Reducing soluble mesothelin by plasma exchange

Abstract

Methods and materials for reducing the level of circulating extracellular mesothelin (also referred to herein as soluble MSLN or sMSLN) by therapeutic plasma exchange (TPE) are provided herein. For example, methods and materials for using TPE to reduce the level of sMSLN in mammals (e.g., humans) identified as having mesothelioma or another solid tumor are provided herein.

Description

[0001] REDUCING SOLUBLE MESOTHELIN BY PLASMA EXCHANGE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority from U.S. Provisional Application Serial No. 63 / 649,774, filed May 20, 2024. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

[0004] TECHNICAL FIELD

[0005] This document relates to methods and materials for reducing the level of circulating extracellular mesothelin (also referred to herein as soluble MSLN or sMSLN) by therapeutic plasma exchange (TPE). For example, this document relates to methods and materials for using TPE to reduce the level of sMSLN in mammals (e.g., humans) identified as having mesothelioma and / or another solid tumor, and also identified as having an elevated level of sMSLN.

[0006] BACKGROUND

[0007] Mesothelin (MSLN) is a cell surface protein that is overexpressed in mesothelioma and other solid tumors. MSLN is the focus of multiple targeted therapies, including antibody drug conjugates (ADCs), T cell receptor (TCR) fusion constructs, and chimeric antigen receptor T-cell (CAR-T) products. The clinical efficacy of these treatments remains limited. One hypothesis is that circulating MSLN, including sMSLN, may bind to MSLN-targeted therapies before these therapies reach the tumor surface.

[0008] SUMMARY

[0009] This document is based, at least in part, on the discovery that therapeutic plasma exchange (TPE) can be used as a treatment to reduce circulating extracellular MSLN in mammals (e.g., humans) having cancer (e.g., mesothelioma and / or other solid tumors). As demonstrated herein, for example, sMSLN was significantly reduced in post-TPE plasma samples obtained from human patients, as compared to the level of sMSLN in pre-TPE plasma samples from the same patients. On average, sMSLN was reduced by about 47% in the post-TPE samples. This document also is based, at least in part, on the development of methods for using TPE in combination with other therapies (e.g.. MSLN-targeted ADCs, TCR fusion constructs, and / or CAR-T therapies) in mammals that have cancer (e.g., in human cancer patients having mesothelioma and / or other solid tumors) and that have elevated levels of sMSLN. Having the abi li ty to identify mammals with mesothelioma and / or other solid tumors as having an elevated level of sMSLN can allow those mammals to be treated in an effective and reliable manner using TPE alone or in combination with another treatment. For example, the methods provided herein can be used to treat a human mesothelioma patient identified as having an elevated level of sMSLN, using TPE to reduce the amount of sMSLN in the patient, and also (e.g., simultaneously or subsequently) treating the patient with one or more therapies targeted to the mesothelioma (e.g., MSLN-targeted ADCs, TCR fusion constructs, and / or CAR-T).

[0010] This document provides methods and materials for treating mammals (e.g., humans) having cancer and having elevated levels of sMSLN. The methods can include, for example, using TPE to reduce the amount of sMSLN in the mammal’s circulation, and treating the mammal with one or more therapies targeted to MSLN. By reducing the amount of sMSLN in the circulation, the effectiveness of therapies targeted to MSLN on the surface of tumor cells (e.g., mesothelioma cells) can be increased.

[0011] In a first aspect, this document features a method for treating cancer. The method can include, or consist essentially of, (a) performing TPE on a mammal identified as (i) having cancer and (ii) having a measured level of sMSLN that is higher than a reference level of sMSLN, and subsequently (b) administering a MSLN-targeted therapy to said mammal. The measured level of sMSLN can have been determined using an enzyme linked immunosorbent assay (ELISA). The measured level of sMSLN can have been determined using nanoflow cytometry. The reference level can be an average level of sMSLN in biological samples from healthy mammals. The MSLN-targeted therapy can include administering, to said mammal, an anti-MSLN antibody, an ADC targeted to MSLN. a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN. The mammal can be a human. The cancer can be mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0012] In another aspect, this document features a method for treating a mammal identified as having cancer. The method can include, or consist essentially of, (a) measuring a level of sMSLN in a biological sample obtained from said mammal, (b) comparing said measured level of said sMSLN to a reference level of sMSLN, (c) when the measured level is greater than said reference level, performing TPE on said mammal, and subsequently (d) administering a MSLN-targeted therapy to said mammal. The measuring can include performing an ELISA. The measuring can include performing nanoflow cytometry. The reference level can be an average level of sMSLN in biological samples from healthy mammals. The MSLN-targeted therapy can include administering, to said mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN. The mammal can be a human. The cancer can be mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0013] In another aspect, this document features a method for treating a mammal identified as having cancer and as having a biological sample with an elevated level of sMSLN. The method can include, or consist essentially of, performing TPE on said mammal and administering a MSLN-targeted therapy to said mammal. The mammal can have been identified as having an elevated level of sMSLN by measuring the level of sMSLN in a biological sample obtained from said mammal, and determining that said level is higher than a reference level of sMSLN. The reference level can be an average level of sMSLN in biological samples from healthy mammals. The MSLN-targeted therapy can include administering, to said mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN. The mammal can be a human. The cancer can be mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0014] In another aspect, this document features a use of TPE and a MSLN-targeted therapy in a method for treating cancer. The method can include, or consist essentially of, (a) performing TPE on a mammal identified as (i) having cancer and (ii) having a measured level of sMSLN that is higher than a reference level of sMSLN, and subsequently (b) administering a MSLN-targeted therapy to said mammal. The measured level of sMSLN can have been determined using an enzyme linked immunosorbent assay (ELISA). The measured level of sMSLN can have been determined using nanoflow cytometry. The reference level can be an average level of sMSLN in biological samples from healthy mammals. The MSLN-targeted therapy can include administering, to said mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN. The mammal can be a human. The cancer can be mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0015] In another aspect, this document features a use of TPE and a MSLN-targeted therapy in a method for treating a mammal identified as having cancer. The method can include, or consist essentially of, (a) measuring a level of sMSLN in a biological sample obtained from said mammal, (b) comparing said measured level of said sMSLN to a reference level of sMSLN. (c) when the measured level is greater than said reference level, performing TPE on said mammal, and subsequently (d) administering a MSLN- targeted therapy to said mammal. The measuring can include performing an ELISA. The measuring can include performing nanoflow cytometry. The reference level can be an average level of sMSLN in biological samples from healthy mammals. The MSLN- targeted therapy can include administering, to said mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN. The mammal can be a human. The cancer can be mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0016] In another aspect, this document features a use of TPE and a MSLN-targeted therapy in a method for treating a mammal identified as having cancer and as having a biological sample with an elevated level of sMSLN. The method can include, or consist essentially of, performing TPE on said mammal and administering a MSLN-targeted therapy to said mammal. The mammal can have been identified as having an elevated level of sMSLN by measuring the level of sMSLN in a biological sample obtained from said mammal, and determining that said level is higher than a reference level of sMSLN. The reference level can be an average level of sMSLN in biological samples from healthy mammals. The MSLN-targeted therapy can include administering, to said mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN. or a CAR-T product targeted to MSLN. The mammal can be a human. The cancer can be mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0018] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0019] DESCRIPTION OF DRAWINGS

[0020] FIG. 1 is a graph plotting sMSLN levels in plasma from humans before and after TPE.

[0021] FIG. 2A is a graph plotting the percent viability of washed Mero-95 cells 48 hours after treatment with 0, 10, 50, or 100 nM anetumab ravtansine (ARav), as indicated, with no pre-treatment prior to the ARav (No pre-Tx), or with pretreatment with recombinant MSLN (rMSLN), DMSO, or one or two protease inhibitors (marimastat (M), TMI-1 (T), or M+T). FIG. 2B is a graph plotting the percent confluence of washed Mero- 95 cells 48 hours after treatment with 0, 10, 50, or 100 nM ARav, as indicated, with no pre-treatment prior to the ARav (No pre-Tx), or with pretreatment with rMSLN, DMSO. or one or two protease inhibitors (M, T, or M+T). FIG. 2C is a graph plotting the percent viability of unwashed Mero-95 cells 48 hours after treatment with 0, 10, 50, or 100 nM ARav, as indicated, with no pre-treatment prior to the ARav (No pre-Tx), or with pretreatment with rMSLN. DMSO, or one or two protease inhibitors (M, T. or M+T). FIG. 2D is a graph plotting the percent viability of unwashed Mero-95 cells 48 hours after treatment with 0, 10, 50, or 100 nM ARav, as indicated, with no pre-treatment prior to the ARav (No pre-Tx), or with pretreatment with rMSLN, DMSO, or one or two protease inhibitors (M, T, or M+T).

[0022] FIG. 3 is a graph plotting the percent viability of Mero-95 cells treated with the indicated concentrations anetumab ravtansine (ARav), with no pre-treatment prior to the ARav (No pre-Tx), or with pretreatment with recombinant MSLN (rMSLN), DMSO, or one or two protease inhibitors (marimastat (M), TMI-1 (T), or M+T). At baseline, the combination M+T resulted in decreased cell viability compared to the control (p=0.0016), rMSLN (p=0.0453), M (p=0.012), T (p=0.014). There was a trend of decreased viability at lOnM, but this was not statistically significant. At 50 and 100 nM, there were no significant differences between the groups. See TABLE 3 for more detailed results. * p < 0.05, ** p < 0.01.

[0023] FIG. 4 is a graph plotting cell confluence of Mero-95 cells treated with the indicated concentrations of anetumab ravtansine (ARav), with no pre-treatment prior to the ARav (No pre-Tx), or with pretreatment with recombinant MSLN (rMSLN), DMSO, or one or two protease inhibitors (marimastat (M). TMI-1 (T), or M+T). At baseline, the combination M+T resulted in decreased confluence compared to rMSLN (p=0.0003), M (p=0.0028), T (p=0.0252). At lOnM of ARav, the addition of rMSLN resulted in higher confluence or less cytotoxicity compared to control DMSO (p=0.0348), T (p=0.0289), M+T (p=0.0206) but not M (p=0.0679). This trend continued at 50 nM. At 100 h nM, there were no significant differences between the groups. See TABLE 4 and FIGS. 5A- 5C for more detailed results. * p < 0.05, ** p < 0.01.

[0024] FIGS. 5A-5C include representative confluence images. FIG. 5A includes images taken before any treatment, about 20 hours after plating 10,000 cells / well. FIG. 5B includes images taken after 20 hours of protease inhibitor pretreatment, prior to adding anetumab ravtansine (ARav) or PBS. From top to bottom: 1% DMSO, no pre-treatment (pre-tx), 10 pM marimastat (M), 10 M TMI-1 (T), 10 pM of combined marimastat and TMI-1 (M+T), and no pre-tx. FIG. 5C includes images taken 48 hours after the addition of ARav or PBS. Top to bottom: 1% DMSO, no pre-treatment (pre-tx), 10 pM marimastat (M), 10 pM TMI-1 (T), 10 pM of combined marimastat and TMI-1 (M+T), and recombinant mesothelin (rMSLN). Left to right: 10 nM ARav, PBS, 50 nM ARav, PBS, 100 nM ARav, PBS.

[0025] DETAILED DESCRIPTION

[0026] This document provides methods and materials for treating mammals (e.g., humans) having cancer and having elevated levels of sMSLN. As noted above, increased levels of extracellular MSLN can reduce the effectiveness of therapies targeted to MSLN, including ADCs. TCR fusion constructs, and CAR-T therapies. For example. sMSLN in the circulation may bind to MSLN-targeted therapies before they reach the tumor surface, reducing the effectiveness of the therapies. Circulating MSLN can include full length MSLN proteins and / or one or more MSLN fragments, depending on whether the full length MSLN protein has been cleaved. By reducing the amount of sMSLN in the circulation, the effectiveness of therapies targeted to MSLN on the surface of tumor cells (e.g., mesothelioma cells) can be increased.

[0027] The methods provided herein can be used to reduce the level of extracellular MSLN, thus boosting the effectiveness of therapies targeted to MSLN. In general, the methods provided herein include the use of TPE to reduce circulating extracellular MSLN in mammals identified as having cancer (e.g., mesothelioma and / or another solid cancer). The methods provided herein also can include administering a MSLN-targeted therapy to a mammal identified as having cancer and as having an elevated level of sMSLN, where the mammal is subjected to TPE before administration of the MSLN-targeted therapy or concurrently with the MSLN-targeted therapy.

[0028] The methods described herein can be used for treatment of any appropriate mammal (e.g., a human, non-human primate, horse, cow, pig, sheep, goat, cat, rabbit, rat, or mouse) identified as being in need thereof. For example, a mammal can be identified as having a cancer (e.g., mesothelioma and / or another solid tumor). The methods provided herein can be used to treat mammals having a cancer in which the level of sMSLN is elevated. Cancers that may have elevated levels of sMSLN and can therefore be treated using the methods described herein include, without limitation, mesothelioma and other solid cancers including, without limitation, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, and cholangiocarcinoma.

[0029] A cancer that is “resistant” to MSLN-targeted therapy in a mammal (e.g., a human) is a cancer that may not respond to treatment with an MSLN-targeted therapy (e.g., an ADC, TCR fusion construct, or CAR-T therapy) in an effective manner. In the methods provided herein, the mammal in need of treatment can be a mammal identified as having an elevated level of sMSLN. In some cases, the methods provided herein can include identifying a mammal as having an elevated level of sMSLN. A mammal in need of the methods provided herein can be identified based on, for example, detection of sMSLN in a biological sample (e.g., a blood, plasma, serum, or urine sample). Having the ability to identify mammals as having a tumor that is resistant to treatment with MSLN- targeted can allow those mammals to be properly identified and treated in an effective and reliable manner. For example, the treatments provided herein in which TPE is combined with one or more MSLN-targeted therapies can be used to treat patients identified as having a tumor resistant to MSLN-targeted therapy. Thus, the methods provided herein can be used to determine which patients are more likely to benefit from MSLN-targeted therapies alone, and which patients are more likely to require additional treatment to reduce sMSLN levels, in addition to treatment with a MSLN-targeted therapy.

[0030] A level of sMSLN in a mammal (e.g., a human) can be determined using any appropriate biological sample from the mammal (e.g., a blood sample, a plasma sample, a serum sample, or a urine sample). An “elevated level” of sMSLN is any level that is greater than a reference level of sMSLN. The reference level of sMSLN can be the average level of sMSLN in control biological samples from a population of corresponding healthy mammals of the same species (e.g., humans that do not have mesothelioma or another solid tumor). An elevated level of sMSLN can be, for example, at least 3% greater, at least 5% greater, at least 10% greater, at least 25% greater, at least 50% greater. 3 to 5% greater, 5 to 10% greater, 10 to 20% greater, 20 to 50% greater, 50 to 100% greater, or more than 100% greater than the reference level of sMSLN.

[0031] Any appropriate method can be used to detect and quantify sMSLN in a biological sample (e.g., a plasma sample) from a mammal with cancer (e.g., a human mesothelioma patient). Suitable methods include, without limitation, enzyme-linked immunosorbent assay (ELISA), and other antibody-based detection methods. In some cases, the methods provided herein can include detecting and / or quantifying sPD-Ll in a sample of body fluid using, for example, immunological techniques. For example, an antibody that binds to an epitope specific for sMSLN can be used to detect sMSLN in a biological fluid sample. In some cases, an antibody directed against sMSLN can bind the polypeptide with an affinity of at least 10‘4M (e.g., at least 10’5, 10'6, 10‘7, 10’8, 10'9, 10‘10, 10-11, or IO’12M).

[0032] ELISA kits and / or antibodies having specific binding affinity for sMSLN can be commercially obtained (e.g.. from Biocare Medical. SKU #3175; Leica Biosystems, SKU #PA0373-U; BioLegend, catalog no. 530101; Rockland Immunochemicals, item no. 200- 301-A88; AbCam, catalog no. abl96235; or Novus Biologicals, catalog no. NB110- 85538), or can be produced using, for example, methods described elsewhere (see, for example, Dong et al., Nature Med. 8:793-800, 2002). In some cases, a sMSLN polypeptide (e.g., a polypeptide comprising or consisting of the extracellular domain of MSLN) can be recombinantly produced, or can be purified from a biological sample, and used to immunize a host animal such as, without limitation, a rabbit, chicken, mouse, guinea pig, or rat. Various adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Monoclonal antibodies can be prepared using a sMSLN polypeptide and hybridoma technology.

[0033] In immunological assays, an antibody having specific binding affinity for sMSLN or a secondary7antibody that binds to such an antibody can be labeled, either directly or indirectly. Suitable labels include, without limitation, radioisotopes (e.g.,125I.131I,35S,3H,32P.33P, or14C), fluorophores (e.g., fluorescein, fluorescein-5-isothiocyanate (FITC). PerCP, rhodamine, or phycoerythrin), luminescent moieties (e.g., QDOT™ nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, CA), compounds that absorb light of a defined wavelength, or enzymes (e.g.. alkaline phosphatase or horseradish peroxidase). In some cases, antibodies can be indirectly labeled by conjugation with biotin and then detected with avidin or streptavidin labeled with a molecule described above. Methods of detecting or quantifying a label depend on the nature of the label, and can include, for example, the use of detectors such as x-ray film, radioactivity' counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers. and densitometers. Combinations of these approaches (including “multi-layer’ assays) can be used to enhance the sensitivity of an assay.

[0034] Immunological assays for detecting sMSLN can be performed in a variety of formats, including sandwich assays (e.g., ELISA assays, sandwich Western blotting assays, or sandwich immunomagnetic detection assays), competition assays (competitive RIA), or bridge immunoassays. See, for example, U.S. Patent Nos. 5,296,347; 4,233,402; 4,098,876; and 4,034,074. Methods of detecting sMSLN generally can include contacting a biological fluid sample (e.g., blood, plasma, serum, or urine) with an antibody that binds to sMSLN and detecting or quantifying binding of sMSLN to the antibody. For example, an antibody having specific binding affinity for sMSLN can be immobilized on a solid substrate and then exposed to the biological sample. In some cases, binding of sMSLN to the antibody on the solid substrate can be detected by exploiting the phenomenon of surface plasmon resonance, which results in a change in the intensity of surface plasmon resonance upon binding that can be detected qualitatively or quantitatively by an appropriate instrument, e.g., a Biacore apparatus (Biacore International AB; Rapsgatan, Sweden). Alternatively, the antibody can be labeled and detected as described above. A standard cur e using known quantities of sMSLN can be generated to aid in the quantitation of sMSLN levels. In some embodiments, a “sandwich’" assay in which a capture antibody or capture binding substrate is immobilized on a solid substrate can be used to detect the presence, absence, or amount of sMSLN. The solid substrate can be contacted with the biological sample such that sMSLN in the sample can bind to the immobilized antibody. The presence of sMSLN bound to the antibody can be determined using a “reporter” antibody having specific binding affinity’ for sMSLN and the methods described above. It is understood that in these sandwich assays, the capture antibody or capture binding substrate should not bind to the same epitope (or range of epitopes in the case of a polyclonal antibody) as the reporter antibody. Thus, if a monoclonal antibody is used as a capture antibody, the reporter antibody can be another monoclonal antibody that binds to an epitope that is either completely physically separated from or only partially overlaps with the epitope to which the capture monoclonal antibody binds, or a polyclonal antibody that binds to epitopes other than or in addition to that to which the capture monoclonal antibody binds.

[0035] Suitable solid substrates to which an antibody (e.g., a capture antibody) or capture binding substrate can be bound include, without limitation, microtiter plates, tubes, membranes such as nylon or nitrocellulose membranes, and beads or particles (e.g., agarose, cellulose, glass, polysty rene, polyacry lamide, magnetic, or magnetizable beads or particles). Magnetic or magnetizable particles can be used when an automated immunoassay system is used.

[0036] Alternative techniques for detecting sMSLN include mass-spectrophotometric techniques such as electrospray ionization (ESI), liquid chromatography-mass spectrometry (LC-MS), and matrix-assisted laser desorption-ionization (MALDI). See, for example, Gevaert et al., Electrophoresis. 22(9): 1645-51, 2001; and Chaurand et al.. J Am Soc Mass Spectrom, 10(2):91- 103, 1999). Mass spectrometers useful for such applications are available from Applied Biosystems (Foster City, CA); Bruker Daltronics (Billerica, MA) and Amersham Pharmacia (Sunny vale, CA). Arrays for detecting polypeptides, two-dimensional gel analysis, and chromatographic separation techniques also can be used to detect sMSLN.

[0037] When a subject is identified as having a level of sMSLN that is elevated above a reference level, the subject can undergo one or more (e.g., one, two, three, four, five, one to three, two to four three to five, or more than five) TPE procedures. TPE also is referred to as plasmapheresis or apheresis, and is a procedure in which blood is removed from a subject, the plasma is removed from the blood and replaced with another fluid, and the remaining blood components and replacement fluid are returned to the subject. The result is that non-cellular substances confined to the plasma are removed from the subject. In some cases, the methods provided herein can include determining the level of sMSLN in the blood or the plasma before and / or after each round of TPE, thus permitting the level of sMSLN to be monitored until it reaches a desired level (e.g., a reference level in healthy mammals, as described above). In some cases, TPE can be repeated until the level of sMSLN is reduced by at least 10% (e.g., at least 15%, at least 20%, at least 50%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 50%, or more than 50%) as compared to a previously measured level of the sMSLN in a sample obtained from the subject before or during treatment (e.g., at an earlier time point during a TPE procedure).

[0038] After the TPE step(s) is complete, the subject can be treated with one or more therapies targeted to MSLN. The one or more MSLN-targeted therapies can include, for example, one or more anti-MSLN antibodies, one or more ADCs, one or more TCRs, one or more CAR-T therapies, or any combination thereof. Anti-MSLN antibodies that can be used as described herein can be polyclonal antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, single chain Fv antibody fragments, Fab fragments, or F(ab)2 fragments that are capable of binding to an epitopic determinant of MSLN (e.g., human MSLN). Examples of anti-MSLN antibodies that can be used as described herein include, without limitation, anetumab (a fully human anti-mesothelin monoclonal antibody, available from Thermo Fisher Scientific, MedChemExpress, and Selleckchem), and BMS-986148 (available from Selleckchem). Examples of ADCs targeted to MSLN that can be used as described herein include, without limitation, anetumab ravtansine (anetumab conjugated to anti -tubulin maytansinoid DM4, also referred to as BAY 94- 9343. previously available from Bayer), BMS-986148 (Bristol Myers Squibb), and DMOT4039A (a humanized anti-mesothelin monoclonal antibody conjugated to the antimitotic agent, monomethyl auristatin E; Genentech).

[0039] A composition containing one or more MSLN-targeted therapies (e.g., one or more anti-MSLN antibodies, one or more ADCs targeted to MSLN. one or more TCR fusion constructs targeted to MSLN. one or more CAR-T therapies targeted to MSLN, or any combination thereof) can be administered to a subject in an amount, at a frequency, and for a duration effective to achieve a desired effect (e.g., to reduce tumor size, reduce cancer cell number, to reduce one or more symptoms of cancer, or to prevent or delay worsening of one or more such symptoms). The one or more MSLN-targeted therapies can be administered in an amount effective to reduce the size of a tumor, reduce the number of cancer cells, or reduce one or more symptoms of cancer in a patient by at least 3% (e.g., at least 5%, at least 10%. at least 20%. at least 50%. 3% to 5%, 5% to 10%.

[0040] 10% to 15%, 15% to 20%, 20% to 25%, 25% to 50%, or more than 50%). In some cases, for example, effective amount of an MSLN-targeted treatment can be an amount that reduces the size of a tumor in a treated mammal by at least 10% as compared to the size of the tumor in the mammal prior to administration of the immunotherapy. The presence or extent of tumors, cancer cells, and cancer symptoms can be evaluated using any appropriate method.

[0041] In some cases, the amounts of one or more MSLN-targeted therapies (e.g., one or more anti-MSLN antibodies, one or more ADCs targeted to MSLN. one or more TCR fusion constructs targeted to MSLN. one or more CAR-T therapies targeted to MSLN, or any combination thereof) administered to a mammal and / or the frequency of administration can be titrated in order to, for example, identify a dosage that is most effective to treat the mammal while having the least amount of adverse effects. For example, an effective amount of a composition containing one or more MSLN-targeted therapies can be any amount that reduces tumor size or reduces cancer symptoms within a mammal, without having significant toxicity in the mammal. If a cancer in a mammal fails to respond to a particular amount, then the amount can be increased by, for example, two-fold, three-fold, five-fold, or ten-fold. After receiving this higher concentration, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments in the dosage can be made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment.

[0042] In some cases, the methods provided herein can include monitoring a treated subject to determine whether or not the therapy is effective. For example, a mammal having a tumor (e.g., a human cancer patient having mesothelioma or another solid tumor) can be monitored to determine whether the tumor has decreased in size after treatment, or whether the number of tumor cells detected in the patient is reduced following treatment.

[0043] In some cases, a method as described herein can include (a) providing a biological sample from a mammal, (b) detecting / quantifying a level of sMSLN in the sample, (c) conducting TPE to reduce the level of sMSLN in the mammal, and (d) administering one or more MSLN-targeted therapies to the mammal. In some cases, the methods provided herein will include only steps (c) and (d). For example, a method can include conducting TPE on a mammal identified as having a level of sMSLN that is greater than a reference level, and then administering an MSLN-targeted therapy to the mammal. The methods described herein can be surprisingly effective, as no clinical intervention has previously been shown to eliminate sMSLN from a subject. The materials and methods provided herein therefore are useful because they can improve the ability of MSLN-targeted therapies to reach their intended target.

[0044] Exemplary Embodiments

[0045] Embodiment 1 is a use of therapeutic plasma exchange (TPE) and a mesothelin- (MSLN-) targeted therapy in a method for treating cancer, wherein the method comprises: (a) performing TPE on a mammal identified as (i) having cancer and (ii) having a measured level of soluble mesothelin (sMSLN) that is higher than a reference level of sMSLN, and subsequently (b) administering a MSLN-targeted therapy to the mammal.

[0046] Embodiment 2 is the use of embodiment 1, wherein the measured level of sMSLN was determined using an enzyme linked immunosorbent assay (ELISA).

[0047] Embodiment 3 is the use of embodiment 1, wherein the measured level of sMSLN was determined using nanoflow cytometry.

[0048] Embodiment 4 is the use of any one of embodiments 1 to 3, wherein the reference level is an average level of sMSLN in biological samples from healthy mammals.

[0049] Embodiment 5 is the use of any one of embodiments 1 to 4. wherein the MSLN- targeted therapy comprises administering, to the mammal, an anti-MSLN antibody, an antibody drug conjugate (ADC) targeted to MSLN, a T cell receptor (TCR) fusion construct targeted to MSLN, or a chimeric antigen receptor T-cell (CAR-T) product targeted to MSLN.

[0050] Embodiment 6 is the use of any one of embodiments 1 to 5, wherein the mammal is a human.

[0051] Embodiment 7 is the use of any one of embodiments 1 to 6, wherein the cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0052] Embodiment 8 is a use of therapeutic plasma exchange (TPE) and a mesothelin- (MSLN-) targeted therapy for treating a mammal identified as having cancer, wherein the method comprises: (a) measuring a level of sMSLN in a biological sample obtained from the mammal, (b) comparing the measured level of the sMSLN to a reference level of sMSLN, (c) when the measured level is greater than the reference level, performing TPE on the mammal, and subsequently (d) administering a MSLN-targeted therapy to the mammal.

[0053] Embodiment 9 is the use of embodiment 8, wherein the measuring comprises performing an ELISA.

[0054] Embodiment 10 is the use of embodiment 8, wherein the measuring comprises performing nanoflow cytometry.

[0055] Embodiment 11 is the use of any one of embodiments 8 to 10, wherein the reference level is an average level of sMSLN in biological samples from healthy mammals.

[0056] Embodiment 12 is the use of any one of embodiments 8 to 11, wherein the MSLN-targeted therapy comprises administering, to the mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN.

[0057] Embodiment 13 is the use of any one of embodiments 8 to 12, wherein the mammal is a human.

[0058] Embodiment 14 is the use of any one of embodiments 8 to 13, wherein the cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0059] Embodiment 15 is a use of therapeutic plasma exchange (TPE) and a mesothelin- (MSLN-) targeted therapy in a method for treating a mammal identified as having cancer and as having a biological sample with an elevated level of sMSLN, wherein the method comprises performing TPE on the mammal and administering a MSLN-targeted therapy to the mammal.

[0060] Embodiment 16 is the use of embodiment 15, wherein the mammal was identified as having an elevated level of sMSLN by measuring the level of sMSLN in a biological sample obtained from the mammal, and determining that the level is higher than a reference level of sMSLN.

[0061] Embodiment 17 is the use of embodiment 16, wherein the reference level is an average level of sMSLN in biological samples from healthy mammals.

[0062] Embodiment 18 is the use of any one of embodiments 15 to 17, wherein the MSLN-targeted therapy comprises administering, to the mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN.

[0063] Embodiment 19 is the use of any one of embodiments 15 to 18, wherein the mammal is a human.

[0064] Embodiment 20 is the use of any one of embodiments 15 to 19, wherein the cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0065] Embodiment 21 is a method for treating cancer, wherein the method comprises: (a) performing therapeutic plasma exchange (TPE) on a mammal identified as (i) having cancer and (ii) having a measured level of soluble mesothelin (sMSLN) that is higher than a reference level of sMSLN, and subsequently (b) administering a MSLN-targeted therapy to the mammal.

[0066] Embodiment 22 is the method of embodiment 21, wherein the measured level of sMSLN was determined using an enzyme linked immunosorbent assay (ELISA).

[0067] Embodiment 23 is the method of embodiment 21. wherein the measured level of sMSLN was determined using nanoflow cytometry.

[0068] Embodiment 24 is the method of any one of embodiments 21 to 23, wherein the reference level is an average level of sMSLN in biological samples from healthy mammals.

[0069] Embodiment 25 is the method of any one of embodiments 21 to 24, wherein the MSLN-targeted therapy comprises administering, to the mammal, an anti-MSLN antibody, an antibody drug conjugate (ADC) targeted to MSLN, a T cell receptor (TCR) fusion construct targeted to MSLN, or a chimeric antigen receptor T-cell (CAR-T) product targeted to MSLN.

[0070] Embodiment 26 is the method of any one of embodiments 21 to 25, w herein the mammal is a human.

[0071] Embodiment 27 is the method of any one of embodiments 21 to 26, wherein the cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0072] Embodiment 28 is a method for treating a mammal identified as having cancer, wherein the method comprises: (a) measuring a level of sMSLN in a biological sample obtained from the mammal, (b) comparing the measured level of the sMSLN to a reference level of sMSLN, (c) when the measured level is greater than the reference level, performing TPE on the mammal, and subsequently (d) administering a MSLN-targeted therapy to the mammal.

[0073] Embodiment 29 is the method of embodiment 28, wherein the measuring comprises performing an ELISA.

[0074] Embodiment 30 is the method of embodiment 28, wherein the measuring comprises performing nanoflow cytometry.

[0075] Embodiment 31 is the method of any one of embodiments 28 to 30, wherein the reference level is an average level of sMSLN in biological samples from healthy mammals.

[0076] Embodiment 32 is the method of any one of embodiments 28 to 31. wherein the MSLN-targeted therapy comprises administering, to the mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN.

[0077] Embodiment 33 is the method of any one of embodiments 28 to 32, wherein the mammal is a human.

[0078] Embodiment 34 is the method of any one of embodiments 28 to 33, wherein the cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0079] Embodiment 35 is a method for treating a mammal identified as having cancer and as having a biological sample with an elevated level of sMSLN, wherein the method comprises performing TPE on the mammal and administering a MSLN-targeted therapy to the mammal.

[0080] Embodiment 36 is the method of embodiment 35, wherein the mammal was identified as having an elevated level of sMSLN by measuring the level of sMSLN in a biological sample obtained from the mammal, and determining that the level is higher than a reference level of sMSLN.

[0081] Embodiment 37 is the method of embodiment 36, wherein the reference level is an average level of sMSLN in biological samples from healthy mammals.

[0082] Embodiment 38 is the method of any one of embodiments 35 to 37, wherein the MSLN-targeted therapy comprises administering, to the mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN. or a CAR-T product targeted to MSLN. Embodiment 39 is the method of any one of embodiments 35 to 38, wherein the mammal is a human.

[0083] Embodiment 40 is the method of any one of embodiments 35 to 39, wherein the cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

[0084] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0085] EXAMPLES

[0086] Example 1 - Use of therapeutic plasma exchange to reduce soluble mesothelin Whole blood samples were collected before and after a 1 plasma volume TPE with albumin as the replacement fluid in patients undergoing routine TPE for medical conditions such as autoimmune diseases and hyperviscosity syndromes. Plasma was separated from whole blood by centrifugation. MSLN levels were measured with an ELISA assay in matched pre- and post-TPE plasma samples. MSLN levels also were measured in anetumab-immunoprecipitated samples. Anetumab was covalently coupled to DYNABEADS® per the manufacturer’s instructions at 5 pg per mg of beads. Three effective concentrations (12.5 pg / ml, 25 pg / ml, and 37.5 pg / ml) of anetumab-conjugated DYNABEADS® were used to immunoprecipitate MSLN from two randomly selected plasma samples from the pre-TPE blood draws. Assays were performed in duplicate. Significance was determined by Wilcoxon signed-rank test.

[0087] Pre- and post-TPE plasma were samples from 16 patients. TPE reduced sMSLN (p<0.001), with an average decrease of 47%, or 14.95 mg / mL (FIG. 1). When anetumab, an anti-MSLN antibody, was added to pre-PLEX plasma, sMSLN was significantly reduced as well (p<0.05), indicating that anetumab was binding to sMSLN. These studies indicated that TPE can reduce sMSLN. The decrease in sMSLN with anetumab indicated that this anti-MSLN antibody binds sMSLN in systemic circulation.

[0088] Example 2 - Effects of anetumab ravtansine on cell vi abi 11 tx and confluence The effects of anetumab ravtansine, an ADC containing a fully human anti- mesothelin monoclonal antibody conjugated to the cytotoxic maytansinoid tubulin inhibitor DM4. were evaluated in the epithelioid mesothelioma cell line, Mero-95. Cells were treated with anetumab ravtansine at concentrations of 0 nM, 10 nM, 50 nM, and 100 nM in the presence of (1) protease inhibitors to stabilize surface MSLN, and (2) rMSLN (a surrogate for sMSLN). The protease inhibitors included marimastat (M). which is a broad spectrum matrix metalloproteinase (MMP) inhibitor, TMI-1 (T), which is an inhibitor of ADAMI 7 and MMPs, and a combination of both (M+T). At baseline (prior to the addition of anetumab ravtansine), there was no significant difference betw een the 1% DMSO control, M, and T with regard to cell viability and cell confluence (FIGS. 2A-2D). However, the M+T combination resulted in decreased cell viability at baseline compared to the control (p=0.0016), rMSLN (p=0.0453), M (p=0.012), T (p=0.014), whether the supernatant was exchanged (FIG. 2A) or not (FIG. 2C). The M+T combination also resulted in decreased confluence compared to rMSLN (p=0.0003), M (p=0.0028), T (p=0.0252) in washed (FIG. 2B) and unwashed (FIG. 2D) cells, suggesting that M+T had a direct cytotoxic effect.

[0089] At 10 nM anetumab ravtansine, the addition of rMSLN resulted in reduced cytotoxicity compared to control DMSO, which was statistically significant with regard to confluence (FIG. 2B; p=0.0348) but not with regard to viability (FIG. 2A; p=0.927). This trend continued at 50 nM of anetumab ravtansine for confluence (FIG. 2B). Also at 10 nM anetumab ravtansine, the cells treated with rMSLN had a higher percentage of confluence compared to T (p=0.0289) and M+T (p=0.0206), but not as compared to M (p=0.0679) (FIGS. 2B and 2D), suggesting that the protease inhibitors may have stabilized surface MSLN to some degree, allowing anetumab ravtansine to be more effective, but this is difficult to put into context knowing that M+T may have direct cytotoxicity. At 100 nM, there w as no significant difference betw een any of the groups for either viability' or confluence, which may have been due to the high ADC concertation (FIGS. 2A-2D)

[0090] Taken together, these studies demonstrated a numeric reduction in cytotoxicity when adding soluble mesothelin to neutralize the anetumab antibody, and numeric enhancements in cytotoxicity when adding the protease inhibitors.

[0091] Example 3 - Evaluating TPE and protease inhibitors (Pls) for reducing soluble mesothelin

[0092] The results in this Example re-present and expand on at least some of the results provided in other Examples. METHODS AND MATERIALS

[0093] Therapeutic Plasma Exchange'. Whole blood samples were collected before and after a one plasma volume of TPE with albumin as the replacement fluid in patients undergoing routine TPE for various medical conditions, such as autoimmune diseases and hyperviscosity syndromes (TABLE 1). The first 8 mL were discarded to avoid contamination. Plasma was separated from whole blood by centrifugation. Plasma waste samples from the procedure itself also were collected. Anticoagulation related to the procedure was achieved with acid citrate dextrose solution A (ACD-A), with or without unfractionated heparin. sMSLN levels were measured with an ELISA assay (RayBiotech; Norcross, GA) in matched pre- and post-TPE plasma samples. Plasma samples were diluted 1 : 10 before applying to the plate. The Wilcoxon signed rank exact test was used to compare sMSLN between pre and post plasma samples.

[0094] TABLE 1

[0095] Patient Characteristics

[0096] Anetumab Immunoprecipitation: The anti-mesothelin antibody anetumab (Invitrogen; Waltham, MA) was covalently coupled to DYNABEADS® (Life Technologies; Waltham, MA) according to the manufacturer’s instructions at 5 pg per mg of beads. Three effective concentrations (12.5 pg / mL. 25 pg / mL, and 37.5 pg / mL) of anetumab-conjugated DYNABEADS* were used to immunoprecipitate MS LN from two randomly selected plasma samples from the pre-TPE blood draws. Assays were performed in duplicate. The Wilcoxon signed-rank test was used to compare MSLN levels before and after immunoprecipitation.

[0097] Protease Inhibitors and Cytotoxicity: The epithelioid mesothelioma cell line Mero-95 was grown in RPMI-1640 supplemented with 10% FBS, 100 U / mL penicillin, and 100 pg / mL streptomycin. For each experiment, cells were plated at a seeding density of 10,000 cells / well for 24 hours before adding protease inhibitors. Protease inhibitors were diluted in growth medium and added to each well and allowed to incubate for 20 hours, using 1% DMSO as a vehicle control. Wells were then washed with PBS before anetumab ravtansine (Selleck Chemicals; Houston, TX) was added at concentrations of 10 nM, 50 nM, or 100 nM, and incubated for 48 hours. To determine the effects of sMSLN on cytotoxicity, anetumab ravtansine was pre-incubated with rMSLN (Creative Biomart; Shirley. NY) prior to being added to Mero-95 cells. Treatment groups for each anetumab ravtansine concentration included:

[0098] (1) no pre-treatment;

[0099] (2) 1% DMSO control;

[0100] (3) rMSLN (3 nM) as a surrogate for sMSLN;

[0101] (4) marimastat (M) 10 pM (Selleck Chemicals);

[0102] (5) TMI-1 (T) 10 pM (Bio-Techne; Minneapolis, MN); and

[0103] (6) combined marimastat and TMI-1 (M+T). each at 10 pM.

[0104] After 48 hours, a WST-8 / l-methoxy PMS (Selleck Chemicals) cytotoxicity assay was conducted to determine viability. For the WST-8 assay, WST-8 at a final concentration of 0.5 rnM was added with 1 -methoxy PMS at a final concentration of 20 pM and incubated in the cell culture chamber for tw o hours. Results were read on a Glomax Explorer plate reader at 450 nm, with a reference wavelength at 650 nm. DMSO at a 1% final concentration was used as a positive control, applied to cells at the same time as the protease inhibitors. Groups were compared with a 2-way ANOVA, and a p value less than 0.05 was considered significant. Graphpad Prism w as used to perform Ordinary Two-way ANOVA with Tukey’s multiple comparisons test to analyze the viability and confluence results. RESULTS

[0105] Therapeutic Plasma Exchange: Pre- and post-TPE plasma samples were evaluated from 15 patients undergoing routine TPE for various medical conditions, including central nervous system (CNS) demyelinating disorders (46.67%), paraneoplastic syndromes (13.33%), paraproteinemia (13.33%), Susac syndrome (13.33%), solid organ transplant rejection (6.67%), and immune encephalitis (6.67%) (TABLE 1). The CNS demyelinating disorders included chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), multiple sclerosis (MS), neuromyelitis optica (NMO), or myelitis. Seven patients were female and eight were male. The mean age was 58 years old (ranging from 25 to 84 years old). Four patients (26.67%) were diagnosed with an active malignancy (melanoma, uterine neuroendocrine malignancy, non-Hodgkins’s lymphoma, and lymphoplasmacytic lymphoma) and three (20%) had a history of cancer (renal cancer carcinoma, anal cancer, and ovarian cancer). In the matched pre- and post-TPE samples, a one plasma volume TPE consistently reduced sMSLN (p = 0.031) with an average decrease of 43.6% or 15.4 ng / mL (TABLE 2). sMSLN levels were decreased for all patients regardless of their oncologic history.

[0106] TABLE 2

[0107] Soluble Mesothelin Levels Pre- and Post- Therapeutic Plasma Exchange Anetumab Immunoprecipitation: In two randomly selected plasma samples, which included one from a patient with a CNS demyelinating disorder and one from a patient with Susac syndrome, it was found that the addition of anetumab-conjugated DYNABEADS® significantly reduced the concentration of sMSLN in plasma samples as detected by a MSLN ELISA (p<0.05). The first plasma sample had a baseline average sMSLN of 0.647 ng / mL, which decreased to an average of 0.229 ng / mL (64.5% decrease) with 12.5 pg / mL of anetumab. 0.352 ng / mL (45.6% decrease) with 25 pg / ml of anetumab, and 0.263 ng / mL (59.4% decrease) with 37.5 pg / mL of anetumab. The second plasma sample had a baseline average sMSLN of 7.231 ng / mL, which decreased to an average of 6.685 ng / mL (7.6% decrease) with 12.5 pg / mL of anetumab, 6.242 ng / mL (13.7% decrease) with 25 pg / mL of anetumab, and 6.703 ng / mL (7.3% decrease) with 37.5 pg / mL of anetumab. These results suggested that anetumab-conjugated DYNABEADS® repeatedly reduced sMSLN from plasma.

[0108] Protease Inhibitors and Cytotoxicity: The cytotoxicity of anetumab ravtansine was evaluated at 0 nM, 10 nM, 50 nM, and 100 nM in the presence of (1) Pls to stabilize surface MSLN. and (2) rMSLN as a surrogate for sMSLN (FIGS. 3-5 and TABLES 3 and 4). The protease inhibitors included marimastat (M), a broad-spectrum matrix metalloproteinase (MMP) inhibitor, TMI-1 (T), an inhibitor of ADAM17 and MMPs, and a combination of both inhibitors (M+T). M and T had been utilized in studies that identified them as proteases responsible for MSLN cleavage; these proteases also reduced MSLN shedding (Liu et al., Commun. Biol., 3: 178, 2020; 10.1038 / s42003-020-01464-5). In addition, M and T were tested on cell lines that express MSLN, demonstrating that both Pls reduced free MSLN in the medium at various concentrations.

[0109] At baseline, prior to the addition of anetumab ravtansine, there was no significant difference between a 1% DMSO control, M, and T on cell viability or on cell confluence. However, the combination of M+T resulted in decreased cell viability compared to no pretreatment (mean difference 48.25%, p=0.0042), control DMSO (mean difference 53%, p=0.0016), rMSLN (mean difference 36.2%, p=0.0453), M (mean difference 43. 1%, p=0.012), and T (mean difference 42.3%, p=0.014). Confluence was decreased compared to no pretreatment (mean difference 31.61%, p=0,0002), rMSLN (mean difference 30.71%, p=0.0003), M (mean difference 25.43%, p=0.0029), and T (mean difference 20.06%, p=0.0252). These results suggested that M+T had a direct cytotoxic effect.

[0110] At 10 nM of anetumab ravtansine, the addition of rMSLN reduced confluence (mean difference 19.21%, p=0.0348) but not viability (p=0.927) compared to the DMSO control. This trend continued with 50 nM of anetumab ravtansine for confluence, but was not significant. Also at 10 nM anetumab ravtansine. the cells treated with rMSLN had a higher percentage of confluence compared to T (mean difference 19.7%, p=0.0289) and M+T (mean difference 20.58%, p=0.0206), but not M (p=0.0679), suggesting that the Pls may have stabilized surface MSLN to some degree, thus allowing anetumab ravtansine to be more effective, but this is difficult to put into context given that M+T may have direct cytotoxicity. At 100 nM of anetumab ravtansine, there were no significant differences between the groups for either viability or confluence, which may have been due to the high ADC concentration or low cell counts. Taken together, these results indicated that high levels of sMSLN represent a mechanism of resistance to anti-MSLN antibody -based therapies, that TMI-1 stabilizes MSLN at the cell surface, and that TPE provides an approach to reduce sMSLN.

[0111] TABLE 3 Viability results

[0112] ARav = anetumab ravtansine; Tx = treatment; rMSLN = recombinant mesothelin;

[0113] M = marimastat; T = TMI-1; M+T = combined marimastat and TMI-1

[0114] TABLE 4

[0115] Confluence results

[0116]

[0117] ARav = anetumab ravtansine; Tx = treatment; rMSLN = recombinant mesothelin;

[0118] M = marimastat; T = TM1-1; M+T = combined marimastat and TM1-1

[0119] Example 4 - Further studies in a prospective cohort The above studies are expanded to a prospective cohort designed to obtain adequate power. In addition, studies are expanded to include MSLN-positive extracellular vesicles (EVs), and to investigate the mechanisms in which circulating MSLN interferes with antibody binding and cytotoxicity. These studies indicate that the use of TPE prior to MSLN-targeted therapies is a mechanism for improving response rates and survival.

[0120] OTHER EMBODIMENTS

[0121] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. Use of therapeutic plasma exchange (TPE) and a mesothelin- (MSLN-) targeted therapy in a method for treating cancer, wherein said method comprises:(a) performing TPE on a mammal identified as (i) having cancer and (ii) having a measured level of soluble mesothelin (sMSLN) that is higher than a reference level of sMSLN, and subsequently(b) administering a MSLN-targeted therapy to said mammal.

2. The use of claim 1, wherein said measured level of sMSLN was determined using an enzyme linked immunosorbent assay (ELISA).

3. The use of claim 1, wherein said measured level of sMSLN was determined using nanoflow cytometry.

4. The use of claim 1. wherein said reference level is an average level of sMSLN in biological samples from healthy mammals.

5. The use of claim 1, wherein said MSLN-targeted therapy comprises administering, to said mammal, an anti-MSLN antibody, an antibody drug conjugate (ADC) targeted to MSLN, a T cell receptor (TCR) fusion construct targeted to MSLN, or a chimeric antigen receptor T-cell (CAR-T) product targeted to MSLN.

6. The use of claim 1. wherein said mammal is a human.

7. The use of claim 1, wherein said cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

8. Use of therapeutic plasma exchange (TPE) and a mesothelin- (MSLN-) targeted therapy for treating a mammal identified as having cancer, wherein said method comprises:(a) measuring a level of sMSLN in a biological sample obtained from said mammal,(b) comparing said measured level of said sMSLN to a reference level of sMSLN,(c) when the measured level is greater than said reference level, performing TPE on said mammal, and subsequently(d) administering a MSLN-targeted therapy to said mammal.

9. The use of claim 8, wherein said measuring comprises performing an ELISA.

10. The use of claim 8, wherein said measuring comprises performing nanoflow cytometry.

11. The use of claim 8, wherein said reference level is an average level of sMSLN in biological samples from healthy mammals.

12. The use of claim 8, wherein said MSLN-targeted therapy comprises administering, to said mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN.

13. The use of claim 8, wherein said mammal is a human.

14. The use of claim 8, wherein said cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

15. Use of therapeutic plasma exchange (TPE) and a mesothelin- (MSLN-) targeted therapy in a method for treating a mammal identified as having cancer and as having a biological sample with an elevated level of sMSLN, wherein said method comprises performing TPE on said mammal and administering a MSLN-targeted therapy to said mammal.

16. The use of claim 15, wherein said mammal was identified as having an elevated level of sMSLN by measuring the level of sMSLN in a biological sample obtained from said mammal, and determining that said level is higher than a reference level of sMSLN.

17. The use of claim 16, wherein said reference level is an average level of sMSLN in biological samples from healthy mammals.

18. The use of claim 15, wherein said MSLN-targeted therapy comprises administering, to said mammal, an anti-MSLN antibody, an ADC targeted to MSLN. a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN.

19. The use of claim 15, wherein said mammal is a human.

20. The use of claim 15, wherein said cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

21. A method for treating cancer, wherein said method comprises:(a) performing therapeutic plasma exchange (TPE) on a mammal identified as (i) having cancer and (ii) having a measured level of soluble mesothelin (sMSLN) that is higher than a reference level of sMSLN, and subsequently(b) administering a MSLN-targeted therapy to said mammal.

22. The method of claim 21. wherein said measured level of sMSLN was determined using an enzy me linked immunosorbent assay (ELISA).

23. The method of claim 21, wherein said measured level of sMSLN was determined using nanoflow cytometry.

24. The method of claim 21, wherein said reference level is an average level of sMSLN in biological samples from healthy mammals.

25. The method of claim 21, wherein said MSLN-targeted therapy comprises administering, to said mammal, an anti-MSLN antibody, an antibody drug conjugate (ADC) targeted to MSLN, a T cell receptor (TCR) fusion construct targeted to MSLN, or a chimeric antigen receptor T-cell (CAR-T) product targeted to MSLN.

26. The method of claim 21, wherein said mammal is a human.

27. The method of claim 21, wherein said cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

28. A method for treating a mammal identified as having cancer, wherein said method comprises:(a) measuring a level of sMSLN in a biological sample obtained from said mammal,(b) comparing said measured level of said sMSLN to a reference level of sMSLN,(c) when the measured level is greater than said reference level, performing TPE on said mammal, and subsequently(d) administering a MSLN-targeted therapy to said mammal.

29. The method of claim 28, wherein said measuring comprises performing an ELISA.

30. The method of claim 28, wherein said measuring comprises performing nanoflow cytometry.

31. The method of claim 28, wherein said reference level is an average level of sMSLN in biological samples from healthy mammals.

32. The method of claim 28, wherein said MSLN-targeted therapy comprises administering, to said mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN.

33. The method of claim 28, wherein said mammal is a human.

34. The method of claim 28, wherein said cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

35. A method for treating a mammal identified as having cancer and as having a biological sample with an elevated level of sMSLN, wherein the method comprises performing TPE on said mammal and administering a MSLN-targeted therapy to said mammal.

36. The method of claim 35, wherein said mammal was identified as having an elevated level of sMSLN by measuring the level of sMSLN in a biological sample obtained from said mammal, and determining that said level is higher than a reference level of sMSLN.

37. The method of claim 36, wherein said reference level is an average level of sMSLN in biological samples from healthy mammals.

38. The method of claim 35, wherein said MSLN-targeted therapy comprises administering, to said mammal, an anti-MSLN antibody, an ADC targeted to MSLN, a TCR fusion construct targeted to MSLN, or a CAR-T product targeted to MSLN.

39. The method of claim 35, wherein said mammal is a human.

40. The method of claim 35, wherein said cancer is mesothelioma, prostate cancer, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, thyroid cancer, kidney cancer, uterine cancer, ovarian cancer, pancreatic cancer, or cholangiocarcinoma.

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