SIGLEC family (Belisle JA et al. Mol Cancer 9:1476-4598, 2010) and by directly binding to subsets of IgG1, IgG3 and IgM antibodies to suppress humoral immune responses. The binding of antibodies disrupts the Fc region, making it less effective for IgG1 and IgG3 antibodies to bind to the Fc-γ-activating receptors FCGR2A (also known as CD32a) and FCGR3A (also known as CD16a) on immune effector cells and / or interfering with the ability of all three antibody classes to bind to 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 anticancer antibodies whose pharmacological activity depends on immune effector mechanisms. In these clinical studies, CA125 levels have been found to be associated 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 studies involving clinically used rituximab antibodies in patients with follicular lymphoma, a 31.4% improvement in 5-year progression-free survival was observed when CA125 levels were within the normal range compared to patients with CA125 levels above the normal range (Prochazka V et al. Int J Hematol 96:58-64, 2012). There is a persistent need in the field to develop agents that can effectively kill tumors in both immune-intact and immunosuppressive microenvironments.

[0004] Mesothelin is a cell surface protein overexpressed in many tumor types, including mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, cholangiocarcinoma, gastric cancer, and endometrial cancer. Several of these tumor types have been found to have humoral immune suppression effects, which potentially diminish the efficacy of antibody-based anti-mesothelin therapies (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 Supplement 32:61, 2014). Specifically, it has been reported that lung cancer, ovarian cancer, pancreatic cancer, and mesothelioma express the humoral immune-suppressive protein CA125 (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 immune suppression observed in a phase 2 clinical trial of the anti-mesothelin antibody amatuximab in first-line mesothelioma using standard of care. In this trial, patients with elevated CA125 levels treated with amatozumab had worse progression-free survival (PFS) and overall survival (OS) outcomes than those with low CA125 levels, supporting the view that humoral immune-dependent anti-mesothelin antibodies will provide lower clinical benefit in humoral immunosuppressive cancers (such as those listed above). There is a need to develop alternative strategies using novel antibody-based agents to potentially overcome this mechanism and provide broader treatment options for patients with and without humoral immunosuppressive cancers. The art requires compositions and methods that effectively kill mesothelin-expressing tumor types exhibiting a humoral immunosuppressive phenotype (e.g., expressing immunosuppressive CA125 protein, etc.) as well as those immune-intact tumor types. Summary of the Invention

[0005] One embodiment is an antibody-drug conjugate (ADC). This antibody-drug conjugate comprises an anti-mesothelin antibody having a complementarity-determining region (CDR) having the amino acid sequences shown in SEQ ID NO: 7-12. One such anti-mesothelin antibody comprises SEQ ID NO: 1 and SEQ ID NO: 2. The ADC also comprises a topoisomerase inhibitor.

[0006] Another embodiment is a method using an anti-mesothelin antibody that does not bind CA125 as part of an antibody-drug conjugate (ADC). This anti-mesothelin antibody comprises a CDR having the amino acids shown in SEQ ID NO: 7-12. The cellular uptake of this anti-mesothelin antibody is greater than that of a CA125-bound anti-mesothelin antibody in a soluble or membrane-bound form. One such CA125-free anti-mesothelin antibody that can be used comprises the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2. The method includes administering the ADC to a person requiring anti-mesothelin therapy. The method may also include using the ADC to detect mesothelin epitopes in target cells.

[0007] Another implementation is a method for treating cancer patients with tumors expressing mesothelin. The patient is treated by administering an antibody-drug conjugate (ADC) comprising an anti-mesothelin antibody and a topoisomerase inhibitor, wherein the anti-mesothelin antibody comprises a CDR having the amino acids shown in SEQ ID NO: 7-12. One such anti-mesothelin antibody that can be used comprises SEQ ID NO: 1 and SEQ ID NO: 2.

[0008] Another embodiment is a bispecific antibody (BSP) comprising a mesothelin-binding moiety and a human cell surface antigen CD3-binding moiety, the mesothelin-binding moiety comprising the amino acid sequence SEQ ID NO: 7-12.

[0009] Another implementation is a method for treating cancers expressing mesothelin in patients. The patient is treated with a bispecific antibody (BSP) comprising a mesothelin-binding moiety and a human cell surface antigen CD3-binding moiety, the mesothelin-binding moiety comprising the amino acid sequence SEQ ID NO: 7-12.

[0010] One aspect of the 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]. Because significantly less antibody is bound to the immunosuppressive CA125 protein, the antibody enjoys enhanced 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 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], wherein the antibody is conjugated to a cytotoxic compound selected for its ability to kill immunologically active and immunosuppressive mesothelin-expressing cancer cells. The cytotoxic compound may be a topoisomerase inhibitor, a microtubule inhibitor, an alkylating agent, or a kinase inhibitor. This conjugate is referred to as a "MES-ADC".

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

[0013] Another aspect of the invention is a method for treating a patient with cancer cells expressing mesothelin, who also expresses elevated levels of CA125 compared to healthy individuals. A MES-ADC antibody is administered to the patient. The MES-ADC antibody consists of amino acid sequences SEQ ID NO:1 and SEQ ID NO:2 and is linked to a cytotoxic topoisomerase inhibitor SN38. Optionally, the antibody and cytotoxin are covalently linked via a cleavable linker.

[0014] Another aspect of the invention is a method for treating a patient with cancer cells expressing mesothelin, who also expresses elevated levels of CA125 compared to healthy individuals. A MES-ADC antibody is administered to the patient. The MES-ADC antibody consists of amino acid sequences SEQ ID NO:1 and SEQ ID NO:2 and is linked to a cytotoxic topoisomerase inhibitor, PNU159682. The antibody-cytotoxin pair is covalently linked via a cleavable linker.

[0015] In one aspect, the MES-ADC comprises an antibody consisting of SEQ ID NO: 1 and SEQ ID NO: 2, linked to a cytotoxic PNU159682 or SN38 topoisomerase inhibitor. Two or more cytotoxic agents can be chemically linked to the antibody via a free cysteine ​​residue generated through partial reduction. The cysteine ​​residue may be native to the immunoglobulin sequence or engineered into the parent antibody sequence.

[0016] In another aspect of the invention, the MES-ADC chemical linker utilizes a cleavable connector, 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, and Ala-Ala-Asn-PAB. TFA salt, 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., their chemical structures are known in the art.

[0017] In another aspect of the invention, the chemical linker of the MES-ADC utilizes a non-enzymatically cleavable linker, 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., whose chemical structures are known in the art.

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

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

[0020] In another aspect of the invention, the cytotoxin is linked to the C-terminus of the heavy chain contained in SEQ ID:2 via a transamidation reaction.

[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, i.e., single-chain anti-CD3 fused to the MES-1 light chain and MES-1 heavy chain, respectively. This bispecific antibody is capable of killing immunologically active and immunosuppressive mesothelin-expressing cancer cells. The single-chain anti-CD3 antibody recognizes CD3... + and / or CD8 + Cell surface antigens expressed on lymphocytes. This bispecific antibody is referred to in this paper as MES-BSP, which stands for Bispecific Antibody Targeting Mesothelin.

[0022] Another aspect of the invention is a bispecific antibody targeting mesothelin, wherein 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. This bispecific antibody can be used to treat cancers expressing mesothelin or other diseases associated with mesothelin expression.

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

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

[0025] Another aspect of the invention is a method for treating a patient with cancer cells expressing mesothelin, who also expresses elevated levels of CA125 compared to healthy individuals. The MES-BSP antibody is administered to the patient. The MES-BSP antibody comprises the amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2 and an antibody that recognizes the human CD8 protein.

[0026] In 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 with a single-chain antibody comprising the amino acid sequence of SEQ ID NO: 6, the single-chain antibody being fused with the N-terminus of the human IgG1 light chain (SEQ ID NO: 1) and / or the N-terminus of the IgG heavy chain (SEQ ID: 2) to produce a bispecific antibody capable of binding mesothelin (Entrez gene ID: 10232) and CD3ε (CD3E) protein (Entrez gene ID: 916), referred herein as MES-BSP.

[0027] In another aspect of the invention, the fusion of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 6 via a linker tether generates a light chain fusion polypeptide composed of a combination of SEQ ID NO: 3 and SEQ ID NO: 2, to generate a functional MES-BSP that can (a) bind mesothelin on target cancer cells in the presence or absence of CA125 and (b) bind CD3E on lymphoid cells, leading to the killing of target cells. The linker can be an amino acid, a polypeptide, or a non-biochemical compound.

[0028] Another aspect of the invention is MES-BSP, which includes complementarity-determining regions (CDRs) having amino acid sequences of SEQ ID NO: 7, 8, 9; SEQ ID NO: 10, 11, 12; SEQ ID NO: 13, 14, 15; and SEQ ID NO: 16, 17, 18, wherein up to three amino acids can be altered within one or more CDRs.

[0029] In another aspect of the invention, the linker in SEQ ID NO: 3 is optimized for (a) canonical antibody formation of the IgG1 heavy chain (SEQ ID NO: 2) and / or (b) improved killing of immunosuppressive tumors by MES-BSP, wherein 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 aspect of the invention, MES-BSP comprises an anti-CD3E single chain genetically linked to the N-terminus of the heavy chain of SEQ ID: 2. This linker is optimized for (a) canonical antibody formation with the IgG1 light chain (SEQ ID NO: 1) and / or (b) improved killing of immunosuppressive tumors by MES-BSP, whereby 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 invention is a stable cell line containing mammalian expression vectors having the nucleotide sequences encoding an MES-1 antibody as shown in SEQ ID NO: 5 and SEQ ID NO: 4, which is genetically linked to a second antibody.

[0032] Another aspect of the invention is an antibody (here referred to as rMES-1) having a CDR amino acid sequence of SEQ ID NO: 7-12 grafted onto a rabbit IgG backbone. The antibody is not bound to the CA125 protein.

[0033] Another aspect of the invention is a method for monitoring mesothelin-expressing tumors in patients using antibodies that do not bind to CA125. A fluid or tissue sample from the patient is contacted with an antibody containing CDR SEQ ID NO: 7-12 (e.g., antibody rMES-1). Mesothelin-positive patients can be treated with MES-ADC or MES-BSP.

[0034] In another aspect, the present invention provides a kit for treating immune-intact and immunosuppressive mesothelin cancers. The kit comprises an antibody containing CDR SEQ ID NO: 7-12, preferably grafted onto a rodent IgG backbone. This antibody can be used to monitor mesothelin expression in tumors via immunohistochemistry (IHC) of biopsy tissue or by flow cytometry of circulating tumor cells (CTCs). When a positive signal is detected, the patient can be treated with a MES-ADC containing the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 chemically linked to a cytotoxin, which may be a topoisomerase inhibitor. Both diagnostic and therapeutic antibodies are provided in the kit.

[0035] In another aspect of the invention, a kit is provided for treating immune-intact and immunosuppressive mesothelin-containing cancers. The kit comprises an antibody containing a CDR preferentially grafted onto a rodent IgG backbone according to SEQ ID NO: 7-12. This antibody can be used to monitor mesothelin expression in tumors via IHC examination of biopsy tissue or by flow cytometry examination of circulating tumor cells (CTCs). When a positive signal is detected, the patient can be treated with the MES-BSP consisting of SEQ ID NO: 2 and SEQ ID NO: 3. Both diagnostic and therapeutic antibodies are provided in the kit.

[0036] Another aspect of the invention is the use of MES-ADC, wherein the MES-ADC is administered as a single agent or in combination with standard care chemotherapy to patients in whom a subset of CA125 is expressed. CA125 expression can be determined using serum analysis or biopsy via methods used by those skilled in the art.

[0037] Another aspect of the invention is the use of MES-BSP, wherein MES-BSP is administered as a single agent or in combination with standard care chemotherapy to a subset of patients in whom CA125 is expressed. CA125 expression is determined using serological analysis or biopsy via methods known in the art.

[0038] Upon reading this specification, those skilled in the art will appreciate that these and other aspects of the invention provide methods, compositions, and kits for improving therapeutic responses in patients with mesothelin-expressing cancers, independent of the immune status of the tumor microenvironment. Tolerance to MES-ADC and / or MES-BSP agents in CA125 inhibition contributes to this therapeutic improvement. Attached Figure Description

[0039] Figure 1A Screening for anti-mesothelin antibodies not bound to immunosuppressive CA125 protein; identification of MES-1 antibodies (SEQ ID NO: 1 and 2). In short, 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 detected with 2.5 µg / mL of different anti-mesothelin antibodies to determine whether they were bound to CA125. Wells were washed and binding was detected via anti-human or anti-rodent Fc-horseradish peroxidase (HRP) conjugated secondary antibody and 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The reaction was terminated with 0.1 NH2SO4, and the plate was read using a Varioskan multi-well plate reader. ™Quantification was performed on the wells at 450 nm using ThermoFisher. As shown in the figure, the antibody MES-1 [Ab-3 (lane 3)] composed of SEQ ID NO: 1 and 2 was not bound by CA125, while all four other anti-mesothelin antibodies tested were bound by CA125. Values ​​were determined from the average of the three copies of the wells. Statistical analysis was performed using the student's T-test. Figure 1B These results demonstrate that, compared to antibodies that bind to CA125 (reference...),... Figure 1A Compared with Ab-4, anti-mesothelin antibodies that were not bound by CA125 (reference) Figure 1A Enhanced uptake of Ab-3. To measure antibody internalization, pHrodo was used. ™ A fluorescence assay (ThermoFisher) was used, in which pH-sensitive fluorescent dyes were conjugated to CA125-resistant Ab-3 (also referred to herein as MES-1) and CA125-sensitive Ab-4 antibodies. Antibody uptake was tested by incubating each antibody with the human ovarian cancer cell line OVCAR3, which expresses mesothelin and membrane-bound CA125. A homogenic CA125 knockdown line (referred to as OVCAR3-KO) was generated via shRNA using the parental line. Uptake was measured in black 96-well microplates over 24 hours using Varioskan assays. ™ Intracellular fluorescence was measured using a plate reader, and the uptake of both Ab-3 and Ab-4 was repeatedly measured. As shown in the figure, Ab-3 (MES-1) showed similar uptake in both cell lines, while Ab-4 showed similar uptake to Ab-3 in OVCAR-KO cells, but not in the CA125-expressing parental OVCAR3, demonstrating that CA125 negatively affects Ab-4 uptake compared to CA125-tolerant Ab-3. Statistical analysis was performed using a two-sided Stellenstein T test.

[0040] Figures 2A-2C Examine cytotoxic payloads and adaptors to develop reformulated MES-1 antibodies for optimal killing of immune-intact and immunosuppressive mesothelin-expressing cancer cells. Figure 2A Chemical structures of various types of cytotoxic payloads (i.e., microtubule inhibitors, DNA alkylating agents, topoisomerase inhibitors, and protein kinase inhibitors, see, for example, Yaghoubi S et al. J Cell Physiol 235:31-64, 2019; Wang RE et al. J Am ChemSoc 137:3229-3232, 2015) were provided, and these types of cytotoxic payloads were tested against immunosuppressive and immune-intact mesothelin-expressing target cells. Figure 2BChemical structures of various enzymatic and non-enzymatic cleavable and non-cleavable linkers were provided, and these linkers were tested to generate MES-ADCs for target cells expressing mesothelin that are immunosuppressive and immune-intact. Figure 2C An overview of the cytotoxicity of potential ADC payloads against mesothelin-expressing cancer cells or control cell lines is provided (Table 1). In short, 5,000 cells / well of mesothelin-positive NCI-N87 (CA125) cells were seeded in 100 µL of RPMI plus 7.5% heat-inactivated fetal bovine serum (FBS) in 96-well tissue culture plates. + Stomach cancer), SW1990 (CA125) + Pancreatic cancer), HAY (CA125) + Mesothelioma), YOU (CA125) - Mesothelioma), OVCAR3 (CA125) + Ovarian cancer and A549 (mesothelin-negative lung cancer) cells were used, along with cytotoxicity levels ranging from 0.0001 ng / mL to 500 ng / mL or negative controls. Cultures were incubated in 5% CO2 at 37°C for 72 hours, followed by quantitative viability staining using crystal violet. Dried staining wells were dissolved in 1% SDS in phosphate-buffered saline (PBS), and the staining was applied using Varioskan. ™ Quantification was performed at 570 nm using a colorimetric densitometer on a multi-well plate reader. Several cytotoxic agents tested significantly killed mesothelin target cells (regardless of CA125 expression) and control cells A549 and CHO (not shown) that did not express mesothelin. As shown in the figure, the microtubule inhibitor MMAE and the topoisomerase inhibitors SN38 and PNU159682 were found to have the best potency, with EC50 ranging from 0.008 ng / mL to 5 ng / mL. Experiments represented at least three wells. Statistical analysis was performed using a two-sided Stellenstein t-test.

[0041] Figures 3A-3C MES-1 and MES-ADC composition, purification and structural analysis. Figure 3A The analysis of the generated CHO-GS and protein A purified MES-1 antibody was performed. Size exclusion (SEC) HPLC analysis of the purified MES-1 showed a highly homogeneous antibody species, which is a prerequisite for producing homogeneous antibody-drug conjugates with the desired drug:antibody ratio (DAR). Figure 3B A schematic diagram of SN38-MES-ADC and PNU-MES-ADC compositions using a pyrolytic connector is provided. Figure 3CThe homogeneity of the DAR of SN38-MES-ADC and PNU-MES-ADC was determined by hydrophobic interaction chromatography (HIC-HPLC) and size exclusion chromatography (SEC-HPLC). Both MES-ADCs were generated via partial reduction of MES-1 and cysteine ​​linkage. The DAR was calculated from the HIC peak area and retention time. As shown in the figure, SN38-MES-ADC and PNU-MES-ADC provide representative curves demonstrating the reproducibility of these two ADCs for generating the desired DAR of 2–6.

[0042] Figures 4A-4E MES-ADC conjugate format and target cell killing in vitro and in vivo. Figure 4A ,from Figure 2C Target cell killing via MES-ADC containing two of the most potent cytotoxic agents, SN38 and PNU159682, was observed in a free cytotoxicity assay. The MES-ADC was generated using a cleavable adapter and tested for killing on various target and control cell lines listed in Table 1. Figure 2C The assay was performed. In short, 5,000 cells / well of mesothelin-positive NCI-N87, SW1990, HAY, YOU, OVCAR3, and CHO-MES cells, along with mesothelin-negative A549 and CHO cells as negative controls, were seeded into 96-well tissue culture plates. Cell plates were seeded in 100 µL of RPMI with 7.5% heat-inactivated fetal bovine serum (FBS) at different MES-ADC concentrations (ranging from 0.01 ng / mL to 100 ng / mL) via limiting dilutions, or in plates seeded with negative control compounds. Cultures were incubated in 5% CO2 at 37°C for 72 h, followed by quantitative analysis of viable cells by crystal violet staining. The dried staining wells were dissolved using 1% SDS in PBS, and the results were analyzed using Varioskan. ™ Quantification was performed at 570 nm using a colorimetric densitometer on a multi-well plate reader. As shown in the figure, SN38-MES-ADC and PNU-MES-ADC exhibited the most significant cell-killing activity in all target cells expressing mesothelin, independent of CA125 expression status, while mesothelin-negative lines A549 or CHO (not shown) were unaffected, demonstrating the selectivity and potency of the two ADCs for mesothelin-expressing cells. We then tested different adapter formats of leader MES-ADCs (… Figure 4BNCI-N87 and SW1990 cells were used as target cells, and PNU-MES-ADC was used as a representative MES-ADC for potency analysis of enzyme-cleavable or non-enzyme-cleavable adapter formats. Cell plates were seeded as described above, and cells were grown by limiting dilution with different concentrations of each PNU-MES-ADC. As shown in the figure, the enzyme-cleavable PNU-MES-ADC was significantly more effective than the non-enzyme-cleavable PNU-MES-ADC (gray and blue lines). A549 mesothelin-negative cells were unaffected by either ADC (not shown), further demonstrating the potency and target specificity of the MES-ADC. Figure 4C We used rMES-1 assay antibody and a commercial CA125 antibody to screen NCI-N87 and SW1990 tumor cell lines and a group of PDX-derived tumors via IHC targeting mesothelin expression to ensure that the expression profiles of these two key proteins were maintained in vivo. Both NCI-N87 and SW1990 cell lines, as tumor fragments derived 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 tumor fragment also expressed robust levels of CA125 (mesothelioma # PXF1118), while the other tumor fragment showed undetectable expression (non-small cell lung cancer (NSCLC) # LXFA983). Figure 4D : In vivo testing of SN38-MES-ADC and PNU-MES-ADC. First, 1×10 7 Tumor cells were injected into the flanks of multiple athymic nude mice (for SW1990 cells) or into the flanks of SCID mice (for N87 cells). This was done to establish measurable tumor lesions (>100 mm). 3 Afterwards, mice were grouped and tumor killing was tested using cleavable and non-cleavable PNU-MES-ADC, SN38-MES-ADC, and PBS (as a negative control). As shown in the figure, the cleavable SN38-MES-ADC and PNU-MES-ADC formats were found to be significantly more effective in vivo than the non-enzymatically cleavable format (not shown) (Top: SN38-MES-ADC at 10 mg / kg). P < 0.049, and at 20 mg / kg P < 0.0003; Figure below: PNU-MES-ADC P < 0.011), reflecting the in vitro target cell killing effect. Figure 4EThis study tested cleavable PNU-MES-ADC on PDX-derived tumor xenografts from immune-intact LXFA983 (low CA125) and immunosuppressive PXF1118 (high CA125) tumors to determine efficacy against these different tumor types. Tumor fragments were implanted into the flanks of athymic nude mice and treated with either PNU-MES-ADC or PBS as controls. As shown in the figure, PNU-MES-ADC was equally effective in killing both types of tumors and inducing regression in both, regardless of the microenvironmental immune status (CA125 expression). P < 0.012). Experiments represent a minimum of three wells for in vitro assays and a minimum of five subjects for in vivo assays. Statistical analysis was performed using a two-sided Steinmann t-test.

[0043] Figure 5 The single and multiple administrations and antitumor effects of cleavable PNU-MES-ADC on CA125-positive, mesothelin-expressing PXF1118 tumors were investigated. Tumor fragments were implanted into the flanks of athymic nude mice and allowed to grow to an average size of 100 mm. 3 Mice with similar tumor sizes were divided into four groups of six mice each 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. Group 4 (arm) received a single dose of 0.75 mg / kg PNU-MES-ADC on day 1 after randomization, and tumor growth was monitored for more than 50 days. As shown in the figure, compared with mice treated with PBS or free drug, a single dose of PNU-MES-ADC was similarly effective in killing tumors, inducing tumor regression, and maintaining a durable response for more than 50 days, as was observed in mice treated with multiple doses of PNU-MES-ADC. P < (0.0061). Each group represented six subjects. Statistical analysis was performed using a two-sided Steinmann t-test.

[0044] Figures 6A-6B MES-BSP and control antibodies were used to assess ADCC activity against immunosuppressive cancer cells. To test the efficacy of MES-BSP in targeting immunosuppressive cancer cells, ADCC assays using primary peripheral blood mononuclear cells (PBMCs) and Jurkat-CD16a ADCC reporter gene assays were employed. Figure 6A MES-BSP, MES-1, and anti-mesothelin meso-Ab-4 were tested (reference). Figure 1ALane 4) Antibody-mediated killing of PBMCs of the human OVCAR3 ovarian cancer cell line expressing mesothelin and producing large amounts of immunosuppressive CA125 protein. In short, 10,000 OVCAR3 cells were seeded per well in a black 96-well plate and incubated overnight in 65 µL RPMI with 7.5% fetal bovine serum and 1% L-glutamine (R7.5). The next day, 35 µL of different concentrations of MES-BSP, MES-1, and meso-Ab-4 were added to each well, along with 2.5 × 10⁻⁶ mcg of medium in R7.5. 7 Remove PBMCs from the plate and incubate it in 5% CO2 at 37°C for 72 hours. Then wash the plate three times with 250 µL R7.5 medium to remove PBMCs and pass it via Cell Titer GLO. ® Follow the manufacturer's instructions (Promega) in Varioskan ™ Viability of adhesive OVCAR3 target cells was quantified on a luminescent plate reader. As shown in the figure, although both MES-1 and MES-BSP antibodies showed tumor cell killing, unlike meso-Ab-4, MES-BSP exhibited significantly higher target cell killing, demonstrating that BSP-formatted MES-1 can provide enhanced killing than parental MES-1 alone when utilizing immune-mediated targeting. To determine the effect of immunosuppressive CA125 protein on ADCC activity, MES-1 and meso-Ab-4 antibodies were tested against OVCAR3 cells and OVCAR3-CA125 knockdown (OVCAR-KO) cell lines generated using the shRNA vector TRCN0000262686 (Sigma-Aldrich) as previously described (Kline JB et al. OncoTarget 8:52045-52060, 2017), as well as the Jurkat-CD16a ADCC reporter cell line, following the manufacturer's instructions (Promega Corp). The Jurkat ADCC system uses luciferase readings, whereby the intensity of the luciferase signal represents the ADCC activity of the antibody against the target cells. For example... Figure 6B As shown, although MES-1 exhibits significantly higher ADCC activity against OVCAR3 than meso-Ab-4, both MES-1 and meso-Ab-4 demonstrate similar ADCC activity against the OVCAR-KO cell line, thus demonstrating the efficacy of naturally occurring CA125-resistant MES-1 parental antibodies in alternative MES-1 formats (i.e., ADC or BSP) as agents capable of effectively killing immunosuppressive tumor cells. MES-BSP was equally effective against both the OVCAR3 and OVCAR-KO lines. All experiments were repeated three times. Statistical analysis was performed using a two-sided Stellenstein t-test.

[0045] Figure 7 MES-BSP was tested in vivo using humanized PBMC nude mice and mesothelin-positive human carcinoma cell lines expressing CA125. On day 0, 8 × 10⁸ cells were implanted into athymic nude mice. 6 Mesothelioma cells. On day 11, when the average lesion size was ~50 mm. 3 At that time, 1×10 was administered intraperitoneally. 7 Personal peripheral blood mononuclear cells (PBMCs). On the second day, mice were administered 3 mg / kg of MES-BSP or PBS, 3 × QOD (every other day), for 3 weeks, and tumor growth was monitored (N=5). As shown in the figure, compared with control mice, MES-BSP resulted in a 70% reduction in CA125-positive mesothelioma tumors (P=0.033). Statistical analysis was performed using a bilateral Steinmann t-test. Detailed Implementation

[0046] The inventors have developed novel therapeutic agents capable of effectively killing mesothelin-positive cancer cells regardless of the immune status of the tumor cell microenvironment. While not wishing to be limited to any particular theory or mechanism of action, the applicants believe that the amino acid sequences encoded by SEQ ID NO: 1 and 2 (referred to herein as MES-1 antibodies) and SEQ ID NO: 2 and 3 (referred herein as MES-BSP antibodies) are resistant to binding to the immunosuppressive CA125 protein, which is resistant to (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, (b) Both antibody-drug conjugate (ADC) uptake and internal cytotoxic agent delivery have negative effects, which are prerequisites for robust target cell killing via ADC (Nicolaides NC et al., US Patent Application 16 / 984,444; Chalouni C and Doll S. J Exp Clin Cancer Res 37:20-32, 2018). This immunosuppressive mechanism appears to involve the direct binding of CA125 to the affected antibody (Nicolaides NC et al., Cancer Biol Ther 13:1-22, 2018).

[0047] In addition, this application provides methods for identifying other antibody-based therapies effective against tumors regardless of their microenvironmental immune status. These methods include testing the binding of parental antibodies to tumor-produced immunosuppressive proteins such as CA125. Such antibodies can be formatted as antibody-drug conjugates or bispecific formats, and their effectiveness in killing tumors with immunosuppressive microenvironments can be optimized through empirical testing using various payloads, gene fusions, and chemical linkers.

[0048] The methods and kits described herein can be used to monitor and confirm the eligibility of patients expressing mesothelin for treatment with MES-ADC or MES-BSP by testing patient tumor cells with antibodies containing the CDR amino acid sequence SEQ ID NO: 7-12 using antigen expression assays known in the art.

[0049] These methods, compositions, and kits can be categorized into two classes of antibody formats. In one class, a MES-ADC has been developed comprising an immunoglobulin light chain (SEQ ID NO: 1) and a heavy chain (SEQ ID NO: 2) chemically linked to a cytotoxic agent. Direct binding of CA125 to the antibody component is low or absent. The number of cytotoxic moieties per antibody molecule [referred to as the drug:antibody ratio (DAR)] can vary depending on the conjugation method, 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 moieties are linked to the antibody via chemical or peptide linkers. Linkers can be of cleavable or non-cleavable types, as known to those skilled in the art. The desired combination of linkers, cytotoxins, and DARs can be determined empirically to optimize the MES-ADC's activity against mesothelin-induced tumor cells in vitro and / or in vivo. Tumor cell viability can be determined using methods employed in the art.

[0050] Suitable cytotoxins include, but are not limited to, those that are safe and preferably biodegradable. These cytotoxins include, but are not limited to: monomethylaurestatin 10, auristatin E, monomethylaurestatin E (MMAE), auristatin F, monomethylaurestatin F, HTI-286, tubulolysin M, maytansine AP-3, maytanol, DM1, DM4, Boc-Val-Dil-Dap-OH, Boc-Val-Dil-Dap-Phe-Ome, Boc-Val-Dil-Dap-Doe, and Boc-Val-Dil-Dap-Nrp. Boc-N-Me-Val-Val-Dil-Dap-OH, tubulolysin IM-1, tubulolysin IM-2, tubulolysin IM-3, dasatinib, pamomycin SA, pamomycin TM, pamomycin MA, pamomycin DM, nemorubicin, PNU-159682, calicacin γ1, N-acetyl-calicacin γ1, α-amaminine, PBD-dimer, etc., the structures of which are known in the art.

[0051] Suitable connectors include, but are not limited to, those that are safe and preferably biodegradable. These connectors include, but are not limited to: 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, and Ala-Ala-Asn-PAB. TFA salt, Fmoc-Ala-Ala-Asn-PAB-PNP, Fmoc-Gly3-Val-Cit-PAB, Fmoc-Gly3-Val-Cit-PAB-PNP, MAC glucoside phenol, Py-ds-Prp-OSu, Py-ds-dmBut-OSu, Py-ds-dmBut-OPFP, Py-ds-Prp-OPFP, SMCC, MAL-HA-OSu, MAL-di-EG-OPFP, MAL-tri-EG-OPFP, MAL-tetra-E G-OPFP, MA-PEG4-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, PEG8-triazole-PABC-peptide-mc, etc., their chemical structures are known in the art.

[0052] One embodiment is a MES-ADC comprising an antibody (SEQ ID NO: 1 and 2) having low CA125 binding and covalently linked to a PNU159682 topoisomerase inhibitor via an MA-PEG4-VC-PAB-DMAE cleavable linker.

[0053] Another implementation is a MES-ADC containing an antibody (SEQ ID NO: 1 and 2) having low CA125 binding and covalently linked to an SN38 topoisomerase inhibitor via a MAC glucuronide or PEG8-triazole-PABC-peptide-mc cleavable linker.

[0054] In another class of antibody formats, MES-BSP (bispecific antibody) comprises the immunoglobulin heavy chain of an MES-1 antibody (SEQ ID NO: 2) and a chimeric light chain (SEQ ID NO: 3) with low CA125 binding. The chimeric light chain is a genetic fusion (in the order of amino to carboxyl) of an anti-CD3 single-chain antibody (SEQ ID NO: 6) and an MES-1 light chain (SEQ ID NO: 1). The anti-CD3 single-chain antibody portion is genetically linked via a spacer region containing any of 20 known natural or modified amino acids, whereby the linker may have 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 empirically to optimize for use in human CD3. + The activity of MES-BSP in killing mesothelin-containing tumor cells in vitro and / or in vivo in the presence of lymphocytes. Tumor cell viability can be determined using any of the various methods known in the art.

[0055] For therapeutic applications, MES-ADC or MES-BSP can be used as a monotherapy or in combination with standard care therapy.

[0056] Compositions can be formed during the execution of these methods. For example, they can be pre-formed and packaged individually and provided to entities with a library of cytotoxins to be screened, or cytotoxins can be linked to MES-1 antibodies using different linkers. Similarly, the components of the assays and methods described herein can be packaged together in a container and sold as a kit. The components of a kit do not necessarily need to be mixed together, but may be. For example, they can be provided in separate or separate containers. Any choice of the detection antibody and MES-ADC or MES-BSP described herein can be formulated as a composition or a kit.

[0057] While several known antibodies are sensitive to CA125 immunosuppression, the compositions described herein can be used to treat patients with mesothelin-expressing cancers with MES-ADCs or MES-BSPs, regardless of the immune status of the tumor microenvironment. Each of these compositions contains one or more sequences from the parental MES-1 sequences (SEQ ID NO: 1-3) that are resistant to CA125 binding, and thus effective in ADC internalization and killing when in the MES-ADC format, or effective in immune-related killing when in the MES-BSP format.

[0058] In some cases, MES-ADCs may need to be formulated in liposomes to enhance their therapeutic window in patients with mesothelin-expressing cancers. Any liposomal formulation used to deliver MES-ADCs can be used to treat patients. Conventional liposomes consist of a lipid bilayer composed of cationic, anionic, or neutral (phospholipid) lipids and cholesterol, surrounding a volume of water. Suitable compositions of liposomes include, but are not limited to, guanidine-cholesterol cationic lipid bis(guanidine)-tren-cholesterol (BGTC) combined with the lipid dioleoylphosphatidylethanolamine (DOPE). Another example of a suitable liposomal formulation is the aminoglycoside lipid dioleoylsuccinylparomomycin (DOSP) bound to the imidazole-based helper lipid MM27. Liposomes can be sterically stable, for example, by using polyethylene glycol-coated liposomes.

[0059] We provide compositions, kits, and methods for identifying patients with mesothelin-positive cancers and treating these patients with MES-ADCs or MES-BSPs. These methods may include a step of diagnosing mesothelin expression in a patient's tumor using a CA125-resistant MES-1 detection antibody containing CDR SEQ ID NO: 7-12. If positive in this assay, the tumor can be treated with a MES-ADC (SEQ ID NO: 1 and 2) or MES-BSP (SEQ ID NO: 2 and 3) linked to a cytotoxin (optionally a topoisomerase inhibitor). The diagnostic and treatment steps may be performed independently or together. Such pre-screening may be optional in cancers with high mesothelin overexpression.

[0060] Another implementation is a MES-ADC containing CDRs (SEQ ID NO: 7-12), whereby any sequence in these CDR sequences can be modified individually or in combination with up to three amino acids, as long as they remain CA125 resistant.

[0061] In another embodiment, the cytotoxic agent of the MES-ADC is SN38 or PNU159682 linked to a cleavable linker. The linker can be a PEG8-triazole-PABC-peptide-mc linker (C...) linked to SN38. 50 H 79 N9O 16 (The entire construct is referred to as SN38-MES-ADC-1), the MAC glucoside phenol-linker connected to SN38 (the entire construct is referred to as SN38-MES-ADC-2), or the MA-PEG4-VC-PAB-DMAE linker connected to PNU159682 (the entire construct is referred to as PNU-MES-ADC).

[0062] 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 acid GGGGS (SEQ ID NO: 20). The linker consists of one or more linker units. As an example, but not limited to, length or amino acid composition, the MES-1 light chain and the anti-CD3 single chain can be linked to one, two, three, or more units. This can be achieved by linking the CD3 single chain to the CD3 single chain. + In the presence of lymphocytes, any method in the art for measuring tumor cell killing, as described herein, is used to determine adapter optimization by achieving optimal killing of mesothelin target cells.

[0063] In yet another embodiment, the linker unit comprises any combination of known natural or modified amino acids and any length that can link the MES-1 light chain to the anti-CD3 single-stranded genetic material. Any combination and any length can be empirically tested to optimize for use in immunologically intact or immunosuppressive microenvironments and human CD3. + Mesothelin-positive lymphocytes kill tumor cells in tumors.

[0064] Another embodiment has a MES-BSP containing the amino acid sequences of SEQ ID NO: 7-18, wherein any sequence in these sequences may be modified individually or in combination by up to three amino acids, provided that they remain resistant to CA125 binding.

[0065] In some implementations, in CD3 + In the presence of lymphocytes, different genetic linkers were used, and functional approaches were employed to optimize the effect of MES-BSP on killing mesothelin-positive tumor cells. The term "effect" typically refers to the interaction between the drug and CD3+ when the drug is incubated alone. + A change of 10% or greater in the killing of target cells compared to lymphocytes. Depending on the antibody and agent used, it can also refer to a change of at least 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75% compared to the control.

[0066] Another embodiment includes a method for screening antibody-drug conjugates (ADCs) that exhibit different cytotoxic and / or linker properties for their pharmacokinetic (PK), pharmacodynamic (PD), or pharmacological (PL) activities, including internalization into cells. In some embodiments, the ADC is added to cells in vitro and target cell killing is tested. ADCs with significant killing activity are suitable for therapeutic testing. Similarly, depending on the antibody and drug used, 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.

[0067] Various terms and expressions (“Terms”) relating to the various aspects described herein are used throughout the specification and claims of this document. Unless otherwise specified, these Terms shall be given their ordinary meaning in the art. Other specially defined Terms shall be interpreted in a manner consistent with the definitions provided.

[0068] As used in this specification and the appended claims, the singular forms “an,” “a,” and “the” also include plural references, unless otherwise expressly stated. For example, reference to “a cell” can include a combination of two or more cells, etc. Reference to “probe” can include a detection antibody, MES-ADC, MES-BSP, or a standalone probe for monitoring the immune status of the tumor microenvironment via any analytical method known in the art.

[0069] The term “about” used when referring to quantities, time periods, and / or similar quantifications is intended to cover variations of up to ±9% from the specified value, as such variations are suitable for performing the disclosed methods. Unless otherwise stated, all values ​​indicating amounts of reagents, such as molecular weight, molar concentration, reaction conditions, percentages, etc., used in the specification and claims should in all cases be understood as quantified using the term “about.” Therefore, unless otherwise indicated, the numerical values ​​listed in the following specification and the listed claims are approximate values ​​and may vary depending on the desired properties of the composition and / or the methods sought to be obtained by the present invention. At least, and not in an attempt to limit the scope of this application, each numerical value should be determined by at least reported significant figures and by common rounding methods known in the art.

[0070] The term "antibody" as used is broad and includes immunoglobulins (also known as "Ig") or antibody molecules, including polyclonal antibodies (also known as pAbs), monoclonal antibodies (also known as mAbs), including mouse, human, humanized, and chimeric mAbs, bispecific antibodies (also known as BSPs), antibody-drug conjugates (also known as ADCs), antibody-fused immunotoxins, and antibody fragments. Generally, an antibody is a protein or polypeptide chain that binds to a specific antigen. An antigen is a structure that is specifically recognized by a given antibody. A typical antibody comprises a heterotetrameric glycosylated protein consisting of two light chains and two heavy chains arranged in a complex linked by disulfide and hydrogen bonds. The term "its disulfide bridge" refers to a disulfide bridge contained within the hinge region of the heavy chain, as is known in the art. Each heavy chain has a variable domain (variable region) (VH) followed by multiple constant domains (called Fc domains). Each light chain has a variable domain (VL) and constant domains; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain VL is aligned with the variable domain of the heavy chain. Any type of antibody light chain is assigned to one of two different types based on the amino acid sequence within its constant structural domain, namely kappa (κ) and lambda (λ).

[0071] The term "single-chain" refers to a single-chain antibody having a structure known in the art.

[0072] Immunoglobulin light chains (LCs) or heavy chains (HCs) contain a "framework" region with three "antigen-binding sites" interrupted, also known as complementarity-determining regions (CDRs), based on reported sequence variability (Wu TT, Kabat EA. J Exp Med 132:211-250, 1970). Typically, an antigen-binding site consists of six CDRs, three located within the VH (CDRH1, CDRH2, CDRH3) and three within the VL (CDRL1, CDRL2, CDRL3) (Kabat EA et al., 5th ed. PHS, National Institutes of Health, Bethesda, Md., 1991).

[0073] "Specific binding" or "specific binding" refers to the binding of an antibody or antigen-binding fragment to an antigen (including sequences contained within the antibody itself) with a greater affinity for other antigens. Typically, specific antibody or antigen-binding fragments bind at an affinity of approximately 5 × 10⁻⁶. -6 M or a smaller equilibrium dissociation constant K D Bind to the target antigen.

[0074] "Antibody derivative" or "alternative format" means an antibody modified by covalent attachment to another molecule as defined above, which is achieved through peptide chemistry (i.e., amidation, etc.), genetic fusion, and / or through post-translational portions (i.e., glycosyl, acetyl, and / or phosphoryl groups) that are not normally associated with the antibody, etc.

[0075] The term "antibody dynamic structure" refers to any structural changes that can affect antibody function, CA125 binding, or intracellular internalization.

[0076] The term "monoclonal antibody (mAb)" refers to an antibody derived from a single-cell clone, including any eukaryotic or prokaryotic cell clone or bacteriophage clone, but not from its method of production. Therefore, the term "monoclonal antibody" is not limited to antibodies produced via hybridoma technology, but can also include those produced via recombinant methods.

[0077] "Fab domain" refers to any antibody sequence at the N-terminus of the disulfide bond region of an antibody hinge, as known in the art.

[0078] "Fc domain" refers to any antibody sequence that is known in the art to be located at the C-terminus of the antibody hinge disulfide bond region and includes that antibody hinge disulfide bond region.

[0079] "Mesothelin" refers to the whole protein or naturally occurring altered form of mesothelin protein (GenBank: AAH03512.1).

[0080] An "antigen" is an entity that an antibody or antibody fragment specifically binds to. This includes binding to an antibody or protein of interest.

[0081] The term "CA125" refers to the gene product produced by the MUC16 gene (HGNC: 15582; OMIM: 606154), which exists in both soluble and membrane-bound forms. It has been reported to bind to antibodies within the Fab domain and influence the humoral immune function of the bound antibodies (Kline JB et al., Oncotarget 8:52045-52060, 2017) as well as ADC uptake.

[0082] The term “CA125 tolerance” refers to an antibody that has low or no binding to the CA125 protein when measured using any method known in the art.

[0083] The terms “CD3” and “CD3E” refer to the CD3-ε protein expressed on human lymphocytes.

[0084] The terms “cancer,” “malignant,” “dysregulation,” and “tumor” are well-known in the art and refer to cells exhibiting unregulated cell growth and morphological characteristics distinct from normal cell types of similar origin, also known as dysregulated cells. Malignancy refers to cancerous cells that are capable of causing disease and / or death. As used, “cancer and tumor” includes both precancerous and malignant types.

[0085] 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, an agent can be shed, secreted, or exported from normal or cancerous cells into biological fluids, including serum, whole blood, plasma, urine, or cellular microfluidics, including tumors.

[0086] As used, the term "level" of a specific 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 the level of protein and / or non-protein agents in vitro or in vivo. Such methods include gel electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), ultradiffusion chromatography, fluid or gel precipitation reactions, absorption spectroscopy, colorimetric assays, spectrophotometry, flow cytometry, immunodiffusion (single or double), solution phase assays, immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, fluorescence resonance energy transfer (FRET), Foster resonance energy transfer, electrochemiluminescence immunoassay, etc. In one embodiment, the level of CA125 is determined using probe-based techniques, as described in more detail.

[0087] The term "humoral immunosuppression" or "humoral immunosuppression" refers to any antibody, antibody fragment, bispecific antibody (BSP), or antibody-drug conjugate (ADC) that directly binds to CA125 and alters its dynamic structure. It has been reported that CA125, produced by malignant cells such as ovarian cancer and mesothelioma (Nicolaides NC et al., Cancer Biol Ther 19:622-630, 2018) and induced by lymphomas from normal peripheral epithelial cells (Sanusi et al., Perit Dial Int. 21:495–500, 2001), can bind to certain antibodies and alter their dynamic structure, thereby affecting their biological activities (including ADCC, CDC, opsonization, internalization and / or PK, PD, and PL profiles).

[0088] The term "antibody-drug conjugate (ADC)" refers to any antibody conjugated or fused with a chemical substance, peptide, nucleic acid, or radionuclide that is toxic to cells.

[0089] The term "cleavable linker" refers to a chemical or amino acid linker that can be cleaved extracellularly or intracellularly by any common mechanism, such as, but not limited to, enzyme or protease digestion, acid degradation, pH, chemical reduction, chemical oxidation, hydrolysis, etc.

[0090] The term "non-cleavable linker" refers to a chemical or amino acid linker that is not normally cleaved outside the cell by common mechanisms such as, but not limited to, enzyme or protease digestion, chemical reduction, chemical oxidation, hydrolysis, etc.

[0091] The terms "enzyme-cleavable linker" and "enzyme-non-cleavable linker" refer to linkers that enzymes or proteases can or cannot cleave.

[0092] The term "bispecific antibody (BSP)" refers to any antibody that can bind to two or more different antigens. A BSP may contain at least, but is not limited to, two full-length antibodies, one full-length antibody and one single-chain antibody, or two single-chain antibodies, each of which binds to a different antigen or a different epitope on the same antigen.

[0093] The term "canonical antibody" refers to an immunoglobulin light chain linked to an immunoglobulin heavy chain, thereby enabling the antibody to specifically recognize the antigen. A canonical antibody may fuse with another antibody capable of specifically recognizing a second antigen.

[0094] The terms “immunosuppressive microenvironment,” “immunosuppressive tumor microenvironment,” and “immunosuppressive 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.

[0095] The terms “immunely active microenvironment,” “immunely active microenvironment,” “immunely active tumor microenvironment,” “immunely intact,” “immune integrity,” and “immunely intact” refer to tumors that do not produce or express factors that suppress cellular or humoral immune function or activity.

[0096] "Immune or immune microenvironmental status" or "microenvironmental immune status" refers to determining whether a tumor is immune-active or immunosuppressive. The term also refers to tumors that produce immunosuppressive proteins, where such tumors are considered to have an immunosuppressive microenvironment.

[0097] The term “antibody-dependent cytotoxicity (ADCC)” refers to an in vitro or in vivo process in which an antibody can bind to an antigen on the cell surface and then bind to immune effector cells via a sequence within the antibody’s Fc domain, thereby causing them to release a toxin that can kill the bound cells.

[0098] 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 then binds to a C1q protein via a sequence within the antibody’s Fc domain, thereby initiating a classical complement cascade that can kill the bound cell.

[0099] The term “internalization” refers to a process in which an antibody, antibody fragment, or ADC can bind to an antigen on the cell surface and then be internalized via a mechanism known to those skilled in the art.

[0100] The term "pharmacokinetics (PK)" refers to the time it takes for an antibody to maintain its steady-state concentration when administered to a subject.

[0101] The term "pharmacodynamics (PD)" refers to the study of the biochemical and physiological effects and mechanisms of action of antibody-based drugs, including the correlation between their effects and their biochemical structure when administered to subjects.

[0102] The term "pharmacology (PL)" refers to the known effects of antibodies on the control or killing of disease cells, either in vitro or in vivo.

[0103] The term "sample" refers to similar liquids, cells, or tissues isolated from a subject, as well as the collective term for liquids, cells, or tissues present within a subject. Fluids can include biological fluids, which include liquid solutions in contact with the subject or biological source, including cell and organoid culture media, urine, saliva, lavage fluids, etc.

[0104] As used, the term "control sample" means any clinically or non-clinically relevant control sample, including, for example, samples from healthy subjects who do not have a specific type of cancer or cells that are different from their parent cells.

[0105] The term "control level" refers to an acceptable or predetermined level of a protein or non-protein reagent used for comparison with the level of the same reagent in a sample derived from a subject or used for in vitro assays.

[0106] As used, the “difference” between the signal of the therapeutic antibody and the control is generally any difference that can be statistically determined using statistical methods commonly used in the art, and is at least 10% or greater 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%.

[0107] The term “inhibition” or “its inhibition” refers to reducing a statistically measurable quantity, or preventing it entirely.

[0108] In the context of antibodies used according to the method and antibody-containing portions (i.e., BSP, ADC, etc.), the term "functional" means that the antibody is able to bind to the antigen or CA125, respectively, and / or is able to bind to and kill target cells in vitro or in vivo.

[0109] The term "target cell" refers to a eukaryotic or prokaryotic cell or cell population that expresses a specific antibody or an antigen containing an antibody moiety.

[0110] The term "therapeutic window" refers to the efficacy of a drug in inhibiting tumor growth within a controlled, tolerable toxic dose.

[0111] The term "pharmaceutically acceptable" refers to a substance that is pharmacologically and toxicologically acceptable for administration to patients and is manufactured using methods known in the art. These include pharmaceutical preparations approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other recognized pharmacopoeias for use in animals and humans. The term "pharmaceuticalally compatible ingredient" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or matrix carrier administered in conjunction with an anticancer agent. "Pharmaceuticalally acceptable carrier" refers to a matrix that does not interfere with the bioactivity of one or more active ingredients and is non-toxic to the host.

[0112] The terms "effective amount" and "therapeuticly effective" are used interchangeably and in the context of administering a drug in an amount sufficient to produce enhanced clinical outcomes in a patient. An effective amount of drug is administered according to the methods described herein in an "effective regimen." The term "effective regimen" refers to a combination of the amount and frequency of administration of a drug sufficient to achieve enhanced clinical outcomes for a patient with a specific cancer. Enhanced efficacy is an improved clinical outcome when a patient is given a drug that overcomes morbidity better than the parent compound or that enhances the clinical outcomes of an effective regimen.

[0113] The terms "patient" and "subject" are used interchangeably to refer to a human being and other non-human animals, including veterinary subjects, receiving treatment with the therapeutic agent. The term "non-human animal" includes all vertebrates. In one implementation, the subject is a human being.

[0114] "Therapeutic agents" are generally free of unwanted contaminants. This means that the agent is usually at least about 50% w / w (weight / weight) pure and is substantially free of interfering proteins and contaminants.

[0115] The term "immune effector cells" refers to any cell that, upon binding to a target cell bound to an antibody, can confer antibody-dependent cytotoxicity (ADCC) or phagocytosis (opsonization), including but not limited to NK cells, bone marrow cells, monocytes, or dendritic cells. Cells may be purified or present in a mixture as peripheral blood mononuclear cells (PBMCs).

[0116] The term "dysregulated cell" refers to any cell that is considered abnormal to its parent cell. These include transformed cells, malignant cells, virus-infected cells, cells that grow autonomously through autoregulation, or prokaryotic pathogens.

[0117] The term "humoral response" refers to ADCC, CDC, opsonization, or the internalization of antibodies into target cells through experimental antibody testing.

[0118] The term "drug" refers to anti-mesothelin ADC or BSP.

[0119] The term "significant" refers to a statistical result in which a p-value is less than 0.05, determined by any number of procedures, including the Stein T test.

[0120] Kits and methods for treating patients with mesothelin-expressing cancers using a combination of detection antibodies, therapeutic MES-ADCs, and MES-BSPs.

[0121] This document provides compositions, kits, and methods for identifying MES-ADCs and MES-BSPs (both referred to as agents) and rMES-1 (referred to as detection antibodies) that can effectively suppress mesothelin-positive cancers, regardless of their microenvironmental immune status. In some embodiments of the methods described herein for identifying optimal MES-ADCs and MES-BSPs, the method involves identifying antibody components of ADCs and / or BSPs that are CA125 tolerant and avoid any of their negative tumor cell-killing activities (i.e., tumor uptake of the ADC, immune responses to bispecific antibodies, etc.). In other embodiments, the CA125-tolerant MES-ADC consists of two or more cytotoxicants linked via cleavable or non-cleavable linkers, and tests the ability of the MES-ADC to exhibit significantly improved cytotoxic effects against immunosuppressive target cells expressing mesothelin, and also against immune-intact target cells. In some embodiments, the MES-ADC cytotoxicants are topoisomerase inhibitors of the SN38 or PNU159682 class. In another embodiment, the MES-ADC comprises a MES-1 antibody conjugated to SN38 via the linker MAC glucosinolate or PEG8-triazole-PABC-peptide-mc at a drug:antibody ratio (DAR) of 2 to 6. In yet another embodiment, the MES-ADC comprises a MES-1 antibody conjugated to PNU159682 via the linker MA-PEG4-VC-PAB-DMAE at a DAR of 2 to 6. Examples are illustrated schematically in… Figure 3BThe kit consists of a MES-ADC that can identify the optimal ADC formulation (cytotoxin and adaptor) using a screening assay employed in the art, employing an ADC killing assay targeting immunosuppressive and immune-intact mesothelin-expressing tumor types. Additionally, the kit consists of an optimal MES-ADC plus an rMES-1 detection antibody, both of which bind to mesothelin in the presence or absence of CA125, to identify patients with mesothelin-expressing cancers for MES-ADC treatment, regardless of the tumor microenvironment's immune status.

[0122] Another embodiment is a method for identifying the optimal MES-BSP. This method involves generating an optimized MES-BSP wherein the MES-BSP light chain contains the amino acids listed in SEQ ID NO: 1 or the heavy chain contains the amino acids listed in SEQ ID NO: 2, and is linked at the N-terminus to an anti-CD3 single-chain antibody (SEQ ID NO: 6). In other embodiments, the MES-BSP comprises a MES-1 light chain fused to an anti-CD3 single chain via a genetically linked spacer region. While the spacer region can consist of any combination and length of natural or modified amino acids, an optional link between the MES-1 light chain and the CD3 single chain is via a genetically encoded adapter unit “GGGGS (SEQ ID NO: 20)”. The link can be via one or more units. The optimal spacer region unit can be determined using assays commonly used in the art, specifically a functional killing assay of immunosuppressive and immune-intact mesothelin-expressing target cells. Examples of screening are discussed in Example 3, and the results are shown in Figure 6. In another implementation, the kit consists of an optimal MES-BSP plus an rMES-1 detection antibody to identify patients with mesothelin-expressing cancers for treatment with MES-BSP, regardless of the tumor microenvironment immune status.

[0123] In methods for identifying optimally active MES-ADC or MES-BSP agents, antibodies are added to cultures of target cells expressing mesothelin, wherein the target cells natively or recombinantly express immunosuppressive proteins. Target cell viability of cultures comparing the responses of cells treated with MES-ADC or MES-BSP to control treatments is monitored using standard kill assays. A change of at least 10% is generally considered a significant effect. 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%, depending on the agent and assay used.

[0124] In some methods used to identify optimal activity of MES-ADC or MES-BSP, the agent is added to a culture of target cells expressing mesothelin, wherein the target cells natively express CA125 or have exogenously added soluble CA125 protein. Cell viability is monitored and compared using standard killing assays to determine the response of cultures treated with MES-ADC or MES-BSP plus CA125 to those treated with control or without CA125. A change in killing intensity of at least 10% is generally considered to have a meaningful effect on the killing of ADCC, CDC, and / or ADC target cells. A meaningful 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%, depending on the agent and assay used.

[0125] This document also provides methods for treating cancer subjects with anti-MES-ADCs or MES-BSPs. For example, patients may have cancers expressing mesothelin, such as, but not limited to, mesothelioma, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer. Several anti-mesothelin antibodies have been reported to bind to CA125 (see Figure 1), which can disrupt their internalization as ADCs or inhibit their immunocytotoxic effects as BSPs, making the use of MES-ADCs or MES-BSPs that are not bound or affected by CA125 a desirable option.

[0126] In some embodiments of methods for treating subjects with MES-ADC or MES-BSP agents, patients with cancer expressing immunosuppressive proteins such as CA125, thus creating an immunosuppressive tumor microenvironment, may be treated alone with MES-ADC or MES-BSP agents or in combination with standard of care. In some embodiments of methods for treating subjects with an immunosuppressive microenvironment, mesothelin-expressing cancers known to express CA125 are described herein. MES-ADC or MES-BSP agents are administered to subjects who have baseline CA125 levels above the normal range. In some embodiments of methods for treating subjects with CA125-expressing cancers described herein, the method involves administering MES-ADC or MES-BSP agents alone. In yet another embodiment, MES-ADC or MES-BSP agents are administered in combination with chemotherapy. Chemotherapy can be any chemotherapeutic agent or biologic agent considered standard of care when treating a subject. In the treatments described herein, CA125 expression levels can be determined by any means known in the art and are defined in the art as being within or above the normal range.

[0127] In other embodiments, the rMES-1 detection antibody is used to identify patients with mesothelin-expressing cancer, and those cancers that are positively bound by rMES are treated with MES-ADC or MES-BSP agents without determining the tumor microenvironment immune status, as MES-ADC or MES-BSP is effective in both immunosuppressive and immunologically intact microenvironments. In some embodiments of the method for treating subjects with mesothelin-expressing cancer described herein, the method involves administering MES-ADC or MES-BSP agents alone. In yet another embodiment, the MES-ADC or MES-BSP agent is administered in combination with chemotherapy. Chemotherapy can be any chemotherapeutic agent or biologic agent considered standard of care when treating the subject.

[0128] In some implementations of the treatment methods 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.

[0129] This approach can be combined with other treatment modalities, such as surgery (e.g., debulking surgery), radiation, targeted therapy, chemotherapy, immunotherapy, and the use of growth factor inhibitors or anti-angiogenic factors. MES-ADC or MES-BSP agents can be administered concurrently to patients receiving surgery, chemotherapy, or radiation therapy. Alternatively, patients may undergo surgery, chemotherapy, or radiation therapy for at least one hour to at most several months before or after administration of MES-ADC or MES-BSP agents, such as before or after standard care therapy. Some implementation schemes of the treatment methods provided herein include administering, in addition to MES-ADC or MES-BSP agents, a therapeutically effective amount of platinum-based chemotherapy and / or folic acid antimetabolites and / or PARP inhibitors to the subject, with or without antibodies against tumor-specific antigens or immune checkpoint proteins.

[0130] In some implementations of the treatment methods described herein, the subject may have already received first-line surgical resection of the tumor, first-line platinum-based therapy, first-line folic acid antimetabolite therapy, first-line platinum and folic acid antimetabolite therapy, PARP inhibitors and / or immune checkpoint inhibitors for the treatment of cancer, and then be given MES-ADC or MES-BSP agents.

[0131] Therapeutic agents (including MES-ADC or MES-BSP agents, folic acid antimetabolites, platinum-based chemotherapy, PARP inhibitors and / or immune checkpoint inhibitors) may be administered in any manner known in the art in accordance with the treatment methods described herein.

[0132] In yet another embodiment, a MES-ADC or MES-BSP agent may be used, comprising the CDR sequence contained in SEQ ID NO: 7-12, according to an IMGT (International ImMunoGeneTics Information System) number outside the standard antibody format. The administration of these modified MES-ADC or MES-BSP agents may be prior to, concurrent with, or after the administration of any other standard care treatment. Treatment may include surgery as well as the current standard care treatment used at the time of treatment.

[0133] Various delivery systems can be used to administer therapeutic agents (including MES-ADC or MES-BSP agents), including intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes, where necessary. Agents can be administered via systemic or local routes of absorption through the epithelial or mucosal skin layers (e.g., oral mucosa, rectal and intestinal mucosa).

[0134] Therapeutic agents can be administered via syringe, catheter, suppository, or any implantable matrix or device.

[0135] The therapeutic agents and pharmaceutical compositions thereof used as described herein may be administered orally in any acceptable dosage form, such as capsules, tablets, aqueous suspensions, solutions, etc.

[0136] Appropriate methods of administration of therapeutic agents include, but are not limited to, intravenous injection and intraperitoneal administration at a final concentration suitable for effective treatment.

[0137] MES-ADC or MES-BSP agents can be administered as a pharmaceutical composition in combination with other drugs, the pharmaceutical composition comprising a therapeutic or preventative effective amount of a therapeutic agent and one or more pharmaceutically acceptable or compatible ingredients.

[0138] The amount of therapeutic agent effective in the treatment or prevention of cancer can be determined using standard clinical techniques. Alternatively, in vitro assays can be used to help identify the optimal dose range required for the MES-ADC or MES-BSP agent. The effective dose can be extrapolated from the dose-response curves of the MES-ADC or MES-BSP agent derived from in vitro cell-based assays, animal models, or other non-human detection systems.

[0139] For example, by determining LD 50 (The lethal dose for 50% of the population) and ED 50 The standard pharmaceutical procedure for determining the toxicity and therapeutic efficacy of a drug in cell cultures or laboratory animals is the dose-to-toxicity ratio (the dose effective in 50% of the population). The dose ratio between toxicity and efficacy is the therapeutic index or therapeutic window and can be expressed as the ratio LD50. 50 / ED 50Agents exhibiting a high therapeutic index are suitable. When an agent exhibits toxic side effects, a delivery system that targets the agent to the affected tissue site can be used to minimize potential damage to non-mesothelin-expressing cells, thereby reducing side effects. Alternatively, if desired, formulations such as, but not limited to, liposome encapsulation can be used to improve the therapeutic index.

[0140] Dosage and administration regimens can vary depending on the concentration of the active drug, which may be required by the subject.

[0141] Another embodiment involves labeling rMES-1 detection antibodies for detecting mesothelin-expressing cells in tumors expressing or not expressing CA125, 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 MES-ADC or MES-BSP agents. Labeling can be any method known in the art for labeling antibodies for diagnostic monitoring.

[0142] Composition of a kit for optimizing the activity of MES-ADC and MES-BSP

[0143] This article also provides kits for preparing optimized MES-ADC and MES-BSP agents suitable for killing immunosuppressive and immune-intact mesothelin-expressing tumors.

[0144] The kit may include a MES-ADC agent containing a cytotoxin linked to an antibody consisting of SEQ ID NO: 1 and 2, which is capable of killing two or more types of mesothelin-expressing cells or tumors regardless of the microenvironmental immune status, wherein the cytotoxin has topoisomerase inhibitory activity and an IC50 value. < 100µM.

[0145] The kit may include a MES-BSP agent containing an antibody or antibody fragment of SEQ ID NO: 7-12 linked to an anti-CD3 single chain (SEQ ID NO: 6), which is capable of killing immunosuppressive and immune-intact mesothelin-expressing cells or tumors, wherein the link between the anti-mesothelin antibody and the anti-CD3 antibody is through an optimized spacer region, which leads to the killing of mesothelin-expressing cells, IC50. < 100µg / mL.

[0146] The foregoing disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by way of reference. A more complete understanding can be obtained by referring to the following specific embodiments, which are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0147] Example 1 - Screening for naturally occurring anti-mesothelin antibodies resistant to proteins produced by immunosuppressive tumors. .

[0148] Several studies have reported that antibodies bound to CA125 are negatively impacted in inducing humoral-mediated immune killing and ADC-mediated target cell killing (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., USPTO application 1698444). In our attempt to identify anti-mesothelin antibodies that are not naturally bound to CA125, we obtained numerous anti-mesothelin antibodies from academic and commercial sources (National Cancer Institute, Creative Biolabs, Novus, etc.) and tested their binding to CA125. Figure 1 shows representative results of an enzyme-linked immunosorbent assay (ELISA) used to screen CA125 binding to different anti-mesothelin antibodies. Recombinant mesothelin was used as a positive control, and human serum albumin (HSA) was used as a negative control. In short, 96-well plates were coated overnight at 4°C with 50 µL / well of 15 KU / 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 for 1 hour at room temperature in 0.05 M phosphate buffer containing 5% bovine serum albumin. The wells were then washed three times with 125 µL of 0.05 M phosphate buffer (pH 7.2) and probed with 2.5 µg / mL of various anti-mesothelin antibodies (meso-Ab1 to meso-Ab5). As shown in Figure 1, several anti-mesothelin antibodies were bound to CA125, while meso-Ab3 (lane 3), composed of MES-1 antibodies (SEQ ID NO:1 and SEQ ID NO:2), unexpectedly was not bound. All antibodies bound mesothelin protein with similar values, while no antibody bound the HSA negative control. These results identify MES-1 as a naturally occurring, CA125-tolerant binding antibody and suggest the potential use of such antibodies for treating cancer cells or tumors that produce this immunosuppressive factor. To confirm the binding of anti-mesothelin antibodies to CA125 Ab-4 (… Figure 1A Compared to ), CA125 binding differentially affects MES-1 ( Figure 1AFor internalization of Ab-3, we used OVCAR3 cells expressing mesothelin and naturally overexpressing membrane-bound CA125 (Kline JB et al., OncoTarget 8:52045-52060, 2017) and an OVCAR3 CA125 knockdown line for internalization assays. Aisgenetic cells (called OVCAR-KO) generated via shRNA produced using a strategy similar to that reported by Kline et al. were used for comparison. Both antibodies were fluorescently labeled using a pH-sensitive pHRodo fluorescent dye system according to the manufacturer's protocol (ThermoFisher, thermofisher.com / adcdiscovery). The antibodies were then repeatedly incubated with OVCAR3 or OVCAR-KO cells in 96-well microplates for 24 hours and treated with Varioskan. ™ ThermoFisher quantifies cellular uptake via fluorescence. For example... Figure 1B As shown, compared to Ab-4, MES-1 Ab (Ab-3) exhibits efficient uptake in both CA125-expressing OVCAR3 cells and CA125-knockdown OVCAR-KO cells, thus supporting the unexpected finding that the sequence encoding the MES-1 antibody is natively CA125-tolerant, making it a qualified antibody-ADC component for treating CA125-expressing tumor cells using the methods taught herein.

[0149] Example 2 - Anti-mesothelin capable of killing both immunosuppressive and immune-intact mesothelin-expressing cancer cells Generation of antibody-drug conjugates (ADCs)

[0150] To determine whether MES-1 is resistant to CA125 and other potential immunosuppressive proteins produced by mesothelin-expressing tumor cells, we configured MES-1 into an ADC format and tested its effectiveness in killing CA125-expressing, immunosuppressive, mesothelin-expressing cancer cells. Here, we provide biochemical analyses of MES-1 and MES-ADC compositions, as well as examples of the efficacy of various MES-ADC formats required for the effective killing of immunosuppressive and immune-intact mesothelin-expressing tumor cells. We also provide the use of CA125-unbound anti-mesothelin antibodies (SEQ ID NO: 1 and SEQ ID NO: 2) linked to a specific payload and adapter type to maximize the killing of immunosuppressive target cells. As discussed above, Nicolaides et al. (Cancer Biol Ther 19:622-630, 2018; Nicolaides et al. USPTO application 16 / 981,444) have reported that CA125 binding to the anti-mesothelin antibody amatoxins leads to humoral immune suppression and reduced ADC killing of any target antigen bound to CA125, due to reduced tumor uptake, compared to those not bound by CA125. To determine the maximum potential efficacy of MES-1 antibodies in ADC formats against tumors with both immune integrity and immunosuppressive microenvironments, we tested several MES-1-based ADCs conjugated with various cytotoxic payloads and multiple adaptor combinations in vitro and then in human tumor xenografts. To generate these ADCs, we first had to generate recombinant MES-1 in a system that would produce the homologous MES-1 protein. Recombinant Chinese hamster ovary (CHO) cells were used for recombinant MES-1 generation because this system had previously been used to generate large quantities of the homologous antibody. cDNA encoding the MES-1 light chain (SEQ ID NO: 4) and MES-1 heavy chain (SEQ ID NO: 5) was synthesized by polymerase chain reaction (PCR), and the PCR fragments were cloned into the pXC vector, which contains two CMV-driven expression cassettes and a glutamine synthase (GS) gene cassette, referred to here as pNAV0047. To generate a stable recombinant fusion protein production cell line, CHOK1SV-GSKO cells containing the knocked-out endogenous GS gene were cultured at 6 × 10⁻⁶ cells / year. 5 Cells / mL were cultured overnight at 37°C in 5% CO2 in CD-CHO medium (Irving Scientific) with 6 mM L-glutamine. The next day, 2.0 × 10⁻⁶ cells / mL were... 7Cells were resuspended in CD-CHO medium with 20 µg of expression plasmid pNAV0047 in 700 µL total volume and transferred to 0.4 cm electroporation cuvettes. Electroporation was performed at 300 V / 900 µF using BioRad GenePulser II. Cells were immediately transferred to flasks containing 30 mL of CD-CHO medium with 6 mM L-glutamine and incubated overnight at 37 °C in 5% CO2 using a shaking incubator. The next day, cells were harvested and resuspended in 30 mL of CD-CHO / SP4 medium containing 50 µM MSX to select high-titer production clones over a two-week selection period. The selected library was then subcloned by limiting dilution, and the production of recombinant MES-1 antibody in the established clones was tested. Productive subclones (expression greater than 1 mg / mL) were amplified, and antibody production and quality were analyzed (target antigen binding was analyzed by ELISA, and protein homogeneity was analyzed by SEC-HPLC). The highest quality clone was then amplified, and the antibody was purified from the culture medium by protein A column affinity chromatography after dialyzing in PBS buffer. The antibody was quantified and its homogeneity analyzed by size exclusion chromatography (SEC-HPLC) and antigen binding. Figure 3A As shown, the MES-1 production system can generate high-quality, homogeneous antibodies, which can be used for the generation of ADCs.

[0151] Here we describe the screening of effective cytotoxic adaptor combinations that are equally effective against both immunosuppressive and immune-intact target cancer cells. To identify the best candidates that meet these criteria, we first tested different classes of cytotoxic agents, including DNA alkylating agents, microtubule inhibitors, and topoisomerase inhibitors. Figure 2A Additionally, we used different connector combinations (disintegrable and non-disintegrable) to determine whether they affected efficacy. Figure 2B To test their efficacy, we used several tumor cell lines expressing mesothelin, a subset of which also co-expressed the immunosuppressive CA125 protein. These cell lines are listed in Table 1.

[0152] Table 1. Cell lines used to test MES-ADC killing effect

[0153]

[0154] The most potent cytotoxic agent identified in our screening was the microtubule inhibitor MMAE (mean EC50). 50 1.39 ng / mL (Li C et al. MAbs 12:1699768, 2020), two topoisomerase inhibitors SN38 (average EC50) 502.44 ng / mL (Meyer-Losic F et al., Clin Cancer Res 14:2145-2155, 2008) and PNU159682 (mean EC50) 50 0.014 ng / mL (here referred to 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 then engineered the lead cytotoxin into an ADC format using a standard cleavable linker and retested them against our tumor cell lines shown in Table 1. While previous reports have confirmed that the drug:antibody ratio (DAR) is an important characteristic of ADC target cell killing, it is also a parameter that is sometimes difficult to control during manufacturing when using partial reduction and chemical conjugation with free cysteine ​​(Farras M et al., Mabs. 12:1702262, 2020). Figure 3B The diagrams provided illustrate SN38-MES-ADC and PNU-MES-ADC. Often, the control of DAR reproducibility is inherent to the starting antibody structure and purity. For example... Figure 3A As shown, purifying the MES-1 antibody from our manufacturing system enables us to generate homogeneous starting antibodies and routinely obtain ADCs with a DAR of 2 to 4 or 4 to 6 when partially denatured. Figure 3C Preliminary cell-killing assays using cleavable linkers with MES-ADCs revealed that SN38- and PNU-MES-ADCs exhibited the most potent targeted killing activity against all mesothelin-expressing cell lines, independent of CA125 status. This may be due to the lack of disruption to cellular uptake, while cell lines not expressing mesothelin remained unaffected. Figure 4A Since PNU-MES-ADC exhibited the most robust killing activity, we next tested PNU-MES-ADC in both the cleavable format (MA-PEG4-VC-PAB-DMAE) and the non-enzymatically cleavable format (MC-EDA), and unexpectedly found that the cleavable format was almost 100 times more potent than the non-enzymatically cleavable format. Figure 4B ).

[0155] To determine the in vivo efficacy of the lead MES-ADC, we next tested the therapeutic efficacy of SN38-MES-ADC and PNU-MES-ADC cleavable and non-enzymatically cleavable formats in mouse xenograft models using tumor cell lines NCI-N87 and SW1990 expressing mesothelin and CA125, as well as patient-derived tumor xenografts (PDXs) with and without CA125 expression. To confirm the expression of mesothelin and CA125 in xenograft and PDX tumors, we used an rMES-1 detection antibody containing SEQ ID NO: 7-12 on a rabbit IgG backbone and a commercial anti-CA125 antibody, respectively, to detect xenograft-derived fragments via immunohistochemistry (IHC). Figure 4C In short, 5µM sections of paraffin-embedded tumor fragments are excised and adhered to glass slides. The sections are dewaxed and prepared for antigen extraction in boiling 10mM sodium citrate at pH 6.0 for 10 minutes, followed by equilibration with phosphate-buffered saline-0.05% Tween-20 (PBS-T). The endogenous peroxidase activity of the sections is then quenched with 0.3% peroxidase / methanol for 10 minutes, and the sections are blocked in 10% goat serum in PBS-T for 1 hour. Next, the slides are rinsed in PBS-T and probed for mesothelin for 1.5 hours with either rMES-1 or rabbit anti-CA125 (Novus) using 3 mg / mL of each primary antibody diluted in blocking buffer, followed by washing, a second blocking for 1 hour, and a 1-hour blocking with a 5µg / mL anti-rabbit horseradish peroxidase (HRP) conjugated secondary antibody. Control slides are incubated without primary antibody. The slides are washed in PBS-T and then processed using eBioscience. ™ DAB advanced chromogenic substrates were exposed as recommended by the manufacturer (ThermoScientific). Finally, the samples were counterstained with hematoxylin, covered with coverslips, and antigen expression was analyzed under an optical microscope. Mesothelin and CA125 (not shown) were found to co-expressed in NCI-N87 and SW1990 xenograft tumors, while non-small cell lung cancer PDX #LXFA983 and mesothelioma PDX #PXF1118 expressed equal levels of mesothelin, and only PDX #PXF118 showed CA125 positivity. Figure 4C (See the image above). We then used these cell lines in in vivo assays in mouse CDX (cell line-derived xenografts) and PDX (patient-derived xenografts).

[0156] For the SW1990 pancreatic cancer CDX model, 1×10 7 Tumor cells were injected into the flanks of multiple athymic nude mice. Tumors with established tumors (136-154 mm) were then injected. 3Mice were randomly assigned to groups and treated intravenously with SN38-MES-ADC at 10 mg / kg or 20 mg / kg or with PBS on days 8, 9, and 10 post-implantation. On day 39, the SN38-MES-ADC treatment group, compared to the vector-treated group, showed a 25% or 53% reduction in tumor growth at 10 mg / kg or 20 mg / kg, respectively, and the differences were statistically significant (P < 0.05 at 10 mg / kg). < 0.049; P at 20 mg / kg < 0.0003) Figure 4D (See the image above).

[0157] For the NCI-N87 gastric cancer CDX model, 1×10 7 Tumor cells were injected into the flanks of multiple SCID mice. The mice with established tumors (126 mm) were targeted. 3 Mice were randomly assigned to groups and treated intravenously as shown below. Following randomization, PNU-MES-ADC in its enzyme-cleavable format was administered at doses of 0.25 mg / kg or 0.5 mg / kg on days 1, 8, 15, 25, 32, and 44. Treatment with the enzyme-cleavable format of PNU-MES-ADC at 0.5 mg / kg reduced tumor growth by 45%, and the difference was statistically significant (p < 0.05). < 0.050) Figure 4D (See figure below). In the same NCI-N87 gastric cancer CDX model, a tumor with an established diameter (126 mm) will be used. 3 Mice were randomly assigned to groups and treated on days 1, 3, 5, 7, 9, 11, 13, and 15 after randomization with SN38-MES-ADC at 20 mg / kg, PNU-MES-ADC in a 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 intravenously with PBS. SN38-MES-ADC treatment reduced tumor growth by 48%, and the difference was statistically significant (p < 0.05). < 0.011 (not shown). PNU-MES-ADC non-enzymatically cleavable format treatment reduced tumor growth by 36%, and the difference was statistically significant (P < 0.011). < (0.033), although not as effective as PNU-MES-ADC with an enzyme-cleavable format.

[0158] For the LXFA983 NSCLC PDX model, tumor fragments were implanted into the flanks of multiple athymic nude mice. The mice with established tumors (128 mm) were then implanted. 3Mice were randomly assigned to groups and treated with PNU-MES-ADC cleavable format at 0.625 mg / kg (days 1, 4, and 8) or 0.4 mg / kg (days 12 and 16) or intravenously with PBS on days 1, 4, 8, 12, and 16 after randomization. PNU-MES-ADC cleavable format induced statistically significant tumor regression (p < 0.05). < 0.0003) Figure 4E (See the image above).

[0159] For the PXF1118 mesothelin PDX model, tumor fragments were implanted into the flanks of multiple athymic nude mice. The mice had established tumors (117-124 mm). 3 Mice were randomly assigned to groups and treated with PNU-MES-ADC cleavable format at 0.625 mg / kg (days 1, 4, and 8) or 0.4 mg / kg (days 12, 16, and 20) or intravenously with PBS on days 1, 4, 8, 12, 16, and 20 after randomization. PNU-MES-ADC cleavable format induced statistically significant tumor regression (p < 0.05). < 0.012) Figure 4E (See the image below).

[0160] Repeated in vivo experiments were conducted using the CA125-expressing PXF1118 PDX line in the model design described above to further determine the efficacy of reduced and single PNU-MES-ADC administration. Briefly, athymic nude mice were prepared as described above and, once established tumors were confirmed, 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 amount in a 0.25 mg / kg PNU-MES-ADC dose), or once on day 1 with 0.75 mg / kg PNU-MES-ADC. Tumor response and health status in mice were tracked for more than 50 days. Figure 5 As shown, on day 49, compared with mice treated with PBS or free PNU159682 (without PN), a single dose of 0.75 mg / kg of cleavable MES-ADC was sufficient to significantly reduce established tumor growth (P < 0.05). < The drug achieved a sustained response similar to that of 0.25 mg / kg PNU-MES-ADC administered three times weekly (0.0061 mg / kg), demonstrating the efficacy of this formulation and composition for treating mesothelin-expressing tumors with or without CA125 expression. The drug treatment was well tolerated in all groups.

[0161] These data support the teaching that compositions containing substances in cleavable formats of PNU-MES-ADC and SN38-MES-ADC can effectively kill tumors in both immunosuppressive and immune-intact tumor microenvironments. The finding that other payloads, such as MMAE microtubule inhibitors, or adaptors, such as non-enzymatically cleavable MC-EDA, are less effective teaches that the use of tumor-targeting ADCs generally cannot simply overcome tumor immunosuppression. Instead, it is necessary to: 1) actively screen for antibodies that do not bind to immunosuppressive factors such as CA125, and 2) screen for optimal payload cytotoxicity, and 3) screen for optimal adaptors, as described in the inventions taught herein.

[0162] Example 3 - Anti-mesothelin capable of killing both immunosuppressive and immune-intact mesothelin-expressing cancer cells Generation of bispecific antibodies (MES-BSP)

[0163] Targeting tumor cells with antibodies typically involves blocking the binding of cytokines to their receptors to inhibit tumor cell growth and / or using humoral-mediated immune killing via opsonization of ADCC, CDC, and / or immune effector cells. As mentioned above, tumor-derived CA125 protein and other tumor-derived proteins (the latter being NCN, JBKLG's personal observation) have the ability to inhibit humoral-mediated antibody killing of target cells. Using antibodies that are naturally resistant to binding to immunosuppressive proteins makes their tumor cell killing possible even in the presence of such proteins. As described in Example 1, anti-mesothelin antibodies were identified by screening for their ability to avoid CA125 binding. To enhance immune-mediated MES-1 killing, we engineered it to potentially enable the use of CD3+ to inhibit the binding of MES-1. +Cytotoxic T cell recruitment to the proximal surface of target cells and subsequent T cell activation for immune-mediated killing are key characteristics of BSP antibody-mediated tumor cell killing (Staerz UD et al., Nature 314;628-631, 1985). The inventors here teach the use of a CA125-unbound anti-mesothelin antibody fused to a second antibody gene, which can bind to cell surface antigens expressed on T lymphocytes, leading to improved immune-mediated tumor cell killing, independent of the immune status of the tumor microenvironment. As an example, we demonstrate the use of a single-chain antibody fused to an MES-1 antibody that can bind to the CD3 antigen on T cells. As discussed in Example 2, Nicolaides et al. (Cancer Biol Ther 19:622-630, 2018) previously demonstrated that the binding of CA125 to the anti-mesothelin antibody amatoxins results in the suppression of humoral immune function. To determine the maximum potential efficacy of BSP-formatted MES-1 antibodies against tumors with both immunocompetent and immunosuppressive microenvironments, we considered several MES-BSP formats that link an anti-CD3 single-chain antibody (SEQ ID NO: 6) to either the MES-1 light chain (SEQ ID NO: 1) or heavy chain (SEQ ID NO: 2). We fused the CD3 single chain to the N-terminus of the MES-1 light chain using the amino acid linker GGGS as shown in SEQ ID NO: 3 and the canonical MES-1 heavy chain (SEQ ID NO: 2). We then generated recombinant MES-BSP as described below and tested its activity against both immunocompetent and CA125-immunosuppressed cancer cell lines.

[0164] To produce high-quality MES-BSP, we engineered recombinant Chinese hamster ovary (CHO) cells to express MES-BSP for large-scale production. A CD3 single-chain antibody was cloned upstream of the mature N-terminal domain of the MES-1 light chain via a genetic adapter encoding the amino acid 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 the pXC vector, which contains two CMV-driven expression cassettes and a glutamine synthase (GS) gene cassette, referred to here as pNAV0071. To generate a stable recombinant fusion protein production cell line, CHOK1SV-GSKO cells containing a knocked-out endogenous GS gene were cultured at 6 × 10⁻⁶ cells / year. 5 Cells / mL were cultured overnight at 37°C in 5% CO2 with 6 mM L-glutamine in CD-CHO (Irving Scientific). The next day, 2.0 × 10⁻⁶ cells / mL were... 7Cells were resuspended in 700 µL of plain CD-CHO with 20 µg of expression plasmid pNAV0071, and then transferred to 0.4 cm electroporation cuvettes and electroporated at 300 V / 900 µF using BioRad GenePulser II. Cells were immediately transferred to flasks containing 30 mL of warm CD-CHO with 6 mM L-glutamine and incubated overnight at 37 °C in 5% CO2 using a shaking incubator. The next day, cells were harvested and resuspended in 30 mL of CD-CHO / SP4 containing 50 µM MSX for selection. Next, the selected library was subcloned by limiting dilution, and recombinant MES-BSP antibodies were generated from the established clones using anti-human Fc-HRP as a probe via ELISA-conditioned medium. Productive subclones (producing greater than 0.5 mg / mL) were amplified, and antibody production and quality (target antigen binding and protein homogeneity) were analyzed. The highest quality clones were then amplified, and the MES-BSP antibody was purified from the culture medium using protein A column affinity chromatography and dialysis in PBS buffer. The MES-BSP antibody was then quantified, and homogeneity was analyzed by SDS-PAGE and antigen binding by ELISA. The efficacy of the high-quality formulation against various tumor cell lines was then tested.

[0165] First, the humoral immune killing effect of MES-BSP was tested by ADCC, and compared with parental MES-1 antibody and humoral immune-suppressive meso-Ab-4 antibody (Ab-4, lane 4, Figure 1) in the presence of immunosuppressive OVCAR3 tumor cell lines expressing mesothelin and naturally overexpressing CA125 protein, and human PBMCs. Figure 6A As shown, MES-BSP exhibited significantly higher killing activity against CA125-producing OVCAR-3 cell lines compared to MES-1 or meso-Ab-4, the latter of which did not show any killing activity. This appears to be CA125-driven, as the ADCC activity of meso-Ab-4 is similar to that of MES-1 in knocking down the ADCC activity of OVCAR-KO cell lines targeting OVCAR3 CA125. Figure 6BIn this assay, the Jurkat-CD16a ADCC reporter cell line was used, and ADCC activity was monitored via luciferase readings according to the manufacturer's instructions (Promega Corp). Previously published and confirmed analog lines exhibited significantly enhanced antibody-mediated humoral responses via CA125-affected antibodies compared 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 CA125-positive tumor cells expressing mesothelin, we used a humanized PBMC mouse model in which athymic nude mice were implanted with mesothelioma-derived tumor cells expressing both mesothelin and CA125. After tumor establishment, mice were implanted with human peripheral blood mononuclear cells (PBMCs) followed by MES-BSP or control treatment. Figure 7 As shown, MES-BSP statistically inhibits tumor growth, demonstrating its efficacy in treating mesothelin-expressing cells within the CA125-mediated immunosuppressive tumor microenvironment. Here, we teach that CA125 may significantly influence the efficacy of antibodies that bind to CA125 and induce immune-mediated killing. Furthermore, we provide a composition of anti-mesothelin BSP antibodies that can effectively kill immunosuppressive tumor cells. sequence list <110> Navrogan <120> Compositions and uses of alternative formatted anti-mesothelin antibodies for cancer treatment <130> 008966.00008 <150> 63 / 084,542 <151> 2020-09-28 <160> 20 <170> FastSEQ for Windows version 4.0 <210> 1 <211> 218 <212> PRT <213> Oryctolagus cuniculus <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser 85 90 95 Asn Asn Trp His Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 2 <211> 453 <212> PRT <213> European rabbit (Oryctolagus cuniculus) <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Leu Gly Phe Tyr 20 25 30 Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser 50 55 60 Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn 100 105 110 Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly 450 <210> 3 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> Antibody sequence modified by fusion <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 130 135 140 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 145 150 155 160 Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln 165 170 175 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Thr Ser Lys Leu 180 185 190 Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205 Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr 210 215 220 Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Cys Gly Thr 225 230 235 240 Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 245 250 255 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 260 265 270 Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln Gln 275 280 285 Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Leu 290 295 300 Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 305 310 315 320 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr 325 330 335 Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala Phe 340 345 350 Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 355 360 365 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 370 375 380 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 385 390 395 400 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 405 410 415 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 420 425 430 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 435 440 445 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 450 455 460 Arg Gly Glu Cys 465 <210> 4 <211> 1464 <212> DNA <213> Artificial sequence <220> <223> Antibody sequence modified by fusion <400> 4 atgtctgtgc ctacccaggt gctgggactg ctgctgctgt ggctgacaga cgcccgctgt 60 caagtgcagt tggtggaatc agggggagga gtcgtgcagc cgggaagatc attgagactg 120 tcgtgcgcgg cgtccggtta caccttcacc cggtatacta tgcactgggt gcgccaggcc 180 cctggcaaat gcctggagtg gatcggttac attaacccga gcagggggta caccaactac 240 aaccagaagg tcaagggccg cttcaccatc tcccgggata actccaagaa caccgcatac 300 ctccaaatga actccctgcg ggccgaagat acggccgtgt actactgtgc ccggtactac 360 gacgaccatt actgccttga ctactggggc cagggcactc tggtgactgt gtccagcggg 420 ggcggtggaa gcgggggggg aggctccgga ggaggcggat cgggtggcgg cggcagcgac 480 atccaaatga cccagtcccc gtcctcactt tccgcatccg tcggcgatcg cgtgaccatt 540 acttgttccg cgtcgtcgtc cgtgagctac atgaactggt atcagcagaa gccaggaaag 600 gccccgaaac tgctgatcta cgacacttcc aagctggctt ctggagtgcc cagcagattc 660 agcggatcag ggtccggtac cgactacacc ttcaccattt cgtccctgca acccgaagat 720 atcgccacct actactgcca gcagtggtcg agcaaccctt ttacgttcgg ctgtggcacc 780 aagctcgaga tcaaaggtgg cggcggtagc gacatccaga tgacacaatc tccttcatct 840 ctcagtgctt ccgtaggaga tagagttact ataacctgtc aagcatctca aaggatctct 900 tcctatctca gttggtatca acagaaaccg ggaaaagtgc ccaaacttct tatctacggt 960 gctagtacac ttgcttccgg ggtcccctca aggttcagcg gcagcggttc tggaacagac 1020 tttaccctga cgatctcaag tctccagcca gaagacgtgg ctacatacta ctgccagtct 1080 tacgcatact tcgatagcaa taactggcac gccttcggtg gcggaaccaa agttgaaata 1140 aaacgaactg tggctgcacc atctgtcttc atcttcccgc catctgatga gcagttgaaa 1200 tctggaactg cctctgttgt gtgcctgctg aataacttct atcccagaga ggccaaagta 1260 cagtggaagg tggataacgc cctccaatcg ggtaactccc aggagagtgt cacagagcag 1320 gacagcaagg acagcaccta cagcctcagc agcaccctga cgctgagcaa agcagactac 1380 gagaaacaca aagtctacgc ctgcgaagtc acccatcagg gcctgagctc gcccgtcaca 1440 aagagcttca acaggggaga gtgt 1464 <210> 5 <211> 1416 <212> DNA <213> Artificial Sequence <220> <223> Modified Antibody Sequence with Leader Sequence <400> 5 atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60 gtgcaactcg tggagtcagg cgggggtctg gttcagccgg gcggcagtct tcggcttagt 120 tgtgccgcaa gcggctttga cctcgggttt tatttctacg cctgttgggt aaggcaggca 180 cctggaaagg gtctggaatg ggtctcttgc atatatacgg caggtagcgg ctccacgtat 240 tacgcaagtt gggccaaggg ccggttcaca atatctaggg acaattccaa aaataccctg 300 tacctgcaaa tgaacagtct cagggctgaa gacactgctg tctactattg cgctcgctca 360 acggctaata cccggtccac ttattacttg aacctctggg gtcagggaac tttggtaaca 420 gtatcatccg catccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 480 acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 540 acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 600 cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 660 acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 720 gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 780 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 840 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 900 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 960 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 1020 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1080 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1140 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1200 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1260 cctcccgtgc tggactccga cggctccttc ttcttatatt caaagctcac cgtggacaag 1320 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1380 cactacacgc agaagagcct ctccctgtct ccgggt 1416 <210> 6 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Antibody sequence modified by fusion <400> 6 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 130 135 140 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 145 150 155 160 Thr Cys Ser Ala Ser Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln 165 170 175 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Thr Ser Lys Leu 180 185 190 Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205 Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr 210 215 220 Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Cys Gly Thr 225 230 235 240 Lys Leu Glu Ile Lys 245 <210> 7 <211> 11 <212> PRT <213> Artificial sequence <220> <223> connector <400> 7 Gln Ala Ser Gln Arg Ile Ser Ser Tyr Leu Ser 1 5 10 <210> 8 <211> 7 <212> PRT <213> Artificial sequence <220> <223> connector <400> 8 Gly Ala Ser Thr Leu Ala Ser 1 5 <210> 9 <211> 13 <212> PRT <213> Artificial sequence <220> <223> connector <400> 9 Gln Ser Tyr Ala Tyr Phe Asp Ser Asn Asn Trp His Ala 1 5 10 <210> 10 <211> 11 <212> PRT <213> Artificial sequence <220> <223> connector <400> 10 Gly Phe Asp Leu Gly Phe Tyr Phe Tyr Ala Cys 1 5 10 <210> 11 <211> 18 <212> PRT <213> Artificial sequence <220> <223> connector <400> 11 Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala 1 5 10 15 Lys Gly <210> 12 <211> 13 <212> PRT <213> Artificial sequence <220> <223> connector <400> 12 Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn Leu 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial sequence <220> <223> connector <400> 13 Gly Tyr Thr Phe Thr Arg Tyr Thr Met His 1 5 10 <210> 14 <211> 20 <212> PRT <213> Artificial sequence <220> <223> connector <400> 14 Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln 1 5 10 15 Lys Val Lys Gly 20 <210> 15 <211> 12 <212> PRT <213> Artificial sequence <220> <223> connector <400> 15 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr 1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial sequence <220> <223> connector <400> 16 Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr 1 5 10 <210> 17 <211> 11 <212> PRT <213> Artificial sequence <220> <223> connector <400> 17 Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser 1 5 10 <210> 18 <211> 9 <212> PRT <213> Artificial sequence <220> <223> connector <400> 18 Gln Gln Trp Ser Ser Asn Pro Phe Thr 1 5 <210> 19 <211> 720 <212> DNA <213> Artificial sequence <220> <223> Modified antibody sequences with modified leader sequences <400> 19 atgtctgtgc ctacccaggt gctgggactg ctgctgctgt ggctgacaga cgcccgctgt 60 gacatacaga tgactcagtc cccttcaagc ctcagtgcat cagtaggtga ccgggttacc 120 attacctgcc aggccagtca acgaatatca tcctacctct cctggtacca gcagaagccg 180 gggaaggtgc ctaagctctt gatctacggc gccagtacgc ttgcaagcgg ggtcccatca 240 cggttctccg gtagtggctc tggaacagat tttacgctca cgatttccag ccttcaaccg 300 gaggatgttg cgacttacta ctgtcaatcc tatgcgtatt tcgattccaa taactggcac 360 gcattcggtg ggggaaccaa agtggagatt aaacgaactg tggctgcacc atctgtcttc 420 atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 480 aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 540 ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 600 agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 660 acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgtctgcac 720 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 20 Gly Gly Gly Gly Ser 1 5

Claims

1. An antibody-drug conjugate comprising an anti-mesothelin antibody and a topoisomerase inhibitor, wherein the anti-mesothelin antibody comprises the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, wherein the topoisomerase inhibitor is SN38 or PNU159682, and wherein the antibody and the topoisomerase inhibitor are covalently linked via a linker.

2. The antibody-drug conjugate according to claim 1, wherein the linker is a cleavable linker.

3. The antibody-drug conjugate according to claim 2, wherein the antibody-drug conjugate is encapsulated in liposomes.

4. The antibody-drug conjugate according to claim 1, wherein the topoisomerase inhibitor is PNU159682.

5. The antibody-drug conjugate according to claim 4, wherein PNU159682 is covalently linked to the anti-mesothelin antibody via the following linker MA-PEG4-VC-PAB-DMAE. 。 6. The antibody-drug conjugate of claim 5, wherein the linker is linked to a cysteine ​​residue in the anti-mesothelin antibody.

7. The antibody-drug conjugate of claim 1, wherein the drug:antibody ratio (DAR) of the antibody-drug conjugate is between 2 and 6, including the end values.

8. The antibody-drug conjugate according to claim 1, wherein the topoisomerase inhibitor is SN38.

9. The antibody-drug conjugate of claim 8, wherein SN38 is covalently linked to the anti-mesothelin antibody via the following linker MAC-glucuronide. 。 10. The antibody-drug conjugate of claim 8, wherein SN38 is covalently linked to the anti-mesothelin antibody via the following linker PEG8-triazole-PABC-peptide-MC. 。 11. The antibody-drug conjugate according to claim 8, wherein SN38 is covalently linked to a cysteine ​​residue in the anti-mesothelin antibody.

12. The antibody-drug conjugate of claim 8, wherein the antibody-drug conjugate has a drug:antibody ratio (DAR) between 2 and 6, including the end values.

13. Use of the pharmaceutical composition in the preparation of a medicament for treating cancer patients with tumors expressing mesothelin, said pharmaceutical composition comprising: An antibody-drug conjugate comprising an anti-mesothelin antibody and a topoisomerase inhibitor, wherein the anti-mesothelin antibody consists of an amino acid sequence as shown in SEQ ID NO:1 and SEQ ID NO:2, wherein the topoisomerase inhibitor is SN38 or PNU159682, and wherein the antibody and the topoisomerase inhibitor are covalently linked via a linker.

14. The use according to claim 13, wherein the cancer is selected from the group consisting of: mesothelioma, breast cancer, lung cancer, colorectal cancer, gastrointestinal cancer, endometrial cancer, cholangiocarcinoma, and pancreatic cancer.

15. The use according to claim 13, wherein The presence of the intermediate cortisol epitope in the cancer patient was detected by contacting a body sample from the cancer patient with an antibody consisting of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO:

2.

16. The use according to claim 13, wherein the cancer patient has a high level of CA125 relative to a healthy population.

17. The use according to claim 13, wherein multiple patients are treated by administering the antibody-drug conjugate, wherein the multiple patients include at least one patient with a high level of CA125 relative to a healthy population and at least one patient with a normal level of CA125.

18. A bispecific antibody comprising a mesothelin-binding moiety and a cell surface antigen CD3-binding moiety, wherein the bispecific antibody comprises the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO:

3.

19. A nucleic acid vector comprising a polynucleotide encoding a bispecific antibody according to claim 18.

20. The nucleic acid vector according to claim 19, wherein the bispecific antibody is encoded by a nucleic acid sequence consisting of SEQ ID NO: 4 and SEQ ID NO:

5.

21. A stable cell line comprising one or more nucleic acids encoding the bispecific antibody according to claim 18.

22. The stable cell line of claim 21, wherein the one or more nucleic acids consist of the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO:

5.

23. Use of a bispecific antibody in the preparation of a medicament for treating cancers expressing mesothelin in patients, said bispecific antibody comprising: Mesothelin-binding region and CD3 cell surface antigen-binding region, The bispecific antibody is composed of the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO:

3.

24. The use according to claim 23, wherein the cancer expressing mesothelin is selected from the group consisting of: mesothelioma, breast cancer, lung cancer, colorectal cancer, gastrointestinal cancer, endometrial cancer, cholangiocarcinoma, and pancreatic cancer.

25. The use according to claim 23, wherein: The patient is tested by contacting a bodily sample with an anti-mesothelin antibody to detect mesothelin epitopes in the patient's cancer.

26. The use according to claim 23, wherein multiple patients are treated by administering the bispecific antibody, wherein the multiple patients include at least one patient with a high level of CA125 relative to a healthy population and at least one patient with a normal level of CA125.

27. The use according to claim 23, wherein the patient has a high level of CA125 relative to a healthy population.

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