WO2019064073A1

WO2019064073A1 - Use of ca125 to predict anti-mesothelin treatment of mesotheliomas

Info

Publication number: WO2019064073A1
Application number: PCT/IB2018/001206
Authority: WO
Inventors: James Bradford KLINE; Nicholas C. NOCOLAIDES; Wenquan Wang; Elizabeth B. Somers
Original assignee: Eisai R&D Management Co., Ltd.
Priority date: 2017-09-27
Filing date: 2018-09-27
Publication date: 2019-04-04

Abstract

Provided herein are methods of identifying and methods of treating a subpopulation of mesothelin-expressing cancer patients predicted to be responsive to treatment regimens including an anti-mesothelin therapeutic agent based on soluble CA125 levels and CA125 direct binding.

Description

. 0 PREDICT ANTI-MESOTHELIN TREATMENT OF MESOTHELIOMAS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application No. 62/564154, filed September 27, 2017, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 27, 2018, is named "104018_001032_SL.txt" and is 9,691 bytes in size.

TECHNICAL FIELD

[0003] The subject matter described herein relates to methods of identifying and methods of treating a subpopulation of cancer patients who would be responsive to treatment regimens that target mesothelin and treatment of such patients using an anti-mesothelin therapeutic agent.

BACKGROUND

[0004] Amatuximab is a chimerized high-affinity monoclonal IgGl/κ antibody targeting mesothelin (Hassan et al. Phase I clinical trial of the chimeric anti-mesothelin monoclonal antibody MORAb-009 in patients with mesothelin-expressing cancers. Clin Cancer Res 2010; 16:6132-6138; Fujisaka et al. Phase I study of amatuximab, a novel monoclonal antibody to mesothelin, in Japanese patients with advanced solid tumors. Invest New Drugs 2015; 33 :380-388). In vitro, amatuximab elicits anti-tumor effects via antibody-dependent cellular cytotoxicity (ADCC) against mesothelin-expressing tumor cells and blocks heterotypic cell adhesion between mesothelin and cells expressing its cognate receptor MUC16/CA125 (Hassan, et al. Preclinical evaluation of MORAb-009, a chimeric antibody targeting tumor- associated mesothelin. Cancer Immun 2007; 7:20-30; Rump et al. Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion. JBiol Chem 2014; 279:9190- 9198; Hassan et al. Inhibition of mesothelin-CA125 interaction in patients with mesothelioma by the anti-mesothelin monoclonal antibody MORAb-009: implications for cancer therapy. Lung Cancer. 2010; 68:455-459). It has been shown to be active in killing mesothelin-positive xenografts in combination with standard chemotherapy. Amatuximab has been tested in a single arm, open-label Phase 2 study, MORAb-009-003 (NCT00738582), in patients with unresectable malignant pleural mesothelioma (MPM) in combination with pemetrexed and cisplatin (Hassan, et al. Phase II clinical trial of amatuximab, a chimeric anti-mesothelin antibody with pemetrexed and cisplatin in advanced unresectable pleural mesothelioma. Clin Cancer Res 2014; 20:5927- 5936). Although the study did not meet the targeted primary endpoint of three month

improvement in progression-free survival (PFS) over historical controls, the median overall survival (OS) of 14.8 months compared favorably with 13.3 months in the fully supplemented subset of patients in the Phase 3 study of cisplatin and pemetrexed (Vogelzang et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 2003; 21 :2636-2644). Moreover, responding patients exhibited an extended OS of greater than 24 months suggesting that the combination of amatuximab plus cisplatin/pemetrexed may be effective in a subgroup of patients.

[0005] A pressing need thus exists for methods for identifying cancer patients likely to be responsive to treatment regimens that target mesothelin-expressing tumors. The methods and kits described herein satisfy this need.

SUMMARY

[0006] Provided herein are methods for predicting response of a subject having mesothelin-expressing mesothelioma to treatment with an anti-mesothelin therapeutic agent. The methods include a step of determining the subject's baseline level of steady state soluble cancer antigen 125 (sCA125) expression. The sCA125 level may be, for example, sCA125 serum level. In some embodiments, a baseline steady state CA125 level greater than 6.5 U/mL and less than about 80 U/mL is predictive of response to the treatment. In some embodiments, a baseline steady state CA125 level greater than the upper limit of normal (ULN) and less than about four times the ULN is predictive of response to the treatment. [0007] In some aspects of the methods for predicting response of a subject having mesothelioma-expressing mesothelioma to treatment with an anti-mesothelin therapeutic agent, the anti-mesothelin therapeutic agent comprises an antibody that specifically binds mesothelin ("an anti-mesothelin antibody"). In some aspects, the anti-mesothelin antibody is:

(a) an antibody comprising SEQ ID NO: l (GYSFTGYTMN) as CDRH1, SEQ ID

NO:2 (LITPYNGAS S YNQ) as CDRH2, SEQ ID NO:3 (GGYDGRGFDY) as CDRH3, SEQ ID NO:4 (SASSSVSYMH) as CDRL1, SEQ ID NO:5

(DTSKLAS) as CDRL2 and SEQ ID NO:6 (QQWSKHPLT) as CDRL3, numbered according to Kabat;

(b) an antibody comprising SEQ ID NO:7 (GYSFTGYT) as CDRH1, SEQ ID

NO: 8 (ITPYNGAS) as CDRH2, SEQ ID NO: 9 (GGYDGRGFDY) as

CDRH3, SEQ ID NO: 10 (SSVSY) as CDRL1, SEQ ID NO: 11 (DTS) as CDRL2 and SEQ ID NO: 12 (QQWSKHPLT) as CDRL3, numbered according to IMGT;

(c) an antibody produced by cells assigned ATCC Accession No. PTA-7553; or (d) an antibody that binds the same epitope as amatuximab.

In some embodiments, the anti-mesothelin antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14. In some aspects, the anti-mesothelin antibody is amatuximab.

[0008] In some aspects of the methods for predicting response of a subject having mesothelioma-expressing mesothelioma to treatment with an anti-mesothelin therapeutic agent, the treatment includes at least a folate antimetabolite and a platinum-based chemotherapy. The folate antimetabolite may be pemetrexed. The platinum-based chemotherapy may be cisplatin.

[0009] Also provided herein are methods of treating a subject having mesothelin- expressing mesothelioma. In some embodiments, the mesothelin-expressing mesothelioma is malignant pleural mesothelioma. In some embodiments of the methods of treatment, the method involves administering to a subject having mesothelin-expressing cancer and a baseline steady state soluble cancer antigen 125 (sCA125) level that is greater than 6.5 U/mL and less than about 80 U/mL an anti-mesothelin therapeutic agent. In some embodiments of the methods of treatment, the method involves administering to a subject having mesothelin-expressing cancer and a baseline steady state soluble cancer antigen 125 (sCA125) level that is greater than the upper limit of normal (ULN) for sCA125 and less than about four times the ULN for sCA125 an anti-mesothelin therapeutic agent. In some embodiments of the methods of treatment, the method involves administering to the subject an anti-mesothelin therapeutic agent, wherein the subject has a baseline steady state soluble cancer antigen 125 (sCA125) level that is greater than 6.5 U/mL and less than about 80 U/mL. In yet other embodiments of the methods of treatment, the method involves administering to the subject an anti-mesothelin therapeutic agent, wherein the subject has a baseline steady state soluble cancer antigen 125 (sCA125) level that is greater than the upper limit of normal (ULN) for sCA125 and less than about four times the ULN for sCA125. In further embodiments of the methods of treatment, the method involves detecting a baseline steady state soluble cancer antigen 125 (sCA125) level in a biological sample obtained from the subject, and administering an anti-mesothelin therapeutic agent to the subject when the subject's baseline sCA125 level is determined to be greater than 6.5 U/mL and less than about 80 U/mL. In still further embodiments of the methods of treatment, the method involves detecting a baseline steady state soluble cancer antigen 125 (sCA125) level in a biological sample obtained from the subject, and administering an anti-mesothelin therapeutic agent to the subject when the subject's baseline sCA125 level is determined to be greater than the upper limit of normal (ULN) for sCA125 and less than about four times the ULN for sCA125. The sCA125 level may be, for example, sCA125 serum level.

[0010] In some aspects of the methods of treatment provided herein, the anti- mesothelin therapeutic agent comprises an anti-mesothelin antibody. In some embodiments, the anti-mesothelin antibody is an antibody that is bound by CA125. In some embodiments, the anti- mesothelin antibody is:

(a) an antibody comprising SEQ ID NO: l (GYSFTGYTMN) as CDRH1, SEQ ID NO:2 (LITPYNGASSYNQ) as CDRH2, SEQ ID NO:3 (GGYDGRGFDY) as CDRH3, SEQ ID NO:4 (SASSSVSYMH) as CDRL1, SEQ ID NO:5 (DTSKLAS) as CDRL2 and SEQ ID NO:6 (QQWSKHPLT) as CDRL3, numbered according to Kabat;

(b) an antibody comprising SEQ ID NO:7 (GYSFTGYT) as CDRH1, SEQ ID NO:8

(ITPYNGAS) as CDRH2, SEQ ID NO: 9 (GGYDGRGFDY) as CDRH3, SEQ ID NO: 10 (SSVSY) as CDRL1, SEQ ID NO: 11 (DTS) as CDRL2 and SEQ ID NO: 12 (QQWSKHPLT) as CDRL3, numbered according to IMGT;

(c) an antibody produced by cells assigned ATCC Accession No. PTA-7553; or

(d) an antibody that binds the same epitope as amatuximab.

In some aspects of the methods of treatment, the anti-mesothelin antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-mesothelin antibody is amatuximab.

[0011] In any of the methods of treatment provided herein, the method may further involve administering at least a folate antimetabolite and a platinum-based chemotherapy to the subject. An exemplary folate antimetabolite is pemetrexed. An exemplary platinum-based chemotherapy is cisplatin.

[0012] Also provided herein are uses of a baseline steady state soluble cancer antigen 125 (sCA125) level in predicting response to treatment with an anti-mesothlin therapeutic agent by a subject having mesothelin-expressing mesothelioma. In some embodiments, the mesothelin- expressing mesothelioma is malignant pleural mesothelioma. In some aspects, the sCA125 level is sCA125 serum level. In accordance with some uses, a baseline sCA125 level that is greater than 6.5 U/mL and less than about 80 U/mL is predictive of response to treatment with an anti- mesothelin therapeutic agent by a subject having mesothelin-expressing mesothelioma. In accordance with some uses, a baseline steady state soluble cancer antigen 125 (sCA125) level greater than the upper limit of normal (ULN) for sCA125 and less than about four times the ULN for sCA125 is predictive of response to treatment with an anti-mesothelin therapeutic agent by a subject having mesothelin-expressing mesothelioma. In some embodiments of such uses, the anti-mesothelin therapeutic agent is an anti-mesothelin antibody. In some embodiments, the anti- mesothelin antibody is an antibody that is bound by CA125. In some embodiments, the anti- mesothelin antibody is:

(e) an antibody comprising SEQ ID NO: l (GYSFTGYTMN) as CDRH1, SEQ ID NO:2 (LITPYNGASSYNQ) as CDRH2, SEQ ID NO:3 (GGYDGRGFDY) as CDRH3, SEQ ID NO:4 (SASSSVSYMH) as CDRL1, SEQ ID NO:5 (DTSKLAS) as CDRL2 and SEQ ID NO:6 (QQWSKHPLT) as CDRL3, numbered according to Kabat; (f) an antibody comprising SEQ ID NO:7 (GYSFTGYT) as CDRH1, SEQ ID NO:8 (ITPYNGAS) as CDRH2, SEQ ID NO: 9 (GGYDGRGFDY) as CDRH3, SEQ ID NO: 10 (SSVSY) as CDRL1, SEQ ID NO: 11 (DTS) as CDRL2 and SEQ ID NO: 12

(QQWSKHPLT) as CDRL3, numbered according to IMGT;

(g) an antibody produced by cells assigned ATCC Accession No. PTA-7553; or

(h) an antibody that binds the same epitope as amatuximab.

In some aspects of the uses disclosed herein, the anti-mesothelin antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-mesothelin antibody is amatuximab.

[0013] In some embodiments of the uses provided herein, the treatment further comprises at least a folate antimetabolite and a platinum-based chemotherapy. An exemplary folate antimetabolite is pemetrexed. An exemplary platinum-based chemotherapy is cisplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1A, IB, 1C, and ID illustrate KM analysis of PFS and OS. Figures 1A and IB show KM analysis of PFS (A) and OS (B) in patients with >57 U/mL or <57 U/mL CA125. To confirm that the effect was not driven by the STAGE IB/II patients in the <57 U/mL CA125 subgroup, KM analysis of PFS (C) and OS (D) in STAGE IB/II only patients with >57 U/mL or <57 U/mL CA125 was performed. As shown patients with CA125 levels <57 U/mL have a statistically improved PFS and OS in both populations. The circles represent censored subjects. All p values are two-sided.

[0015] Figure 2 illustrates the effect of exogenous sCA125 on amatuximab as compared to farletuzumab ADCC activity using Jurkat-Luc reporter assay.

[0016] Figures 3A, 3B, 3C, and 3D illustrate the effect of CA125 on amatuximab ADCC in OVCAR3 parental vs OVCAR3-KD1 cells whose CA125 is knocked down via shRNA. Figure 3A shows FACS analysis of cell surface CA125 expression. Figure 3B shows glycan analysis via PAS staining. Figure 3C shows FACS analysis of cell surface mesothelin expression. Figure 3D shows ADCC activation of Jurkat-Luc cells by OVCAR3 parental and OVCAR3-KD1 cells. [0017] Figures 4A, 4B, 4C, and 4D. Figure 4A shows the CA125 effect on direct amatuximab and fragment binding. Figure 4B shows engagement with CD 16a Fc-γ receptor on Jurkat-CD16a cells in the presence or absence of CA125 in duplicate. Figure 4C shows engagement of amatuximab with CD 16a, CD32a and CD64a Fc-γ activating receptors in the presence or absence of CA125. Figure 4D shows engagement with FcRn receptor in the presence or absence of CA125.

[0018] Figure 5 illustrates the binding data of various antibodies that are biotinylated and used as probe in ELISA to measure for CA125 binding. As shown and depicted by the bar graph in Figure 6, only 7 of 16 antibody probes were found to significantly bind wells coated with CA125. The last 2 columns are a combination of M9-bio (MORAb-009-biotinylated) and M3-bio (MORAb-003 -biotinylated) probing human albumin and wells not coated with antigen (last two columns, respectively).

[0019] Figure 6 illustrates binding of only a subset of human, humanized or chimeric antibodies to CA125. As shown, only humanized MORAb-003 (M3-bio) and its (Fab')₂ fragment (M3-FAB2-bio); along with chimerized MORAb-009 (M9-bio) and rituximab (Rituximab-B); and fully human MORAb-022 (M22-bio), Total human IgM (Total-huIgM-B) and a human IgM clone (IgM-clonal-B) were able to significantly bind CA125. Bars above the dotted red line have statistical significance in binding as compared to controls.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0020] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0021] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0022] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±5% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0023] The term "antibody" as used herein is meant in a broad sense and includes immunoglobulin or antibody molecules including polyclonal antibodies, monoclonal antibodies including murine, human, human-adapted, humanized and chimeric monoclonal antibodies and antibody fragments. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Intact antibodies are heterotetrameric glycoproteins, composed of two identical light chains and two identical heavy chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (variable region) (VH) followed by a number of constant domains (constant regions). Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0024] Immunoglobulins can be assigned to five major classes or isotypes, depending upon the type of constant domain possessed by its heavy chain, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0025] An immunoglobulin light chain variable region or heavy chain variable region consists of a "framework" region interrupted by three "antigen-binding sites". The antigen- binding sites are defined using various terms as follows: (i) the term Complementarity

Determining Regions (CDRs) is based on sequence variability (Wu and Kabat, J. Exp. Med. 132:211-250, 1970). Generally, the antigen-binding site has six CDRs; three in the VH (CDRHl, CDRH2, CDRH3), and three in the VL (CDRL1, CDRL2, CDRL3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The "IMGT-CDRs" as proposed by Lefranc (Lefranc et al., Dev.

Comparat. Immunol. 27:55-77, 2003) are based on the comparison of V domains from

immunoglobulins and T-cell receptors. The International ImMunoGeneTics (EVIGT) database (http://www_imgt_org) provides a standardized numbering and definition of these regions. The correspondence between CDRs and EVIGT delineations is described in Lefranc et al., Dev.

Comparat. Immunol. 27:55-77, 2003.

[0026] Antigen-binding fragments are any proteinaceous structure that may exhibit binding affinity for a particular antigen. Some antigen-binding fragments are composed of portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule. For example, antigen-binding fragments may comprise at least one variable region (either a heavy chain or light chain variable region) or one or more CDRs of an antibody known to bind a particular antigen. Examples of suitable antigen-binding fragments include, without limitation diabodies and single-chain molecules as well as Fab, F(ab')₂, Fc, Fabc, and Fv molecules, single chain (Sc) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like. All antibody isotypes may be used to produce antigen-binding fragments. Additionally, antigen-binding fragments may include non-antibody proteinaceous frameworks that may successfully incorporate polypeptide segments in an orientation that confers affinity for a given antigen of interest, such as protein scaffolds.

Antigen-binding fragments may be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies. The phrase "an antibody or antigen-binding fragment thereof may be used to denote that a given antigen-binding fragment incorporates one or more amino acid segments of the antibody referred to in the phrase.

[0027] "Specific binding" or "specifically binds" refers to the binding of an antibody or antigen-binding fragment to an antigen with greater affinity than for other antigens. Typically, the antibody or antigen-binding fragment binds to the antigen with an equilibrium dissociation constant KD of about 5xl0^"8 M or less, for example about 5xl0^"9 M or less, about lxlO^"9 M or less, about lxlO^"10 M or less, or about lx 10^"11 M or less.

[0028] An "antibody derivative" means an antibody, as defined above, that is modified by covalent attachment of a heterologous molecule such as, e.g., by attachment of a heterologous polypeptide (e.g., a cytotoxin) or therapeutic agent (e.g., a chemotherapeutic agent), or by glycosylation, deglycosylation, acetylation or phosphorylation not normally associated with the antibody, and the like.

[0029] The term "monoclonal antibody" refers to an antibody that is derived from a single cell clone, including any eukaryotic or prokaryotic cell clone, or a phage clone, and not the method by which it is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology.

[0030] An "antigen" is an entity to which an antibody specifically binds. For example, mesothelin is the antigen to which an anti-mesothelin antibody specifically binds.

[0031] The term "CA125" refers to the gene product produced by MUC16 gene (HGNC: 15582; OMIM: 606154), which can be in membrane bound form or soluble form shed from the surface of epithelial cells and various malignant cells. The product is a highly O- glycosylated mucin representing heterogeneous protein species found in systemic circulation and on cells (Yin, B. W. T., Lloyd, K. O. Molecular cloning of the CA125 ovarian cancer antigen: identification as a new mucin, MUC16. J. Biol. Chem. 276: 27371-27375, 2001).

[0032] The terms "cancer" and "tumor" are well known in the art and refer to the presence, e.g., in a subject, of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone within a subject, or may be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term "cancer" includes pre-malignant as well as malignant cancers.

[0033] As used herein, the term "mesothelin" refers to the glycosylphosphatidyl inositol (GPI)-linked protein having UniProt Q13421 [296-606]. Variants, for example, naturally occurring allelic variants or sequences containing at least one amino acid substitution, are encompassed by the term mesothelin as used herein.

[0034] As used herein, the term "not bound to a cell" refers to a protein that is not attached to the cellular membrane of a cell, such as a cancerous cell. For example, a protein that is not bound to a cell may be shed, secreted or exported from normal or cancerous cells, for example, from the surface of cancerous cells, into biological fluids. In a particular embodiment, CA125 not bound to a cell is unbound to any cell and is freely floating or solubilized in biological fluids, e.g., urine, serum or the soluble tumor microenvironment.

[0035] Antibodies used in immunoassays to determine the level of expression of a specified protein, such as for example, CA125, may be labeled with a detectable label. The term "labeled", with regard to the binding agent or antibody, is intended to encompass direct labeling of the binding agent or antibody by coupling (i.e., physically linking) a detectable substance to the binding agent or antibody, as well as indirect labeling of the binding agent or antibody by reactivity with another reagent that is directly labeled. An example of indirect labeling includes detection of a primary antibody using a fluorescently labeled secondary antibody. In one embodiment, the antibody is labeled, e.g., radio-labeled, chromophore-labeled, fluorophore- labeled, or enzyme-labeled. In another embodiment, the antibody is an antibody derivative (e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair (e.g., biotin-streptavidin), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain).

[0036] The "level" of a specified protein, as used herein, refers to the level of the protein as determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion

chromatography, fluid or gel precipitation reactions, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), solution phase assay, Immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and electrochemiluminescence immunoassay (exemplified below), and the like. In one embodiment, proteomic methods, e.g., mass spectrometry, are used. Mass spectrometry is an analytical technique that consists of ionizing chemical compounds to generate charged molecules (or fragments thereof) and measuring their mass-to-charge ratios. In a typical mass spectrometry procedure, a sample is obtained from a subject, loaded onto the mass spectrometry, and its components (e.g., CA125) are ionized by different methods (e.g., by impacting them with an electron beam), resulting in the formation of charged particles (ions). The mass-to-charge ratio of the particles is then calculated from the motion of the ions as they transit through electromagnetic fields. In another

embodiment, matrix-associated laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) or surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), which involves the application of a sample, such as urine or serum, to a protein-binding chip (Wright, G.L., Jr., et al. (2002) Expert Rev Mol Diagn 2:549; Li, J., et al. (2002) Clin Chem 48: 1296; Laronga, C, et al. (2003) Dis Markers 19:229; Adam, B.L., et al. (2002) Cancer Res 62:3609; Tolson, J., et al. (2004) Lab Invest 84:845; Xiao, Z., et al. (2001) Cancer Res 61 :6029), can be used to determine the level of CA125. Furthermore, in vivo techniques for determination of the level of a molecular marker (e.g., CA125) include

introducing into a subject a labeled antibody directed against the marker, which binds to and transforms the marker into a detectable molecule. The presence, level, or location of the detectable marker in a subject may be determined using standard imaging techniques. In a preferred embodiment, the level of a molecular marker (e.g., CA125) is determined using antibody -based techniques, as described in more detail herein.

[0037] The term "baseline level" with respect to a molecular marker refers to an initial determination of the amount or level of that marker in a subject or a biological sample obtained from a subject. For example, a baseline level of a biomarker may be the level of the marker determined upon or following diagnosis with mesothelioma, upon or following surgical resection of the mesothelioma, or upon or following initiation or completion of a first-line or other therapy for mesothelioma. [0038] As used herein, a "mesothelin-expressing mesothelioma" includes any type of mesothelioma characterized in that the cancer cells express or present on their surface mesothelin. A mesothelioma may have been, but is not required to have been, clinically diagnosed as expressing mesothelin to be encompassed by the term "mesothelin-expressing mesothelioma" as used herein.

[0039] As used herein, a subject who is "afflicted with" or "having mesothelioma" is one who is clinically diagnosed with mesothelioma at any stage by a qualified clinician, or one who exhibits one or more signs or symptoms of such a cancer and is subsequently clinically diagnosed with such a cancer by a qualified clinician. A non-human subject that serves as an animal model of mesothelin-expressing mesothelioma may also fall within the scope of a subject "afflicted with mesothelin-expressing mesothelioma."

[0040] The term "sample" as used herein refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject.

Biological fluids are typically liquids at physiological temperatures and may include naturally occurring fluids present in, withdrawn from, expressed or otherwise extracted from a subject or biological source. Certain biological fluids derive from particular tissues, organs or localized regions and certain other biological fluids may be more globally or systemically situated in a subject or biological source. Examples of biological fluids include blood, serum and serosal fluids, plasma, lymph, urine, cerebrospinal fluid, saliva, ocular fluids, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, gynecological fluids, ascites fluids such as those associated with non-solid tumors, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage and the like. Biological fluids may also include liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium, lavage fluids and the like.

[0041] In some embodiments, only a portion of the sample is subjected to an assay for determining the level of a molecular marker, or various portions of the sample are subjected to various assays for determining the level of a molecular marker. Also, in many embodiments, the sample may be pre-treated by physical or chemical means prior to the assay. For example, samples may be subjected to centrifugation, dilution and/or treatment with a solubilizing substance (e.g., guanidine treatment) prior to assaying the samples for a molecular marker. Such techniques serve to enhance the accuracy, reliability and reproducibility of the assays.

[0042] The term "control sample," as used herein, refers to any clinically relevant control sample, including, for example, a sample from a healthy subject not afflicted with mesothelioma, a sample from a subject having a less severe or slower progressing mesothelioma than the subject to be assessed, a sample from a subject having some other type of cancer or disease, and the like. A control sample may include a sample derived from one or more subjects. A control sample may also be a sample made at an earlier timepoint from the subject to be assessed. For example, the control sample could be a sample taken from the subject to be assessed before the onset of mesothelioma, at an earlier stage of disease, or before the

administration of treatment or of a portion of treatment. The control sample may also be a sample from an animal model, or from a tissue or cell lines derived from the animal model, of mesothelioma. The level of a molecular marker in a control sample that consists of a group of measurements may be determined based on any appropriate statistical measure, such as, for example, measures of central tendency including average, median, or modal values.

[0043] The term "control level" refers to an accepted or pre-determined level of a molecular marker which is used to compare with the level of the molecular marker in a sample derived from a subject. In one embodiment, the control level of a molecular marker is based on the level of the molecular marker in sample(s) from a subject(s) having slow disease progression. In another embodiment, the control level of a molecular marker is based on the level in a sample from a subject(s) having rapid disease progression. In another embodiment, the control level of a molecular marker is based on the level of the molecular marker in a sample(s) from an unaffected, i.e., non-diseased, subject(s), i.e., a subject who does not have mesothelioma. In yet another embodiment, the control level of a molecular marker is based on the level of the molecular marker in a sample from a subject(s) prior to the administration of a therapy for mesothelioma. In another embodiment, the control level of a molecular marker is based on the level of the molecular marker in a sample(s) from a subject(s) having mesothelioma that is not contacted with a test compound. In another embodiment, the control level of a molecular marker is based on the level of the molecular marker in a sample(s) from a subject(s) not having mesothelioma that is contacted with a test compound. In one embodiment, the control level of a molecular marker is based on the level of the molecular marker in a sample(s) from an animal model of mesothelioma, a cell, or a cell line derived from the animal model of mesothelioma. In one embodiment, the control is a standardized control, such as, for example, a control which is predetermined using an average of the levels of a molecular marker from a population of subjects having no mesothelioma. In still other embodiments of the invention, a control level of a molecular marker is based on the level of the molecular marker in a non-cancerous sample(s) derived from the subject having mesothelioma.

[0044] As used herein, "a difference" between the level of a molecular marker in a sample from a subject (i.e., a test sample) and the level of the molecular marker in a control sample refers broadly to any clinically relevant and/or statistically significant difference in the level of the molecular marker in the two samples. For example, "an increase" in the level of a molecular marker may refer to a level in a test sample that is about two, and more preferably about three, about four, about five, about six, about seven, about eight, about nine, about ten or more times more than the level of the molecular marker in the control sample. An increase may also refer to a level in a test sample that is preferably at least about 1.5, and more preferably about two, about three, about four, about five or more standard deviations above the average level of the molecular marker in the control sample.

[0045] As used herein, the term "contacting the sample" with a specific binding agent, e.g., an antibody, includes exposing the sample, or any portion thereof with the agent or antibody, such that at least a portion of the sample comes into contact with the agent or antibody. The sample or portion thereof may be altered in some way, such as by subjecting it to physical or chemical treatments (e.g., dilution or guanidine treatment), prior to the act of contacting it with the agent or antibody.

[0046] The term "inhibit" or "inhibition of means to reduce by a measurable amount, or to prevent entirely.

[0047] The term "deplete," in the context of the effect of an anti-mesothelin therapeutic agent on mesothelin-expressing cells, refers to a reduction in the number of, or elimination of, the mesothelin-expressing cells. [0048] The term "functional," in the context of an antibody to be used in accordance with the methods described herein, indicates that the antibody is (1) capable of binding to antigen and/or (2) depletes or inhibits the proliferation of antigen-expressing cells.

[0049] The term "treatment" or "treat" or "positive therapeutic response" refers to slowing, stopping, or reversing the progression of a mesothelin-expressing cancer in a patient, as evidenced by a decrease or elimination of a clinical or diagnostic symptom of the disease, by administration of an anti-mesothelin therapeutic agent to the subject after the onset of a clinical or diagnostic symptom of the mesothelin-expressing cancer at any clinical stage. Treatment can include, for example, a decrease in the severity of a symptom, the number of symptoms, or frequency of relapse.

[0050] The phrase "responsive to treatment with an anti-mesothelin therapeutic agent" is intended to mean that the candidate subject (i.e., an individual with mesothelin-expressing cancer), following administration of the anti-mesothelin therapeutic agent, would have a positive therapeutic response with respect to the mesothelin-expressing cancer.

[0051] The term "pharmaceutically acceptable" refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability and includes properties and/or substances approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "pharmaceutically compatible ingredient" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or vehicle with which an anti-mesothelin antibody is administered. "Pharmaceutically acceptable carrier" refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered.

[0052] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and, in the context of the administration of a pharmaceutical agent, refer to the amount of the agent that is sufficient to inhibit the occurrence or ameliorate one or more clinical or diagnostic symptoms of a mesothelin-expressing cancer in a patient. A therapeutically effective amount of an agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antigen-binding fragment thereof to elicit a desired response in the individual. Such results may include, but are not limited to, the treatment of a mesothelin-expressing mesothelioma, as determined by any means suitable in the art. An effective amount of an agent is administered according to the methods described herein in an "effective regimen." The term "effective regimen" refers to a combination of amount of the agent and dosage frequency adequate to accomplish treatment of a mesothelin-expressing cancer.

[0053] The terms "patient" and "subject" are used interchangeably to refer to humans and other non-human animals, including veterinary subjects, that receive diagnostic, prophylactic or therapeutic treatment. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dog, cat, horse, cow, chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a human.

[0054] Therapeutic agents are typically substantially pure from undesired contaminants. This means that an agent is typically at least about 50% w/w (weight/weight) pure as well as substantially free from interfering proteins and contaminants. Sometimes the agents are at least about 80% w/w and, more preferably at least 90 or about 95% w/w pure. However, using conventional protein purification techniques, homogeneous peptides of at least 99% purity w/w can be obtained.

Methods for identifying a subject having a cancer predicted to respond to treatment with an anti-mesothelin therapeutic agent

[0055] Provided herein are methods for identifying a subject having a mesothelin- expressing cancer predicted to respond to treatment with an anti-mesothelin therapeutic agent. In some embodiments of the methods for identifying a subject having mesothelin-expressing cancer predicted to respond to treatment with an anti-mesothelin therapeutic agent described herein, the mesothelin-expressing cancer predicted to respond to treatment with an anti-mesothelin therapeutic agent is mesothelioma. In some embodiments, the mesothelioma is malignant pleural mesothelioma. The subject may have received a platinum-based, folate antimetabolite-based, or platinum- and folate antimetabolite-based first-line therapy.

[0056] The methods for identifying a subject having a mesothelin-expressing cancer predicted to respond to treatment with an anti-mesothelin therapeutic agent as described herein involve determining a baseline level of steady state soluble cancer antigen 125 (sCA125) expression of the subject. In some embodiments, the baseline level of sCA125 is the baseline serum CA125 level. As is understood by those skilled in the art, the upper limit of normal (ULN) for sCA125 varies depending upon the assay employed. For example, the upper limit of normal for sCA125 in the Immulite® assay for CA125 exemplified herein is currently established to be about 21 units per milliliter (U/ml). In other such sCA125 assays, however, exemplified by the Abbott Architect, Beckman Access and the like, the upper limit of normal for sCA125 is established to be about 35 U/ml. In some embodiments of the methods for identifying a subject having a mesothelin-expressing cancer predicted to respond to treatment with an anti-mesothelin therapeutic agent provided herein, a baseline sCA125 level that is less than about four times the ULN for sCA125, preferably less than about three times the ULN for sCA125, and more preferably less than about two times the ULN for sCA125 is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. In some embodiments of the methods for identifying a subject having a mesothelin-expressing cancer predicted to respond to treatment with an anti-mesothelin therapeutic agent provided herein, a baseline sCA125 level that is greater than the ULN but less than about four times the ULN for sCA125, preferably less than about three times the ULN for sCA125, and more preferably less than about two times the ULN for sCA125 is indicative of a subject who would benefit from treatment with an anti- mesothelin therapeutic agent. In some embodiments of the methods for identifying a subject having a mesothelin-expressing cancer predicted to respond to treatment with an anti-mesothelin therapeutic agent provided herein, a baseline sCA125 level that is less than about 80 units/mL, less than about 70 units/mL, more preferably less than about 60 units/mL, in some embodiments less than about 57 units/mL, and in some embodiments less than about 50 units/mL is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. In some embodiments of the methods for identifying a subject having a mesothelin-expressing cancer predicted to respond to treatment with an anti-mesothelin therapeutic agent provided herein, a baseline sCA125 level that is greater than about 6.5 units/mL, 8 units/mL, 10 units/mL, more preferably 21 units/mL, and less than about 80 units/mL, about 70 units/mL, more preferably about 60 units/mL, in some embodiments about 57 units/mL, and in some

embodiments about 50 units/mL is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. In some aspects, a baseline sCA125 level is greater than 6.5 U/mL and less than about 60 U/mL is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. In some aspects, a baseline sCA125 level is greater than 6.5 U/mL and less than about 57 U/mL is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. In some aspects, a baseline sCA125 level is greater than about 8 U/mL and less than about 57 U/mL is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent.

[0057] In the methods for identifying a subject having a mesothelin-expressing cancer predicted to respond to treatment with an anti-mesothelin therapeutic agent described herein, sCA125 expression level may be determined by any means known in the art. For example, the level of sCA125 expression may be determined using an antibody to detect protein expression, nucleic acid hybridization, quantitative RT-PCR, western blot analysis, radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion,

electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS), or ELISA assay. The step of determining expression level of CA125 may be performed ex vivo or in vivo.

[0058] For ex vivo assessments, the biological sample used in determining the baseline level of sCA125 may be may be derived from whole blood, serum, plasma, pleural effusions, ascites, tissues (e.g., surgically resected tumor tissue, biopsies, including fine needle aspiration), histological preparations, and the like. The sample on which the assay is performed can be fixed or frozen to permit histological sectioning. Preferably, the excised tissue samples are fixed in aldehyde fixatives such as formaldehyde, paraformaldehyde, glutaraldehyde; or heavy metal fixatives such as mercuric chloride. More preferably, the excised tissue samples are fixed in formalin and embedded in paraffin wax prior to incubation with the antibody. Optionally, FFPE specimens can be treated with citrate, EDTA, enzymatic digestion or heat to increase accessibility of epitopes. Alternatively, a protein fraction can be isolated from cells from known or suspected mesothelin-expressing cancer and analyzed by ELISA, Western blotting, immunoprecipitation or the like. In another variation, cells can be analyzed for expression of mesothelin by FACS analysis. In a further variation, mRNA can be extracted from cells from known or suspected mesothelin-expressing cancer. The mRNA or a nucleic acid derived therefrom, such as a cDNA can then be analyzed by hybridization to a nucleic probe binding to DNA encoding CA125.

[0059] For example, the step of determining expression level of CA125 may involve determining the level of CA125 expression in a biological sample of the mesothelioma tissue obtained from the subject. CA125 expression levels may be determined by an immunoassay in which a sample containing cells known or suspected to be from a cancer (e.g., mesothelioma) is contacted with an anti-CA125 antibody or antigen-binding fragment. After contact, the presence or absence of a binding event of the antibody or antigen-binding fragment to the cells in the specimen is determined. The binding is related to the presence or absence of the antigen expressed on cancerous cells in this specimen. Generally, the sample is contacted with a labeled specific binding partner of the anti-CA125 antibody or antigen-binding fragment capable of producing a detectable signal. Alternatively, the anti-CA125 antibody or fragment itself can be labeled. Examples of types of labels include enzyme labels, radioisotopic labels, nonradioactive labels, fluorescent labels, toxin labels and chemoluminescent labels. Many such labels are readily known to those skilled in the art. For example, suitable labels include, but should not be considered limited to, radiolabels, fluorescent labels (such as DyLight® 649), epitope tags, biotin, chromophore labels, ECL labels, or enzymes. More specifically, the described labels include ruthenium, ^luIn-DOTA, ^U1ln- diethylenetriaminepentaacetic acid (DTP A), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, poly-histidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazin dyes, phenanthridine dyes, rhodamine dyes, Alexafluor® dyes, and the like. Detection of a signal from the label indicates the presence of the antibody or fragment specifically bound to CA125 in the sample.

[0060] In another variation, CA125 expression level in known or suspected

mesothelioma can be detected in vivo by administering a labeled anti-CA125 antibody or antigen-binding fragment thereof to a patient and detecting the antibody or fragment by in vivo imaging.

[0061] The level of CA125 in a biological sample can (but need not) be determined with respect to one or more standards. The standards can be historically or contemporaneously determined. The standard can be, for example, a biological sample known not to be cancerous from a different subject. The standard can also be the patient sample under analysis contacted with an irrelevant antibody (e.g., an antibody raised to a bacterial antigen).

[0062] The presence of detectable signal from binding of an anti-CA125 antibody or fragment to CA125 relative to a standard (if used) indicates the presence of CA125 in the biological sample, and the level of detectable binding provides an indication of the level of expression of CA125. The level (intensity) of expression can be used as a measure of the total expression in the sample.

[0063] The baseline level of sCA125 may be either a measurement of the sCA125 level in the subject at a single timepoint or may involve measurement of CA125 levels in the subject at two, three, four, five, or more points in time (e.g., serial CA125 determinations). Determination of the baseline level of sCA125 in the subject may be performed upon diagnosis, upon surgical resection, upon initiation of first-line therapy, upon completion of first-line therapy, upon initiation of second-line therapy, upon completion of second-line therapy, and/or upon symptomatic progression, serologic progression, and/or radiologic progression of the cancer.

[0064] In some embodiments of the methods for identifying a subject having mesothelioma predicted to respond to treatment with an anti-mesothelin therapeutic agent, the anti-mesothelin therapeutic agent is an antibody that specifically binds to mesothelin, preferably to mesothelin expressed on mesothelioma cells; antigen-binding fragments of such an antibody; derivatives; and variants thereof. An exemplary antibody that specifically binds to mesothelin may be an antibody selected from the group consisting of:

(a) an antibody comprising SEQ ID NO: 1 (GYSFTGYTMN) as CDRH1, SEQ ID

NO:2 (LITP YNGAS S YNQ) as CDRH2, SEQ ID NO:3 (GGYDGRGFDY) as CDRH3, SEQ ID NO:4 (SASSSVSYMH) as CDRL1, SEQ ID N0 5

(b) an antibody comprising SEQ ID NO:7 (GYSFTGYT) as CDRH1, SEQ ID

NO: 8 (ITP YNGAS) as CDRH2, SEQ ID NO: 9 (GGYDGRGFDY) as

CDRH3, SEQ ID NO: 10 (SSVSY) as CDRL1, SEQ ID NO: 1 1 (DTS) as CDRL2 and SEQ ID NO: 12 (QQWSKHPLT) as CDRL3, numbered according to IMGT; (c) an antibody produced by cells assigned ATCC Accession No. PTA-7553; or

(d) an antibody that binds the same epitope as amatuximab.

In some embodiments, the antibody that specifically binds to mesothelin comprises a mature light chain variable region comprising the amino acid sequence of SEQ ID NO: 13 :

DIELTQS PAIMSAS PGEKVTMTCSAS S SVSYMHWYQQKSGTS PKRWI YDTSKLA SGVPGRFSGSGSGNSYSLT I S SVEAEDDATYYCQQWSKHPLT FGSGTKVE IKRT VAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDS TYSLS S TLTLSKADYEKHKVYACEVTHQGLS S PVTKS FNRGEC* .

In some embodiments, the antibody that specifically binds to mesothelin comprises a mature heavy chain variable region comprising the amino acid SEQ ID NO: 14:

QVQLQQSGPELEKPGASVKI SCKASGYS FTGYTMNWVKQSHGKSLEWI GL I TPY NGAS SYNQKFRGKATLTVDKS S S TAYMDLLSLTSEDSAVYFCARGGYDGRGFDY WGSGTPVTVS SAS TKGPSVFPLAPS SKS TSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHT FPAVLQS SGLYSLS SWTVPS S SLGTQTYI CNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCK VSNKALPAP IEKT I SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLS PGK* .

[0065] In some embodiments, the antibody that specifically binds to mesothelin comprises a mature light chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a mature heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14. An example of such an antibody is MORAb-009 (USAN: amatuximab).

Amatuximab is a humanized monoclonal antibody directed against mesothelin. Chinese hamster ovary (CHO) cells producing MORAb-009 have been deposited with the ATCC (10801

University Boulevard, Manassas, VA 20110) on April 24, 2006 and assigned accession no. PTA- 7553.

[0066] Other useful antibodies that specifically bind to mesothelin comprise mature light and heavy chain variable regions having at least 90% and preferably at least 95% or 99% sequence identity to SEQ ID NO: 13 and SEQ ID NO: 14, respectively. Other useful anti- mesothelin antibodies or derivatives thereof can competitively inhibit binding of amatuximab to mesothelin, as determined, for example, by immunoassay. Competitive inhibition means that an antibody, when present in at least a two-fold and preferably five-fold excess, inhibits binding of amatuximab to mesothelin by at least 50%, more typically at least 60%, yet more typically at least 70%), and most typically at least 75%, at least 80%>, at least 85%>, at least 90%, or at least 95%.

[0067] The anti-mesothelin therapeutic agent may also be a derivative of an anti- mesothelin antibody. Typical modifications include, e.g., glycosylation, deglycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, and the like.

Additionally, the derivative may contain one or more non-classical amino acids.

[0068] In some embodiments of the methods for identifying a subject having mesothelioma predicted to respond to treatment with an anti-mesothelin therapeutic agent described herein, the subject may have received surgical resection of the mesothelioma, first-line platinum-based therapy, first-line folate antimetabolite-based therapy, and/or first-line platinum and folate antimetabolite-based therapy for treatment of the mesothelioma prior to determining the baseline level of sCA125. In some embodiments of the methods in which the subject received surgical resection of the mesothelioma, first-line platinum-based therapy, first-line folate antimetabolite-based therapy, and/or first-line platinum and folate antimetabolite-based therapy for treatment of the mesothelioma prior to determining the baseline level of sCA125, the subject may have exhibited symptomatic progression, serologic progression, and/or radiologic progression of the mesothelioma prior to the step of determining the baseline level of sCA125.

Methods of Treatment

[0069] Also provided herein are methods of treating a subject with mesothelin- expressing cancer, for example, mesothelin-expressing mesothelioma.

[0070] In some embodiments of the methods of treating a subject with mesothelin- expressing cancer described herein, an anti-mesothelin therapeutic agent is administered to a subject having a baseline sCA125 level that is less than about four times the ULN for sCA125, preferably less than about three times the ULN for sCA125, and more preferably less than about two times the ULN for sCA125. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer provided herein, an anti-mesothelin therapeutic agent is administered to a subject having a baseline sCA125 level that is greater than the ULN but less than about four times the ULN for sCA125, preferably less than about three times the ULN for sCA125, and more preferably less than about two times the ULN for sCA125. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer provided herein, an anti-mesothelin therapeutic agent is administered to a subject having a baseline sCA125 level that is less than about 80 units/mL, less than about 70 units/mL, more preferably less than about 60 units/mL, in some embodiments less than about 57 units/mL, and in some embodiments less than about 50 units/mL. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer provided herein, an anti-mesothelin therapeutic agent is administered to a subject having a baseline sCA125 level that is greater than about 6.5 units/mL, 8 units/mL, 10 units/mL, more preferably 21 units/mL, and less than about 80 units/mL, about 70 units/mL, more preferably about 60 units/mL, in some embodiments about 57 units/mL, and in some embodiments about 50 units/mL. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer provided herein, an anti-mesothelin therapeutic agent is administered to a subject having a baseline sCA125 level that is greater than about 6.5 units/mL and less than about 60 units/mL, more preferably less than about 57 units/mL. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer provided herein, an anti-mesothelin therapeutic agent is administered to a subject having a baseline sCA125 level that is greater than about 8 units/mL and less than about 60 units/mL, more preferably less than about 57 units/mL.

[0071] In some embodiments of the methods of treating a subject with mesothelin- expressing cancer described herein, an anti-mesothelin therapeutic agent is administered to the subject, wherein the subject has a baseline sCA125 level that is less than about four times the ULN for sCA125, preferably less than about three times the ULN for sCA125, and more preferably less than about two times the ULN for sCA125. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer provided herein, an anti-mesothelin therapeutic agent is administered to the subject, wherein the subject has a baseline sCA125 level that is greater than the ULN but less than about four times the ULN for sCA125, preferably less than about three times the ULN for sCA125, and more preferably less than about two times the ULN for sCA125. In some embodiments of the methods of treating a subject with mesothelin- expressing cancer provided herein, an anti-mesothelin therapeutic agent is administered to the subject, wherein the subject has a baseline sCA125 level that is less than about 80 units/mL, less than about 70 units/mL, more preferably less than about 60 units/mL, in some embodiments less than about 57 units/mL, and in some embodiments less than about 50 units/mL. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer provided herein, an anti-mesothelin therapeutic agent is administered to the subject, wherein the subject has a baseline sCA125 level that is greater than about 6.5 units/mL, 8 units/mL, 10 units/mL, more preferably 21 units/mL, and less than about 80 units/mL, about 70 units/mL, more preferably about 60 units/mL, in some embodiments about 57 units/mL, and in some

embodiments about 50 units/mL. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer provided herein, an anti-mesothelin therapeutic agent is administered to the subject, wherein the subject has a baseline sCA125 level that is greater than about 6.5 units/mL and less than about 60 units/mL, preferably less than about 57 U/mL. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer provided herein, an anti-mesothelin therapeutic agent is administered to the subject, wherein the subject has a baseline sCA125 level that is greater than about 8 units/mL and less than about 60 units/mL, preferably less than about 57 U/mL.

[0072] In some embodiments of the methods of treating a subject with mesothelin- expressing cancer described herein, the method involves detecting a baseline level of sCA125 in a biological sample obtained from the subject and administering an anti-mesothelin therapeutic agent to the subject when the baseline sCA125 level is determined to be less than about four times the ULN for sCA125, preferably less than about three times the ULN for CA125, and more preferably less than about two times the ULN for CA125. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer described herein, the method involves detecting a baseline level of sCA125 in a biological sample obtained from the subject and administering an anti-mesothelin therapeutic agent to the subject when the baseline sCA125 level is determined to be greater than the ULN but less than about four times the ULN for sCA125, preferably less than about three times the ULN for CA125, and more preferably less than about two times the ULN for CA125. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer described herein, the method involves detecting a baseline level of sCA125 in a biological sample obtained from the subject and administering an anti-mesothelin therapeutic agent to the subject when the baseline sCA125 level is determined to be greater than the ULN but less than about four times the ULN for sCA125, preferably less than about three times the ULN for CA125, and more preferably less than about two times the ULN for CA125. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer described herein, the method involves detecting a baseline level of sCA125 in a biological sample obtained from the subject and administering an anti-mesothelin therapeutic agent to the subject when the baseline sCA125 level is determined to be less than about 80 units/mL, less than about 70 units/mL, more preferably less than about 60 units/mL, in some embodiments less than about 57 units/mL, and in some embodiments less than about 50 units/mL. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer described herein, the method involves detecting a baseline level of sCA125 in a biological sample obtained from the subject and administering an anti-mesothelin therapeutic agent to the subject when the baseline sCA125 level is determined to be greater than about 6.5 units/mL, 8 units/mL, 10 units/mL, more preferably 21 units/mL, and less than about 80 units/mL, about 70 units/mL, more preferably about 60 units/mL, in some embodiments about 57 units/mL, and in some embodiments about 50 units/mL. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer described herein, the method involves detecting a baseline level of sCA125 in a biological sample obtained from the subject and administering an anti-mesothelin therapeutic agent to the subject when the baseline sCA125 level is determined to be greater than about 6.5 units/mL and less than about 60 units/mL, preferably less than about 57 units/mL. In some embodiments of the methods of treating a subject with mesothelin-expressing cancer described herein, the method involves detecting a baseline level of sCA125 in a biological sample obtained from the subject and administering an anti-mesothelin therapeutic agent to the subject when the baseline sCA125 level is determined to be greater than about 8 units/mL and less than about 60 units/mL, preferably less than about 57 units/mL.

[0073] In the methods of treatment described herein, CA125 expression level may be determined by any means known in the art, as described supra. [0074] In some embodiments of the methods of treatment described herein, the anti- mesothelin therapeutic agent is an antibody that specifically binds to mesothelin, preferably to mesothelin expressed on mesothelioma cells; antigen-binding fragments of such an antibody; derivatives; and variants thereof. An exemplary antibody that specifically binds to mesothelin may be an antibody selected from the group consisting of:

(a) an antibody comprising SEQ ID NO: l (GYSFTGYTMN) as CDRH1, SEQ ID NO:2 (LITP YNGAS S YNQ) as CDRH2, SEQ ID NO: 3 (GGYDGRGFDY) as CDRH3, SEQ ID NO:4 (SASSSVSYMH) as CDRL1, SEQ ID NO:5 (DTSKLAS) as CDRL2 and SEQ ID NO: 6 (QQWSKHPLT) as CDRL3, numbered according to Kabat;

(b) an antibody comprising SEQ ID NO:7 (GYSFTGYT) as CDRH1, SEQ ID NO:8 (ITP YNGAS) as CDRH2, SEQ ID NO: 9 (GGYDGRGFDY) as CDRH3, SEQ ID NO: 10 (SSVSY) as CDRL1, SEQ ID NO: 11 (DTS) as CDRL2 and SEQ ID NO: 12 (QQWSKHPLT) as CDRL3, numbered according to IMGT;

(c) an antibody produced by cells assigned ATCC Accession No. PTA-7553; or

(d) an antibody that binds the same epitope as amatuximab.

In some embodiments, the antibody that specifically binds to mesothelin comprises a mature light chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and/or a mature heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In preferred embodiments of the methods of treatment described herein, the anti-mesothelin therapeutic agent is amatuximab. As described supra, other useful antibodies that specifically bind to mesothelin comprise mature light and heavy chain variable regions having at least 90% and preferably at least 95% or 99% sequence identity to SEQ ID NO: 13 and SEQ ID NO: 14, respectively. Other useful anti-mesothelin antibodies or derivatives thereof can competitively inhibit binding of amatuximab to mesothelin, as determined, for example, by immunoassay. A derivative of an anti-mesothelin antibody can also be used in the practice of present methods. Typical modifications include, e.g., glycosylation, deglycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, and the like. Additionally, the derivative may contain one or more non-classical amino acids. [0075] The present methods can be combined with other means of treatment such as surgery (e.g., debulking surgery), radiation, targeted therapy, chemotherapy, immunotherapy, use of growth factor inhibitors, or anti-angiogenesis factors. An anti-mesothelin therapeutic agent can be administered concurrently to a patient undergoing surgery, chemotherapy or radiation therapy treatments. Alternatively, a patient can undergo surgery, chemotherapy or radiation therapy prior or subsequent to administration of the anti-mesothelin therapeutic agent by at least an hour and up to several months, for example at least an hour, five hours, 12 hours, a day, a week, a month, or three months, prior or subsequent to administration of the anti-mesothelin therapeutic agent. Some embodiments of the methods of treatment provided herein involve administration of a therapeutically effective amount of a platinum-based chemotherapy and/or a folate antimetabolite to the subject in addition to the anti-mesothelin therapeutic agent. For example, some embodiments of the methods of treatment provided herein involve administration of therapeutically effective amounts of amatuximab, a platinum-based chemotherapy, and a folate antimetabolite. In some embodiments, the platinum-based chemotherapy is cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, Nedaplatin, Triplatin, Lipoplatin, or combinations thereof. In certain embodiments, the platinum-based chemotherapy is any other platinum-based chemotherapy known in the art. In some embodiments, the folate antimetabolite is pemetrexed. The platinum -based chemotherapy may be administered to the subject once every week, once every two weeks, once every three weeks, or once every four weeks. The folate antimetabolite may be administered to the subject once every week, once every two weeks, once every three weeks, or once every four weeks. In embodiments in which both a folate antimetabolite and a platinum-based chemotherapy are administered to the subject as part of the treatment regimen, the anti-mesothelin therapeutic agent, the platinum-based chemotherapy, and the folate antimetabolite may be administered sequentially in any order or simultaneously.

[0076] In some embodiments of the methods of treatment described herein, the subject may have received first line surgical resection of the tumor, first-line platinum-based therapy, first-line folate antimetabolite-based therapy, and/or first-line platinum and folate antimetabolite- based therapy for treatment of the mesothelin-expressing cancer prior to determining the baseline level of sCA125. In some embodiments of the methods in which the subject received first line surgical resection of the tumor, first-line platinum-based therapy, first-line folate antimetabolite- based therapy, and/or first-line platinum and folate antimetabolite-based therapy for treatment of the mesothelin-expressing cancer prior to determining the baseline level of sCA125, the subject may have exhibited symptomatic progression, serologic progression, and/or radiologic progression of the mesothelin-expressing cancer prior to the step of determining the baseline level of sCA125.

[0077] Administration of the therapeutic agents (including the anti-mesothelin therapeutic agent, the folate antimetabolite, and/or the platinum-based chemotherapy) in accordance with the methods of treatment described herein may be by any means known in the art.

[0078] Various delivery systems can be used to administer the therapeutic agents (including the anti-mesothelin therapeutic agent, the folate antimetabolite, and/or the platinum- based chemotherapy) including intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The agents can be administered, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, and the like). Administration can be systemic or local.

[0079] The therapeutic agents can be administered by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non- porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. The therapeutic agents and pharmaceutical compositions thereof for use as described herein may be administered orally in any acceptable dosage form such as capsules, tablets, aqueous suspensions, solutions or the like.

[0080] Preferred methods of administration of the therapeutic agents include but are not limited to intravenous injection and intraperitoneal administration.

[0081] Alternatively, the therapeutic agents can be delivered in a controlled release system. For example, a pump can be used (see Langer, 1990, Science 249: 1527-1533; Sefton, 1989, CRC Crit. Ref Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321 :574). Alternatively, polymeric materials can be used (see Medical Applications of Controlled Release (Langer & Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen & Ball eds., Wiley, New York, 1984); Ranger & Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23 :61. See also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71 : 105.) Other controlled release systems are discussed, for example, in Langer, supra.

[0082] The therapeutic agents can be administered as pharmaceutical compositions comprising a therapeutically or prophylactically effective amount of the therapeutic agent(s) and one or more pharmaceutically acceptable or compatible ingredients. For example, the

pharmaceutical composition typically includes one or more pharmaceutical carriers (e.g., sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions (e.g., phosphate buffered saline) and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, pH buffering agents (e.g., amino acids) and/or solubilizing or stabilizing agents (e.g., nonionic surfactants such as tween or sugars such as sucrose, trehalose or the like). In some aspects, the formulation of amatuximab contains amatuximab, glutamate, and sorbitol.

[0083] The pharmaceutical compositions provided herein can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid preparations. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the antibody, typically in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulations correspond to the mode of administration. [0084] Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. When necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or a concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. When the pharmaceutical composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0085] The amount of the therapeutic agent that is effective in the treatment or prophylaxis of mesothelin-expressing cancer can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation also depends on the route of administration, and the stage of the cancer, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the ICso (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.

[0086] For example, toxicity and therapeutic efficacy of the agents can be determined in cell cultures or experimental animals by standard pharmaceutical procedures for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Agents that exhibit large therapeutic indices are preferred. When an agent exhibits toxic side effects, a delivery system that targets the agent to the site of affected tissue can be used to minimize potential damage to non-mesothelin-expressing cells and, thereby, reduce side effects.

[0087] In some embodiments, the subject can be administered an anti-mesothelin therapeutic agent described herein in a daily dose range of about 0.01 μg to about 500 mg per kg of the weight of the subject. Typically, the dosage of the therapeutic agent (e.g., the anti- mesothelin therapeutic agent, preferably amatuximab) administered to a patient with mesothelin- expressing cancer is about 0.1 mg/kg to about 100 mg/kg of the subject's body weight. More typically, the dosage administered to a subject is about 1.25 mg/kg to about 12.5 mg/kg of the subject's body weight, or even more typically about 2.5 mg/kg to about 10.0 mg/kg of the subject's body weight. In some embodiments, the dosage of the anti-mesothelin therapeutic agent, preferably amituximab, administered to a subject having mesothelin-expressing mesothelioma is about 5.0 mg/kg to about 7.5 mg/kg of the subject's body weight. In some embodiments of the methods of treatment described herein, the anti-mesothelin therapeutic agent is administered to the subject once every week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments of the methods of treatment described herein, the anti-mesothelin therapeutic agent is administered to the subject on days 1 and 8 of a 21-day cycle. In some embodiments, the dosage of the folate antimetabolite administered to a subject having mesothelin-expressing cancer is about 50 mg/m² to about 750 mg/m²of the subject's body weight, preferably about 400 mg/m² to about 600 mg/m². In some embodiments, the dosage of the platinum-based chemotherapy administered to a subject having mesothelin-expressing cancer is about 50 mg/m² to about 250 mg/m²of the subject's body weight, preferably about 75 mg/m² to about 150 mg/m².

[0088] For effective treatment, one skilled in the art may recommend a dosage schedule and dosage amount of the therapeutic agent(s) adequate for the subject being treated. In embodiments in which both a folate antimetabolite and a platinum-based chemotherapy are administered to the subject as part of the treatment regimen, the folate antimetabolite may be administered before, after, or simultaneously with the platinum-based chemotherapy.

[0089] The dosing may occur less frequently if the compositions are formulated in sustained delivery vehicles. The dosage schedule may also vary depending on the active drug concentration, which may depend on the needs of the subject.

Kits

[0090] Further provided herein are kits for identifying a subject having mesothelin- expressing cancer that will be responsive to treatment with an anti-mesothelin therapeutic agent. In some embodiments, the kits contain an anti-CA125 antibody, a vessel for containing the antibody when not in use, and instructions for using the anti-CA125 antibody for determining the level of CA125 of a subject. The instructions may specify that a baseline sCA125 level is less than about four times the upper limit of normal (ULN) for sCA125, preferably less than about three times the ULN, and more preferably less than about two times the ULN for sCA125, is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. In some embodiments, the instructions may specify that a baseline sCA125 level is greater than the ULN but less than about four times the upper limit of normal (ULN) for sCA125, preferably less than about three times the ULN, and more preferably less than about two times the ULN for sCA125, is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. Alternatively, the instructions may specify that a baseline sCA125 level that is less than about 80 units/mL, less than about 70 units/mL, more preferably less than about 60 units/mL, more preferably less than about 57 units/mL, and in some embodiments less than about 50 units/mL, is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. In some embodiments, the instructions may specify that a baseline sCA125 level that is greater than about 6.5 units/mL, 8 units/mL, 10 units/mL, more preferably 21 units/mL, and less than about 80 units/mL, about 70 units/mL, more preferably about 60 units/mL, more preferably about 57 units/mL, and in some

embodiments about 50 units/mL, is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the molecular marker assay(s). Such kits can also, or alternatively, contain a detection reagent that contains a reporter group suitable for direct or indirect detection of antibody binding.

[0091] Also provided herein are kits for treating a subject having mesothelioma that will be responsive to treatment with an anti-mesothelin therapeutic agent comprising the anti- mesothelin therapeutic agent, a vessel for containing the anti-mesothelin therapeutic agent when not in use, and instructions for use of the anti-mesothelin therapeutic agent. Amituximab is the preferred anti-mesothelin therapeutic agent in the kits. The instructions may specify that a baseline sCA125 level is less than about four times the upper limit of normal (ULN) for sCA125, preferably less than about three times the ULN, and more preferably less than about two times the ULN for sCA125, is indicative of a subject who would benefit from treatment with an anti- mesothelin therapeutic agent. In some embodiments, the instructions may specify that a baseline sCA125 level is greater than the ULN but less than about four times the upper limit of normal (ULN) for sCA125, preferably less than about three times the ULN, and more preferably less than about two times the ULN for sCA125, is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. Alternatively, the instructions may specify that a baseline sCA125 level that is less than about 80 units/mL, less than about 70 units/mL, more preferably less than about 60 units/mL, more preferably less than about 57 units/mL, and in some embodiments less than about 50 units/mL, is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. In some embodiments, the instructions may specify that a baseline sCA125 level that is greater than about 6.5 units/mL, 8 units/mL, 10 units/mL, more preferably 21 units/mL, and less than about 80 units/mL, about 70 units/mL, more preferably about 60 units/mL, more preferably about 57 units/mL, and in some

embodiments about 50 units/mL, is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. In some embodiments, the instructions may specify that a baseline sCA125 level that is greater than about 6.5 units/mL and less than about 60 units/mL, more preferably about 57 units/mL, is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. In some embodiments, the instructions may specify that a baseline sCA125 level that is greater than about 8 units/mL and less than about 60 units/mL, more preferably about 57 units/mL, is indicative of a subject who would benefit from treatment with an anti-mesothelin therapeutic agent. In some embodiments, the kits for treating a subject having mesothelioma that will be responsive to treatment with an anti- mesothelin therapeutic agent contain the anti-mesothelin therapeutic agent as well as an anti- CA125 antibody, a vessel for containing the anti-CA125 antibody when not in use, and instructions for using the anti-CA125 antibody for determining a baseline level of sCA125 in a biological sample obtained from the subject.

[0092] The kits for treating a subject having mesothelioma that will be responsive to treatment with an anti-mesothelin therapeutic agent also may contain additional therapeutic agents (e.g., a platinum-based chemotherapy and/or a folate antimetabolite) as described herein. Examples of platinum-based chemotherapies for inclusion in the kits include, but are not limited to, cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, Nedaplatin, Triplatin, Lipoplatin, or combinations thereof. An example of a folate antimetabolite is pemetrexed. The therapeutic agents can be in any of a variety of forms suitable for distribution in a kit. Forms of the therapeutic agents suitable for distribution in the kits can include a liquid, powder, tablet, suspension and the like formulation for providing the therapeutic agent. The kits can also include a pharmaceutically acceptable diluent (e.g., sterile water) for injection, reconstitution or dilution of the therapeutic agent(s). One or more additional containers may enclose elements, such as reagents or buffers, to be used in the molecular marker assay(s). Such kits can also, or alternatively, contain a detection reagent that contains a reporter group suitable for direct or indirect detection of antibody binding.

[0093] Kits also typically contain a label or instructions for use in the methods described herein. The label or instruction refers to any written or recorded material that is attached to, or otherwise accompanies a kit at any time during its manufacture, transport, sale or use. It can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The label or instruction can also encompass advertising leaflets and brochures, packaging materials, instructions, audio or videocassettes, computer discs, as well as writing imprinted directly on the pharmaceutical kits.

[0094] Illustrative Embodiments

[0095] Provided here are illustrative embodiments of the disclosed technology. These embodiments are illustrative only and do not limit the scope of the present disclosure or of the claims attached hereto.

[0096] Embodiment 1. A method for predicting response of a subject having mesothelin-expressing mesothelioma to treatment with an anti-mesothelin therapeutic agent comprising determining a baseline level of steady state soluble cancer antigen 125 (sCA125) expression of the subject, wherein a baseline steady state CA125 level greater than 6.5 U/mL and less than about 80 U/mL is predictive of response to the treatment. [0097] Embodiment 2. A method for predicting response of a subject having mesothelin-expressing mesothelioma to treatment with an anti-mesothelin therapeutic agent comprising determining a baseline level of steady state soluble cancer antigen 125 (sCA125) expression of the subject, wherein a baseline steady state CA125 level greater than the upper limit of normal (ULN) and less than about four times the ULN is predictive of response to the treatment.

[0098] Embodiment 3. The method according to Embodiment 1 or 2 wherein the anti- mesothelin therapeutic agent comprises:

(a) an antibody comprising SEQ ID NO: 1 (GYSFTGYTMN) as CDRH1, SEQ ID NO:2 (LITP YNGAS S YNQ) as CDRH2, SEQ ID NO:3 (GGYDGRGFDY) as CDRH3, SEQ ID NO:4 (SASSSVSYMH) as CDRL1, SEQ ID NO:5 (DTSKLAS) as CDRL2 and SEQ ID NO: 6 (QQWSKHPLT) as CDRL3, numbered according to Kabat;

(b) an antibody comprising SEQ ID NO:7 (GYSFTGYT) as CDRH1, SEQ ID NO:8 (ITP YNGAS) as CDRH2, SEQ ID NO: 9 (GGYDGRGFDY) as CDRH3, SEQ ID NO: 10 (SSVSY) as CDRL1, SEQ ID NO: 11 (DTS) as CDRL2 and SEQ ID NO: 12 (QQWSKHPLT) as CDRL3, numbered according to EVIGT;

(c) an antibody produced by cells assigned ATCC Accession No. PTA-7553; or

(d) an antibody that binds the same epitope as amatuximab.

[0099] Embodiment 4. The method according to Embodiment 3 wherein the antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14.

[00100] Embodiment 5. The method according to Embodiment 3 or 4 wherein the anti- mesothelin therapeutic agent comprises amatuximab.

[0100] Embodiment 6. The method according to any preceding Embodiment wherein the treatment further comprises at least a folate antimetabolite and a platinum-based

chemotherapy. [0101] Embodiment 7. The method according to Embodiment 6 wherein the folate antimetabolite comprises pemetrexed and the platinum-based chemotherapy comprises cisplatin.

[0102] Embodiment 8. A method of treating a subject having mesothelin-expressing mesothelioma, the method comprising administering to a subject having mesothelin-expressing cancer and a baseline steady state soluble cancer antigen 125 (sCA125) level that is greater than 6.5 U/mL and less than about 80 U/mL an anti-mesothelin therapeutic agent.

[0103] Embodiment 9. A method of treating a subject having mesothelin-expressing mesothelioma, the method comprising administering to a subject having mesothelin-expressing cancer and a baseline steady state soluble cancer antigen 125 (sCA125) level that is greater than the upper limit of normal (ULN) for sCA125 and less than about four times the ULN for sCA125 an anti-mesothelin therapeutic agent.

[0104] Embodiment 10. A method of treating a subject having mesothelin-expressing mesothelioma, the method comprising administering to the subject an anti-mesothelin therapeutic agent, wherein the subject has a baseline steady state soluble cancer antigen 125 (sCA125) level that is greater than 6.5 U/mL and less than about 80 U/mL.

[0105] Embodiment 11. A method of treating a subject having mesothelin-expressing mesothelioma, the method comprising administering to the subject an anti-mesothelin therapeutic agent, wherein the subject has a baseline steady state soluble cancer antigen 125 (sCA125) level that is greater than the upper limit of normal (ULN) for sCA125 and less than about four times the ULN for sCA125.

[0106] Embodiment 12. A method of treating a subject having mesothelin-expressing mesothelioma, the method comprising:

detecting a baseline steady state soluble cancer antigen 125 (sCA125) level in a biological sample obtained from the subject, and

administering an anti-mesothelin therapeutic agent to the subject when the subject's baseline sCA125 level is determined to be greater than 6.5 U/mL and less than about 80 U/mL.

[0107] Embodiment 13. A method of treating a subject having mesothelin-expressing mesothelioma, the method comprising:

detecting a baseline steady state soluble cancer antigen 125 (sCA125) level in a biological sample obtained from the subject, and administering an anti-mesothelin therapeutic agent to the subject when the subject's baseline sCA125 level is determined to be greater than the upper limit of normal (ULN) for sCA125 and less than about four times the ULN for sCA125.

[0108] Embodiment 14. The method according to any one of Embodiments 8 to 13 wherein the anti-mesothelin therapeutic agent comprises:

(b) an antibody comprising SEQ ID NO:7 (GYSFTGYT) as CDRH1, SEQ ID NO:8

(ITPYNGAS) as CDRH2, SEQ ID NO: 9 (GGYDGRGFDY) as CDRH3, SEQ ID NO: 10 (SSVSY) as CDRL1, SEQ ID NO: 11 (DTS) as CDRL2 and SEQ ID NO: 12

(QQWSKHPLT) as CDRL3, numbered according to IMGT;

(c) an antibody produced by cells assigned ATCC Accession No. PTA-7553; or

(d) an antibody that binds the same epitope as amatuximab.

[0109] Embodiment 15. The method according to Embodiment 14 wherein the antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14.

[0110] Embodiment 16. The method according to any one of Embodiments 8 to 15 wherein the anti-mesothelin therapeutic agent comprises amatuximab.

[0111] Embodiment 17. The method according to any one of Embodiments 8 to 16, further comprising administering at least a folate antimetabolite and a platinum-based chemotherapy to the subject.

[0112] Embodiment 18. The method according to Embodiment 17 wherein the folate antimetabolite comprises pemetrexed and the platinum-based chemotherapy comprises cisplatin.

[0113] Embodiment 19. The method according to any one of Embodiments 14 to 18 wherein the anti-mesothelin antibody is an antibody that is bound by CA125.

[0114] Embodiment 20. The method according to any preceding Embodiment wherein the mesothelin-expressing mesothelioma is malignant pleural mesothelioma. [0115] Embodiment 21. The method according to any preceding Embodiment wherein the sCA125 level is a sCA125 serum level.

[0116] Embodiment 22. The method according to any preceding Embodiment wherein the ULN for sCA125 is 21 U/mL.

[0117] Embodiment 23. Use of a baseline steady state soluble cancer antigen 125 (sCA125) level greater than 6.5 U/mL and less than about 80 U/mL in predicting response to treatment with an anti-mesothelin therapeutic agent by a subject having mesothelin-expressing mesothelioma.

[0118] Embodiment 24. Use of a baseline steady state soluble cancer antigen 125 (sCA125) level greater than the upper limit of normal (ULN) for sCA125 and less than about four times the ULN for sCA125 in predicting response to treatment with an anti-mesothelin therapeutic agent by a subject having mesothelin-expressing mesothelioma.

[0119] Embodiment 25. The use according to Embodiment 23 or 24 wherein the treatment further comprises at least a folate antimetabolite and a platinum-based chemotherapy.

[0120] Embodiment 26. The use according to Embodiment 25 wherein the folate antimetabolite comprises pemetrexed and the platinum-based chemotherapy comprises cisplatin.

[0121] Embodiment 27. The use according to any one of Embodiments 23 to 26 wherein the mesothelin-expressing mesothelioma is malignant pleural mesothelioma.

[0122] Embodiment 28. The use according to any one of Embodiments 23 to 27 wherein the anti-mesothelin therapeutic agent comprises:

(a) an antibody comprising SEQ ID NO: 1 (GYSFTGYTMN) as CDRH1, SEQ ID NO:2 (LITPYNGASSYNQ) as CDRH2, SEQ ID NO:3 (GGYDGRGFDY) as CDRH3, SEQ ID NO:4 (SASSSVSYMH) as CDRL1, SEQ ID NO:5 (DTSKLAS) as CDRL2 and SEQ ID NO:6 (QQWSKHPLT) as CDRL3, numbered according to Kabat;

(b) an antibody comprising SEQ ID NO:7 (GYSFTGYT) as CDRH1, SEQ ID NO:8 (ITPYNGAS) as CDRH2, SEQ ID NO: 9 (GGYDGRGFDY) as CDRH3, SEQ ID NO: 10 (SSVSY) as CDRLl, SEQ ID NO: 11 (DTS) as CDRL2 and SEQ ID NO: 12 (QQWSKHPLT) as CDRL3, numbered according to EVIGT;

(c) an antibody produced by cells assigned ATCC Accession No. PTA-7553; or

(d) an antibody that binds the same epitope as amatuximab. [0123] Embodiment 29. The use according to Embodiment 28 wherein the antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14.

[0124] Embodiment 30. The use according to any one of Embodiments 23 to 29 wherein the anti-mesothelin therapeutic agent comprises amatuximab.

[0125] Embodiment 31. The use according to any one of Embodiments 28 to 30 wherein the anti-mesothelin therapeutic antibody is an antibody that is bound by CA125.

[0126] Embodiment 32. The use according to any one of Embodiments 23 to 31 wherein the sCA125 level is a sCA125 serum level.

[0127] Embodiment 33. The use according to any one of Embodiments 23 to 32 wherein the ULN for sCA125 is 21 U/mL.

[0128] The following example is provided to further describe some of the embodiments disclosed herein. The example is intended to illustrate, not to limit, the disclosed embodiments.

[0129] Examples

[0130] Example 1. Clinical Trial

[0131] Patients with histologically confirmed, chemotherapy-naive malignant pleural mesothelioma (MPM) who were not candidates for curative surgery were assessed for eligibility. The study was approved by the Institutional Review Boards of participating institutions and informed consent was obtained prior to enrolment. The trial was registered at clinicaltrials.gov (identifier NCT00738582).

[0132] Eligibility criteria included age > 18 years, epithelial type or biphasic (mixed) MPM with low sarcomatous content, radiographically measurable disease, Karnofsky

performance status (KPS) score of >70, adequate bone marrow reserve [absolute neutrophil count (ANC) >1.5 x 109/L; platelet count >100 ^χ 109/L; hemoglobin > 9 g/dL], hepatic function [bilirubin <1.5 times the upper limit of normal (ULN); alanine transaminase (ALT) and aspartate transaminase (AST) <2.5 χ ULN; alkaline phosphatase <3.0 χ ULN], and normal renal function (serum creatinine <2.0 mg/dL and a calculated creatinine clearance >60 ml/min) based on the standard Cockcroft and Gault formula. Pregnant women were not eligible, and all men and women of reproductive potential were required to use an approved method of birth control. Patients were excluded if they had predominantly mesothelioma of the sarcomatous type, disease located primarily in the peritoneum, prior systemic therapy or radiotherapy, known central nervous system tumor involvement, treatment within three months of the start of the trial with other immunomodulatory therapy and known hypersensitivity to any of the following:

monoclonal antibodies or biologic therapy, pemetrexed, cisplatin or other platinum containing compounds.

[0133] Amatuximab 5 mg/kg by intravenous infusion over 1 hour was administered on days 1 and 8 with pemetrexed (500 mg/m² by intravenous infusion over 10 minutes) and cisplatin (75 mg/m² by intravenous infusion over 2 hours) administered on day 1 of 21 -day cycles for up to six cycles. Patients with objective response or stable disease continued to receive amatuximab maintenance (5 mg/kg on days 1 and 8 of a 21-day cycle) until disease progression. Supportive treatment included premedication with acetaminophen 650 mg and diphenhydramine 25 to 50 mg 30 minutes prior to amatuximab infusion; folic acid (350 μg to 1 mg PO daily) starting at least five days prior to the first dose of pemetrexed; vitamin B12 (1 mg approximately every 9 weeks); dexamethasone 4 mg PO twice daily on the day before, the day of and the day following pemetrexed.

[0134] Tumor measurements were performed at baseline and thereafter on day 8 of every third cycle starting with cycle 3 until disease progression or treatment discontinuation. Response was assessed using the modified RECIST for the assessment of response in malignant pleural mesothelioma (24). A complete blood count and a comprehensive metabolic panel were performed on days 1 and 8 of each cycle during the combination therapy phase and on day 1 of each cycle during the amatuximab maintenance phase. Safety was assessed using the Common Terminology Criteria for Adverse Events (CTCAE) scale, version 3.0.

[0135] 89 patients were enrolled. Twelve patients were enrolled over the design- specified target of 77, under the a priori assumption of an approximate 10% loss-to-followup and non-evaluability for tumor assessment. All patients received at least one dose of amatuximab. Eighty three (93%) patients had at least one post-baseline imaging assessment and were evaluable for efficacy. One patient withdrew consent after their third cycle. [0136] The median age was 67 years (range, 46 -80 years), 78% were male, 89%> were Caucasian, and 93% had a KPS score >80. Eighty-eight percent had stage III/IV disease and 89%> had epithelial histology. Sixty-three percent reported prior exposure to asbestos.

[0137] Forty patients (45%>) completed 6 cycles of the combination therapy phase. Reasons for discontinuation of combination therapy included adverse events (35%>), progressive disease (12%>), investigator discretion (3%>) and other (5%>). Fifty-six patients (63%>) entered the amatuximab maintenance phase. Reasons for discontinuation of maintenance therapy were progressive disease (73%), adverse events (5%), and other (4%). A median of five cycles of combination therapy (range, 1-6) and six cycles of maintenance therapy (range, 1-30) were administered. Sixteen patients received >10 cycles and eight patients received >20 cycles of amatuximab maintenance, the longest being 52 cycles.

[0138] Example 2

[0139] Quantitation of TSAs from patient serum

[0140] Pre-treatment serum samples obtained from subjects in the Phase 2 trial were evaluated for CA125, CA19-9 and CA15-3 levels using the Roche COBAS 6000 platform. The clinical diagnostic assay used within this study establishes CA125 upper limit of normal (ULN) at 21 U/mL. Testing was performed in a CLIA accredited laboratory at Aspira Labs (Trumbull, CT).

[0141] All assays employed an electrochemiluminescence immunoassay format.

Assays were single-step sandwich assay incorporating a streptavidin-coated microparticle, a biotinylated-capture antibody and a ruthenylated-detection antibody.

[0142] All protocol and informed consent documentation received institutional review board approval in accordance with the Declaration of Helsinki. Detailed study information and written informed consent was provided to each patient before any study-specific screening. All patients consented to having post hoc biomolecular studies.

[0143] Statistical analysis of CA125 and clinical response

[0144] Cut-point analysis of baseline CA125 in the Phase 2 population was performed using maximal chi-square methodology (Miller R, Siegmund D. Maximally selected chi square statistics. Biometrics 1982; 38: 1011-1016) and log-rank tests were used to compare progression- free survival (PFS) and overall survival (OS) among subgroups by CA125. All reported p values are two-sided unless otherwise specified. Kaplan-Meier curves were generated on PFS and OS and median times were derived. The hazard ratio (HR) (>57 U/mL : <57 U/mL CA125) is estimated based on a Cox proportional hazards model.

[0145] Cell culture

[0146] The human ovarian cancer cell lines OVCAR3 and Jurkat were all obtained from ATCC. Both were grown in complete RPMI medium as suggested by the vendor. All cells were maintained at 37°C/5% CO2.

[0147] CA125 preparations

[0148] CA125 preparations were obtained from primary patient ascites, pooled and purified by filtration, affinity and size exclusion at Morphotek (Exton, PA) or the independent vendors Lee Biosolutions (Maryland Heights, MO), Antibodies-online (Atlanta, GA). Purified preparations were done as described (Kline, et al. Tumor antigen CA125 suppresses antibody- dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc receptor engagement. Oncotarget. 2017; 8:52045-52060).

[0149] Generation of recombinant Jurkat cell lines

[0150] Jurkat-CD16a lines were generated by electroporation using equivalent amounts of plasmids encoding open reading frames of human CD 16a and FcsR (CD 16 receptor complex) and selection with 400 μg/mL G418 and 2.5 μg/mL blasticidin. Surface expression of CD16a was confirmed by flow cytometry as previously described (Kline, et al. Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc receptor engagement. Oncotarget. 2017; 8:52045-52060).

[0151] shRNA knockdown of mCA125 expression in cancer cells

[0152] CA125 knockdown was performed using shRNA Mission Lentiviral particles as recommended by the vendor (Sigma-Aldrich, St. Louis, MO) as previously described (Kline, et al. Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc receptor engagement. Oncotarget. 2017; 8:52045-52060). Vectors targeting different regions of the mRNA were used for cell transductions and stable integration. Single cell subclones were expanded and screened for CA125 knockdown and mesothelin expression by flow cytometry with the OC125 antibody (Abeam, Cambridge, UK) or MORAb-009 (Fujisaka, et al. Phase I study of amatuximab, a novel monoclonal antibody to mesothelin, in Japanese patients with advanced solid tumors. Invest New Drugs 2015; 33 :380- 388), respectively, followed by staining with FITC-conjugated secondary antibody. Clonal cell lines were monitored over multiple passages for continued suppression. Effective shRNA constructs used in this study were TRCN0000262688 (KD1 : 5'- CCGGTCACATCTCCAATGGTTATTACTCGAGT

AATAACCATTGGAGATGTGATTTTTG-3') (SEQ ID NO: 15) and TRCN0000262686 (KD3 : 5 ' - CCGGT-GCCGTTC ACCCTC AACTTTACTCGA

GTAAAGTTGAGGGTGAACGGCATTTTTG-3') (SEQ ID NO: 16). Cell lines were characterized using the assays previously described to confirm growth and morphological similarities between CA125 knock down lines (OVCAR3-KD1) and parental OVCAR3 cells (Kline, et al. Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity

(ADCC) via direct antibody binding and suppressed Fc receptor engagement. Oncotarget. 2017; 8:52045-52060).

[0153] Periodic acid-Schiff (PAS) staining

[0154] OVCAR3 and OVCAR3-KD1 cells were grown to 80% confluence in 24 well plates, washed once in DPBS without Ca⁺⁺ or Mg⁺⁺ and fixed in 0.2% glutaraldehyde in DPBS for 30 minutes. All subsequent steps were done at room temperature. Wells were washed twice with DPBS and prepared for PAS staining as recommended by the vendor (Therm oFisher, Waltham, MA). Briefly, cells were incubated with 50% methanol / 50% DPBS for 30 minutes then washed twice with 3% acetic acid in DPBS for 10 minutes. Next, oxidation reagent was added for 15 minutes and cells were then washed three times with 3% acetic acid for 5 minutes each. Glycoprotein stain reagent was added for 15 minutes then removed and cells were treated with reduction agent for 5 minutes. Finally, cells were rinsed once with 3% acetic acid and then dFbO and imaged with the EVOS imaging system (Thermo-Fisher, Waltham, MA). [0155] Antibody-dependent cellular cytotoxicity

[0156] ADCC assays were conducted using Jurkat-Luc (Promega) effector cells as previously described (Kline, et al. Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc receptor engagement.

Oncotarget. 2017; 8:52045-52060). Percent ADCC inhibition was calculated as 1 - (sCA125- treated / untreated) x 100%.

[0157] Biological Rolling Assay (BRA assay)

[0158] BRA assays were conducted as described (Kline, et al. Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc receptor engagement. Oncotarget. 2017; 8:52045-52060). Briefly, U-bottom microplates (untreated) were coated overnight with antibody and antibody fragments in 100 μΐ^ containing 10 μg/mL protein in 0.2 M bicarbonate buffer, pH 9.4 at 4°C. Wells were washed and blocked with 200 μΐ. PBS/1% BSA for 1 hr at room temp. Next, 2.5xl0⁴ Jurkat-CD16a cells were added with or without CA125 to wells in a total volume of 100 μΐ^ in ADCC assay buffer (Promega). Microplates were placed at 37°C/5% CO2 for 5 hr then imaged using an EVOS imaging system.

[0159] ELISA analysis of amatuximab-CA125 binding

[0160] ELISAs for antibodies, antibody fragments and CD 16a, CD32a and CD64a receptors were carried out as previously described (Kline, et al. Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc receptor engagement. Oncotarget. 2017; 8:52045-52060). ELISA for FcRn was carried out similarly with the exception of probing and washing using powdered milk instead of BSA and PBS buffer pH 5.5 instead of pH 7.2. Recombinant human CD16a-, CD32a- and CD64a-biotin probes were purchased from Sino Biological Inc. Recombinant human FcRn was purchased from R&D Systems. Antibodies, antibody fragments and FcRn were biotinylated using sulfo-tag conjugation as described (Meso Scale Diagnostics Inc). All reactions were done in at least triplicate. [0161] Statistical analysis on laboratory experiments

[0162] Mean values were compared using an unpaired Student's two-tailed t test. All p values are two-sided.

[0163] Results

[0164] Serum CA125 and tumor measurements by computerized tomography (CT) scan were conducted on all patients eligible for MORAb-009-003 Phase 2 clinical study entry. The sum of the longest diameter (SLD) of target lesions for each patient was measured and documented prior to first treatment based on independent review to support the primary endpoint of EORTC modified PFS by RECIST vl .O (Therasse et al., J. Natl. Cancer Inst. 2000, 92: 205- 216). Analysis of serum CA125 was conducted for all eligible trial patients (N = 89) with 75 subjects having baseline measurable CA125 levels available. Baseline CA125 values ranged from 1 U/mL to 1266 U/mL with 29 (38.7%) having CA125 above the normal range. Serum cut- point analysis of baseline CA125 using maximal chi-square methodology (Miller R, Siegmund D. Maximally selected chi square statistics. Biometrics 1982; 38: 1011-1016) and log-rank tests were used to compare PFS and OS among CA125 subgroups (Kaplan & Meier, J. Am. Stat. Assoc. 1958; 53 : 457-481). These analyses identified 57 U/mL to be the optimal threshold of CA125 in patients within this population. Kaplan-Meier (KM) analysis showed that patients with <57 U/mL CA125 had a statistically significant improvement in PFS (HR = 0.43, p = 0.0062) and OS (FIR = 0.40, p = 0.0022) as compared to those with >57 U/mL (Figure 1). Demographics found that the <57 U/mL CA125 patient population had nine patients with STAGE IB and STAGE II disease, which has been reported to be a positive prognostic factor in MPM (Pass et al., J. Thorac. Oncol. 2014, 9: 856-864). To determine the impact of these patients on the CA125 clinical outcome relationship between the <57 U/mL and >57 U/mL groups, KM analysis was conducted on patients with only Stage III/IV disease. Results found that patients with <57 U/mL CA125 still had a significant improvement in PFS (HR = 0.42, p = 0.0074) and OS (HR = 0.38, p = 0.0016) as compared to those above this threshold. Optimal cut-point analysis of two other prominent TSAs used in clinical assessment of mesothelioma, CA15-3 and CA19-9 (Pass, et al. Supplementary prognostic variables for pleural mesothelioma: A report from the IASLC staging committee. J Thorac Oncol 2014; 9:856-864) showed no correlation with response.

[0165] As reported by Kline et al., baseline CA125 levels did not correlate with measurable radiographic disease bulk in patients with ovarian cancer (Kline, et al. Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc receptor engagement. Oncotarget. 2017; 8:52045-52060). In this study, SLD at baseline ranged from 13 to 334 mm with a mean SLD of 95 mm as determined by independent review. Analysis found that no significant correlation to baseline CA125 levels occurred

(Pearson correlation coefficient of -0.112/0.046 for original/natural log scale, respectively) corroborating the findings in ovarian cancer and consistent with the scenario that MPM tumors may yield high or low levels of CA125 regardless of measurable disease by radiology.

[0166] Soluble CA125 inhibits amatuximab-mediated ADCC

[0167] To determine if soluble CA125 can bind and perturb amatuximab ADCC, ADCC assays using mesothelin-positive tumor cell lines and exogenously added or

endogenously produced CA125 were conducted. The ability of exogenously added soluble CA125 to perturb ADCC was tested by adding patient-derived CA125 to amatuximab ADCC assays. As shown in Figure 2, CA125 was effective in suppressing the ADCC activity on mesothelin-positive tumor cells similar to that shown for farletuzumab (Kline, et al. Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc receptor engagement. Oncotarget. 2017; 8:52045-52060). The isogenic human ovarian cancer cells lines OVCAR3 and OVCAR3-KD1, the latter of which has knocked-down levels of endogenous CA125 via shRNA as previously described and shown in Figure 3A (Kline, et al. Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc receptor engagement.

Oncotarget. 2017; 8:52045-52060) were then employed. Cells were stained with Periodic acid- Schiff (PAS) to determine if decreased CA125 expression had a meaningful effect on the overall glycoprotein profile of OVCAR3 (Wong et al., 2003, J. Biol. Chem., 278: 28619-28634).

Interestingly, OVCAR3-KD1 cells had a significant reduction in staining when CA125 was suppressed, suggesting that it is one of the major gly can-containing proteins within these lines (Figure 3B). Flow cytometry found both lines express similar levels of mesothelin (Figure 3C). When using the Jurkat-Luc ADCC reporter cell line, amatuximab was shown to mediate a significant increase in ADCC signaling against OVCAR3-KD1 cells as compared to parental (Figure 3D). No ADCC effect was observed in either line when an irrelevant mAb was used.

[0168] CA125 binds to amatuximab and perturbs CD16a Fc-γ receptor binding

[0169] CA125 has been previously shown to bind IgGl-type antibodies and alter their ability to engage with CD16a Fc-γ receptor on cells and at the molecular level (Kline, et al. Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc receptor engagement. Oncotarget. 2017; 8:52045-52060; Gunn, et al. Mucosal Immunol. 2016, 9: 1549-1558). To determine if CA125 exerts its suppression on ADCC activity via a similar mechanism, amatuximab-CA125 binding assays using biotinylated amatuximab and derived fragments as probes were conducted. As shown in Figure 4A, amatuximab is able to bind to CA125 and appears to bind within the (Fab')₂ domain.

[0170] The effect of CA125 on blocking amatuximab-CD16a binding was next tested using cell-based and ELISA-based assays. The biological rolling assay (BRA) was employed to determine if CA125 can perturb the engagement of amatuximab to CD16a, which is ectopically expressed by Jurkat-CD16a cells. The BRA assay utilizes microwell plates coated with amatuximab which are then seeded with Jurkat-CD16a with or without soluble CA125 (Kline, et al. Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc receptor engagement. Oncotarget. 2017). If CA125 perturbs cell surface Jurkat-CD16a-amatuxumab binding, the cells will not adhere to the amatuximab-coated well walls and cluster at the bottom of the U-shaped well. As shown in Figure 4B, Jurkat-CD16a cells can bind to amatuximab but are inhibited upon exposure to CA125. This inhibition appears to occur via binding to the (Fab')₂ domain as shown in Figure 4A and allosterically inhibiting the binding to CD 16a, and to a lesser extent CD32a as shown by ELISA (Figure 4C. The CA125 perturbation of CD16a and CD32a appeared specific and not a universal effect to Fc binding proteins as CA125 did not affect binding to the high affinity CD64a Fc-γ receptor or to the low affinity FcRn receptor which also binds to the antibody Fc domain (Figure 4D). (O'Shannessy, et al. Correlation of FCGRT genomic structure with serum immunoglobulin, albumin and farletuzumab pharmacokinetics in patients with first relapsed ovarian cancer. Genomics 2017; 109:251-257).

[0171] Example 3. Binding of biotinylated antibodies to test for binding to CA125 via ELISA

[0172] 15KU/ml of human CA125 was used to coat 96-well ELISA plates. Wells were blocked with 5% BSA in PBS (pH 7.2). Wells were washed per protocol and probed with 5 ug/ml biotin-labeled antibody. Wells were washed and secondary probed with streptavidin- horseradish peroxidase (HRP). Wells were washed and TMB substrate added for 5-10 min. Reaction was stopped with sulfuric acid. Plates were read at 450nm OD. As shown in Figures 5 and 6, CA125 can bind to MORAb-009, MORAb-003 and its (Fab')₂, MORAb-022, rituximab (Biovision), total human IgM (Sigma, St. Louis, MO) and a monoclonal human IgM antibody (ATCC). Conjugates of CA125-binding antibodies were also tested and found to retain binding by CA125 (not shown). ELISAs were carried out as previously described (Kline et al., 2017). Antibodies and fragments were biotinylated using sulfo-tag conjugation as described (Meso Scale Diagnostics Inc). All reactions were done in at least triplicate and mean values were analyzed using students T-test.

Claims

What is claimed:

1. A method for predicting response of a subject having mesothelin-expressing mesothelioma to treatment with an anti-mesothelin therapeutic agent comprising determining a baseline level of steady state soluble cancer antigen 125 (sCA125) expression of the subject, wherein a baseline steady state CA125 level greater than 6.5 U/mL and less than about 80 U/mL or a baseline steady state CA125 level greater than the upper limit of normal (ULN) and less than about four times the ULN is predictive of response to the treatment.

2. The method according to claim 1 wherein the anti-mesothelin therapeutic agent comprises:

(c) an antibody produced by cells assigned ATCC Accession No. PTA-7553; or

(d) an antibody that binds the same epitope as amatuximab.

3. The method according to claim 2 wherein the antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14.

4. The method according to claim 2 wherein the anti-mesothelin therapeutic agent comprises amatuximab.

5. The method according to claim 1 wherein the treatment further comprises at least a folate antimetabolite and a platinum-based chemotherapy.

6. The method according to claim 5 wherein the folate antimetabolite comprises pemetrexed and the platinum-based chemotherapy comprises cisplatin.

7. A method of treating a subject having mesothelin-expressing mesothelioma, the method comprising administering to a subject having mesothelin-expressing mesothelioma and a baseline steady state soluble cancer antigen 125 (sCA125) level that is greater than 6.5 U/mL and less than about 80 U/mL or that is greater than the upper limit of normal (ULN) for sCA125 and less than about four times the ULN for sCA125 an anti-mesothelin therapeutic agent.

8. A method of treating a subject having mesothelin-expressing mesothelioma, the method comprising: detecting a baseline steady state soluble cancer antigen 125 (sCA125) level in a biological sample obtained from the subject, and administering an anti-mesothelin therapeutic agent to the subject when the subject's baseline sCA125 level is determined to be greater than 6.5 U/mL and less than about 80 U/mL or greater than the upper limit of normal (ULN) for sCA125 and less than about four times the ULN for sCA125.

9. The method according to claim 7 or 8 wherein the anti-mesothelin therapeutic agent comprises:

(e) an antibody comprising SEQ ID NO: l (GYSFTGYTMN) as CDRH1, SEQ ID NO:2 (LITPYNGASSYNQ) as CDRH2, SEQ ID NO:3 (GGYDGRGFDY) as CDRH3, SEQ ID NO:4 (SASSSVSYMH) as CDRL1, SEQ ID NO:5 (DTSKLAS) as CDRL2 and SEQ ID NO:6 (QQWSKHPLT) as CDRL3, numbered according to Kabat;

(f) an antibody comprising SEQ ID NO:7 (GYSFTGYT) as CDRH1, SEQ ID NO:8

(g) an antibody produced by cells assigned ATCC Accession No. PTA-7553; or

(h) an antibody that binds the same epitope as amatuximab.

10. The method according to claim 9 wherein the antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14.

11. The method according to claim 7, 8 or 9 wherein the anti-mesothelin therapeutic agent comprises amatuximab.

12. The method according to claim 7, 8, or 9, further comprising administering at least a folate antimetabolite and a platinum -based chemotherapy to the subject.

13. The method according to claim 12 wherein the folate antimetabolite comprises pemetrexed and the platinum-based chemotherapy comprises cisplatin.

14. The method according to any one of claims 9 to 13 wherein the anti-mesothelin antibody is an antibody that is bound by CA125.

15. The method according to any preceding claim wherein the mesothelin-expressing mesothelioma is malignant pleural mesothelioma.

16. The method according to any preceding claim wherein the sCA125 level is a sCA125 serum level.

17. The method according to any preceding claim wherein the ULN for sCA125 is 21 U/mL.

18. Use of a baseline steady state soluble cancer antigen 125 (sCA125) level greater than 6.5 U/mL and less than about 80 U/mL or greater than the upper limit of normal (ULN) for sCA125 and less than about four times the ULN for sCA125 in predicting response to treatment with an anti-mesothelin therapeutic agent by a subject having mesothelin-expressing mesothelioma.

19. The use according to claim 18 wherein the treatment further comprises at least a folate antimetabolite and a platinum-based chemotherapy.

20. The use according to claim 19 wherein the folate antimetabolite comprises pemetrexed and the platinum-based chemotherapy comprises cisplatin.

21. The use according to any one of claims 18 to 20 wherein the mesothelin-expressing mesothelioma is malignant pleural mesothelioma.

22. The use according to any one of claims 18 to 21 wherein the anti-mesothelin therapeutic agent comprises:

(c) an antibody produced by cells assigned ATCC Accession No. PTA-7553; or

(d) an antibody that binds the same epitope as amatuximab.

23. The use according to claim 22 wherein the antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14.

24. The use according to any one of claims 18 to 23 wherein the anti-mesothelin therapeutic agent comprises amatuximab.

25. The use according to any one of claims 22 to 24 wherein the anti-mesothelin therapeutic antibody is an antibody that is bound by CA125.

26. The use according to any one of claims 18 to 25 wherein the sCA125 level is a sCA125 serum level.

27. The use according to any one of claims 18 to 26 wherein the ULN for sCA125 is 21 U/mL.