EP4314831A2 - Tgf-beta-inhibitoren und verwendung davon - Google Patents

Tgf-beta-inhibitoren und verwendung davon

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Publication number
EP4314831A2
EP4314831A2 EP22723227.9A EP22723227A EP4314831A2 EP 4314831 A2 EP4314831 A2 EP 4314831A2 EP 22723227 A EP22723227 A EP 22723227A EP 4314831 A2 EP4314831 A2 EP 4314831A2
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EP
European Patent Office
Prior art keywords
tgfβ
tumor
circulating
cancer
inhibitor
Prior art date
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Pending
Application number
EP22723227.9A
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English (en)
French (fr)
Inventor
Si Tuen Lee-Hoeflich
Christopher Brueckner
Constance MARTIN
Ryan Faucette
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Scholar Rock Inc
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Scholar Rock Inc
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Publication date
Application filed by Scholar Rock Inc filed Critical Scholar Rock Inc
Priority claimed from PCT/US2022/022063 external-priority patent/WO2022204581A2/en
Publication of EP4314831A2 publication Critical patent/EP4314831A2/de
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00113—Growth factors
    • A61K39/001134—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • TGF-BETA INHIBITORS AND USE THEREOF RELATED APPLICATIONS [1] This Application claims the benefit of and priority to US Provisional Applications 63/166,824 filed March 26, 2021; 63/202,260 filed June 3, 2021; 63/302,999 filed January 25, 2022; and 63/313,386 filed February 24, 2022, each entitled “TGF-BETA INHIBITORS AND USE THEREOF,” the contents of which are expressly incorporated herein by reference in their entirety.
  • FIELD [2] The instant application relates generally to TGF ⁇ inhibitors and therapeutic use thereof, as well assays for diagnosing, monitoring, prognosticating, and treating disorders, including cancer.
  • Transforming growth factor beta 1 is a member of the TGF ⁇ superfamily of growth factors, along with two other structurally related isoforms, namely, TGF ⁇ 2 and TGF ⁇ 3, each of which is encoded by a separate gene.
  • TGF ⁇ isoforms function as pleiotropic cytokines that regulate cell proliferation, differentiation, immunomodulation (e.g., adaptive immune response), and other diverse biological processes both in homeostasis and in disease contexts.
  • the three TGF ⁇ isoforms signal through the same cell-surface receptors and trigger similar canonical downstream signal transduction events that include the SMAD2/3 pathway.
  • TGF ⁇ has been implicated in the pathogenesis and progression of a number of disease conditions, such as cancer, fibrosis, and immune disorders. In many cases, such conditions are associated with dysregulation of the extracellular matrix (ECM). For these and other reasons, TGF ⁇ has been an attractive therapeutic target for the treatment of immune disorders, various proliferative disorders, and fibrotic conditions. However, observations from preclinical studies, including in rats and dogs, have revealed serious toxicities associated with systemic inhibition of TGF ⁇ s in vivo, and to date, there are no TGF ⁇ therapeutics available in the market which are deemed both safe and efficacious. [5] Dose-limiting toxicities noted with inhibition of the TGF ⁇ pathway have remained a major concern in the development of anti-TGF ⁇ therapies.
  • cardiovascular abnormalities include cardiovascular abnormalities, skin lesions, epithelial oral hyperplasia, and gingival bleeding (Vitsky 2009; Lonning 2011; Stauber 2014; Mitra 2020). Although many of these toxicities are either reversible or manageable, the cardiovascular lesions such as inflammation, hemorrhage or hyperplasia in the valves, aortic arch and associated arteries of the heart, are not reversible and therefore continue to be key safety issues when developing TGF ⁇ inhibitors (Stauber 2014; Anderton 2011; Mitra 2020).
  • TGF ⁇ 1 inhibitors that are both i) isoform-specific; and, ii) capable of broadly targeting multiple TGF ⁇ 1 signaling complexes that are associated with different presenting molecules, as therapeutic agents for conditions driven by multifaceted TGF ⁇ 1 effects and dysregulation thereof.
  • a non-limiting example of such an isoform-specific inhibitor is a TGF ⁇ 1-selective antibody, e.g., Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, or Ab34 disclosed herein.
  • TGF ⁇ 1-selective antibody e.g., Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, or Ab34 disclosed herein.
  • PCT/US2019/041373 discloses that isoform-selective, high affinity antibodies capable of targeting large latent complexes (LLCs) of TGF ⁇ 1 may be effective to treat TGF ⁇ 1-related indications, such as diseases involving abnormal gene expression (e.g., TGFB1, Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, Loxl2, CCL2 and Mmp2), diseases involving ECM dysregulation (e.g., fibrosis, myelofibrosis and solid tumor), diseases characterized by increased immunosuppressive cells (e.g., Tregs, MDSCs and/or M2 macrophages), diseases involving mesenchymal transition, diseases involving proteases, diseases related to abnormal stem cell proliferation and/or differentiation.
  • diseases involving abnormal gene expression e.g., TGFB1, Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, Loxl2, CCL2 and Mmp2
  • TGF ⁇ 1 inhibitors were shown to overcome tumor primary resistance (i.e., present before treatment initiation) to an immunotherapy (e.g., checkpoint inhibitors), where the tumor is infiltrated with immunosuppressive cell types, such as regulatory T cells, M2-type macrophages, and/or myeloid-derived suppressive cells (tumor-associated MDSCs).
  • immunosuppressive cell types such as regulatory T cells, M2-type macrophages, and/or myeloid-derived suppressive cells (tumor-associated MDSCs).
  • a reduction in the number of tumor- associated immunosuppressive cells e.g., MDSCs
  • a corresponding increase in the number of anti-tumor effector T cells were observed.
  • TGF ⁇ inhibitors that target more than one isoform.
  • ALK5 antagonists such as Galunisertib (LY2157299 monohydrate); monoclonal antibodies (such as neutralizing antibodies) that inhibit all three isoforms (“pan-inhibitor” antibodies) (see, for example, WO 2018/134681); monoclonal antibodies that preferentially inhibit two of the three isoforms (e.g., antibodies against TGF ⁇ 1/2 (for example WO 2016/161410) and TGF ⁇ 1/3 (for example WO 2006/116002 and WO 2020/051333); integrin inhibitors such as antibodies that bind to ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins and inhibit downstream activation of TGF ⁇ .
  • TGF ⁇ 1 and/or TGF ⁇ 3 e.g., PLN-74809
  • engineered molecules e.g., fusion proteins
  • ligand traps for example, WO 2018/029367; WO 2018/129331 and WO 2018/158727.
  • TME tumor microenvironment
  • compositions comprising TGF ⁇ inhibitors and methods for selecting suitable subjects to treat with TGF ⁇ inhibitors, as well as related methods of treatments and monitoring treatment parameters such as efficacy and target engagement.
  • the disclosure provides better and more targeted therapeutics and treatment modalities, including improved ways of identifying candidates for treatment and/or monitoring treatment efficacy, e.g., patients or patient populations who are likely to benefit from the TGF ⁇ inhibitor therapy.
  • Related methods, including therapeutic regimens, and methods for manufacturing such inhibitors are encompassed herein.
  • the selection of particular subjects and TGF ⁇ inhibitors for therapeutic use is aimed to achieve in vivo efficacy.
  • the present disclosure provides, inter alia, i) enhanced methods for analysis aimed to provide better characterization of the cellular architecture within and surrounding a tumor; ii) improved methods for determining circulatory TGF ⁇ levels aimed to achieve greater accuracy; iii) improved methods for assessing circulating MDSC levels, including identification of LRRC33 as a novel marker for circulating MDSC cells and improved surface markers for identification of mMDSC and gMDSC sub-populations; and iv) additional biomarkers, including p-Smad2, which are useful for predicting and monitoring therapeutic efficacy, as well as determining target engagement.
  • the pharmaceutical composition and/or treatment regimen disclosed herein comprises a TGF ⁇ inhibitor and one or more additional therapies such as a genotoxic therapy and/or a checkpoint inhibitor therapy.
  • the one or more additional therapies may be administered in conjunction with the TGF ⁇ inhibitor, either as a separate molecular entity administered separately, as a single formulation (e.g., an admixture), or as part of a single molecular entity, e.g., an engineered multifunctional construct that functions as both a checkpoint inhibitor and a TGF ⁇ inhibitor.
  • the checkpoint inhibitor therapy is an antibody or an antigen-binding fragment that targets PD-1, PD-L1, CTLA-4, or LAG3.
  • the genotoxic therapy is a chemotherapy or a radiation therapy.
  • the TGF ⁇ inhibitor and the one or more additional therapies are administered concurrently, separately, or sequentially.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1 inhibitor, such as apitegromab or Ab6.
  • the TGF ⁇ inhibitor is any one of the antibodies or antigen-binding fragments disclosed in PCT/JP2015/006323, the content of which is hereby incorporated by reference in its entirety.
  • the TGF ⁇ inhibitor is GYM329.
  • the TGF ⁇ inhibitor is an inhibitor of TGF ⁇ 1 and TGF ⁇ 2, such as NIS793/XOMA-089 or GC1008.
  • the TGF ⁇ inhibitor is an inhibitor of TGF ⁇ 1 and TGF ⁇ 3, such as M7824 (bintrafusp alpha) or AVID200.
  • the TGF ⁇ inhibitor is a pan TGF ⁇ inhibitor (i.e., an agent that inhibits TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3), including GC1008 or derivative thereof, SAR439459, LY3022859, or an agent that blocks a ligand-binding domain of a TGF ⁇ receptor.
  • the TGF ⁇ inhibitor is an agent that binds the RGD motif present in latent TGF ⁇ 1 and/or TGF ⁇ 3, e.g., a low molecular weight (small molecule) compound or an antibody or antigen-binding fragment.
  • the TGF ⁇ inhibitor is an RNA-based inhibitor of TGF ⁇ 1 expression.
  • the TGF ⁇ inhibitor is a soluble ligand trap such as M7824 (bintrafusp alpha) or AVID200.
  • the disclosure provides a method of treating, predicting, and/or monitoring therapeutic efficacy of a TGF ⁇ inhibitor treatment in a subject by measuring or monitoring circulating TGF ⁇ levels (e.g., circulating TGF ⁇ 1 levels, e.g., circulating latent TGF ⁇ 1 levels).
  • circulating TGF ⁇ levels e.g., circulating TGF ⁇ 1 levels, e.g., circulating latent TGF ⁇ 1 levels.
  • circulating TGF ⁇ e.g., from a blood sample obtained from a subject, as a biomarker to determine and monitor therapeutic efficacy and/or target engagement, and to guide decisions on treatment.
  • circulating and circulatory as in “circulating TGF ⁇ ” and “circulatory TGF ⁇ ” may be used interchangeably.
  • the disclosure provides a method of treating, predicting, and/or monitoring therapeutic efficacy of a TGF ⁇ inhibitor treatment in a subject, the method comprising (i) determining a level of circulating TGF ⁇ in the subject prior to administering a TGF ⁇ inhibitor; (ii) administering to the subject a therapeutically effective amount of the TGF ⁇ inhibitor; and (iii) determining a level of circulating TGF ⁇ in the subject after administration, wherein an increase in circulating TGF ⁇ after the administration as compared to before the administration indicates therapeutic efficacy.
  • the increase is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more.
  • the treatment alters the level of circulating TGF ⁇ . In some embodiments, continued treatment is contingent on an observed increase in circulating TGF ⁇ .
  • the disclosure encompasses a method of determining target engagement and/or therapeutic efficacy of a TGF ⁇ inhibitor treatment in a subject, wherein the treatment comprises (i) determining the circulating TGF ⁇ level in a sample obtained from the subject prior to administering the TGF ⁇ inhibitor; (ii) administering a first dose of the TGF ⁇ inhibitor to the subject; and (iii) determining the circulating TGF ⁇ level in a sample obtained from the subject after the administration, wherein an increase in the circulating TGF ⁇ level after the administration as compared to before the administration is indicative of target engagement and/or therapeutic efficacy.
  • the method comprises administering to the subject a second dose of the TGF ⁇ inhibitor if an increase in the circulating TGF ⁇ level after the administration as compared to before the administration is observed.
  • the treatment is continued if an increase in the circulating TGF ⁇ level after the administration as compared to before the administration is observed.
  • the increase is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more.
  • the treatment may further comprise administering to the subject one or more additional therapies, e.g., a genotoxic therapy and/or immunotherapy, wherein the one or more additional therapies are administered concurrently (e.g., simultaneously), separately, or sequentially.
  • the additional therapy may comprise a checkpoint inhibitor therapy.
  • the disclosure provides an improved method for measuring a circulating TGF ⁇ level from a blood sample or a sample derived from blood, the method comprising processing the sample at a temperature of 2-8 ⁇ C in a sample tube comprising an anticoagulant.
  • the anticoagulant is citrate-theophylline-adenosine-dipyridamole (CTAD).
  • the tube is coated with a citrate- theophylline-adenosine-dipyridamole (CTAD) solution, a 0.11 M buffered trisodium citrate solution, 15 M theophylline, 3.7 M adenosine, and 0.198 M dipyridamole, wherein the solution has a pH of 5.0.
  • CAD citrate- theophylline-adenosine-dipyridamole
  • the sample processing comprises one or more centrifugation steps at a speed of greater than 100xg and/or one or more centrifugation steps at a speed of below 15000xg.
  • the sample processing comprises a centrifugation protocol comprising: i) a first step of 10 minutes at 150xg and a second step of 20 minutes at 2500xg; or ii) a first step of 10 minutes at 2500xg and a second step of 20 minutes at 2500xg; or iii) a first step of 10 minutes at 1500xg and a second step of 5 minutes at 12000xg.
  • the method comprises analyzing a level of plasma factor 4 (PF4) in the same sample from which the circulating TGF ⁇ level is determined, such that the PF4 level provides quality control for the sample.
  • PF4 plasma factor 4
  • a sample is only used to determine a circulating TGF ⁇ level if the PF4 level in the same sample is below a concentration indicative of plasma activation. In some embodiments, a PF4 level of greater than 500 ng/ml is indicative of plasma activation. Thus, in some embodiments, a sample is used to assess a circulating TGF ⁇ level only if the sample has a PF4 level of 500 ng/ml or less.
  • the disclosure provides a method of treating, predicting, and/or monitoring therapeutic efficacy of a TGF ⁇ inhibitor treatment in a subject, the method comprising (i) determining a level of phosphorylated Smad2 (P-Smad2) nuclear translocation in a tumor sample obtained from the subject prior to administering a TGF ⁇ inhibitor (pre-treatment tumor sample); (ii) administering to the subject one or more doses of the TGF ⁇ inhibitor; and (iii) determining a level of P-Smad2 nuclear translocation in a tumor sample obtained from the subject after the administration (post-treatment tumor sample); wherein a decrease in P-Smad2 nuclear translocation after the administration as compared to before the administration indicates therapeutic efficacy.
  • P-Smad2 phosphorylated Smad2
  • the treatment is continued if a decrease in P-Smad2 nuclear translocation is observed in the post-treatment tumor sample.
  • the decrease is by at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold.
  • the level of P-Smad2 nuclear translocation is determined by nuclear masking.
  • the disclosure provided herein involves the use of circulating MDSC levels as a predictive biomarker to improve the diagnosis, monitoring, patient selection, prognosis, and/or continued treatment of a subject being administered a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1-selective inhibitor such as Ab6) by monitoring circulating MDSC levels.
  • a TGF ⁇ inhibitor e.g., a TGF ⁇ 1-selective inhibitor such as Ab6
  • the disclosure also encompasses methods of determining therapeutic efficacy and therapeutic agents (e.g., compositions) or regiments for use in subjects with cancer by measuring levels of circulating MDSCs.
  • therapeutic agents e.g., compositions
  • the instant inventors have discovered that reversal of or overcoming an immunosuppressive phenotype, e.g., in a cancer or related condition that manifests dysregulation of the ECM, such as by administration of a TGF ⁇ inhibitor, can be indicated by analyzing circulating MDSC levels, e.g., in a sample obtained from a subject, e.g., in blood or a blood component, e.g., prior to the time point when a reduction in tumor volume or other biomarkers might be used to confirm treatment efficacy.
  • circulatory MDSCs are characterized by cell-surface expression of LRRC33.
  • sub-populations of circulatory MDSCs are measured, such as m-MDSCs and/or g-MDSCs.
  • the terms circulating and circulatory (as in “circulating MDSCs” and “circulatory MDSCs”) may be used interchangeably.
  • Tumor-associated MDSC cells may contribute to TGF ⁇ 1-mediated immunosuppression in the tumor microenvironment.
  • Tumor biopsy may reveal an immune profile of a tumor microenvironment (TME); however, in addition to being invasive, biopsy- based information may be inaccurate or skewed because tumor-infiltrating lymphocytes (TILs) may not be uniformly present within the whole tumor, and therefore, depending on which portion of the tumor is sampled by biopsy, results may vary.
  • TME tumor microenvironment
  • data presented herein now establish the correlation between tumor-associated (e.g., intratumoral) MDSC levels and circulatory MDSC levels, raising the possibility that MDSCs measured in blood samples (e.g., whole blood or a blood component, e.g., PBMCs) may serve as a surrogate to more accurately predict patient populations that are likely to benefit from certain therapeutic regimens.
  • blood samples e.g., whole blood or a blood component, e.g., PBMCs
  • LRRC33 as a novel cell-surface marker for MDSCs in circulation (e.g., blood samples). This observation raises the possibility that surface LRRC33 expression may be used as a blood-based predictive biomarker.
  • the methods disclosed herein employ circulating MDSCs as an early biomarker to predict the efficacy of combination therapy comprising a TGF ⁇ inhibitor.
  • Data disclosed herein show that after TGF ⁇ 1 inhibitor treatment, there is a marked reduction in circulating MDSC levels relative to baseline, which can be a reduction in mMDSC levels and/or gMDSC levels.
  • Such circulating MDSC levels can be measured in blood or a blood component, which can be detected well before antitumor efficacy outcome can readily be obtained, in some cases shortening the timeline by weeks.
  • the disclosure provides the use of circulating MDSCs as a predictive biomarker for the patient’s responsiveness to a cancer therapy, e.g., a combination therapy.
  • the level of circulating MDSC cells may be determined within 1-10 weeks, e.g., 3-6 weeks, following administration of a dose of a TGF ⁇ inhibitor, optionally within 3 weeks or at about 3 weeks following administration of the dose of TGF ⁇ inhibitor. In some embodiments, the level of circulating MDSC cells may be determined within 2 weeks following administration of the dose of TGF ⁇ inhibitor. In some embodiments, the level of circulating MDSC cells may be determined at about 10 days following administration of the dose of TGF ⁇ inhibitor. [25] Cancer immunotherapy may harness or enhance the body’s immunity to combat cancer.
  • low levels of circulating MDSCs in subjects with cancer indicate that the body has retained or restored disease-fighting immunity (e.g., antitumor activity), more specifically, lymphocytes such as CD8+ T cells, which can be mobilized to attack malignant cells.
  • disease-fighting immunity e.g., antitumor activity
  • lymphocytes such as CD8+ T cells
  • reduced levels of circulating MDSCs upon TGF ⁇ inhibitor treatment may indicate pharmacodynamic effects of TGF ⁇ inhibition (e.g., TGF ⁇ 1 inhibition) and serve as an early predictive biomarker for therapeutic efficacy when treated with a cancer therapy such as checkpoint inhibitors.
  • non-selective TGF ⁇ inhibitor may be administered infrequently or intermittently, for example on an “as-needed” basis.
  • circulating MDSC levels may be monitored periodically in order to determine that the effects of overcoming immunosuppression are sufficiently maintained, so as to ensure antitumor effects of the cancer therapy.
  • MDSC levels become elevated, this may indicate that the patient may benefit from additional dose(s) of a TGF ⁇ inhibitor.
  • the TGF ⁇ inhibitor targets TGF ⁇ 1/2 signaling.
  • the TGF ⁇ inhibitor targets TGF ⁇ 1/3 signaling.
  • the TGF ⁇ inhibitor targets TGF ⁇ 1/2/3 signaling.
  • disclosed herein are methods of treating cancer (also described herein in the context of compositions for use in treating cancer or cancer treatments). Also disclosed are methods of predicting, determining, or monitoring therapeutic efficacy in subjects with cancer, e.g., monitoring a patient’s responsiveness to treatment and/or making continued treatment decisions based on the monitored parameters.
  • the cancer is an immune excluded cancer and/or a myeloproliferative disorder, wherein the myeloproliferative disorder may be myelofibrosis.
  • the cancer is a TGF ⁇ 1-positive cancer.
  • the TGF ⁇ 1-positive cancer may co-express TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3.
  • the TGF ⁇ 1-positive cancer may be a TGF ⁇ 1-dominant tumor.
  • the TGF ⁇ 1-positive cancer may be a TGF ⁇ 1-dominant tumor and may co-express TGF ⁇ 1, TGF ⁇ 2, and/or TGF ⁇ 3.
  • the TGF ⁇ 1-positive cancer may be a TGF ⁇ 1-dominant tumor and may co-express TGF ⁇ 1 and TGF ⁇ 2.
  • the TGF ⁇ 1-positive cancer may be a TGF ⁇ 1-dominant tumor and may co-express TGF ⁇ 1 and TGF ⁇ 3.
  • Such cancer includes advanced cancer, e.g., metastatic cancer (e.g., metastatic solid tumors) and cancer with a locally advanced tumor (e.g., locally advanced solid tumors).
  • the treatment comprises administering to the subject a TGF ⁇ inhibitor in an amount sufficient to reduce circulating MDSC levels.
  • the disclosure encompasses a method of predicting or determining therapeutic efficacy in a subject having cancer comprising the steps of determining circulating MDSC levels in the subject prior to administering a TGF ⁇ inhibitor (alone or in conjunction with a cancer therapy), administering to the subject a therapeutically effective amount of the TGF ⁇ inhibitor (alone or in conjunction with a cancer therapy), and determining circulating MDSC levels in the subject after the administration, wherein a reduction in circulating MDSC levels after administration, as compared to circulating MDSC levels before administration, predicts therapeutic efficacy.
  • the disclosure encompasses a method of determining therapeutic efficacy of a TGF ⁇ inhibitor treatment in a subject, wherein the treatment comprises (i) determining the circulating MDSC level in a sample obtained from the subject prior to administering the TGF ⁇ inhibitor; (ii) administering a first dose of the TGF ⁇ inhibitor to the subject; and (iii) determining the circulating MDSC level in a sample obtained from the subject after the administration, wherein a reduction in the circulating MDSC level after the administration as compared to before the administration is indicative of therapeutic efficacy.
  • the method comprises administering to the subject a second dose of the TGF ⁇ inhibitor if a reduction in the circulating MDSC level after the administration as compared to before the administration is observed.
  • the treatment is continued if a reduction in the circulating MDSC level after the administration as compared to before the administration is observed.
  • the disclosure encompasses a combination therapy comprising a dose of a TGF ⁇ inhibitor and a checkpoint inhibitor therapy and/or a genotoxic therapy for use in the treatment of cancer, wherein the treatment comprises concurrent (e.g., simultaneous), separate, or sequential administration of a dose of the TGF ⁇ inhibitor, wherein a reduction in circulating MDSC level after the administration as compared to before the administration has been determined.
  • the disclosure encompasses a TGF ⁇ inhibitor for use in the treatment of cancer in a subject, wherein the subject is administered a dose of the TGF ⁇ inhibitor, and wherein the TGF ⁇ inhibitor reduces or reverses immune suppression in the cancer, wherein said reduced or reversed immune suppression has been determined by a reduction in the circulating MDSC level in the subject measured after the administration of the TGF ⁇ inhibitor as compared to the circulating MDSC level measured in the subject prior to administering the dose of the TGF ⁇ inhibitor.
  • the disclosure encompasses a method of treating advanced cancer in a human subject comprising the steps of selecting a subject with advanced cancer and administering a combination therapy comprising a TGF ⁇ inhibitor and a checkpoint inhibitor therapy, wherein the advanced cancer comprises a locally advanced tumor and/or metastatic cancer with primary resistance to a checkpoint inhibitor therapy, wherein the subject has elevated circulating MDSC levels.
  • the combination therapy reduces the circulating MDSC level in the subject.
  • continued treatment is contingent on an observed reduction in the subject’s circulating MDSC level.
  • the circulating MDSC level may be a level of mMDSC and/or gMDSC.
  • mMDSCs are identified by cell surface markers of CD11b+, HLA-DR-/low, CD14+, CD15-, CD33+/high, and CD66b-.
  • gMDSCs are identified by cell surface markers of CD11b+, HLA- DR-, CD14-, CD15+, CD33+/low, and CD66b+.
  • the reduction in a circulating MDSC level may be a reduction of at least 10%.
  • the disclosure encompasses a method of treating, predicting, determining, and/or monitoring therapeutic efficacy of a cancer treatment in a subject administered a TGF ⁇ inhibitor alone or in combination with one or more additional cancer therapies (e.g., a checkpoint inhibitor therapy and/or a genotoxic therapy), the method comprising the steps of: (i) obtaining a pre-treatment biopsy sample from the subject, (ii) determining a level of tumor-associated CD8+ cells in the pre-treatment biopsy sample, (iii) administering the treatment to the subject, (iv) obtaining a post-treatment biopsy sample from the subject, and (v) determining a level of tumor-associated CD8+ cells in the post-treatment biopsy sample, wherein the levels of tumor-associated CD8+ cells in the biopsy samples are determined by immunohistochemical analysis of individual tumor nests within the tumor.
  • additional cancer therapies e.g., a checkpoint inhibitor therapy and/or a genotoxic therapy
  • a subject having an immune inflamed tumor characterized by a pre-treatment biopsy sample having greater than 5% CD8+ cells in individual tumor nests is selected for the treatment.
  • the immune inflamed tumor is characterized by having greater than 5% CD8+ cells in greater than 50% of the individual tumor nests detected.
  • a subject having an immune excluded tumor characterized by a pre-treatment biopsy sample having less than 5% intratumor CD8+ cells and greater than 5% margin CD8+ cells is selected for treatment.
  • the immune excluded tumor is characterized by having less than 5% CD8+ cells in individual tumor nests.
  • the immune excluded tumor is characterized by having less than 5% CD8+ cells in greater than 50% of the individual tumor nests detected.
  • therapeutic efficacy can be monitored by comparing the levels of CD8+ cells in the pre-treatment biopsy sample and the post-treatment biopsy sample such that a change in CD8+ level in the post- treatment biopsy sample as compared to the CD8+ level in the pre-treatment indicates therapeutic efficacy.
  • an increase of intratumor CD8+ cells e.g., an increase of CD8+ cells inside tumor nests
  • any one of the biomarkers disclosed herein may be used in conjunction with one or more of the other biomarkers provided herein.
  • circulating TGF ⁇ may be monitored in combination with one or more biomarkers disclosed herein, e.g., circulating MDSC, tumor-associated CD8+ cell, intratumor or circulating cytokines, and/or p-Smad2 nuclear translocation.
  • treatment efficacy and/or continued treatment may be contingent on observed changes in two or more sets of biomarkers.
  • the methods and compositions for use of the present disclosure comprise treating or selecting for treatment a subject having a cancer, wherein the cancer may be a highly metastatic cancer and/or a solid cancer.
  • the subject has melanoma, triple-negative breast cancer, HER2-positive breast cancer colorectal cancer (e.g., microsatellite stable-colorectal cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), pancreatic cancer, bladder cancer, kidney cancer (e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC), including clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC, uterine cancer, prostate cancer, stomach cancer (e.g., gastric cancer), or thyroid cancer.
  • HER2-positive breast cancer colorectal cancer e.g., microsatellite stable-colorectal cancer
  • lung cancer e.g., non-small cell lung cancer or small cell lung cancer
  • pancreatic cancer e.g., bladder cancer
  • kidney cancer e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC)
  • the methods and compositions for use of the present disclosure comprise treating or selecting for treatment a subject having a cancer that is resistant to immunotherapy.
  • the subject may be treatment-na ⁇ ve (e.g., has not previously received a cancer therapy), may have primary resistance to an immunotherapy (i.e., resistance is present before treatment initiation), or may have acquired resistance to an immunotherapy (i.e., resistance as a result of at least one dose of treatment).
  • the immunotherapy is a checkpoint inhibitor therapy, e.g., an anti-PD-1 or anti-PD-L1 antibody.
  • the methods and compositions for use according to the present disclosure encompass providing treatment to a treatment-na ⁇ ve subject.
  • the methods and compositions for use according to the present disclosure encompass providing treatment to a subject who has previously received a cancer therapy or who is currently receiving cancer therapy.
  • a previous cancer therapy may be the same cancer therapy to be administered according to the invention.
  • the cancer therapy may be checkpoint inhibitor (CPI) therapy.
  • CPI checkpoint inhibitor
  • the methods and compositions for use according to the present disclosure encompass providing treatment to a cancer subject wherein the cancer is or is suspected of being immune suppressive (e.g., having a tumor with an immune excluded or immunosuppressive phenotype).
  • the methods and compositions for use according to the present disclosure encompass providing treatment to a subject having a cancer with a high response rate to checkpoint inhibitor therapy (e.g., overall response rate of greater than 30%, greater 40%, greater than 50%, or greater).
  • a high response rate to checkpoint inhibitor therapy e.g., overall response rate of greater than 30%, greater 40%, greater than 50%, or greater.
  • cancer with high response rates to checkpoint inhibitor therapy examples include, but are not limited to, microsatellite instability-colorectal cancer (MSI-CRC), renal cell carcinoma (RCC), melanoma (e.g., metastatic melanoma), Hodgkin’s lymphoma, NSCLC, cancer with high microsatellite instability (MSI-H), cancer with mismatch repair deficiency (dMMR), primary mediastinal large B-cell lymphoma (PMBCL), and Merkel cell carcinoma (e.g., as reported in Haslam et al., JAMA Network Open.2019;2(5): e192535).
  • MSI-CRC microsatellite instability-colorectal cancer
  • RCC renal cell carcinoma
  • melanoma e.g., metastatic melanoma
  • Hodgkin’s lymphoma NSCLC
  • MSI-H cancer with high microsatellite instability
  • dMMR cancer with mismatch repair deficiency
  • the methods and compositions for use according to the present disclosure encompass providing treatment to a subject having a cancer with a low response rate to checkpoint inhibitor therapy (e.g., overall response rate of 30% or less, 20% or less, or 10%, or less).
  • the subject may be treatment-na ⁇ ve.
  • the subject may be resistant to checkpoint inhibitor therapy. Examples of cancer with low response rates to checkpoint inhibitor therapy include, but are not limited to, ovarian cancer, gastric cancer, and triple-negative breast cancer.
  • the methods and compositions for use according to the present disclosure encompass providing treatment to a subject having a solid cancer.
  • the solid cancer is selected from melanoma (e.g., metastatic melanoma), renal cell carcinoma, breast cancer, e.g., triple-negative breast cancer, HER2-positive breast cancer, colorectal cancer, e.g., microsatellite stable-colorectal cancer and colon adenocarcinoma, lung cancer (e.g., metastatic non-small cell lung cancer, small cell lung cancer), esophageal cancer, pancreatic cancer, bladder cancer, kidney cancer, e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC), including clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC, uterine cancer, e.g., uterine corpus endometrial carcinoma, prostate cancer, stomach cancer (e.g., gastric cancer), head and neck cancer, e.g., head and neck squamous cell cancer, urothelial
  • a TGF ⁇ inhibitor of the present disclosure may be used to treat, including to improve rates or ratios of complete verses partial responses among the responders of a cancer therapy. Typically, even in cancer types where response rates to a cancer therapy (e.g., a checkpoint inhibitor therapy) are relatively high (e.g., ⁇ 30% responders), complete response rates are low.
  • the TGF ⁇ inhibitors of the present disclosure may therefore be used to increase the fraction of complete responders within the responder population.
  • the TGF ⁇ inhibitor is Ab6.
  • the TGF ⁇ inhibitor of the present disclosure does not inhibit TGF ⁇ 2 signaling at a therapeutically effective dose.
  • the TGF ⁇ inhibitor does not inhibit TGF ⁇ 3 signaling at a therapeutically effective dose. In some embodiments, the TGF ⁇ inhibitor does not inhibit TGF ⁇ 2 signaling and TGF ⁇ 3 signaling at a therapeutically effective dose. [47] In various embodiments, the TGF ⁇ inhibitor is a TGF ⁇ 1-selective inhibitor. In some embodiments, the TGF ⁇ inhibitor may bind TGF ⁇ 1 with an affinity of 0.5 nM or greater (KD ⁇ 0.5 nM) with a dissociation rate of no more than 10.0E-4 (1/s) as measured by SPR. More preferably, the TGF ⁇ inhibitor is an activation inhibitor of TGF ⁇ 1.
  • the activation inhibitor may be a monoclonal antibody or an antigen-binding fragment thereof that binds the latent lasso region of a latent TGF ⁇ 1 complex.
  • the TGF ⁇ inhibitor is Ab4, Ab5, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, or Ab34.
  • the TGF ⁇ inhibitor is Ab6 or a variant thereof (e.g., a variant of Ab6 as used herein is one that retains at least 80%, 90%, 95% or greater sequence similarity to Ab6 and/or retains one or more binding and/or therapeutic properties of Ab6, so as to achieve a desired therapeutic effect).
  • the methods and compositions for use disclosed herein comprise use of a TGF ⁇ inhibitor disclosed herein in conjunction (e.g., in combination) with a checkpoint inhibitor and/or a genotoxic therapy, wherein the checkpoint inhibitor is an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4-antibody, anti-LAG3 antibody, or an antigen-binding fragment thereof; and/or the genotoxic therapy is a chemotherapy or a radiation therapy, wherein optionally, the chemotherapy is a PARP inhibitor therapy.
  • the methods and compositions for use disclosed herein comprise use of a TGF ⁇ inhibitor disclosed herein in conjunction with at least one additional therapy.
  • the at least one additional therapy is a cancer therapy, such as an immunotherapy, a genotoxic therapy, including chemotherapy and radiation therapy (including radiotherapeutic agents), an engineered immune cell therapy (e.g., CAR-T therapy), a cancer vaccine therapy, and/or an oncolytic viral therapy.
  • the at least one additional therapy is chemotherapy or radiation therapy (including radiotherapeutic agents).
  • the at least one additional cancer therapy is a checkpoint inhibitor therapy.
  • the checkpoint inhibitor may comprise an agent targeting programmed cell death protein 1 (PD-1) or programmed cell death protein 1 ligand (PD-L1).
  • the checkpoint inhibitor may comprise an anti-PD-1 or anti-PD- L1 antibody.
  • the TGF ⁇ inhibitors disclosed herein may be used in conjunction with at least one additional therapy selected from: a PD-1 antagonist (e.g., a PD-1 antibody), a PDL1 antagonist (e.g., a PDL1 antibody), a PD-L1 or PDL2 fusion protein, a CTLA4 antagonist (e.g., a CTLA4 antibody), a GITR agonist e.g., a GITR antibody), an anti-ICOS antibody, an anti-ICOSL antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-OX40 antibody (OX40 agonist), an anti-CD27 antibody, an anti-CD70 antibody, an anti-CD47 antibody, an anti-41BB antibody, an anti-PD-1 antibody, an anti-CD20 antibody, an anti-CD3 antibody, an anti-PD-1/anti-PDL1 bispecific or multispecific antibody, an anti-CD3/anti-
  • FIG.1 shows tumor MDSC levels measured in MBT-2 tumors.
  • FIG.2 shows tumor volume and circulating G-MDSC and M-MDSC levels in MBT-2 mice.
  • FIG.3 shows tumor volume in MBT-2 mice across treatment groups.
  • FIG.4 shows baseline level of circulating MDSCs in non-tumor bearing mice.
  • FIG.5 shows levels of circulating MDSCs in tumor-bearing mice.
  • FIG.6 shows a comparison of circulating MDSC levels in non-tumor bearing mice and tumor-bearing mice.
  • FIG. 7A shows a comparison of circulating M-MDSC and G-MDSC levels on days 3-10; FIG.
  • FIG. 7B shows time-course of changes in circulating M-MDSC and G-MDSC levels from days 3-10.
  • FIG.8 is a plot of circulating MDSC level and tumor volume on day 10 across treatment groups.
  • FIG.9A shows tumor MDSC levels in different treatment groups;
  • FIG.9B shows a comparison of circulating G-MDSC levels and tumor MDSC levels on day 10 across treatment groups.
  • FIG.10 shows correlation of tumor MDSC levels to circulating MDSC levels.
  • FIG.11 shows tumor G-MDSC and tumor CD8+ cells across all treatment groups.
  • FIG.12A shows circulating gMDSC and mMDSC levels in whole blood of mice bearing MBT2 tumors;
  • FIG.12A shows circulating gMDSC and mMDSC levels in whole blood of mice bearing MBT2 tumors;
  • FIG.12A shows circulating gMDSC and mMDSC levels in whole blood of mice bearing MBT2 tumors;
  • FIG. 12B shows intratumoral gMDSC and mMDSC levels in mice bearing MBT2 tumors.
  • FIG. 13A shows circulating TGF ⁇ 1 levels (pg/mL) in MBT-2 mice;
  • FIG. 13B shows plasma levels of Ab6 ( ⁇ g/mL, left) and TGF ⁇ 1 (pg/mL, right);
  • FIG.13C shows correlation of plasma levels of Ab6 ( ⁇ g/mL) and TGF ⁇ 1 (pg/mL) in MBT-2 mice treated with AB6 alone or in combination with an anti-PD1 antibody.
  • FIG. 14 shows plasma platelet factor 4 levels (ng/mL) in MBT-2 mice (right) and sample outliers as determined by interquartile range (left).
  • FIG. 14 shows plasma platelet factor 4 levels (ng/mL) in MBT-2 mice (right) and sample outliers as determined by interquartile range (left).
  • FIG. 15 shows identified sample outliers (left) and outlier-corrected levels (pg/mL) of circulatory TGF ⁇ 1 (right).
  • FIG.16 shows circulatory TGF ⁇ levels in NHP following a single dose of Ab6.
  • FIG.17 shows circulatory TGF ⁇ levels in rats following a single dose of Ab6.
  • FIG. 18 shows an exemplary sample collection and processing method for evaluating circulating TGF ⁇ 1 levels in blood.
  • FIG.19A shows circulating TGF ⁇ 1 levels in blood samples as evaluated under various sample processing conditions.
  • FIG. 19B shows platelet factor 4 (PF4) levels in blood samples as evaluated under various sample processing conditions.
  • FIG.20 shows correlation of circulating TGF ⁇ 1 levels and PF4 levels in blood samples as evaluated under various sample processing conditions.
  • FIG. 21A shows PF4 levels in blood samples as evaluated under various sample processing conditions;
  • FIG.21B and FIG.21C show exemplary outlier analysis based on measurement of PF4 levels.
  • FIG.22 shows PF4 vs. TGF ⁇ 1 levels pre-dose and 1 hour post-dose.
  • FIG.23A shows fold change in TGF ⁇ levels over time in subjects treated with 80-240 mg of Ab6;
  • FIG.23B shows fold change in TGF ⁇ levels over time in subjects treated with 800 mg of Ab6;
  • FIG.23C shows fold change in TGF ⁇ levels over time in subjects treated with 1600 mg of Ab6.
  • FIG.24 shows a P-Smad2 IHC analysis of melanoma samples.
  • FIG.25 shows pSmad-2 signaling in MBT2 tumors following treat with Ab6-mIgG1.
  • FIG.26A shows tissue compartment data of bladder cancer samples;
  • FIG.26B shows tissue compartment data of melanoma samples.
  • FIG.27 shows density of CD8+ cells in bladder cancer samples as analyzed based on tumor nest.
  • FIG. 28 shows immune phenotype analysis of a single bladder cancer sample based on density of CD8+ cells measured in tumor nests.
  • FIG. 29A shows average percentages of CD8+ cells and immune phenotyping in bladder cancer and melanoma samples, as analyzed by tumor compartments (left) and tumor nests (right); underlined phenotype reflects differences between analyses;
  • FIG. 29B shows average percentages of CD8+ cells and immune phenotyping in bladder cancer and melanoma samples, as analyzed by tumor compartments (left) and tumor nests (right); underlined phenotype reflects differences between analyses;
  • FIG.29C shows tumor nest data and immune phenotyping for individual tumor nests identified from bladder cancer samples;
  • FIG.29D shows tumor nest CD8+ data and immune phenotyping for bladder cancer and melanoma samples;
  • FIG.29E shows percent CD8+ cells in tumor, tumor margin, and stroma compartments of commercially available bladder cancer samples.
  • FIG. 30A shows representative CD8+ staining in bladder cancer samples
  • FIG.30B shows subdivision of CD8+ staining in the tumor margin compartment
  • FIG.30C shows subdivision of CD8+ staining in the tumor margin compartment of a bladder sample.
  • FIG.31 shows comparison of compartment CD8+ ratio and absolute percent CD8 positivity.
  • FIG.32 shows comparison of CD8+ cell density and absolute percent CD8 positivity.
  • FIG.33 shows tumor depth of bladder samples.
  • FIG.34 shows CD8 density in a melanoma sample.
  • FIGs.35A-c show exemplary analysis of MDSC by signal filtering.
  • FIGs.36A-C shows identification of tumor MDSC populations in various solid cancer samples.
  • FIGs.37A-C shows analysis of gMDSC and mMDSC populations in various solid cancer samples.
  • FIG.38 shows a schematic of an exemplary pathology analysis of tumor tissue sample.
  • FIG.39 shows a schematic of an exemplary pathology analysis of tumor tissue sample.
  • FIG.40 shows a schematic of an exemplary TGF ⁇ inhibitor treatment regimen.
  • FIG. 41 illustrates identification of three binding regions (Region 1, Region 2, and Region 3) following statistical analyses. Region 1 overlaps with a region called “Latency Lasso” within the prodomain of proTGF ⁇ 1, while Regions 2 and 3 are within the growth factor domain.
  • FIG.42 depicts various domains and motifs of proTGF ⁇ 1, relative to the three binding regions involved in Ab6 binding. Sequence alignment among the three isoforms is also provided.
  • DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Definitions [93] In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.
  • Advanced cancer advanced malignancy
  • advanced cancer or “advanced malignancy” as used herein has the meaning understood in the pertinent art, e.g., as understood by oncologists in the context of diagnosing or treating subjects/patients with cancer.
  • Advanced malignancy with a solid tumor can be locally advanced or metastatic.
  • locally advanced cancer is used to describe a cancer (e.g., tumor) that has grown outside the organ it started in but has not yet spread to distant parts of the body. Thus, the term includes cancer that has spread from where it started to nearby tissue or lymph nodes.
  • Affinity is the strength of binding of a molecule (such as an antibody) to its ligand (such as an antigen). It is typically measured and reported by the equilibrium dissociation constant (K D ). In the context of antibody-antigen interactions, KD is the ratio of the antibody dissociation rate (“off rate” or Koff), how quickly it dissociates from its antigen, to the antibody association rate (“on rate” or Kon) of the antibody, how quickly it binds to its antigen.
  • an antibody with an affinity of ⁇ 5 nM has a K D value that is 5 nM or lower (i.e., 5 nM or higher affinity) determined by a suitable in vitro binding assay.
  • suitable in vitro assays can be used to measure K D values of an antibody for its antigen, such as Biolayer Interferometry (BLI) and Solution Equilibrium Titration (e.g., MSD-SET).
  • affinity is measured by surface plasmon resonance (e.g., Biacore®).
  • An antibody with a suitable affinity in a surface plasmon resonance assay may have, e.g., a K D of at most about 1 nM, e.g., at most about 0.5 nM, e.g., at most about 0.5, 0.4, 0.3, 0.2, 0.15 nM, or less.
  • a K D of at most about 1 nM, e.g., at most about 0.5 nM, e.g., at most about 0.5, 0.4, 0.3, 0.2, 0.15 nM, or less.
  • the term refers to an immunoglobulin molecule that specifically binds to a target antigen, and includes, for instance, chimeric, humanized, fully human, and multispecific antibodies (including bispecific antibodies).
  • An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some instances can include fewer chains such as antibodies naturally occurring in camelids which can comprise only heavy chains.
  • Antibodies can be derived solely from a single source, or can be “chimeric,” that is, different portions of the antibody can be derived from two different antibodies.
  • Antibodies, or antigen binding portions thereof can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • antibodies includes monoclonal antibodies, multispecific antibodies such as bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), respectively. In some embodiments, the term also encompasses peptibodies.
  • Antigen broadly includes any molecules comprising an antigenic determinant within a binding region(s) to which an antibody or a fragment specifically binds. An antigen can be a single-unit molecule (such as a protein monomer or a fragment) or a complex comprised of multiple components.
  • An antigen provides an epitope, e.g., a molecule or a portion of a molecule, or a complex of molecules or portions of molecules, capable of being bound by a selective binding agent, such as an antigen binding protein (including, e.g., an antibody).
  • a selective binding agent may specifically bind to an antigen that is formed by two or more components in a complex.
  • the antigen is capable of being used in an animal to produce antibodies capable of binding to that antigen.
  • An antigen can possess one or more epitopes that are capable of interacting with different antigen binding proteins, e.g., antibodies.
  • a suitable antigen is a complex (e.g., multimeric complex comprised of multiple components in association) containing a proTGF dimer in association with a presenting molecule.
  • a proTGF dimer in association with a presenting molecule.
  • Each monomer of the proTGF dimer comprises a prodomain and a growth factor domain, separated by a furin cleavage sequence. Two such monomers form the proTGF dimer complex.
  • This in turn is covalently associated with a presenting molecule via disulfide bonds, which involve a cysteine residue present near the N-terminus of each of the proTGF monomer.
  • This multi-complex formed by a proTGF dimer bound to a presenting molecule is generally referred to as a large latent complex.
  • An antigen complex suitable for screening antibodies or antigen-binding fragments includes a presenting molecule component of a large latent complex.
  • presenting molecule component may be a full-length presenting molecule or a fragment(s) thereof.
  • Minimum required portions of the presenting molecule typically contain at least 50 amino acids, but more preferably at least 100 amino acids of the presenting molecule polypeptide, which comprises two cysteine residues capable of forming covalent bonds with the proTGF ⁇ 1 dimer.
  • Antigen-binding portion/fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TGF ⁇ 1).
  • Antigen binding portions include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • an antigen-binding portion of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • Non-limiting examples of antigen-binding portions include: (i) Fab fragments, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the VH and CH1 domains;; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (v) single-chain Fv (scFv) molecules (see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc.
  • Fab fragments a monovalent fragment consisting of the VL, VH, CL and CH1 domains
  • F(ab')2 fragments a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • dAb fragments see, e.g., Ward et al., (1989) Nature 341: 544-546
  • minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)).
  • CDR complementarity determining region
  • antigen binding portion of an antibody includes a “single chain Fab fragment” otherwise known as an “scFab,” comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N- terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids.
  • Bias In the context of the present disclosure, the term “bias” (as in “biased binding”) refers to skewed or uneven affinity towards or against a subset of antigens to which an antibody is capable of specifically binding. For example, an antibody is said to have bias when the affinity for one antigen complex and the affinity for another antigen complex are not equivalent. Context-independent antibodies according to the present disclosure have equivalent affinities towards such antigen complexes (i.e., unbiased or uniform).
  • Binding region As used herein, a “binding region” is a portion of an antigen that, when bound to an antibody or a fragment thereof, can form an interface of the antibody-antigen interaction.
  • BLI Biolayer Interferometry
  • Cancer refers to the physiological condition in multicellular eukaryotes that is typically characterized by unregulated cell proliferation and malignancy. The term broadly encompasses, solid and liquid malignancies, including tumors, blood cancers (e.g., leukemias, lymphomas and myelomas), as well as myelofibrosis.
  • Cell-associated proTGF ⁇ 1 The term refers to TGF ⁇ 1 or its signaling complex (e.g., pro/latent TGF ⁇ 1) that is membrane-bound (e.g., tethered to cell surface).
  • TGF ⁇ 1 that is presented by GARP or LRRC33 is a cell-associated TGF ⁇ 1.
  • GARP and LRRC33 are transmembrane presenting molecules that are expressed on cell surface of certain cells.
  • GARP-proTGF ⁇ 1 and LRRC33- may be collectively referred to as “cell-associated” (or “cell-surface”) proTGF ⁇ 1 complexes, that mediate cell proTGF ⁇ 1-associated (e.g., immune cell-associated) TGF ⁇ 1 activation/signaling.
  • the term also includes recombinant, purified GARP-proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1 complexes in solution (e.g., in vitro assays) which are not physically attached to cell membranes.
  • Average KD values of an antibody (or its fragment) to a GARP-proTGF ⁇ 1 complex and an LRRC33- proTGF ⁇ 1 complex may be calculated to collectively represent affinities for cell-associated (e.g., immune cell- associated) proTGF ⁇ 1 complexes. See, for example, Table 5, column (G).
  • cell-associated proTGF ⁇ 1 may be a target for internalization (e.g., endocytosis) and/or cell killing such as ADCC, ADCP, or ADC-mediated depletion of the target cells expressing such cell surface complexes.
  • checkpoint inhibitors refer to immune checkpoint inhibitors and carries the meaning as understood in the art.
  • a “checkpoint inhibitor therapy” or “checkpoint blockade therapy” is one that targets a checkpoint molecule to partially or fully alter its function.
  • a checkpoint is a receptor molecule on a T cell or NK cell, or a corresponding cell surface ligand on an antigen-presenting cell (APC) or tumor cell.
  • API antigen-presenting cell
  • immune checkpoints are activated in immune cells to prevent inflammatory immunity developing against the “self”. Therefore, changing the balance of the immune system via checkpoint inhibition may allow it to be fully activated to detect and eliminate the cancer.
  • CTLA-4 cytotoxic T-lymphocyte antigen-4
  • PD-1 programmed cell death protein 1
  • PD-L1 programmed cell death receptor ligand 1
  • TIM3 T-cell immunoglobulin domain and mucin domain-3
  • LAG3 lymphocyte-activation gene 3
  • KIR killer cell immunoglobulin-like receptor
  • GITR glucocorticoid-induced tumor necrosis factor receptor
  • Ig V-domain immunoglobulin-containing suppressor of T-cell activation
  • Non-limiting examples of checkpoint inhibitors include: Nivolumab, Pembrolizumab, cemiplimab, BMS-936559, Atezolizumab, Avelumab, Durvalumab, Ipilimumab, Tremelimumab, IMP-321 (Eftilagimod alpha or ImmuFact®), BMS-986016 (Relatlimab), budigalimab (ABBV-181), and Lirilumab.
  • Keytruda® is one example of anti-PD-1 antibodies
  • Opdivo® is one example of an anti-PD-L1 antibody.
  • CBT checkpoint blockade therapy
  • CPI checkpoint inhibitor therapy
  • Clinical benefit As used herein, the term “clinical benefits” is intended to include both efficacy and safety of a therapy. Thus, therapeutic treatment that achieves a desirable clinical benefit is both efficacious (e.g., achieves therapeutically beneficial effects) and safe (e.g., with tolerable or acceptable levels of toxicities or adverse events).
  • Combination therapy “Combination therapy” refers to treatment regimens for a clinical indication that comprise two or more therapeutic agents.
  • the term refers to a therapeutic regimen in which a first therapy comprising a first composition (e.g., active ingredient) is administered in conjunction with at least a second therapy comprising a second composition (active ingredient) to a patient, intended to treat the same or overlapping disease or clinical condition.
  • the term may further encompass a therapeutic regimen in which a first therapy comprising a first composition (e.g., active ingredient) is administered in conjunction with a second therapy comprising a second composition (e.g., active ingredient such as a checkpoint inhibitor), a third therapy comprising a third composition (e.g., active ingredient such as a chemotherapy), or more (e.g., additional distinct active ingredients).
  • the first, second, and (optionally additional) compositions may act on the same cellular target, or discrete cellular targets.
  • the phrase “in conjunction with,” in the context of combination therapies, means that therapeutic effects of a first therapy overlaps temporally and/or spatially with therapeutic effects of a second and additional therapy in the subject receiving the combination therapy.
  • the first, second, and/or additional compositions may be administered concurrently (e.g., simultaneously), separately, or sequentially.
  • the combination therapies may be formulated as a single formulation for concurrent administration, or as separate formulations, for sequential, concurrent, or simultaneous administration of the therapies.
  • a combinatorial epitope is an epitope that is recognized and bound by a combinatorial antibody at a site (i.e., antigenic determinant) formed by non-contiguous portions of a component or components of an antigen, which, in a three-dimensional structure, come together in close proximity to form the epitope.
  • a site i.e., antigenic determinant
  • antibodies of the disclosure may bind an epitope formed by two or more components (e.g., portions or segments) of a pro/latent TGF ⁇ 1 complex.
  • a combinatory epitope may comprise amino acid residue(s) from a first component of the complex, and amino acid residue(s) from a second component of the complex, and so on. Each component may be of a single protein or of two or more proteins of an antigenic complex.
  • a combinatory epitope is formed with structural contributions from two or more components (e.g., portions or segments, such as amino acid residues) of an antigen or antigen complex.
  • Compete or cross-compete; cross-block The term “compete” when used in the context of antigen binding proteins (e.g., an antibody or antigen binding portion thereof) that compete for the same epitope means competition between antigen binding proteins as determined by an assay in which the antigen binding protein being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein to a common antigen (e.g., TGF ⁇ 1 or a fragment thereof).
  • a common antigen e.g., TGF ⁇ 1 or a fragment thereof.
  • ⁇ solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay solid phase direct biotin-avidin EIA
  • solid phase direct labeled assay solid phase direct labeled sandwich assay.
  • a competing antigen binding protein when present in excess, it will inhibit (e.g., reduce) specific binding of a reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70- 75% or 75% or more.
  • binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more when the competing antibody is present in excess.
  • an SPR e.g., Biacore
  • Biacore Biacore
  • a BLI e.g., Octet®
  • a BLI e.g., Octet® assay is used to determine competition
  • a first antibody or antigen-binding portion thereof and a second antibody or antigen- binding portion thereof “cross-block” with each other with respect to the same antigen for example, as assayed by Biolayer Interferometry (such as Octet®) or by surface plasmon resonance (such as Biacore System), using standard test conditions, e.g., according to the manufacturer’s instructions (e.g., binding assayed at room temperature, ⁇ 20-25°C).
  • the first antibody or fragment thereof and the second antibody or fragment thereof may have the same epitope.
  • first antibody or fragment thereof and the second antibody or fragment thereof may have non-identical but overlapping epitopes.
  • first antibody or fragment thereof and the second antibody or fragment thereof may have separate (different) epitopes which are in close proximity in a three-dimensional space, such that antibody binding is cross-blocked via steric hindrance.
  • Cross-block means that binding of the first antibody to an antigen prevents binding of the second antibody to the same antigen, and similarly, binding of the second antibody to an antigen prevents binding of the first antibody to the same antigen.
  • Antibody binning may be carried out to characterize and sort a set (e.g., “a library”) of monoclonal antibodies made against a target protein or protein complex (i.e., antigen). Such antibodies against the same target are tested against all other antibodies in the library in a pairwise fashion to evaluate if antibodies block one another’s binding to the antigen. Closely related binning profiles indicate that the antibodies have the same or closely related (e.g., overlapping) epitope and are “binned” together.
  • an antibody that binds the same epitope as Ab6 binds a proTGF ⁇ 1 complex such that the epitope of the antibody includes one or more amino acid residues of Region 1, Region 2 and Region 3, identified as the binding region of Ab6.
  • CDR Complementary determining region
  • CDR1 CDR1
  • CDR2 CDR2
  • CDR3 CDR3
  • CDR set refers to a group of three CDRs that occur in a single variable region that can bind the antigen. The exact boundaries of these CDRs have been defined differently according to different systems.
  • These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs.
  • Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (1995) FASEB J. 9: 133-139 and MacCallum (1996) J. Mol. Biol. 262(5): 732-45.
  • a conformational epitope is an epitope that is recognized and bound by a conformational antibody in a three-dimensional conformation, but not in an unfolded peptide of the same amino acid sequence.
  • a conformational epitope may be referred to as a conformation-specific epitope, conformation- dependent epitope, or conformation-sensitive epitope.
  • a corresponding antibody or fragment thereof that specifically binds such an epitope may be referred to as conformation-specific antibody, conformation-selective antibody, or conformation-dependent antibody. Binding of an antigen to a conformational epitope depends on the three-dimensional structure (conformation) of the antigen or antigen complex.
  • Constant region An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.
  • Context-biased antibodies refer to a type of conformational antibodies that binds an antigen with differential affinities when the antigen is associated with (i.e.., bound to or attached to) an interacting protein or a fragment thereof.
  • a context-biased antibody that specifically binds an epitope within proTGF ⁇ 1 may bind LTBP1-proTGF ⁇ 1, LTBP3-proTGF ⁇ 1, GARP-proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1 with different affinities.
  • an antibody is said to be “matrix-biased” if it has higher affinities for matrix- associated proTGF ⁇ 1 complexes (e.g., LTBP1-proTGF ⁇ 1 and LTBP3-proTGF ⁇ 1) than for cell-associated proTGF ⁇ 1 complexes (e.g., GARP-proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1).
  • matrix-associated proTGF ⁇ 1 complexes e.g., LTBP1-proTGF ⁇ 1 and LTBP3-proTGF ⁇ 1
  • cell-associated proTGF ⁇ 1 complexes e.g., GARP-proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1
  • Relative affinities of [matrix-associated complexes] [cell-associated complexes] may be obtained by taking average KD values of the former, taking average KD values of the latter, and calculating the ratio of the two, as exemplified herein.
  • Context-independent a context-independent antibody that binds proTGF ⁇ 1 has equivalent affinities across the four known presenting molecule-proTGF ⁇ 1 complexes, namely, LTBP1-proTGF ⁇ 1, LTBP3-proTGF ⁇ 1, GARP-proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1.
  • Context-independent antibodies disclosed in the present application may also be characterized as unbiased.
  • context- independent antibodies show equivalent (i.e., no more than five-fold bias in) affinities, such that relative ratios of measured KD values between matrix-associated complexes and cell-associated complexes are no greater than 5 as measured by a suitable in vitro binding assay, such as surface plasmon resonance, Biolayer Interferometry (BLI), and/or solution equilibrium titration (e.g., MSD-SET).
  • a suitable in vitro binding assay such as surface plasmon resonance, Biolayer Interferometry (BLI), and/or solution equilibrium titration (e.g., MSD-SET).
  • surface plasmon resonance is used.
  • ECM-associated TGF ⁇ 1/proTGF ⁇ 1 The term refers to TGF ⁇ 1 or its signaling complex (e.g., pro/latent TGF ⁇ 1) that is a component of (e.g., deposited into) the extracellular matrix.
  • TGF ⁇ 1 that is presented by LTBP1 or LTBP3 is an ECM-associated TGF ⁇ 1, namely, LTBP1-proTGF ⁇ 1 and LTBP3-proTGF ⁇ 1, respectively.
  • LTBPs are critical for correct deposition and subsequent bioavailability of TGF ⁇ in the ECM, where fibrillin (Fbn) and fibronectin (FN) are believed to be the main matrix proteins responsible for the association of LTBPs with the ECM.
  • fibrillin (Fbn) and fibronectin (FN) are believed to be the main matrix proteins responsible for the association of LTBPs with the ECM.
  • matrix-associated latent complexes are enriched in connective tissues, as well as certain disease-associated tissues, such as tumor stroma and fibrotic tissues.
  • Effective amount refers to the ability or an amount to sufficiently produce a detectable change in a parameter of a disease, e.g., a slowing, pausing, reversing, diminution, or amelioration in a symptom or downstream effect of the disease. The term encompasses but does not require the use of an amount that completely cures a disease.
  • an “effective amount” may be a dosage or dosing regimen that achieves a statistically significant clinical benefit (e.g., efficacy) in a patient population.
  • a statistically significant clinical benefit e.g., efficacy
  • Ab6 has been shown to be efficacious at doses as low as 3 mg/kg and as high as 30 mg/kg in preclinical models.
  • minimum effective dose or “minimum effective amount” refers to the lowest amount, dosage, or dosing regimen that achieves a detectable change in a parameter of a disease, e.g., a statistically significant clinical benefit.
  • references herein to a dose of an agent may be a therapeutically effective dose, as described herein.
  • a dose of a TGF ⁇ 1 inhibitor may be a therapeutically effective dose, as described herein.
  • the term “pharmacological active dose (PAD)” may be used to refer to effective dosage.
  • Effective amounts may be expressed in terms of doses being administered or in terms of exposure levels achieved as a result of administration (e.g., serum concentrations).
  • Effective tumor control may be used to refer to a degree of tumor regression achieved in response to treatment, where, for example, the tumor is regressed by a defined fraction (such as ⁇ 25%) of an endpoint tumor volume.
  • effective tumor control is achieved if the tumor is reduced to less than 500 mm 3 assuming the threshold of ⁇ 25%. Therefore, effective tumor control encompasses complete regression.
  • effective tumor control can be measured by objective response, which includes partial response (PR) and complete response (CR) as determined by art-recognized criteria, such as RECIST v1.1 and corresponding iRECIST (iRECIST v1.1).
  • effective tumor control in clinical settings also includes stable disease, where tumors that are typically expected to grow at certain rates are prevented from such growth by the treatment, even though shrinkage is not achieved.
  • Effector T cells are T lymphocytes that actively respond immediately to a stimulus, such as co-stimulation and include, but are not limited to, CD4+ T cells (also referred to as T helper or Th cells) and CD8+ T cells (also referred to as cytotoxic T cells). Th cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surfaces. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including Th1, Th2, Th3, Th17, Th9, or TFh, which secrete different cytokines to facilitate different types of immune responses. Signaling from the APC directs T cells into particular subtypes. Cytotoxic (Killer). Cytotoxic T cells (TC cells, CTLs, T-killer cells, killer T cells), on the other hand, destroy virus-infected cells and cancer cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein at their surfaces.
  • Cytotoxic effector cell e.g., CD8+ cells
  • endpoints represent the measures of predetermined parameters indicative of treatment effects.
  • suitable endpoints may include overall survival, disease-free survival (DFS), event-free survival (EFS), progression-free survival (PFS), objective response rate (ORR), complete response (CR), partial response (PR), time to progression (TTP), as well as patient-reported outcomes (e.g., symptom assessment) and biomarker assessment such as blood or body fluid-based assessments.
  • DFS disease-free survival
  • EFS event-free survival
  • PFS progression-free survival
  • ORR objective response rate
  • CR complete response
  • PR partial response
  • TTP time to progression
  • patient-reported outcomes e.g., symptom assessment
  • biomarker assessment such as blood or body fluid-based assessments.
  • epithelial hyperplasia refers to the undesired toxicity resulting from TGF ⁇ inhibition which may include, but is not limited to, abnormal growth of epithelial cells in the oral cavity, esophagus, breast, and ovary.
  • Epitope may be also referred to as an antigenic determinant, is a molecular determinant (e.g., polypeptide determinant) that can be specifically bound by a binding agent, immunoglobulin, or T-cell receptor.
  • Epitope determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three- dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope recognized by an antibody or an antigen-binding fragment of an antibody is a structural element of an antigen that interacts with CDRs (e.g., the complementary site) of the antibody or the fragment.
  • An epitope may be formed by contributions from several amino acid residues, which interact with the CDRs of the antibody to produce specificity.
  • An antigenic fragment can contain more than one epitope.
  • an antibody may specifically bind an antigen when it recognizes its target antigen in a complex mixture of proteins and/or macromolecules. For example, antibodies are said to “bind to the same epitope” if the antibodies cross-compete (one prevents the binding or modulating effect of the other).
  • Extended Latency Lasso refers to a portion of the prodomain that comprises Latency Lasso and Alpha-2 Helix, e.g., LASPPSQGEVPPGPLPEAVLALYNSTR (SEQ ID NO: 127).
  • Extended Latency Lasso further comprises a portion of Alpha-1 Helix, e.g., LVKRKRIEA (SEQ ID NO: 132) or a portion thereof.
  • Fibrosis The term “fibrosis” or “fibrotic condition/disorder” refers to the process or manifestation characterized by the pathological accumulation of extracellular matrix (ECM) components, such as collagens, within a tissue or organ.
  • ECM extracellular matrix
  • Finger-1 (of TGF ⁇ 1 Growth Factor): As used herein, “Finger-1” is a domain within the TGF ⁇ 1 growth factor domain.
  • Finger-1 of human proTGF ⁇ 1 contains the following amino acid sequence: CVRQLYIDFRKDLGWKWIHEPKGYHANFC (SEQ ID NO: 124). In the 3D structure, the Finger-1 domain comes in close proximity to Latency Lasso.
  • Finger-2 (of TGF ⁇ 1 Growth Factor): As used herein, “Finger-2” is a domain within the TGF ⁇ 1 growth factor domain. In its unmutated form, Finger-2 of human proTGF ⁇ 1 contains the following amino acid sequence: CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 125). Finger-2 includes the “binding region 6”, which spatially lies in close proximity to Latency Lasso.
  • GARP-proTGF ⁇ 1 complex refers to a protein complex comprising a pro-protein form or latent form of a transforming growth factor- ⁇ 1 (TGF ⁇ 1) protein and a glycoprotein- A repetitions predominant protein (GARP) or fragment or variant thereof.
  • TGF ⁇ 1 transforming growth factor- ⁇ 1
  • GARP glycoprotein- A repetitions predominant protein
  • a pro-protein form or latent form of TGF ⁇ 1 protein may be referred to as “pro/latent TGF ⁇ 1 protein”.
  • a GARP- TGF ⁇ 1 complex comprises GARP covalently linked with pro/latent TGF ⁇ 1 via one or more disulfide bonds.
  • a GARP-TGF ⁇ 1 complex comprises GARP non- covalently linked with pro/latent TGF ⁇ 1.
  • a GARP-TGF ⁇ 1 complex is a naturally-occurring complex, for example a GARP-TGF ⁇ 1 complex in a cell.
  • the term “hGARP” denotes human GARP.
  • High-affinity As used herein, the term “high-affinity” as in “a high-affinity proTGF ⁇ 1 antibody” refers to in vitro binding activities having a KD value of ⁇ 5 nM, more preferably ⁇ 1 nM.
  • a high-affinity, context- independent proTGF ⁇ 1 antibody encompassed by the disclosure herein has a K D value of ⁇ 5 nM, more preferably ⁇ 1 nM, towards each of the following antigen complexes: LTBP1-proTGF ⁇ 1, LTBP3-proTGF ⁇ 1, GARP-proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1.
  • Human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term "human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • Humanized antibody refers to antibodies, which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like,” i.e., more similar to human germline variable sequences.
  • a non-human species e.g., a mouse
  • One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences.
  • humanized antibody is an antibody, or a variant, derivative, analog or fragment thereof, which immunospecifically binds to an antigen of interest and which comprises an FR region having substantially the amino acid sequence of a human antibody and a CDR region having substantially the amino acid sequence of a non-human antibody.
  • substantially in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR.
  • a humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody also comprises at least a portion of an immunoglobulin Fc region, typically that of a human immunoglobulin.
  • a humanized antibody contains the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • a humanized antibody only contains a humanized light chain. In some embodiments a humanized antibody only contains a humanized heavy chain. In specific embodiments a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.
  • Immune-excluded or immuno-excluded tumor As used herein, tumors characterized as “immune excluded” are devoid of or substantially devoid of intratumoral anti-tumor lymphocytes.
  • tumors with poorly infiltrated T cells may have T cells that surround the tumor, e.g., the external perimeters of a tumor mass and/or near the vicinity of vasculatures (“perivascular”) of a tumor, which nevertheless fail to effectively swarm into the tumor to exert cytotoxic function against cancer cells.
  • tumors fail to provoke a strong immune response (so-called “immune desert” tumors) such that few T cells are present near and in the tumor environment.
  • tumors that are infiltrated with anti-tumor lymphocytes are sometimes characterized as “hot” or “inflamed” tumors; such tumors tend to be more responsive to and therefore are the target of immune checkpoint blockade therapies (“CBTs”).
  • CBTs immune checkpoint blockade therapies
  • Immune safety As used herein, the term refers to safety assessment related to immune responses (immune activation), Acceptable immune safety criteria include no significant cytokine release as determined by in vitro or in vivo cytokine release testing (e.g., assays); and no significant platelet aggregation, activation as determined with human platelets. Statistical significance in these studies may be determined against a suitable control as reference. For example, for a test molecule which is a human monoclonal antibody, a suitable control may be an immunoglobulin of the same subtype, e.g., an antibody of the same subtype known to have a good safety profile in a human.
  • Immunosuppression, immune suppression, immunosuppressive The terms refer to the ability to suppress immune cells, such as T cells, NK cells and B cells.
  • the gold standard for evaluating immunosuppressive function is the inhibition of T cell activity, which may include antigen-specific suppression and non-specific suppression.
  • Regulatory T cells (Tregs) and MDSCs may be considered immunosuppressive cells.
  • M2-polarized macrophages e.g., disease-localized macrophages such as TAMs and FAMs
  • Immunological memory Immunological memory refers to the ability of the immune system to quickly and specifically recognize an antigen that the body has previously encountered and initiate a corresponding immune response.
  • Immunological memory is responsible for the adaptive component of the immune system, special T and B cells — the so-called memory T and B cells.
  • Antigen-na ⁇ ve T cells expand and differentiate into memory and effector T cells after they encounter their cognate antigen within the context of an MHC molecule on the surface of a professional antigen presenting cell (e.g., a dendritic cell).
  • a professional antigen presenting cell e.g., a dendritic cell.
  • the single unifying theme for all memory T cell subtypes is that they are long-lived and can quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen. By this mechanism they provide the immune system with "memory" against previously encountered pathogens.
  • Memory T cells may be either CD4+ or CD8+ and usually express CD45RO. In a preclinical setting, immunological memory may be tested in a tumor rechallenge paradigm.
  • Inhibit or inhibition of means to reduce by a measurable amount, and can include but does not require complete prevention or inhibition.
  • Isoform-non-specific The term “isoform non-specific” refers to an agent’s ability to bind to more than one structurally related isoforms.
  • An isoform-non-specific TGF ⁇ inhibitor exerts its inhibitory activity toward more than one isoform of TGF ⁇ , such as TGF ⁇ 1/3, TGF ⁇ 1/2, TGF ⁇ 2/3, and TGF ⁇ 1/2/3.
  • Isoform-specific The term “isoform specificity” refers to an agent’s ability to discriminate one isoform over other structurally related isoforms (i.e., isoform selectivity).
  • An isoform-specific TGF ⁇ inhibitor exerts its inhibitory activity towards one isoform of TGF ⁇ but not the other isoforms of TGF ⁇ at a given concentration.
  • an isoform-specific TGF ⁇ 1 antibody selectively binds TGF ⁇ 1.
  • a TGF ⁇ 1-specific inhibitor preferentially targets (binds thereby inhibits) the TGF ⁇ 1 isoform over TGF ⁇ 2 or TGF ⁇ 3 with substantially greater affinity.
  • the selectivity in this context may refer to at least a 10-fold, 100-fold, 500-fold, 1000-fold, or greater difference in respective affinities as measured by an in vitro binding assay such as BLI (Octet®) or preferably SPR (Biacore®).
  • the selectivity is such that the inhibitor when used at a dosage effective to inhibit TGF ⁇ 1 in vivo does not inhibit TGF ⁇ 2 and TGF ⁇ 3.
  • a TGF ⁇ 1-selective inhibitor is a pharmacological agent that interferes with the function or activities of TGF ⁇ 1, but not of TGF ⁇ 2 and/or TGF ⁇ 3, irrespective of the mechanism of action.
  • Isolated An “isolated” antibody as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities. In some embodiments, an isolated antibody is substantially free of other unintended cellular material and/or chemicals.
  • LLC large latent complex
  • a large latent complex is a presenting molecule-proTGF ⁇ 1 complex, such as LTBP1-proTGF ⁇ 1, LTBP3-proTGF ⁇ 1, GARP- proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1.
  • Such complexes may be formed in vitro using recombinant, purified components capable of forming the complex.
  • LAP Latency associated peptide
  • Latency Lasso As used herein, “Latency Lasso,” sometimes also referred to as Latency Loop, is a domain flanked by Alpha-1 Helix and the Arm within the prodomain of proTGFb1. In its unmutated form, Latency Lasso of human proTGF ⁇ 1 comprises the amino acid sequence: LASPPSQGEVPPGPL (SEQ ID NO: 126) which is spanned by Region 1 identified in FIG.41. As used herein, the term Extended Latency Lasso region” refers to the Latency Lasso together with its immediate C-terminal motif referred to as Alpha-2 Helix ( ⁇ 2-Helix) of the prodomain.
  • the proline residue that is at the C-terminus of the Latency Lasso provides the perpendicular “turn” like an “elbow” that connects the lasso loop to the ⁇ 2-Helix.
  • Certain high affinity TGF ⁇ 1 activation inhibitors bind at least in part to Latency Lasso or a portion thereof to confer the inhibitory potency (e.g., the ability to block activation), wherein optionally the portion of the Latency Lasso is ASPPSQGEVPPGPL (SEQ ID NO: 170).
  • the antibodies of the present disclosure bind a proTGF ⁇ 1 complex at ASPPSQGEVPPGPL (SEQ ID NO: 170) or a portion thereof.
  • Certain high affinity TGF ⁇ 1 activation inhibitors bind at least in part to Extended Latency Lasso or a portion thereof to confer the inhibitory potency (e.g., the ability to block activation), wherein optionally the portion of the Extended Latency Lasso is KLRLASPPSQGEVPPGPLPEAVL (SEQ ID NO: 142).
  • the term “localized” refers to anatomically isolated or isolatable abnormalities, such as solid malignancies, as opposed to systemic disease.
  • LRRC33-proTGF ⁇ 1 complex refers to a complex between a pro-protein form or latent form of transforming growth factor- ⁇ 1 (TGF ⁇ 1) protein and a Leucine-Rich Repeat-Containing Protein 33 (LRRC33; also known as Negative Regulator of Reactive Oxygen Species or NRROS) or fragment or variant thereof.
  • a LRRC33-TGF ⁇ 1 complex comprises LRRC33 covalently linked with pro/latent TGF ⁇ 1 via one or more disulfide bonds. In nature, such covalent bonds are formed with cysteine residues present near the N-terminus (e.g., amino acid position 4) of a proTGF ⁇ 1 dimer complex.
  • a LRRC33-TGF ⁇ 1 complex comprises LRRC33 non-covalently linked with pro/latent TGF ⁇ 1.
  • a LRRC33-TGF ⁇ 1 complex is a naturally-occurring complex, for example a LRRC33-TGF ⁇ 1 complex in a cell.
  • the term “hLRRC33” denotes human LRRC33.
  • LRRC33 and LRRC33-containing complexes on cell surface may be internalized.
  • LRRC33 is expressed on a subset of myeloid cells, including M2- polarized macrophages (such as TAMs) and MDSCs.
  • MDSCs that express LRRC33 on cell surface include tumor- associated MDSCs and circulatory MDSCs.
  • LRRC33-expressing tumor-associated MDSCs may include gMDSCs.
  • LRRC33-expressing MDSCs in circulation may include g-MDSCs.
  • LTBP1-proTGF ⁇ 1 complex refers to a protein complex comprising a pro-protein form or latent form of transforming growth factor- ⁇ 1 (TGF ⁇ 1) protein and a latent TGF- beta binding protein 1 (LTBP1) or fragment or variant thereof.
  • a LTBP1-TGF ⁇ 1 complex comprises LTBP1 covalently linked with pro/latent TGF ⁇ 1 via one or more disulfide bonds. In nature, such covalent bonds are formed with cysteine residues present near the N-terminus (e.g., amino acid position 4) of a proTGF ⁇ 1 dimer complex.
  • a LTBP1-TGF ⁇ 1 complex comprises LTBP1 non-covalently linked with pro/latent TGF ⁇ 1.
  • a LTBP1-TGF ⁇ 1 complex is a naturally-occurring complex, for example a LTBP1-TGF ⁇ 1 complex in a cell.
  • the term “hLTBP1” denotes human LTBP1.
  • LTBP3-proTGF ⁇ 1 complex refers to a protein complex comprising a pro-protein form or latent form of transforming growth factor- ⁇ 1 (TGF ⁇ 1) protein and a latent TGF- beta binding protein 3 (LTBP3) or fragment or variant thereof.
  • a LTBP3-TGF ⁇ 1 complex comprises LTBP3 covalently linked with pro/latent TGF ⁇ 1 via one or more disulfide bonds. In nature, such covalent bonds are formed with cysteine residues present near the N-terminus (e.g., amino acid position 4) of a proTGF ⁇ 1 dimer complex.
  • a LTBP3-TGF ⁇ 1 complex comprises LTBP1 non-covalently linked with pro/latent TGF ⁇ 1.
  • a LTBP3-TGF ⁇ 1 complex is a naturally-occurring complex, for example a LTBP3-TGF ⁇ 1 complex in a cell.
  • the term “hLTBP3” denotes human LTBP3.
  • M2 macrophages represent a subset of activated or polarized macrophages and include disease-associated macrophages in both fibrotic and tumor microenvironments.
  • Cell-surface markers for M2-polarized macrophages typically include CD206 and CD163 (i.e., CD206+/CD163+).
  • M2-polarized macrophages may also express cell-surface LRRC33. Activation of M2 macrophages is promoted mainly by IL-4, IL-13, IL-10 and TGF ⁇ ; they secrete the same cytokines that activate them (IL-4, IL-13, IL-10 and TGF ⁇ ).
  • TGF ⁇ extracellular matrix
  • LTBP1-proTGF ⁇ 1 and LTBP3-proTGF ⁇ 1 may be collectively referred to as “ECM- associated” (or “matrix-associated”) proTGF ⁇ 1 complexes, that mediate ECM-associated TGF ⁇ 1 activation/signaling.
  • ECM-associated or “matrix-associated” proTGF ⁇ 1 complexes, that mediate ECM-associated TGF ⁇ 1 activation/signaling.
  • the term also includes recombinant, purified LTBP1-proTGF ⁇ 1 and LTBP3-proTGF ⁇ 1 complexes in solution (e.g., in vitro assays) which are not physically attached to a matrix or substrate.
  • MTD Maximally tolerated dose
  • the term MTD generally refers to, in the context of safety/toxicology considerations, the highest amount of a test article (such as a TGF ⁇ 1 inhibitor) evaluated with no-observed- adverse-effect level (NOAEL).
  • NOAEL no-observed- adverse-effect level
  • the NOAEL for Ab6 in rats was the highest dose evaluated (100 mg/kg), suggesting that the MTD for Ab6 is >100 mg/kg, based on a four-week toxicology study.
  • the NOAEL for Ab6 in non-human primates was the highest dose evaluated (300 mg/kg), suggesting that the MTD for Ab6 in the non-human primates is >300 mg/kg, based on a four-week toxicology study.
  • MSD Meso-Scale Discovery
  • ECL electrochemiluminescence
  • high binding carbon electrodes are used to capture proteins (e.g., antibodies).
  • the antibodies can be incubated with particular antigens, which binding can be detected with secondary antibodies that are conjugated to electrochemiluminescent labels.
  • light intensity can be measured to quantify analytes in the sample.
  • Myelofibrosis also known as osteomyelofibrosis, is a relatively rare bone marrow proliferative disorder (e.g., cancer), Myelofibrosis is generally characterized by the proliferation of an abnormal clone of hematopoietic stem cells in the bone marrow and other sites results in fibrosis, or the replacement of the marrow with scar tissue.
  • myelofibrosis encompasses primary myelofibrosis (PMF), also be referred to as chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary mean that in these cases the disease is of unknown or spontaneous origin), as well as secondary types of myelofibrosis, such as myelofibrosis that develops secondary to polycythemia vera (PV) or essential thrombocythaemia (ET).
  • PMF primary myelofibrosis
  • cIMF chronic idiopathic myelofibrosis
  • PV polycythemia vera
  • ET essential thrombocythaemia
  • Myelofibrosis is a form of myeloid metaplasia, which refers to a change in cell type in the blood-forming tissue of the bone marrow, and often the two terms are used synonymously.
  • myelofibrosis is characterized by mutations that cause upregulation or overactivation of the downstream JAK pathway.
  • Myeloid cells In hematopoiesis, myeloid cells are blood cells that arise from a progenitor cell for granulocytes, monocytes, erythrocytes, or platelets (the common myeloid progenitor, that is, CMP or CFU-GEMM), or in a narrower sense also often used, specifically from the lineage of the myeloblast (the myelocytes, monocytes, and their daughter types), as distinguished from lymphoid cells, that is, lymphocytes, which come from common lymphoid progenitor cells that give rise to B cells and T cells.
  • a progenitor cell for granulocytes, monocytes, erythrocytes, or platelets
  • lymphoid cells that is, lymphocytes, which come from common lymphoid progenitor cells that give rise to B cells and T cells.
  • a human neutrophil can be identified by at least one (e.g., all) of the cell surface markers CD11b + , CD14-, CD15 + , and CD66b + .
  • a human neutrophil is LOX-1-.
  • a human neutrophil is HLA-DR -/med .
  • a classical human monocyte can be identified by at least one (e.g., all) of the cell surface markers CD14 + CD15 ⁇ CD16 ⁇ HLA-DR + .
  • a classical human monocyte is CD33 + and/or CD11b + .
  • a classical human monocyte is CD16-.
  • an intermediate human monocyte can be identified by at least one (e.g., all) of the cell surface markers CD14 + CD15 ⁇ CD16 + HLA-DR + .
  • a non-classical human monocyte can be identified by at least one (e.g., all) of the cell surface markers CD14 ⁇ CD15 ⁇ CD16 + HLA-DR + .
  • a human M1 macrophage can be identified by at least one (e.g., all) of the cell surface markers CD15 ⁇ CD16 + CD80 + HLA-DR +/high CD33 + .
  • a human M1 macrophage is CD66b-.
  • a human M1 macrophage is CD11b + .
  • a human M1 macrophage is CD14-.
  • a human M2 macrophage can be identified by at least one (e.g., all) of the cell surface markers CD11b + and CD15 ⁇ .
  • a human M2 macrophage is CD206 + .
  • a human M2 macrophage is CD163 + . In some embodiments, a human M2 macrophage is HLA-DR + . In some embodiments, a human M2 macrophage is CD14-. In some embodiments, a human M2 macrophage is CD33 + . In some embodiments, a human M2 macrophage is CD66b-.
  • MDSCs Myeloid-derived suppressor cells
  • MDSCs include at least two categories of cells termed i) “granulocytic” (G-MDSC) or polymorphonuclear (PMN-MDSC), which are phenotypically and morphologically similar to neutrophils; and ii) monocytic (M-MDSC) which are phenotypically and morphologically similar to monocytes.
  • G-MDSC granulocytic
  • PMN-MDSC polymorphonuclear
  • M-MDSC monocytic
  • suitable cell surface markers for identifying MDSCs may include one or more of CD11b, CD33, CD14, CD15, HLA-DR and CD66b.
  • human G- MDSCs/PMN-MDSCs typically express the cell-surface markers CD11b, CD33, CD15 and CD66b.
  • human G-MDSCs may express low levels of the CD33 cell surface marker.
  • human G-MDSCs/PMN-MDSCs may express LOX-1 and/or Arginase.
  • human M-MDSCs typically express the cell surface markers CD11b, CD33 and CD14.
  • both human G-MDSCs/PMN-MDSCs and M-MDSCs may also exhibit low levels or undetectable levels of HLA-DR.
  • human G-MDSCs may be HLA-DR-.
  • G-MDSCs may be differentiated from M-MDSCs based on the presence or absence of certain cell surface marker (e.g., CD14, CD15, and/or CD66b).
  • G-MDSCs may be identified by the presence or elevated expression of surface markers CD11b, CD33, CD15, CD66b, and/or LOX-1, and the absence of CD14, whereas M-MDSCs may be identified by the presence or elevated expression of surface markers CD11b, CD33, and/or CD14, and the absence of CD15.
  • M-MDSCs may be CD66b-.
  • MDSCs may be characterized by the ability to suppress immune cells, such as T cells, NK cells and B cells. Immune suppressive functions of MDSCs may include inhibition of antigen-non-specific function and inhibition of antigen-specific function.
  • MDSCs including tumor-associated MDSCs and MDSCs in circulation, can express cell surface LRRC33 and/or LRRC33-proTGF ⁇ 1.
  • a signal intensity of a cell surface marker may be categorized, or binned, as “low”, “medium”, or “high” based on normalization of signal intensity to reduce background and bleed through signals.
  • a signal intensity of a cell surface marker may be categorized based on cutoff thresholds provided in Table 38A.
  • a signal intensity of a cell surface marker may be determined by binary intensity selection.
  • the binary intensity selection comprises categorizing a signal intensity measured for a particular cell surface marker as “positive” or “negative.”
  • a signal intensity of a cell surface marker may be categorized based on the cutoff thresholds provided in Table 38B.
  • signal intensities of a set of surface markers may be determined by sequential application of signal filtering, where the signal intensity threshold for one or more surface markers is determined before the threshold is determined for one or more additional surface markers.
  • Myofibroblasts are cells with certain phenotypes of fibroblasts and smooth muscle cells and generally express vimentin, alpha-smooth muscle actin ( ⁇ -SMA; human gene ACTA2) and paladin.
  • ⁇ -SMA alpha-smooth muscle actin
  • Pan-TGF ⁇ inhibitor/pan-inhibition of TGF ⁇ refers to any agent that is capable of inhibiting or antagonizing all three isoforms of TGF ⁇ . Such an inhibitor may be a small molecule inhibitor of TGF ⁇ isoforms, such as those known in the art.
  • pan-TGF ⁇ antibody which refers to any antibody capable of binding to each of TGF ⁇ isoforms, i.e., TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3.
  • a pan-TGF ⁇ antibody binds and neutralizes activities of all three isoforms, i.e., TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3.
  • the antibody 1D11 (or the human analog fresolimumab (GC1008)) is a well-known example of a pan-TGF ⁇ antibody that neutralizes all three isoforms of TGF ⁇ .
  • pan-TGF ⁇ inhibitors examples include galunisertib (LY2157299 monohydrate), which is an antagonist for the TGF ⁇ receptor I kinase/ALK5 that mediates signaling of all three TGF ⁇ isoforms.
  • galunisertib LY2157299 monohydrate
  • perivascular literally translates to around the blood vessels.
  • the term “perivascular infiltration” refers to a mode of entry for tumor-infiltrating immune cells (e.g., lymphocytes) via the vasculature of a solid tumor.
  • Potency refers to activity of a drug, such as an inhibitory antibody (or fragment) having inhibitory activity, with respect to concentration or amount of the drug to produce a defined effect.
  • a drug such as an inhibitory antibody (or fragment) having inhibitory activity
  • concentration or amount of the drug to produce a defined effect For example, an antibody capable of producing certain effects at a given dosage is more potent than another antibody that requires twice the amount (dosage) to produce equivalent effects.
  • Potency may be measured in cell- based assays, such as TGF ⁇ activation/inhibition assays, whereby the degree of TGF ⁇ activation, such as activation triggered by integrin binding, can be measured in the presence or absence of test article (e.g., inhibitory antibodies) in a cell-based system.
  • Preclinical model refers to a cell line or an animal that exhibits certain characteristics of a human disease which is used to study the mechanism of action, efficacy, pharmacology, and toxicology of a drug, procedure, or treatment before it is tested on humans.
  • cell-based preclinical studies are referred to as “in vitro” studies, whereas animal-based preclinical studies are referred to as “in vivo” studies.
  • Predictive biomarkers provide information on the probability or likelihood of response to a particular therapy. Typically, a predictive biomarker is measured before and after treatment, and the changes or relative levels of the marker in samples collected from the subject indicates or predicts therapeutic benefit.
  • Presenting molecules in the context of the present disclosure refer to proteins that form covalent bonds with latent pro-proteins (e.g., proTGF ⁇ 1) and tether (“present”) the inactive complex to an extracellular niche (such as ECM or immune cell surface) thereby maintaining its latency until an activation event occurs.
  • Known presenting molecules for proTGF ⁇ 1 include: LTBP1, LTBP3, GARP and LRRC33, each of which can form a presenting molecule-proTGF ⁇ 1 complex (i.e., LLC), namely, LTBP1-proTGF ⁇ 1, LTBP3-proTGF ⁇ 1, GARP-proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1, respectively.
  • LTBP1 and LTBP3 are components of the extracellular matrix (ECM); therefore, LTBP1-proTGF ⁇ 1 and LTBP3-proTGF ⁇ 1 may be collectively referred to as “ECM-associated” (or “matrix-associated”) proTGF ⁇ 1 complexes, that mediate ECM-associated TGF ⁇ 1 signaling/activities.
  • ECM-associated or “matrix-associated” proTGF ⁇ 1 complexes, that mediate ECM-associated TGF ⁇ 1 signaling/activities.
  • GARP and LRRC33 are transmembrane proteins expressed on cell surface of certain cells; therefore, GARP-proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1 may be collectively referred to as “cell- associated” (or “cell-surface”) proTGF ⁇ 1 complexes, that mediate cell-associated (e.g., immune cell-associated) TGF ⁇ 1 signaling/activities.
  • cell-associated e.g., immune cell-associated
  • ProTGF ⁇ 1 The term “proTGF ⁇ 1” as used herein is intended to encompass precursor forms of inactive TGF ⁇ 1 complex that comprises a prodomain sequence of TGF ⁇ 1 within the complex. Thus, the term can include the pro-, as well as the latent- forms of TGF ⁇ 1.
  • pro/latent TGF ⁇ 1 may be used interchangeably.
  • the “pro” form of TGF ⁇ 1 exists prior to proteolytic cleavage at the furin site. Once cleaved, the resulting form is said to be the “latent” form of TGF ⁇ 1.
  • the “latent” complex remains non-covalently associated until further activation trigger, such as integrin-driven activation event.
  • the proTGF ⁇ 1 complex is comprised of dimeric TGF ⁇ 1 pro-protein polypeptides, linked with disulfide bonds.
  • the latent dimer complex is covalently linked to a single presenting molecule via the cysteine residue at position 4 (Cys4) of each of the proTGF ⁇ 1 polypeptides.
  • the adjective “latent” may be used generally/broadly to describe the “inactive” state of TGF ⁇ 1, prior to integrin-mediated or other activation events.
  • the proTGF ⁇ 1 polypeptide contains a prodomain (LAP) and a growth factor domain (SEQ ID NO: 119).
  • Regression tumor regression: Regression of tumor or tumor growth can be used as an in vivo efficacy measure. For example, in preclinical settings, median tumor volume (MTV) and Criteria for Regression Responses Treatment efficacy may be determined from the tumor volumes of animals remaining in the study on the last day. Treatment efficacy may also be determined from the incidence and magnitude of regression responses observed during the study.
  • a PR response is defined as the tumor volume that is 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm 3 for one or more of these three measurements.
  • a CR response is defined as the tumor volume that is less than 13.5 mm 3 for three consecutive measurements during the course of the study.
  • an animal with a CR response at the termination of a study may be additionally classified as a tumor-free survivor (TFS).
  • TFS tumor-free survivor
  • the term “effective tumor control” may be used to refer to a degree of tumor regression achieved in response to treatment, where, for example, the tumor volume is reduced to ⁇ 25% of the endpoint tumor volume in response to treatment. For instance, in a particular model, if the endpoint tumor volume is 2,000 mm 3 , effective tumor control is achieved if the tumor is reduced to less than 500 mm 3 . Therefore, effective tumor control encompasses complete regression, as well as partial regression that reaches the threshold reduction.
  • Resistance to a particular therapy may be due to the innate characteristics of the disease such as cancer (“primary resistance”, i.e., present before treatment initiation), or due to acquired phenotypes that develop over time following the treatment (“acquired resistance”).
  • Primary resistance i.e., present before treatment initiation
  • Patients who do not show therapeutic response to a therapy e.g., those who are non-responders or poorly responsive to the therapy
  • Patients who have never previously received a treatment and do not show a therapeutic response to the treatment are said to have primary resistance.
  • Patients who initially show therapeutic response to a therapy but later lose effects e.g., progression or recurrence despite continued therapy) are said to have acquired resistance to the therapy.
  • RECIST Response Evaluation Criteria in Solid Tumors
  • iRECIST RECIST is a set of published rules that define when tumors in cancer patients improve ("respond"), stay the same (“stabilize”), or worsen ("progress") during treatment. The criteria were published in February 2000 by an international collaboration including the European Organisation for Research and Treatment of Cancer (EORTC), National Cancer Institute of the United States, and the National Cancer Institute of Canada Clinical Trials Group.
  • Response criteria are as follows: Complete response (CR): Disappearance of all target lesions; Partial response (PR): At least a 30% decrease in the sum of the LD of target lesions, taking as reference the baseline sum LD; Stable disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment started; Progressive disease (PD): At least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions.
  • CR Complete response
  • PR Partial response
  • SD Stable disease
  • PD Progressive disease
  • iRECIST provides a modified set of criteria that takes into account immune-related response (see: www.ncbi.nlm.nih.gov/pmc/articles/PMC5648544/ contents of which are incorporated herein by reference).
  • the RECIST and iRECIST criteria are standardized, may be revised from time to time as more data become available, and are well understood in the art.
  • Response rate The term response rate (as in “low response rates”) as used herein carries the ordinary meaning as understood by the skilled person in medicine, such as oncologists.
  • a response rate is the proportion (e.g., fraction or percentage) of subjects in a patient population who shows clinical improvement upon receiving a treatment (e.g., pharmacological intervention) and may include complete response and partial response.
  • clinical improvement may include tumor shrinkage (e.g., partial response) or disappearance (e.g., complete response).
  • ORR objective response rate
  • the FDA defines ORR as the proportion of patietns with tumor size reduction of a predefined amount and for a minimum time period. See: “Clinical Trial Endpoints for the Approval of Cander Drugs and Biologics – Guidance for Industry” published by U.S.
  • Solid tumor refers to proliferative disorders resulting in an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (non-cancerous), or malignant (cancerous). Solid tumors include tumors of advanced malignancies, such as locally advanced solid tumors and metastatic cancer.
  • Solid tumors are typically comprised of multiple cell types, including, without limitation, cancerous (malignant) cells, stromal cells such as CAFs, and infiltrating leukocytes, such as macrophages, MDSCs and lymphocytes.
  • Solid tumors to be treated with an isoform-selective inhibitor of TGF ⁇ 1, such as those described herein, are typically TGF ⁇ 1-positive (TGF ⁇ 1+) tumors, which may include multiple cell types that produce TGF ⁇ 1.
  • the TGF ⁇ 1+ tumor may also co-express TGF ⁇ 3 (i.e., TGF ⁇ 3- positive).
  • certain tumors are TGF ⁇ 1/3-co-dominant.
  • such tumors are caused by cancer of epithelial cells, e.g., carcinoma.
  • such tumors include ovarian cancer, breast cancer, bladder cancer, pancreatic cancer, e.g., pancreatic adenocarcinoma, prostate cancer, e.g., prostate adenocarcinoma, melanoma, e.g., skin cutaneous melanoma, lung cancer, e.g., lung squamous cell carcinoma and lung adenocarcinoma, liver cancer (e.g., liver hepatocellular carcinoma), uterine cancer, e.g., uterine corpus endometrial carcinoma, kidney cancer, e.g., renal clear cell carcinoma, head and neck cancer, e.g., head and neck squamous cell carcinoma, colon cancer, e.g., colon adenocarcinoma, esophageal carcinoma, and tenosynovial giant cell tumor (TGCT).
  • pancreatic cancer
  • a solid tumor treated herein exhibits elevated TGF ⁇ 1 expression as compared to other tumor types and exhibits a reduced responsiveness to mainline therapy, e.g., genotoxic therapy.
  • TGF ⁇ inhibitors e.g., Ab6
  • one or more genotoxic therapies e.g., chemotherapy and/or radiation therapy, including radiotherapeutic agents
  • Specific binding means that an antibody, or antigen binding portion thereof, exhibits a particular affinity for a particular structure (e.g., an antigenic determinant or epitope) in an antigen (e.g., a KD measured by Biacore®).
  • an antibody, or antigen binding portion thereof specifically binds to a target, e.g., TGF ⁇ 1, if the antibody has a K D for the target of at least about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or less.
  • the term “specific binding to an epitope of proTGF ⁇ 1”, “specifically binds to an epitope of proTGF ⁇ 1”, “specific binding to proTGF ⁇ 1”, or “specifically binds to proTGF ⁇ 1” as used herein, refers to an antibody, or antigen binding portion thereof, that binds to proTGF ⁇ 1 and has a dissociation constant (K D ) of 1.0 x 10 -8 M or less, as determined by suitable in vitro binding assays, such as surface plasmon resonance and Biolayer Interferometry (BLI).
  • kinetic rate constants e.g., K D
  • K D are determined by surface plasmon resonance (e.g., a Biacore system).
  • an antibody, or antigen binding portion thereof can specifically bind to both human and a non-human (e.g., mouse) orthologues of proTGF ⁇ 1.
  • an antibody may also “selectively” (i.e., “preferentially”) bind a target antigen if it binds that target with a comparatively greater strength than the strength of binding shown to other antigens, e.g., a 10-fold, 100-fold, 1000-fold, or greater comparative affinity for a target antigen (e.g., TGF ⁇ 1) than for a non-target antigen (e.g., TGF ⁇ 2 and/or TGF ⁇ 3).
  • a target antigen e.g., TGF ⁇ 1
  • a non-target antigen e.g., TGF ⁇ 2 and/or TGF ⁇ 3
  • an isoform-selective inhibitor exhibits no detectable binding or potency towards other isoforms or counterparts.
  • an antibody that binds specifically to a set of antigens may have high affinity toward said antigens but may not distinguish said antigens from one another (i.e., the antibody is specific but not selective).
  • an antibody that binds to an antigen with a particularly high affinity as compared to other antigens may be considered selective for said antigen. For instance, an antibody that binds to antigen X with 1000- fold higher affinity as compared to antigen Y may be considered an antibody that is selective for antigen X over antigen Y.
  • an antibody that specifically binds an antigen with high affinity generally refers to a KD of 1.0 x 10 -8 M or less.
  • Subject in the context of therapeutic applications refers to an individual who receives or is in need of clinical care or intervention, such as treatment, diagnosis, etc. Suitable subjects include vertebrates, including but not limited to mammals (e.g., human and non-human mammals). Where the subject is a human subject, the term “patient” may be used interchangeably.
  • a patient population or “patient subpopulation” is used to refer to a group of individuals that falls within a set of criteria, such as clinical criteria (e.g., disease presentations, disease stages, susceptibility to certain conditions, responsiveness to therapy, etc.), medical history, health status, gender, age group, genetic criteria (e.g., carrier of certain mutation, polymorphism, gene duplications, DNA sequence repeats, etc.) and lifestyle factors (e.g., smoking, alcohol consumption, exercise, etc.).
  • clinical criteria e.g., disease presentations, disease stages, susceptibility to certain conditions, responsiveness to therapy, etc.
  • medical history e.g., medical history, health status, gender, age group
  • genetic criteria e.g., carrier of certain mutation, polymorphism, gene duplications, DNA sequence repeats, etc.
  • lifestyle factors e.g., smoking, alcohol consumption, exercise, etc.
  • TGF ⁇ 1-related indication is a TGF ⁇ 1-associated disorder and means any disease or disorder, and/or condition, in which at least part of the pathogenesis and/or progression is attributable to TGF ⁇ 1 signaling or dysregulation thereof. Certain TGF ⁇ 1-associated disorders are driven predominantly by the TGF ⁇ 1 isoform. Subjects having a TGF ⁇ 1-related indication may benefit from inhibition of the activity and/or levels TGF ⁇ 1. Certain TGF ⁇ 1-related indications are driven predominantly by the TGF ⁇ 1 isoform.
  • TGF ⁇ 1-related indications include, but are not limited to: fibrotic conditions (such as organ fibrosis, and fibrosis of tissues involving chronic inflammation), proliferative disorders (such as cancer, e.g., solid tumors and myelofibrosis), disease associated with ECM dysregulation (such as conditions involving matrix stiffening and remodeling), disease involving mesenchymal transition (e.g., EndMT and/or EMT), disease involving proteases, disease with aberrant gene expression of certain markers described herein. These disease categories are not intended to be mutually exclusive.
  • fibrotic conditions such as organ fibrosis, and fibrosis of tissues involving chronic inflammation
  • proliferative disorders such as cancer, e.g., solid tumors and myelofibrosis
  • ECM dysregulation such as conditions involving matrix stiffening and remodeling
  • mesenchymal transition e.g., EndMT and/or EMT
  • proteases disease with aberrant gene expression of certain markers described herein.
  • TGF ⁇ inhibitor refers to any agent capable of antagonizing biological activities, signaling or function of TGF ⁇ growth factor (e.g., TGF ⁇ 1, TGF ⁇ 2 and/or TGF ⁇ 3).
  • TGF ⁇ growth factor e.g., TGF ⁇ 1, TGF ⁇ 2 and/or TGF ⁇ 3
  • the term is not intended to limit its mechanism of action and includes, for example, neutralizing inhibitors, receptor antagonists, soluble ligand traps, TGF ⁇ activation inhibitors, and integrin inhibitors (e.g., antibodies that bind to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibit downstream activation of TGF ⁇ .
  • integrin inhibitors e.g., antibodies that bind to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or
  • TGF ⁇ inhibitors that are isoform-selective and non-selective inhibitors.
  • the latter include, for example, small molecule receptor kinase inhibitors (e.g., ALK5 inhibitors), antibodies (such as neutralizing antibodies) that preferentially bind two or more isoforms, and engineered constructs (e.g., fusion proteins) comprising a ligand-binding moiety.
  • these antibodies may include or may be engineered to include a mutation or modification that causes an extended half-life of the antibody.
  • such mutations or modifications may be within the Fc domain of the antibodies (e.g., Fc-modified antibodies).
  • the mutation is so-called YTE mutation.
  • TGF ⁇ inhibitors also include antibodies that are capable of reducing the availability of latent proTGF ⁇ which can be activated in the niche, for example, by inducing antibody-dependent cell mediated cytotoxicity (ADCC), and/or antibody-dependent cellular phagocytosis (ADPC), as well as antibodies that result in internalization of cell-surface complex comprising latent proTGF ⁇ , thereby removing the precursor from the plasma membrane without depleting the cells themselves.
  • ADCC antibody-dependent cell mediated cytotoxicity
  • ADPC antibody-dependent cellular phagocytosis
  • TGF ⁇ 1-positive cancer/tumor The term, as used herein, refers to a cancer/tumor with aberrant TGF ⁇ 1 expression (overexpression).
  • TGF ⁇ 1 tumor-associated macrophages
  • CAFs cancer-associated fibroblasts
  • Tregs regulatory T cells
  • MDSCs myeloid-derived suppressor cells
  • ECM extracellular matrix
  • Therapeutic window refers to a dosage range that produces therapeutic response without causing significant/observable/unacceptable adverse effect (e.g., within adverse effects that are acceptable or tolerable) in subjects. Therapeutic window may be calculated as a ratio between minimum effective concentrations (MEC) to the minimum toxic concentrations (MTC).
  • MEC minimum effective concentrations
  • MTC minimum toxic concentrations
  • a TGF ⁇ 1 inhibitor that achieves in vivo efficacy at 10 mg/kg dosage and shows tolerability or acceptable toxicities at 100 mg/kg provides at least a 10-fold (e.g., 10x) therapeutic window.
  • a pan-inhibitor of TGF ⁇ that is efficacious at 10 mg/kg but causes adverse effects at less than the effective dose is said to have “dose-limiting toxicities.”
  • the maximally tolerated dose may set the upper limit of the therapeutic window.
  • Ab6 was shown to be efficacious at dosage ranging between about 3-30 mg/kg/week and was also shown to be free of observable toxicities associated with pan-inhibition of TGF ⁇ at dosage of at least 100 or 300 mg/kg/week for 4 weeks in rats or non-human primates. Based on this, Ab6 shows at minimum a 3.3-fold and up to 100-fold therapeutic window.
  • the concept of therapeutic window may be expressed in terms of safety factors.
  • Toxicity refers to unwanted in vivo effects in subjects (e.g., patients) associated with a therapy administered to the subjects (e.g., patients), such as undesirable side effects and adverse events. “Tolerability” refers to a level of toxicities associated with a therapy or therapeutic regimen, which can be reasonably tolerated by patients, without discontinuing the therapy due to the toxicities. Typically, toxicity/toxicology studies are carried out in one or more preclinical models prior to clinical development to assess safety profiles of a drug candidate (e.g., monoclonal antibody therapy).
  • a drug candidate e.g., monoclonal antibody therapy
  • Toxicity/toxicology studies may help determine the “no-observed-adverse-effect level (NOAEL)” and the “maximally tolerated dose (MTD)” of a test article, based on which a therapeutic window may be deduced.
  • NOAEL no-observed-adverse-effect level
  • MTD maximum tolerated dose
  • a species that is shown to be sensitive to the particular intervention should be chosen as a preclinical animal model in which safety/toxicity study is to be carried out.
  • suitable species include rats, dogs, and cynos. Mice are reported to be less sensitive to pharmacological inhibition of TGF ⁇ and may not reveal toxicities that are potentially dangerous in other species, including human, although certain studies report toxicities observed with pan-inhibition of TGF ⁇ in mice.
  • the NOAEL for Ab6 in rats was the highest dose evaluated (100 mg/kg), suggesting that the MTD is >100 mg/kg, based on a four-week toxicology study.
  • the MTD of Ab6 in non-human primates is >300 mg/kg based on a four-week toxicology study.
  • a species that is shown to be sensitive to the particular intervention should be chosen as a preclinical animal model in which safety/toxicology study is to be carried out.
  • suitable species include, but are not limited to, rats, dogs, and cynos.
  • Treat/treatment includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor.
  • the term is intended to broadly mean: causing therapeutic benefits in a patient by, for example, enhancing or boosting the body’s immunity; reducing or reversing immune suppression; reducing, removing or eradicating harmful cells or substances from the body; reducing disease burden (e.g., tumor burden); preventing recurrence or relapse; prolonging a refractory period, and/or otherwise improving survival.
  • the term includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.
  • the term may also refer to: i) the ability of a second therapeutic to reduce the effective dosage of a first therapeutic so as to reduce side effects and increase tolerability; ii) the ability of a second therapy to render the patient more responsive to a first therapy; and/or iii) the ability to effectuate additive or synergistic clinical benefits.
  • TAMs are polarized/activated macrophages with pro-tumor phenotypes (M2-like macrophages).
  • TAMs can be either marrow-originated monocytes/macrophages recruited to the tumor site or tissue-resident macrophages which are derived from erythro-myeloid progenitors. Differentiation of monocytes/macrophages into TAMs is influenced by a number of factors, including local chemical signals such as cytokines, chemokines, growth factors and other molecules that act as ligands, as well as cell-cell interactions between the monocytes/macrophages that are present in the niche (tumor microenvironment).
  • monocytes/macrophages can be polarized into so-called “M1” or “M2” subtypes, the latter being associated with more pro-tumor phenotype.
  • M1 macrophages typically express cell surface HLA-DR, CD68 and CD86
  • M2 macrophages typically express cell surface HLA-DR, CD68, CD163 and CD206.
  • Tumor-associated, M2-like macrophages can express cell surface LRRC33 and/or LRRC33-proTGF ⁇ 1.
  • Tumor microenvironment refers to a local disease niche, in which a tumor (e.g., solid tumor) resides in vivo.
  • the TME may comprise disease-associated molecular signature (a set of chemokines, cytokines, etc.), disease-associated cell populations (such as TAMs, CAFs, MDSCs, etc.) as well as disease-associated ECM environments (alterations in ECM components and/or structure).
  • variable region refers to a portion of the light and/or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. In certain embodiments, variable regions of different antibodies differ extensively in amino acid sequence even among antibodies of the same species. The variable region of an antibody typically determines specificity of a particular antibody for its target. [183] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about.” The term “about” means ⁇ 10% of the recited value.
  • a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase “at least one,” in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, e.g., 10-20, 1-10, 30-40, etc.
  • TGF ⁇ Transforming Growth Factor-beta
  • TGF ⁇ superfamily can be categorized into at least three subclasses by structural similarities: TGF ⁇ s, Growth- Differentiation Factors (GDFs) and Bone-Morphogenetic Proteins (BMPs).
  • GDFs Growth- Differentiation Factors
  • BMPs Bone-Morphogenetic Proteins
  • the TGF ⁇ subclass is comprised of three highly conserved isoforms, namely, TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3, which are encoded by three separate genes in human.
  • the TGF ⁇ s are thought to play key roles in diverse processes, such as inhibition of cell proliferation, extracellular matrix (ECM) remodeling, and immune homeostasis.
  • ECM extracellular matrix
  • TGF ⁇ 1 for T cell homeostasis is demonstrated by the observation that TGF ⁇ 1-/- mice survive only 3-4 weeks, succumbing to multi-organ failure due to massive immune activation (Kulkarni, A.B., et al., Proc Natl Acad Sci U S A, 1993. 90(2): p. 770-4; Shull, M.M., et al., Nature, 1992.359(6397): p.693-9). The roles of TGF ⁇ 2 and TGF ⁇ 3 are less clear.
  • TGF ⁇ RI and TGF ⁇ RII Whilst the three TGF ⁇ isoforms have distinct temporal and spatial expression patterns, they signal through the same receptors, TGF ⁇ RI and TGF ⁇ RII, although in some cases, for example for TGF ⁇ 2 signaling, type III receptors such as betaglycan are also required (Feng, X.H. and R. Derynck, Annu Rev Cell Dev Biol, 2005.21: p.659-93; Massague, J., Annu Rev Biochem, 1998. 67: p. 753-91).
  • TGF ⁇ RI/II Ligand-induced oligomerization of TGF ⁇ RI/II triggers the phosphorylation of SMAD transcription factors, resulting in the transcription of target genes, such as Col1a1, Col3a1, ACTA2, and SERPINE1 (Massague, J., J. Seoane, and D. Wotton, Genes Dev, 2005. 19(23): p. 2783- 810).
  • target genes such as Col1a1, Col3a1, ACTA2, and SERPINE1 (Massague, J., J. Seoane, and D. Wotton, Genes Dev, 2005. 19(23): p. 2783- 810).
  • SMAD-independent TGF ⁇ signaling pathways have also been described, for example in cancer or in the aortic lesions of Marfan mice (Derynck, R. and Y.E.
  • TGF ⁇ signaling pathway dysregulation has been implicated in multiple diseases, several drugs that target the TGF ⁇ pathway have been developed and tested in patients, but with limited success.
  • Dysregulation of the TGF ⁇ signaling has been associated with a wide range of human diseases. Indeed, in a number of disease conditions, such dysregulation may involve multiple facets of TGF ⁇ function.
  • Diseased tissue such as fibrotic and/or inflamed tissues and tumors, may create a local environment in which TGF ⁇ activation can cause exacerbation or progression of the disease, which may be at least in part mediated by interactions between multiple TGF ⁇ -responsive cells, which are activated in an autocrine and/or paracrine fashion, together with a number of other cytokines, chemokines and growth factors that play a role in a particular disease setting.
  • a tumor microenvironment contains multiple cell types expressing TGF ⁇ 1, such as activated myofibroblast-like fibroblasts, stromal cells, infiltrating macrophages, MDSCs and other immune cells, in addition to cancer (i.e., malignant) cells.
  • TME represents a heterogeneous population of cells expressing and/or responsive to TGF ⁇ 1 but in association with more than one types of presenting molecules, e.g., LTBP1, LTBP3, LRRC33 and GARP, within the niche.
  • CBT checkpoint blockade therapies
  • tumor exclusion a phenomenon referred to as “immune exclusion” was coined to describe a tumor environment from which anti-tumor effector T cells (e.g., CD8+ T cells) are kept away (hence “excluded”) by immunosuppressive local cues.
  • TGF ⁇ pathway activation in mediating primary resistance to CBT.
  • transcriptional profiling and analysis of pretreatment melanoma biopsies revealed an enrichment of TGF ⁇ -associated pathways and biological processes in tumors that are non-responsive to anti-PD-1 CBT.
  • effector cells which would otherwise be capable of attacking cancer cells by recognizing cell-surface tumor antigens, are prevented from gaining access to the site of cancer cells.
  • cancer cells evade host immunity and immuno-oncologic therapeutics, such as checkpoint inhibitors, that exploit and rely on such immunity.
  • checkpoint inhibitors such as checkpoint inhibitors
  • Such tumors show resistance to checkpoint inhibition, such as anti-PD-1 and anti-PD-L1 antibodies, presumably because target T cells are blocked from entering the tumor hence failing to exert anti-cancer effects.
  • transcriptional profiling and analysis of pretreatment melanoma biopsies revealed an enrichment of TGF ⁇ -associated pathways and biological processes in tumors that are non- responsive to anti-PD-1 CBT.
  • similar analyses of tumors from metastatic urothelial cancer patients revealed that lack of response to PD-L1 blockade with atezolizumab was associated with transcriptional signatures of TGF ⁇ signaling, particularly in tumors wherein CD8+ T cells appear to be excluded from entry into the tumor.
  • the critical role of TGF ⁇ signaling in mediating immune exclusion resulting in anti-PD-(L)1 resistance has been verified in the EMT-6 syngeneic mouse model of breast cancer.
  • TGF ⁇ activates CAFs, inducing extracellular matrix production and promotion of tumor progression. Finally, TGF ⁇ induces EMT, thus supporting tissue invasion and tumor metastases.
  • Mammals have distinct genes that encode and express the three TGF ⁇ growth factors, TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3, all of which signal through the same heteromeric TGF ⁇ receptor complex. Despite the common signaling pathway, each TGF ⁇ isoform appears to have distinct biological functions, as evidenced by the non-overlapping TGF ⁇ knockout mouse phenotypes.
  • TGF ⁇ isoforms are expressed as inactive prodomain-growth factor complexes, in which the TGF ⁇ prodomain, also called latency-associated peptide (LAP), wraps around its growth factor and holds it in a latent, non-signaling state. Furthermore, latent TGF ⁇ is co-expressed with latent TGF ⁇ - binding proteins and forms large latent complexes (LLCs) through disulfide linkage. Association of latent TGF ⁇ with Latent TGF ⁇ Binding Protein-1 (LTBP1) or LTBP3 enables tethering to extracellular matrix, whereas association to the transmembrane proteins GARP or LRRC33 enables elaboration on the surface of Tregs or macrophages, respectively.
  • LTBP1 Latent TGF ⁇ Binding Protein-1
  • GARP or LRRC33 association to the transmembrane proteins
  • latent TGF ⁇ 1 and latent TGF ⁇ 3 are activated by a subset of ⁇ V integrins, which bind a consensus RGD sequence on LAP, triggering a conformational change to release the growth factor.
  • the mechanism by which latent TGF ⁇ 2 is activated is less clear as it lacks a consensus RGD motif.
  • TGF ⁇ 1 release by proteolytic cleavage of LAP has also been implicated as an activation mechanism, but its biological relevance is less clear. [198] Although the pathogenic role of TGF ⁇ activation is clear in several disease states, it is equally clear that therapeutic targeting of the TGF ⁇ pathway has been challenging due to the pleiotropic effects that result from broad and sustained pathway inhibition.
  • TGF ⁇ R1 TGF ⁇ type I receptor kinase ALK5
  • TGF ⁇ R1 TGF ⁇ type I receptor kinase ALK5
  • MDSCs Circulating/circulatory MDSCs as a biomarker
  • MDSCs are a heterogeneous population of cells named for their myeloid origin and their main immune suppressive function (Gabrilovich. Cancer Immunol Res. 2017 Jan; 5(1): 3–8). MDSCs generally exhibit high plasticity and strong capacity to reduce cytotoxic functions of T cells and natural killer (NK) cells, including their ability to promote T regulatory cell (Treg) expansion and in turn suppress T effector cell function (Gabrilovich et al., Nat Rev Immunol. (2012) 12:253–68).
  • NK natural killer
  • MDSCs are typically classified into two subsets, monocytic (m-MDSCs) and granulocytic (G-MDSCs or PMN-MDSCs), based on their expression of surface markers (Consonni et al., Front Immunol.2019 May 3; 10:949).
  • Suppressive G-MDSCs can be characterized by their production of reactive oxygen species (ROS) as the major mechanism of immune suppression.
  • ROS reactive oxygen species
  • M-MDSCs mediate immune suppression primarily by upregulating the inducible nitric oxide synthase gene (iNOS) and produce nitric oxide (NO) as well as an array of immune suppressive cytokines (Youn and Garilovich, Eur J Immunol.
  • MDSCs have been implicated in various diseases, such as chronic inflammation, infection, autoimmune diseases, and graft-versus-host diseases.
  • diseases such as chronic inflammation, infection, autoimmune diseases, and graft-versus-host diseases.
  • MDSCs have become an immune population of interest in cancer due to their role in inducing T cell tolerance through checkpoint blockade molecules such as the programmed death-ligand 1 (PD-L1) and the cytotoxic T-lymphocyte antigen 4 (CTLA4) (Trovato et al., J Immunother Cancer.2019 Sep 18;7(1):255).
  • PD-L1 programmed death-ligand 1
  • CTLA4 cytotoxic T-lymphocyte antigen 4
  • MDSCs have generally been characterized as favoring tumor progression by mechanisms in addition to immune suppression, including promoting tumor angiogenesis.
  • Many human cancers are known to show elevated levels of MDSCs in biopsies from patients, as compared to healthy controls (reviewed, for example, in Elliott et al., (2017) Frontiers in Immunology, Vol.8, Article 86).
  • These human cancers include but are not limited to bladder cancer, colorectal cancer, prostate cancer, breast cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, lung cancer, melanoma, NSCLC, ovarian cancer, pancreatic cancer, and renal cell carcinoma.
  • the compositions and methods according to the present disclosure may be applied to one or more of these cancers.
  • circulating MDSC levels may be determined by detecting or measuring LRRC33-positive cells in a blood sample, identifying LRRC33 as a novel blood-based biomarker for circulating MDSCs.
  • LRRC33-positive cells in a blood sample collected from a patient may be detected or measured by a FACS-based assay using an antibody that binds cell-surface LRRC33.
  • the LRRC33-expressing cells in a blood sample collected from a subject having cancer are G-MDSCs. While MDSCs are derived from bone marrow- originated monocytes, cell-surface expression of LRRC33 appears to be narrowly restricted to MDSCs, and not monocytes, in circulation. This recognition raises a new possibility of using LRRC33 as a blood-based marker for circulating MDSCs.
  • LRRC33 expression may be determined by any of the antibodies disclosed in WO/2018/208888 and WO/2018/081287, the contents of which are incorporated herein in their entirety. Applicant has now established a correlation between circulatory MDSC levels and tumor-associated MDSC levels.
  • LRRC33 levels measured in blood samples may serve as an effective surrogate to assess tumor immune-phenotype, such as immunosuppression, without the need for more invasive procedures such as tumor biopsy.
  • the present disclosure provides methods of treating cancer, predicting, or determining efficacy, and/or confirming pharmacological response by monitoring the levels of circulating MDSCs in a sample obtained from a patient (e.g., in the blood or a blood component of a patient) receiving a TGF ⁇ inhibitor, e.g., a TGF ⁇ 1-selective inhibitor (such as a selective pro- or latent-TGF ⁇ 1 inhibitor, e.g., Ab6), isoform-non- selective TGF ⁇ inhibitors (such as low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors), and/or an integrin inhibitor (and integrin inhibitors (e.g., antibodies that bind to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5,
  • the circulating MDSCs may be measured within 1, 2, 3, 4, 5, 6, or 7 days, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks (e.g., preferably less than 6 weeks) following administration of a treatment to a subject, e.g., administration of a therapeutic dose of a TGF ⁇ inhibitor.
  • the TGF ⁇ treatment may be administered alone or in conjunction with an additional cancer therapy.
  • the treatment may be administered to subjects with an immunosuppressive cancer or a myeloproliferative disorder.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1-selective antibody or antigen- binding fragment thereof encompassed in the current disclosure (e.g., Ab6).
  • the TGF ⁇ 1- selective antibody or antigen-binding fragment does not inhibit TGF ⁇ 2 and TGF ⁇ 3 at a therapeutically effective dose.
  • the TGF ⁇ inhibitor is an isoform-non-selective TGF ⁇ inhibitor (such as low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, and ligand traps, e.g., TGF ⁇ 1/3 inhibitors).
  • the TGF ⁇ inhibitor is an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • integrin inhibitors include the anti- ⁇ V ⁇ 8 integrin antibodies provided in WO2020051333, the disclosure of which is incorporated by reference.
  • the additional cancer therapy may include chemotherapy, radiation therapy (including radiotherapeutic agents), cancer vaccine or immunotherapy including checkpoint inhibitor therapies such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies.
  • the checkpoint inhibitor therapy is selected from the group consisting of ipilimumab (e.g., Yervoy®); nivolumab (e.g., Opdivo®); pembrolizumab (e.g., Keytruda®); avelumab (e.g., Bavencio®); cemiplimab (e.g., Libtayo®); atezolizumab (e.g., Tecentriq®); budigalimab (e.g., ABBV-181); and durvalumab (e.g., Imfinzi®).
  • ipilimumab e.g., Yervoy®
  • nivolumab e.g., Opdivo®
  • pembrolizumab e.g., Keytruda®
  • avelumab e.g., Bavencio®
  • cemiplimab e
  • a combination cancer therapy comprises Ab6 and at least one checkpoint inhibitor (such as those listed above).
  • a combination of Ab6 and a checkpoint inhibitor is used for the treatment of cancer in a human patient in amounts effective to treat the cancer.
  • the TGF ⁇ treatment may further or alternatively include a second checkpoint inhibitor.
  • the TGF ⁇ treatment may further or alternatively include a chemotherapy (e.g., a genotoxic therapy or radiation therapy).
  • a chemotherapy e.g., a genotoxic therapy or radiation therapy.
  • cancers of the epithelia e.g., carcinoma.
  • cancer types include ovarian cancer, breast cancer, bladder cancer, pancreatic cancer, e.g., pancreatic adenocarcinoma, prostate cancer, e.g., prostate adenocarcinoma, melanoma, e.g., skin cutaneous melanoma, lung cancer, e.g., lung squamous cell carcinoma and lung adenocarcinoma, liver cancer (e.g., liver hepatocellular carcinoma), uterine cancer, e.g., uterine corpus endometrial carcinoma, kidney cancer, e.g., renal clear cell carcinoma, head and neck cancer, e.g., head and neck squamous cell carcinoma, colon cancer, e.g., colon adenocarcinoma, es
  • the cancer is a cancer having elevated TGF ⁇ 1 levels associated with ROS (e.g., elevated ROS).
  • ROS may induce an increase in TGF ⁇ levels (e.g., TGF ⁇ 1 levels) which may be reduced by a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 inhibitor) disclosed herein.
  • TGF ⁇ inhibitors e.g., Ab6
  • one or more genotoxic therapies e.g., chemotherapy and/or radiation therapy, including radiotherapeutic agents
  • such a cancer may have elevated TGF ⁇ levels, e.g., elevated TGF ⁇ activity, as indicated by direct measurement and/or one or more changes in downstream gene regulation (e.g., in one or more genes involved in DNA repair).
  • a cancer such as one of the cancers listed above, may have elevated TGF ⁇ signaling as indicated by upregulation of one or more genes associated with non-homologous end joining (NHEJ), e.g., Cyclin Dependent Kinase Inhibitor 1A (CDKN1A), or downregulation of one or more genes relating to alternative end joining, e.g., LIG1 (DNA ligase 1), PARP1, and/or POLQ.
  • NHEJ non-homologous end joining
  • CDKN1A Cyclin Dependent Kinase Inhibitor 1A
  • LIG1 DNA ligase 1
  • PARP1 DNA ligase 1
  • POLQ POLQ
  • the present disclosure also provides methods of using measurements of circulating MDSCs in treating cancer in subjects administered a TGF ⁇ inhibitor alone or in conjunction with an immunotherapy. Furthermore, the descriptions presented herein provide support for the circulating MDSC population as an early predictive marker of efficacy, particularly in cancer subjects treated with a TGF ⁇ inhibitor and checkpoint inhibitor combination therapy, e.g., at a time point before other markers of treatment efficacy, such as a reduction in tumor volume, can be detected.
  • a TGF ⁇ inhibitor e.g., a TGF ⁇ 1-selective inhibitor such as Ab6, an isoform-non- selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ inhibitor e.g., a TGF ⁇ 1-selective inhibitor such as Ab6
  • an isoform-non- selective inhibitor e.g., low molecular weight ALK5 antagonists
  • TGF ⁇ 1 and/or TGF ⁇ 3 are administered concurrently (e.g., simultaneously), separately, or sequentially to a checkpoint inhibitor therapy such that the amount (e.g., dose) of TGF ⁇ 1 inhibition administered is sufficient to reduce circulating MDSC levels by at least 10%, at least 15%, at least 20%, at least 25%, or more, as compared to baseline MDSC levels.
  • Circulating MDSC levels may be measured prior to or after each treatment or each dose of the TGF ⁇ inhibitor such that a decrease of at least 10%, at least 15%, at least 20%, at least 25%, or more in circulating MDSC levels may be indicative or predictive of treatment efficacy.
  • the level of circulating MDSCs may be used to determine disease burden (e.g., as measured by a change in relative tumor volume before and after a treatment regimen). In some embodiments, the level of circulating mMDSCs may be used to determine disease burden (e.g., as measured by a change in relative tumor volume before and after a treatment regimen). [212] In certain embodiments, a decrease in circulating MDSC levels (e.g., mMDSC levels) may be indicative of a decrease in disease burden (e.g., a decrease in relative tumor volume).
  • circulating MDSC levels may be measured prior to and following administration of a first dose of a TGF ⁇ inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non-selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform
  • TGF ⁇ inhibitors e.g., Ab6, isoform-non-selective TGF ⁇ inhibitors, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor, e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • TGF ⁇ inhibitors e.g., Ab6, isoform-non-selective TGF ⁇ inhibitors, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e
  • selective inhibition of TGF ⁇ 1 and/or TGF ⁇ 3 may be used to reduce tumor volume, such that administration of the TGF ⁇ inhibitor reduces circulating MDSC levels by at least 10%, at least 20%, at least 25%, or more, as compared to circulating MDSC levels (e.g., circulating mMDSC levels) prior to administration.
  • circulating MDSC levels e.g., circulating mMDSC levels
  • reduction in circulating MDSC levels is indicative or predictive of pharmacological effects and further warrants administration of a second or more dose(s) of the TGF ⁇ inhibitor.
  • reduction in circulating mMDSC levels is indicative or predictive of pharmacological effects and further warrants administration of a second or more dose(s) of the TGF ⁇ inhibitor.
  • the first dose of the TGF ⁇ inhibitor is the very first dose of TGF ⁇ inhibitor received by the patient.
  • the first dose of the TGF ⁇ inhibitor is the first dose of a given treatment regimen comprising more than one dose of TGF ⁇ inhibitor.
  • circulating MDSC levels may be measured prior to and after combination treatment comprising a TGF ⁇ inhibitor (e.g., Ab6) and a checkpoint inhibitor therapy, administered concurrently (e.g., simultaneously), separately, or sequentially, and a reduction in the circulating MDSC levels is indicative or predictive of therapeutic efficacy.
  • TGF ⁇ inhibitor e.g., Ab6
  • checkpoint inhibitor therapy administered concurrently (e.g., simultaneously), separately, or sequentially, and a reduction in the circulating MDSC levels is indicative or predictive of therapeutic efficacy.
  • the reduction of circulating MDSC levels following the combination treatment of a TGF ⁇ inhibitor such as a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non-selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1-selective inhibitor
  • the reduction of circulating mMDSC levels following the combination treatment of a checkpoint inhibitor therapy and a TGF ⁇ inhibitor such as a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, or an isoform-non-selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, an antibody that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3,
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab
  • the reduction of circulating gMDSC levels following the combination treatment of a checkpoint inhibitor therapy and a TGF ⁇ inhibitor such as a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, or an isoform-non- selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, an antibody that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins,
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1-selective inhibitor
  • levels of circulating MDSCs may be used to predict, determine, and monitor pharmacological effects of treatment comprising a dose of TGF ⁇ inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non-selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrin
  • TGF ⁇ inhibitor such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non-se
  • a baseline circulating MDSC level may be measured before administering an initial treatment (e.g., a first dose of a TGF ⁇ inhibitor).
  • a low baseline circulating MDSC level is predictive of better response to the TGF ⁇ inhibitor treatment.
  • a low baseline circulating mMDSC level is predictive of better response to the TGF ⁇ inhibitor treatment.
  • a low baseline circulating mMDSC level is predictive of better response to the TGF ⁇ inhibitor treatment.
  • a patient administered the TGF ⁇ inhibitor treatment has a low baseline circulating mMDSC level.
  • circulating MDSCs may be measured within six weeks following administration of the initial treatment. In certain embodiments, circulating MDSC levels may be measured within thirty days following administration of the initial dose of TGF ⁇ inhibitor. In some embodiments, MDSC levels may be measured within or at about three weeks following administration of the initial dose of TGF ⁇ inhibitor. In some embodiments, MDSC levels may be measured within or at about two weeks following administration of the initial dose of TGF ⁇ inhibitor. In some embodiments, MDSC levels may be measured within or at about ten days following administration of the initial dose of TGF ⁇ inhibitor. In certain embodiments, the MDSCs are circulating mMDSCs. In certain embodiments, the MDSCs are circulating gMDSCs.
  • circulating MDSC levels may be used to select, inform treatment, and/or predict response in patients who have not received a checkpoint inhibitor treatment previously.
  • Patients diagnosed with a cancer type with reported high response rates to checkpoint inhibitor therapy e.g., overall response rate of greater than 30%, greater 40%, greater than 50%, or greater, as reported in the art
  • patients diagnosed with a cancer type with reported high response rates to checkpoint inhibitor therapy e.g., overall response rate of greater than 30%, greater 40%, greater than 50%, or greater, as reported in the art
  • circulating MDSCs may be used in conjunction with immunohistochemistry, flow cytometry, and/or in vivo imaging methods known in the art to determine the immune phenotype of the tumor.
  • Patients with cancers exhibiting an immune-excluded or immunosuppressive phenotype may be selected to receive a TGF ⁇ inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non- selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ 1-selective inhibitor e.g., Ab6
  • the circulating MDSCs are circulating mMDSCs. In some embodiments, the circulating MDSCs are circulating gMDSCs. In some embodiments, patients exhibiting an immune-excluded or immunosuppressive phenotype are treated with a TGF ⁇ inhibitor.
  • circulating MDSC levels e.g., circulating mMDSC levels
  • patients diagnosed with a cancer type with reported low response rates to checkpoint inhibitor therapy (e.g., overall response rate of 30% or less, 20% or less, or 10%, or less, as reported in the art) who have not received a checkpoint inhibitor therapy previously may be treated with a combination of a TGF ⁇ inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non- selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrin
  • treatment response in these patients may be predicted by monitoring circulating MDSC levels.
  • treatment response in these patients may be predicted by monitoring circulating mMDSC levels.
  • treatment response in these patients may be predicted by monitoring circulating gMDSC levels.
  • treatment is continued based on the circulating MDSC levels.
  • circulating MDSC levels may be used for selecting, informing treatment in, and predicting response in patients who are resistant to checkpoint inhibitor therapy or who do not tolerate checkpoint inhibitor therapy (e.g., due to adverse effects).
  • TGF ⁇ inhibitor therapy such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non- selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, and ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or
  • a TGF ⁇ inhibitor therapy such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non- selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.
  • patients with either primary resistance or acquired resistance to checkpoint inhibitor may be administered a TGF ⁇ inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non-selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ 1-selective inhibitor e.g., Ab6
  • an isoform-non-selective inhibitor e
  • a reduction of at least 10%, at least 15%, at least 20%, at least 25%, or more in circulating MDSC levels may be indicative of response to the TGF ⁇ inhibitor therapy.
  • a reduction of at least 10%, at least 15%, at least 20%, at least 25%, or more in circulating MDSC levels may indicate pharmacological effects of a treatment, e.g., with a TGF ⁇ inhibitor.
  • a decrease in circulating MDSC levels may be indicative of a decrease in tumor size.
  • a decrease in circulating mMDSC levels may be indicative of a decrease in tumor size. In certain embodiments, a decrease in circulating gMDSC levels may be indicative of a decrease in tumor size.
  • a chart summarizing exemplary treatment regimens is provided in FIG.40. [219] Most TGF ⁇ inhibitors currently in development are not isoform-selective. These include pan-inhibitors of TGF ⁇ , and inhibitors that target TGF ⁇ 1/2 and TGF ⁇ 1/3. Approaches taken to manage possible toxicities associated with such inhibitors include careful dosing regimens to hit a narrow window in which both efficacy and acceptable safety profiles may be achieved.
  • This may include sparing of an isoform non-selective inhibitor, which may include infrequent dosing and/or reducing dosage per administration. For instance, in lieu of weekly dosing of a biologic TGF ⁇ inhibitor, monthly dosing may be considered. Another example is to dose only in an initial phase of a combination immunotherapy so as to avoid or minimize toxicities associated with TGF ⁇ inhibition.
  • a combination therapy comprising a cancer therapy (such as checkpoint inhibitor therapy) and an isoform-non-selective TGF ⁇ inhibitor may result in a greater risk of toxicity as compared to a TGF ⁇ 1-selective inhibitor (e.g.
  • the isoform-non-selective TGF ⁇ inhibitor may be administered infrequently or intermittently, for example on an “as-needed” basis.
  • circulating MDSC levels may be monitored periodically in order to determine that the effects of overcoming immunosuppression are sufficiently maintained, so as to ensure antitumor effects of the cancer therapy.
  • MDSCs become elevated, it indicates that the patient benefits from additional doses of a TGF ⁇ inhibitor.
  • Such approach may help reduce unnecessary risk and adverse events associated with TGF ⁇ inhibition, non-isoform-selective inhibitors in particular.
  • the TGF ⁇ inhibitor targets TGF ⁇ 1/2.
  • the TGF ⁇ inhibitor targets TGF ⁇ 1/3. In some embodiments, the TGF ⁇ inhibitor targets TGF ⁇ 1/2/3. In some embodiments, the TGF ⁇ inhibitor selectively targets TGF ⁇ 1.
  • the intermittent dosing regimen comprises the following steps: measuring circulating MDSCs (e.g., circulating mMDSCs and/or circulating gMDSCs) in a first sample collected from the patient prior to a TGF ⁇ inhibitor treatment; administering a TGF ⁇ inhibitor to the patient treated with a cancer therapy, wherein the cancer therapy is optionally a checkpoint inhibitor therapy and/or a chemotherapy; measuring circulating MDSCs (e.g., circulating mMDSCs and/or circulating gMDSCs) in a second sample collected from the patient after the TGF ⁇ inhibitor treatment; continuing with the cancer therapy if the second sample shows reduced levels of circulating MDSCs as compared
  • the intermittent dosing regimen further comprises measuring circulating MDSCs (e.g., circulating mMDSCs and/or circulating gMDSCs) in a third sample; and, administering to the patient an additional dose of a TGF ⁇ inhibitor, if the third sample shows elevated levels of circulating MDSC levels as compared to the second sample.
  • the TGF ⁇ inhibitor is an isoform-non-selective inhibitor.
  • the sample is blood or a blood component sample.
  • the isoform-non-selective inhibitor inhibits TGF ⁇ 1/2/3, TGF ⁇ 1/2 or TGF ⁇ 1/3.
  • Baseline circulating MDSC levels are likely to be elevated in cancer patients as compared to healthy individuals, and subjects with immunosuppressive cancers may have even more elevated circulating MDSC levels.
  • a TGF ⁇ inhibitor therapy such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), an isoform-non-selective inhibitor (e.g., low molecular weight ALK5 antagonists), neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3 (e.g., GC1008 and variants), antibodies that bind TGF ⁇ 1/3, ligand traps (e.g., TGF ⁇ 1/3 inhibitors), and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1
  • TGF ⁇ inhibitor therapy such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6)
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), an isoform-non- selective inhibitor (e.g., low molecular weight ALK5 antagonists), neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3 (e.g., GC1008 and variants), antibodies that bind TGF ⁇ 1/3, ligand traps (e.g., TGF ⁇ 1/3 inhibitors), and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), an isoform-non- selective inhibitor (e.g., low molecular weight ALK5 antagonists), neutralizing antibodies
  • TGF ⁇ inhibitor e.g., selective inhibition of TGF ⁇ 1 and/or TGF ⁇ 3 is administered to a subject with cancer such that the dose of the TGF ⁇ inhibitor is sufficient to reduce or reverse immune suppression in the cancer as indicated by a reduction of circulating MDSC levels (e.g., a reduction of circulating mMDSC levels) and/or a change in the levels of tumor-associated immune cells measured after administering the TGF ⁇ inhibitor treatment as compared to levels measured before administration.
  • a reduction of circulating MDSC levels e.g., a reduction of circulating mMDSC levels
  • a change in the levels of tumor-associated immune cells measured after administering the TGF ⁇ inhibitor treatment as compared to levels measured before administration.
  • levels of circulating MDSC and/or tumor-associated immune cells are measured before and after administration of a TGF ⁇ inhibitor treatment such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), an isoform-non- selective inhibitor (e.g., low molecular weight ALK5 antagonists), neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3 (e.g., GC1008 and variants), antibodies that bind TGF ⁇ 1/3, ligand traps (e.g., TGF ⁇ 1/3 inhibitors), and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ 1-selective inhibitor e.g., Ab6
  • an isoform-non- selective inhibitor e.g.
  • a reduction of circulating MDSC levels and/or change(s) in the levels of tumor-associated immune cells measured after treatment as compared to levels measure before treatment indicates reduction or reversal of immune suppression in the cancer.
  • a reduction of circulating mMDSC levels and/or change(s) in the levels of tumor-associated immune cells measured after treatment as compared to levels measure before treatment indicates reduction or reversal of immune suppression in the cancer.
  • Circulating MDSC levels may be determined in a sample such as a whole blood sample or a blood component (e.g., PBMCs).
  • PBMCs blood component
  • the sample is fresh whole blood or a blood component of a sample that has not been previously frozen.
  • circulating MDSCs may be collected by drawing peripheral blood into heparinized tubes.
  • peripheral blood mononuclear cells may be isolated using, e.g., elutriation, magnetic beads separation, or density gradient centrifugation methods (e.g., Ficoll-Paque®) known in the art.
  • MDSCs may be separated from peripheral blood mononuclear cells by surface marker selection (e.g., using immunofluorescence, e.g., flow cytometry/FACS analysis or immunohistochemistry).
  • G-MDSCs and M-MDSCs may be further distinguished using surface markers provided herein.
  • Immune cell markers may be used to determine whether a cancer has an immune-excluded phenotype, and/or may be used in determining treatment efficacy or treatment regimen, alone or in combination with other circulating biomarkers such as circulating MDSCs. If the tumor is determined to have an immune-excluded phenotype, cancer therapy (such as CBT) alone may not be efficacious. Without being bound by theory, the tumor may lack sufficient cytotoxic cells within the tumor environment for effective CBT treatment alone. Thus, an alternative and/or add-on therapy with a TGF ⁇ inhibitor (such as those described herein) may reduce immuno- suppression, thereby providing an improved treatment alone or rendering the resistant tumor more responsive to a cancer therapy.
  • cancer therapy such as CBT
  • TGF ⁇ inhibitor such as those described herein
  • immune cell markers are measured in biopsies (e.g., core needle biopsies).
  • patients having an immune-excluded tumor are administered a treatment comprising one or more TGF ⁇ inhibitor (e.g., TGF ⁇ 1 inhibitor, e.g., Ab6).
  • patients having an immune-excluded tumor are administered a treatment comprising one or more TGF ⁇ inhibitor (e.g., TGF ⁇ 1 inhibitor, e.g., Ab6) inhibitor and monitored for improvement in condition (e.g., increased immune cell penetration into a tumor, reduced tumor volume, etc.).
  • a patient exhibiting an improvement in condition after a first round of treatment is administered one or more additional rounds of treatment.
  • tumor-associated immune cells that may be used to indicate the immune contexture of a tumor/cancer microenvironment include, but are not limited to, cytotoxic T cells and tumor-associated macrophages (TAMs), as well as tumor-associated MDSCs.
  • TAMs tumor-associated macrophages
  • Biomarkers to detect cytotoxic T cell levels may include, but are not limited to, the CD8 glycoprotein, granzyme B, perforin, and IFN ⁇ , of which the latter three markers may also be indicative of activated cytotoxic T cells.
  • cytotoxic T cells e.g., activated cytotoxic T cells
  • increased levels of cytotoxic T cells, e.g., activated cytotoxic T cells, detected within the tumor microenvironment may be indicative of reduction or reversal of immune suppression.
  • an increase in CD8 expression and perforin, granzyme B, and/or IFN ⁇ expression by tumor-associated immune cells may be indicative of reduction or reversal of immune suppression in the cancer.
  • decreased levels of TAMs or tumor-associated MDSCs detected within the tumor microenvironment may be indicative of reduced or reversal of immune suppression.
  • a decrease of HLA-DR, CD68, CD163, and CD206 expression by tumor-associated immune cells may indicate reduced or reversal of immune suppression in the cancer.
  • tumor-associated immune cells e.g., CD8+ T cells
  • the immune contexture of a tumor may be characterized by the density, location, organization, and/or functional orientation of tumor-infiltrating immune cells.
  • markers may be used to determine the immune phenotype of a tumor, e.g., to determine if a tumor is immune excluded, inflamed, or desert.
  • cytotoxic T cells e.g., in a patient sample, may be used to determine whether a cancer has an immune-excluded phenotype, and/or may be used in determining treatment efficacy or treatment regimen, alone or in combination with other biomarkers such as circulating MDSCs.
  • CD8 expression and/or the distribution of CD8 expression in a tumor sample may be used.
  • CD8 expression may be examined in a sample to determine distribution in the tumor (i.e., tumor compartment), stroma (i.e., stroma compartment), and margin (i.e., margin compartment; identified, e.g., by assessing the region approximately 10- 100 ⁇ m, or 25-75 ⁇ m, or 30-60 ⁇ m, e.g., 50 ⁇ m, between tumor and stroma).
  • tumor, stroma, and/or margin compartments within the tumor may be identified using histological methods (e.g., pathologist assessment, pathologist-trained machine learning algorithms, and/or immunohistochemistry).
  • CD8+ T cells in a tumor compartment may be referred to as “tumor-associated CD8+ cells”.
  • CD8+ T cells in a stroma compartment may be referred to as “stroma-associated CD8+ cells”.
  • CD8+ T cells in a margin compartment may be referred to as “margin-associated CD8+ cells”.
  • CD8 distribution may be determined in a tumor nest (e.g., a mass of cells extending from a common center seen in a cancerous growth), the stroma surrounding the tumor nest, and the margin between the tumor nest and its surrounding stroma (identified, e.g., by assessing the region approximately 10-100 ⁇ m, or 25-75 ⁇ m, or 30-60 ⁇ m, e.g., 50 ⁇ m, between the tumor nest and the surrounding stroma).
  • tumor nests may be identified using histological methods (e.g., pathologist assessment, pathologist-trained machine learning algorithms, and/or immunohistochemistry).
  • one or more tumor nests may be found within a tumor compartment.
  • a tumor may comprise multiple (e.g., at least 5, at least 10, at least 20, at least 25, at least 50, or more) tumor nests.
  • stroma or “stroma compartment” refers to the stroma surrounding the tumor
  • margin or “margin compartment” refers to the margin between the tumor and the stroma surround the tumor.
  • the structural interface between the tumor/tumor nest and the surrounding stroma is determined by imaging analysis. A margin can then be defined as the region surrounding the interface in either direction by a predetermined distance, for example, 10-100 ⁇ m.
  • this distribution may be used prior to administering a TGF ⁇ inhibitor, such as a TGF ⁇ 1 inhibitor (e.g., Ab6) to select a patient for treatment and/or predict and/or determine the likelihood of a therapeutic response (e.g., an anti-tumor response) to an anti-cancer therapy comprising an anti-TGF ⁇ inhibitor.
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1 inhibitor (e.g., Ab6)
  • a therapeutic response e.g., an anti-tumor response
  • an anti-cancer therapy comprising an anti-TGF ⁇ inhibitor.
  • cytotoxic T cells e.g., less than 5% CD8+ T cells
  • cytotoxic T cells e.g., greater than 5% CD8+ T cells
  • this patient may also have limited benefit from TGF inhibitor therapy (without being bound by theory, this may be because immune cells have already infiltrated the tumor).
  • the subject’s cancer may exhibit an immune-excluded phenotype, in which cytotoxic T cells (e.g., CD8+ T cells) are observed clustered primarily in or near the margin, e.g., at the border between the margin and the tumor, and not significantly infiltrated into the tumor itself (e.g., less than 5% CD8+ T cells in the tumor compartment and greater than 10% CD8+ T cells in the margin and/or stroma compartment).
  • cytotoxic T cells e.g., CD8+ T cells
  • the subject’s cancer may exhibit an immune-excluded phenotype, in which cytotoxic T cells (e.g., CD8+ T cells) are observed clustered primarily in or near the margin, e.g., at the border between the margin and the tumor (or peri-vasculature), and not significantly infiltrated into the tumor core itself (e.g., less than 5% CD8+ T cells in the tumor compartment and greater than 5% CD8+ T cells in the margin and/or stroma compartment).
  • cytotoxic T cells e.g., CD8+ T cells
  • the subject’s cancer may exhibit an immune-excluded phenotype, in which cytotoxic T cells (e.g., CD8+ T cells) are observed clustered primarily in or near the margin, e.g., at the border between the margin and the tumor, and not significantly infiltrated into the tumor itself (e.g., less than 5%, less than 10%, less than 15%, or fewer CD8+ T cells in the tumor compartment and greater than 5%, greater than 10%, greater than 15%, or more CD8+ T cells in the margin and/or stroma compartment).
  • CD8+ content in tumor compartments may be based on any of the methods described in Ziai et al. (PLoS One.
  • an immune-excluded phenotype is characterized by determining a cluster score of cytotoxic T cells (e.g., CD8+ T cells) within a tumor-associated compartment, e.g., in the tumor, in the margin near the external perimeters of a tumor mass, and/or in the vicinity of tumor vasculatures.
  • cytotoxic T cells e.g., CD8+ T cells
  • the cluster score of cytotoxic T cells can be determined based on the homogeneity of immune cells in a particular tumor-associated compartment, such that a compartment containing highly uniform distribution of cytotoxic T cells (e.g., CD8+ T cells) yields a high cluster score.
  • tumors exhibiting an immune-excluded phenotype may be characterized by lower densities of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor as compared to densities outside of the tumor (e.g., the external perimeters of a tumor mass and/or near the vicinity of vasculatures of a tumor).
  • the immune-excluded phenotype is characterized by cytotoxic T cells (e.g., CD8+ T cells) in the tumor stroma that are located in close vicinity (e.g., less than 100 ⁇ m) to the tumor.
  • the immune-excluded phenotype is characterized by cytotoxic T cells (e.g., CD8+ T cells) capable of infiltrating the tumor nest and locating at a close distance (e.g., less than 100 ⁇ m) to the tumor.
  • CD8+ T cells can be observed in clusters within a tumor near intratumoral blood vessels as determined for example by endothelial markers.
  • levels of tumor-infiltrating cytotoxic T cells may be determined from a tumor biopsy sample obtained from the subject.
  • tumor biopsy samples e.g., core needle biopsies, may be obtained at least 28 days prior to and at least 100 days following treatment administration.
  • tumor biopsy samples e.g., core needle biopsies
  • tumor biopsy samples may be obtained about 21 days to about 45 days following treatment administration.
  • tumor biopsy samples may be obtained via core needle biopsy.
  • treatment is continued if an increase is detected.
  • the immune phenotype of a subject’s cancer may be determined by measuring the cell densities of cytotoxic T cells (e.g., percent of CD8+ T cells per square millimeter or other defined square distance) in a tumor biopsy sample.
  • the immune phenotype of a subject’s cancer may be determined by comparing the densities of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor to that outside the tumor (e.g., to cells in the margin, e.g., at the external perimeters of a tumor mass and/or near the vicinity of vasculatures of a tumor). In some embodiments, the immune phenotype of a subject’s cancer may be determined by comparing the percentage of CD8+ lymphocytes inside the tumor to that outside the tumor.
  • cytotoxic T cells e.g., CD8+ T cells
  • the immune phenotype of a subject’s cancer may be determined by comparing the cluster or dispersion of cytotoxic T cells (e.g., average number of CD8+ T cells surrounding other CD8+ T cells) in the tumor, stroma, or margin. In certain embodiments, the immune phenotype of a subject’s cancer may be determined by measuring the average distance from cytotoxic T cells (e.g., CD8+ T cells) in the stroma to the tumor. In certain embodiments, the immune phenotype of a subject’s cancer may be determined by measuring the average depth of cytotoxic T cell (e.g., CD8+ T cell) penetration into the tumor nest. Cell counts and density may be determined using immunostaining and computerized or manual measurement protocols.
  • cytotoxic T cells e.g., average number of CD8+ T cells surrounding other CD8+ T cells
  • the immune phenotype of a subject’s cancer may be determined by measuring the average distance from cytotoxic T cells (e.g., CD8+ T
  • levels of cytotoxic T cells may be measured using immunohistochemical analysis of tumor biopsy samples.
  • levels of cytotoxic T cells e.g., CD8+ T cells
  • levels of cytotoxic T cells may be determined at least 28 days prior to and/or at least 100 days following administering a TGF ⁇ therapy.
  • levels of cytotoxic T cells may be determined up to about 45 days (e.g., about 21 days to about 45 days) following administering a TGF ⁇ therapy.
  • levels of cytotoxic T cells are determined 5, 10, 15, 20, 25, 30, or more days prior to and/or at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 days following administering a TGF ⁇ therapy (or at any time point in between).
  • a tumor with lower levels of cytotoxic T cells e.g., CD8+ T cells
  • cytotoxic T cell levels e.g., CD8+ T cells
  • outside the tumor e.g., the external perimeters of a tumor and/or near the vicinity of vasculatures of a tumor
  • immune-excluded tumors may also have higher levels of cytotoxic T cells (e.g., CD8+ T cells) in the tumor stroma as compared to inside the tumor.
  • immune-excluded tumors may be identified by determining the ratio of cytotoxic T cell density (e.g., CD8+ T cells) inside the tumor to outside of the tumor, wherein the ratio is less than 1.
  • immune-excluded tumors may be identified by determining the cytotoxic T cell density ratio inside the tumor to density in the tumor margin, wherein the ratio is less than 1.
  • immune-excluded tumors may be identified by determining the cell density ratio inside the tumor to density in the tumor stroma, wherein the ratio is less than 1.
  • immune-excluded tumors may be identified by comparing the absolute number, percentage, and/or density of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor to outside the tumor (e.g., margin and/or stroma).
  • the absolute number, percentage, and/or density of cytotoxic T cells (e.g., CD8+ T cells) outside the tumor is at least 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, or 10-fold greater than inside the tumor in an immune-excluded tumor.
  • an immune-excluded tumor comprises less than 5% CD8+ T cells inside the tumor and greater than 10% CD8+ T cells in the tumor margin and/or stroma.
  • immune-excluded tumors may be identified by comparing a ratio of compartmentalized cytotoxic T cell density (e.g., density of CD8+ cells inside the tumor to density in the tumor margin and/or stroma) and the ratio of whole tissue cytotoxic T cell density (e.g., CD8+ cells inside the tumor to CD8+ cells in the entire tumor tissue or biopsy), wherein the compartmentalized ratio is greater than the whole tissue ratio.
  • a tumor with increased cell density of cytotoxic T cells (e.g., CD8+ T cells) at an average distance of about 100 ⁇ m or less outside of the tumor may be identified as an immune-excluded tumor.
  • cytotoxic T cell density (e.g., CD8+ T cells) may be used in conjunction with one or more parameters, such as average CD8+ cluster score.
  • an average CD8+ clustering score of 50% or less in the tumor indicates immune exclusion.
  • a tumor with lower levels of CD8+ T cells inside (e.g., core of) the tumor as compared to CD8+ T cells outside the tumor e.g., peripheries of the tumor, e.g., the external perimeters of a tumor and/or near the vicinity of vasculatures of a tumor, e.g., in the tumor margin and/or stroma
  • an immune-excluded tumor e.g., CD8+ T cells
  • an immune-excluded tumor comprises less than 5%, less than 10%, or less than 15% CD8+ T cells inside the tumor and/or inside one or more tumor nests and greater than 5%, greater than 10%, or greater than 15% CD8+ T cells outside the tumor and/or outside one or more tumor nests. In some embodiments, an immune-excluded tumor comprises less than 5% CD8+ T cells inside the tumor and/or inside one or more tumor nests and greater than 5% CD8+ T cells outside of the tumor and/or outside one or more tumor nests.
  • an immune-excluded tumor comprises less than 10% CD8+ T cells inside the tumor and/or inside one or more tumor nests and greater than 10% CD8+ T cells outside of the tumor and/or outside one or more tumor nests. In some embodiments, an immune-excluded tumor comprises less than 15% CD8+ T cells inside the tumor and/or inside one or more tumor nests and greater than 15% CD8+ T cells outside of the tumor and/or outside one or more tumor nests.
  • a tumor with higher levels of CD8+ T cells inside the tumor as compared to CD8+ T cells outside the tumor may be identified as an immune-inflamed tumor.
  • an immune-inflamed tumor comprises greater than 5% CD8+ T cells inside the tumor.
  • an immune-inflamed tumor comprises greater than 10% CD8+ T cells inside the tumor and/or inside one or more tumor nests.
  • an immune-inflamed tumor comprises greater than 15% CD8+ T cells inside the tumor and/or inside one or more tumor nests.
  • a tumor with low levels of CD8+ T cells both inside and outside the tumor may be identified as an immune desert tumor.
  • an immune desert tumor comprises less than 5% CD8+ T cells inside the tumor and less than 10% CD8+ T cells in the tumor margin and/or stroma.
  • an immune desert tumor comprises less than 5% CD8+ T cells inside the tumor (and/or inside one or more tumor nests) and less than 5% CD8+ T cells in the tumor margin and/or stroma.
  • CD8+ content in tumor compartments may be determined based on any of the methods described in Ziai et al. (PLoS One.2018; 13(1): e0190158), Massi et al. (J Immunother Cancer.2019 Nov 15;7(1):308), Sharma et al. (Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3967-72), or Echarti et al. (Cancers (Basel). 2019 Sep; 11(9): 1398), the contents of which are hereby incorporated in their entirety. In some embodimehnts, any of these methods may be used to determine the immune phenotype of the tumor.
  • the immune phenotype of a subject’s cancer may be determined by average percent CD8 positivity (i.e., percentage of CD8+ lymphocytes) as measured over multiple (e.g., at least 5, at least 15, at least 25, at least 50, or more) tumor nests of a tumor (e.g., in one or more tumor biopsy samples).
  • the immune phenotype of a given tumor nest may be determined by comparing the CD8 positivity inside the tumor nest to the CD8 positivity outside the tumor nest (e.g., in the tumor nest margin and/or the tumor nest stroma).
  • a tumor nest may be identified as immune inflamed if the CD8 positivity inside the tumor nest is greater than 5%.
  • a tumor nest may be identified as immune excluded if the CD8 positivity inside the tumor nest is less than 5% and the CD8 positivity in the tumor nest margin is greater than 5%. In certain embodiments, a tumor nest may be identified as an immune desert if the CD8 positivity inside the tumor nest is less than 5% and CD8 positivity in the tumor nest margin is less than 5%. In certain embodiments, a subject’s cancer may be identified immune inflamed if greater than 50% of the total tumor area analyzed comprises tumor nests exhibiting immune inflamed phenotype. In certain embodiments, a subject’s cancer may be identified as immune excluded if greater than 50% of the total tumor area analyzed comprises tumor nests exhibiting immune excluded phenotype.
  • a subject’s cancer may be identified as an immune desert if greater than 50% of the total tumor area analyzed comprises tumor nests exhibiting immune desert phenotype. In certain embodiments, a subject’s cancer may be identified based on determination of CD8 positivity from more than one sample (e.g., at least three samples, e.g., four samples) taken from the same tumor. [239] In certain embodiments, tumor biopsy samples may be obtained by core needle biopsy. In certain embodiments, three to five samples (e.g., four samples) may be taken from the same tumor.
  • the needle may be inserted along a single trajectory, wherein multiple samples (e.g., three to five samples, e.g., four samples) may be taken at different tumors depths along the same needle trajectory. In certain embodiments, samples taken at different tumor depths may be used to analyze combined CD8 positivity over multiple tumor nests. In certain embodiments, the combined CD8 positivity determined in these samples may be representative of CD8 positivity in the rest of the tumor. In certain embodiments, the combined CD8 positivity determined in these samples may be used to identify immune phenotype of a subject’s cancer.
  • the immune phenotype of a subject’s tumor may be determined by combined analysis of the absolute number, percentage, ratio, and/or density of CD8+ cells in the tumor and the combined CD8 positivity (i.e., percentage of CD8+ lymphocytes) across tumor nests throughout the tumor.
  • tumor compartments may be identified, determined, and/or analyzed for markers such as CD8 content manually, e.g., by a pathologist inspection of tumor samples.
  • tumor compartments may be identified, determined, and/or analyzed for markers such as CD8 content by digital analysis, e.g., by using a software or computer program for automated identification.
  • a skilled artisan may use such a software or computer program for automated identification of tumor nests and the boundaries between a tumor nest, stroma compartment, and/or tumor margin compartment.
  • a software or computer program may be used to evaluate the distribution of suitable markers such as CD8+ T cells in the identified tumor nest, stromal compartment, and/or tumor margin compartment.
  • the software or computer program may be based on one or more machine learning algorithms.
  • the one or more machine learning algorithms may be based initially on manual classification of reference samples, e.g., by a trained pathologist.
  • the software or computer program may use a neural network approach with machine learning based on reference samples categorized manually, e.g., by a pathologist.
  • Exemplary softwares or computer programs include any software or computer program that has the capability of intaking an image (e.g., microscope images of a tumor sample comprising immune staining), processing and analyzing the image, and segmenting the tumor compartments in the image based on specific parameters (e.g., nuclear staining, fibroblast staining, CD8+ staining, other biomarkers).
  • the softwares or computer program may be any of those provided by Visiopharm, HALO (Indica Labs), CellProfiler Analyst, Aperio Image Analysis, Zeiss ZEN Inicis, or ImageJ.
  • Such programs may advantageously achieve sufficient resolution for visualizing certain characteristics of individual tumor nests within a solid tumor (e.g., boundaries for tumor nest, stroma, and/or margin compartmenrs), as opposed to analyzing substantially the entire tumor as a whole.
  • a subject whose cancer exhibits an immune-excluded phenotype may be more responsive to a therapy comprising administration of a TGF ⁇ inhibitor (e.g., Ab6).
  • a subject is identified for treatment.
  • such a subject is administered a treatment comprising a TGF inhibitor, such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), an isoform-non-selective inhibitor (e.g., low molecular weight ALK5 antagonists), neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3 (e.g., GC1008 and variants), antibodies that bind TGF ⁇ 1/3, ligand traps (e.g., TGF ⁇ 1/3 inhibitors), and/or an integrin inhibitor (e.g., an antibodies that bind to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibit downstream activation of TGF ⁇ .
  • a TGF inhibitor such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), an isoform-non-selective inhibitor (e.g.,
  • a subject whose cancer exhibits an immune-excluded phenotype may be more responsive to a combination therapy comprising a TGF ⁇ inhibitor, such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), an isoform-non-selective inhibitor (e.g., low molecular weight ALK5 antagonists), neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3 (e.g., GC1008 and variants), antibodies that bind TGF ⁇ 1/3, ligand traps (e.g., TGF ⁇ 1/3 inhibitors), and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1-selective inhibitor (e.g.,
  • the additional cancer therapy may comprise chemotherapy, radiation therapy (including radiotherapeutic agents), a cancer vaccine, or an immunotherapy comprising a checkpoint inhibitor such as an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody.
  • the checkpoint inhibitor therapy is selected from the group consisting of ipilimumab (e.g., Yervoy®); nivolumab (e.g., Opdivo®); pembrolizumab (e.g., Keytruda®); avelumab (e.g., Bavencio®); cemiplimab (e.g., Libtayo®); atezolizumab (e.g., Tecentriq®); budigalimab (e.g., ABBV-181); and durvalumab (e.g., Imfinzi®).
  • ipilimumab e.g., Yervoy®
  • nivolumab e.g., Opdivo®
  • pembrolizumab e.g., Keytruda®
  • avelumab e.g., Bavencio®
  • cemiplimab e
  • a subject whose cancer exhibits an immune-excluded phenotype is administered a combination therapy comprising a TGF ⁇ inhibitor, such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), and an additional cancer therapy, e.g., a checkpoint inhibitor.
  • a subject whose cancer exhibits an immune-excluded phenotype may be more responsive to a combination therapy comprising a TGF ⁇ inhibitor, such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), and a checkpoint inhibitor therapy (e.g., a PD1 or PDL1 antibody).
  • a subject is identified for receiving the combination therapy.
  • such a subject is identified for receiving the combination therapy prior to receiving the checkpoint inhibitor therapy alone. In some embodiments, such a subject is identified for receiving the combination therapy prior to receiving either the checkpoint inhibitor therapy or the TGF ⁇ inhibitor alone. In some embodiments, such a subject is treatment-na ⁇ ve. In some embodiments, such a subject has previously received a checkpoint inhibitor therapy and is non-responsive to the checkpoint inhibitor therapy. In some embodiments, such a subject has cancer that exhibits an immune-excluded phenotype. In some embodiments, such a subject has previously received a checkpoint inhibitor therapy and is directly given a combination therapy (e.g., bypassing the need to first try treatment with a checkpoint inhibitor alone).
  • a combination therapy e.g., bypassing the need to first try treatment with a checkpoint inhibitor alone.
  • such a subject is administered a combination therapy comprising a TGF ⁇ inhibitor, such as a TGF ⁇ 1- selective inhibitor (e.g., Ab6), and an additional cancer therapy, e.g., a PD1 or PDL1 antibody.
  • a subject whose cancer exhibits an immune-excluded phenotype may be selected for treatment and/or monitored during and/or after administration of the therapy comprising a TGF ⁇ inhibitor, such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6).
  • patient selection and/or treatment efficacy is determined by measuring the level of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor as compared to the level of cytotoxic T cells (e.g., CD8+ T cells) outside the tumor (e.g., in the margin).
  • cytotoxic T cells e.g., CD8+ T cells
  • an increase in the levels of tumor-infiltrating cytotoxic T cells (e.g., CD8+ T cells) inside the tumor relative to outside the tumor (e.g., margin and/or stroma) following administration of a TGF ⁇ inhibitor therapy (e.g., Ab6), alone or in combination with an additional therapy (e.g., a checkpoint inhibitor therapy), may indicate a therapeutic response (e.g., anti-tumor response).
  • a TGF ⁇ inhibitor therapy e.g., Ab6
  • an additional therapy e.g., a checkpoint inhibitor therapy
  • an increase of at least 10%, 15%, 20%, 25%, or more in tumor-infiltrating cytotoxic T cell levels following TGF ⁇ inhibitor treatment (e.g., Ab6) as compared to tumor-infiltrating cytotoxic T cell levels before the treatment may be indicative of therapeutic response (e.g., anti-tumor response).
  • an increase of at least 10%, 15%, 20%, 25%, or more in total tumor area comprising immune inflamed tumor nests may be indicative of therapeutic response.
  • levels of cytolytic proteins such as perforin or granzyme B or proinflammatory cytokines such as IFN ⁇ expressed by the tumor-infiltrating cytotoxic T cells may also be measured to determine the activation status of the tumor-infiltrating cytotoxic T cells.
  • an increase of at least 1.5-fold, or 2-fold, or 5-fold, or more in cytolytic protein levels may be indicative of therapeutic response (e.g., anti-tumor response).
  • a change of at least a 1.5-fold, 2-fold, 5-fold, or 10-fold, or more increase in IFN ⁇ levels may be indicative of a therapeutic response (e.g., anti-tumor response).
  • treatment is continued if an increase in tumor-infiltrating cytotoxic T cells (e.g., CD8+ T cells) is detected.
  • cytotoxic T cells e.g., CD8+ T cells
  • a subject whose cancer exhibits an immune-inflamed phenotype may be more responsive to a therapy comprising a checkpoint inhibitor without a TGF ⁇ inhibitor than would a subject having an immune-excluded phenotype.
  • the checkpoint inhibitor therapy is selected from the group consisting of ipilimumab (e.g., Yervoy®); nivolumab (e.g., Opdivo®); pembrolizumab (e.g., Keytruda®); avelumab (e.g., Bavencio®); cemiplimab (e.g., Libtayo®); atezolizumab (e.g., Tecentriq®); budigalimab (e.g., ABBV-181); and durvalumab (e.g., Imfinzi®).
  • ipilimumab e.g., Yervoy®
  • nivolumab e.g., Opdivo®
  • pembrolizumab e.g., Keytruda®
  • avelumab e.g., Bavencio®
  • cemiplimab e
  • immune phenotyping of a subject’s tumor may be determined from a tumor biopsy sample (e.g., core needle biopsy sample), for example histologically, using one or more parameters such as, but not limited to, distribution of cytotoxic T cells (e.g., CD8+ T cells), percentage of cytotoxic T cells (e.g., CD8+ T cells) in the tumor versus stromal compartment, and percentage of cytotoxic T cells (e.g., CD8+ T cells) in the tumor margin.
  • cytotoxic T cells e.g., CD8+ T cells
  • percentage of cytotoxic T cells e.g., CD8+ T cells
  • CD8+ T cells percentage of cytotoxic T cells in the tumor versus stromal compartment
  • percentage of cytotoxic T cells e.g., CD8+ T cells
  • the present disclosure also provides improved methods, where needle biopsy is employed for tumor analysis.
  • the risk of bias inherent to needle biopsy may be significantly reduced by collecting adjacent tumor samples, for example, at least three, but preferably four samples collected from adjacent tumor tissue (e.g., from the same tumor). This may be carried out from a single needle insertion point, by, for example, altering the angle and/or the depth of insertion.
  • a sample may be analyzed for its distribution of cytotoxic T cells (e.g., CD8+ T cells) using a method such as CD8 immunostaining.
  • the distribution of cytotoxic T cells may be relatively uniform (e.g., distribution is homogeneous throughout the sample, e.g., CD8 density across tumor nests have a variance of 10% or lower).
  • a tumor nest refers to a mass of cells extending from a common center of a cancerous growth.
  • a tumor nest may comprise cells interspersed in stroma.
  • a sample such as a sample with an even distribution of cytotoxic T cells (e.g., CD8 T cells), may be analyzed to determine the percentages of cytotoxic T cells (e.g., CD8+ T cells) in the tumor and in the stroma.
  • a high percentage (e.g., greater than 5%) of cytotoxic T cells (e.g., CD8+ T cells) in the tumor and a low percentage (e.g., less than 5%) of cytotoxic T cells (e.g., CD8+ T cells) in the stroma may be indicative of an inflamed tumor phenotype.
  • a low percentage of cytotoxic T cells (e.g., CD8+ T cells) in both the tumor and the stroma e.g., combined tumor and stroma CD8 percentage of less than 5%
  • a poorly immunogenic tumor phenotype e.g., an immune desert phenotype
  • a low percentage (e.g., less than 5%) of cytotoxic T cells (e.g., CD8+ T cell cells) in the tumor and a high percentage (e.g., greater than 5%) of cytotoxic T cells (e.g., CD8+ T cell cells) in the stroma may be indicative of an immune-excluded tumor phenotype.
  • a tumor-to-stroma CD8 ratio may be determined by dividing CD8 percentage in the tumor over the percentage in the stroma.
  • a tumor-to-stroma CD8 ratio of greater than 1 may be indicative of an inflamed tumor phenotype.
  • a tumor-to-stroma CD8 ratio of less than 1 may be indicative of an immune-excluded tumor.
  • percentages of cytotoxic T cells may be determined by immunohistochemical analysis of CD8 immunostaining.
  • a sample such as a sample with uneven distribution of cytotoxic T cells (e.g., CD8 density across tumor nests have a variance of greater than 10%), may be analyzed to determine the margin-to- stroma CD8 ratio.
  • such ratio may be calculated by dividing CD8 density in the tumor margin over CD8 density in the tumor stroma.
  • an immune excluded tumor exhibits a margin-to-stroma CD8 ratio of greater than 0.5 and less than 1.5.
  • a sample having a margin-to-stroma CD8 ratio of greater than 1.5 may be further analyzed to determine and/or confirm immune phenotyping (e.g., to determine and/or confirm whether the tumor has an immune-excluded phenotype) by evaluating tumor depth.
  • tumor depth may be measured in increments of 20 ⁇ m-200 ⁇ m (e.g., 100 ⁇ m).
  • tumor depth may be determined by pathological analysis and/or digital image analysis.
  • a significant tumor depth may be indicated by a distance of about 2-fold or greater than the depth of the tumor margin.
  • a tumor sample may have a tumor margin depth of 100 ⁇ m and a tumor depth measurement of greater than 200 ⁇ m, such sample would have a tumor depth score of greater than 2, and would therefore have significant tumor depth.
  • significant tumor depth may be indicated by a ratio of 2 or greater as determined by dividing tumor depth by the depth of the tumor margin.
  • tumor depth may be measured in increments corresponding to the depth of the tumor margin. For instance, the tumor depth of a tumor nest having a tumor margin of 100 ⁇ m may be measured in increments of 100 ⁇ m.
  • a tumor sample with significant tumor depth may exhibit shallow penetration by cytotoxic T cells (e.g., the tumor sample having greater than 5% CD8 T cells but does not exhibit tumor penetration beyond one tumor depth increment). In certain embodiments, a tumor sample with significant tumor depth that exhibits shallow CD8 penetration may be indicative of an immune excluded tumor.
  • a tumor phenotype analysis may be conducted according to any part of the exemplary flow chart shown in FIG.38, e.g., using all the steps in that figure.
  • a subject whose cancer exhibits an immune excluded phenotype may be selected for TGF ⁇ inhibitor therapy (e.g., a TGF ⁇ 1 inhibitor such as Ab6).
  • a subject whose cancer exhibits an immune excluded phenotype may be more responsive to a TGF ⁇ inhibitor therapy (e.g., a TGF ⁇ 1 inhibitor such as Ab6).
  • a subject whose cancer exhibits an immune-excluded phenotype may be more responsive to a combination therapy comprising a TGF ⁇ inhibitor, such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), and a second cancer therapy, e.g., a checkpoint inhibitor therapy (e.g., a PD1 or PDL1 antibody).
  • a TGF ⁇ inhibitor therapy e.g., a TGF ⁇ 1 inhibitor such as Ab6
  • a second cancer therapy e.g., a checkpoint inhibitor therapy (e.g., a PD1 or PDL1 antibody).
  • a response to TGF ⁇ inhibitor therapy may be monitored and/or determined using parameters such as any of the ones described above.
  • a change in a distribution of cytotoxic T cells (e.g., CD8+ T cells) in a pre-treatment tumor sample as compared to a corresponding post-treatment sample from the corresponding tumor may be indicative of a therapeutic response to treatment.
  • a change (e.g., increase) of at least 1-fold e.g., 1.1- fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, or greater
  • a change (e.g., increase) of 1.5-fold or greater in the tumor-to- stroma CD8 density ratio between the pre-treatment and post-treatment tumor samples may be indicative of a therapeutic response.
  • the tumor-to-stroma CD8 density ratio may be determined by dividing CD8 cell density in the tumor nest over CD8 cell density in the tumor stroma.
  • a change (e.g., increase) of 1.5-fold or greater in the density of cytotoxic T cells (e.g., CD8+ T cells) in the tumor margin between the pre-treatment and post-treatment tumor samples may be indicative of a therapeutic response.
  • a change (e.g., increase) of 1.5-fold or greater in the tumor depth score of pre-treatment and post-treatment tumor samples may be indicative of a therapeutic response.
  • the TGF ⁇ inhibitor therapy (e.g., a TGF ⁇ 1 inhibitor such as Ab6) achieves at least a 2-fold, e.g., 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or a greater degree of increase in the number of intratumoral T cells, e.g., when used in conjunction with a checkpoint inhibitor such as a PD-(L)1 antibody, relative to pre-treatment.
  • treatment with a TGF ⁇ inhibitor therapy e.g., a TGF ⁇ 1 inhibitor such as Ab6
  • a TGF ⁇ 1 inhibitor such as Ab6 may be continued if a therapeutic response is observed.
  • the pre-treatment and post-treatment samples have comparable tumor depth scores (e.g., variance of less than 0.25 in tumor depth scores of pre-treatment and post-treatment tumor samples) and the samples may be analyzed to determine therapeutic response according to one or more of the parameters described above.
  • the pre-treatment and post-treatment samples have comparable total and compartmental areas (e.g., variance of less than 0.25 in analyzable total and compartmental area of pre- treatment and post-treatment tumor samples) and the samples may be analyzed to determine therapeutic response according to one or more of the parameters described above.
  • percent necrosis in a tumor sample may be assessed by histological and/or digital image analysis, which may reflect the presence or activities of cytotoxic cells in the tumor.
  • percent necrosis in tumor samples may be compared in pre-treatment and post-treatment tumor samples collected from a subject administered a TGF ⁇ inhibitor (e.g., Ab6).
  • TGF ⁇ inhibitor e.g., Ab6
  • increase of greater than 10% in percent necrosis e.g., the proportion of necrotic area to total tissue area in a tumor sample
  • TGF ⁇ inhibitor therapy e.g., TGF ⁇ 1 inhibitor such as Ab6.
  • an increase of 10% or greater in percent necrosis in or near the center of the tumor may be indicative of a therapeutic response.
  • a therapeutic response may be determined according to any part of the exemplary flow chart shown in FIG.39.
  • an increased level of tumor-infiltrating cytotoxic T cells e.g., CD8+ T cells
  • activated cytotoxic T cells following TGF ⁇ inhibitor therapy (e.g., a TGF ⁇ 1 inhibitor such as Ab6) may indicate conversion of an immune-excluded tumor microenvironment toward an immune-infiltrated or “inflamed” microenvironment.
  • an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more in tumor-associated cytotoxic T cell levels following TGF ⁇ inhibitor treatment (e.g., Ab6) as compared to tumor-associated cytotoxic T cell levels before the treatment may be indicative of a reduction or reversal of immune suppression in the cancer.
  • tumor-associated cytotoxic T cell levels following TGF ⁇ inhibitor treatment e.g., Ab6
  • an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more in tumor area comprising immune inflamed tumor nests may be indicative of a reduction or reversal of immune suppression in the cancer.
  • levels of cytolytic proteins such as perforin or granzyme B or proinflammatory cytokines such as IFN ⁇ expressed by the tumor-associated cytotoxic T cells may be measured to determine the activation status of the tumor-associated cytotoxic T cells.
  • an increase of at least 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, or 2-fold, or 5-fold, or more in cytolytic protein levels may be indicative of reduction or reversal of immune suppression in the cancer.
  • a change of at least a 1.5-fold, 2-fold, 5-fold, or 10-fold, or more increase in IFN ⁇ levels may be indicative of a reduction or reversal of immune suppression in the cancer.
  • TGF ⁇ inhibitor therapy e.g., a TGF ⁇ 1 inhibitor such as Ab6
  • treatment with the TGF ⁇ inhibitor therapy is continued if such a reduction or reversal of immune suppression in the cancer is detected.
  • TMEs Tumor-associated lymphocytes associated with TMEs
  • M2 tumor-associated macrophages
  • Most of these cells are monocyte-derived cells that originate in the bone marrow.
  • Intratumoral (e.g., tumor-associated) levels of immunosuppressive cells such as TAMs and MDSCs may also be measured to determine the status of immune suppression in a cancer.
  • a decrease of at least 10%, 15%, 20%, 25%, or more in the level of TAMs may be indicative of reduced or reversal of immune suppression.
  • tumor- associated immune cells may be measured from a biopsy sample from the subject prior to and following TGF ⁇ inhibitor treatment (e.g., Ab6). In certain embodiments, biopsy samples may be obtained between 28 days and 130 days following treatment administration.
  • TGF ⁇ inhibitor treatment e.g., Ab6
  • biopsy samples may be obtained between 28 days and 130 days following treatment administration.
  • TIME is usually associated with the clinical outcome of cancer patients and has been used for estimating cancer prognosis (see, for example, Fridman et al., (2017) Nat Rev Clin Oncol.14(12): 717-734) “The immune contexture in cancer prognosis and treatment”).
  • tissue samples from tumors are collected (e.g., biopsy such as core needle biopsy) for TIL analyses.
  • TILs are analyzed by FACS-based methods.
  • TILs are analyzed by immunohistochemical (IHC) methods.
  • TILs are analyzed by so-called digital pathology (see, for example, Saltz et al., (2016) Cell Reports 23, 181-193.
  • tumor biopsy samples may be used in various DNA- and/or RNA-based assays (e.g. RNAseq or Nanostring) to evaluate the tumor immune contexture.
  • Circulating/circulatory latent-TGF ⁇ may serve as a target engagement biomarker.
  • an activation inhibitor is selected as a therapeutic candidate, for example, such biomarker may be employed to evaluate or confirm in vivo target engagement by monitoring the levels of circulating TGF beta (circulating TGF ⁇ ) before and after administration.
  • a target engagement marker comprising circulating latent TGF ⁇ (e.g., circulating latent TGF ⁇ 1) is measured in a sample.
  • circulating TGF ⁇ 1 in a blood sample e.g., plasma and/or serum
  • a blood sample e.g., plasma and/or serum
  • total circulating TGF ⁇ e.g., total circulating TGF ⁇ 1
  • ELISA enzyme-linked immunosorbent assay
  • reagents such as antibodies that specifically bind the latent form of TGF ⁇ (e.g., TGF ⁇ 1) may be employed to specifically measure circulatory latent TGF ⁇ 1.
  • a majority of the measured circulating TGF ⁇ (e.g., circulating TGF ⁇ 1) is released from a latent complex.
  • the total circulating TGF ⁇ (e.g., circulating TGF ⁇ 1) measured is equivalent to dissociated latent TGF ⁇ (e.g., latent TGF ⁇ 1) in addition to any free TGF ⁇ (e.g., TGF ⁇ 1) present prior to acid treatment, which is known to be only a small fraction of circulating TGF ⁇ 1.
  • circulating latent TGF ⁇ e.g., circulating latent TGF ⁇ 1
  • circulating latent TGF ⁇ e.g., circulating latent circulating TGF ⁇ 1
  • circulating TGF ⁇ can be measured from a blood sample by any of the methods described in or adapted from Mussbacher et al., PLos One. 2017 Dec 8; 12(12):e0188921 and Mancini et al. Transl Res. 2018 Feb; 192: 15-29, the contents of which are hereby incorporated by reference in their entirety.
  • FIG. 1 Aspects of the present disclosure include improved assays for measuring circulatory TGF ⁇ levels. Such assays comprise a sample collection step, sample processing step and measuring step.
  • Sample collection comprises placing a blood sample obtained from a subject (e.g., cancer patient) into a container (e.g., collection tube).
  • the collection tube is a sterile, evacuated glass or plastic tube containing anticoagulant.
  • such tube is about 13 mm times 75 mm in size and has a capacity of about 2.7 mL.
  • the collection tube contains an anticoagulant solution which includes a form of sodium citrate.
  • the anticoagulant solution is so-called CTAD.
  • the CTAD contains buffered trisodium citrate solution, theophylline, adenosine and dipyrudamole.
  • the CTAD may contain 0.11M buffered trisodium citrate solution (pH about 5.0), 15M theophylline, 3.7M adenosine and 0.198M dipyridamole.
  • Such collection tubes may contain an internal silicone coating to minimize contact activation.
  • Such tubes may be equipped with a closing means (e.g., cap or stopper) aimed to protect users from blood which might splatter when the tube is opened.
  • a closing means e.g., cap or stopper
  • Such closure may be a rubber stopper, which may be recessed inside the plastic shield, preventing exposure to blood present on the stopper.
  • Examples of commercially available collection tubes include BD VacutainerTM CTAD Blood Collection Tubes, which is equipped with a HemogardTM closure. The manufacture’s product description suggests that upon collection of blood into the tube, the samples be centrifuged at 1500g for 15 minutes at room/ambient temperature (18-25°C).
  • circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1) in a blood sample is measured by collecting the blood sample in a collection tube that comprises (containing or coated with) an anticoagulant.
  • the collection tube comprises a citrate coating.
  • the collection tube is coated with a solution comprising 0.1-0.5 M buffered trisodium citrate.
  • the collection tube is coated with a solution comprising 10-20 M theophylline.
  • the collection tube is coated with a solution comprising 2-5 M adenosine. In some embodiments, the collection tube is coated with a solution comprising 0.1-0.25 M dipyridamole. In some embodiments, the collection tube is coated with a solution having a pH of 4.0-6.0. In some embodiments, the collection tube is coated with an anticoagulant selected from citrate-theophylline-adenosine-dipyridamole (CTAD), citrate (e.g., sodium citrate), acid-citrate-dextrose (ACD), ethylenediaminetetraacetic acid (EDTA), and heparin. In some embodiments, the collection tube is coated with CTAD.
  • CAD citrate-theophylline-adenosine-dipyridamole
  • ACD acid-citrate-dextrose
  • EDTA ethylenediaminetetraacetic acid
  • heparin heparin. In some embodiments, the collection tube is coated with CTAD.
  • the collection tube is coated with a CTAD solution comprising about 0.11M buffered trisodium citrate solution, about 15 M theophylline, about 3.7 M adenosine, and about 0.198 M dipyridamole.
  • the CTAD solution has a pH of about 5.0.
  • the collection tube is glass.
  • the collection tube has a silicone coating.
  • the collection tube has a Hemogard TM closure.
  • the collection tube has a volume capacity of 2-3 mL (e.g., 2.7 mL).
  • the collection tube is sterile.
  • the collection tube is a BD Vacutainer TM CTAD blood collection tube (Macey et al. Clin Chem.2002 Jun;48(6 Pt 1):891-9).
  • Sample processing refers to any handling or processing of a biological sample (e.g., blood sample) following the sample collection step discussed above.
  • the sample processing step may include, for example, centrifugation, fractionation or separation of sample, pipetting, mechanical agitation (e.g., shaking or mixing), etc.
  • sample processing is carried out to prepare platelet-poor plasma (PP).
  • a PPP fraction may be prepared from a blood sample for the measurement of circulatory TGF ⁇ 1 levels.
  • processing the blood sample comprises incubation and/or centrifugation at a temperature that is lower than room temperature. In some embodiments, processing the blood sample comprises incubation and/or centrifugation at a temperature that is lower than 20 °C, lower than 15 °C, lower than 10 °C, lower than 5 °C, or lower. In some embodiments, processing the blood sample comprises incubation and/or centrifugation at 2-8 °C. In some embodiments, processing the blood sample comprises incubation and/or centrifugation at about 4 °C.
  • processing the blood sample comprises one or more incubation steps as described in Example 4. [269] In some embodiments, processing the blood sample comprises one or more centrifugation steps. In some embodiments, processing the blood sample comprises one or more centrifugation steps carried out at about 4 °C. In some embodiments, processing the blood sample comprises a centrifugation step at a speed of below 1500xg, below 1000xg, below 800xg, below 400xg, below 250xg, below 200xg, or lower. In some embodiments, processing the blood sample comprises a centrifugation step at a speed of about 150xg.
  • processing the blood sample comprises a centrifugation step at a speed of above 1500xg, above 2000xg, above 2500xg, above 5000xg, above 7500xg, above 10000xg, above 12000xg, or higher.
  • processing the blood sample comprises a centrifugation step at a speed of about 2500xg.
  • processing the blood sample comprises a centrifugation step at a speed of about 12000xg.
  • processing the blood sample comprises a first centrifugation step at a speed below 1000xg, and a second centrifugation step at a speed above 2000xg, optionally with one or both steps at about 4 °C.
  • processing the blood sample comprises a first centrifugation step at a speed of about 150xg, and a second centrifugation step at a speed of about 2000xg. In some embodiments, processing the blood sample comprises a first centrifugation step at a speed below 2500xg, and a second centrifugation step at a speed above 10000xg. In some embodiments, processing the blood sample comprises a first centrifugation step at a speed of about 1500xg, and a second centrifugation step at a speed of about 12000xg.
  • processing the blood sample comprises a first centrifugation step at a speed of between 1000xg to 5000xg, and a second centrifugation step at a speed of between 1000xg to 5000xg. In some embodiments, processing the blood sample comprises a first step and a second centrifugation step, wherein the two centrifugation steps are carried out at the same speed. In some embodiments, processing the blood sample comprises a first centrifugation step at a speed of about 2500xg, and a second centrifugation step at a speed of about 2500xg.
  • processing the blood sample comprises one or more centrifugation steps carried out for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, or longer.
  • the blood sample is processed by a first centrifugation step for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, or longer, followed by a second centrifugation step for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, or longer.
  • processing the blood sample comprises a first centrifugation step for about 10 minutes, and a second centrifugation step for about 20 minutes.
  • processing the blood sample comprises a first centrifugation step for about 10 minutes, and a second centrifugation step for about 5 minutes.
  • processing the blood sample comprises transferring the supernatant portion of the sample to a separate tube after the first centrifugation step, and further processing the supernatant in a second centrifugation step.
  • the supernatant portion of the sample following the second centrifugation step is used for measuring circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1) levels.
  • TGF ⁇ (e.g., circulating latent TGF ⁇ 1) levels may be determined using Bio-Plex ProTM TGF- ⁇ Assays (Strauss et al.
  • processing the blood sample comprises a first centrifugation step of 100-500xg for 5-25 minutes, and a second centrifugation step of 1000-3000xg for 10-40 minutes, each step is optionally carried out at about 4°C.
  • processing the blood sample comprises a first centrifugation step of 1000- 3000xg for 5-25 minutes, and a second centrifugation step of 1000-3000xg for 10-40 minutes, each step is optionally carried out at about 4°C.
  • processing the blood sample comprises a first centrifugation step of 1000-3000xg for 5-25 minutes, and a second centrifugation step of 5000-15000xg for 2-10 minutes, each step is optionally carried out at about 4°C.
  • processing the blood sample comprises a first centrifugation step of 1500xg for 10 minutes, and a second centrifugation step of 12000xg for 5 minutes, optionally with one or both steps carried out at about 4°C.
  • the blood sample is processed by a first centrifugation step of 2500xg for 10 minutes, followed by a second centrifugation step of 2500xg for 10 minutes, optionally with one or both steps at about 4°C.
  • one or more additional centrifugation step is applied.
  • the present disclosure provides methods of determining and monitoring the level of circulating latent TGF ⁇ in a sample obtained from a patient, such that unwanted or inadvertent TGF ⁇ activation associated with sample processing and preparation is reduced.
  • the methods disclosed herein may be used to determine or monitor the level of circulating latent TGF ⁇ 1, e.g., by using sample collection methods disclosed herein and/or by normalzing to control markers of platelet activation during collection, e.g., PF4 levels.
  • the resulting samples may be used to carry out one or more measuring steps for circulatory TGF ⁇ .
  • the present disclosure provides, in various embodiments, a method for measuring circuating TGF ⁇ levels in a blood sample, wherein the method comprises a collection step and a processing step, each of which is carried out at 2-8°C using a CTAD collection tube.
  • the processing step may comprise two centrifugation steps as described above, to generate a PPP fraction from the blood sample.
  • the resulting PPP is used to measure TGF ⁇ levels.
  • total TGF ⁇ levels which include both the active and latent TGF ⁇ forms, are measured.
  • active TGF ⁇ (mature growth factor) levels are measured.
  • latent TGF ⁇ levels are measured.
  • a majority of the TGF ⁇ measured in an acidified sample is from circulating latent TGF ⁇ .
  • the level of the TGF ⁇ 1 isoform is selectively measured.
  • the measuring step may include acidification of the sample to release TGF ⁇ (i.e., mature growth factor) from the latent complex (i.e., proTGF ⁇ , such as proTGF ⁇ 1).
  • ELISA-based methods may be employed to then capture and detect/quantitate TGF ⁇ present in the sample.
  • circulatory TGF ⁇ levels may serve as a predictive biomarker.
  • circulatory TGF ⁇ levels may serve as a predictive biomarker for therapeutic response to a checkpoint inhibitor therapy.
  • high baseline levels of circulatory TGF ⁇ levels e.g., in the plasma
  • may be predictive of poor therapeutic response to a checkpoint inhibitor therapy e.g., pembrolizumab
  • the treatment regimen may include administration of a therapy that includes a TGF ⁇ inhibitor, such as TGF ⁇ 1 inhibitor.
  • TGF ⁇ inhibitors include, for example, monoclonal antibodies that bind the latent form of TGF ⁇ (i.e., proTGF ⁇ , such as proTGF ⁇ 1) thereby preventing the release of the growth factor, such as Ab6 and other anitbodies that work by the same mechanism of action (see, for example, WO 2000/014460, WO 2000/041390, PCT/2021/012930, WO 2018/013939, WO 2020/160291).
  • the TGF ⁇ inhibitors include neutralizing antibodies and engineered constructs that incorporate an antigen-binding fragment thereof.
  • the TGF ⁇ inhibitors also include so-called ligand traps, which comprise the ligand binding fragment(s) of the TGF ⁇ receptor(s). Examples of ligand traps include M7824 (bintrafusp alpha) and AVID200.
  • the TGF ⁇ inhibitors also include low molecular weight receptor kinase inhibitors, such as ALK5 inhibitors.
  • the patient being administered the treatment regimen is diagnosed with, at risk of developing, or suspected to have a TGF ⁇ -related disease, such as cancer, myeloproliferative disorders (such as myelofibrosis), fibrosis and immune disorders.
  • the present disclosure provides a TGF ⁇ inhibitor for use in the treatment of a TGF ⁇ -related disease in a subject, wherein the treatment comprises administration of a composition comprising a TGF ⁇ inhibitor in an amount sufficient to treat the disease, wherein the treatment further comprises determination of circulatory TGF ⁇ levels in accordance with the disclosure herein.
  • the treatment further comprises determination of circulatory MDSCs.
  • circulatory MDSC levels are determined by measuring cell-surface marker(s).
  • the cell-surface marker is LRRC33.
  • the patient is a cancer patient, wherein optionally the cancer comprises a solid tumor, such as locally advanced or metastatic tumor.
  • the patient previously received a cancer therapy, wherein the cancer therapy is checkpoint inhibitor, radiation therapy and/or chemotherapy.
  • the subject was unresponsive or refractory to the cancer therapy, wherein optionally the cancer therapy comprises a checkpoint inhibitor (e.g., chechpoint inhibitor- resistant).
  • the tumor is refractory to the cancer therapy.
  • the patient is na ⁇ ve to a cancer therapy, e.g., a checkpoint inhibitor (i.e., a checipoint inhibitor-na ⁇ ve patient).
  • the checkpoint inhibitor-na ⁇ ve patient is diagnosed with a type of cancer that has statistically shown to have low response rates (e.g., below 30%, below 25%, below 20%, below 15%, etc.) to checkpoint inhibitors, such as anti-PD-(L)1.
  • the solid tumor has an immune excluded phenotype.
  • the solid tumor has low expression of PD-L1.
  • the present disclosure provides methods of treating a TGF ⁇ -related disorder, comprising monitoring the level of circulating TGF ⁇ , e.g., circulating latent TGF ⁇ (e.g., TGF ⁇ 1) in a sample obtained from a patient (e.g., in the blood, e.g., plasma and/or serum, of a patient) receiving a TGF ⁇ inhibitor.
  • circulating TGF ⁇ e.g., circulating latent TGF ⁇ (e.g., TGF ⁇ 1) may be measured in plasma samples collected from the subject.
  • measuring TGF ⁇ e.g., circulating latent TGF ⁇ (e.g., TGF ⁇ 1) from the plasma may reduce the risk of inadvertently activating TGF ⁇ , such as that observed during serum preparations and/or processing.
  • the present disclosure includes a TGF ⁇ inhibitor for use in the treatment of diseases such as cancer, myelofibrosis, and fibrosis, in a subject, wherein the treatment comprises a step of measuring circulating TGF ⁇ levels from a plasma sample collected from the subject. Such samples may be collected before and/or after administration of a TGF ⁇ inhibitor to treat such diseases.
  • the level of circulating TGF ⁇ may be monitored alone or in conjunction with one or more of the biomarkers disclosed herein (e.g., MDSCs).
  • circulating TGF ⁇ e.g., circulating latent TGF ⁇ 1
  • the TGF ⁇ inhibitor may be administered alone or in conjunction with an additional cancer therapy.
  • the treatment may be administered to a subject afflicted with a TGF ⁇ -related cancer or myeloproliferative disorder.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1-selective antibody or antigen-binding fragment thereof encompassed in the current disclosure (e.g., Ab6).
  • the TGF ⁇ inhibitor is an isoform-non-selective TGF ⁇ inhibitor (such as low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, and ligand traps, e.g., TGF ⁇ 1/3 inhibitors).
  • the TGF ⁇ inhibitor is an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ . e.g., selective inhibition of TGF ⁇ 1 and/or TGF ⁇ 3).
  • the additional cancer therapy may comprise chemotherapy, radiation therapy (including radiotherapeutic agents), a cancer vaccine, or an immunotherapy, such as a checkpoint inhibitor therapy, e.g., an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody.
  • the checkpoint inhibitor therapy is selected from the group consisting of ipilimumab (e.g., Yervoy®); nivolumab (e.g., Opdivo®); pembrolizumab (e.g., Keytruda®); avelumab (e.g., Bavencio®); cemiplimab (e.g., Libtayo®); atezolizumab (e.g., Tecentriq®); budigalimab (ABBV-181); and durvalumab (e.g., Imfinzi®).
  • ipilimumab e.g., Yervoy®
  • nivolumab e.g., Opdivo®
  • pembrolizumab e.g., Keytruda®
  • avelumab e.g., Bavencio®
  • cemiplimab e.g., Lib
  • circulating TGF ⁇ may be measured in a sample obtained from a subject (e.g., whole blood or a blood component).
  • the circulating latent TGF ⁇ levels may be measured within 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 21, 22, 25, 28, 30, 35, 40, 45, 48, 50, or 56 days following administration of the TGF ⁇ inhibitor to a subject, e.g., up to 56 days after administration of a therapeutic dose of a TGF ⁇ inhibitor.
  • the circulating latent TGF ⁇ levels may be measured about 8 to about 672 hours following administration of a therapeutic dose of a TGF ⁇ inhibitor.
  • the circulating latent TGF ⁇ levels may be measured about 72 to about 240 hours (e.g., about 72 to about 168 hours, about 84 to about 156 hours, about 96 to about 144 hours, about 108 to about 132 hours) following administration of a therapeutic dose of a TGF ⁇ inhibitor.
  • the circulating latent TGF ⁇ levels may be measured about 120 hours following administration of a therapeutic dose of a TGF ⁇ inhibitor.
  • the circulating latent TGF ⁇ levels may be measured by any method known in the art (e.g., ELISA).
  • circulating TGF ⁇ levels are measured from a blood sample (e.g., a plasma sample).
  • the present disclosure encompasses a method of treating cancer in a subject, wherein the treatment comprises determining a level of circulating TGF ⁇ in the subject prior to administering a TGF ⁇ inhibitor, administering to the subject a therapeutically effective amount of the TGF ⁇ inhibitor, and determining a level of circulating TGF ⁇ in the subject after administration.
  • the circulating TGF ⁇ level is determined or has been determined by processing a blood sample from the subject below room temperature in a sample tube coated with an anticoagulant.
  • a method of treating a cancer or other TGF-related disorder comprises administering a TGF ⁇ inhibitor (e.g., an anti-TGF ⁇ 1 antibody) to a patient in need thereof and confirming the level of target engagement by the inhibitor.
  • determining the level of target engagement comprises determining the levels of circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1) in a sample obtained from a patient (e.g., in the blood or a blood component of a patient) receiving the TGF ⁇ inhibitor.
  • an increase in circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1) after administration of the TGF inhibitor indicates target engagement.
  • the present disclosure provides a method of determining targeting engagement in a subject having cancer, comprising determining a level of circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1) in the subject prior to administering a TGF ⁇ inhibitor, administering to the subject a therapeutically effective amount of the TGF ⁇ inhibitor, and determining a level of circulating TGF ⁇ in the subject after administration.
  • a level of circulating TGF ⁇ e.g., circulating latent TGF ⁇ 1
  • an increase in circulating TGF ⁇ levels e.g., circulating latent TGF ⁇ 1 levels
  • an increase in circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1) after administration of the TGF ⁇ inhibitor indicates target engagement, wherein the increase is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, or more over baseline level.
  • the circulating TGF ⁇ levels are determined or have been determined by processing a blood sample from the subject below room temperature in a sample tube coated with an anticoagulant.
  • further therapeutically effective amount of the TGF ⁇ inhibitor are administered if target engagement is detected.
  • the present disclosure also provides methods of using circulating TGF ⁇ levels (e.g., circulating latent TGF ⁇ 1 levels) to predict therapeutic response, as well as for informing further treatment decisions (e.g., by continuing treatment if an increase is observed).
  • an additional dose of the TGF ⁇ inhibitor e.g., an anti-TGF ⁇ 1 antibody
  • the method of determining therapeutic efficacy comprises determining a level of circulating TGF ⁇ in the subject prior to administering a TGF ⁇ inhibitor, administering to the subject a therapeutically effective amount of the TGF ⁇ inhibitor, and determining a level of circulating TGF ⁇ in the subject after administration.
  • circulating TGF ⁇ levels are measured from a blood sample.
  • the circulating TGF ⁇ levels are determined or have been determined by processing the blood sample from the subject below room temperature in a sample tube coated with an anticoagulant.
  • further therapeutically effective amount of the TGF ⁇ inhibitor are administered if efficacy is detected.
  • levels of circulating TGF ⁇ e.g., circulating latent TGF ⁇ 1 are determined to inform treatment and predict therapeutic efficacy in subjects administered a TGF ⁇ inhibitor such as a TGF ⁇ 1-selective inhibitor described herein.
  • a TGF ⁇ inhibitor e.g., Ab6 is administered alone or concurrently (e.g., simultaneously), separately, or sequentially with an additional cancer therapy, e.g., a checkpoint inhibitor therapy, such that the amount of TGF ⁇ 1 inhibition administered is sufficient to increase the levels of circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1) as compared to baseline levels.
  • Circulating TGF ⁇ levels e.g., circulating latent TGF ⁇ 1
  • Circulating TGF ⁇ levels may be measured prior to or after each treatment such that an increase in circulating latent-TGF ⁇ levels (e.g., latent TGF ⁇ 1) following the treatment indicates therapeutic efficacy.
  • circulating TGF ⁇ levels may be measured prior to and after the administration of a TGF ⁇ inhibitor (e.g., Ab6) and an increase in circulating TGF ⁇ levels (e.g., latent TGF ⁇ 1) following the treatment predicts therapeutic efficacy.
  • treatment is continued if an increase in circulating TGF ⁇ is detected.
  • circulating TGF ⁇ levels may be measured prior to and following administration of a first dose of a TGF ⁇ inhibitor such as a TGF ⁇ 1 inhibitor described herein (e.g., Ab6), and an increase in circulating TGF ⁇ levels (e.g., circulating latent TGF ⁇ 1) following the administration predicts therapeutic efficacy and further warrants administration of a second or more dose(s) of the TGF ⁇ inhibitor.
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1 inhibitor described herein (e.g., Ab6)
  • circulating TGF ⁇ levels may be measured prior to and after a combination treatment of TGF ⁇ inhibitor such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), and an additional therapy (e.g., a checkpoint inhibitor therapy), administered concurrently (e.g., simultaneously), separately, or sequentially, and a change in circulating latent-TGF ⁇ levels following the treatment predicts therapeutic efficacy.
  • TGF ⁇ inhibitor such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6)
  • an additional therapy e.g., a checkpoint inhibitor therapy
  • treatment is continued if an increase is detected.
  • an increase in circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1) after administration of the TGF ⁇ inhibitor indicates therapeutic efficacy, wherein the increase is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, or more, as compared to baseline.
  • the increase in circulating TGF ⁇ levels (e.g., circulating latent TGF ⁇ 1) following a combination treatment may warrant continuation of treatment.
  • circulating TGF ⁇ levels are measured from a blood sample, wherein the blood sample is optionally processed below room temperature in a sample tube coated with an anticoagulant.
  • the current disclosure provides a method of treating a cancer in a subject, comprising administering a second dose of a TGF ⁇ inhibitor to a subject having an elevated level of circulating TGF ⁇ after receiving a first dose the TGF ⁇ inhibitor, wherein the level of TGF ⁇ has been measured by processing a blood sample from the subject below room temperature in a sample tube coated with an anticoagulant.
  • the current disclosure provides a method of treating a cancer in a subject comprising determining a level of circulating TGF ⁇ in the subject prior to administering a TGF ⁇ inhibitor, administering to the subject a first dose of TGF ⁇ inhibitor, determining a level of circulating TGF ⁇ in the subject after administration, and administering a second dose of the TGF ⁇ inhibitor to the subject if the level of circulating TGF ⁇ is elevated.
  • measuring the level of circulating TGF ⁇ comprises processing a blood sample from the subject below room temperature in a sample tube coated with an anticoagulant.
  • the level of circulating TGF ⁇ after the first dose of the TGF ⁇ inhibitor is elevated by at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more as compared to baseline (e.g., the level of circulating TGF ⁇ before the first dose of the TGF ⁇ inhibitor).
  • the circulating TGF ⁇ is latent TGF ⁇ .
  • the circulating TGF ⁇ is circulating TGF ⁇ 1.
  • the cancer comprises a solid tumor, wherein optionally the solid tumor is selected from: melanoma (e.g., metastatic melanoma), triple-negative breast cancer, HER2-positive breast cancer, colorectal cancer (e.g., microsatellite stable-colorectal cancer), lung cancer (e.g., metastatic non-small cell lung cancer, small cell lung cancer), esophageal cancer, pancreatic cancer, bladder cancer, kidney cancer (e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC), including clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC, uterine cancer, prostate cancer, stomach cancer (e.g., gastric cancer), head and neck squamous cell cancer, urothelial carcinoma (e.g., metastatic urothelial carcinoma), hepatocellular carcinoma, or thyroid cancer.
  • melanoma e.g., metastatic mel
  • the current disclosure encompasses a method of treating a TGF ⁇ -related disorder comprising administering a therapeutically effective amount of a TGF ⁇ inhibitor to a subject having a TGF ⁇ -related disorder, wherein the therapeutically effective amount is an amount sufficient to increase the level of circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1).
  • the TGF ⁇ inhibitor is a TGF ⁇ activation inhibitor.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1 inhibitor (e.g., Ab6).
  • the circulating TGF ⁇ is latent TGF ⁇ 1.
  • the therapeutically effective amount of the TGF ⁇ inhibitor is between 0.1-30 mg/kg per dose. In some embodiments, therapeutically effective amount of the TGF ⁇ inhibitor (e.g., Ab6) is between 1-30 mg/kg per dose. In some embodiments, the therapeutically effective amount of the TGF ⁇ inhibitor (e.g., Ab6) is between 5-20 mg/kg per dose. In some embodiments, the therapeutically effective amount of the TGF ⁇ inhibitor (e.g., Ab6) is between 3-10 mg/kg per dose. In some embodiments, the therapeutically effective amount of the TGF ⁇ inhibitor (e.g., Ab6) is between 1-10 mg/kg per dose.
  • the therapeutically effective amount of the TGF ⁇ inhibitor (e.g., Ab6) is between 2-7 mg/kg per dose. In some embodiments, the therapeutically effective amount of the TGF ⁇ inhibitor (e.g., Ab6) is about 2-6 mg/kg per dose. In some embodiments, the therapeutically effective amount of the TGF ⁇ inhibitor (e.g., Ab6) is about 1 mg/kg per dose. In some embodiments, doses are administered about every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) is dosed weekly, every 2 weeks, every 3 weeks, every 4 weeks, monthly, every 6 weeks, every 8 weeks, bi-monthly, every 10 weeks, every 12 weeks, every 3 months, every 4 months, every 6 months, every 8 months, every 10 months, or once a year.
  • circulating TGF ⁇ levels are measured from a blood sample (e.g., a plasma sample, serum sample, etc.).
  • total circulatory TGF ⁇ 1 e.g., circulating latent TGF ⁇ 1 in blood samples collected from patients may range between about 2-200 ng/mL at baseline, although the measured amounts vary depending on the individuals, health status, and the exact assays being employed.
  • total circulatory TGF ⁇ 1 in blood samples collected from patients may range between about 1 ng/mL to about 10 ng (e.g., about 1000 pg/mL to about 7000 pg/mL).
  • the level of circulating latent TGF ⁇ (e.g., latent TGF ⁇ 1) following administration of a TGF ⁇ inhibitor (e.g., Ab6) is increased by at least 1.5-fold (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or more) as compared to circulating latent TGF ⁇ levels prior to the administration (e.g., baseline).
  • circulating TGF ⁇ levels are measured from a blood sample (e.g., a plasma sample, serum sample, etc.).
  • circulating TGF ⁇ levels may be used to monitor target engagement and pharmacological activity of a TGF ⁇ inhibitor in a subject receiving a TGF ⁇ inhibitor therapy (e.g., a TGF ⁇ activation inhibitor, e.g., Ab6).
  • circulating TGF ⁇ levels e.g., circulating latent TGF ⁇ 1 levels
  • a first dose of TGF ⁇ inhibitor e.g., Ab6
  • circulating TGF ⁇ levels may be measured prior to and after administration of a first dose of TGF ⁇ inhibitor (e.g., Ab6) such that an increase in circulating TGF ⁇ levels (e.g., circulating latent TGF ⁇ 1) following the administration indicates therapeutic efficacy.
  • TGF ⁇ inhibitor e.g., Ab6
  • treatment is continued if an increase in circulating TGF ⁇ levels (e.g., circulating latent TGF ⁇ 1) following administration of a TGF ⁇ inhibitor (e.g., Ab6) is detected.
  • circulating TGF ⁇ levels e.g., circulating latent TGF ⁇ 1 are measured from a blood sample (e.g., a plasma sample, serum sample, etc.).
  • circulating TGF ⁇ levels may be measured prior to and after administration of a first dose of a TGF ⁇ inhibitor (e.g., Ab6), and an increase in circulating TGF ⁇ levels (e.g., circulating latent TGF ⁇ 1) after the administration indicates target engagement, treatment response, and/or further warrants administration of a second or more dose(s) of the TGF ⁇ inhibitor.
  • a TGF ⁇ inhibitor e.g., Ab6
  • circulating TGF ⁇ levels may be measured prior to and after administration of a first dose of a combination treatment comprising a checkpoint inhibitor therapy and a TGF ⁇ inhibitor such as a TGF ⁇ 1- selective inhibitor (e.g., Ab6), and an increase in circulating TGF ⁇ levels (e.g., circulating latent TGF ⁇ 1) after the administration indicates target engagement, treatment response, and/or further warrants continuation of treatment.
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1- selective inhibitor (e.g., Ab6)
  • the combination therapy comprises a checkpoint inhibitor therapy and a TGF ⁇ inhibitor such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), an isoform-non-selective inhibitor (e.g., low molecular weight ALK5 antagonists), neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3 (e.g., GC1008 and variants), antibodies that bind TGF ⁇ 1/3, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ 1-selective inhibitor e.g., Ab6
  • an isoform-non-selective inhibitor e.g., low molecular weight ALK5 antagonists
  • circulating TGF ⁇ levels are measured from a blood (e.g., plasma sample, serum sample, etc.).
  • circulating TGF ⁇ can be circulating TGF ⁇ 1 or circulating latent TGF ⁇ 1.
  • the circulating TGF ⁇ 1 or circulating latent TGF ⁇ 1 is measured from a blood sample collected from the subject.
  • the blood sample is processed below room temperature in a sample tube containing or coated with an anticoagulant.
  • Smad2 phosphorylation [297] According to the present disclosure, activation of Smad2 may serve as a marker for target engagement and/or therapeutic efficacy.
  • Smad2 activation is detected by measuring a level of Smad2 phosphorylation (p-Smad2) and/or p-Smad2 nuclear translocation.
  • p-Smad2 levels and/or p-Smad2 nuclear translocation is measured by immunohistochemistry.
  • p-Smad2 nuclear translocation is determined by nuclear masking analysis.
  • a method for determining therapeutic efficacy in a subject being treated for cancer comprises determining a level of p-Smad2 nuclear translocation in a tumor sample obtained from the subject prior to administering a therapy comprising a TGF ⁇ inhibitor; administering to the subject one or more doses of the TGF ⁇ inhibitor; and determining a level of p-Smad2 nuclear translocation in a tumor sample obtained from the subject after the administration; wherein a decrease in p-Smad2 nuclear translocation after the administration as compared to before the administration indicates therapeutic efficacy.
  • the p-Smad2 nuclear translocation after the administration is decreased by at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 3- fold, at least 4-fold, at least 5-fold, or more as compared to the p-Smad2 nuclear translocation before the administration.
  • one or more additional doses of the treatment is administered if a decrease in p-Smad2 nuclear translocation is observed.
  • a method for determining target engagement in a subject having cancer comprises determining a level of p-Smad2 nuclear translocation in a tumor sample obtained from the subject prior to administering a therapy comprising a TGF ⁇ inhibitor; administering to the subject one or more doses of the TGF ⁇ inhibitor; determining a level of p-Smad2 nuclear translocation in a tumor sample obtained from the subject after the administration; and wherein a decrease in p-Smad2 nuclear translocation after the administration as compared to before the administration indicates target engagement of the TGF ⁇ inhibitor.
  • the p- Smad2 nuclear translocation after the administration is decreased by at least 1.3-fold, at least 1.5-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, or more as compared to the p-Smad2 nuclear translocation before the administration.
  • one or more additional doses of the treatment is administered if a decrease in p-Smad2 nuclear translocation is observed.
  • a method for treating cancer in a subject or a TGF ⁇ inhibitor for use in treating cancer comprises determining a level of p-Smad2 nuclear translocation in a tumor sample obtained from the subject prior to administering a therapy comprising a TGF ⁇ inhibitor; administering to the subject a first dose of the TGF ⁇ inhibitor; determining a level of p-Smad2 nuclear translocation in a tumor sample obtained from the subject after the administration; and administering to the subject one or more additional doses of the TGF ⁇ inhibitor if the p-Smad2 nuclear translocation after the administration of the first dose is decreased as compared to the p-Smad2 nuclear translocation before the administration of the first dose.
  • the p-Smad2 nuclear translocation after the administration is decreased by at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more as compared to the p-Smad2 nuclear translocation before the administration.
  • the treatment comprises administering one or more additional doses of the treatment if a decrease in p-Smad2 nuclear translocation is observed.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1 inhibitor, e.g., Ab6.
  • Cytokines play an important role in normal immune responses, but when the immune system is triggered to become hyperactive, the positive feedback loop of cytokine production can lead to a “cytokine storm” or hypercytokinemia, a situation in which excessive cytokine production causes an immune response that can damage organs, especially the lungs and kidneys, and even lead to death. Such condition is characterized by markedly elevated proinflammatory cytokines in the serum.
  • TGF ⁇ -directed therapies do not target a specific T cell receptor or its ligand
  • Applicant of the present disclosure reasoned that it was prudent to carry out immune safety assessment, including, for example, in vitro cytokine release assays, in vivo cytokine measurements from plasma samples of non-human primate treated with a TGF ⁇ inhibitor, and platelet assays using human platelets. Exemplary assays were previously described, for instance in PCT/US2021/012969 at Example 23.
  • one or more of the cytokines IL-2, TNF ⁇ , IFN ⁇ , IL-1 ⁇ , CCL2 (MCP-1), and IL-6 may be assayed, e.g., by exposure to peripheral blood mononuclear cell (PBMC) constituents from heathy donors.
  • PBMC peripheral blood mononuclear cell
  • Cytokine response after exposure to an antibody disclosed herein, e.g., Ab6 may be compared to release after exposure to a control, e.g., an IgG isotype negative control antibody. Cytokine activation may be assessed in plate- bound and/or soluble assay formats.
  • Levels of IFN ⁇ , IL-2, IL-1 ⁇ , TNF ⁇ , IL-6, and CCL2 should not exceed 10-fold, e.g., 8-, 6-, 4-, or 2-fold the activation in the negative control.
  • a positive control may also be used to confirm cytokine activation in the sample, e.g., in the PBMCs.
  • these in vitro cytokine release results may be further confirmed in vivo, e.g., in an animal model such as a monkey toxicology study, e.g., a 4-week GLP repeat-dose monkey study as described in PCT/US2021/012969 at Example 24.
  • Human platelets have been reported to express GARP, which can form TGF ⁇ 1 LLCs (Tran et al., 2009. Proc Natl Acad Sci U S A.106(32): 13445–13450).
  • an antibody disclosed herein e.g., Ab6
  • platelet activation is evaluated in vitro, as described in Example 23.
  • platelet aggregation, binding, and activation may be assessed in human whole blood or platelet-rich plasma from healthy donors.
  • Platelet aggregation and binding after exposure to an antibody disclosed herein, e.g., Ab6 may be compared to exposure to a negative control, e.g., saline solution, or a reference sample, e.g., a buffered solution. In certain embodiments, platelet aggregation and binding do not exceed 10% above the aggregation in the negative control. In some embodiments, platelet activation following exposure to an antibody disclosed herein, e.g., Ab6, may be compared to exposure to a positive control, e.g., adenosine diphosphate (ADP).
  • ADP adenosine diphosphate
  • the activation status of platelets may be determined by surface expression of activation markers e.g., CD62P (P-Selectin) and GARP detectable by flow cytometry. Platelet activation should not exceed 10% above the activation in the negative control.
  • in vitro platelet response results may be further confirmed in vivo, e.g., in an animal model such as a monkey toxicology study, e.g., a 4-week GLP repeat-dose monkey study.
  • selection of an antibody or an antigen-binding fragment thereof for therapeutic use may include: identifying an antibody or antigen-binding fragment that meets the criteria of one or more of those described herein; carrying out an in vivo efficacy study in a suitable preclinical model to determine an effective amount of the antibody or the fragment; carrying out an in vivo safety/toxicology study in a suitable model to determine an amount of the antibody that is safe or toxic (e.g., MTD, NOAEL, or any art-recognized parameters for evaluating safety/toxicity); and, selecting the antibody or the fragment that provides at least a three-fold therapeutic window (preferably 6-fold, more preferably a 10-fold therapeutic window, even more preferably a 15-fold therapeutic window).
  • a three-fold therapeutic window preferably 6-fold, more preferably a 10-fold therapeutic window, even more preferably a 15-fold therapeutic window.
  • the in vivo efficacy study is carried out in two or more suitable preclinical models that recapitulate human conditions.
  • preclinical models comprise a TGF ⁇ 1-positive cancer, which may optionally comprise an immunosuppressive tumor.
  • the immunosuppressive tumor may be resistant to a cancer therapy such as CBT, chemotherapy and radiation therapy (including a radiotherapeutic agent).
  • the preclinical models are selected from MBT-2, Cloudman S91 and EMT6 tumor models.
  • Identification of an antibody or antigen-binding fragment thereof for therapeutic use may further include carrying out an immune safety assay, which may include, but is not limited to, measuring cytokine release and/or determining the impact of the antibody or antigen-binding fragment on platelet binding, activation, and/or aggregation.
  • cytokine release may be measured in vitro using PBMCs or in vivo using a preclinical model such as non-human primates.
  • the antibody or antigen-binding fragment thereof does not induce a greater than 10-fold release in IL-6, IFN ⁇ , and/or TNF ⁇ levels as compared to levels in an IgG control sample in the immune safety assessment.
  • assessment of platelet binding, activation, and aggregation may be carried out in vitro using PBMCs.
  • the antibody or antigen-binding fragment thereof does not induce a more than 10% increase in platelet binding, activation, and/or aggregation as compared to buffer or isotype control in the immune safety assessment.
  • the selected antibody or the fragment may be used in the manufacture of a pharmaceutical composition comprising the antibody or the fragment.
  • Such pharmaceutical composition may be used in the treatment of a TGF ⁇ indication in a subject as described herein.
  • the TGF ⁇ indication may be a proliferative disorder, e.g., a TGF ⁇ 1-positive cancer.
  • the invention includes a method for manufacturing a pharmaceutical composition comprising a TGF ⁇ inhibitor, wherein the method includes the step of selecting a TGF ⁇ inhibitor which is tested for immune safety as assessed by immune safety assessment comprising cytokine release assays and optionally further comprising a platelet assay.
  • the TGF ⁇ inhibitor selected by the method does not trigger unacceptable levels of cytokine release (e.g., no more than 10-fold, but more preferably within 2.5-fold as compared to control such as IgG control).
  • the TGF ⁇ inhibitor selected by the method does not cause unacceptable levels of platelet aggregation, platelet activation and/or platelet binding.
  • TGF ⁇ inhibitor is then manufactured at large-scale, for example 250L or greater, e.g., 1000L, 2000L, 3000L, 4000L or greater, for commercial production of the pharmaceutical composition comprising the TGF ⁇ inhibitor.
  • Cancer / malignancies [309] Various cancers involve TGF ⁇ activities, e.g., TGF ⁇ 1 activities, and may be treated with the antibodies, compositions, and methods of the present disclosure.
  • the term “cancer” comprises any of various malignant neoplasms, optionally associated with TGF ⁇ 1-positive cells.
  • Such malignant neoplasms are characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths.
  • the source of TGF ⁇ 1 may vary and may include the malignant (cancer) cells themselves, as well as their surrounding or support cells/tissues, including, for example, the extracellular matrix, various immune cells, and any combinations thereof.
  • Examples of cancer which may be treated in accordance with the present disclosure include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include, but are not limited to, anal carcinoma; bile duct cancer; brain tumor (including glioblastoma); breast cancer, e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC), ductal carcinoma in situ (DCIS); cervical cancer; colorectal cancer; endometrial or uterine carcinoma; esophageal cancer; gastric or gastrointestinal cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumors (GIST); head and neck cancer, e.g.
  • anal carcinoma bile duct cancer
  • brain tumor including glioblastoma
  • breast cancer e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC), ductal carcinoma in situ (DCIS); cervical cancer; colorectal cancer; endometrial or uterine carcinoma; esophageal cancer; gastric or gastrointestinal cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumors (GIST); head and neck cancer, e.g.
  • HNSCC head and neck squamous cell cancer
  • liver cancer e.g., hepatocellular carcinoma (HCC)
  • lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), metastatic NSCLC, adenocarcinoma of the lung, and squamous carcinoma of the lung; melanoma; ovarian cancer; pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC);penile carcinoma; prostate cancer, e.g., castration- resistant prostate cancer (CRPC); renal cell carcinoma (RCC), e.g., clear cell RCC; cancer of the peritoneum; salivary gland carcinoma; thyroid cancer; urothelial carcinoma (UC) of the bladder and urinary tract, including metastatic UC (mUC); urothelial bladder cancer, muscle-invasive bladder cancer (MIBC), and non-muscle-invasive bladder cancer (NMIBC); myeloproliferative neoplasms (
  • a cancer which may be treated in accordance with the present disclosure includes one having high tumor mutational burden.
  • Affirmative identification of cancer as “TGF ⁇ 1-positive” is not required for carrying out the therapeutic methods described herein but is encompassed in some embodiments.
  • certain cancer types are known to be or suspected, based on credible evidence, to be associated with TGF ⁇ 1 signaling.
  • Cancers may be localized (e.g., solid tumors) or systemic.
  • localized refers to anatomically isolated or isolatable abnormalities/lesions, such as solid malignancies, as opposed to systemic disease (e.g., so-called liquid tumors or blood cancers).
  • Certain cancers such as certain types of leukemia (e.g., myelofibrosis) and multiple myeloma, for example, may have both a localized component (for instance the bone marrow) and a systemic component (for instance circulating blood cells) to the disease.
  • cancers may be systemic, such as hematological malignancies.
  • TGF ⁇ 1-positive include but are not limited to, all types of lymphomas/leukemias, carcinomas and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus.
  • lymphomas/leukemias such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancre
  • the cancer may be an advanced cancer, such as a locally advanced solid tumor and metastatic cancer.
  • the cancer may be a cancer having elevated TGF ⁇ 1 levels associated with reactive oxygen species (ROS).
  • the cancer may be a cancer having elevated ROS levels and expressing high levels of TGF ⁇ 1.
  • Antibodies or antigen-binding fragments thereof encompassed by the present disclosure may be used in the treatment of cancer, including, without limitation: myelofibrosis, melanoma, adjuvant melanoma, renal cell carcinoma (RCC), including clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC, bladder cancer, colorectal cancer (CRC) (e.g., microsatellite-stable CRC, mismatch repair deficient colorectal cancer), colon cancer, rectal cancer, anal cancer, breast cancer, triple-negative breast cancer (TNBC), HER2- negative breast cancer, HER2-positive breast cancer, BRCA-mutated breast cancer, hematologic malignancies, non-small cell carcinoma, non-small cell lung cancer/carcinoma (NSCLC), small cell lung cancer/carcinoma (SCLC), extensive-stage small cell lung cancer (ES-SCLC), lymphoma (classical Hodgkin’s
  • any cancer e.g., patients with such cancer
  • TGF ⁇ 1 in which TGF ⁇ 1 is overexpressed or is at least a predominant isoform, as determined by, for example biopsy
  • TGF ⁇ 1 may be either growth promoting or growth inhibitory.
  • SMAD4 wild type tumors may experience inhibited growth in response to TGF ⁇ , but as the disease progresses, constitutively activated type II receptor is typically present. Additionally, there are SMAD4- null pancreatic cancers.
  • antibodies, antigen binding portions thereof, and/or compositions of the present disclosure are designed to selectively target components of TGF ⁇ signaling pathways that function uniquely in one or more forms of cancer.
  • Leukemias, or cancers of the blood or bone marrow that are characterized by an abnormal proliferation of white blood cells, i.e., leukocytes can be divided into four major classifications including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia or acute myeloid leukemia (AML) (AML with translocations between chromosome 10 and 11 [t(10, 11)], chromosome 8 and 21 [t(8;21)], chromosome 15 and 17 [t(15;17)], and inversions in chromosome 16 [inv(16)]; AML with multilineage dysplasia, which includes patients who have had a prior myelodysplastic syndrome (MDS) or myeloproliferative disease that transforms
  • any one of the above referenced TGF ⁇ 1-positive cancer may also be TGF ⁇ 3- positive.
  • tumors that are both TGF ⁇ 1-positive and TGF ⁇ 3-positive may be TGF ⁇ 1/TGF ⁇ 3 co-dominant.
  • such cancer is carcinoma comprising a solid tumor.
  • such tumors are breast carcinoma.
  • the breast carcinoma may be of triple-negative genotype (triple-negative breast cancer).
  • subjects with TGF ⁇ 1-positive cancer have elevated levels of MDSCs.
  • such tumors may comprise MDSCs recruited to the tumor site resulting in an increased number of MDSC infiltrates.
  • elevated levels of MDSCs may be detected in the blood (i.e., circulating MDSCs).
  • subjects with breast cancer show elevated levels of C- Reactive Protein (CRP), an inflammatory marker associated with recurrence and poor prognosis.
  • subjects with breast cancer show elevated levels of IL-6.
  • CRP C- Reactive Protein
  • the TGF ⁇ inhibitors of the disclosure may be used to treat patients suffering from chronic myeloid leukemia, which is a stem cell disease, in which the BCR/ABL oncoprotein is considered essential for abnormal growth and accumulation of neoplastic cells. Imatinib is an approved therapy to treat this condition; however, a significant fraction of myeloid leukemia patients show Imatinib-resistance.
  • TGF ⁇ inhibition achieved by the inhibitor such as those described herein may potentiate repopulation/expansion to counter BCR/ABL-driven abnormal growth and accumulation of neoplastic cells, thereby providing clinical benefit.
  • TGF ⁇ inhibitors such as those described herein may be used to treat multiple myeloma.
  • Multiple myeloma is a cancer of B lymphocytes (e.g., plasma cells, plasmablasts, memory B cells) that develops and expands in the bone marrow, causing destructive bone lesions (i.e., osteolytic lesion).
  • the disease manifests enhanced osteoclastic bone resorption, suppressed osteoblast differentiation (e.g., differentiation arrest) and impaired bone formation, characterized in part, by osteolytic lesions, osteopenia, osteoporosis, hypercalcemia, as well as plasmacytoma, thrombocytopenia, neutropenia and neuropathy.
  • the TGF ⁇ inhibitor therapy described herein may be effective to ameliorate one or more such clinical manifestations or symptoms in patients.
  • the TGF ⁇ 1 inhibitor may be administered to patients who receive additional therapy or therapies to treat multiple myeloma, including those listed elsewhere herein.
  • multiple myeloma may be treated with a TGF ⁇ inhibitor such as an isoform-specific context-independent inhibitor, e.g., Ab6, in combination with a myostatin inhibitor (such as an antibody disclosed in WO 2017/049011, e.g., apitegromab, also known as SRK-015) or an IL-6 inhibitor.
  • a TGF ⁇ inhibitor such as an isoform-specific context-independent inhibitor, e.g., Ab6
  • a myostatin inhibitor such as an antibody disclosed in WO 2017/049011, e.g., apitegromab, also known as SRK-015
  • an IL-6 inhibitor such as an antibody disclosed in WO 2017/049011, e.g., apitegromab, also known as SRK-015
  • the TGF ⁇ inhibitor may be used in conjunction with traditional multiple myeloma therapies, such as bortezomib, lenalidomide, carfilzomib, pomalidomide, thalidomide, doxorubicin, corticosteroids (e.g., dexamethasone and prednisone), chemotherapy (e.g., melphalan), radiation therapy (including radiotherapeutic agents), stem cell transplantation, plitidepsin, elotuzumab, Ixazomib, masitinib, and/or panobinostat.
  • traditional multiple myeloma therapies such as bortezomib, lenalidomide, carfilzomib, pomalidomide, thalidomide, doxorubicin, corticosteroids (e.g., dexamethasone and prednisone), chemotherapy (e.g., melphalan), radiation therapy (including radiotherapeutic agents),
  • carcinomas which may be treated by the methods of the present disclosure include, but are not limited to, papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and sinonasal undifferentiated carcinoma.
  • sarcomas include, but are not limited to, soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosar
  • TGF ⁇ inhibitors such as those described herein may be suited for treating malignancies involving cells of neural crest origin.
  • Cancers of the neural crest lineage include, but are not limited to: melanoma (cancer of melanocytes), neuroblastoma (cancer of sympathoadrenal precursors), ganglioneuroma (cancer of peripheral nervous system ganglia), medullary thyroid carcinoma (cancer of thyroid C cells), pheochromocytoma (cancer of chromaffin cells of the adrenal medulla), and MPNST (cancer of Schwann cells).
  • antibodies and methods of the disclosure may be used to treat one or more types of cancer or cancer-related conditions that may include, but are not limited to, colon cancer, renal cancer, breast cancer, malignant melanoma, urothelial carcinoma, and glioblastoma (Schlingensiepen et al., 2008. Cancer Res.177: 137- 50; Ouhtit et al., 2013. J Cancer.4 (7): 566-572.
  • Immunological Characteristics [322] Under normal conditions, regulatory T cells (Tregs) represent a small subset of the overall CD4-positive lymphocyte population and play key roles for maintaining immune system in homeostasis. In nearly all cancers, however, the number of Tregs is markedly increased.
  • Tregs play an important role in dampening immune responses in healthy individuals, an elevated number of Tregs in cancer has been associated with poor prognosis. Elevated Tregs in cancer may dampen the host’s anti-cancer immunity and may contribute to tumor progression, metastasis, tumor recurrence and/or treatment resistance.
  • human ovarian cancer ascites are infiltrated with Foxp3+ GARP+ Tregs (Downs-Canner et al., Nat Commun. 2017, 8: 14649).
  • Tregs positively correlated with a more immunosuppressive and more aggressive phenotype in advanced hepatocellular carcinoma (Kalathil et al., Cancer Res.2013, 73(8): 2435-44).
  • Tregs can suppress the proliferation of effector T cells.
  • Tregs exert contact-dependent inhibition of immune cells (e.g., na ⁇ ve CD4+ T cells) through the production of TGF ⁇ 1.
  • immune cells e.g., na ⁇ ve CD4+ T cells
  • Increasing lines of evidence suggest the role of macrophages in tumor/cancer progression. The present disclosure encompasses the notion that this is in part mediated by TGF ⁇ activation, especially TGF ⁇ 1 activation, in the tumor microenvironment.
  • Bone marrow-derived monocytes are recruited to tumor sites in response to tumor-derived cytokines/chemokines (such as CCL2, CCL3 and CCL4), where monocytes undergo differentiation and polarization to acquire pro-cancer phenotype (e.g., M2-biased or M2-like macrophages, TAMs).
  • tumor-derived cytokines/chemokines such as CCL2, CCL3 and CCL4
  • monocytes undergo differentiation and polarization to acquire pro-cancer phenotype
  • monocytes isolated from human PBMCs can be induced to polarize into different subtypes of macrophages, e.g., M1 (pro-fibrotic, anti-cancer) and M2 (pro-cancer).
  • M1 pro-fibrotic, anti-cancer
  • M2 pro-cancer
  • M2c and M2d subtypes are found to express elevated LRRC33 on the cell surface.
  • macrophages can be further skewed or activated by certain cytokine exposure, such as M-CSF, resulting in a marked increase in LRRC33 expression, which coincides with TGF ⁇ 1 expression.
  • Increased levels of circulating M-CSF i.e., serum M-CSF concentrations
  • myeloproliferative disease e.g., myelofibrosis
  • TAM macrophage
  • MDSC infiltrate are associated with poor prognosis.
  • the TGF ⁇ inhibitors such as those encompassed herein can be used in the treatment of cancer that is characterized by elevated levels of pro-cancer macrophages and/or MDSCs.
  • the TGF ⁇ inhibitors such as those encompassed herein can be used in the treatment of cancer that is characterized by elevated levels of MDSCs regardless of levels of other macrophages.
  • the LRRC33-arm of the inhibitors may at least in part mediate its inhibitory effects against disease- associated immunosuppressive myeloid cells, e.g., M2-macrophages and MDSCs.
  • tumor-associated MDSCs are also elevated in established tumors (about 10-12% of CD45+ cells) and are markedly reduced (to negligible levels) by inhibiting both PD-1 and TGF ⁇ 1 in the treated animals.
  • a majority of tumor-infiltrating M2 macrophages and MDSCs express cell-surface LRRC33 and/or LRRC33-proTGF ⁇ 1 complex.
  • cell-surface expression of LRRC33 appears to be highly regulated.
  • TGF ⁇ inhibitors described herein are capable of becoming rapidly internalized in cells expressing LRRC33 and proTGF ⁇ 1, and the rate of internalization achieved with the TGF ⁇ inhibitor is significantly higher than that with a reference antibody that recognizes cell- surface LRRC33. Similar results are obtained from primary human macrophages. These observations show that Ab6 can promote internalization upon binding to its target, LRRC33-proTGF ⁇ 1, thereby removing the LRRC33- containing complexes from the cell surface.
  • target engagement by a TGF ⁇ inhibitor of the present disclosure e.g., Ab6 may induce antibody-dependent downregulation of the target protein (e.g., cell-associated proTGF ⁇ 1 complexes).
  • the TGF ⁇ inhibitors of the disclosure may inhibit the LRRC33 arm of TGF ⁇ 1 via dual mechanisms of action: i) blocking the release of mature growth factor from the latent complex; and, ii) removing LRRC33-proTGF ⁇ 1 complexes from cell-surface via internalization.
  • the antibodies may target cell- associated latent proTGF ⁇ 1 complexes, augmenting the inhibitory effects on the target cells, such as M2 macrophages (e.g., TAMs), MDSCs, and Tregs.
  • the antibodies that are capable of targeting multiple arms of TGF ⁇ 1 function, such as those described herein, should provide a particular functional advantage.
  • Many human cancers are known to cause elevated levels of MDSCs in patients, as compared to healthy control (reviewed, for example, in Elliott et al., (2017) “Human tumor-infiltrating myeloid cells: phenotypic and functional diversity” Frontiers in Immunology, Vol.8, Article 86).
  • PBMC peripheral blood mononuclear cell
  • tissue samples e.g., tumor biopsy
  • frequency of or changes in the number of MDSCs may be measured as: percent (%) of total PBMCs, percent (%) of CD14+ cells, percent (%) of CD45+ cells; percent (%) of mononuclear cells, percent (%) of total cells, percent (%) of CD11b+ cells, percent (%) of monocytes, percent (%) of non-lymphocytic MNCs, percent (%) of KLA-DR cells, using suitable cell surface markers (phenotype).
  • macrophage infiltration into a tumor may also signify effectiveness of a therapy.
  • effector T cells e.g., CD8+ T cells
  • Intratumoral effector T cells may lead to recruitment of phagocytic monocytes/macrophages that clean up cell debris.
  • TGF ⁇ 1 inhibitors of the present disclosure may be used to promote effector T-cell infiltration into tumors.
  • the TGF ⁇ 1 inhibitors of the present disclosure may be used to increase non-M2 macrophages associated with tumor.
  • CBT checkpoint blockade therapy
  • Retrospective analysis of urothelial cancer and melanoma tumors has recently implicated TGF ⁇ activation as a potential driver of primary resistance, very likely via multiple mechanisms including exclusion of cytotoxic T cells from the tumor as well as their expansion within the tumor microenvironment (immune exclusion).
  • TGF ⁇ pathway inhibition As a promising avenue for overcoming primary resistance to CBT.
  • therapeutic targeting of the TGF ⁇ pathway has been hindered by dose-limiting preclinical cardiotoxicities, most likely due to inhibition of signaling from one or more TGF ⁇ isoforms.
  • Many tumors lack of primary response to CBT.
  • CD8+ T cells are commonly excluded from the tumor parenchyma, suggesting that tumors may co-opt the immunomodulatory functions of TGF ⁇ signaling to generate an immunosuppressive microenvironment.
  • TGF ⁇ inhibition may unblock the immunosuppression and enable effector T cells (particularly cytotoxic CD8+ T cells) to access and kill target cancer cells.
  • TGF ⁇ inhibition may also promote CD8+ T cell expansion. Such expansion may occur in the lymph nodes and/or in the tumor (intratumorally).
  • TGF ⁇ 1 activation involving regulatory T cells and activated macrophages. It has been reported that TGF ⁇ directly promotes Foxp3 expression in CD4+ T cells, thereby converting them into a regulatory (immunosuppressive) phenotype (i.e., Treg). Moreover, Tregs suppress effector T cell proliferation, thereby reducing immune responses. This process is shown to be TGF ⁇ 1-dependent and likely involves GARP-associated TGF ⁇ 1 signaling. Observations in both humans and animal models have indicated that an increase in Tregs in TME is associated with poor prognosis in multiple types of cancer.
  • M2-polarized macrophages exposed to tumor-derived factors such as M-CSF dramatically upregulate cell-surface expression of LRRC33, which is a presenting molecule for TGF ⁇ 1 (see, for example: PCT/US2018/031759).
  • TAMs tumor-associated macrophages
  • a number of solid tumors are characterized by having tumor stroma enriched with myofibroblasts or myofibroblast-like cells.
  • TGF ⁇ 1 activation is mediated via ECM-associated presenting molecules, e.g., LTBP1 and LTBP3 in the tumor stroma.
  • ECM-associated presenting molecules e.g., LTBP1 and LTBP3 in the tumor stroma.
  • Selective inhibition of TGF ⁇ activation such as TGF ⁇ 1 inhibition, may be sufficient to overcome primary resistance to CBT.
  • an isoform-selective inhibitor of TGF ⁇ 1 may achieve isoform specificity and inhibit latent TGF ⁇ 1 activation.
  • TGF ⁇ pathway such as the TGF ⁇ 1 pathway
  • Pleiotropic effects associated with broad TGF ⁇ pathway inhibition have hindered therapeutic targeting of the TGF ⁇ pathway.
  • Most experimental therapeutics to date e.g., galunisertib, LY3200882, fresolimumab
  • Most experimental therapeutics to date lack selectivity for a single TGF ⁇ isoform, potentially contributing to the dose-limiting toxicities observed in nonclinical and clinical studies.
  • Genetic data from knockout mice and human loss-of-function mutations in the TGF ⁇ 2 or TGF ⁇ 3 genes suggest that the cardiac toxicities observed with nonspecific TGF ⁇ inhibitors may be due to inhibition of TGF ⁇ 2 or TGF ⁇ 3.
  • TGF ⁇ 1 activation with such an antibody has an improved safety profile and is sufficient to elicit robust antitumor responses when combined with PD-1 blockade, enabling the evaluation of the TGF ⁇ 1 inhibitor efficacy at clinically tractable dose levels.
  • TGF ⁇ 1 inhibitor e.g., Ab6
  • a checkpoint inhibitor may have profound effects on the intratumoral immune contexture (e.g., increased levels of tumor-associated CD8+ T cells). These may include an unexpected enrichment of Treg cells by the combination treatment with anti-PD-1/TGF ⁇ 1 inhibitor.
  • the TGF ⁇ inhibitor/anti-PD-1 combination treatment may also beneficially impact the immunosuppressive myeloid compartment. Therefore, a therapeutic strategy that includes targeting of these important immunosuppressive cell types may have a greater effect than targeting a single immunosuppressive cell type (i.e., only Treg cells) in the tumor microenvironment.
  • the TGF ⁇ 1 inhibitors of the present disclosure may be used to reduce tumor- associated immunosuppressive cells, such as M2 macrophages and MDSCs.
  • TGF ⁇ inhibitors such as selective TGF ⁇ 1 inhibitors may be used to counter primary resistance to CBT, thereby rendering the tumor/cancer more susceptible to the CBT.
  • Such effects may be applicable to treating a wide spectrum of malignancy types, where the cancer/tumor is TGF ⁇ 1-positive.
  • tumor/cancer may further express additional isoform, such as TGF ⁇ 3.
  • Non-limiting examples of the latter may include certain types of carcinoma, such as breast cancer.
  • selection criteria for identifying or selecting a patient or patient populations/sub-populations for which the TGF ⁇ 1 inhibitors are likely to achieve clinical benefit.
  • suitable phenotypes of human tumors include: i) a subset(s) are shown to be responsive to CBT (e.g., PD-(L)1 axis blockade); ii) evidence of immune exclusion; and/or, iii) evidence of TGFB1 expression and/or TGF ⁇ signaling.
  • CBT e.g., PD-(L)1 axis blockade
  • evidence of immune exclusion e.g., PD-(L)1 axis blockade
  • evidence of TGFB1 expression and/or TGF ⁇ signaling e.g., TGFB1 expression and/or TGF ⁇ signaling.
  • Various cancer types fit the profile, including, for example, melanoma and bladder cancer.
  • TGF ⁇ inhibitors such as those described herein may be used in the treatment of melanoma.
  • the types of melanoma that may be treated with such inhibitors include, but are not limited to, Lentigo maligna, Lentigo maligna melanoma, Superficial spreading melanoma, Acral lentiginous melanoma, Mucosal melanoma, Nodular melanoma, Polypoid melanoma, and Desmoplastic melanoma.
  • the melanoma is a metastatic melanoma.
  • the melanoma is a cutaneous melanoma.
  • PD-1 antibodies e.g., nivolumab and pembrolizumab
  • nivolumab and pembrolizumab have now become the standard of care for certain types of cancer such as advanced melanoma, which have demonstrated significant activity and durable response with a manageable toxicity profile.
  • LRRC33-expressing cells such as myeloid cells, including myeloid precursors, MDSCs and TAMs, may create or support an immunosuppressive environment (such as TME and myelofibrotic bone marrow) by inhibiting T cells (e.g., T cell depletion), such as CD4 and/or CD8 T cells, which may at least in part underline the observed anti-PD-1 resistance in certain patient populations. Indeed, evidence suggests that resistance to anti-PD-1 monotherapy was marked by failure to accumulate CD8+ cytotoxic T cells and reduced Teff/Treg ratio.
  • the present inventors have recognized that there is a bifurcation among certain cancer patients, such as a melanoma patient population, with respect to LRRC33 expression levels: one group exhibits high LRRC33 expression (LRRC33 high ), while the other group exhibits relatively low LRRC33 expression (LRRC33 low ).
  • the disclosure includes the notion that the LRRC33 high patient population may represent those who are poorly responsive to or resistant to immune checkpoint inhibitor therapy.
  • agents that inhibit LRRC33 such as those described herein, may be particularly beneficial for the treatment of cancer, such as melanoma, lymphoma, and myeloproliferative disorders, that is resistant to checkpoint inhibitor therapy (e.g., anti- PD-1).
  • a cancer/tumor is intrinsically resistant to or unresponsive to an immune checkpoint inhibitor (e.g., primary resistance).
  • an immune checkpoint inhibitor e.g., primary resistance
  • the inventors of the present disclosure contemplate that this may be at least partly due to upregulation of TGF ⁇ 1 signaling pathways, which may create an immunosuppressive microenvironment where checkpoint inhibitors fail to exert their effects. TGF ⁇ 1 inhibition may render such cancer more responsive to checkpoint inhibitor therapy.
  • Non-limiting examples of cancer types which may benefit from a combination of an immune checkpoint inhibitor and a TGF ⁇ 1 inhibitor include: myelofibrosis, melanoma, renal cell carcinoma, bladder cancer, colon cancer, hematologic malignancies, non-small cell carcinoma, non-small cell lung cancer/carcinoma (NSCLC), lymphoma (classical Hodgkin’s and non- Hodgkin’s), head and neck cancer, urothelial cancer e.g., metastatic urothelial carcinoma), cancer with high microsatellite instability, cancer with mismatch repair deficiency, gastric cancer, renal cancer, and hepatocellular cancer.
  • any cancer in which TGF ⁇ 1 is overexpressed, is co-expressed with TGF ⁇ 3, or is the dominant isoform over TGF ⁇ 2/3, as determined by, for example biopsy, may be treated with a TGF ⁇ inhibitor in accordance with the present disclosure.
  • a cancer/tumor becomes resistant over time. This phenomenon is referred to as acquired resistance. Like primary resistance, in some embodiments, acquired resistance is at least in part mediated by TGF ⁇ 1-dependent pathways. TGF ⁇ inhibitors described herein may be effective in restoring anti- cancer immunity in these cases.
  • the TGF ⁇ inhibitors of the present disclosure may be used to reduce recurrence of tumor.
  • the TGF ⁇ inhibitors of the present disclosure may be used to enhance durability of cancer therapy such as CBT.
  • the term “durability” used in the context of therapies refers to the time between clinical effects (e.g., tumor control) and tumor re-growth (e.g., recurrence). Presumably, durability and recurrence may correlate with secondary or acquired resistance, where the therapy to which the patient initially responded stops working.
  • the TGF ⁇ inhibitors of the present disclosure may be used to increase the duration of time the cancer therapy remains effective.
  • the TGF ⁇ inhibitors of the present disclosure may be used to reduce the probability of developing acquired resistance among the responders of the therapy.
  • the TGF ⁇ inhibitors of the present disclosure may be used to enhance progression-free survival in patients.
  • the TGF ⁇ inhibitors described herein may be used to improve disease-free survival time in patients.
  • the TGF ⁇ inhibitors of the present disclosure may be effective for improving patient-reported outcomes, reduced complications, faster time to treatment completion, more durable treatment, longer time between retreatment, etc.
  • the TGF ⁇ inhibitors of the present disclosure may be used to improve overall survival in patients. [347]
  • the TGF ⁇ inhibitors of the present disclosure may be used to improve rates or ratios of complete verses partial responses among the responders of a cancer therapy. Typically, even in cancer types where response rates to a cancer therapy (such as CBT) are relatively high (e.g., ⁇ 35%), CR rates are quite low.
  • TGF ⁇ inhibitors of the present disclosure are therefore used to increase the fraction of complete responders within the responder population.
  • the TGF ⁇ inhibitor may be also effective to enhance or augment the degree of partial response among partial responders.
  • clinical endpoints for the TGF ⁇ inhibitors described herein include those described in the 2018 Food and Drug Administration Guidelines for Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologics, the content of which is incorporated herein in its entirety.
  • combination therapy comprising an immune checkpoint inhibitor and an LRRC33 inhibitor (such as those described herein) may be used with the methods disclosed herein and may be effective to treat such cancer.
  • high LRRC33-positive cell infiltrate in tumors, or otherwise sites/tissues with abnormal cell proliferation, may serve as a biomarker for host immunosuppression and immune checkpoint resistance.
  • effector T cells may be precluded from the immunosuppressive niche which limits the body’s ability to combat cancer.
  • Tregs that express GARP-presented TGF ⁇ 1 suppress effector T cell proliferation.
  • TGF ⁇ 1 is likely a key driver in the generation and maintenance of an immune inhibitory disease microenvironment (such as TME), and multiple TGF ⁇ 1 presentation contexts are relevant for tumors.
  • the combination therapy may achieve more favorable Teff/Treg ratios.
  • the antibodies, or antigen binding portions thereof, that specifically bind a GARP- TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and/or a LRRC33-TGF ⁇ 1 complex, as described herein, may be used in methods for treating cancer in a subject in need thereof, said method comprising administering the antibody, or antigen binding portion thereof, to the subject such that the cancer is treated.
  • the cancer is colon cancer.
  • the cancer is melanoma.
  • the cancer is bladder cancer.
  • the cancer is head and neck cancer.
  • the cancer is lung cancer.
  • the antibodies, or antigen binding portions thereof, that specifically bind a GARP- TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and/or a LRRC33-TGF ⁇ 1 complex, as described herein, may be used in methods for treating solid tumors.
  • solid tumors may be desmoplastic tumors, which are typically dense and hard for therapeutic molecules to penetrate. By targeting the ECM component of such tumors, such antibodies may “loosen” the dense tumor tissue to disintegrate, facilitating therapeutic access to exert its anti-cancer effects.
  • additional therapeutics such as any known anti-tumor drugs, may be used in combination.
  • isoform-specific, context-independent antibodies for fragments thereof that are capable of inhibiting TGF ⁇ 1 activation may be used in conjunction with the chimeric antigen receptor T-cell (“CAR-T”) technology as cell-based immunotherapy, such as cancer immunotherapy for combatting cancer.
  • CAR-T chimeric antigen receptor T-cell
  • the antibodies, or antigen binding portions thereof, that specifically bind a GARP- TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and/or a LRRC33-TGF ⁇ 1 complex, as described herein, may be used in methods for inhibiting or decreasing solid tumor growth in a subject having a solid tumor, said method comprising administering the antibody, or antigen binding portion thereof, to the subject such that the solid tumor growth is inhibited or decreased.
  • the solid tumor is a colon carcinoma tumor.
  • the antibodies, or antigen binding portions thereof useful for treating a cancer is an isoform-specific, context-independent inhibitor of TGF ⁇ 1 activation.
  • such antibodies target a GARP-TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and a LRRC33- TGF ⁇ 1 complex. In some embodiments, such antibodies target a GARP-TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, and a LTBP3-TGF ⁇ 1 complex. In some embodiments, such antibodies target a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and a LRRC33-TGF ⁇ 1 complex. In some embodiments, such antibodies target a GARP-TGF ⁇ 1 complex and a LRRC33-TGF ⁇ 1 complex.
  • TGF ⁇ inhibitors such as context-independent, isoform-specific inhibitors of TGF ⁇ 1, in the treatment of cancer comprising a solid tumor in a subject.
  • TGF ⁇ inhibitors may inhibit the activation of TGF ⁇ 1.
  • TGF ⁇ inhibitors comprise an antibody or antigen-binding portion thereof that binds a proTGF ⁇ 1 complex. The binding can occur when the complex is associated with any one of the presenting molecules, e.g., LTBP1, LTBP3, GARP or LRRC33, thereby inhibiting release of mature TGF ⁇ 1 growth factor from the complex.
  • the solid tumor is characterized by having stroma enriched with CD8+ T cells making direct contact with CAFs and collagen fibers.
  • a tumor may create an immuno-suppressive environment that prevents anti-tumor immune cells (e.g., effector T cells) from effectively infiltrating the tumor, limiting the body’s ability to fight cancer. Instead, such cells may accumulate within or near the tumor stroma.
  • anti-tumor immune cells e.g., effector T cells
  • TGF ⁇ 1 inhibitors disclosed herein may unblock the suppression so as to allow effector cells to reach and kill cancer cells, for example, used in conjunction with an immune checkpoint inhibitor.
  • TGF ⁇ is contemplated to play multifaceted roles in a tumor microenvironment, including tumor growth, host immune suppression, malignant cell proliferation, vascularity, angiogenesis, migration, invasion, metastasis, and chemo-resistance.
  • Each “context” of TGF ⁇ 1 presentation in the environment may therefore participate in the regulation (or dysregulation) of disease progression.
  • the GARP axis is particularly important in Treg response that regulates effector T cell response for mediating host immune response to combat cancer cells.
  • the LTBP1/3 axis may regulate the ECM, including the stroma, where cancer-associated fibroblasts (CAFs) play a role in the pathogenesis and progression of cancer.
  • CAFs cancer-associated fibroblasts
  • TGF ⁇ 1-expressing cells infiltrate the tumor, creating or contributing to an immunosuppressive local environment. The degree by which such infiltration is observed may correlate with worse prognosis. In some embodiments, higher infiltration is indicative of poorer treatment response to another cancer therapy, such as immune checkpoint inhibitors.
  • TGF ⁇ 1-expressing cells in the tumor microenvironment comprise immunosuppressive immune cells such as Tregs and/or myeloid cells.
  • the myeloid cells include, but are not limited to, macrophages, monocytes (tissue resident or bone marrow-derived), and MDSCs.
  • LRRC33-expressing cells in the TME are myeloid-derived suppressor cells (MDSCs).
  • MDSC infiltration e.g., solid tumor infiltrate
  • MDSCs may underline at least one mechanism of immune escape, by creating an immunosuppressive niche from which host’s anti-tumor immune cells become excluded.
  • MDSCs are mobilized by inflammation-associated signals, such as tumor-associated inflammatory factors, Opon mobilization, MDSCs can influence immunosuppressive effects by impairing disease-combating cells, such as CD8+ T cells and NK cells.
  • TGF ⁇ inhibitor such as those described herein may be administered to patients with immune evasion (e.g., compromised immune surveillance) to restore or boost the body’s ability to fight the disease (such as a cancer or tumor). As described in more detail herein, this may further enhance (e.g., restore or potentiate) the body’s responsiveness or sensitivity to another therapy, such as cancer therapy.
  • immune evasion e.g., compromised immune surveillance
  • elevated frequencies (e.g., number) of circulating MDSCs in patients are predictive of poor responsiveness to checkpoint blockade therapies, such as PD-1 antagonists and PD-L1 antagonists.
  • resistance to PD-1 checkpoint blockade in inflamed head and neck carcinoma associates with expression of GM-CSF and Myeloid Derived Suppressor Cell (MDSC) markers.
  • HNC inflamed head and neck carcinoma
  • MDSC Myeloid Derived Suppressor Cell
  • LRRC33 or LRRC33-TGF ⁇ complexes represent a novel target for cancer immunotherapy due to selective expression on immunosuppressive myeloid cells. Therefore, without intending to be bound by particular theory, targeting this complex may enhance the effectiveness of standard-of-care checkpoint inhibitor therapies in the patient population.
  • TGF ⁇ inhibitors such as the isoform-specific TGF ⁇ 1 inhibitor described herein, for the treatment of cancer that comprises a solid tumor.
  • Such treatment comprises administration of a TGF ⁇ inhibitor encompassed by the disclosure, e.g., Ab6, to a subject diagnosed with cancer that includes at least one localized tumor (solid tumor) in an amount effective to treat the cancer.
  • the subject is further treated with a cancer therapy, such as CBT, chemotherapy, and/or radiation therapy (such as a radiotherapeutic agent).
  • a cancer therapy such as CBT, chemotherapy, and/or radiation therapy (such as a radiotherapeutic agent).
  • the TGF ⁇ inhibitor increases the rate/fraction of a primary responder patient population to the cancer therapy.
  • the TGF ⁇ inhibitor increases the degree of responsiveness of primary responders to the cancer therapy.
  • the TGF1 inhibitor increases the ratio of complete responders to partial responders to the cancer therapy.
  • the TGF ⁇ inhibitor increases the durability of the cancer therapy such that the duration before recurrence and/or before the cancer therapy becomes ineffective is prolonged.
  • the TGF ⁇ inhibitor reduces occurrences or probability of acquired resistance to the cancer therapy among primary responders.
  • cancer progression may be at least in part driven by tumor-stroma interaction.
  • CAFs may contribute to this process by secretion of various cytokines and growth factors and ECM remodeling.
  • Factors involved in the process include but are not limited to stromal-cell-derived factor 1 (SCD-1), MMP2, MMP9, MMP3, MMP-13, TNF- ⁇ , TGF ⁇ 1, VEGF, IL-6, M-CSF.
  • CAFs may recruit TAMs by secreting factors such as CCL2/MCP-1 and SDF- 1/CXCL12 to a tumor site; subsequently, a pro-TAM niche (e.g., hyaluronan-enriched stromal areas) is created where TAMs preferentially attach.
  • a pro-TAM niche e.g., hyaluronan-enriched stromal areas
  • TAMs preferentially attach e.g., hyaluronan-enriched stromal areas
  • the antibodies, or antigen binding portions thereof, that specifically bind a GARP- TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and/or a LRRC33-TGF ⁇ 1 complex, as described herein, are administered to a subject having cancer or a tumor, either alone or in combination with an additional agent, e.g., an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist).
  • additional agent e.g., an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist).
  • Other combination therapies which are included in the disclosure are the administration of an antibody, or antigen binding portion thereof, described herein, with radiation (radiation therapy, including radiotherapeutic agents), or a chemotherapeutic agent (chemotherapy).
  • Exemplary additional agents to use with an anti-TGF ⁇ inhibitor include, but are not limited to, a PD-1 antagonist (e.g., a PD-1 antibody), a PDL1 antagonist (e.g., a PDL1 antibody), a PD-L1 or PDL2 fusion protein, a CTLA4 antagonist (e.g., a CTLA4 antibody), a GITR agonist e.g., a GITR antibody), an anti-ICOS antibody, an anti-ICOSL antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody, an anti- LAG3 antibody, an anti-OX40 antibody (OX40 agonist), an anti-CD27 antibody, an anti-CD70 antibody, an anti- CD47 antibody, an anti-41BB antibody, an anti-PD-1 antibody, an anti-CD20 antibody, an anti-CD3 antibody, an anti-CD3/anti-CD20 bispecific or multispecific antibody, an anti-HER2 antibody, an anti-CD79b antibody
  • oncolytic viruses examples include, adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus, senecavirus, enterovirus and vaccinia.
  • the oncolytic virus is engineered for tumor selectivity.
  • determination or selection of therapeutic approach for combination therapy that suits particular cancer types or patient population may involve the following: a) considerations regarding cancer types for which a standard-of-care therapy is available (e.g., immunotherapy-approved indications); b) considerations regarding treatment-resistant subpopulations (e.g., immune excluded); and c) considerations regarding cancers/tumors that are or generally suspected to be “TGF ⁇ 1 pathway-active” or otherwise at least in part TGF ⁇ 1- dependent (e.g., TGF ⁇ 1 inhibition-sensitive). For example, many cancer samples show that TGF ⁇ 1 is the predominant isoform by, for instance, TCGA RNAseq.
  • DNA- and/or RNA-based assays may be used to evaluate the level of TGF ⁇ signaling (e.g. TGF ⁇ 1 signaling) in tumor samples.
  • TGF ⁇ signaling e.g. TGF ⁇ 1 signaling
  • over 50% e.g., over 50%, 60%, 70%, 80% and 90%
  • samples from each tumor type are positive for TGF ⁇ 1 isoform expression.
  • the cancers/tumors that are “TGF ⁇ 1 pathway-active” or otherwise at least in part TGF ⁇ 1-dependent contain at least one Ras mutation, such as mutations in K-ras, N-ras and/or H-ras.
  • the cancer/tumor comprises at least one K-ras mutation.
  • Confirmation of TGF ⁇ 1 expression in clinical samples collected from patients (such as biopsy samples) is not prerequisite to TGF ⁇ 1 inhibition therapy, where the particular condition has been generally known or suspected to involve the TGF ⁇ pathway.
  • a TGF ⁇ inhibitor such as those described herein is administered in conjunction with checkpoint inhibitory therapy to patients diagnosed with cancer for which one or more checkpoint inhibitor therapies are approved or shown effective.
  • bladder urothelial carcinoma such as metastatic urothelial carcinoma
  • squamous cell carcinoma such as head & neck
  • kidney clear cell carcinoma kidney papillary cell carcinoma
  • liver hepatocellular carcinoma lung adenocarcinoma, skin cutaneous melanoma, and stomach adenocarcinoma.
  • the poor responsiveness is due to primary resistance.
  • the cancer that is resistant to checkpoint blockade shows downregulation of TCF7 expression.
  • TCF7 downregulation in checkpoint inhibition-resistant tumor may be correlated with a low number of intratumoral CD8+ T cells.
  • a TGF ⁇ inhibitor such as those described herein may be used in the treatment of chemotherapy- or radiotherapy-resistant cancers.
  • a TGF ⁇ 1 inhibitor e.g., Ab6
  • chemotherapy and/or radiation therapy such as a radiotherapeutic agent
  • the use of the TGF ⁇ 1 inhibitor is advantageous where the cancer (patient) is resistant to such therapy.
  • such cancer comprises quiescent tumor propagating cancer cells (TPCs), in which TGF ⁇ signaling controls their reversible entry into a growth arrested state, which protects TPCs from chemotherapy or radiation therapy (such as a radiotherapeutic agent).
  • TPCs with compromised fail to enter quiescence and thus rendered susceptible to chemotherapy and/or radiation therapy (such as a radiotherapeutic agent).
  • radiation therapy such as a radiotherapeutic agent.
  • cancer includes various carcinomas, e.g., squamous cell carcinomas. See, for example, Brown et al., (2017) “TGF- ⁇ -Induced Quiescence Mediates Chemoresistance of Tumor-Propagating Cells in Squamous Cell Carcinoma.” Cell Stem Cell. 21(5):650-664.
  • a TGF ⁇ inhibitor such as an isoform-selective TGF ⁇ 1 inhibitor (e.g., Ab6) may be used to treat (e.g., reduce) anemia in a subject, e.g., in a cancer patient.
  • an isoform-selective TGF ⁇ 1 inhibitor e.g., Ab6
  • a TGF ⁇ inhibitor such as an isoform-selective TGF ⁇ 1 inhibitor (e.g., Ab6) may be used in combination with a BMP inhibitor (e.g., a BMP6 inhibitor, e.g., a RGMc inhibitor, e.g., any of the RGMc inhibitor disclosed in WO/2020/086736, the content of which is hereby incorporated in its entirety) to treat (e.g., reduce) anemia, e.g., in the subject.
  • a BMP inhibitor e.g., a BMP6 inhibitor, e.g., a RGMc inhibitor, e.g., any of the RGMc inhibitor disclosed in WO/2020/086736, the content of which is hereby incorporated in its entirety
  • anemia e.g., in the subject.
  • the anemia results from reduced or impaired red blood cell production (e.g., as a result of myelofibrosis or cancer), iron restriction (e.g., as a result of cancer or treatment-induced anemia, such as chemotherapy-induced anemia), or both.
  • the combination of a TGF ⁇ inhibitor and a BMP inhibitor (antagonist) may be administered at a therapeutically effective amount or amounts that is/are sufficient to relieve one or more anemia-related symptom and/or complication in the subject, e.g., a cancer patient.
  • the combination of a TGF ⁇ inhibitor and a BMP inhibitor (antagonist) may be administered at a therapeutically effective amount that is sufficient to increase or normalize red blood cell production and/or reduce iron restriction.
  • TGF ⁇ 1 inhibitors e.g., Ab6
  • BMP inhibitors e.g., a BMP6 inhibitor, e.g., a RGMc inhibitor
  • iron-deficiency anemia e.g., chemotherapy-induced anemia
  • the treatment for anemia further comprises administering one or more JAK inhibitor (e.g., Jak1/2 inhibitor, Jak1 inhibitor, and/or Jak2 inhibitor).
  • the BMP inhibitor is an antagonist of the kinase associated with the BMP receptor (e.g., type I receptor and/or type II receptor).
  • the BMP inhibitor is a “ligand trap” that binds (or sequesters) the BMP growth factor(s), including BMP6.
  • the BMP inhibitor is an antibody that neutralizes the BMP growth factor(s), including BMP6.
  • the BMP inhibitor is an inhibitor of a BMP6 co-receptor, such as RGMc.
  • RGMc a BMP6 co-receptor
  • such inhibitor may include an antibody that binds RGMa/c. (Bierer et al. AAPS J.2015 Jul;17(4): 930-938). More preferably, such inhibitor is an antibody that selectively binds RGMc (see, for example, WO 2020/086736).
  • TGF ⁇ -Inhibitor e.g., Ab6
  • the identification/screening/selection of suitable indications and/or patient populations for which TGF ⁇ inhibitors, such as those described herein, are likely to have advantageous therapeutic benefits comprise: i) whether the disease is driven by or dependent predominantly on the TGF ⁇ 1 isoform over the other isoforms in human (or at least co-dominant); ii) whether the condition (or affected tissue) is associated with an immunosuppressive phenotype (e.g., an immune-excluded tumor); and, iii) whether the disease involves both matrix-associated and cell-associated TGF ⁇ 1 function.
  • an immunosuppressive phenotype e.g., an immune-excluded tumor
  • TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3 Differential expression of the three known TGF ⁇ isoforms, namely, TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3, has been observed under normal (healthy; homeostatic) as well as disease conditions in various tissues. Nevertheless, the concept of isoform selectivity has neither been fully exploited nor robustly achieved with conventional approaches that favor pan-inhibition of TGF ⁇ across multiple isoforms. Moreover, expression patterns of the isoforms may be differentially regulated, not only in normal (homeostatic) vs abnormal (pathologic) conditions, but also in different subpopulations of patients.
  • TGF ⁇ 1 and TGF ⁇ 3 are often co-dominant (co-expressed at similar levels) in certain murine syngeneic cancer models (e.g., EMT-6 and 4T1) that are widely used in preclinical studies (e.g., see PCT/US2019/041373 at FIG.21B).
  • TGF ⁇ 1 isoform(s) predominantly expressed under homeostatic conditions may not be the disease-associated isoform(s).
  • tonic TGF ⁇ signaling appears to be mediated mainly by TGF ⁇ 3.
  • TGF ⁇ 1 appears to become markedly upregulated in disease conditions, such as lung fibrosis.
  • TGF ⁇ isoforms may be beneficial to test or confirm relative expression of TGF ⁇ isoforms in clinical samples so as to select suitable therapeutics to which the patient is likely to respond.
  • determination of relative isoform expression may be made post-treatment.
  • patients’ responsiveness e.g., clinical response/benefit
  • relative expression levels of TGF ⁇ isoforms may be correlated with relative expression levels of TGF ⁇ isoforms.
  • TGF ⁇ 3 inhibition may in fact be harmful.
  • mice treated with an isoform-selective inhibitor of TGF ⁇ 3 manifest exacerbation of fibrosis.
  • a significant increase of collagen deposits in liver sections of these animals suggest that inhibition of TGF ⁇ 3 in fact may result in greater dysregulation of the ECM. Without being bound by theory, this suggests that TGF ⁇ 3 inhibition may promote a pro- fibrotic phenotype.
  • a hallmark of pro-fibrotic phenotypes is increased deposition and/or accumulation of collagens in the ECM, which is associated with increased stiffness of tissue ECMs. This has been observed during pathological progression of cancer, fibrosis and cardiovascular disease. Consistent with this, Applicant previously demonstrated the role of matrix stiffness on integrin-dependent activation of TGF ⁇ , using primary fibroblasts grown on silicon- based substrates with defined stiffness (e.g., 5 kPa, 15 kPa or 100 kPa) (see WO 2018/129329). Matrices with greater stiffness enhanced TGF ⁇ 1 activation, and this was suppressed by isoform-specific inhibitors of TGF ⁇ 1.
  • TGF ⁇ 3 may exert opposing effects to TGF ⁇ 1 inhibition by creating a pro-tumor microenvironment, where greater stiffness of the tissue matrix may support cancer progression.
  • TGF- ⁇ -associated extracellular matrix genes link cancer-associated fibroblasts to immune evasion and immunotherapy failure
  • pro-fibrotic effects of TGF ⁇ 3 inhibition observed in a fibrosis model may be applicable to cancer contexts.
  • TGF ⁇ inhibitors with inhibitory potency against TGF ⁇ 3 may not only be ineffective in treating cancer but may in fact be detrimental.
  • TGF ⁇ 3 inhibition is avoided in patients suffering from a cancer type that is statistically highly metastatic.
  • Cancer types that are typically considered highly metastatic include, but are not limited to, colorectal cancer, lung cancer, bladder cancer, kidney cancer (e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC), including clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC, uterine cancer, prostate cancer, stomach cancer, and thyroid cancer.
  • TGF ⁇ 3 inhibition may be best avoided in patients having or are at risk of developing a fibrotic condition and/or cardiovascular disease.
  • Such patients at risk of developing a fibrotic condition and/or cardiovascular disease include, but are not limited to, those with metabolic disorders, such as NAFLD and NASH, obesity, and type 2 diabetes.
  • TGF ⁇ 3 inhibition may be best avoided in patients diagnosed with or at risk of developing myelofibrosis.
  • Those at risk of developing myelofibrosis include those with one or more genetic mutations implicated in the pathogenesis of myelofibrosis.
  • TGF ⁇ 2 As an exercise-induced adipokine, which stimulated glucose and fatty acid uptake in vitro, as well as tissue glucose uptake in vivo; which improved metabolism in obese mice; and, which reduced high fat diet-induced inflammation.
  • lactate a metabolite released from muscle during exercise, stimulated TGF ⁇ 2 expression in human adipocytes and that a lactate-lowering agent reduced circulating TGF ⁇ 2 levels and reduced exercise-stimulated improvements in glucose tolerance.
  • a TGF ⁇ inhibitor may be used in treating a subject that does not have inhibitory activity towards the TGF ⁇ 2 isoform, e.g., to avoid a potentially harmful impact on one or more metabolic functions of a treated subject.
  • a potential link between cancer and various metabolic conditions has been recognized. For example, as reviewed by Braun et al., an enhanced risk of cancer mortality is associated with metabolic syndrome among men (Braun et al. Int J Biol Sci. 2011; 7(7): 1003–1015). Similarly, the authors noted “metabolic dysregulation may play an important role in the etiology and progression of certain cancer types and worse outcome for some cancers.
  • a TGF ⁇ inhibitor may be used in the treatment of a TGF ⁇ -related indication (e.g., cancer) in a subject, wherein, the TGF ⁇ inhibitor inhibits TGF ⁇ 1 but does not inhibit TGF ⁇ 2 at the therapeutically effective dose administered.
  • a TGF ⁇ -related indication e.g., cancer
  • the subject benefits from improved metabolism after such treatment, wherein optionally, the subject has or is at risk of developing a metabolic disease, such as obesity, high fat diet-induced inflammation, and glucose dysregulation (e.g., diabetes).
  • a metabolic disease such as obesity, high fat diet-induced inflammation, and glucose dysregulation (e.g., diabetes).
  • the TGF ⁇ -related indication is cancer, wherein optionally the cancer comprises a solid tumor, such as locally advanced cancer and metastatic cancer.
  • the TGF ⁇ inhibitor is TGF ⁇ 1-selective (e.g., it does not inhibit TGF ⁇ 2 and/or TGF ⁇ 3 signaling at a therapeutically effective dose).
  • a TGF ⁇ 1-selective inhibitor is selected for use in treating a cancer patient.
  • such a treatment avoids TGF ⁇ 3 inhibition to reduce the risk of exacerbating ECM dysregulation (which may contribute to tumor growth and invasiveness) and ii) avoids TGF ⁇ 2 inhibition to reduce the risk of increasing metabolic burden in the patients.
  • Related methods for selecting a TGF ⁇ inhibitor for therapeutic use are also encompassed herein.
  • the disclosure includes methods for selecting a TGF ⁇ inhibitor for use in the treatment of cancer, wherein the TGF ⁇ inhibitor has no or little inhibitory potency against TGF ⁇ 3 (e.g., the TGF ⁇ inhibitor does not target TGF ⁇ 3).
  • the TGF ⁇ inhibitor is a TGF ⁇ 1-selective inhibitor (e.g., antibodies or antigen binding fragments that do not inhibit TGF ⁇ 2 and/or TGF ⁇ 3 signaling at therapeutically effective doses). It is contemplated that this selection strategy may reduce the risk of exacerbating ECM dysregulation in cancer patients and still provide benefits of TGF ⁇ 1 inhibition to treat cancer.
  • the cancer patients are also treated with a cancer therapy, such as immune checkpoint inhibitors.
  • the cancer patient is at risk of developing a metabolic disease, such as fatty liver, obesity, high fat diet-induced inflammation, and glucose or insulin dysregulation (e.g., diabetes).
  • the present disclosure also includes related methods for selecting and/or treating suitable patient populations who may be candidates for receiving a TGF ⁇ inhibitor capable of inhibiting TGF ⁇ 3.
  • Such methods include use of a TGF ⁇ inhibitor capable of inhibiting TGF ⁇ 3 for the treatment of cancer in subjects who are not diagnosed with a fibrotic disorder (such as organ fibrosis), who are not diagnosed with myelofibrosis, who are not diagnosed with a cardiovascular disease and/or those who are not at risk of developing such conditions.
  • a TGF ⁇ inhibitor capable of inhibiting TGF ⁇ 3 for the treatment of cancer in subjects, wherein the cancer is not considered to be highly metastatic.
  • the TGF ⁇ inhibitor capable of inhibiting TGF ⁇ 3 may include pan-inhibitors of TGF ⁇ (such as low molecular weight antagonists of TGF ⁇ receptors, e.g., ALK5 inhibitors, and neutralizing antibodies that bind TGF ⁇ 1/2/3), isoform-non-selective inhibitors such as antibodies that bind TGF ⁇ 1/3 and engineered fusion proteins capable of binding TGF ⁇ 1/3, e.g., ligand traps, and integrin inhibitors (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • pan-inhibitors of TGF ⁇ such as low molecular weight antagonists of TGF ⁇ receptors, e.g., ALK5 inhibitors, and neutralizing antibodies that bind TGF ⁇ 1/2/3
  • TGF ⁇ 3 inhibition may in fact be disease-promoting suggests that patients who have been previously treated with or currently undergoing treatment with a TGF ⁇ inhibitor with inhibitory activity towards TGF ⁇ 3 may benefit from additional treatment with a TGF ⁇ 1-selective inhibitor to counter the possible pro- fibrotic effects of the TGF ⁇ 3 inhibitor.
  • the disclosure includes a TGF ⁇ 1-selective inhibitor for use in the treatment of cancer in a subject, wherein the subject has been treated with a TGF ⁇ inhibitor that inhibits TGF ⁇ 3 in conjunction with a checkpoint inhibitor, comprising the step of: administering to the subject a TGF ⁇ 1-selective inhibitor, wherein optionally the cancer is a metastatic cancer, a desmoplastic tumor, myelofibrosis, and/or, wherein the subject has a fibrotic disorder or is at risk of developing a fibrotic disorder and/or cardiovascular disease, wherein optionally the subject at risk of developing a fibrotic disorder or cardiovascular disease suffers from a metabolic condition, wherein optionally the metabolic condition is NAFLD, NASH, obesity or diabetes.
  • the isoform-selective TGF ⁇ 1 inhibitors are particularly advantageous for the treatment of diseases in which the TGF ⁇ 1 isoform is predominantly expressed relative to the other isoforms (e.g., referred to as TGF ⁇ 1-dominant).
  • TGF ⁇ 1-dominant e.g., a non-limiting list of human cancer clinical samples with relative expression levels of TGFB1 (left), TGFB2 (center) and TGFB3 (right) were previously described in PCT/US2019/041373, e.g., at FIGs.20 and 21A. Each horizontal line across the three isoforms represents a single patient.
  • TGF ⁇ 1 expression is significantly higher in most of these human tumors/cancers than the other two isoforms across many tumor/cancer types, suggesting that TGF ⁇ 1-selective inhibition may be beneficial in these disease types.
  • TGF ⁇ 1-selective inhibition may be beneficial in these disease types.
  • these lines of evidence support the notion that selective inhibition of TGF ⁇ 1 activity may overcome primary resistance to CBT.
  • Generation of highly selective TGF ⁇ 1 inhibitors will also enable evaluation of whether such an approach will address key safety issues observed with pan-TGF ⁇ inhibition, which will be important for assessment of their therapeutic utility.
  • TGF ⁇ 1 inhibitors may not be efficacious, particularly in cancer types in which TGF ⁇ 1 is co-dominant with another isoform or in which TGF ⁇ 2 and/or TGF ⁇ 3 expression is significantly greater than TGF ⁇ 1.
  • TGF ⁇ inhibitors e.g., TGF ⁇ 1 inhibitors, such as a TGF ⁇ 1-selective inhibitor (e.g., Ab6), used in conjunction with a checkpoint inhibitor (e.g., anti-PD-1 antibody), is capable of causing significant tumor regression in the EMT-6 model, which is known to express both TGF ⁇ 1 and TGF ⁇ 3 at similar levels.
  • Such tumor may include, for example, cancers of epithelial origin, i.e., carcinoma (e.g., basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, and adenocarcinoma).
  • carcinoma e.g., basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, and adenocarcinoma
  • TGF ⁇ 1 is predominantly the disease-associated isoform
  • TGF ⁇ 3 supports homeostatic function in the tissue, such as epithelia.
  • the methods of treatment herein comprise the administration of a TGF ⁇ inhibitor that does not inhibit TGF ⁇ 3, e.g., using a TGF ⁇ 1-selective antibody, e.g., Ab6.
  • TGF ⁇ 1-selective antibody e.g., Ab6.
  • TGF ⁇ 1 inhibition therapy can be used in conjunction with one or more of these cancer therapies to increase anti-tumor effects.
  • combination therapy is aimed at converting “cold” tumors (e.g., poorly immunogenic tumors) into “hot” tumors by promoting neo-antigens and facilitating effector cells to attack the tumor.
  • any one or more of the antibodies or fragments thereof described herein may be used to treat poorly immunogenic tumor (e.g., an “immune-excluded” tumor) sensitized with a cancer therapy aimed to promote T cell immunity.
  • poorly immunogenic tumor e.g., an “immune-excluded” tumor
  • a cancer therapy aimed to promote T cell immunity.
  • the immunosuppressive tumor environment may be mediated in a TGF ⁇ 1-dependent fashion.
  • adjunct therapy comprising a TGF ⁇ 1 inhibitor may overcome the immunosuppressive phenotype, allowing T cell infiltration, proliferation, and anti-tumor function, thereby rendering such tumor more responsive to cancer therapy such as CBT.
  • the second inquiry is drawn to identification or selection of patients who have immunosuppressive tumor(s), who are likely to benefit from a TGF ⁇ inhibitor therapy, e.g., a TGF ⁇ 1 inhibitor such as Ab6.
  • a TGF ⁇ inhibitor therapy e.g., a TGF ⁇ 1 inhibitor such as Ab6.
  • the presence or the degree of frequencies of effector T cells in a tumor is indicative of anti-tumor immunity. Therefore, detecting anti-tumor cells such as CD8+ cells in a tumor provides useful information for assessing whether the patient may benefit from a CBT and/or TGF ⁇ 1 inhibitor therapy.
  • Detection may be carried out by known methods such as immunohistochemical analysis of tumor biopsy samples, including digital pathology methods.
  • non-invasive imaging methods are being developed which will allow the detection of cells of interest (e.g., cytotoxic T cells) in vivo.
  • cells of interest e.g., cytotoxic T cells
  • PNAS 111(3): 1108-1113
  • Tavare et al. (2015) J Nucl Med 56(8): 1258-1264
  • Rashidian et al. (2017) J Exp Med 214(8): 2243-2255
  • Beckford Vera et al. (2018) PLoS ONE 13(3): e0193832
  • Tavare et al. (2015) Cancer Res 76(1): 73-82, each of which is incorporated herein by reference.
  • antibodies or antibody-like molecules engineered with a detection moiety can be infused into a patient, which then will distribute and localize to sites of the particular marker (for instance CD8+).
  • a detection moiety e.g., radiolabel
  • CD8+ sites of the particular marker
  • cancer therapy such as CBT
  • Add-on therapy with a TGF ⁇ inhibitor such as those described herein may reduce immuno-suppression thereby rendering the cancer therapy- resistant tumor more responsive to a cancer therapy.
  • Non-invasive in vivo imaging techniques may be applied in a variety of suitable methods for purposes of diagnosing patients; selecting or identifying patients who are likely to benefit from TGF ⁇ inhibitor therapy, e.g., a TGF ⁇ inhibitor therapy; and/or, monitoring patients for therapeutic response upon treatment.
  • Any cells with a known cell-surface marker may be detected/localized by virtue of employing an antibody or similar molecules that specifically bind to the cell marker.
  • cells to be detected by the use of such techniques are immune cells, such as cytotoxic T lymphocytes, regulatory T cells, MDSCs, tumor-associated macrophages, NK cells, dendritic cells, and neutrophils.
  • Antibodies or engineered antibody-like molecules that recognize such markers can be coupled to a detection moiety.
  • Non-limiting examples of suitable immune cell markers include monocyte markers, macrophage markers (e.g., M1 and/or M2 macrophage markers), CTL markers, suppressive immune cell markers, MDSC markers (e.g., markers for G- and/or M-MDSCs), including but are not limited to: CD8, CD3, CD4, CD11b, CD33, CD163, CD206, CD68, CD14, CD15, CD66b, CD34, CD25, and CD47.
  • the in vivo imaging comprises T cell tracking, such as cytotoxic CD8-positive T cells.
  • any one of the TGF ⁇ inhibitors of the present disclosure may be used in the treatment of cancer in a subject with a solid tumor, wherein the treatment comprises: i) carrying out an in vivo imaging analysis to detect T cells in the subject, wherein optionally the T cells are CD8+ T cells, and if the solid tumor is determined to be an immune-excluded solid tumor based on the in vivo imaging analysis of step (i), then, administering to the subject a therapeutically effective amount of a TGF ⁇ inhibitor, e.g., Ab6.
  • the subject has received a CBT, wherein optionally the solid tumor is resistant to the CBT.
  • the subject is administered with a CBT in conjunction with the TGF ⁇ 1 inhibitor, as a combination therapy.
  • the combination may comprise administration of a single formulation that comprises both a checkpoint inhibitor and a TGF ⁇ inhibitor.
  • the TGF ⁇ inhibitor may be a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non-selective inhibitor, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 and variants, antibodies that bind TGF ⁇ 1/3, ligand traps, e.g., TGF ⁇ 1/3 inhibitors, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibit
  • the combination therapy may comprise administration of a first formulation comprising a checkpoint inhibitor and a second formulation comprising a TGF ⁇ inhibitor, wherein the TGF ⁇ inhibitor may be a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non-selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 or variants, an antibody that bind TGF ⁇ 1/3, a ligand trap, e.g., a TGF ⁇ 1/3 inhibitor, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇
  • a TGF ⁇ 1 inhibitor such as a TGF ⁇ 1-selective inhibitor
  • the in vivo imaging comprises MDSC tracking, such as G-MDSCs and M-MDSCs.
  • MDSCs may be enriched at a disease site (such as fibrotic tissues and solid tumors) at the baseline.
  • a disease site such as fibrotic tissues and solid tumors
  • Upon therapy e.g., TGF ⁇ 1 inhibitor therapy
  • fewer MDSCs may be observed, as measured by reduced intensity of the label (such as radioisotope and fluorescence), indicative of therapeutic effects.
  • the in vivo imaging comprises tracking or localization of LRRC33-positive cells.
  • LRRC33-positive cells include, for example, MDSCs and activated M2-like macrophages (e.g., TAMs and activated macrophages associated with fibrotic tissues).
  • LRRC33-positive cells may be enriched at a disease site (such as fibrotic tissues and solid tumors) at the baseline.
  • a disease site such as fibrotic tissues and solid tumors
  • Upon therapy e.g., TGF ⁇ 1 inhibitor therapy
  • fewer cells expressing cell surface LRRC33 may be observed, as measured by reduced intensity of the label (such as radioisotope and fluorescence), indicative of therapeutic effects.
  • the in vivo imaging comprises the use of PET-SPECT, MRI and/or optical fluorescence/bioluminescence in order to detect target of interest (e.g., molecules or entities which can be bound by the labeled reagent, such as cells and tissues expressing appropriate marker(s)).
  • labeling of antibodies or antibody-like molecules with a detection moiety may comprise direct labeling or indirect labeling.
  • the detection moiety may be a tracer.
  • the tracer may be a radioisotope, wherein optionally the radioisotope may be a positron-emitting isotope.
  • the radioisotope is selected from the group consisting of: 18 F. 11 C, 13 N, 15 O, 68 Ga, 177 Lu, 18 F and 89 Zr.
  • such methods may be employed to carry out in vivo imaging with the use of labeled antibodies in immune-PET.
  • such in vivo imaging is performed for monitoring a therapeutic response to the TGF ⁇ 1 inhibition therapy in the subject.
  • the therapeutic response may comprise conversion of an immune excluded tumor into an inflamed tumor, which correlates with increased immune cell infiltration into a tumor. This may be visualized by increased intratumoral immune cell frequency or degree of detection signals, such as radiolabeling and fluorescence.
  • the disclosure includes a method for treating cancer which may comprise the following steps: i) selecting a patient diagnosed with cancer comprising a solid tumor, wherein the solid tumor is or is suspected to be an immune excluded tumor; and, ii) administering to the patient an antibody or the fragment encompassed herein in an amount effective to treat the cancer.
  • the patient has received, or is a candidate for receiving a cancer therapy such as immune checkpoint inhibition therapies (e.g., PD-(L)1 antibodies), chemotherapies, radiation therapies, engineered immune cell therapies, and cancer vaccine therapies.
  • immune checkpoint inhibition therapies e.g., PD-(L)1 antibodies
  • chemotherapies chemotherapies
  • radiation therapies e.g., engineered immune cell therapies, and cancer vaccine therapies.
  • the selection step (i) comprises detection of immune cells or one or more markers thereof, wherein optionally the detection comprises a tumor biopsy analysis, serum marker analysis, and/or in vivo imaging.
  • the patient is diagnosed with cancer for which a CBT has been approved, wherein optionally, statistically a similar patient population with the particular cancer shows relatively low response rates to the approved CBT, e.g., under 25%.
  • the response rates for the CBT may be between about 10-25%, for example about 10-15%.
  • Such cancer may include, for example, ovarian cancer, gastric cancer, and triple- negative breast cancer.
  • the TGF ⁇ inhibitors of the present disclosure may be used in the treatment of such cancer, where the subject has not yet received a CBT.
  • the TGF ⁇ 1 inhibitor may be administered to the subject in combination with a CBT.
  • the subject may receive or may have received additional cancer therapy, such as chemotherapy and radiation therapy (including a radiotherapeutic agent).
  • additional cancer therapy such as chemotherapy and radiation therapy (including a radiotherapeutic agent).
  • In vivo imaging techniques described above may be employed to detect, localize, and/or track certain MDSCs in a patient diagnosed with a TGF ⁇ -associated disease, such as cancer. Healthy individuals have no or low frequency of MDSCs in circulation. With the onset of or progression of such a disease, elevated levels of circulating and/or disease-localized MDSCs may be detected.
  • CCR2-positive M-MDSCs have been reported to accumulate to tissues with inflammation and may cause progression of fibrosis in the tissue (such as pulmonary fibrosis), and this is shown to correlate with TGF ⁇ 1 expression.
  • MDSCs are enriched in a number of solid tumors (including triple-negative breast cancer) and in part contribute to the immunosuppressive phenotype of the TME. Therefore, treatment response to TGF ⁇ inhibition, such as TGF ⁇ 1 inhibition, according to the present disclosure may be monitored by localizing or tracking circulating MDSCs. Reduction of or low frequency of circulating MDSC levels is typically indicative of therapeutic benefits or better prognosis.
  • the current disclosure provides methods of predicting and monitoring therapeutic efficacy of TGF ⁇ inhibitor therapy, e.g., combination therapy of a TGF ⁇ 1 inhibitor and a checkpoint inhibitor, by measuring circulating MDSCs in the blood or a blood component of the subject.
  • the current disclosure also provides methods of selecting patients, e.g., patients with immunosuppressive cancers and determining treatment regimens based on levels of circulating MDSCs measured.
  • the TGF ⁇ inhibitor may be a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1-selective inhibitor, e.g., Ab6, an isoform-non-selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 or variants, an antibody that bind TGF ⁇ 1/3, a ligand trap, e.g., a TGF ⁇ 1/3 inhibitor, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ 1-selective inhibitor e.g., Ab6
  • an isoform-non-selective inhibitor e.
  • the TGF ⁇ inhibitors of the present disclosure may be used in the treatment of cancer in a subject, wherein the cancer is characterized by immune suppression, wherein the cancer optionally comprises a solid tumor that is TGF ⁇ 1-positive and TGF ⁇ 3-positive.
  • the carcinoma is breast carcinoma, wherein optionally the breast carcinoma is triple-negative breast cancer (TNBC).
  • TNBC triple-negative breast cancer
  • Such treatment can further comprise a cancer therapy, including, without limitation, chemotherapies, radiation therapies, cancer vaccines, engineered immune cell therapies (such as CAR-T), and immune checkpoint blockade therapies, such as anti-PD(L)-1 antibodies.
  • the TGF ⁇ inhibitor may be a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1- selective inhibitor, e.g., Ab6, or an isoform-non-selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 or variants, an antibody that bind TGF ⁇ 1/3, a ligand trap, e.g., a TGF ⁇ 1/3 inhibitor, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ 1- selective inhibitor e.g., Ab6, or an isoform-non-selective inhibitor, e.g., a
  • a cold tumor is identified, in which few effector cells are present both inside and outside the tumor or is known to be a type of cancer characterized as poorly immunogenic (e.g., a tumor characterized as an immune desert).
  • a subject/patient with such a tumor is treated with an immune-sensitizing cancer therapy, such as chemotherapy, radiation therapy (such as a radiotherapeutic agent), oncolytic viral therapy, and cancer vaccine, in order to elicit stronger T cell response to tumor antigens (e.g., neo-antigens).
  • This step may convert the cold tumor into an “immune excluded” tumor.
  • the subject optionally further receives a CBT, such as anti-PD-(L)1.
  • a CBT such as anti-PD-(L)1.
  • the subject is further treated with a TGF ⁇ 1 inhibitor, such as the antibodies disclosed herein. This may convert the cold or immune excluded tumor into an “inflamed” or “hot” tumor, which confers responsiveness to immunotherapy.
  • TGF ⁇ 1 inhibitor such as the antibodies disclosed herein.
  • Non-limiting examples of poorly immunogenic cancers include breast cancer (such as TNBC), prostate cancer (such as Castration resistant prostate cancer (CRPC)) and pancreatic cancer (such as pancreatic adenocarcinoma (PDAC)).
  • TNBC breast cancer
  • prostate cancer such as Castration resistant prostate cancer (CRPC)
  • pancreatic cancer such as pancreatic adenocarcinoma (PDAC)
  • PDAC pancreatic adenocarcinoma
  • the plasmin-plasminogen axis has been implicated in certain tumorigenesis, invasion and/or metastasis, of various cancer types, carcinoma in particular, such as breast cancer. Therefore, it is possible that the TGF ⁇ inhibitors such as those described herein may exert the inhibitory effects via this mechanism in tumors or tumor models, such as EMT6, involving the epithelia. Indeed, Plasmin-dependent destruction or remodeling of epithelia may contribute to the pathogenesis of conditions involving epithelial injuries and invasion/dissemination of carcinoma. The latter may be triggered by epithelial to mesenchymal transition (“EMT”).
  • EMT epithelial to mesenchymal transition
  • the TGF ⁇ inhibitors of the present disclosure may be used in the treatment of anemia in a subject in need thereof.
  • the subject is diagnosed with cancer.
  • the subject is diagnosed with a myeloproliferative disorder (e.g., myelofibrosis).
  • a TGF ⁇ inhibitor (e.g., Ab6) is used alone to treat anemia.
  • the TGF ⁇ inhibitor is used in combination with an additional agent, e.g., a BMP antagonist (e.g., a BMP6 inhibitor, e.g., a RGMc inhibitor).
  • a combination comprising a TGF ⁇ 1 inhibitor (e.g., Ab6) and a BMP antagonist (e.g., a BMP6 inhibitor, e.g., a RGMc inhibitor) is used to improve anemia resulting from insufficient erythrocyte production, iron deficiency, and/or chemotherapy.
  • the treatment for anemia further comprises administering one or more JAK inhibitor (e.g., Jak1/2 inhibitor, Jak1 inhibitor, and/or Jak2 inhibitor).
  • JAK inhibitor e.g., Jak1/2 inhibitor, Jak1 inhibitor, and/or Jak2 inhibitor.
  • the disclosure includes a method for selecting a patient population or a subject who is likely to respond to a therapy comprising a TGF ⁇ inhibitor such as those described herein. Subjects selected according to such methods may be the subjects treated according to the various aspects of the present disclosure.
  • Such method may comprise the steps of: providing a biological sample (e.g., clinical sample) collected from a subject, determining (e.g., measuring or assaying) relative levels of TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3 in the sample, and, administering to the subject a composition comprising a TGF ⁇ inhibitor, such as a TGF ⁇ 1 inhibitor described herein, if TGF ⁇ 1 is the dominant isoform over TGF ⁇ 2 and TGF ⁇ 3; and/or, if TGF ⁇ 1 is significantly overexpressed or upregulated as compared to control.
  • a biological sample e.g., clinical sample
  • determining e.g., measuring or assaying
  • TGF ⁇ inhibitor such as a TGF ⁇ 1 inhibitor described herein
  • such method comprises the steps of obtaining information on the relative expression levels of TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3 which was previously determined; identifying a subject to have TGF ⁇ 1-positive, preferably TGF ⁇ 1-dominant, disease; and administering to the subject a composition comprising a TGF ⁇ inhibitor disclosed herein.
  • a subject has a disease (such as cancer) that is resistant to a therapy (such as cancer therapy).
  • a therapy such as cancer therapy
  • such subject shows intolerance to the therapy and therefore has or is likely to discontinue the therapy. Addition of the TGF ⁇ inhibitor to the therapeutic regimen may enable reducing the dosage of the first therapy and still achieve clinical benefits in combination.
  • the TGF ⁇ inhibitor may delay or reduce the need for surgeries.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1 inhibitor described herein, e.g., Ab6.
  • Relative levels of the isoforms may be determined by RNA-based assays and/or protein-based assays, which are well-known in the art.
  • the step of administration may also include another therapy, such as immune checkpoint inhibitors, or other agents provided elsewhere herein.
  • Such methods may optionally include a step of evaluating a therapeutic response by monitoring changes in relative levels of TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3 at two or more time points.
  • clinical samples (such as biopsies) are collected both prior to and following administration.
  • clinical samples are collected multiple times following treatment to assess in vivo effects over time.
  • the third inquiry interrogates the breadth of TGF ⁇ function, such as TGF ⁇ 1 function, involved in a particular disease.
  • TGF ⁇ 1 function such as TGF ⁇ 1 function
  • this may be represented by the number of TGF ⁇ 1 contexts, namely, which presenting molecule(s) mediate disease-associated TGF ⁇ 1 function.
  • TGF ⁇ 1-specific, broad- context inhibitors are advantageous for the treatment of diseases that involve both an ECM component and an immune component of TGF ⁇ 1 function.
  • Such disease may be associated with dysregulation in the ECM as well as perturbation in immune cell function or immune response.
  • the TGF ⁇ 1 inhibitors described herein are capable of targeting ECM-associated TGF ⁇ 1 (e.g., presented by LTBP1 or LTBP3) as well as immune cell-associated TGF ⁇ 1 (e.g., presented by GARP or LRRC33).
  • Such inhibitors inhibit all four of the therapeutic targets (e.g., “context-independent” inhibitors): GARP-associated pro/latent TGF ⁇ 1; LRRC33-associated pro/latent TGF ⁇ 1; LTBP1-associated pro/latent TGF ⁇ 1; and, LTBP3-associated pro/latent TGF ⁇ 1, so as to broadly inhibit TGF ⁇ 1 function in these contexts.
  • Whether or not a particular condition of a patient involves or is driven by multiple aspects of TGF ⁇ 1 function may be assessed by evaluating expression profiles of the presenting molecules, in a clinical sample collected from the patient.
  • Various assays are known in the art, including RNA-based assays and protein-based assays, which may be performed to obtain expression profiles.
  • Relative expression levels (and/or changes/alterations thereof) of LTBP1, LTBP3, GARP, and LRRC33 in the sample(s) may indicate the source and/or context of TGF ⁇ 1 activities associated with the condition. For instance, a biopsy sample taken from a solid tumor may exhibit high expression of all four presenting molecules.
  • the disclosure includes a method for determining (e.g., testing or confirming) the involvement of TGF ⁇ 1 in the disease, relative to TGF ⁇ 2 and TGF ⁇ 3.
  • the method further comprises a step of: identifying a source (or context) of disease-associated TGF ⁇ 1.
  • the source/context is assessed by determining the expression of TGF ⁇ presenting molecules, e.g., LTBP1, LTBP3, GARP and LRRC33 in a clinical sample taken from patients. In some embodiments, such methods are performed ex post facto.
  • TGF ⁇ presenting molecules e.g., LTBP1, LTBP3, GARP and LRRC33 in a clinical sample taken from patients. In some embodiments, such methods are performed ex post facto.
  • LRRC33-positive cells Applicant of the present disclosure has recognized that there can be a significant discrepancy between RNA expression and protein expression of LRRC33. In particular, while a select cell type appears to express LRRC33 at the RNA level, only a subset of such cells express the LRRC33 protein on the cell-surface.
  • LRRC33 expression may be highly regulated via protein trafficking/localization, for example, in terms of plasma membrane insertion and rapid internalization. Therefore, in certain embodiments, LRRC33 protein expression may be used as a marker associated with a diseased tissue (such as tumor tissues) enriched with, for example, activated/M2-like macrophages and MDSCs.
  • a diseased tissue such as tumor tissues
  • a PD-(L)1 antibody e.g., a PD-(L)1 antibody.
  • Non-limiting examples of useful checkpoint inhibitors include: ipilimumab (Yervoy®); nivolumab (Opdivo®); pembrolizumab (Keytruda®); avelumab (Bavencio®); cemiplimab (Libtayo®); atezolizumab (Tecentriq®); budigalimab (ABBV-181); durvalumab (Imfinzi®), etc.
  • a cancer treatment method may include a checkpoint inhibitor for use in the treatment of cancer in a subject, wherein the treatment comprises administration of a checkpoint inhibitor to the subject who is treated with a TGF ⁇ inhibitor or vice versa (i.e., administration of a TGF ⁇ inhibitor to a subject who is treated with a checkpoint inhibitor), wherein, upon treatment of the TGF ⁇ inhibitor, circulating MDSC levels in a sample collected from the subject are reduced, as compared to prior to the treatment.
  • the sample may be a blood sample or a sample of blood component.
  • the checkpoint inhibitor may be a PD-1 antibody.
  • the checkpoint inhibitor may be a PD-L1 antibody.
  • the checkpoint inhibitor may be a CTLA4 antibody.
  • the checkpoint inhibitor is selected from the group consisting of ipilimumab (e.g., Yervoy®); nivolumab (e.g., Opdivo®); pembrolizumab (e.g., Keytruda®); avelumab (e.g., Bavencio®); cemiplimab (e.g., Libtayo®); atezolizumab (e.g., Tecentriq®); budigalimab (ABBV-181); and durvalumab (e.g., Imfinzi®).
  • ipilimumab e.g., Yervoy®
  • nivolumab e.g., Opdivo®
  • pembrolizumab e.g., Keytruda®
  • avelumab e.g., Bavencio®
  • cemiplimab e.g., Libt
  • a cancer treatment method may include a checkpoint inhibitor for use in the treatment of cancer in a subject who is poorly responsive to the checkpoint inhibitor, or wherein the subject has a cancer with primary resistant to the checkpoint inhibitor, wherein the treatment comprises administering to the subject a TGF ⁇ inhibitor, measuring circulating MDSC levels before and after the administration of the TGF ⁇ inhibitor, and if circulating MDSCs are reduced after the TGF ⁇ inhibitor administration, further administering a checkpoint inhibitor to the subject in an amount sufficient to treat cancer.
  • the checkpoint inhibitor may be a PD-1 antibody.
  • the checkpoint inhibitor may be a PD-L1 antibody.
  • the checkpoint inhibitor may be a CTLA4 antibody.
  • the checkpoint inhibitor is selected from the group consisting of ipilimumab (e.g., Yervoy®); nivolumab (e.g., Opdivo®); pembrolizumab (e.g., Keytruda®); avelumab (e.g., Bavencio®); cemiplimab (e.g., Libtayo®); atezolizumab (e.g., Tecentriq®); budigalimab (ABBV-181); and durvalumab (e.g., Imfinzi®).
  • ipilimumab e.g., Yervoy®
  • nivolumab e.g., Opdivo®
  • pembrolizumab e.g., Keytruda®
  • avelumab e.g., Bavencio®
  • cemiplimab e.g., Libt
  • the TGF ⁇ inhibitor is an isoform-selective inhibitor of TGF ⁇ 1, wherein optionally the inhibitor is an activation inhibitor of TGF ⁇ 1 or neutralizing antibody that selectively binds TGF ⁇ 1; or an isoform-non-selective inhibitor (e.g., inhibitors of TGF ⁇ 1/2/3, TGF ⁇ 1/3, TGF ⁇ 1/2).
  • Combination Therapy [419] Disclosed herein are pharmaceutical compositions of a TGF ⁇ inhibitor, e.g., an antibody or antigen-binding portion thereof, described herein, and related methods used as, or referring to, combination therapies for treating subjects who may benefit from TGF ⁇ inhibition in vivo.
  • such subjects may receive combination therapies that include a first composition comprising at least one TGF ⁇ inhibitor, e.g., Ab6, in conjunction with at least a second composition comprising at least one additional therapeutic intended to treat the same or overlapping disease or clinical condition.
  • a second composition comprising at least one additional therapeutic intended to treat the same or overlapping disease or clinical condition.
  • an additional third composition comprising at least one additional therapeutic intended to treat the same or overlapping disease or clinical condition.
  • the TGF ⁇ inhibitor may be a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1-selective inhibitor (e.g., one which does not inhibit TGF ⁇ 2 and/or TGF ⁇ 3 signaling at a therapeutically effective dose), e.g., Ab6, or an isoform- non-selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that bind two or more of TGF ⁇ 1/2/3, e.g., GC1008 or variants, an antibody that bind TGF ⁇ 1/3, ligand trap, e.g., a TGF ⁇ 1/3 inhibitor, and/or an integrin inhibitor (e.g., an antibody that binds to ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • the first, second, and third compositions may both act on the same cellular target, or discrete cellular targets.
  • the first, second, and third compositions may treat or alleviate the same or overlapping set of symptoms or aspects of a disease or clinical condition.
  • the first, second, and third compositions may treat or alleviate a separate set of symptoms or aspects of a disease or clinical condition.
  • the combination therapy may comprise more than three compositions, which may act on the same target or discrete cellular targets, and which may treat or alleviate the same or overlapping set of symptoms or aspects of a disease or clinical condition.
  • the first composition may treat a disease or condition associated with TGF ⁇ signaling, while the second composition may treat inflammation or fibrosis associated with the same disease, etc.
  • the first composition may treat a disease or condition associated with TGF ⁇ signaling, while the second and third compositions may have anti-neoplastic effects and/or help reverse immune suppression.
  • the first composition may be a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 inhibitor described herein), the second composition may be a checkpoint inhibitor, and the third composition may be a checkpoint inhibitor distinct from the second composition.
  • a first composition comprising a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 inhibitor described herein) is combined with a checkpoint inhibitor and a chemotherapeutic agent.
  • a first composition comprising a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 inhibitor described herein) is combined with two distinct checkpoint inhibitors and a chemotherapeutic agent.
  • Such combination therapies may be administered in conjunction with each other.
  • the phrase “in conjunction with,” in the context of combination therapies means that therapeutic effects of a first therapy overlap temporally and/or spatially with therapeutic effects of a second therapy in the subject receiving the combination therapy.
  • the first, second, and/or additional compositions may be administered concurrently (e.g., simultaneously), separately, or sequentially.
  • the combination therapies may be formulated as a single formulation for concurrent or simultaneous administration, or as separate formulations for concurrent (e.g., simultaneous), separate, or sequential administration of the therapies.
  • a combination therapy may comprise two or more therapies (e.g., compositions) given in a single bolus or administration, or in a single patient visit (e.g., to or with a medical professional) but in two or more separate boluses or administrations, or in separate patient visits (and, e.g., in two or more separate boluses or administrations).
  • the therapies may be given less than about 5 minutes apart, or 1 minute apart.
  • the therapies may be given less than about 30 minutes or 1 hour apart (e.g., in a single patient visit). In some embodiments, the therapies may be given more than about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hour, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 1 day, about 2 day, about 3 days, about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, or more, apart. In some embodiments, the therapies may be given more than about 1 day apart (e.g., in separate visits). The therapies may be given within 3 months (e.g., within 1 month) of one another.
  • a therapy may be given according to the dosing schedule of one or more approved therapeutics for treating the condition (e.g., administered at the same frequency as for an approved checkpoint inhibitor or other chemotherapeutic agent).
  • the TGF ⁇ inhibitor e.g., a TGF ⁇ 1 inhibitor described herein
  • the TGF ⁇ inhibitor may be administered in an amount of about 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered in an amount of about 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less, e.g., at a frequency of once every two weeks, three weeks, or any multiples of two weeks or three weeks (e.g., once every four weeks, once every six weeks), wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered alone or in combination with a checkpoint inhibitor therapy, (e.g., any approved checkpoint inhibitor therapy, including, but not limited to, antibodies or other agents against cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed cell death receptor ligand 1 (PD-L1), T- cell immunoglobulin domain and mucin domain-3 (TIM3), lymphocyte-activation gene 3 (LAG3), killer cell immunoglobulin-like receptor (KIR), glucocorticoid-induced tumor necrosis factor receptor (GITR)
  • the TGF ⁇ inhibitor may be administered alone at 3000 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 2400 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 2000 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 1600 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 800 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 240 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 80 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at an amount of less than 80 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at an amount of less than 80 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered every six weeks at a dose of 50-3000 mg, e.g., 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg, 1000-3000 mg, 1500-2500 mg, 2000-3000 mg.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 3000 mg once every four weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 2400 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 2000 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 1600 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 800 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 240 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 80 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at an amount of less than 80 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at an amount of less than 80 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In some embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered every four weeks at a dose of 50-3000 mg, e.g., 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250- 2500 mg, 1000-3000 mg, 1500-2500 mg, 2000-3000 mg.
  • the TGF ⁇ inhibitor may be administered alone at 3000 mg once every three weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD- (L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 2400 mg once every three weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 2000 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD- (L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 1600 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD- (L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 800 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD- (L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 240 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD- (L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 80 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at an amount of less than 80 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at an amount of less than 80 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at less than 80 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered every three weeks at a dose of 50-3000 mg, e.g., 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg, 1000-3000 mg, 1500-2500 mg, 2000- 3000 mg.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 3000 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 2400 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 2000 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 1600 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 800 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 240 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 80 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at an amount of less than 80 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at an amount of less than 80 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at less than 80 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered every two weeks at a dose of 50-3000 mg, e.g., 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg, 1000-3000 mg, 1500-2500 mg, 2000-3000 mg.
  • a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 inhibitor described herein, e.g, Ab6) may be administered in combination or in conjunction with a genotoxic therapy.
  • a subject receiving a genotoxic therapy may have elevated levels of TGF ⁇ 1, for example where administration of the genotoxic therapy may cause an increase in TGF ⁇ 1 levels in the subject.
  • administration of a TGF ⁇ inhibitor e.g., a TGF ⁇ 1 inhibitor described herein, e.g, Ab6 in combination or in conjunction with a genotoxic therapy may provide added therapeutic benefit by binding to this increased TGF ⁇ 1 following treatment with the genotoxic therapy.
  • the genotoxic therapy may be a chemotherapy and/or a radiation therapy.
  • a TGF ⁇ inhibitor e.g., a TGF ⁇ 1 inhibitor described herein, e.g, Ab6
  • checkpoint inhibitor therapy e.g., an anti-PD-(L)1 therapy
  • the subject has not previously received a checkpoint inhibitor therapy.
  • checkpoint inhibitors include, but are not limited to, nivolumab (Opdivo®, anti-PD-1 antibody), pembrolizumab (Keytruda®, anti-PD-1 antibody), cemiplimab (Libtayo®, anti-PD-1 antibody), budigalimab (ABBV-181, anti-PD-1 antibody); BMS-936559 (anti-PD-L1 antibody), atezolizumab (Tecentriq®, anti-PD-L1 antibody), avelumab (Bavencio®, anti- PD-L1 antibody), durvalumab (Imfinzi®, anti-PD-L1 antibody), ipilimumab (Yervoy®, anti-CTLA4 antibody), tremelimumab (anti-CTLA4 antibody), IMP-321 (eftilgimod alpha or “ImmuFact®”, anti-LAG3 large molecule), BMS- 986016 (Relatlimab, anti-
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered alone to a subject having advanced solid cancer. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with a checkpoint inhibitor therapy to a subject having advanced solid cancer. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with a checkpoint inhibitor therapy to a subject having advanced solid cancer, wherein the subject is a non-responder to prior checkpoint inhibitor therapy. In some embodiments, the subject has non-small cell lung cancer (NSCLC), melanoma (MEL), or urothelial carcinoma (UC), including metastatic urothelial carcinoma (mUC).
  • NSCLC non-small cell lung cancer
  • MEL melanoma
  • UC urothelial carcinoma
  • mUC metastatic urothelial carcinoma
  • the subject has ovarian cancer, colorectal cancer (CRC), bladder cancer, renal cell carcinoma (RCC), including clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC, or head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC) or oropharynx cancer)).
  • CRC colorectal cancer
  • RCC renal cell carcinoma
  • HNSCC head and neck squamous cell carcinoma
  • HNSCC head and neck squamous cell carcinoma
  • the subject has esophageal cancer, gastric cancer, hepatocellular carcinoma (HCC), triple-negative breast cancer (TNBC), cervical cancer, endometrial cancer, basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (CSCC), merkel cell carcinoma (MCC), small-cell lung cancer (SCLC), primary mediastinal large B-cell lymphoma (PMBCL), Hodgkin’s lymphoma, microsatellite instability high cancer (MSI-H) (e.g., MSI-H CRC), mismatch repair deficient cancer (dMMR)(e.g., dMMR CRC), tumor mutational burden-high (TMB-H) cancer, or malignant pleural mesothelioma (MPM).
  • MSI-H microsatellite instability high cancer
  • dMMR mismatch repair deficient cancer
  • TMB-H tumor mutational burden-high
  • MMB-H malignant pleural mesothelioma
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with a checkpoint inhibitor therapy to a subject having urothelial carcinoma (UC), including metastatic urothelial carcinoma (mUC), melanoma (MEL), or non-small cell lung cancer NSCLC.
  • UC urothelial carcinoma
  • MEL metastatic urothelial carcinoma
  • NSCLC non-small cell lung cancer
  • the subject is a non-responder to checkpoint inhibitor therapy.
  • the checkpoint inhibitor therapy is pembrolizumab (e.g., Keytruda®).
  • the checkpoint inhibitor therapy is nivolumab (e.g., Opdivo®).
  • the checkpoint inhibitor therapy is cemiplimab (e.g., Libtayo®).
  • the checkpoint inhibitor therapy is atezolizumab (e.g., Tecentriq®). In certain embodiments, the checkpoint inhibitor therapy is avelumab (e.g., Bavencio®). In certain embodiments, the checkpoint inhibitor therapy is durvalumab (e.g., Imfinzi®). In certain embodiments, the checkpoint inhibitor therapy is budigalimab (e.g., ABBV-181).
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with pembrolizumab at a frequency of once every two weeks, three weeks, or any multiples of two weeks or three weeks (e.g., once every four weeks, once every six weeks).
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with pembrolizumab to a subject having NSCLC, UC, MEL, esophageal cancer, gastric cancer, HNSCC, HCC, cervical cancer, SCLC, PMBCL, Hodgkin’s lymphoma, MSI-H or dMMR cancer, or TMB-H cancer.
  • the subject is a non-responder to pembrolizumab.
  • the subject has not received pembrolizumab previously.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with pembrolizumab to a subject having NSCLC who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject having NSCLC has not received pembrolizumab previously. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with pembrolizumab to a subject having MEL who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject having MEL has not received pembrolizumab previously.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with pembrolizumab to a subject having UC or mUC who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject having UC or mUC has not received pembrolizumab previously.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with nivolumab at a frequency of once every two weeks, three weeks, or any multiples of two weeks or three weeks (e.g., once every four weeks, once every six weeks).
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with nivolumab to a subject having NSCLC, UC, MEL, esophageal cancer, HNSCC, HCC, RCC, Hodgkin’s lymphoma, MSI-H or dMMR CRC, or MPM.
  • the subject is a non-responder to nivolumab.
  • the subject has not received nivolumab previously.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with cemiplimab at a frequency of once every two weeks, three weeks, or any multiples of two weeks or three weeks (e.g., once every four weeks, once every six weeks).
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with cemiplimab to a subject having BCC or CSCC.
  • the subject is a non-responder to cemiplimab.
  • the subject has not received cemiplimab previously.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with atezolizumab at a frequency of once every two weeks, three weeks, or any multiples of two weeks or three weeks (e.g., once every four weeks, once every six weeks).
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with atezolizumab to a subject having NSCLC, MEL, HCC, TNBC, or SCLC.
  • the subject is a non-responder to atezolizumab.
  • the subject has not received atezolizumab previously.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with avelumab at a frequency of once every two weeks, three weeks, or any multiples of two weeks or three weeks (e.g., once every four weeks, once every six weeks).
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with avelumab to a subject having UC or MCC.
  • the subject is a non-responder to avelumab.
  • the subject has not received avelumab previously.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with durvalumab at a frequency of once every two weeks, three weeks, or any multiples of two weeks or three weeks (e.g., once every four weeks, once every six weeks).
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with durvalumab to a subject having NSCLC or SCLC.
  • the subject is a non-responder to durvalumab.
  • the subject has not received durvalumab previously.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less in combination with budigalimab (e.g., ABBV-181), e.g., at a dose of 250 mg, 375 mg, or 500 mg at a frequency of once every two weeks, three weeks, or any multiples of two weeks or three weeks (e.g., once every four weeks, once every six weeks).
  • budigalimab (e.g., ABBV-181) is administered at 250 mg once every two weeks.
  • budigalimab (e.g., ABBV- 181) is administered at 375 mg once every three weeks. In certain embodiments, budigalimab (e.g., ABBV-181) is administered at 500 mg once every four weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with budigalimab to a subject having a locally advanced or metastatic solid tumor. See, e.g., NCT 03821935 (Study to determine the safety, tolerability, pharmacokinetics and recommended phase 2 dose (RP2D) of ABBV-151 as a single agent and in combination with ABBV-181 in participants with locally advanced or metastatic solid tumors.
  • R2D phase 2 dose
  • budigalimab is administered once every four weeks.
  • the TGF ⁇ inhibitor e.g., Ab6
  • the TGF ⁇ inhibitor is administered in combination with budigalimab to a subject having triple-negative breast cancer (TNBC), pancreatic adenocarcinoma, urothelial cancer, or Hepatocellular carcinoma (HCC).
  • the TGF ⁇ inhibitor e.g., Ab6
  • the TGF ⁇ inhibitor is administered in combination with budigalimab to a subject having non-small cell lung cancer or head and neck squamous cell carcinoma. Italiano et al. Cancer Immunol Immunother. 2022 Feb; 71(2):417-431.
  • combination therapies produce synergistic effects in the treatment of a disease.
  • the term “synergistic” refers to effects that are greater than additive effects (e.g., greater efficacy) of each monotherapy in aggregate.
  • combination therapies comprising a pharmaceutical composition described herein produce efficacy that is overall equivalent to that produced by another therapy (such as monotherapy of a second agent) but are associated with fewer unwanted adverse effect or less severe toxicity associated with the second agent, as compared to the monotherapy of the second agent.
  • such combination therapies allow lower dosage of the second agent but maintain overall efficacy.
  • Such combination therapies may be particularly suitable for patient populations where a long-term treatment is warranted and/or involving pediatric patients.
  • the disclosure provides pharmaceutical compositions and methods for use in, and as, combination therapies for the reduction of TGF ⁇ 1 protein activation and the treatment or prevention of diseases or conditions associated with TGF ⁇ 1 signaling, as described herein.
  • the methods or the pharmaceutical compositions may further comprise a second therapy.
  • the methods or pharmaceutical compositions disclosed herein may further comprise a third therapy.
  • the second therapy and/or the third therapy may be useful in treating or preventing diseases or conditions associated with TGF ⁇ 1 signaling.
  • the second therapy and/or the third therapy may diminish or treat at least one symptom(s) associated with the targeted disease.
  • the first, second, and third therapies may exert their biological effects by similar or unrelated mechanisms of action; or either one or both of the first and second therapies may exert their biological effects by a multiplicity of mechanisms of action.
  • the second therapy and a TGF ⁇ inhibitor disclosed herein e.g., a TGF ⁇ 1-selective inhibitor disclosed herein
  • the second therapy, the third therapy, and a TGF ⁇ inhibitor disclosed herein are present in a single formulation or in separate formulations contained within in a single package or kit.
  • the second therapy, and a TGF ⁇ inhibitor disclosed herein are comprised in a single molecule, e.g., in a bispecific antibody or other multispecific construct or, wherein the checkpoint inhibitor is a small molecule, in an antibody-drug conjugate.
  • the second therapy, the third therapy, and a TGF ⁇ inhibitor disclosed herein are comprised in a single molecule, e.g., in a bispecific antibody or other multispecific construct or, wherein the checkpoint inhibitor is a small molecule, in an antibody-drug conjugate.
  • a TGF ⁇ inhibitor disclosed herein e.g., a TGF ⁇ 1-selective inhibitor disclosed herein
  • the checkpoint inhibitor is a small molecule, in an antibody-drug conjugate.
  • engineered constructs with TGF ⁇ inhibitory activities include M7824 (Bintrafusp alfa) and AVID200.
  • M7824 is a bifunctional fusion protein composed of 2 extracellular domains of TGF- ⁇ RII (a TGF- ⁇ “trap”) fused to a human IgG1 monoclonal antibody against PD-L1.
  • AVID200 is an engineered TGF- ⁇ ligand trap comprised of TGF- ⁇ receptor ectodomains fused to a human Fc domain.
  • the pharmaceutical compositions described herein may have the first and second therapies in the same pharmaceutically acceptable carrier or in a different pharmaceutically acceptable carrier for each described embodiment. It further should be understood that the first and second therapies may be administered concurrently (e.g., simultaneously), separately, or sequentially within described embodiments.
  • the one or more anti-TGF ⁇ antibodies, or antigen binding portions thereof, of the disclosure may be used in conjunction with one or more of additional therapeutic agents.
  • additional therapeutic agents which can be used with an anti-TGF ⁇ antibody of the disclosure include, but are not limited to: cancer vaccines, engineered immune cell therapies, chemotherapies, radiation therapies (e.g., radiotherapeutic agents), a modulator of a member of the TGF ⁇ superfamily, such as a myostatin inhibitor (e.g., a myostatin inhibitor disclosed in WO2016/073853 and WO2017/049011, the contents of which are hereby incorporated in their entirety), and a GDF11 inhibitor; a VEGF agonist; a VEGF inhibitor (such as bevacizumab); an IGF1 agonist; an FXR agonist; a CCR2 inhibitor; a CCR5 inhibitor; a dual CCR2/CCR5 inhibitor; CCR4 inhibitor, a lysyl oxidase-like-2 inhibitor; an ASK1 inhibitor; an Acetyl-CoA Carboxylase (ACC) inhibitor; a p38 kinase inhibitor;
  • additional therapeutic agents which can be used with the TGF ⁇ inhibitors include, but are not limited to, an indoleamine 2,3-dioxygenase (IDO) inhibitor, an arginase inhibitor, a tyrosine kinase inhibitor, Ser/Thr kinase inhibitor, a dual-specific kinase inhibitor.
  • IDO indoleamine 2,3-dioxygenase
  • arginase inhibitor e.g., a tyrosine kinase inhibitor
  • Ser/Thr kinase inhibitor a dual-specific kinase inhibitor.
  • such an agent may be a PI3K inhibitor, a PKC inhibitor, or a JAK inhibitor.
  • CPI checkpoint inhibitor
  • the current disclosure includes use of a TGF ⁇ inhibitor, e.g., Ab6, as a potential anti-cancer therapy alone or in combination with other therapies for the treatment of solid tumors and rare hematological malignancies for which TGF ⁇ signaling dysregulation has been implicated as a mediator of the disease process.
  • combination therapy comprising a TGF ⁇ inhibitor, e.g., Ab6, and at least one additional agent may be efficacious in patients with advanced solid tumors such as cutaneous melanoma, urothelial carcinoma (UC), non-small cell lung cancer (NSCLC), and head and neck cancer.
  • UC urothelial carcinoma
  • NSCLC non-small cell lung cancer
  • combination therapy comprising a TGF ⁇ inhibitor, e.g., Ab6, and at least one additional agent may be efficacious in patients with immune-excluded tumors such as non-small cell lung cancer, melanoma, renal cell carcinoma, triple-negative breast cancer, gastric cancer, microsatellite stable-colorectal cancer, pancreatic cancer, small cell lung cancer, HER2-positive breast cancer, or prostate cancer.
  • the at least one additional agent e.g., cancer therapy agent used in a method or composition disclosed herein is a checkpoint inhibitor.
  • the at least one additional agent is selected from the group consisting of a PD-1 antagonist, a PD-L1 antagonist, a PD-L1 or PD-L2 fusion protein, a CTLA4 antagonist, a GITR agonist, an anti-ICOS antibody, an anti-ICOSL antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-OX40 antibody (OX40 agonist), an anti- CD27 antibody, an anti-CD70 antibody, an anti-CD47 antibody, an anti-41BB antibody, an anti-PD-1 antibody, an oncolytic virus, and a PARP inhibitor.
  • checkpoint inhibitors include, but are not limited to, nivolumab (Opdivo®, anti-PD-1 antibody), pembrolizumab (Keytruda®, anti-PD-1 antibody), cemiplimab (Libtayo®, anti-PD-1 antibody), budigalimab (ABBV-181, anti-PD-1 antibody), BMS-936559 (anti-PD-L1 antibody), atezolizumab (Tecentriq®, anti-PD-L1 antibody), avelumab (Bavencio®, anti-PD-L1 antibody), durvalumab (Imfinzi®, anti-PD-L1 antibody), ipilimumab (Yervoy®, anti-CTLA4 antibody), tremelimumab (anti-CTLA4 antibody), IMP-321 (eftilgimod alpha or “ImmuFact®”, anti-LAG3 large molecule), BMS-986016 (Relatlimab, anti-LA
  • the TGF ⁇ inhibitors disclosed herein is used in the treatment of cancer in a subject who is a poor responder or non-responder of a checkpoint inhibition therapy, such as those listed herein.
  • the checkpoint inhibitor and a TGF ⁇ inhibitor e.g., a TGF ⁇ 1- selective inhibitor disclosed herein
  • the disclosure encompasses use of a TGF ⁇ inhibitor, e.g., Ab6, in combination with at least one checkpoint inhibitor therapy for the treatment of solid tumors and/or hematological malignancies for which TGF ⁇ signaling dysregulation has been implicated as a mediator of the disease process.
  • the combination therapy may be administered to patients who are not responsive to checkpoint inhibitor therapy (e.g., anti-PD-1 or anti-PD-L1 therapy).
  • checkpoint inhibitor therapy e.g., anti-PD-1 or anti-PD-L1 therapy.
  • Such patients may include, but are not limited to, those diagnosed with non-small cell lung cancer, urothelial bladder carcinoma, melanoma, triple-negative breast cancer, or other advance solid cancers.
  • the combination therapy may comprise a TGF ⁇ inhibitor, e.g., Ab6, and a checkpoint inhibitor therapy (e.g., pembrolizumab).
  • the combination therapy may be administered to immunotherapy-na ⁇ ve patients (e.g., patients who have not previously received a checkpoint inhibitor therapy) diagnosed with a cancer that has received FDA approval for treatment with a checkpoint inhibitor therapy.
  • Such cancer may be gastric cancer (e.g., metastatic gastric cancer), urothelial bladder carcinoma, lung cancer, triple-negative breast cancer, renal cell carcinoma, including clear cell RCC or papillary RCC, cervical cancer, or head and neck squamous cell carcinoma.
  • the combination therapy may comprise a TGF ⁇ inhibitor, e.g., Ab6, and a checkpoint inhibitor therapy (e.g., pembrolizumab).
  • the combination therapy may further comprise an additional agent, e.g., an additional checkpoint inhibitory and/or another chemotherapeutic agent.
  • the combination therapy may be administered to immunotherapy-na ⁇ ve patients (e.g., patients who have not previously received a checkpoint inhibitor therapy) diagnosed with a cancer that has not received FDA approval for treatment with a checkpoint inhibitor therapy.
  • Such cancer may be a microsatellite-stable colorectal cancer or pancreatic cancer.
  • the combination therapy may comprise a TGF ⁇ inhibitor, e.g., Ab6, a checkpoint inhibitor therapy (e.g., pembrolizumab), and at least one chemotherapeutic agent (e.g., axitinib, paclitaxel, cisplatin, and/or 5- fluorouracil).
  • a checkpoint inhibitor therapy e.g., pembrolizumab
  • at least one chemotherapeutic agent e.g., axitinib, paclitaxel, cisplatin, and/or 5- fluorouracil
  • the checkpoint inhibitor therapy may be pembrolizumab, nivolumab, and/or atezolizumab.
  • the combination therapy is administered to patients who have cancers characterized as exhibiting an immune-excluded phenotype.
  • additional analyses of a patient’s cancer may be carried out to further inform treatment, and such analyses may use known cancer-specific markers including microsatellite instability levels, PD-1 and/or PD-L1 expression level, and/or the presence of mutations in known cancer driver genes such as EGFR, ALK, ROS1, BRAF.
  • tumor PD-L1 expression may be used as a biomarker of therapeutic response.
  • the TGF ⁇ inhibitor e.g., Ab6
  • the TGF ⁇ inhibitor e.g., Ab6
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered in an amount of about 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less, e.g., at a frequency of once every six weeks, once every four weeks, once every three weeks, once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered alone or in combination with a checkpoint inhibitor therapy, e.g., an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 3000 mg once every three weeks.
  • the TGF ⁇ inhibitor may be administered alone at 3000 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 2400 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 2000 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 1600 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 800 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 240 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 80 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at an amount of less than 80 mg once every six weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at an amount of less than 80 mg once every six weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered every six weeks at a dose of 50-3000 mg, e.g., 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg, 1000-3000 mg, 1500-2500 mg, 2000-3000 mg.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 3000 mg once every four weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 2400 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 2000 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 1600 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 800 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 240 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 80 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at an amount of less than 80 mg once every four weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at an amount of less than 80 mg once every four weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In some embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered every four weeks at a dose of 50-3000 mg, e.g., 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250- 2500 mg, 1000-3000 mg, 1500-2500 mg, 2000-3000 mg.
  • the TGF ⁇ inhibitor may be administered alone at 3000 mg once every three weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD- (L)1 therapy.
  • the TGF ⁇ inhibitor may be administered alone at 2400 mg once every three weeks. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 2000 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD- (L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 1600 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD- (L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 800 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD- (L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 240 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD- (L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 80 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti- PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at an amount of less than 80 mg once every three weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at an amount of less than 80 mg once every three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at less than 80 mg at a frequency of any multiples of three weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered every three weeks at a dose of 50-3000 mg, e.g., 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg, 1000-3000 mg, 1500-2500 mg, 2000- 3000 mg.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 3000 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 3000 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 2400 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2400 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 2000 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 2000 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 1600 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 1600 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 800 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 800 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 240 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 240 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at 80 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at 80 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered alone at an amount of less than 80 mg once every two weeks.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at an amount of less than 80 mg once every two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered at less than 80 mg at a frequency of any multiples of two weeks, wherein the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with an anti-PD-(L)1 therapy.
  • the TGF ⁇ inhibitor (e.g., Ab6) may be administered every two weeks at a dose of 50-3000 mg, e.g., 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-2000 mg, 250-2500 mg, 1000-3000 mg, 1500-2500 mg, 2000-3000 mg.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with pembrolizumab to a subject having NSCLC, UC, MEL, esophageal cancer, gastric cancer, HNSCC, HCC, cervical cancer, SCLC, PMBCL, Hodgkin’s lymphoma, MSI-H or dMMR cancer, or TMB-H cancer.
  • the subject is a non-responder to pembrolizumab.
  • the subject has not received pembrolizumab previously.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with pembrolizumab to a subject having NSCLC who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject having NSCLC has not received pembrolizumab previously. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with pembrolizumab to a subject having MEL who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject having MEL has not received pembrolizumab previously.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with pembrolizumab to a subject having UC or mUC who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject having UC or mUC has not received pembrolizumab previously. [451] In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with nivolumab to a subject having NSCLC, UC, MEL, esophageal cancer, HNSCC, HCC, RCC, Hodgkin’s lymphoma, MSI-H or dMMR CRC, or MPM.
  • the subject is a non-responder to nivolumab. In certain embodiments, the subject has not received nivolumab previously.
  • the TGF ⁇ inhibitor e.g., Ab6
  • the TGF ⁇ inhibitor is administered in combination with cemiplimab to a subject having BCC or CSCC. In certain embodiments, the subject is a non-responder to cemiplimab. In certain embodiments, the subject has not received cemiplimab previously.
  • the TGF ⁇ inhibitor e.g., Ab6 is administered in combination with atezolizumab to a subject having NSCLC, MEL, HCC, TNBC, or SCLC.
  • the subject is a non-responder to atezolizumab. In certain embodiments, the subject has not received atezolizumab previously.
  • the TGF ⁇ inhibitor e.g., Ab6
  • the TGF ⁇ inhibitor is administered in combination with avelumab to a subject having UC or MCC. In certain embodiments, the subject is a non-responder to avelumab. In certain embodiments, the subject has not received avelumab previously.
  • the TGF ⁇ inhibitor e.g., Ab6 is administered in combination with durvalumab to a subject having NSCLC or SCLC. In certain embodiments, the subject is a non-responder to durvalumab.
  • the subject has not received durvalumab previously.
  • the TGF ⁇ inhibitor e.g., Ab6
  • the TGF ⁇ inhibitor is administered in combination with a checkpoint inhibitor therapy to a subject having a solid tumor for which a checkpoint inhibitor therapy has been approved.
  • the subject has a tumor type that has been approved for treatment with a combination of a checkpoint inhibitor therapy and a chemotherapy.
  • the subject has a tumor type that typically exhibits immune exclusion in more than 50% of the tumor area (e.g., tumor nests).
  • the immune excluded tumor types include triple-negative breast cancer or renal cell carcinoma.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with a checkpoint inhibitor therapy to a subject having a solid tumor for which a checkpoint inhibitor monotherapy has been approved.
  • the subject has a tumor type that typically exhibits immune exclusion in more than 50% of the tumor area (e.g., tumor nests), such as non-small cell lung cancer, urothelial carcinoma, gastric cancer, and renal cell carcinoma.
  • the subject has a tumor type that typically exhibits immune exclusion in less than 50% of the tumor area (e.g., tumor nests), such as small-cell lung cancer or melanoma.
  • the TGF ⁇ inhibitor (e.g., Ab6) is administered in combination with a checkpoint inhibitor therapy to a subject having a solid tumor for which a checkpoint inhibitor has not been approved.
  • the subject has a tumor type that typically exhibits immune exclusion in more than 50% of the tumor area (e.g., tumor nests), such as microsatellite stable colorectal cancer, pancreatic cancer, and prostate cancer.
  • TGF ⁇ inhibitor therapy e.g., Ab6
  • TGF ⁇ inhibitors may be used in conjunction (e.g., in combination) with a checkpoint inhibitor therapy for the treatment of cancer in a subject, wherein the cancer comprises an immunosuppressive tumor, and wherein the immunosuppressive tumor is resistant to checkpoint inhibitor therapy.
  • Non-limiting examples of immunosuppressive tumors include acute myeoid leukemia, adrenocortical cancer, brain lower grade glioma, cholangiocarcinoma, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, kidney chromophobe, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectum adenocarcinoma, sarcoma, testicular germ cell tumor, thymoma, thyroid carcinoma, uterine carcinosarcoma, uterine corpus endometrioid carcinoma, or uveal melanoma.
  • the at least one additional agent binds a T-cell costimulation molecule, such as inhibitory costimulation molecules and activating costimulation molecules.
  • the at least one additional agent is selected from the group consisting of an anti-CD40 antibody, an anti-CD38 antibody, an anti- KIR antibody, an anti-CD33 antibody, an anti-CD137 antibody, and an anti-CD74 antibody.
  • the at least one additional therapy is radiation.
  • the at least one additional agent is a radiotherapeutic agent.
  • the at least one additional agent is a chemotherapeutic agent.
  • the chemotherapeutic agent is Taxol.
  • the at least one additional agent is an anti-inflammatory agent. In some embodiments, the at least one additional agent inhibits the process of monocyte/macrophage recruitment and/or tissue infiltration. In some embodiments, the at least one additional agent is an inhibitor of hepatic stellate cell activation. In some embodiments, the at least one additional agent is a chemokine receptor antagonist, e.g., CCR2 antagonists and CCR5 antagonists. In some embodiments, such chemokine receptor antagonist is a dual specific antagonist, such as a CCR2/CCR5 antagonist. In some embodiments, the at least one additional agent to be administered as combination therapy is or comprises a member of the TGF ⁇ superfamily of growth factors or regulators thereof.
  • such agent is selected from modulators (e.g., inhibitors and activators) of GDF8/myostatin and GDF11.
  • such agent is an inhibitor of GDF8/myostatin signaling.
  • such agent is a monoclonal antibody that specifically binds a pro/latent myostatin complex and blocks activation of myostatin.
  • the monoclonal antibody that specifically binds a pro/latent myostatin complex and blocks activation of myostatin does not bind free, mature myostatin; see, for example, WO 2017/049011.
  • an additional therapy comprises cell therapy, such as CAR-T therapy and CAR-NK therapy.
  • an additional therapy comprises administering an anti-VEGF therapy, such as a VEGF inhibitor, e.g., bevacizumab.
  • a VEGF inhibitor e.g., bevacizumab
  • inhibitors of TGF ⁇ contemplated herein may be used in conjunction with (e.g., combination therapy, add-on therapy, etc.) a VEGF inhibitor (e.g., bevacizumab) for the treatment of solid cancer (e.g., ovarian cancer).
  • a VEGF inhibitor e.g., bevacizumab
  • hematopoietic cancers hematopoietic cancers.
  • an additional therapy is a cancer vaccine.
  • peptide-based cancer vaccines Numerous clinical trials that tested peptide-based cancer vaccines have targeted hematological malignancies (cancers of the blood), melanoma (skin cancer), breast cancer, head and neck cancer, gastroesophageal cancer, lung cancer, pancreatic cancer, prostate cancer, ovarian cancer, and colorectal cancers.
  • the antigens included peptides from HER2, telomerase (TERT), survivin (BIRC5), and Wilms’ tumor 1 (WT1).
  • Several trials also used “personalized” mixtures of 12-15 distinct peptides. That is, they contain a mixture of peptides from the patient’s tumor that the patient exhibits an immune response against.
  • Some trials are targeting solid tumors, glioma, glioblastoma, melanoma, and breast, cervical, ovarian, colorectal, and non-small lung cell cancers and include antigens from MUC1, IDO1 (Indoleamine 2,3- dioxygenase), CTAG1B, and two VEGF receptors, FLT1 and KDR.
  • the IDO1 vaccine is tested in patients with melanoma in combination with the immune checkpoint inhibitor ipilimumab and the BRAF (gene) inhibitor vemurafenib.
  • Non-limiting examples of tumor antigens useful as cancer vaccines include: NY-ESO-1, HER2, HPV16 E7 (Papillomaviridae#E7), CEA (Carcinoembryonic antigen), WT1, MART-1, gp100, tyrosinase, URLC10, VEGFR1, VEGFR2, surviving, MUC1 and MUC2.
  • Activated immune cells primed by such cancer vaccine may, however, be excluded from the TME in part through TGF ⁇ 1-dependent mechanisms. To overcome the immunosuppression, use of TGF ⁇ 1 inhibitors of the present disclosure may be considered so as to unleash the potential of the vaccine.
  • Combination therapies contemplated herein may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
  • use of an isoform-specific inhibitor of TGF ⁇ 1 described herein may render those who are poorly responsive or not responsive to a therapy (e.g., standard of care) more responsive.
  • use of an isoform-specific inhibitor of TGF ⁇ 1 described herein may allow reduced dosage of the therapy (e.g., standard of care) which still produces equivalent clinical efficacy in patients but fewer or lesser degrees of drug- related toxicities or adverse events.
  • inhibitors of TGF ⁇ contemplated herein may be used in conjunction with (e.g., combination therapy, add-on therapy, etc.) a selective inhibitor of myostatin (GDF8).
  • the selective inhibitor of myostatin is an inhibitor of pro/latent myostatin activation. See, for example, the antibodies disclosed in WO 2017/049011, such as apitegromab.
  • Advantages of TGF ⁇ 1 Inhibitors as a Therapeutic It has been recognized that various diseases involve heterogeneous populations of cells as sources of TGF ⁇ 1 that collectively contribute to the pathogenesis and/or progression of the disease.
  • TGF ⁇ 1-containing complexes More than one types of TGF ⁇ 1-containing complexes (“contexts”) likely coexist within the same disease microenvironment.
  • diseases may involve both an ECM (or “matrix”) component of TGF ⁇ 1 signaling (e.g., ECM dysregulation) and an immune component of TGF ⁇ 1 signaling.
  • ECM or “matrix” component of TGF ⁇ 1 signaling
  • immune component of TGF ⁇ 1 signaling e.g., ECM dysregulation
  • selectively targeting only a single TGF ⁇ 1 context e.g., TGF ⁇ 1 associated with one particular type of presenting molecule
  • broadly inhibitory TGF ⁇ 1 antagonists are desirable for therapeutic use.
  • inhibitory antibodies that broadly targeted multiple latent complexes of TGF ⁇ 1 exhibited skewed binding profiles among the target complexes (see, for example, WO 2018/129329 and WO 2019/075090).
  • the inventors therefore set out to identify more uniformly inhibitory antibodies that selectively inhibit TGF ⁇ 1 activation, irrespective of particular presenting molecule linked thereto. It was reasoned that particularly for immune-oncology applications, it is advantageous to potently inhibit both matrix-associated TGF ⁇ 1 and immune cell-associated TGF ⁇ 1.
  • context-independent inhibitors of TGF ⁇ 1 are used in the treatments and methods disclosed herein to target the pro/latent forms of TGF ⁇ 1.
  • the inhibitor targets ECM-associated TGF ⁇ 1 (LTBP1/3-TGF ⁇ 1 complexes).
  • the inhibitor targets immune cell- associated TGF ⁇ 1.
  • GARP-presented TGF ⁇ 1 such as GARP-TGF ⁇ 1 complexes expressed on Treg cells and LRRC33-TGF ⁇ 1 complexes expressed on macrophages and other myeloid/lymphoid cells, as well as certain cancer cells.
  • Such antibodies may include isoform-specific inhibitors of TGF ⁇ 1 that bind and prevent activation (or release) of mature TGF ⁇ 1 growth factor from a pro/latent TGF ⁇ 1 complex in a context-independent manner, such that the antibodies can inhibit activation (or release) of TGF ⁇ 1 associated with multiple types of presenting molecules.
  • the present disclosure provides antibodies capable of blocking ECM-associated TGF ⁇ 1 (LTBP-presented and LTBP3-presented complexes) and cell-associated TGF ⁇ 1 (GARP-presented and LRRC33- presented complexes).
  • ECM-associated TGF ⁇ 1 LTBP-presented and LTBP3-presented complexes
  • GAP-presented and LRRC33- presented complexes cell-associated TGF ⁇ 1
  • TGF ⁇ 1 the pathogenesis and/or progression of certain human conditions appear to be predominantly driven by or dependent on TGF ⁇ 1 activities.
  • many such diseases and disorders involve both an ECM component and an immune component of TGF ⁇ 1 function, suggesting that TGF ⁇ 1 activation in multiple contexts (e.g., mediated by more than one type of presenting molecules) is involved.
  • TGF ⁇ 1 activation in multiple contexts (e.g., mediated by more than one type of presenting molecules) is involved.
  • TGF ⁇ 1 activation in multiple contexts (e.g., mediated by more than one type of presenting molecules) is involved.
  • TGF ⁇ 1 activation in multiple contexts (e.g., mediated by more than one type of presenting molecules) is involved.
  • TGF ⁇ 1 activation in multiple contexts (e.g., mediated by more than one type of presenting molecules) is involved.
  • TGF ⁇ 1 activation in multiple contexts (e.g., mediated by more than one type of presenting molecules) is involved.
  • TGF ⁇ 1 tumor microenvironment
  • FME fibrotic microenvironment
  • TGF ⁇ 1 activities of one context may in turn regulate or influence TGF ⁇ 1 activities of another context, raising the possibility that when dysregulated, this may result in exacerbation of disease conditions. Therefore, it is desirable to broadly inhibit across multiple modes of TGF ⁇ 1 function (i.e., multiple contexts) while selectively limiting such inhibitory effects to the TGF ⁇ 1 isoform.
  • TGF ⁇ inhibitors such as the TGF ⁇ 1 inhibitors described herein, may be used to inhibit TGF ⁇ 1 associated with immunosuppressive cells.
  • the immunosuppressive cells include regulatory T-cells (Tregs), M2 macrophages/tumor-associated macrophages, and MDSCs.
  • TGF ⁇ inhibitors of the current disclosure may inhibit, reduce, or reverse immunosuppressive phenotype at a disease site such as the tumor microenvironment.
  • the TGF ⁇ 1 inhibitor inhibits TGF ⁇ 1 associated with a cell expressing the GARP- TGF ⁇ 1 complex or the LRRC33-TGF ⁇ 1 complex, wherein optionally the cell may be a T-cell, a fibroblast, a myofibroblast, a macrophage, a monocyte, a dendritic cell, an antigen presenting cell, a neutrophil, a myeloid- derived suppressor cell (MDSC), a lymphocyte, a mast cell, or a microglial cell.
  • the T-cell may be a regulatory T cell (e.g., immunosuppressive T cell).
  • the neutrophil may be an activated neutrophil.
  • the macrophage may be an activated (e.g., polarized) macrophage, including profibrotic and/or tumor-associated macrophages (TAM), e.g., M2c subtype and M2d subtype macrophages.
  • TAM tumor-associated macrophages
  • macrophages are exposed to tumor-derived factors (e.g., cytokines, growth factors, etc.) which may further induce pro-cancer phenotypes in macrophages.
  • tumor-derived factor e.g., cytokines, growth factors, etc.
  • such tumor-derived factor is CSF-1/M-CSF.
  • the cell expressing the GARP-TGF ⁇ 1 complex or the LRRC33-TGF ⁇ 1 complex is a cancer cell, e.g., circulating cancer cells and tumor cells.
  • TGF ⁇ inhibitors suitable for the therapeutic use and related methods disclosed herein include small molecule (i.e., low molecular weight) antagonists and biologics. Such inhibitors include isoform-selective inhibitors and isoform-non-selective inhibitors.
  • Biologics inhibitors include antibodies, antigen-binding fragments thereof, antibody-based or immunoglobulin-like molecules, as well as other engineered constructs, typically fusion proteins, such as ligand traps.
  • Ligand traps typically include a ligand-binding moiety that is derived from ligand-binding portion or portions of TGF ⁇ receptor(s).
  • Such biologics may be multifunctional constructs, such as bi-functional fusion proteins and bispecific antibodies.
  • methods disclosed herein may employ one or more of the following: low molecular weight antagonists of TGF ⁇ receptors, e.g., ALK5 antagonists, such as Galunisertib (LY2157299 monohydrate); monoclonal antibodies (such as neutralizing antibodies) that inhibit all three isoforms (“pan-inhibitor” antibodies) (see, for example, WO 2018/134681); monoclonal antibodies that preferentially inhibit two of the three isoforms (e.g., antibodies against T 2 (for example WO 2016/161410) and TGF ⁇ 1/3 (for example WO 2006/116002); and engineered molecules (e.g., fusion proteins) such as ligand traps (for example, WO 2018/029367; WO 2018/129331 and WO 2018/158727).
  • ALK5 antagonists such as Galunisertib (LY2157299 monohydrate
  • methods disclosed herein may employ one or more of the TGF ⁇ inhibitors disclosed in Batlle and Massague (Immunity, 2019. Apr 16;50(4):924-940), the content of which is incorporated herein in its entirety.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1-selective inhibitor, such as a TGF ⁇ 1-selective inhibitor disclosed herein, e.g., Ab6.
  • the TGF ⁇ 1-selective inhibitor is one disclosed in PCT/US2019/041390, PCT/US2019/041373, or PCT/US2021/012930, the contents of each of which are hereby incorporated by reference in their entirety.
  • the low molecular weight antagonists of TGF ⁇ receptors may include Vactosertib (TEW-7197, EW-7197), LY3200882, PF-06952229, AZ 12601011, and/or AZ 12799734.
  • the neutralizing pan-TGF ⁇ antibody is GC1008, also known as fresolimumab, or a derivative thereof. In some embodiments, such antibody comprises the sequence in accordance with the disclosure of WO/2018/134681. In some embodiments, the pan-TGF ⁇ antibody is SAR439459 or a derivative thereof.
  • the TGF ⁇ 1/2 antibodies include XPA-42-089 or a derivative thereof.
  • the antibody is a neutralizing antibody that specifically binds both TGF ⁇ 1 and TGF ⁇ 3. In some embodiments such antibody preferentially binds TGF ⁇ 1 over TGF ⁇ 3.
  • the antibody comprises the sequence in accordance with the disclosure of WO/2006/116002. In some embodiments, the antibody is 21D1.
  • the antibody is a neutralizing antibody that specifically binds both TGF ⁇ 1 and TGF ⁇ 2. In some embodiments, the antibody comprises the sequence in accordance with the disclosure of WO/2017/161410. In some embodiments, the antibody is XOMA-089, or NIS-793.
  • the antibody is an activation inhibitor antibody that is selective for TGF ⁇ 1.
  • the antibody comprises the sequence in accordance with the disclosure of WO/2015/015003, WO/2019/075090 or WO/2017/115345. [485] In some embodiments, the antibody is a neutralizing antibody that is selective for TGF ⁇ 1. In some embodiments, the antibody comprises the sequence in accordance with the disclosure of WO/2013/134365 or WO/2018/043734. [486] In some embodiments, the TGF ⁇ inhibitor is a ligand trap. In some embodiments, the ligand trap comprises the structure in accordance with the disclosure of WO/2018/158727. In some embodiments, the ligand trap comprises the structure in accordance with the disclosure of WO 2018/029367; WO 2018/129331.
  • the ligand trap is a construct known as CTLA4- TGFbRII.
  • the ligand trap is a bi-functional fusion protein comprising a checkpoint inhibitor function and a TGF ⁇ inhibitor function.
  • the bi-functional fusion protein is a construct known as M7824 or PDL1-TGFbRII.
  • the TGF ⁇ inhibitor is a receptor based TGF ⁇ trap, e.g., AVID200. [487]
  • the TGF ⁇ inhibitor is an integrin inhibitor.
  • the TGF ⁇ inhibitor is an inhibitor of an integrin such as ⁇ V ⁇ 1, ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, and/or ⁇ 8 ⁇ 1.
  • Integrin inhibitors include small molecule inhibitors and antibodies that bind to an integrin and/or inhibit the binding of an integrin to the RGD motif of proTGF ⁇ 1 and/or proTGF ⁇ 3.
  • the TGF ⁇ inhibitor is an inhibitor of latent TGF ⁇ (e.g., latent TGF ⁇ 1 or latent TGF ⁇ 3).
  • the TGF ⁇ inhibitor is an inhibitor that binds the RGD motif of proTGF ⁇ 1 and/or proTGF ⁇ 3.
  • the TGF ⁇ inhibitor is an LTBP-selective inhibitor, such as an LTBP-selective inhibitor disclosed in PCT/US2020/015915.
  • the TGF ⁇ inhibitor is an LTBP1-selective inhibitor.
  • the TGF ⁇ inhibitor is an LTBP3-selective inhibitor.
  • the TGF ⁇ inhibitor is an LTBP-1 and LTBP3-selective inhibitor.
  • Isoform-Selective Antibodies of proTGF ⁇ 1 [490]
  • the therapeutic use and related methods in accordance with the present disclosure are carried out with an isoform-selective inhibitor of TGF ⁇ 1, e.g., Ab6 (the sequence of which is as disclosed in PCT/US2019/041373, the contents of which are herein incorporated by reference in its entirety).
  • Applicant previously disclosed improved antibodies which embody all or most of the following features: 1) selectivity towards TGF ⁇ 1 is maintained to minimize unwanted toxicities associated with pan-inhibition (“isoform- selectivity”) (see, for example, PCT/US2017/021972); 2) exhibit broad binding activities across various biological contexts, or, both matrix-associated and cell-associated categories (“context-independent”) (see, for example, WO 2018/129329); 3) achieve more even or unbiased affinities across multiple antigen complexes (“uniformity”); 4) show strong binding activities for each of the antigen complexes, (“high-affinity”) and have robust inhibitory activities for each context (“potency”) (see, for example, PCT/US2019/041373); and, 5) the preferred mechanism of action is to inhibit the activation step so the inhibitor can target a tissue-tethered, latent TGF ⁇ 1 complex, so as to preemptively prevent downstream activation events to achieve durable effects, rather than to directly target soluble/free growth factors (“durability”).
  • TGF ⁇ 1 inhibitors are highly potent and highly selective inhibitor of latent TGF ⁇ 1 activation.
  • Data presented therein demonstrated, inter alia, that this mechanism of isoform-selective inhibition is sufficient to overcome primary resistance to anti-PD-1 in syngeneic mouse models that closely recapitulate some of the features of primary resistance to CBT found in human cancers.
  • Preferred antibodies and corresponding nucleic acid sequences that encode such antibodies useful for carrying out the present disclosure include one or more of the CDR amino acid sequences shown in Tables 1 and 2.
  • Each set of the H-CDRs (H-CDR1, H-CDR2 and H-CDR3) listed in Table 1 can be combined with the L-CDRs (L-CDR1, L-CDR2 and L-CDR3) provided in Table 2.
  • the disclosure provides an antibody or antigen binding fragment thereof, e.g., an isolated antibody or antigen-binding fragment thereof, comprising six CDRs (e.g., an H-CDR1, an H-CDR2, an H-CDR3, an L-CDR1, an L-CDR2 and an L-CDR3) as listed in Table 1, and wherein the L-CDR1 comprises QASQDITNYLN (SEQ ID NO: 78), the L-CDR2 comprises DASNLET (SEQ ID NO: 79), and the L-CDR3 comprises QQADNHPPWT (SEQ ID NO: 6), wherein optionally, the H-CDR1 may comprise FTFSSFSMD (SEQ ID NO: 80); the H-CDR-2 may comprise YISPSADTIYYADSVKG (SEQ ID NO: 76); and/or, the H-CDR3 may comprise ARGVLDYGDMLMP (SEQ ID NO: 3).
  • CDRs e.g., an H-CDR1, an H
  • the antibody or the fragment comprises H-CDR1 having the amino acid sequence FTFSSFSMD (SEQ ID NO: 80), H-CDR2 having the amino acid sequence YISPSADTIYYADSVKG (SEQ ID NO: 76), and H-CDR-3 having the amino acid sequence ARGVLDYGDMLMP (SEQ ID NO: 3); L-CDR1 having the amino acid sequence QASQDITNYLN (SEQ ID NO: 78), L-CDR2 having the amino acid sequence DASNLET (SEQ ID NO: 79), and L-CDR3 having the amino acid sequence QQADNHPPWT (SEQ ID NO: 6).
  • Commonly used systems include but are not limited to: Kabat numbering system, IMTG numbering system, Chothia numbering system, and others such as the numbering scheme described by Lu et al., (Lu X et al., MAbs.2019 Jan;11(1):45-57).
  • 6 CDR sequences of Ab6 as defined by four different numbering systems are exemplified below. Any art-recognized CDR numbering systems may be used to define CDR sequences of the antibodies of the present disclosure.
  • Table 3 Six CDRs of an exemplary antibody (Ab6) based on four numbering schemes [496] Amino acid sequences of the heavy chain variable domain and the light chain variable domain of exemplary antibodies of the present disclosure are provided in Table 4.
  • the isoform-selective TGF ⁇ 1 inhibitor of the present disclosure may be an antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and the VL sequences are selected from any one of the sets of VH and VL sequences listed in Table 4 below. Table 4.
  • an antibody or an antigen-binding fragment thereof that comprises a heavy chain variable domain and a light chain variable domain, wherein, the heavy chain variable domain has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% and 100%) sequence identity with any one of the sequences selected from the group consisting of: Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34; and, wherein the light chain variable domain has at least 90% identity with any one of the sequences selected from Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34, wherein, optionally, the heavy chain variable domain may optionally
  • the heavy chain variable domain of the antibody or the fragment has at least 90% sequence identity with SEQ ID NO: 7, and wherein optionally, the light chain variable domain of the antibody or the fragment has at least 90% sequence identity with SEQ ID NO: 8. In some embodiments, the heavy chain variable domain of the antibody or the fragment has at least 95% sequence identity with SEQ ID NO: 7, and wherein optionally, the light chain variable domain of the antibody or the fragment has at least 95% sequence identity with SEQ ID NO: 8. In some embodiments, the heavy chain variable domain of the antibody or the fragment has at least 98% sequence identity with SEQ ID NO: 7, and wherein optionally, the light chain variable domain of the antibody or the fragment has at least 98% sequence identity with SEQ ID NO: 8.
  • the heavy chain variable domain of the antibody or the fragment has 100% sequence identity with SEQ ID NO: 7, and wherein optionally, the light chain variable domain of the antibody or the fragment has 100% sequence identity with SEQ ID NO: 8.
  • an antibody or an antigen-binding fragment thereof disclosed herein comprises 6 CDRs from, or the full sequences of, the heavy and light chain variable domains of SEQ ID Nos: 7 and 8, respectively.
  • the antibody or an antigen-binding fragment thereof comprises heavy and light chain variable domain sequences with at least 90% sequence identity (e.g., at least 95% identity) to SEQ ID NOs: 7 and 8, respectively.
  • the antibody or an antigen-binding fragment thereof may comprise a set of 6 respective H- and L- CDRs selected from those set out in Tables 1 and 2 above.
  • the antibody or antigen-binding fragment thereof comprises a set of 6 respective H- and L- CDRs as set out in Table 3 (e.g., using the system of Lu et al.).
  • the antibody or an antigen-binding fragment thereof used in the context of the present disclosure may comprise heavy and light chain variable domains with at least 90% sequence identity (e.g., at least 95% identity) to SEQ ID Nos: 7 and 8, respectively, and specifically binds a proTGF ⁇ 1 complex at (i) a first binding region comprising at least a portion of Latency Lasso (SEQ ID NO: 126); and ii) a second binding region comprising at least a portion of Finger-1 (SEQ ID NO: 124); characterized in that when bound to the proTGF ⁇ 1 complex in a solution, the antibody or the fragment protects the binding regions from solvent exposure as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS).
  • HDX-MS hydrogen-deuterium exchange mass spectrometry
  • the first binding region may comprise PGPLPEAV (SEQ ID NO: 134) or a portion thereof and the second binding region may comprise RKDLGWKW (SEQ ID NO: 143) or a portion thereof.
  • protection of the binding region refers to protein-protein interactions, such as antibody-antigen binding, the degree by which a protein (e.g., a region of a protein containing an epitope) is exposed to a solvent as assessed by an HDX-MS-based assay of protein-protein interactions. Protection of binding may be determined by the level of proton exchange occurring at a binding site, which is inversely correlates with the degree of binding/interaction.
  • the binding region is “protected” from being exposed to the solvent because the protein-protein interaction precludes the binding region from being accessible by the surrounding solvent.
  • the protected region is thus indicative of a site of interaction.
  • the antibody or the fragment may further bind the proTGF ⁇ 1 complex at one or more of the following binding regions or a portion thereof: LVKRKRIEA (SEQ ID NO: 132); LASPPSQGEVPPGPL (SEQ ID NO: 126); LALYNSTR (SEQ ID NO: 135); REAVPEPVL (SEQ ID NO: 136); YQKYSNNSWR (SEQ ID NO: 137); RKDLGWKWIHE (SEQ ID NO: 144); HEPKGYHANF (SEQ ID NO: 145); LGPCPYIWS (SEQ ID NO: 139); ALEPLPIV (SEQ ID NO: 140); and, VGRKPKVEQL (SEQ ID NO: 141).
  • LVKRKRIEA SEQ ID NO: 132
  • LASPPSQGEVPPGPL SEQ ID NO: 126
  • LALYNSTR SEQ ID NO: 135)
  • REAVPEPVL SEQ ID NO: 136
  • the antibody or antigen-binding fragments may further be characterized in that it cross-blocks (cross-competes) for binding to TGF ⁇ 1 (e.g., to pro- and/or latent- TGF ⁇ 1) with an antibody having the heavy chain variable domain of SEQ ID NO: 7, and the light chain variable domain of SEQ ID NO: 8.
  • the antibody that cross-blocks or cross-competes comprises heavy and light chain variable domains that are at least about 90% (e.g., 95% or 99%) identical to those of SEQ ID NOs 7 and 8, respectively.
  • the antibody or antigen binding portion thereof, that specifically binds to a GARP- TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and/or a LRRC33-TGF ⁇ 1 complex comprises a heavy chain variable domain amino acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 7, and a light chain variable domain amino acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 8.
  • the antibody or antigen binding portion thereof comprises a heavy chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 7, and a light chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 8.
  • any of the antibodies of the disclosure that specifically bind to a GARP-TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and/or a LRRC33-TGF ⁇ 1 complex include any antibody (including antigen binding portions thereof) having one or more CDR (e.g., CDRH or CDRL) sequences substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3.
  • CDR e.g., CDRH or CDRL
  • the antibodies may include one or more CDR sequences as shown in Table 1 containing up to 5, 4, 3, 2, or 1 amino acid residue variations as compared to the corresponding CDR region in any one of SEQ ID NOs: 3, 6, 76, 78, 79, 80, 81, 82, 8384, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99.
  • one or more of the six CDR sequences contain up to three (3) amino acid changes as compared to the sequences provided in Table 1.
  • Such antibody variants comprising up to 3 amino acid changes per CDR are encompassed by the present disclosure.
  • such variant antibodies are generated by the process of optimization, such as affinity maturation.
  • the complete amino acid sequences for the heavy chain variable region and light chain variable region of the antibodies listed in Table 4 (e.g., Ab6), as well as nucleic acid sequences encoding the heavy chain variable region and light chain variable region of certain antibodies are provided below:
  • Ab6 – Light chain variable region amino acid sequence DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTIS SLQPEDIATYYCQQADNHPPWTFGGGTKVEIK SEQ ID NO
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • one or more conservative mutations can be introduced into the CDRs or framework sequences at positions where the residues are not likely to be involved in an antibody-antigen interaction.
  • such conservative mutation(s) can be introduced into the CDRs or framework sequences at position(s) where the residues are not likely to be involved in interacting with a GARP-TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and a LRRC33- TGF ⁇ 1 complex as determined based on the crystal structure.
  • likely interface e.g., residues involved in an antigen-antibody interaction
  • a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
  • amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. [506]
  • the antibodies provided herein comprise mutations that confer desirable properties to the antibodies.
  • the antibodies provided herein may comprise a stabilizing ‘Adair’ mutation (Angal et al., “A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline resulting in an IgG1-like (CPPCP (SEQ ID NO: 43)) hinge sequence.
  • any of the antibodies may include a stabilizing ‘Adair’ mutation or the amino acid sequence CPPCP (SEQ ID NO: 43).
  • Isoform-specific, context-independent inhibitors of TGF ⁇ 1 of the present disclosure may optionally comprise antibody constant regions or parts thereof.
  • a VL domain may be attached at its C-terminal end to a light chain constant domain like C ⁇ or C ⁇ .
  • a VH domain or portion thereof may be attached to all or part of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and any isotype subclass.
  • Antibodies may include suitable constant regions (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)).
  • antibodies within the scope of this may disclosure include VH and VL domains, or an antigen binding portion thereof, combined with any suitable constant regions.
  • such antibodies may or may not include the framework region of the antibodies of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, and 8.
  • antibodies that specifically bind to a GARP-TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3- TGF ⁇ 1 complex, and a LRRC33-TGF ⁇ 1 complex are murine antibodies and include murine framework region sequences.
  • such antibodies bind to a GARP-TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and a LRRC33-TGF ⁇ 1 complex with relatively high affinity, e.g., with a KD less than 10 -9 M, 10 -10 M, 10 -11 M or lower.
  • such antibodies may bind a GARP-TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and/or a LRRC33-TGF ⁇ 1 complex with an affinity between 5 pM and 1 nM, e.g., between 10 pM and 1 nM, e.g., between 10 pM and 500 pM.
  • the disclosure also includes antibodies or antigen binding fragments that compete with any of the antibodies described herein for binding to a GARP-TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and/or a LRRC33-TGF ⁇ 1 complex and that have a KD value of 1 nM or lower (e.g., 1 nM or lower, 500 pM or lower, 100 pM or lower).
  • 1 nM or lower e.g., 1 nM or lower, 500 pM or lower, 100 pM or lower.
  • affinity and binding kinetics of the antibodies that specifically bind to a GARP-TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3- TGF ⁇ 1 complex, and/or a LRRC33-TGF ⁇ 1 complex can be tested using any suitable method including but not limited to biosensor-based technology (e.g., OCTET® or Biacore®) and solution equilibrium titration-based technology (e.g., MSD-SET).
  • biosensor-based technology e.g., OCTET® or Biacore®
  • MSD-SET solution equilibrium titration-based technology
  • affinity and binding kinetics are measured by SPR, such as Biacore systems.
  • such antibodies dissociate from each of the aforementioned large latent complex with an OFF rate of 10e-4 or less.
  • inhibitors of cell-associated TGF ⁇ 1 include antibodies or fragments thereof that specifically bind such complex (e.g., GARP-pro/latent TGF ⁇ 1 and LRRC33-pro/latent TGF ⁇ 1) and trigger internalization of the complex.
  • This mode of action causes removal or depletion of the inactive TGF ⁇ 1 complexes (e.g., GARP- proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1) from the cell surface (e.g., Treg, macrophages, etc.), hence reducing TGF ⁇ 1 available for activation.
  • such antibodies or fragments thereof bind the target complex in a pH-dependent manner such that binding occurs at a neutral or physiological pH, but the antibody dissociates from its antigen at an acidic pH; or, dissociation rates are higher at acidic pH than at neutral pH.
  • Such antibodies or fragments thereof may function as recycling antibodies.
  • Antibodies Competing with the Preferred Antibodies of TGF ⁇ 1 [511] Aspects of the disclosure relate to antibodies that compete or cross-compete with any of the antibodies provided herein.
  • a first antibody binds to an epitope (e.g., an epitope of a GARP-proTGF ⁇ 1 complex, a LTBP1-proTGF ⁇ 1 complex, a LTBP3-proTGF ⁇ 1 complex, and a LRRC33-proTGF ⁇ 1 complex) in a manner sufficiently similar to or overlapping with the binding of a second antibody, such that the result of binding of the first antibody with its epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody.
  • an epitope e.g., an epitope of a GARP-proTGF ⁇ 1 complex, a LTBP1-proTGF ⁇ 1 complex, a LTBP3-proTGF ⁇ 1 complex, and a LRRC33-proTGF ⁇ 1 complex
  • a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope.
  • each antibody detectably inhibits the binding of the other antibody with its epitope or ligand whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing antibodies are within the scope of this disclosure.
  • cross-blocking and “cross-competing” may be used interchangeably.
  • Two different monoclonal antibodies (or antigen-binding fragments) that bind the same antigen may be able to simultaneously bind to the antigen if the binding sites are sufficiently further apart in the three-dimensional space such that each binding does not interfere with the other binding.
  • two different monoclonal antibodies may have binding regions of an antigen that are the same or overlapping, in which case, binding of the first antibody may prevent the second antibody from being able to bind the antigen, or vice versa. In the latter case, the two antibodies are said to “cross-block” with each other with respect to the same antigen.
  • Antibody “binning” experiments are useful for classifying multiple antibodies that are made against the same antigen into various “bins” based on the relative cross-blocking activities. Each “bin” therefore represents a discrete binding region(s) of the antigen. Antibodies in the same bin by definition cross-block each other.
  • Binning can be examined by standard in vitro binding assays, such as Biacore or Octet®, using standard test conditions, e.g., according to the manufacturer’s instructions (e.g., binding assayed at room temperature, ⁇ 20-25°C).
  • an antibody, or antigen binding portion thereof binds at or near the same epitope as any of the antibodies provided herein.
  • an antibody, or antigen binding portion thereof binds near an epitope if it binds within 15 or fewer amino acid residues of the epitope.
  • any of the antibody, or antigen binding portion thereof, as provided herein binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues of an epitope that is bound by any of the antibodies provided herein.
  • an antibody, or antigen binding portion thereof competes or cross-competes for binding to any of the antigens provided herein (e.g., a GARP-TGF ⁇ 1 complex, a LTBP1-TGF ⁇ 1 complex, a LTBP3-TGF ⁇ 1 complex, and/or a LRRC33-TGF ⁇ 1 complex) with an equilibrium dissociation constant, KD, between the antibody and the protein of less than 10 -8 M.
  • an antibody competes or cross-competes for binding to any of the antigens provided herein with a K D in a range from 10 -12 M to 10 -9 M.
  • an anti-TGF ⁇ 1 antibody, or antigen binding portion thereof that competes for binding with an antibody, or antigen binding portion thereof, described herein.
  • one method is to identify the epitope to which the antigen binds, or “epitope mapping.”
  • epitope mapping There are many suitable methods for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999.
  • epitope mapping can be used to determine the sequence to which an antibody binds.
  • the epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence).
  • the epitope is a TGF ⁇ 1 epitope that is only available for binding by the antibody, or antigen binding portion thereof, described herein, when the TGF ⁇ 1 is in a GARP-proTGF ⁇ 1 complex, a LTBP1-proTGF ⁇ 1 complex, a LTBP3-proTGF ⁇ 1 complex, or a LRRC33-proTGF ⁇ 1 complex.
  • Peptides of varying lengths can be isolated or synthesized (e.g., recombinantly) and used for binding assays with an antibody.
  • the epitope to which the antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody.
  • the gene fragment expression assays the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined.
  • the gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids.
  • the binding of the antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis.
  • Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays.
  • mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding.
  • domain swapping experiments can be performed using a mutant of a target antigen in which various fragments of the GARP-proTGF ⁇ 1 complex, a LTBP1-proTGF ⁇ 1 complex, a LTBP3-proTGF ⁇ 1 complex, and/or a proLRRC33-TGF ⁇ 1 complex have been replaced (swapped) with sequences from a closely related, but antigenically distinct protein, such as another member of the TGF ⁇ protein family (e.g., GDF11).
  • competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition assays are well known to those of skill in the art.
  • a pharmaceutical composition may be made by a process comprising a step of: selecting an antibody or antigen-binding fragment thereof, which cross-competes with an antibody having a heavy chain variable domain of SEQ ID NO: 7 and a light chain variable domain of SEQ ID NO: 8 for binding to TGF ⁇ 1 (e.g., to pro-TGF ⁇ 1 and/or latent TGF ⁇ 1).
  • a pharmaceutical composition may be made by the process comprising a step of: selecting an antibody or antigen-binding fragment thereof, which cross-competes with the antibody selected from the group consisting of Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33 and Ab34; and, formulating into a pharmaceutical composition.
  • the antibody selected by the process is a high-affinity binder characterized in that the antibody or the antigen-binding fragment is capable of binding to each of human LLCs (e.g., hLTBP1-proTGF ⁇ 1, hLTBP3- proTGF ⁇ 1, hGARP-proTGF ⁇ 1 and hLRRC33-proTGF ⁇ 1) with a K D of ⁇ 1 nM, as measured by solution equilibrium titration.
  • human LLCs e.g., hLTBP1-proTGF ⁇ 1, hLTBP3- proTGF ⁇ 1, hGARP-proTGF ⁇ 1 and hLRRC33-proTGF ⁇
  • Such cross-competing antibodies may be used in the treatment of TGF ⁇ 1-related indications a subject in accordance with the present disclosure.
  • Naturally-occurring antibody structural units typically comprise a tetramer. Each such tetramer typically is composed of two identical pairs of polypeptide chains, each pair having one full-length “light” (in certain embodiments, about 25 kDa) and one full-length “heavy” chain (in certain embodiments, about 50-70 kDa).
  • the amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that typically is responsible for antigen recognition.
  • each chain typically defines a constant region that can be responsible for effector function.
  • Human antibody light chains are typically classified as kappa and lambda light chains.
  • Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the isotype of the antibody.
  • An antibody can be of any type (e.g., IgM, IgD, IgG, IgA, IgY, and IgE) and class (e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgM 1 , IgM 2 , IgA 1 , and IgA 2 ).
  • variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids (see, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety)).
  • the variable regions of each light/heavy chain pair typically form the antigen binding site.
  • the variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs.
  • both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol.196: 901-917; Chothia et al., (1989) Nature 342: 878-883.
  • the CDRs of a light chain can also be referred to as CDR-L1, CDR-L2, and CDR-L3, and the CDRs of a heavy chain can also be referred to as CDR-H1, CDR-H2, and CDR-H3.
  • an antibody can comprise a small number of amino acid deletions from the carboxy end of the heavy chain(s).
  • an antibody comprises a heavy chain having 1-5 amino acid deletions in the carboxy end of the heavy chain.
  • definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and/or solving the structure of the antibody-ligand complex.
  • an "affinity matured" antibody is an antibody with one or more alterations in one or more CDRs thereof, which result in an improvement in the affinity of the antibody for antigen compared to a parent antibody, which does not possess those alteration(s).
  • Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities (e.g., KD of ⁇ 10 -9 M-10 -12 M range) for the target antigen.
  • Affinity matured antibodies are produced by procedures known in the art. Marks et al., (1992) Bio/Technology 10: 779-783 describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by Barbas, et al., (1994) Proc Nat. Acad. Sci. USA 91: 3809-3813; Schier et al., (1995) Gene 169: 147- 155; Yelton et al., (1995) J.
  • CDR-grafted antibody refers to antibodies, which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
  • chimeric antibody refers to antibodies, which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.
  • the term "framework” or "framework sequence” refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations.
  • the six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4.
  • a framework region represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain.
  • a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 1 (H-FR1) having the following amino acid sequence with optionally 1, 2 or 3 amino acid changes: EVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 147).
  • the Gly residue at position 16 may be replaced with an Arg (R); and/or, the Ala residue at position 23 may be replaced with a Thr (T).
  • the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 2 (H-FR2) having the following amino acid sequence with optionally 1, 2 or 3 amino acid changes: WVRQAPGKGLEWVS (SEQ ID NO: 148).
  • the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 3 (H-FR3) having the following amino acid sequence with optionally 1, 2 or 3 amino acid changes: RFTISRDNAKNSLYLQMNSLRAEDTAVYYC (SEQ ID NO: 149).
  • the Ser residue at position 12 may be replaced with a Thr (T).
  • the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 4 (H-FR4) having the following amino acid sequence with optionally 1, 2 or 3 amino acid changes: WGQGTLVTVSS (SEQ ID NO: 150).
  • the antibody or antigen-binding fragment thereof comprises a light chain framework region 1 (L-FR1) having the following amino acid sequence with optionally 1, 2 or 3 amino acid changes: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 151).
  • the antibody or antigen-binding fragment thereof comprises a light chain framework region 2 (L-FR2) having the following amino acid sequence with optionally 1, 2 or 3 amino acid changes: WYQQKPGKAPKLLIY (SEQ ID NO: 152).
  • the antibody or antigen-binding fragment thereof comprises a light chain framework region 3 (L-FR3) having the following amino acid sequence with optionally 1, 2 or 3 amino acid changes: GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 153).
  • the antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (L-FR4) having the following amino acid sequence with optionally 1, 2 or 3 amino acid changes: FGGGTKVEIK (SEQ ID NO: 154).
  • the antibody, or antigen binding portion thereof comprises a heavy chain immunoglobulin constant domain of a human IgM constant domain, a human IgG constant domain, a human IgG1 constant domain, a human IgG2 constant domain, a human IgG2A constant domain, a human IgG2B constant domain, a human IgG2 constant domain, a human IgG3 constant domain, a human IgG3 constant domain, a human IgG4 constant domain, a human IgA constant domain, a human IgA1 constant domain, a human IgA2 constant domain, a human IgD constant domain, or a human IgE constant domain.
  • the antibody, or antigen binding portion thereof comprises a heavy chain immunoglobulin constant domain of a human IgG1 constant domain or a human IgG4 constant domain. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain immunoglobulin constant domain of a human IgG4 constant domain. In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain immunoglobulin constant domain of a human IgG4 constant domain having a backbone substitution of Ser to Pro that produces an IgG1-like hinge and permits formation of inter-chain disulfide bonds.
  • the antibody or antigen binding portion thereof further comprises a light chain immunoglobulin constant domain comprising a human Ig lambda constant domain or a human Ig kappa constant domain.
  • the antibody is an IgG having four polypeptide chains which are two heavy chains and two light chains.
  • the antibody is a humanized antibody, a diabody, or a chimeric antibody.
  • the antibody is a humanized antibody.
  • the antibody is a human antibody.
  • the antibody comprises a framework having a human germline amino acid sequence.
  • the antigen binding portion is a Fab fragment, a F(ab')2 fragment, a scFab fragment, or an scFv fragment.
  • the antibody contains one or more amino acid modifications in the Fc region.
  • modifications in the Fc region may provide altered properties, such as altered half life in circulation, e.g., by altering affinity for Fc receptors such as FcRn (Front Immunol.2019 Jun 7;10:1296).
  • modifications to the Fc region of the antibody provides increased Fc ⁇ R binding.
  • modifications in the Fc region provide improved antibody effector function.
  • modifications in the Fc region provide increased in vivo half-life for the antibody.
  • the half life of the antibody may be increased by increasing its affinity for binding to FcRn at low pH (e.g., pH ⁇ 6.5).
  • such Fc modifications may include Met252Tyr, Ser254Thr, and Thr256Glu substitutions (see, e.g., US7083784).
  • the term "germline antibody gene” or “gene fragment” refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin (see, e.g., Shapiro et al., (2002) Crit. Rev. Immunol.22(3): 183-200; Marchalonis et al., (2001) Adv. Exp. Med. Biol.484: 13-30).
  • neutralizing refers to counteracting the biological activity of an antigen (e.g., target protein) when a binding protein specifically binds to the antigen.
  • the neutralizing binding protein binds to the antigen/ target, e.g., cytokine, kinase, growth factor, cell surface protein, soluble protein, phosphatase, or receptor ligand, and reduces its biologically activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%. 96%, 97%. 98%, 99% or more.
  • a neutralizing antibody to a growth factor specifically binds a mature, soluble growth factor that has been released from a latent complex, thereby preventing its ability to bind its receptor to elicit downstream signaling.
  • the mature growth factor is TGF ⁇ 1 or TGF ⁇ 3.
  • binding protein includes any polypeptide that specifically binds to an antigen (e.g., TGF ⁇ 1), including, but not limited to, an antibody, or antigen binding portions thereof, and a bispecific or multispecific construct that comprises an antigen binding region (e.g., a region capable of binding TGF ⁇ 1) and a region capable of binding one or more additional antigens or additional epitopes on a single antigen.
  • an antigen e.g., TGF ⁇ 1
  • a bispecific or multispecific construct that comprises an antigen binding region (e.g., a region capable of binding TGF ⁇ 1) and a region capable of binding one or more additional antigens or additional epitopes on a single antigen.
  • an antigen binding region e.g., a region capable of binding TGF ⁇ 1
  • additional antigens or additional epitopes e.g., a single antigen.
  • Examples include a DVD-IgTM, a TVD-Ig, a RAb-Ig,
  • a binding protein may also comprise an antibody-drug conjugate, e.g., wherein a second agent (e.g., a small molecule checkpoint inhibitor) is linked to an antibody or antigen-binding fragment thereof capable of binding TGF ⁇ 1 (e.g., capable of binding pro- and/or latent-TGF ⁇ 1) [544]
  • a second agent e.g., a small molecule checkpoint inhibitor
  • an antibody or antigen-binding fragment thereof capable of binding TGF ⁇ 1 (e.g., capable of binding pro- and/or latent-TGF ⁇ 1)
  • the term "monoclonal antibody” or “mAb” when used in a context of a composition comprising the same may refer to an antibody preparation obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen.
  • each mAb is directed against a single determinant on the antigen.
  • the modifier "monoclonal” is not to be construed as requiring production of the antibody by any particular method.
  • the term "recombinant human antibody,” as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section II C, below), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom, H.R. (1997) TIB Tech.
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • “Dual Variable Domain Immunoglobulin” or “DVD-IgTM” and the like include binding proteins comprising a paired heavy chain DVD polypeptide and a light chain DVD polypeptide with each paired heavy and light chain providing two antigen binding sites.
  • Each binding site includes a total of 6 CDRs involved in antigen binding per antigen binding site.
  • a DVD-IgTM is typically has two arms bound to each other at least in part by dimerization of the CH3 domains, with each arm of the DVD being bispecific, providing an immunoglobulin with four binding sites. DVD-IgTM are provided in US Patent Publication Nos.2010/0260668 and 2009/0304693, each of which are incorporated herein by reference including sequence listings.
  • “Triple Variable Domain Immunoglobulin” or “TVD-Ig” and the like are binding proteins comprising a paired heavy chain TVD binding protein polypeptide and a light chain TVD binding protein polypeptide with each paired heavy and light chain providing three antigen binding sites. Each binding site includes a total of 6 CDRs involved in antigen binding per antigen binding site.
  • a TVD binding protein may have two arms bound to each other at least in part by dimerization of the CH3 domains, with each arm of the TVD binding protein being trispecific, providing a binding protein with six binding sites.
  • Receptor-Antibody Immunoglobulin or “RAb-Ig” and the like are binding proteins comprising a heavy chain RAb polypeptide, and a light chain RAb polypeptide, which together form three antigen binding sites in total.
  • One antigen binding site is formed by the pairing of the heavy and light antibody variable domains present in each of the heavy chain RAb polypeptide and the light chain RAb polypeptide to form a single binding site with a total of 6 CDRs providing a first antigen binding site.
  • Each the heavy chain RAb polypeptide and the light chain RAb polypeptide include a receptor sequence that independently binds a ligand providing the second and third “antigen” binding sites.
  • a RAb-Ig typically has two arms bound to each other at least in part by dimerization of the CH3 domains, with each arm of the RAb-Ig being trispecific, providing an immunoglobulin with six binding sites.
  • RAb-Igs are described in US Patent Application Publication No. 2002/0127231, the entire contents of which including sequence listings are incorporated herein by reference).
  • the present disclosure provides, in part, novel antibodies and antigen-binding fragments that may be used alone, linked to one or more additional agents (e.g., as ADCs), or as part of a larger macromolecule (e.g., a bispecific antibody, dual-specific antibody, or as a multispecific antibody, or as part of a construct further comprising a ligand trap, e.g., in combination with a TGFB ligand trap such as M7824 (Merck) and AVID200 (Forbius)), or as part of a bifunctional or multifunctional engineered construct (e.g., fusion proteins and ligand traps) and may be administered as part of pharmaceutical compositions or combination therapies.
  • additional agents e.g., as ADCs
  • a larger macromolecule e.g., a bispecific antibody, dual-specific antibody, or as a multispecific antibody, or as part of a construct further comprising a ligand trap, e.g., in combination with a TGFB
  • bispecific antibody refers to full-length antibodies that are generated by quadroma technology (see Milstein, C. and Cuello, A.C. (1983) Nature 305(5934): p.537-540), by chemical conjugation of two different monoclonal antibodies (see Staerz, U.D. et al., (1985) Nature 314(6012): 628-631), or by knob-into-hole or similar approaches, which introduce mutations in the Fc region that do not inhibit CH3-CH3 dimerization (see Holliger, P.
  • a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC).
  • a bispecific antibody has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen it binds to.
  • a bispecific antibody comprising two binding arms directed toward TGF ⁇ 1 and PD-1 may be used to combine a TGF ⁇ 1 inhibitor (Ab6 or Ab6-derived binding moiety) and a checkpoint inhibitor (e.g., an anti-PD1 antibody or moiety).
  • a bispecific antibody may be used as an exemplary form of treatment for patients selected to receive a TGF ⁇ 1 inhibitor and checkpoint inhibitor combination therapy.
  • a dual- specific binding protein has two identical antigen binding arms, with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.
  • the term “multispecific antibody” refers to an antibody or antigen binding fragment that displays binding specificity for two or more epitopes, where each binding site differs and recognizes a different epitope (on the same or different antigens).
  • a bispecific antibody is an exemplary type of multispecific antibody. Higher order multispecifics (i.e., antibodies exhibiting more than two specificities) include but are not limited to trispecific antibodies in TriMAb, triple body, and tribody formats.
  • the term "Kon,” as used herein, is intended to refer to the on rate constant for association of a binding protein (e.g., an antibody) to the antigen to form the, e.g., antibody/antigen complex as is known in the art.
  • the “Kon” also is known by the terms “association rate constant,” or “ka,” as used interchangeably herein. This value indicating the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen also is shown by the equation: Antibody (“Ab”) + Antigen (“Ag”) ⁇ Ab-Ag.
  • Koff is intended to refer to the off rate constant for dissociation of a binding protein (e.g., an antibody) from the, e.g., antibody/antigen complex as is known in the art.
  • the “Koff” also is known by the terms “dissociation rate constant” or “kd” as used interchangeably herein. This value indicates the dissociation rate of an antibody from its target antigen or separation of Ab-Ag complex over time into free antibody and antigen as shown by the equation: Ab + Ag ⁇ Ab-Ag.
  • equilibrium dissociation constant refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (koff) by the association rate constant (kon).
  • the association rate constant, the dissociation rate constant, and the equilibrium dissociation constant are used to represent the binding affinity of a binding protein, e.g., antibody, to an antigen.
  • Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence– based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium.
  • Crystal and “crystallized” as used herein, refer to a binding protein (e.g., an antibody), or antigen binding portion thereof, that exists in the form of a crystal. Crystals are one form of the solid state of matter, which is distinct from other forms such as the amorphous solid state or the liquid crystalline state.
  • Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen/antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well-understood in the field.
  • the fundamental unit, or building block, that is repeated in a crystal is called the asymmetric unit. Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined crystallographic symmetry provides the "unit cell" of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giege, R. and Ducruix, A.
  • linker is used to denote polypeptides comprising two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions.
  • linker polypeptides are well known in the art (see, e.g., Holliger, P. et al., (1993) Proc. Natl. Acad. Sci. USA 90: 6444- 6448; Poljak, R.J. et al., (1994) Structure 2:1121-1123).
  • linkers include, but are not limited to, ASTKGPSVFPLAP (SEQ ID NO: 44), ASTKGP (SEQ ID NO: 45); TVAAPSVFIFPP (SEQ ID NO: 46); TVAAP (SEQ ID NO: 47); AKTTPKLEEGEFSEAR (SEQ ID NO: 48); AKTTPKLEEGEFSEARV (SEQ ID NO: 49); AKTTPKLGG (SEQ ID NO: 50); SAKTTPKLGG (SEQ ID NO: 51); SAKTTP (SEQ ID NO: 52); RADAAP (SEQ ID NO: 53); RADAAPTVS (SEQ ID NO: 54); RADAAAAGGPGS (SEQ ID NO: 55); RADAAAA(G4S)4 (SEQ ID NO: 56); SAKTTPKLEEGEFSEARV (SEQ ID NO: 57); ADAAP (SEQ ID NO: 58); ADAAPTVSIFPP (SEQ ID NO: 59); QPKAAP (SEQ ID NO: 60); QPKAAPS (
  • Label and “detectable label” or “detectable moiety” mean a moiety attached to a specific binding partner, such as an antibody or an analyte, e.g., to render the reaction between members of a specific binding pair, such as an antibody and an analyte, detectable, and the specific binding partner, e.g., antibody or analyte, so labeled is referred to as “detectably labeled.”
  • a specific binding partner such as an antibody or an analyte
  • the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods).
  • marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 18 F.
  • labels commonly employed for immunoassays include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety. Use of “detectably labeled” is intended to encompass the latter type of detectable labeling.
  • the binding affinity of an antibody, or antigen binding portion thereof, to an antigen is determined using BLI (e.g., an Octet® assay).
  • a BLI e.g., Octet® assay is an assay that determines one or more a kinetic parameters indicative of binding between an antibody and antigen.
  • an Octet® system (FortéBio®, Menlo Park, CA) is used to determine the binding affinity of an antibody, or antigen binding portion thereof, to presenting molecule-proTGF ⁇ 1 complexes.
  • binding affinities of antibodies may be determined using the FortéBio Octet® QKe dip and read label free assay system utilizing bio-layer interferometry.
  • antigens are immobilized to biosensors (e.g., streptavidin-coated biosensors) and the antibodies and complexes (e.g., biotinylated presenting molecule-proTGF ⁇ 1 complexes) are presented in solution at high concentration (50 ⁇ g/mL) to measure binding interactions.
  • the binding affinity of an antibody, or antigen binding portion thereof, to a presenting molecule-proTGF ⁇ 1 complex is determined using the protocol outlined herein. Characterization of Exemplary Antibodies against proTGF ⁇ 1 Binding profiles [559]
  • Exemplary antibodies according to the present disclosure include those having enhanced binding activities (e.g., subnanomolar KD).
  • a class of high-affinity, context-independent antibodies capable of selectively inhibiting TGF ⁇ 1 activation.
  • context independent is used herein with a greater degree of stringency as compared to previous more general usage.
  • the term confers a level of uniformity in relative affinities (i.e., unbias) that the antibody can exert towards different antigen complexes.
  • the context-independent antibody of the present disclosure is capable of targeting multiple types of TGF ⁇ 1 precursor complexes (e.g., presenting molecule-proTGF ⁇ 1 complexes) and of binding to each such complex with equivalent affinities (i.e., no greater than three-fold differences in relative affinities across the complexes) with KD values lower than 10 nM, preferably lower than 5 nM, more preferably lower than 1 nM, even more preferably lower than 100 pM, as measured by, for example, MSD-SET.
  • KD values lower than 10 nM preferably lower than 5 nM, more preferably lower than 1 nM, even more preferably lower than 100 pM, as measured by, for example, MSD-SET.
  • many antibodies encompassed by the disclosure have KD values in a sub-nanomolar range.
  • the antibodies are capable of specifically binding to each of the human presenting molecule- proTGF ⁇ 1 complexes (sometimes referred to as “Large Latency Complex” which is a ternary complex comprised of a proTGF ⁇ 1 dimer coupled to a single presenting molecule), namely, LTBP1-proTGF ⁇ 1, LTBP3-proTGF ⁇ 1, GARP-proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1.
  • purified protein complexes are used as antigens (e.g., antigen complexes) to evaluate or confirm the ability of an antibody to bind the antigen complexes in suitable in vitro binding assays.
  • BLI-based binding assays are widely used in the art for measuring affinities and kinetics of antibodies to antigens. It is a label-free technology in which biomolecular interactions are analyzed on the basis of optical interference.
  • One of the proteins for example, an antibody being tested, can be immobilized on the biosensor tip. When the other protein in solution, for example, an antigen, becomes bound to the immobilized antibody, it causes a shift in the interference pattern, which can be measured in real-time.
  • BLI is a kinetic measure that reveals the dynamics of the system. Due to its ease of use and fast results, BLI-based assays such as the Octet® system (available from FortéBio®/Molecular Devices®, Fremont California), are particularly convenient when used as an initial screening method to identify and separate a pool of “binders” from a pool of “non-binders” or “weak binders” in the screening process. [562] BLI-based binding assays revealed that the novel antibodies are characterized as “context- balanced/context-independent” antibodies when binding affinity is measured by Octet®.
  • Non-limiting examples of context-independent TGF ⁇ 1 antibodies and KD values measured by BLI [565] The disclosure provides a class of high-affinity, context-independent antibodies, each of which is capable of binding with equivalent affinities to each of the four known presenting molecule-proTGF ⁇ 1 complexes, namely, LTBP1-proTGF ⁇ 1, LTBP3-proTGF ⁇ 1, GARP-proTGF ⁇ 1, and LRRC33-proTGF ⁇ 1.
  • the antibody binds each of the presenting molecule-proTGF ⁇ 1 complexes with equivalent or higher affinities, as compared to the previously described reference antibody, Ab3.
  • such antibody specifically binds each of the aforementioned complexes with an affinity (determined by K D ) of ⁇ 5 nM as measured by a suitable in vitro binding assay, such as Biolayer Interferometry and surface plasmon resonance.
  • the antibody or the fragment binds a human LTBP1-proTGF ⁇ 1 complex with an affinity of ⁇ 5 nM, ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 5 nM or ⁇ 0.5 nM.
  • the antibody or the fragment binds a human LTBP3-proTGF ⁇ 1 complex with an affinity of ⁇ 5 nM, ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 5 nM or ⁇ 0.5 nM. In some embodiments, the antibody or the fragment binds a human GARP-proTGF ⁇ 1 complex with an affinity of ⁇ 5 nM, ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 5 nM or ⁇ 0.5 nM.
  • the antibody or the fragment binds a human LRRC33-proTGF ⁇ 1 complex with an affinity of ⁇ 5 nM, ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM or ⁇ 0.5 nM.
  • such antibody is human- and murine-cross-reactive.
  • the antibody or the fragment binds a murine LTBP1-proTGF ⁇ 1 complex with an affinity of ⁇ 5 nM, ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 5 nM or ⁇ 0.5 nM.
  • the antibody or the fragment binds a murine LTBP3- proTGF ⁇ 1 complex with an affinity of ⁇ 5 nM, ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM or ⁇ 0.5 nM. In some embodiments, the antibody or the fragment binds a murine GARP-proTGF ⁇ 1 complex with an affinity of ⁇ 5 nM, ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM or ⁇ 0.5 nM.
  • the antibody or the fragment binds a murine LRRC33-proTGF ⁇ 1 complex with an affinity of ⁇ 5 nM, ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM or ⁇ 0.5 nM.
  • the proTGF ⁇ 1 antibodies of the present disclosure have particularly high affinities for matrix- associated proTGF ⁇ 1 complexes.
  • the average K D value of the matrix-associated complexes i.e., LTBP1-proTGF ⁇ 1 and LTBP3-proTGF ⁇ 1 is ⁇ 1 nM or ⁇ 0.5 nM.
  • the proTGF ⁇ 1 antibodies of the present disclosure have high affinities for cell-associated proTGF ⁇ 1 complexes.
  • the average KD value of the cell-associated complexes i.e., GARP- proTGF ⁇ 1 and LRRC33-proTGF ⁇ 1
  • the high-affinity proTGF ⁇ 1 antibodies of the present disclosure are characterized by their uniform (unbiased) affinities towards the all four antigen complexes (compare, for example, to Ab3). No single antigen complex among the four known presenting molecule-proTGF ⁇ 1 complexes described herein deviates significantly in KD.
  • the antibody or the fragment shows unbiased or uniform binding profiles, characterized in that the difference (or range) of affinities of the antibody or the fragments across the four proTGF ⁇ 1 antigen complexes is no more than five-fold between the lowest and the highest KD values. In some embodiments, the relative difference (or range) of affinities is no more than three-fold. [570] The concept of “uniformity” or lack of bias is further illustrated in Table 5.
  • Average KD values between the two matrix-associated and cell-associated complexes are calculated, respectively (see columns (D) and (G)). These average KD values can then be used to ask whether bias in binding activities exists between complexes associated with matrix vs. complexes associated with cell surface (e.g., immune cells). Bias may be expressed as “fold-difference” in the average KD values, as illustrated in Table 5.
  • the high-affinity, context-independent proTGF ⁇ 1 antibodies encompassed by the present disclosure are remarkably unbiased in that many show no more than three-fold difference in average KD values between matrix- and cell-associated complexes (compare this to 25+ fold bias in Ab3).
  • a class of context-independent monoclonal antibodies or fragments is provided, each of which is capable of binding with equivalent affinities to each of the following presenting molecule-proTGF ⁇ 1 complexes with an affinity of ⁇ 1 nM as measured by Biolayer Interferometry or surface plasmon resonance: LTBP1-proTGF ⁇ 1, LTBP3-proTGF ⁇ 1, GARP-proTGF ⁇ 1, and LRRC33-proTGF ⁇ 1.
  • Such antibody specifically binds each of the aforementioned complexes with an affinity of ⁇ 5 nM as measured by Biolayer Interferometry or surface plasmon resonance, wherein the monoclonal antibody or the fragment shows no more than a three-fold bias in affinity towards any one of the above complexes relative to the other complexes, and wherein the monoclonal antibody or the fragment inhibits release of mature TGF ⁇ 1 growth factor from each of the proTGF ⁇ 1 complexes but not from proTGF ⁇ 2 or proTGF ⁇ 3 complexes.
  • antibodies with fast “on” rate (“Kon”) which would be reflected in binding measurements obtained by BLI, may provide relevant parameters for evaluating neutralizing antibodies (e.g., antibodies that directly target and must rapidly sequester the active, soluble growth factor itself for them to function as effective inhibitors).
  • neutralizing antibodies e.g., antibodies that directly target and must rapidly sequester the active, soluble growth factor itself for them to function as effective inhibitors.
  • the same may not necessarily apply for antibodies that function as activation inhibitors, such as those disclosed herein.
  • the mechanism of action of the novel TGF ⁇ 1 inhibitors of the present disclosure is via the inhibition of the activation step, which is achieved by targeting the tissue/cell-tethered latent complex, as opposed to sequestration of soluble, post-activation growth factor.
  • an activation inhibitor of TGF ⁇ 1 targets the inactive precursor localized to respective tissues (e.g., within the ECM, immune cell surface, etc.) thereby preemptively prevent the mature growth factor from being released from the complex.
  • This mechanism of action is thought to allow the inhibitor to achieve target saturation (e.g., equilibrium) in vivo, without the need for rapidly competing for transient growth factor molecules against endogenous receptors as required by conventional neutralizing inhibitors. [573] Taking this difference in the mechanism of action into consideration, further evaluation of binding properties was carried out by the use of another mode of in vitro binding assays that allows the determination of affinity at equilibrium.
  • MSD-SET-based binding assays may be performed, as exemplified in Table 6 below.
  • Solution equilibrium titration (“SET”) is an assay whereby binding between two molecules (such as an antigen and an antibody that binds the antigen) can be measured at equilibrium in a solution.
  • MSD-based SET is a useful mode of determining dissociation constants for particularly high-affinity protein-protein interactions at equilibrium (see, for example: Ducata et al., (2015) J Biomolecular Screening 20(10): 1256-1267).
  • the SET-based assays are particularly useful for determining KD values of antibodies with sub-nanomolar (e.g., picomolar) affinities. Table 6.
  • Table 6 also includes three previously described TGF ⁇ 1-selective antibodies (C1, C2 and Ab3) as reference antibodies.
  • C1 and C2 were first disclosed in PCT/US2017/021972 published as WO 2017/156500 (corresponding to “Ab1” and “Ab2” therein), and Ab3 was described in PCT/US2018/012601 published as WO 2018/129329 (corresponding to “Ab3” therein).
  • binding activities of the novel antibodies according to the present disclosure are significantly higher than the previously identified reference antibodies.
  • novel TGF ⁇ 1 antibodies are “context-independent” in that they bind to each of the human LLC complexes with equivalent affinities (e.g., ⁇ sub-nanomolar range, e.g., with KD of ⁇ 1 nM).
  • equivalent affinities e.g., ⁇ sub-nanomolar range, e.g., with KD of ⁇ 1 nM.
  • the high-affinity, context-independent binding profiles suggest that these antibodies may be advantageous for use in the treatment of TGF ⁇ 1-related indications that involve dysregulation of both the ECM-related and immune components, such as cancer.
  • protein complexes that comprise one of the presenting molecules such as those shown above may be employed as antigen (presenting molecule-TGF ⁇ 1 complex, or an LLC).
  • Test antibodies are allowed to form antigen-antibody complex in solution.
  • Antigen- antibody reaction mixtures are incubated to allow an equilibrium to be reached; the amount of the antigen- antibody complex present in the assay reactions can be measured by suitable means well known in the art.
  • SET-based assays are less affected by on/off rates of the antigen-antibody complex, allowing sensitive detection of very high affinity interactions.
  • certain high-affinity inhibitors of TGF ⁇ 1 show a sub-nanomolar (e.g., picomolar) range of affinities across all large latent complexes tested, as determined by SET-based assays.
  • a class of context-independent monoclonal antibodies or fragments is provided, each of which is capable of binding with equivalent affinities to each of the following human presenting molecule- proTGF ⁇ 1 complexes with a K D of ⁇ 1 nM as measured by a solution equilibrium titration assay, such as MSD- SET: hLTBP1-proTGF ⁇ 1, hLTBP3-proTGF ⁇ 1, hGARP-proTGF ⁇ 1, and hLRRC33-proTGF ⁇ 1.
  • Such antibody specifically binds each of the aforementioned complexes with a K D of ⁇ 1 nM as measured by MSD-SET, and wherein the monoclonal antibody or the fragment inhibits release of mature TGF ⁇ 1 growth factor from each of the proTGF ⁇ 1 complexes but not from proTGF ⁇ 2 or proTGF ⁇ 3 complexes.
  • such antibody or the fragment binds each of the aforementioned complexes with a KD of 500 pM or less (i.e., ⁇ 500 pM), 250 pM or less (i.e., ⁇ 250 pM), or 200 pM or less (i.e., ⁇ 200 pM).
  • such antibody or the fragment binds each of the aforementioned complexes with a KD of 100 pM or less (i.e., ⁇ 100 pM).
  • the antibody or the fragment does not bind to free TGF ⁇ 1 growth factor which is not associated with the prodomain complex.
  • the antibody or the fragment does not bind to LTBP1/TGF ⁇ 2 or LTBP3/TGF ⁇ 3 LLCs. This can be tested or confirmed by suitable in vitro binding assays known in the art, such as biolayer interferometry.
  • such antibodies or the fragments are also cross-reactive with murine (e.g., rat and/or mouse) and/or non-human primate (e.g., cyno) counterparts.
  • murine e.g., rat and/or mouse
  • non-human primate e.g., cyno
  • Ab6 is capable of binding with high affinity to each of the large latent complexes of multiple species, including: human, murine, rat, and cynomolgus monkey, as exemplified in Table 7 below. Table 7.
  • Non-limiting example of a TGF ⁇ 1 antibody with cross-species reactivities as measured by MSD-SET (“h” denotes human; “m” denotes murine)
  • SPR Surface plasmon resonance
  • the SPR-based biosensors (such as Biacore systems) can be used in determination of active concentration as well as characterization of molecular interactions in terms of both affinity and chemical kinetics.
  • K OFF slow off rates
  • Ab6 which is an activation inhibitor of TGF ⁇ 1
  • the off rate of an antibody may therefore be an important binding kinetics criterion for selection consideration for a therapeutic antibody to be manufactured and for use in human therapy described herein.
  • the invention includes a TGF ⁇ inhibitor which is an antibody or antigen-binding fragment thereof, for use in the treatment of cancer in a subject (according to the present disclosure), wherein the antibody or the fragment with a K OFF of less than 10.0E-4 (1/s) is selected, wherein optionally the selected antibody has a KD of less than 0.5 nM as measured by SPR.
  • the invention further includes a method for manufacturing a pharmaceutical composition comprising a TGF ⁇ inhibitor which is an antibody or antigen-binding fragment thereof, for use in the treatment of cancer in a subject (according to the present disclosure), the method comprising the step of selecting an antibody or antigen- binding fragment which has a KOFF of less than 10.0E-4 (1/s) and optionally has a KD of less than 0.5 nM as measured by SPR.
  • Potency [584]
  • Antibodies disclosed herein may be broadly characterized as “functional antibodies” for their ability to inhibit TGF ⁇ 1 signaling.
  • a functional antibody confers one or more biological activities by virtue of its ability to bind a target protein (e.g., antigen), in such a way as to modulate its function.
  • Functional antibodies therefore broadly include those capable of modulating the activity/function of target molecules (i.e., antigen).
  • modulating antibodies include inhibiting antibodies (or inhibitory antibodies) and activating antibodies.
  • the present disclosure is drawn to antibodies which can inhibit a biological process mediated by TGF ⁇ signaling associated with multiple contexts of TGF ⁇ 1.
  • Inhibitory agents used to carry out the present disclosure are intended to be TGF ⁇ 1-selective and not to target or interfere with TGF ⁇ 2 and TGF ⁇ 3 when administered at a therapeutically effective dose (dose at which sufficient efficacy is achieved within acceptable toxicity levels).
  • the novel antibodies of the present disclosure have enhanced inhibitory activities (potency) as compared to previously identified activation inhibitors of TGF ⁇ 1.
  • potency of an inhibitory antibody may be measured in suitable cell-based assays, such as CAGA reporter cell assays described herein.
  • cultured cells such as heterologous cells and primary cells, may be used for carrying out cell-based potency assays.
  • Cells that express endogenous TGF ⁇ 1 and/or a presenting molecule of interest may be used.
  • exogenous nucleic acids encoding protein(s) of interest such as TGF ⁇ 1 and/or a presenting molecule of interest, such as LTBP1, LTBP3, GARP and LRRC33, may be introduced into such cells for expression, for example by transfection (e.g., stable transfection or transient transfection) or by viral vector-based infection.
  • LN229 cells are employed for such assays.
  • the cells expressing TGF ⁇ 1 and a presenting molecule of interest are grown in culture, which “present” the large latent complex either on cell surface (when associated with GARP or LRRC33) or deposit into the ECM (when associated with an LTBP).
  • a presenting molecule of interest e.g., LTBP1, LTBP3, GARP or LRRC33
  • Activation of TGF ⁇ 1 may be triggered by integrin, expressed on another cell surface.
  • the integrin- expressing cells may be the same cells co-expressing the large latent complex or a separate cell type. Reporter cells are added to the assay system, which incorporates a TGF ⁇ -responsive element.
  • the degree of TGF ⁇ activation may be measured by detecting the signal from the reporter cells (e.g., TGF ⁇ -responsive reporter genes, such as luciferase coupled to a TGF ⁇ -responsive promoter element) upon TGF ⁇ activation.
  • the reporter cells e.g., TGF ⁇ -responsive reporter genes, such as luciferase coupled to a TGF ⁇ -responsive promoter element
  • inhibitory activities of the antibodies can be determined by measuring the change (reduction) or difference in the reporter signal (e.g., luciferase activities as measured by fluorescence readouts) either in the presence or absence of test antibodies.
  • Such assays are exemplified in PCT/US2019/041373 at Example 2.
  • the inhibitory potency (IC50) of the novel antibodies of the present disclosure calculated based on cell-based reporter assays for measuring TGF ⁇ 1 activation (such as LN229 cell assays described elsewhere herein) may be 5 nM or less, measured against each of the hLTBP1-proTGF ⁇ 1, hLTBP3- proTGF ⁇ 1, hGARP-proTGF ⁇ 1 and hLRRC33-proTGF ⁇ 1 complexes.
  • the antibodies have an IC 50 of 2 nM or less (i.e., ⁇ 2 nM) measured against each of the LLCs.
  • the IC 50 of the antibody measured against each of the LLC complexes is 1nM or less. In some embodiments, the antibody has an IC 50 of less than 1 nM against each of the hLTBP1-proTGF ⁇ 1, hLTBP3-proTGF ⁇ 1, hGARP-proTGF ⁇ 1 and hLRRC33-proTGF ⁇ 1 complexes. Table 8. Inhibitory potencies (in IC50) of select antibodies as measured by reporter cell assays [587] Activation of TGF ⁇ 1 may be triggered by an integrin-dependent mechanism or protease-dependent mechanism.
  • the inhibitory activities (e.g., potency) of the antibodies according to the present disclosure may be evaluated for the ability to block TGF ⁇ 1 activation induced by one or both of the modes of activation.
  • the reporter cell assays described above are designed to measure the ability of the antibodies to block or inhibit integrin- dependent activation of TGF ⁇ 1 activation.
  • Inhibitory potency may also be assessed by measuring the ability of the antibodies to block protease-induced activation of TGF ⁇ 1.
  • Non-limiting embodiments of such assays were previously disclosed. See, e.g., PCT/US2019/041373 at Example 3.
  • the isoform-selective inhibitor according to the present disclosure is capable of inhibiting integrin- dependent activation of TGF ⁇ 1 and protease-dependent activation of TGF ⁇ 1.
  • Such inhibitor may be used to treat a TGF ⁇ 1-related indication characterized by EDM dysregulation involving protease activities.
  • TGF ⁇ 1-related indication may be associated with elevated myofibroblasts, increased stiffness of the ECM, excess or abnormal collagen deposition, or any combination thereof.
  • Such conditions include, for example, fibrotic disorders and cancer comprising a solid tumor (such as metastatic carcinoma) or myelofibrosis.
  • potency may be evaluated in suitable in vivo models as a measure of efficacy and/or pharmacodynamics effects. For example, if the first antibody is efficacious in an in vivo model at a certain concentration, and the second antibody is equally efficacious at a lower concentration than the first in the same in vivo model, then, the second antibody can be said to me more potent than the first antibody.
  • Any suitable disease models known in the art may be used to assess relative potencies of TGF ⁇ 1 inhibitors, depending on the particular indication of interest, e.g., cancer models and fibrosis models. Preferably, multiple doses or concentrations of each test antibody are included in such studies.
  • PD pharmacodynamics
  • TGF ⁇ signaling pathway include, without limitation, phosphorylation of SMAD2/3 and expression of downstream effector genes, the transcription of which is sensitive to TGF ⁇ activation, such as those with a TGF ⁇ -responsive promoter element (e.g., Smad-binding elements).
  • the antibodies of the present disclosure are capable of completely blocking disease-induced SMAD2/3 phosphorylation in preclinical fibrosis models when the animals are administered at a dose of 3 mg/kg or less.
  • the antibodies of the present disclosure are capable of reducing and/or completely blocking disease-induced SMAD2/3 phosphorylation. In some embodiments, the antibodies of the present disclosure are capable of reducing and/or completely blocking disease-induced SMAD2 phosphorylation (e.g., regardless of any change in SMAD3) , e.g., in the nucleus. In some embodiments, reduction is measured as a ratio of phosphorylated SMAD2/3 over total SMAD2/3. In some embodiments, reduction is measured as a ratio of phosphorylated SMAD2 over total SMAD2. In some embodiments, the antibodies of the present disclosure are capable of reducing nuclear localization of phosphorylated SMAD2, as measured, for example, by IHC.
  • measurement of nuclear localization of phosphorylated Smad2 can use one or more digital image analysis parameters to identify degrees of P-Smad2 nucleus staining intensities (e.g., a digital image analysis parameter that allows visualization and measurement of IHC signal intensity of an individual nucleus, e.g., a nucleus mask).
  • P-Smad2 positive nuclei can be sorted into categories (e.g., 1+, 2+, 3+) based on chromogenic intensity relative to normalized scanning parameters.
  • the analysis can allow for data to be considered in multiple contexts (e.g., % Nuclear Positivity, H-Score, Density), and can be combined with additional analysis parameters (e.g., compartment area, total number of quantified cells, number of P-Smad2 positive cells, and/or number or percent of 0 P-Smad2, 1+ P-Smad2, 2+ P-Smad2, or 3+ P-Smad2 cells).
  • additional analysis parameters e.g., compartment area, total number of quantified cells, number of P-Smad2 positive cells, and/or number or percent of 0 P-Smad2, 1+ P-Smad2, 2+ P-Smad2, or 3+ P-Smad2 cells.
  • Pseudocolor image masks can be applied to each staining category for visualization and QC purposes.
  • Exemplary tools for analyzing nuclear masking include, but are not limited to, software developed by Visiopharm, Indica Labs (e.g., HALO® imaging analysis platform), and Flagship Bioscience
  • measuring SMAD2 phosphorylation e.g., nuclear SMAD2 phosphorylation (without measuring SMAD3) may improve the accurate detection of a treatment-related effect (e.g., therapeutic efficacy and/or target engagement).
  • a treatment-related effect e.g., therapeutic efficacy and/or target engagement.
  • P-Smad2 nuclear translocation may be used in a method of determining therapeutic efficacy in a subject administered one or more doses of a TGF ⁇ inhibitor (e.g., Ab6).
  • a level of P-Smad2 nuclear translocation is determined in a tumor sample obtained from the subject before and after administering one or more doses of a TGF ⁇ inhibitor (e.g., Ab6), wherein a decrease in P-Smad2 nuclear translocation after the administration as compared to before the administration indicates therapeutic efficacy.
  • the P-Smad2 nuclear translocation may be determined using immunohistochemistry.
  • the P-Smad2 nuclear translocation may be determined using nuclear masking.
  • the P-Smad2 nuclear translocation may be determined using a digital image analysis tool, such as one developed by Flagship Biosciences, Visiopharm, or Indica Labs.
  • one or more additional doses of the TGF ⁇ inhibitor is administered if a decrease is detected.
  • P-Smad2 nuclear translocation may be used in a method of determining target engagement in a subject administered one or more doses of a TGF ⁇ inhibitor (e.g., Ab6).
  • a level of P-Smad2 nuclear translocation is determined in a tumor sample obtained from the subject before and after administering one or more doses of a TGF ⁇ inhibitor (e.g., Ab6), wherein a decrease in P-Smad2 nuclear translocation after the administration as compared to before the administration indicates target engagement.
  • the P-Smad2 nuclear translocation may be determined using immunohistochemistry. In some embodiments, the P-Smad2 nuclear translocation may be determined using nuclear masking. In some embodiments, the P-Smad2 nuclear translocation may be determined using a digital image analysis tool, such as one developed by Flagship Biosciences, Visiopharm, or Indica Labs.
  • one or more additional doses of the TGF ⁇ inhibitor e.g., Ab6 is administered if a decrease is detected.
  • P-Smad2 nuclear translocation may be used in conjunction with other biomarkers, such as tumor CD8+ cells (e.g., percent CD8+ cells in various tumor compartments, e.g., tumor nests, stroma, margin), to determine therapeutic efficacy and/or target engagement in a subject administered one or more doses of a TGF ⁇ inhibitor (e.g., Ab6).
  • TGF ⁇ inhibitor e.g., Ab6
  • therapeutic efficacy and/or target engagement may be indicated by, inter alia, a decrease in P-Smad2 nuclear translocation in a tumor sample obtained from the subject after the administration of a TGF ⁇ inhibitor (e.g., Ab6) as compared to before the administration.
  • therapeutic efficacy and/or target engagement may be indicated by a decrease in P-Smad2 nuclear translocation and an increase in CD8+ cells in a tumor sample obtained from the subject after the administration as compared to before the administration.
  • therapeutic efficacy and/or target engagement may be indicated by a decrease in P-Smad2 nuclear translocation and an increase in total tumor area comprising immune-inflamed tumor nests in a tumor sample obtained from the subject after the administration as compared to before the administration.
  • more than one additional marker is used to assess therapeutic efficacy and/or target engagement in conjunction with P-Smad2 nuclear translocation.
  • the TGF ⁇ inhibitor e.g., Ab6 is administered in conjunction with a checkpoint inhibitor therapy.
  • additional doses of the TGF ⁇ inhibitor may be administered to patients whose tumors show therapeutic efficacy and/or target engagement.
  • the antibodies of the present disclosure are capable of significantly suppressing fibrosis-induced expression of a panel of marker genes including Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, Loxl2, when the animals are administered at a dose of 10 mg/kg or less in the UUO model of kidney fibrosis.
  • the selection process of an antibody or antigen-binding fragment thereof for therapeutic use may therefore include identifying an antibody or fragment that shows sufficient inhibitory potency.
  • the selection process may include a step of carrying out a cell-based TGF ⁇ 1 activation assay to measure potency (e.g., IC 50 ) of one or more test antibodies or fragments thereof, and, selecting a candidate antibody or fragment thereof that shows desirable potency.
  • potency e.g., IC 50
  • the selected antibody or the fragment may then be used in the treatment of a TGF ⁇ 1-related indication described herein.
  • Binding regions [596] In the context of the present disclosure, “binding region(s)” of an antigen provides a structural basis for the antibody-antigen interaction.
  • a “binding region” refers to the areas of interface between the antibody and the antigen, such that, when bound to the proTGF ⁇ 1 complex (“antigen”) in a physiological solution, the antibody or the fragment protects the binding region from solvent exposure, as determined by suitable techniques, such as hydrogen-deuterium exchange mass spectrometry (HDX-MS). Identification of binding regions is useful in gaining insight into the antigen-antibody interaction and the mechanism of action for the particular antibody. Identification of additional antibodies with similar or overlapping binding regions may be facilitated by cross-blocking experiments that enable epitope binning. Optionally, X-ray crystallography may be employed to identify the exact amino acid residues of the epitope that mediate antigen-antibody interactions.
  • HDX-MS is a widely used technique for exploring protein conformation or protein-protein interactions in solution. This method relies on the exchange of hydrogens in the protein backbone amide with deuterium present in the solution. By measuring hydrogen-deuterium exchange rates, one can obtain information on protein dynamics and conformation (reviewed in: Wei et al., (2014) “Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications.” Drug Discov Today.19(1): 95-102; incorporated by reference).
  • the application of this technique is based on the premise that when an antibody-antigen complex forms, the interface between the binding partners may occlude solvent, thereby reducing or preventing the exchange rate due to steric exclusion of solvent.
  • the present disclosure includes antibodies or antigen-binding fragments thereof that bind a human LLC at a region (“binding region”) comprising Latency Lasso or a portion thereof. Latency Lasso is a protein module within the prodomain. It is contemplated that many potent activation inhibitors may bind this region of a proTGF ⁇ 1 complex in such a way that the antibody binding would “lock in” the growth factor thereby preventing its release.
  • this is the section of the complex where the butterfly-like elongated regions of the growth factor (e.g., corresponding to, for example, Finger-1 and Finger-2) closely interact with the cage-like structure of the prodomain.
  • an antibody that tightly wraps around the binding regions identified may effectively prevent the proTGF ⁇ 1 complex from disengaging (i.e., releasing the growth factor), thereby blocking activation.
  • binding regions of proTGF ⁇ 1 can be determined.
  • a portion on proTGF ⁇ 1 identified to be important in binding an antibody or fragment includes at least a portion of the prodomain and at least a portion of the growth factor domain.
  • Antibodies or fragments that bind a first binding region (“Region 1” in FIG.41) comprising at least a portion of Latency Lasso are preferable. More preferably, such antibodies or fragments further bind a second binding region (“Region 2” in FIG.41) comprising at least a portion of the growth factor domain at Finger-1 of the growth factor domain. Such antibodies or fragments may further bind a third binding region (“Region 3” in FIG. 41) comprising at least a portion of Finger-2 of the growth factor domain. [600] Additional regions within the proTGF ⁇ 1 may also contribute, directly or indirectly, to the high-affinity interaction of these antibodies disclosed herein.
  • Regions that are considered important for mediating the high- affinity binding of the antibody to the proTGF ⁇ 1 complex may include, but are not limited to: LVKRKRIEA (SEQ ID NO: 132); LASPPSQGEVP (SEQ ID NO: 133); PGPLPEAV (SEQ ID NO: 134); LALYNSTR (SEQ ID NO: 135); REAVPEPVL (SEQ ID NO: 136); YQKYSNNSWR (SEQ ID NO: 137); RKDLGWKWIHEPKGYHANF (SEQ ID NO: 138); LGPCPYIWS (SEQ ID NO: 139); ALEPLPIV (SEQ ID NO: 140); and, VGRKPKVEQL (SEQ ID NO: 141) (based on the native sequence of human proTGF ⁇ 1).
  • the high- affinity antibody of the present disclosure may bind an epitope that comprises at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL (“Region 1”) (SEQ ID NO: 142).
  • the high-affinity antibody of the present disclosure may bind an epitope that comprises at least one residue of the amino acid sequence RKDLGWKWIHEPKGYHANF (“Region 2”) (SEQ ID NO: 138).
  • the high-affinity antibody of the present disclosure may bind an epitope that comprises at least one residue of the amino acid sequence VGRKPKVEQL (“Region 3”) (SEQ ID NO: 141).
  • the high-affinity antibody of the present disclosure may bind an epitope that comprises at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL (“Region 1”) (SEQ ID NO: 142) and at least one residue of the amino acid sequence RKDLGWKWIHEPKGYHANF (“Region 2”) (SEQ ID NO: 138).
  • the high-affinity antibody of the present disclosure may bind an epitope that comprises at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL (“Region 1”) (SEQ ID NO: 142) and at least one residue of the amino acid sequence VGRKPKVEQL (“Region 3”) (SEQ ID NO: 141).
  • the high-affinity antibody of the present disclosure may bind an epitope that comprises at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL (“Region 1”) (SEQ ID NO: 142), at least one residue of the amino acid sequence RKDLGWKWIHEPKGYHANF (“Region 2”) (SEQ ID NO: 138), and, at least one residue of the amino acid sequence VGRKPKVEQL (“Region 3”) (SEQ ID NO: 141).
  • such epitope may further include at least one amino acid residues from a sequence selected from the group consisting of: LVKRKRIEA (SEQ ID NO: 132); LASPPSQGEVP (SEQ ID NO: 133); PGPLPEAV (SEQ ID NO: 134); LALYNSTR (SEQ ID NO: 135); REAVPEPVL (SEQ ID NO: 136); YQKYSNNSWR (SEQ ID NO: 137); RKDLGWKWIHEPKGYHANF (SEQ ID NO: 138); LGPCPYIWS (SEQ ID NO: 139); ALEPLPIV (SEQ ID NO: 140); and, VGRKPKVEQL (SEQ ID NO: 141).
  • LVKRKRIEA SEQ ID NO: 132
  • LASPPSQGEVP SEQ ID NO: 133
  • PGPLPEAV SEQ ID NO: 134
  • LALYNSTR SEQ ID NO: 135)
  • REAVPEPVL SEQ ID NO:
  • binding regions identified in structural studies using four representative isoform- selective TGF ⁇ 1 antibodies are found to be overlapping, pointing to certain regions within the proTGF ⁇ 1 complex that may be particularly important in maintaining latency of the proTGF ⁇ 1 complex.
  • antibodies or fragments thereof may be selected at least in part on the basis of their binding region(s) that include the overlapping portions identified across multiple inhibitors described herein.
  • These overlapping portions of binding regions include, for example, SPPSQGEVPPGPLPEAVL (SEQ ID NO: 165), WKWIHEPKGYHANF (SEQ ID NO: 166), and PGPLPEAVL (SEQ ID NO: 167).
  • the high-affinity, isoform-selective TGF ⁇ 1 inhibitor according to the present disclosure may bind a proTGF ⁇ 1 complex (e.g., human LLCs) at an epitope that comprises one or more amino acid residues of SPPSQGEVPPGPLPEAVL (SEQ ID NO: 165), WKWIHEPKGYHANF (SEQ ID NO: 166), and/or PGPLPEAVL (SEQ ID NO: 167).
  • any of the antibody or antigen-binding fragment encompassed by the present disclosure such as antibodies or fragments of Categories 1 through 5 disclosed herein, may bind one or more of the binding regions identified herein.
  • Such antibodies may be used in the treatment of a TGF ⁇ 1 indication in a subject as described herein. Accordingly, selection of an antibody or antigen-binding fragment thereof suitable for therapeutic use in accordance with the present disclosure may include identifying or selecting an antibody or a fragment thereof that binds SPPSQGEVPPGPLPEAVL (SEQ ID NO: 165), WKWIHEPKGYHANF (SEQ ID NO: 166), PGPLPEAVL (SEQ ID NO: 167), or any portion(s) thereof.
  • SPPSQGEVPPGPLPEAVL SEQ ID NO: 165
  • WKWIHEPKGYHANF SEQ ID NO: 166
  • PGPLPEAVL SEQ ID NO: 167
  • Non-limiting examples of protein domains or motifs of human proTGF ⁇ 1 as previously described are provided in Table 9. Table 9. Select protein domains/motifs of human TGF ⁇ 1-related polypeptides
  • TGF ⁇ inhibitors The development of TGF ⁇ inhibitors remains challenging due to the need to identify a therapy with the desired pharmacological effects and sufficient therapeutic window, which also eliminates on-target toxicities.
  • the majority of TGF ⁇ inhibitors including monoclonal antibodies and small molecule kinase inhibitors (SMIs), non- selectively target either multiple TGF ⁇ isoforms or the TGF ⁇ receptor, which mediates signaling from all three TGF ⁇ isoforms.
  • SMIs small molecule kinase inhibitors
  • these inhibitors have not demonstrated promising clinical data in cancer patients mainly due to a lack of efficacy (Akhurst 2017; Cohn 2014; Voelker 2017), an unfavorable safety profile, or both (Tolcher 2017; Volker 2017; Cohn 2014).
  • the toxicities associated with these molecules include cardiovascular abnormalities, epithelial hyperplasia, gastrointestinal abnormalities, and skin lesions. Each of these toxicities have been characterized in multiple animal species (e.g., rodents, dogs, and cynomolgus monkeys) in studies ranging in duration from 1–2 weeks up to 6-months (Lonning 2011; Stauber 2014; Mitra 2020). Amongst these toxicities, the irreversible cardiovascular inflammatory lesions, hemorrhage and hyperplasia in heart valves, and arterial lesions that include the aorta and coronary arteries, are of major concern.
  • pan-inhibitors of TGF ⁇ capable of antagonizing multiple isoforms have been known to cause a number of toxicities, including, for example, cardiovascular toxicities (cardiac lesions, most notably valvulopathy) reported across multiple species including dogs and rats.
  • cardiovascular toxicities cardiac lesions, most notably valvulopathy
  • hyperplasia in aortic valve, right AV valve, and left AV valve include inflammation in aortic valve, left AV valve, and ascending aorta; hemorrhage in ascending aorta, aortic valve and left AV valve; connective tissue degeneration in ascending aorta (see for example, Strauber et al., (2014) “Nonclinical safety evaluation of a Transforming Growth Factor ⁇ receptor I kinase inhibitor in Fischer 344 rats and beagle dogs” J. Clin. Pract 4(3): 1000196).
  • WO 2017/156500 disclosed an isoform-selective inhibitor of TGF ⁇ 1 activation, which, when administered at a dose of up to 100 mg/kg per week for 4 weeks in rats, no test article-related toxicities were observed, establishing the NOAEL for the antibody as the highest dose tested, i.e., 100 mg/kg.
  • the novel antibody according to the present disclosure has the maximally tolerated dose (MTD) of >100 mg/kg when dosed weekly for at least 4 weeks.
  • the novel antibody according to the present disclosure has the no-observed-adverse-effect level (NOAEL) of up to 100 mg/kg when dosed weekly for at least 4 weeks.
  • NOAEL no-observed-adverse-effect level
  • Suitable animal models to be used for conducting safety/toxicology studies for TGF ⁇ inhibitors and TGF ⁇ 1 inhibitors include, but are not limited to: rats, dogs, cynos, and mice.
  • the minimum effective amount of the antibody based on a suitable preclinical efficacy study is below the NOAEL. More preferably, the minimum effective amount of the antibody is about one-third or less of the NOAEL. In certain embodiments, the minimum effective amount of the antibody is about one-sixth or less of the NOAEL.
  • the minimum effective amount of the antibody is about one-tenth or less of the NOAEL.
  • the disclosure encompasses an isoform-selective antibody capable of inhibiting TGF ⁇ 1 signaling, which, when administered to a subject, does not cause cardiovascular or known epithelial toxicities at a dose effective to treat a TGF ⁇ 1-related indication.
  • the antibody has a minimum effective amount of about 3-10 mg/kg administered weekly, biweekly or monthly.
  • the antibody causes no to minimum toxicities at a dose that is at least six-times the minimum effective amount (e.g., a six-fold therapeutic window).
  • the antibody causes no to minimum toxicities at a dose that is at least ten- times the minimum effective amount (e.g., a ten-fold therapeutic window). Even more preferably, the antibody causes no to minimum toxicities at a dose that is at least fifteen-times the minimum effective amount (e.g., a fifteen- fold therapeutic window).
  • Therapeutic agents that engage immune cells pose the potential risk of activating immune cells when administered to patients. In selecting a TGF ⁇ inhibitor for therapeutic use, it is therefore important to determine or confirm that a candidate inhibitor does not trigger a proinflammatory cytokine response (e.g., cytokine release) in human peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • Proinflammatory cytokines include, for example, IFN ⁇ , IL-2, IL-1 ⁇ , TNF ⁇ , CCL2 and IL-6.
  • acceptable levels of cytokine release triggered by a test agent are within 2.5-fold of the response as compared to vehicle control (e.g., IgG).
  • the present disclosure provides a TGF ⁇ inhibitor for use in the treatment of a TGF ⁇ -related condition (e.g., cancer, myelofibrosis, fibrosis, etc.) in a human patient, which includes i) selection of a TGF ⁇ inhibitor, which has been shown not to trigger unsafe levels of proinflammatory cytokine release in human PBMCs; and, ii) administration of a composition comprising a therapeutically effective amount of the TGF ⁇ inhibitor to the patient, to treat the condition,
  • the TGF ⁇ inhibitor does not trigger unsafe levels of cytokine release from human PBMCs at an amount that is at least three times the therapeutically effective amount.
  • the therapeutically effective amount of the TGF ⁇ inhibitor does not cause unsafe levels of cytokine release in human PBMCs.
  • Human platelets have been reported to express latent TGF ⁇ 1. Pharmacological intervention that targets platelets may cause unwanted effects on platelet function, such as platelet aggregation and activation, which could result in blood coagulation dysregulation. Therefore, it is important to determine or confirm that a candidate inhibitor does not cause unwanted platelet activation or interfere with the normal function of platelets.
  • the present disclosure provides a TGF ⁇ inhibitor for use in the treatment of a TGF ⁇ -related condition (e.g., cancer, myelofibrosis, fibrosis, etc.) in a human patient, which includes i) selection of a TGF ⁇ inhibitor, which has been shown not to cause platelet aggregation or activation; and, ii) administration of a composition comprising a therapeutically effective amount of the TGF ⁇ inhibitor to the patient, to treat the condition,
  • the TGF ⁇ inhibitor does not cause spontaneous or ADP-induced platelet activation in a dose-dependent manner at an amount that is at least three times the therapeutically effective amount.
  • the TGF ⁇ inhibitor does not cause platelet activation.
  • the TGF ⁇ inhibitor does not inhibit ADP-induced platelet activation in a dose-dependent manner at an amount that is at least three times the therapeutically effective amount.
  • at least five times the therapeutically effective amount of the TGF ⁇ inhibitor does not inhibit platelet activation.
  • the present disclosure includes a TGF ⁇ inhibitor for use in the treatment of cancer in a human patient, wherein the treatment comprises: i) selecting a TGF ⁇ inhibitor shown to be both efficacious and safe in a preclinical model(s), and, ii) administering to the human patient an effective dose of the TGF ⁇ inhibitor, wherein optionally the TGF ⁇ inhibitor is effective to reduce tumor burden when used in conjunction with a checkpoint inhibitor, wherein further optionally the TGF ⁇ inhibitor does not trigger platelet activation in human blood samples and does not cause inflammatory cytokine release in PBMCs at doses greater than a minimum efficacious dose; and, further optionally the TGF ⁇ inhibitor does not cause unacceptable adverse events as evaluated in a standard toxicology study in one or more preclinical models in which NOAEL is at least 10 times the minimum efficacious dose.
  • selection of an antibody or an antigen-binding fragment thereof for therapeutic use may include: selecting an antibody or antigen-binding fragment that meets the criteria of one or more of Categories 1-5 described herein; carrying out an in vivo efficacy study in a suitable preclinical model to determine an effective amount of the antibody or the fragment; carrying out an in vivo safety/toxicology study in a suitable model to determine an amount of the antibody that is safe or toxic (e.g., MTD, NOAEL, cytokine release, effects on platelets, or any art-recognized parameters for evaluating safety/toxicity); and, selecting the antibody or the fragment that provides at least a three- fold therapeutic window (preferably 6-fold, more preferably a 10-fold therapeutic window, even more preferably a 15-fold therapeutic window).
  • a three- fold therapeutic window preferably 6-fold, more preferably a 10-fold therapeutic window, even more preferably a 15-fold therapeutic window.
  • the in vivo efficacy study is carried out in two or more suitable preclinical models that recapitulate human conditions.
  • preclinical models comprise TGF ⁇ 1-positive cancer, which may optionally comprise an immunosuppressive tumor.
  • the immunosuppressive tumor may be resistant to a cancer therapy such as CBT, chemotherapy and radiation therapy (such as a radiotherapeutic agent).
  • the preclinical models are selected from MBT-2, Cloudman S91 and EMT6 tumor models.
  • the selected antibody or the fragment may be used in the manufacture of a pharmaceutical composition comprising the antibody or the fragment.
  • Such pharmaceutical composition may be used in the treatment of a TGF ⁇ 1 indication in a subject as described herein.
  • the TGF ⁇ 1 indication may be a proliferative disorder and/or a fibrotic disorder.
  • Mechanism of action Antibodies of the present disclosure that are useful as therapeutics are inhibitory antibodies of TGF ⁇ 1. Further, the antibodies are activation inhibitors, that is, the antibodies block the activation step of TGF ⁇ 1, rather than directly chasing after already activated growth factor. [627] In a broad sense, the term “inhibiting antibody” refers to an antibody that antagonizes or neutralizes the target function, e.g., growth factor activity.
  • certain inhibitory antibodies of the present disclosure are capable of inhibiting mature growth factor release from a latent complex, thereby reducing growth factor signaling.
  • Inhibiting antibodies include antibodies targeting any epitope that reduces growth factor release or activity when associated with such antibodies. Such epitopes may lie on the prodomains of TGF ⁇ proteins (e.g., TGF ⁇ 1), growth factors or other epitopes that lead to reduced growth factor activity when bound by antibody. Inhibiting antibodies of the present disclosure include, but are not limited to, TGF ⁇ 1-inhibiting antibodies. In some embodiments, inhibitory antibodies of the present disclosure specifically bind a combinatory epitope, i.e., an epitope formed by two or more components/portions of an antigen or antigen complex.
  • a combinatory epitope i.e., an epitope formed by two or more components/portions of an antigen or antigen complex.
  • a combinatorial epitope may be formed by contributions from multiple portions of a single protein, i.e., amino acid residues from more than one non-contiguous segments of the same protein.
  • a combinatorial epitope may be formed by contributions from multiple protein components of an antigen complex.
  • inhibitory antibodies of the present disclosure specifically bind a conformational epitope (or conformation-specific epitope), e.g., an epitope that is sensitive to the three-dimensional structure (i.e., conformation) of an antigen or antigen complex.
  • the mechanism of action underlining the inhibitory antibodies acts upstream of TGF ⁇ 1 activation and ligand-receptor interaction.
  • high-affinity, isoform-specific, context- independent inhibitors of TGF ⁇ 1 suitable for carrying out the present disclosure should preferably target the inactive (e.g., latent) precursor TGF ⁇ 1 complex (e.g., a complex comprising pro/latent TGF ⁇ 1) prior to its activation, in order to block the activation step at its source (such as in a disease microenvironment, e.g., TME).
  • inactive (e.g., latent) precursor TGF ⁇ 1 complex e.g., a complex comprising pro/latent TGF ⁇ 1
  • TME disease microenvironment
  • such inhibitors target with equivalent affinities both ECM-associated and cell surface-tethered pro/latent TGF ⁇ 1 complexes, rather than free ligands that are transiently available for receptor binding.
  • the mechanism of action achieved by the antibodies of the present disclosure may further contribute to enhanced durability of effect, as well as overall greater potency and safety.
  • these antibodies may exert additional inhibitory activities toward cell-associated TGF ⁇ 1 (LRRC33-proTGF ⁇ 1 and GARP-proTGF ⁇ 1).
  • LRRC33-binding antibodies tend to become internalized upon binding to cell-surface LRRC33. Whether the internalization is actively induced by antibody binding, or alternatively, whether this phenomenon results from natural (e.g., passive) endocytic activities of macrophages is unclear.
  • the high-affinity, isoform-selective TGF ⁇ 1 inhibitor, Ab6 is capable of becoming rapidly internalized in cells transfected with LRRC33 and proTGF ⁇ 1, and the rate of internalization achieved with Ab6 is significantly higher than that with a reference antibody that recognizes cell-surface LRRC33. Similar results are obtained from primary human macrophages. These observations raise the possibility that Ab6 can induce internalization upon binding to its target, LRRC33-proTGF ⁇ 1, thereby removing the LRRC33-containing complexes from the cell surface. At the disease loci, this may reduce the availability of activatable latent LRRC33-proTGF ⁇ 1 levels.
  • the isoform-selective TGF ⁇ 1 inhibitors may inhibit the LRRC33 arm of TGF ⁇ 1 via two parallel mechanisms of action: i) blocking the release of mature growth factor from the latent complex; and, ii) removing LRRC33-proTGF ⁇ 1 complexes from cell-surface via internalization. It is possible that similar inhibitory mechanisms of action may apply to GARP-proTGF ⁇ 1.
  • the antibody is a pH-sensitive antibody that binds its antigen with higher affinity at a neutral pH (such as pH of around 7) than at an acidic pH (such as pH of around 5). Such antibodies may have higher dissociation rates at acidic conditions than neutral or physiological conditions.
  • the ratio between dissociation rates measured at an acidic pH and dissociation rates measured at neutral pH may be at least 1.2.
  • the ratio is at least 1.5.
  • the ratio is at least 2.
  • Such pH-sensitive antibodies may be useful as recycling antibodies.
  • the antibody may trigger antibody-dependent internalization of (hence removal of) membrane-bound proTGF ⁇ 1 complexes (associated with LRRC33 or GARP).
  • an acidic intracellular compartment such as lysosome, the antibody-antigen complex dissociates, and the free antibody may be transported back to the extracellular domain.
  • selection of an antibody or an antigen-binding fragment for therapeutic use may be in part based on the ability to induce antibody-dependent internalization and/or pH-dependency of the antibody.
  • Antigen Complexes and Components Thereof [635]
  • the novel antibodies of the present disclosure specifically bind each of the four known human large latency complexes (e.g., hLTBP1-proTGF ⁇ 1, hLTBP3-proTGF ⁇ 1, hGARP-proTGF ⁇ 1 and hLRRC33-proTGF ⁇ 1), selectively inhibits TGF ⁇ 1 activation.
  • Screening e.g., identification and selection of such antibodies involves the use of suitable antigen complexes, which are typically recombinantly produced.
  • suitable antigen complexes which are typically recombinantly produced.
  • Useful protein components that may comprise such antigen complexes are provided, including TGF ⁇ isoforms and related polypeptides, fragments and variants, presenting molecules (e.g., LTBPs, GARP, LRRC33) and related polypeptides, fragments and variants. These components may be expressed, purified, and allowed to form a protein complex (such as large latent complexes), which can be used in the process of antibody screening.
  • the screening may include positive selection, in which desirable binders are selected from a pool or library of binders and non-binders, and negative selection, in which undesirable binders are removed from the pool.
  • at least one matrix-associated complex e.g., LTBP1- proTGF ⁇ 1 and/or LTBP1-proTGF ⁇ 1
  • at least one cell-associated complex e.g., GARP-proTGF ⁇ 1 and/or LRRC33-proTGF ⁇ 1
  • the TGF ⁇ 1 comprises a naturally occurring mammalian amino acid sequence.
  • the TGF ⁇ 1 comprises a naturally occurring human amino acid sequence.
  • the TGF ⁇ 1 comprises a human, a monkey, a rat or a mouse amino acid sequence.
  • an antibody, or antigen binding portion thereof, described herein does not specifically bind to TGF ⁇ 2.
  • an antibody, or antigen binding portion thereof, described herein does not specifically bind to TGF ⁇ 3.
  • an antibody, or antigen binding portion thereof, described herein does not specifically bind to TGF ⁇ 2 or TGF ⁇ 3.
  • an antibody, or antigen binding portion thereof, described herein specifically binds to a TGF ⁇ 1 comprising the amino acid sequence set forth in SEQ ID NO: 23.
  • TGF ⁇ 2, and TGF ⁇ 3 amino acid sequence are set forth in SEQ ID NOs: 27 and 21, respectively.
  • an antibody, or antigen binding portion thereof, described herein specifically binds to a TGF ⁇ 1 comprising a non-naturally-occurring amino acid sequence (otherwise referred to herein as a non-naturally-occurring TGF ⁇ 1).
  • a non-naturally-occurring TGF ⁇ 1 may comprise one or more recombinantly generated mutations relative to a naturally-occurring TGF ⁇ 1 amino acid sequence.
  • a TGF ⁇ 1, TGF ⁇ 2, or TGF ⁇ 3 amino acid sequence comprises the amino acid sequence as set forth in SEQ ID NOs: 13-24, as shown in Table 11. In some embodiments, a TGF ⁇ 1, TGF ⁇ 2, or TGF ⁇ 3 amino acid sequence comprises the amino acid sequence as set forth in SEQ ID NOs: 25-32, as shown in Table 12.
  • TGF ⁇ 1 prodomain + growth factor domain
  • antigenic protein complexes may comprise one or more presenting molecules, such as LTBP proteins (e.g., LTBP1, LTBP2, LTBP3, and LTBP4), GARP proteins, LRRC33 proteins, or fragment(s) thereof.
  • LTBP proteins e.g., LTBP1, LTBP2, LTBP3, and LTBP4
  • GARP proteins e.g., GARP proteins
  • LRRC33 proteins e.g., LTBP proteins, GARP proteins, LRRC33 proteins, or fragment(s) thereof.
  • a minimum required fragment suitable for carrying out the embodiments disclosed herein includes at least 50 amino acids, preferably at least 100 amino acids, of a presenting molecule protein, comprising at least two cysteine residues capable of forming disulfide bonds with a proTGF ⁇ 1 complex.
  • Cys residues form covalent bonds with Cysteine resides present near the N-terminus of each monomer of the proTGF ⁇ 1 complex.
  • the N-terminal so-called “Alpha-1 Helix” of each monomer comes in close proximity to each other , setting the distance between the two cysteine residues (one from each helix) required to form productive covalent bonds with a corresponding pair of cysteines present in a presenting molecule (see, for example, Cuende et al., (2015) Sci. Trans. Med. 7: 284ra56).
  • LTBPs e.g., LTBP1, LTBP3 and LTBP4
  • cysteine residues separated by the right distance, which will allow proper disulfide bond formation with a proTGF ⁇ 1 complex in order to preserve correct conformation of the resulting LLC.
  • LTBPs e.g., LTBP1, LTBP3 and LTBP4
  • An antibody, or antigen binding portion thereof, as described herein, is capable of binding to a LTBP1- TGF ⁇ 1 complex.
  • the LTBP1 protein is a naturally-occurring protein or fragment thereof. In some embodiments, the LTBP1 protein is a non-naturally occurring protein or fragment thereof. In some embodiments, the LTBP1 protein is a recombinant protein. Such recombinant LTBP1 protein may comprise LTBP1, alternatively spliced variants thereof and/or fragments thereof. Recombinant LTBP1 proteins may also be modified to comprise one or more detectable labels. In some embodiments, the LTBP1 protein comprises a leader sequence (e.g., a native or non-native leader sequence).
  • a leader sequence e.g., a native or non-native leader sequence
  • the LTBP1 protein does not comprise a leader sequence (i.e., the leader sequence has been processed or cleaved).
  • detectable labels may include, but are not limited to biotin labels, polyhistidine tags, myc tags, HA tags and/or fluorescent tags.
  • the LTBP1 protein is a mammalian LTBP1 protein.
  • the LTBP1 protein is a human, a monkey, a mouse, or a rat LTBP1 protein.
  • the LTBP1 protein comprises an amino acid sequence as set forth in SEQ ID NOs: 35 and 36 in Table 12.
  • the LTBP1 protein comprises an amino acid sequence as set forth in SEQ ID NO: 39 in Table 14.
  • An antibody, or antigen binding portion thereof, as described herein, is capable of binding to a LTBP3- TGF ⁇ 1 complex.
  • the LTBP3 protein is a naturally-occurring protein or fragment thereof.
  • the LTBP3 protein is a non-naturally occurring protein or fragment thereof.
  • the LTBP3 protein is a recombinant protein.
  • Such recombinant LTBP3 protein may comprise LTBP3, alternatively spliced variants thereof and/or fragments thereof.
  • the LTBP3 protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP3 protein does not comprise a leader sequence (i.e., the leader sequence has been processed or cleaved). Recombinant LTBP3 proteins may also be modified to comprise one or more detectable labels. Such detectable labels may include, but are not limited to biotin labels, polyhistidine tags, myc tags, HA tags and/or fluorescent tags. In some embodiments, the LTBP3 protein is a mammalian LTBP3 protein. In some embodiments, the LTBP3 protein is a human, a monkey, a mouse, or a rat LTBP3 protein.
  • a leader sequence e.g., a native or non-native leader sequence
  • the LTBP3 protein does not comprise a leader sequence (i.e., the leader sequence has been processed or cleaved).
  • Recombinant LTBP3 proteins may also be modified to
  • the LTBP3 protein comprises an amino acid sequence as set forth in SEQ ID NOs: 33 and 34 in Table 12.
  • the LTBP1 protein comprises an amino acid sequence as set forth in SEQ ID NO: 40 in Table 14.
  • An antibody, or antigen binding portion thereof, as described herein, is capable of binding to a GARP-TGF ⁇ 1 complex.
  • the GARP protein is a naturally-occurring protein or fragment thereof.
  • the GARP protein is a non-naturally occurring protein or fragment thereof.
  • the GARP protein is a recombinant protein. Such a GARP may be recombinant, referred to herein as recombinant GARP.
  • Some recombinant GARPs may comprise one or more modifications, truncations and/or mutations as compared to wild type GARP.
  • Recombinant GARPs may be modified to be soluble.
  • the GARP protein comprises a leader sequence (e.g., a native or non-native leader sequence).
  • the GARP protein does not comprise a leader sequence (i.e., the leader sequence has been processed or cleaved).
  • recombinant GARPs are modified to comprise one or more detectable labels.
  • detectable labels may include, but are not limited to biotin labels, polyhistidine tags, flag tags, myc tags, HA tags and/or fluorescent tags.
  • the GARP protein is a mammalian GARP protein. In some embodiments, the GARP protein is a human, a monkey, a mouse, or a rat GARP protein. In some embodiments, the GARP protein comprises an amino acid sequence as set forth in SEQ ID NOs: 37-38 in Table 12. In some embodiments, the GARP protein comprises an amino acid sequence as set forth in SEQ ID NOs: 41 and 42 in Table 14. In some embodiments, the antibodies, or antigen binding portions thereof, described herein do not bind to TGF ⁇ 1 in a context-dependent manner, for example binding to TGF ⁇ 1 would only occur when the TGF ⁇ 1 molecule was complexed with a specific presenting molecule, such as GARP.
  • the antibodies, and antigen-binding portions thereof bind to TGF ⁇ 1 in a context-independent manner.
  • the antibodies, or antigen-binding portions thereof bind to TGF ⁇ 1 when bound to any presenting molecule: GARP, LTBP1, LTBP3, and/or LRRC33.
  • An antibody, or antigen binding portion thereof, as described herein, is capable of binding to a LRRC33- TGF ⁇ 1 complex.
  • the LRRC33 protein is a naturally-occurring protein or fragment thereof.
  • the LRRC33 protein is a non-naturally occurring protein or fragment thereof.
  • the LRRC33 protein is a recombinant protein.
  • Such a LRRC33 may be recombinant, referred to herein as recombinant LRRC33.
  • Some recombinant LRRC33 proteins may comprise one or more modifications, truncations and/or mutations as compared to wild type LRRC33.
  • Recombinant LRRC33 proteins may be modified to be soluble.
  • the ectodomain of LRRC33 may be expressed with a C-terminal His-tag in order to express soluble LRRC33 protein (sLRRC33; see, e.g., SEQ ID NO: 73).
  • the LRRC33 protein comprises a leader sequence (e.g., a native or non-native leader sequence).
  • the LRRC33 protein does not comprise a leader sequence (i.e., the leader sequence has been processed or cleaved).
  • recombinant LRRC33 proteins are modified to comprise one or more detectable labels.
  • detectable labels may include, but are not limited to biotin labels, polyhistidine tags, flag tags, myc tags, HA tags and/or fluorescent tags.
  • the LRRC33 protein is a mammalian LRRC33 protein.
  • the LRRC33 protein is a human, a monkey, a mouse, or a rat LRRC33 protein.
  • the LRRC33 protein comprises an amino acid sequence as set forth in SEQ ID NOs: 72, 73, and 74 in Table 14. Table 13. Exemplary LTBP amino acid sequences Table 14. Exemplary GARP and LRRC33 amino acid sequences
  • compositions and Formulations [646] The disclosure further provides pharmaceutical compositions used as a medicament suitable for administration in human and non-human subjects.
  • One or more high-affinity, context-independent antibodies encompassed by the disclosure can be formulated or admixed with a pharmaceutically acceptable carrier (excipient), including, for example, a buffer, to form a pharmaceutical composition.
  • a pharmaceutically acceptable carrier including, for example, a buffer
  • Such formulations may be used for the treatment of a disease or disorder that involves TGF ⁇ signaling.
  • such formulations may be used for immuno-oncology applications.
  • the pharmaceutical compositions of the disclosure may be administered to patients for alleviating a TGF ⁇ - related indication (e.g., fibrosis, immune disorders, and/or cancer).
  • “Acceptable” means that the carrier is compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.
  • Examples of pharmaceutically acceptable excipients (carriers), including buffers, would be apparent to the skilled artisan and have been described previously. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.
  • a pharmaceutical composition described herein contains more than one antibody that specifically binds a GARP-proTGF ⁇ 1 complex, a LTBP1-proTGF ⁇ 1 complex, a LTBP3-proTGF ⁇ 1 complex, and a LRRC33-proTGF ⁇ 1 complex where the antibodies recognize different epitopes/residues of the complex.
  • the pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover).
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • compositions that comprise an antibody or fragment thereof according to the present disclosure, and a pharmaceutically acceptable excipient.
  • the antibody or a molecule comprising an antigen-binding fragment of such antibody can be formulated into a pharmaceutical composition suitable for human administration.
  • the pharmaceutical formulation may include one or more excipients.
  • the pharmaceutical composition is typically formulated to a final concentration of the active biologic (e.g., monoclonal antibody, engineered binding molecule comprising an antigen-binding fragment, etc.) to be between about 20 mg/mL and about 200 mg/mL.
  • the active biologic e.g., monoclonal antibody, engineered binding molecule comprising an antigen-binding fragment, etc.
  • the final concentration (wt/vol) of the formulations may range between about 20-200, 20-180, 20-160, 20-150, 20-120, 20-100, 20-80, 20-70, 20-60, 20-50, 20-40, 30-200, 30- 180, 30-160, 30-150, 30-120, 30-100, 30-80, 30-70, 30-60, 30-50, 30-40, 40-200, 40-180, 40-160, 40-150, 40-120, 40-100, 40-80, 40-70, 40-60, 40-50, 50-200, 50-180, 50-160, 50-150, 50-120, 50-100, 50-80, 50-70, 50-60, 60- 200, 60-180, 60-160, 60-150, 60-120, 60-100, 60-80, 60-70, 70-200, 70-180, 70-160, 70-150, 70-120, 70-100, 70- 80 mg/mL.
  • the final concentration of the biologic in the formulation is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg/mL.
  • suitable buffers include but are not limited to: phosphate buffer, citric buffer, and histidine buffer.
  • the final pH of the formulation is typically between pH 5.0 and 8.0.
  • the pH of the pharmaceutical composition may be about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8.
  • a surfactant such as nonionic detergent, approved for the use in pharmaceutical formulations.
  • surfactants include, for example, polysorbates, such as Polysorbate 20 (TweenTM-20), Polysorbate 80 (Tween-80) and NP-40.
  • the pharmaceutical composition of the present disclosure may comprise a stabilizer.
  • Suitable stabilizers include but are not limited to: sucrose, maltose, sorbitol, as well as certain amino acids such as histidine, glycine, methionine and arginine.
  • the pharmaceutical composition of the present disclosure may contain one or any combinations of the following excipients: Sodium Phosphate, Arginine, Sucrose, Sodium Chloride, Tromethamine, Mannitol, Benzyl Alcohol, Histidine, Sucrose, Polysorbate 80, Sodium Citrate, Glycine, Polysorbate 20, Trehalose, Poloxamer 188, Methionine, Trehalose, Hyaluronidase, Sodium Succinate, Potassium Phosphate, Disodium Edetate, Sodium Chloride, Potassium Chloride, Maltose, Histidine Acetate, Sorbitol, Pentetic Acid, Human Serum Albumin, Pentetic Acid.
  • excipients Sodium Phosphate, Arginine, Sucrose, Sodium Chloride, Tromethamine, Mannitol, Benzyl Alcohol, Histidine, Sucrose, Polysorbate 80, Sodium Citrate, Glycine, Polysorbate 20, Treha
  • the pharmaceutical composition of the present disclosure may contain a preservative.
  • the pharmaceutical composition of the present disclosure is typically presented as a liquid or a lyophilized form.
  • the products can be presented in vial (e.g., glass vial). Products available in syringes, pens, or autoinjectors may be presented as pre-filled liquids in these container/closure systems.
  • the pharmaceutical composition described herein comprises liposomes containing an antibody that specifically binds a GARP-proTGF ⁇ 1 complex, a LTBP1-proTGF ⁇ 1 complex, a LTBP3-proTGF ⁇ 1 complex, and a LRRC33-proTGF ⁇ 1 complex, which can be prepared by any suitable method, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos.4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat.
  • liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • liposomes with targeting properties are selected to preferentially deliver or localize the pharmaceutical composition to certain tissues or cell types.
  • certain nanoparticle-based carriers with bone marrow-targeting properties may be employed, e.g., lipid-based nanoparticles or liposomes.
  • compositions of the disclosure may comprise or may be used in conjunction with an adjuvant. It is contemplated that certain adjuvant can boost the subject's immune responses to, for example, tumor antigens, and facilitate T effector function, DC differentiation from monocytes, enhanced antigen uptake and presentation by APCs, etc.
  • Suitable adjuvants include but are not limited to retinoic acid-based adjuvants and derivatives thereof, oil-in-water emulsion-based adjuvants, such as MF59 and other squalene- containing adjuvants, Toll-like receptor (TRL) ligands (e.g., CpGs), ⁇ -tocopherol (vitamin E) and derivatives thereof.
  • TRL Toll-like receptor
  • CpGs CpGs
  • vitamin E ⁇ -tocopherol
  • the antibodies described herein may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Exemplary techniques have been described previously, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).
  • the pharmaceutical composition described herein can be formulated in sustained-release format.

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EP22723227.9A 2021-03-26 2022-03-25 Tgf-beta-inhibitoren und verwendung davon Pending EP4314831A2 (de)

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