EP4348260A2 - Tgf-beta inhibitoren und deren therapeutische verwendung - Google Patents

Tgf-beta inhibitoren und deren therapeutische verwendung

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Publication number
EP4348260A2
EP4348260A2 EP22733495.0A EP22733495A EP4348260A2 EP 4348260 A2 EP4348260 A2 EP 4348260A2 EP 22733495 A EP22733495 A EP 22733495A EP 4348260 A2 EP4348260 A2 EP 4348260A2
Authority
EP
European Patent Office
Prior art keywords
tgfβ
antibody
antigen
seq
cancer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22733495.0A
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English (en)
French (fr)
Inventor
Lu Gan
Thomas SCHURPF
George CORICOR
Justin Jackson
Si Tuen Lee-Hoeflich
Christopher Brueckner
Constance MARTIN
Ryan Faucette
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Scholar Rock Inc
Original Assignee
Scholar Rock Inc
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Publication date
Priority claimed from PCT/US2022/022063 external-priority patent/WO2022204581A2/en
Application filed by Scholar Rock Inc filed Critical Scholar Rock Inc
Publication of EP4348260A2 publication Critical patent/EP4348260A2/de
Pending legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule

Definitions

  • the instant application relates generally to TGF ⁇ inhibitors and therapeutic use thereof, as well as related assays for diagnosing, monitoring, prognosticating, and treating disorders, including cancer and fibrosis.
  • TGF ⁇ 1 Transforming growth factor beta 1 (TGF ⁇ 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. These 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 such as cancer, 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.
  • ECM extracellular matrix
  • 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 (also referred to as SRK-181 ), Ab21 , Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, or Ab34 disclosed herein.
  • WO 2020/014460 discloses that isoform-selective, high affinity antibodies capable of targeting the 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
  • 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 ⁇ receptors include 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 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins and inhibit downstream activation of TGF ⁇ .
  • ALK5 antagonists such as Galunisertib (LY2157299 monohydrate
  • monoclonal antibodies such as neutral
  • 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.
  • cancer therapies such as chemotherapy, radiation therapy and immune checkpoint inhibitors
  • these therapies are effective in only a small fraction of patient populations. Majority of patients either do not respond (primary resistance) or become nonresponsive (acquired resistance) to these therapies.
  • checkpoint inhibitors typically have response rates of less than 30%.
  • TME tumor microenvironment
  • TGF ⁇ 1 -selective inhibitors e.g., monoclonal antibodies or antigen- binding fragments thereof
  • ECM extracellular matrix
  • fibrotic disorders such as organ fibrosis, and fibrosis involving chronic inflammation
  • proliferative disorders such as cancer, e.g., solid tumors and myelofibrosis
  • EndMT endothelial-to-mesenchymal transition
  • EMT epithelial-to-mesenchymal transition
  • proteases disease with aberrant gene expression of certain markers described herein.
  • the TGF ⁇ 1-selective inhibitors may be used in conjunction with another therapy as combination therapies (e.g., add-on therapies).
  • combination therapies e.g., add-on therapies.
  • Methods for treating such disease or disorders comprising administration of the TGF ⁇ 1 -selective inhibitor in a subject, either as monotherapy or combination therapy, are encompassed by the disclosure.
  • the present disclosure provides the use of TGF ⁇ 1 -selective inhibitors in cancer treatment, both as monotherapy and in conjunction with additional agents as combination or add-on/adjunct therapies.
  • the combination or adjunct therapies may comprise a TGF ⁇ 1 -selective inhibitor and a cancer therapy to which the cancer is resistant or nonresponsive, including, for example, checkpoint inhibitor therapy, chemotherapy and radiation therapy.
  • the TGF ⁇ 1 -selective inhibitor is SRK-181 or an antibody or engineered construct comprising antigen-binding fragments (e.g., the 6 CDRs) of Ab6.
  • an effective amount of TGF ⁇ 1 -selective inhibitor such as SRK-181 is used to treat cancer in patients.
  • SRK-181 is administered to a patient either as monotherapy or combination therapy at 240-3000 mg per dose every 2 weeks or 3 weeks, so as to reduce or slow tumor growth.
  • an effective amount of the TGF ⁇ 1 -selective inhibitor, such as SRK-181 is sufficient to achieve stable disease (SD).
  • an effective amount of the TGF ⁇ 1 -selective inhibitor, such as SRK-181 is sufficient to achieve partial response (PR).
  • isoform-selective inhibitors of TGF ⁇ 1 activation with advantageous features that can be used for the treatment of fibrosis, e.g., lung fibrosis.
  • the methods disclosed and claimed herein are based on in vivo data from multiple preclinical fibrosis models, which demonstrate surprisingly effective therapeutic results, such as reduction in the amount of collagen present in a fibrotic tissue, reduction in the amount of new collagen synthesis, and/or reduction in the amount of phosphorylated Smad2 in a fibrotic tissue.
  • the instant invention provides novel therapeutic dosing strategies, including a loading / maintenance dosing strategy demonstrated to be surprisingly therapeutically effective in vivo.
  • the cancer to be treated with the TGF ⁇ 1-selective inhibitor is characterized by increased alternative end-joining DNA repair or impaired double-strand break repair.
  • the cancer to be treated with the TGF ⁇ 1-selective inhibitor, either as monotherapy or combination or adjunct therapy comprises a solid tumor that is resistant or nonresponsive to checkpoint inhibitor therapy, chemotherapy, radiation therapy, or any combinations thereof.
  • the cancer to be treated with the TGF ⁇ 1-selective inhibitor is ovarian cancer, renal cell carcinoma, breast cancer (such as triple-negative breast cancer), prostate cancer, or esophagus cancer.
  • the cancer to be treated with the TGF ⁇ 1-selective inhibitor may be carcinoma, wherein optionally the carcinoma is a basal cell carcinoma, squamous cell carcinoma, transitional cell carcinoma, renal cell carcinoma, adenocarcinoma.
  • the basal cell carcinoma is basal cell carcinoma of the skin.
  • the squamous cell carcinoma (SCC) is squamous cell carcinoma of the skin (cutaneous SCC), SCC of the lung, SCC of the esophagus, SCC of the head and neck.
  • the transitional cell carcinoma is a transitional cell carcinoma of the kidney.
  • the adenocarcinoma is breast adenocarcinoma, colorectal adenocarcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, or prostate adenocarcinoma.
  • the cancer to be treated with the TGF ⁇ 1-selective inhibitor is: uterine corpus endometrial carcinoma (UCEC), thyroid carcinoma (THCA), testicular germ cell tumors (TGCT), skin cutaneous melanoma (SKCM), prostate adenocarcinoma (PRAD), ovarian serous cystadenocarcinoma (OV), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), liver hepatocellular carcinoma (LIHC), kidney renal clear cell carcinoma (KIRC), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme (GMB), esophageal carcinoma (ESCA), colon adenocarcinoma (COAD), breast invasive carcinoma (BRCA), or bladder urothelial carcinoma (BLCA).
  • UCEC uterine corpus endometrial carcinoma
  • THCA thyroid carcinoma
  • TGCT testicular germ cell tumors
  • SKCM skin cutaneous mela
  • a TGF ⁇ 1 -selective inhibitor (such as SRK-181 ) is used in the treatment of cancer in a subject who is treated with a background therapy comprising a checkpoint inhibitor, chemotherapy and/or radiation therapy.
  • a genotoxic therapy (such as chemotherapy and/or radiation therapy) is used in the treatment of cancer in a subject, who is treated with a TGF ⁇ 1-selective inhibitor (such as SRK-181).
  • a TGF ⁇ 1-selective inhibitor such as SRK-181.
  • a TGF ⁇ 1-selective inhibitor and a genotoxic therapy are used as combination therapy in the treatment of cancer in a subject, wherein the genotoxic therapy comprises chemotherapy and/or radiation therapy.
  • a TGF ⁇ 1-selective inhibitor is used as monotherapy in the treatment of cancer in a subject, wherein optionally the TGF ⁇ 1-selective inhibitor is SRK-181, an antibody that comprises an antigen- binding fragment of Ab6, a variant thereof, or an engineered construct comprising the same.
  • the subject has a cancer for which no checkpoint inhibitor is approved by a regulatory authority such as the FDA, EMA and MHLW.
  • the subject has a carcinoma.
  • the carcinoma is a basal cell carcinoma, squamous cell carcinoma, transitional cell carcinoma, renal cell carcinoma, adenocarcinoma.
  • the basal cell carcinoma is basal cell carcinoma of the skin.
  • the squamous cell carcinoma is squamous cell carcinoma of the skin (cutaneous SCC), SCC of the lung, SCC of the esophagus, SCC of the head and neck.
  • the transitional cell carcinoma is a transitional cell carcinoma of the kidney.
  • the adenocarcinoma is breast adenocarcinoma, colorectal adenocarcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, or prostate adenocarcinoma.
  • the subject has ovarian cancer, e.g., ovarian carcinoma.
  • a subject or patient to be administered with is naive to checkpoint inhibitor therapy, chemotherapy and/or radiation therapy.
  • a subject or patient to be administered with is a nonresponder to a checkpoint inhibitor therapy, chemotherapy and/or radiation therapy.
  • the cancer therapy, genotoxic agent, chemotherapy, radiation therapy, TGF ⁇ inhibitor and/or the TGF ⁇ 1- selective inhibitor is used to treat cancer in the subject who may further receive a checkpoint inhibitor therapy, wherein optionally the checkpoint inhibitor therapy comprises an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • the present disclosure also provides i) enhanced methods for image 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; and/or, iii) LRRC33 as a potential blood- based biomarker indicative of immunosuppression, and/or treatment, e.g., cancer treatment, that incorporates i), ii), and/or iii).
  • LRRC33 as a potential blood- based biomarker indicative of immunosuppression, and/or treatment, e.g., cancer treatment, that incorporates i), ii), and/or iii).
  • one or more of these features may be employed as part of diagnostic and/or therapeutic regimen for subjects (e.g., patients) either as monotherapy or combination/adjunct therapy to treat cancer.
  • the present disclosure relates to compositions comprising TGF ⁇ inhibitors and methods for selecting suitable TGF ⁇ inhibitors for treating certain patient populations, as well as related treatments using the TGF ⁇ inhibitors.
  • 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 TGF ⁇ inhibitors for therapeutic use is aimed to achieve in vivo efficacy while controlling potential risk, e.g., toxicities known to be associated with pan-inhibition of TGF ⁇ .
  • the present disclosure is based, at least in part, on an unexpected finding that concurrent inhibition of the TGF ⁇ 1/3 isoforms attenuated efficacy of a TGF ⁇ 1 -selective inhibitor in vivo, e.g., in conditions with dysregulated ECM (e.g., involving ECM dysregulation, e.g., alterations in ECM structure and/or composition), suggesting that TGF ⁇ 3 inhibition may be detrimental.
  • dysregulated ECM e.g., involving ECM dysregulation, e.g., alterations in ECM structure and/or composition
  • ECM dysregulation may involve changes in one or more gene markers selected from Collagen I (Col1a1), Collagen III (Col3a1), Fibronectin 1 (Fn1), Lysyl Oxidase (Lox), Lysyl Oxidase-like 2 (Loxl2), Smooth muscle actin (Acta2), Matrix metalloprotease (Mmp2), and Integrin alpha 11 (Itga11 ).
  • ECM dysregulation may be identified by an increase in Acta2, alone or in combination with one or more markers, e.g., the markers mentioned above.
  • disorders involving ECM dysregulation may include certain cancers (e.g., metastatic cancer), fibrotic conditions, and/or cardiovascular diseases.
  • the fibrotic conditions and/or cardiovascular diseases include, but are not limited to, metabolic disorders such as NAFLD, NASH, obesity, and type 2 diabetes.
  • disorders involving ECM dysregulation may include myelofibrosis.
  • ECM dysregulation has been linked to disease progression, such as increased invasiveness and metastasis, as well as increased fibrotic features which are common to tumor stroma. The observation that TGF ⁇ 3 inhibition may in fact exacerbate ECM dysregulation in vivo raises the possibility that TGF ⁇ 3 inhibitory activities found in a number of TGF ⁇ antagonists may increase risk to cancer patients.
  • the disclosure includes, in some embodiments, methods comprising selecting and/or administering a TGF ⁇ inhibitor that does not target TGF ⁇ 3 signaling for therapeutic use.
  • the TGF ⁇ inhibitor 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.
  • the TGF ⁇ inhibitor does not inhibit TGF ⁇ 2 signaling and TGF ⁇ 3 signaling at a therapeutically effective dose.
  • such inhibitor is TGF ⁇ 1 -selective.
  • TGF ⁇ inhibitor that does not inhibit TGF ⁇ 3 for producing a medicament.
  • the medicament may be for a cancer therapy.
  • such inhibitor is TGF ⁇ 1-selective.
  • selection of TGF ⁇ inhibitors for therapeutic use may involve testing a candidate TGF ⁇ inhibitor for immune safety. Such tests may include cytokine release assays and may further include platelet assays.
  • a candidate TGF ⁇ inhibitor selected to be produced at large scale and used in, e.g, cancer treatment does not trigger cytokine release (described herein) or platelet aggression (described herein).
  • such inhibitor is TGF ⁇ 1 -selective.
  • the disclosure provides a method of manufacturing a pharmaceutical composition comprising a TGF ⁇ inhibitor, wherein the method comprises the steps of: i) selecting a TGF ⁇ inhibitor that meets immune safety criteria characterized by: no significant cytokine release triggered as compared to control (such as IgG) in in vitro cytokine release assays and/or in vivo study in which serum concentrations of such cytokines are measured in response to administration of the TGF ⁇ inhibitor; and/or, no significant binding to, aggregation/activation of human platelets, wherein the TGF ⁇ inhibitor is efficacious in one or more preclinical animal models at a dose below MTD or NOAEL as determined in a preclinical toxicology study; ii) producing the TGF ⁇ inhibitor, e.g., an inhibitor selected as described herein, in a culture (e.g., bioreactor) with a volume of 250L or greater, optionally further comprising: iii) formulating into a pharmaceutical composition comprising the TGF
  • the pharmaceutical composition and/or treatment regimen disclosed herein may further comprise a checkpoint inhibitor (e.g., as a cancer therapy agent, e.g., a PD-1 antibody, a PD-L1 antibody, or a CTLA-4 antibody) 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.
  • a cancer therapy agent e.g., checkpoint inhibitor
  • TGF ⁇ inhibitor e.g., as a cancer therapy agent
  • these components may be provided as a single molecular entity.
  • 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 inhibitor, e.g., a TGF ⁇ 1 -selective inhibitor such as Ab6) by monitoring circulating MDSC levels.
  • a TGF ⁇ inhibitor e.g., a TGF ⁇ 1 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.
  • circulatory MDSCs are g-MDSCs. In some embodiments, circulatory MDSCs are m- MDSCs.
  • circulatory MDSCs are g-MDSCs and m-MDSCs. In some embodiments, circulatory MDSCs are characterized by cell-surface expression of LRRC33.
  • 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.
  • Applicant showed that MDSCs were indeed enriched in solid tumors and that inhibition of TGF ⁇ 1 in conjunction with a checkpoint inhibitor treatment significantly reduced intratumoral MDSCs, which correlated with slowed tumor growth and, in some cases, achieved complete regression in multiple preclinical tumor models (PCT/US2019/04133).
  • effectiveness of such combination therapy was observed over the course of weeks to months (for example, 6-12 weeks) by monitoring tumor growth.
  • 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
  • TILs tumor-infiltrating lymphocytes
  • 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.
  • the degree of tumor burden e.g. , the size of tumor
  • response to the therapy e.g., therapeutic effects
  • LRRC33 as a novel cell-surface marker for MDSCs in circulation (e.g., blood samples). This observation raises the possibility that LRRC33 may be used as a blood-based predictive biomarker.
  • the instant inventors identify 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, e.g., as 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 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.
  • 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.
  • the likelihood of patient’s responsiveness to cancer immunotherapy may be assessed by measuring circulating MDSCs, e.g., in blood or a blood component, as an indicator of TGF ⁇ (e.g., TGF ⁇ 1)-mediated immunosuppression.
  • the circulating MDSCs are characterized by expression of one or more of the following markers: CD11b, CD33, CD14, CD15, LOX-1 , CD66b, and HLA-DR lo/-
  • the circulating MDSCs are G-MDSCs.
  • a combination therapy comprising a cancer therapy (such as checkpoint inhibitor) and a TGF ⁇ inhibitor that is not selective for TGF ⁇ 1 (non-selective TGF ⁇ inhibitor)
  • a cancer therapy such as checkpoint inhibitor
  • a TGF ⁇ inhibitor that is not selective for TGF ⁇ 1
  • the 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, 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.
  • the TGF ⁇ inhibitor selectively targets TGF ⁇ 1 signaling.
  • a second TGF ⁇ 1-selective inhibitor is used to further reduce the frequency of exposure to a non-TGF ⁇ 1 selective inhibitor.
  • sparing of TGF ⁇ inhibitors with anti-TGF ⁇ 3 activities may be especially useful for treating patients who are diagnosed with a type of cancer known to be highly metastatic, myelofibrotic, and/or those having or are at risk of developing a fibrotic condition.
  • TGF ⁇ inhibitors that do not target TGF ⁇ 3 mat be useful for treating patients who are diagnosed with or who are at risk of developing a condition involving dysregulated ECM.
  • the condition involving dysregulated ECM may be cancer.
  • the condition with dysregulated ECM may be a fibrotic condition such as myelofibrosis.
  • the disclosure herein includes a TGF ⁇ inhibitor for use in the treatment of cancer wherein the inhibitor does not inhibit TGF ⁇ 3 and wherein the patient has a metastatic cancer or myelofibrosis, or the patient has or is at risk of developing a fibrotic condition, wherein optionally the fibrotic condition is non-alcoholic steatohepatitis (NASH).
  • the inhibitor may not inhibit TGF ⁇ 3 and the patient (subject) may have a metastatic cancer or myelofibrosis, or the patient may have or be at risk of developing a fibrotic condition, wherein optionally the fibrotic condition is NASH.
  • the TGF ⁇ inhibitor that does not inhibit TGF ⁇ 3 may be Ab6 or an antibody comprising heavy chain complementarity determining regions (CDRs) comprising amino acid sequences of SEQ ID NO: 1001 (H-CDR1 ), SEQ ID NO: 1002 (H-CDR2), SEQ ID NO: 1003 (H-CDR3), and light chain CDRs comprising amino acid sequences of SEQ ID NO: 1004 (L-CDR1 ), SEQ ID NO: 1005 (L-CDR2), and SEQ ID NO: 1006 (L-CDR3), as defined by the IMTG numbering system.
  • CDRs heavy chain complementarity determining regions
  • a preferred TGF ⁇ inhibitor may be TGF ⁇ 1 -selective. It may bind the target with an affinity of 0.5 nM or greater (K D ⁇ 0.5 nM) with a dissociation rate of no more than 10.0E-4 (1/s) as measured by SPR. More preferably, such TGF ⁇ inhibitor may be 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 antibody 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 TGF ⁇ 1 inhibitors include monoclonal antibodies (including immunoglobulins and antigen-binding fragments or portions thereof) that exhibit slow dissociation rates (i.e., off-rates, k OFF ).
  • the disclosure is based at least on the recognition that treatment of chronic and progressive disease such as fibrosis, and in particular lung fibrosis, may require inhibitors with superior durability, which may be reflected on the dissociation rate of such antibody.
  • the affinity of an antibody to its antigen is typically measured as the equilibrium dissociation constant, or K D .
  • the ratio of the experimentally measured off- and on-rates ( k OFF /k ON ) can be used to calculate the K D value.
  • the k OFF value represents the antibody dissociation rate, which indicates how quickly it dissociates from its bound antigen, whilst the k ON value represents the antibody association rate which provides how quickly it binds to its antigen.
  • the latter is typically concentration-dependent, while the former is concentration-independent.
  • the K D value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the K D value (lower concentration) and thus the higher the affinity of the antibody.
  • a higher affinity antibody may have a lower k OFF rate, a higher k ON rate, or both.
  • Both the k OFF and k ON rates contribute to the overall affinity of a particular antibody to its antigen, and relative importance or impact of each component may depend on the mechanism of action of the antibody.
  • neutralizing antibodies which bind mature growth factors (e.g., soluble, transient TGF ⁇ 1 ligand liberated from a latent complex), must compete with the endogenous high-affinity receptors for ligand binding in vivo. Because the ligand-receptor interaction is a local event and because the ligand is short-lived, such antibodies must be capable of rapidly targeting and sequestering the soluble growth factor before the ligand finds its cellular receptor - thereby activating the TGF ⁇ 1 signaling pathway - in the tissue. Therefore, for ligand-targeting neutralizing antibodies to be potent, the ability to bind the target growth factor fast, .i.e., high association rates (k ON ), may be especially important.
  • activation inhibitors antibodies that inhibit the TGF ⁇ 1 signaling by preventing the release of mature growth factor from the latent complex
  • activation inhibitors may preferentially benefit from having slow dissociation rates once the antibody is engaged with the target antigen (e.g., proTGF ⁇ 1 complexes).
  • target antigen e.g., proTGF ⁇ 1 complexes.
  • such antibodies do not directly compete with cellular receptors; rather, they work upstream of the signaling by targeting inactive precursor forms (e.g., latent proTGF ⁇ 1 complexes) that remain dormant within a tissue environment thereby preemptively preventing the activation of TGF ⁇ 1.
  • Such antibodies may exert their inhibitory activity by preventing mature growth factor from being liberated from the latent complex.
  • such antibodies may function like a “clamp” to lock the active growth factor in the prodomain cage structure to keep it in an inactive (e.g., “latent”) state.
  • structural analyses, including epitope mapping provided insight into the molecular mechanism underlining the ability of these antibodies to block TGF ⁇ 1 activation.
  • the Latency Lasso region of the prodomain may be a particularly useful target.
  • antibodies that are able to remain bound to the target are expected to be advantageous in achieving superior in vivo potency, due to enhanced durability of effects and/or avidity.
  • Applicant of the present disclosure sought to identify isoform-selective activation inhibitors of TGF ⁇ 1 with particularly low k OFF values as compared to previously described antibodies.
  • preferred antibodies have high affinities (e.g., a K D of sub- nanomolar to picomolar range) primarily attributable to a slow dissociation rate (k OFF ), as opposed to fast association rate (k ON ).
  • such antibodies bind an epitope that comprises at least a portion of Latency Lasso.
  • the present disclosure provides an isoform-selective inhibitor of TGF ⁇ 1 activation, wherein the inhibitor is a monoclonal antibody or an antigen-binding fragment thereof, which selectively inhibits TGF ⁇ 1 activation; wherein the monoclonal antibody binds human LTBP1-proTGF ⁇ 1 and/or human LTBP3-proTGF ⁇ 1 with a monovalent dissociation rate of 10.0e-04 or less, as measured by a surface plasmon resonance (SPR)-based technique, and optionally with a K D value of ⁇ 1 .0 nM; and, wherein the antibody or the antigen-binding fragment comprises the following six CDRs: an H-CDR1 comprising GFTFADYA (SEQ ID NO: 276); an H-CDR2 comprising ISGSG(X 1 )AT, wherein optionally the X 1 is A or K (SEQ ID NO: 277); an H-CDR3 comprising VSSG(X 1 )WD(X
  • the antibody comprises the six CDR sequences of Ab46 or Ab50.
  • compositions such as pharmaceutical compositions (e.g., formulations, medicament) that are suitable for administration to human patients, comprising at least one of the antibodies or fragment thereof in accordance with the present disclosure, and an excipient.
  • pharmaceutical compositions e.g., formulations, medicament
  • the antibodies or fragment thereof in accordance with the present disclosure can be used in the manufacture of such medicament.
  • 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 TGF ⁇ inhibitor is a TGF ⁇ 1 selective inhibitor.
  • 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 combination with a cancer therapy), administering to the subject a therapeutically effective amount of the TGF ⁇ inhibitor (alone or in combination 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 cancer treatment in a subject, wherein the treatment comprises administering to the subject a combination therapy comprising a dose of a TGF ⁇ inhibitor and a cancer therapy, the method comprising the steps of (i) determining the circulating MDSC level in a sample obtained from the subject prior to administering the TGF ⁇ inhibitor, (ii) determining the circulating MDSC level in a sample obtained from the subject after administration of the TGF ⁇ inhibitor, and (iii) determining whether the level determined in step (ii) is reduced compared to the level determined in step (i), such reduction being indicative of therapeutic efficacy of the cancer treatment
  • the dose of the TGF ⁇ inhibitor and the cancer therapy in the combination therapy are for concurrent (e.g., simultaneous), separate, or sequential administration.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1- selective inhibitor, e.g., Ab4, Ab5, Ab6, Ab21 , Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31 , Ab32, Ab33, and Ab34.
  • the TGF ⁇ inhibitor is Ab6.
  • the disclosure includes a method of treating cancer in a subject, comprising the steps of determining circulating MDSC levels in the subject prior to administering a TGF ⁇ inhibitor, administering to the subject a first therapeutically effective dose of the TGF ⁇ inhibitor, determining circulating MDSC levels in the subject after administering the TGF ⁇ inhibitor, and administering to the subject a second therapeutically effective dose of the TGF ⁇ inhibitor or combination therapy if the circulating MDSC levels measured after administering the first therapeutically effective dose of the TGF ⁇ inhibitor are reduced as compared to the circulating MDSC levels measured prior to administering the first therapeutically effective dose of the TGF ⁇ 1 inhibitor.
  • a combination therapy comprising a second cancer therapy is administered concurrently, sequentially, or simultaneously with the first therapeutically effective dose of the TGF ⁇ inhibitor and the combination therapy is continued if the circulating MDSC levels measured after administering the first therapeutically effective dose of the combination therapy are reduced as compared to the circulating MDSC levels measured prior to administering the first therapeutically effective dose.
  • a second cancer therapy e.g., checkpoint inhibitor therapy
  • the disclosure encompasses a cancer therapy agent for use in the treatment of cancer in a subject, wherein the subject has received a dose of a TGF ⁇ inhibitor and wherein the circulating MDSC level in the subject measured after administration of the TGF ⁇ inhibitor has been determined to be reduced as compared to the circulating MDSC level measured in the subject prior to administering the dose of the TGF ⁇ inhibitor.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1 -selective inhibitor, e.g., Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31 , Ab32, Ab33, and Ab34.
  • the TGF ⁇ inhibitor is Ab6.
  • the disclosure encompasses a combination therapy comprising a dose of a TGF ⁇ inhibitor and a cancer therapy agent for use in the treatment of cancer, wherein the treatment comprises concurrent (e.g., simultaneous), separate, or sequential administration to a subject of a dose of the TGF ⁇ inhibitor and the cancer therapy agent, and wherein the circulating MDSC level in the subject measured after the administration of the TGF ⁇ inhibitor has been determined to be reduced as compared to the circulating MDSC level measured in the subject prior to administering the dose of the TGF ⁇ inhibitor.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1 - selective inhibitor, e.g., Ab4, Ab5, Ab6, Ab21 , Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31 , Ab32, Ab33, and Ab34.
  • the TGF ⁇ inhibitor is Ab6.
  • the disclosure encompasses a TGF ⁇ inhibitor for use in the treatment of cancer in a subject, wherein the subject has received at least a first dose of the TGF ⁇ inhibitor, and wherein the treatment comprises administering a further dose of the TGF ⁇ inhibitor, provided that the circulating MDSC level in the subject measured after the administration of the at least first dose of the TGF ⁇ inhibitor is reduced as compared to the circulating MDSC level measured in the subject prior to administering a dose of the TGF ⁇ inhibitor.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1 -selective inhibitor, e.g., Ab4, Ab5, Ab6, Ab21 , Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31 , Ab32, Ab33, and Ab34.
  • the TGF ⁇ inhibitor is Ab6.
  • 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 TGF ⁇ inhibitor is a TGF ⁇ 1 -selective inhibitor, e.g., Ab4, Ab5, Ab6, Ab21 , Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31 , Ab32, Ab33, and Ab34.
  • the TGF ⁇ inhibitor is Ab6.
  • the disclosure encompasses a method of treating advanced cancer in a human subject comprising the steps of selecting a subject with advanced cancer comprising a locally advanced tumor and/or metastatic cancer with primary resistance to a checkpoint inhibitor therapy, administering a TGF ⁇ inhibitor, and administering to the subject a checkpoint inhibitor therapy.
  • the cancer may be advanced cancer. It may comprise a locally advanced tumor and/or metastatic cancer with primary resistance to a checkpoint inhibitor therapy.
  • the cancer therapy may comprise a checkpoint inhibitor therapy.
  • the subject may be a human subject.
  • the cancer has elevated circulating MDSC levels.
  • treatment reduces the level of circulating MDSCs.
  • continued treatment is contingent on an observed reduction in circulating MDSCs.
  • 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 another cancer therapy (e.g., checkpoint inhibitor).
  • the method comprises the steps of determining the levels of tumor-associated immune cells (e.g., CD8+ T cells and tumor-associated macrophages) in the subject prior to administering a treatment, administering the treatment to the subject, and determining the levels of tumor-associated immune cells in the subject after administering the treatment, wherein a change in the level of one or more tumor-associated immune cell populations after inhibitor administration, as compared to the levels of tumor-associated immune cells before administration, indicates therapeutic efficacy.
  • tumor-associated immune cells e.g., CD8+ T cells and tumor-associated macrophages
  • treatment alters the level of tumor-associated immune cells.
  • continued treatment is contingent on an observed change in tumor-associated immune cells.
  • the tumor-associated immune cell levels are monitored in combination with monitoring circulating MDSC levels and treatment efficacy and/or continued treatment is contingent on observed changes in both sets of biomarkers.
  • the disclosure provides a checkpoint inhibitor and a TGF ⁇ 1 inhibitor for use in the treatment of cancer in a subject in need thereof, wherein the treatment comprises administration of a checkpoint inhibitor and a TGF ⁇ 1 inhibitor in amounts effective to treat cancer, wherein optionally the checkpoint inhibitor is a PD-(L)1 inhibitor, wherein further optionally the PD-(L)1 inhibitor is budigalimab; wherein optionally the TGF ⁇ 1 inhibitor is a TGF ⁇ 1 -selective inhibitor, wherein further optionally the TGF ⁇ 1 -selective inhibitor is SRK-181 ; and, wherein optionally the cancer comprises a solid tumor of immunosuppressive phenotype
  • the disclosure provides a checkpoint inhibitor for use in the treatment of cancer in a subject in need thereof, wherein the treatment comprises administration of a checkpoint inhibitor to the subject treated with a TGFb1 inhibitor, in amounts effective to treat cancer, wherein optionally the checkpoint inhibitor is a PD-(L)1 inhibitor, wherein further optionally the PD-(L)1 inhibitor is budigalimab; wherein optionally the TGF ⁇ 1 inhibitor is a TGF ⁇ 1-selective inhibitor, wherein further optionally the TGF ⁇ 1 -selective inhibitor is SRK-181 ; and, wherein optionally the cancer comprises a solid tumor of immunosuppressive phenotype.
  • the disclosure provides a TGF ⁇ 1 inhibitor for use in the treatment of cancer in a subject in need thereof, wherein the treatment comprises administration of a TGF ⁇ 1 inhibitor to the subject treated with a checkpoint inhibitor, in amounts effective to treat cancer, wherein optionally the checkpoint inhibitor is a PD- (L)1 inhibitor, wherein further optionally the PD-(L)1 inhibitor is budigalimab; wherein optionally the TGF ⁇ 1 inhibitor is a TGF ⁇ 1 -selective inhibitor, wherein further optionally the TGF ⁇ 1 -selective inhibitor is SRK-181 ; and, wherein optionally the cancer comprises a solid tumor of immunosuppressive phenotype.
  • the checkpoint inhibitor is a PD- (L)1 inhibitor, wherein further optionally the PD-(L)1 inhibitor is budigalimab
  • the TGF ⁇ 1 inhibitor is a TGF ⁇ 1 -selective inhibitor, wherein further optionally the TGF ⁇ 1 -selective inhibitor is SR
  • the disclosure encompasses methods of treating, predicting, determining, and/or monitoring therapeutic efficacy of a cancer treatment in a subject.
  • the method comprises measuring levels of CD8+ cells in the tumor (or in one or more tumor nests within the tumor) and the surrounding stroma and/or margin compartments in one or more tumor samples obtained from the subject.
  • the method comprises identifying the immune phenotype of the subject’s cancer based on the level of CD8+ cells inside the tumor or tumor nest(s) as compared to the level of CD8+ cells outside of the tumor or tumor nest(s) (e.g., the surrounding stroma and/or margin compartments).
  • the cancer treatment comprises a TGF ⁇ inhibitor, e.g., a TGF ⁇ 1 inhibitor, e.g., Ab4, Ab5, Ab6, Ab21 , Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31 , Ab32, Ab33, or Ab34.
  • the cancer treatment comprises Ab6.
  • the cancer treatment comprises an immune checkpoint inhibitor.
  • the cancer treatment comprises a TGF ⁇ 1 inhibitor (e.g., Ab6) and an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, or a CTLA-4 antibody).
  • the disclosure provides a method of treating, predicting, and/or monitoring therapeutic efficacy of a cancer treatment in a subject administered a TGF ⁇ inhibitor alone or in combination with another cancer therapy (e.g., checkpoint inhibitor).
  • the method comprises the steps of determining the levels of circulating latent TGF ⁇ in the subject prior to administering a treatment, administering the treatment to the subject, and determining the levels of circulating latent TGF ⁇ in the subject after administering the treatment, wherein a change (e.g., increase) in circulating latent TGF ⁇ after inhibitor administration, as compared to circulating latent TGF ⁇ before administration, indicates therapeutic efficacy.
  • treatment alters the level of circulating latent TGF ⁇ .
  • continued treatment is contingent on an observed change (e.g., increase) in circulating latent TGF ⁇ .
  • the circulating latent TGF ⁇ is monitored in combination with monitoring circulating MDSC levels and/or tumor-associated immune cell levels.
  • treatment efficacy and/or continued treatment is contingent on observed changes in two or more sets of biomarkers.
  • the methods and compositions disclosed herein for use in treating cancer that involve a determination of circulating MDSC levels (and optionally also the assessment of a change in the level of one or more tumor-associated immune cell populations) may further comprise the assessment of the level of circulating latent TGF ⁇ , as described herein.
  • compositions comprising a therapeutically effective dose of a TGF ⁇ inhibitor for use in treating cancer, wherein the TGF ⁇ inhibitor is administered if a reduction in circulating MDSC levels are determined (alone or in combination with a change in circulating latent TGF ⁇ ) after administration of a previous dose of a TGF ⁇ inhibitor.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1 -selective inhibitor, e.g., Ab6.
  • continued treatment is contingent on an observed change in circulating latent TGF ⁇ .
  • the circulating latent TGF ⁇ is monitored in combination with monitoring circulating MDSC levels and/or tumor-associated immune cell levels.
  • treatment efficacy and/or continued treatment is contingent on observed changes in two or more sets of biomarkers.
  • the disclosure provides a method of treating cancer, comprising administering to a subject a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 inhibitor) in a therapeutically effective amount that does not cause a significant release of one or more cytokines selected from interferon gamma (IFN ⁇ ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF ⁇ ), interleukin 1 beta (IL-13), and chemokine C-C motif ligand 2 (CCL2) / monocyte chemoattractant protein 1 (MCP-1 ).
  • a TGF ⁇ inhibitor e.g., a TGF ⁇ 1 inhibitor
  • cytokines selected from interferon gamma (IFN ⁇ ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF ⁇ ), interleukin 1 beta (
  • the method does not induce a significant increase in platelet binding, activation, and/or aggregation.
  • the cancer has elevated circulating MDSC levels.
  • treatment with a therapeutically effective amount of the TGF ⁇ inhibitor e.g., a TGF ⁇ 1 inhibitor reduces the level of circulating MDSCs.
  • continued treatment is contingent on an observed reduction in circulating MDSCs.
  • the disclosure provides a method for identifying whether a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 inhibitor) will be tolerated in a patient, comprising contacting a cell culture or fluid sample with the TGF ⁇ inhibitor and determining whether it causes a significant release of one or more cytokines selected from interferon gamma (IFN ⁇ ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF ⁇ ), interleukin 1 beta (IL- 1 ⁇ ) and chemokine C-C motif ligand 2 (CCL2) / monocyte chemoattractant protein 1 (MCP-1 ), wherein a significant release indicates the TGF ⁇ inhibitor will not be well tolerated.
  • cytokines selected from interferon gamma (IFN ⁇ ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF ⁇ ), interleukin 1 beta (IL- 1 ⁇ ) and chemokine C-C
  • the method may comprise monitoring cytokine release in an in vitro cytokine release assay.
  • the assay is in peripheral blood mononuclear cells (PBMCs) or whole blood, optionally wherein the PBMCs or whole blood are obtained from the subject prior to administering a TGF ⁇ inhibitor therapy.
  • PBMCs peripheral blood mononuclear cells
  • the disclosure encompasses a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 -selective inhibitor) for use in the treatment of cancer by administering to a subject a dose of said TGF ⁇ inhibitor, wherein said TGF ⁇ inhibitor does not cause a significant release of one or more cytokines selected from interferon gamma (IFN ⁇ ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF ⁇ ), interleukin 1 beta (IL-1 ⁇ ) and chemokine C-C motif ligand 2 (CCL2) / monocyte chemoattractant protein 1 (MCP-1 ).
  • cytokines selected from interferon gamma (IFN ⁇ ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF ⁇ ), interleukin 1 beta (IL-1 ⁇ ) and chemokine C-C motif ligand 2 (CCL2) / monocyte chemoattrac
  • the disclosure encompasses a combination therapy comprising a dose of a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 inhibitor) and a cancer therapy agent (e.g., a checkpoint inhibitor therapy) for use in the treatment of cancer, wherein the treatment comprises simultaneous, concurrent, or sequential administration to a subject of a dose of the TGF ⁇ inhibitor and the cancer therapy agent, wherein said TGF ⁇ inhibitor does not cause a significant release of one or more cytokines selected from interferon gamma (IFN ⁇ ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF ⁇ ), interleukin 1 beta (IL-1 ⁇ ) and chemokine C-C motif ligand 2 (CCL2) / monocyte chemoattractant protein 1 (MCP-1 ).
  • the TGF ⁇ inhibitor for use in the treatment of cancer is administered in a therapeutically effective amount that is sufficient to reduce circulating MDSCs.
  • the disclosure provides a method for determining whether a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 inhibitor) causes a significant increase in platelet binding, activation and/or aggregation following exposure of the sample to said TGF ⁇ inhibitor, which method comprises measuring platelet binding, activation and/or aggregation in a plasma or whole blood sample.
  • a TGF ⁇ inhibitor e.g., a TGF ⁇ 1 inhibitor
  • the disclosure encompasses a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 inhibitor) for use in the treatment of cancer by administering to a subject a dose of said TGF ⁇ inhibitor, wherein said TGF ⁇ inhibitor does not cause a significant increase in platelet binding, activation and/or aggregation.
  • the disclosure encompasses a combination therapy comprising a dose of a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 inhibitor) and a cancer therapy agent (e.g., a checkpoint inhibitor therapy) for the treatment of cancer, wherein the treatment comprises concurrent (e.g., simultaneous), separate, or sequential administration to a subject of a dose of the TGF ⁇ inhibitor and the cancer therapy agent, wherein said TGF ⁇ inhibitor does not cause a significant increase in platelet binding, activation and/or aggregation.
  • the TGF ⁇ inhibitor for use is administered in a therapeutically effective amount that is sufficient to reduce circulating MDSCs.
  • the subject may have a cancer, e.g., a highly metastatic 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
  • the disclosure provides a method of making a TGF ⁇ inhibitor for treating cancer in a subject, comprising the steps of selecting a TGF ⁇ inhibitor which satisfies one or more, or e.g., all of, the following criteria: a) the TGF ⁇ inhibitor is efficacious in one or more preclinical models, b) the TGF ⁇ inhibitor does not cause valvulopathies or epithelial hyperplasia in toxicology studies in one or more animal species at a dose at least greater than a minimum efficacious dose, c) the TGF ⁇ inhibitor does not induce significant cytokine release from human PBMCs or whole blood in an in vitro cytokine release assay at the minimum efficacious dose as determined in the one or more preclinical models of (a), d) the TGF ⁇ inhibitor does not induce a significant increase in platelet binding, activation, and/or aggregation at the minimum efficacious dose as determined in the one or more preclinical models of (a), and e) the
  • the methods of the present disclosure may be used to select and treat patients exhibiting resistance to immunotherapy, e.g., to checkpoint inhibitor therapy.
  • the patient or subject referred to in the methods and compositions for use disclosed herein may have resistance to immunotherapy, e.g., checkpoint inhibitor therapy.
  • Patient populations encompassed by the current disclosure may be treatment-naive (e.g., may have not received previous cancer therapy), have primary resistance (i.e., present before treatment initiation), or have acquired resistance to an immunotherapy, e.g., checkpoint inhibitor therapy.
  • the disclosure encompasses a TGF ⁇ 1 -selective inhibitor for use in the treatment of cancer wherein the treatment comprises the steps of selecting a subject whose cancer is highly metastatic and administering to the subject an isoform-selective TGF ⁇ 1 inhibitor.
  • the highly metastatic cancer comprises 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, small cell 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 (e.g., gastric cancer), or thyroid cancer.
  • colorectal cancer e.g., microsatellite stable-colorectal cancer
  • lung cancer e.g., non-small cell lung cancer, small cell lung cancer
  • bladder cancer e.g., kidney cancer (e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC), including clear cell RCC, papillary RCC, chro
  • the disclosure encompasses a TGF ⁇ 1 -selective inhibitor for use in the treatment of cancer in a subject wherein the treatment comprises the steps of selecting a subject having a myelofibrotic disorder, or is at risk of developing a myelofibrotic disorder, and administering to the subject the TGF ⁇ 1-selective inhibitor in an amount effective to treat the cancer.
  • the disclosure encompasses a method of treating cancer in a subject, wherein the subject has previously, is currently, or will be treated with a TGF ⁇ inhibitor that inhibits TGF ⁇ 3, e.g., in conjunction with a checkpoint inhibitor.
  • TGF ⁇ inhibitor that inhibits TGF ⁇ 3, e.g., in conjunction with a checkpoint inhibitor.
  • These patients may have reduced dosage or treatment frequency by monitoring circulating MDSC levels and only administering treatment when MDSC levels rise. These patients may also have reduced dosage or treatment frequency by adding in one or more doses of a TGF ⁇ 1 or TGF ⁇ 1/2 inhibitor.
  • the patient may have been previously treated with a TGF ⁇ inhibitor that inhibits TGF ⁇ 3 in conjunction with a checkpoint inhibitor.
  • TGF ⁇ 1 or TGF ⁇ 1/2 inhibitors for use in treating cancer in a subject are provided, wherein the subject has previously, is currently, or will be treated with a TGF ⁇ inhibitor that inhibits TGF ⁇ 3, e.g., in conjunction with a checkpoint inhibitor.
  • the cancer is a metastatic cancer, a desmoplastic tumor, or myelofibrosis.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1 -selective inhibitor, e.g., Ab6 or a variant thereof, e.g., Ab4, Ab5, Ab6, Ab21 , Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31 , Ab32, Ab33, and Ab34.
  • the TGF ⁇ inhibitor is Ab6.
  • the TGF ⁇ inhibitor is isoform-non-selective and inhibits TGF ⁇ 1/2/3 or TGF ⁇ 1/3.
  • the disclosure encompasses an isoform-non-selective TGF ⁇ inhibitor for the treatment of cancer comprising the steps of selecting a subject who is not diagnosed with a fibrotic disorder or who is not at high risk of developing a fibrotic disorder, e.g., a subject who does not exhibit elevated MDSC levels as compared to a control sample, and administering to the subject the isoform-non-selective TGF ⁇ inhibitor in an amount effective to treat the cancer.
  • the isoform-non-selective TGF ⁇ inhibitor is an antibody (or agent) that inhibits TGF ⁇ 1/2/3 or TGF ⁇ 1/3.
  • the isoform-non-selective TGF ⁇ inhibitor is an engineered construct comprising a TGF ⁇ receptor ligand-binding moiety.
  • the present disclosure encompasses a TGF ⁇ inhibitor for use in an intermittent dosing regimen for cancer immunotherapy in a patient, wherein the intermittent dosing regimen comprises the following steps: measuring circulating MDSCs 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; measuring circulating MDSCs 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 to the first sample; measuring circulating MDSCs 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 isoform-non-selective inhibitor inhibits TGF ⁇ 1/2/3, TGF ⁇ 1/2 or TGF ⁇ 1/3.
  • the sample is a blood sample or a blood component.
  • the TGF ⁇ inhibitor may be a TGF ⁇ 1 -selective inhibitor, e.g., an anti- TGF ⁇ 1 antibody having a sequence as disclosed below, e.g., Ab4, Ab5, Ab6, Ab21 , Ab 22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31 , Ab32, Ab33, and Ab34.
  • the TGF ⁇ inhibitor is Ab6.
  • the TGF ⁇ inhibitors disclosed herein are well tolerated in preclinical safety/toxicology studies in doses up to 100, 200, or 300 mg/kg when dosed weekly for at least 4 weeks. Such studies may be carried out in animal models that are known to be sensitive to TGF ⁇ inhibition, such as rats and non-human primates.
  • the TGF ⁇ inhibitors disclosed herein do not cause observable toxicities associated with pan-inhibition of TGF ⁇ . Observable toxicities may include cardiovascular toxicities (e.g., valvulopathy). Other observable toxicities include epithelial hyperplasia. Yet further observable toxicities are known in the art.
  • the TGF ⁇ inhibitors disclosed herein do not induce significant cytokine release or platelet aggregation, binding, or activation.
  • the TGF ⁇ inhibitor may not induce significant cytokine release (e.g., as determined by a method described herein).
  • the TGF ⁇ inhibitor may not cause a significant increase in platelet binding, activation and/or aggregation (e.g., as determined by a method described herein).
  • the TGF ⁇ inhibitor may be or may have been determined by a method described herein not to induce significant cytokine release and not to cause a significant increase in platelet binding, activation and/or aggregation.
  • the TGF ⁇ inhibitors disclosed herein achieve a sufficient therapeutic window in that effective amounts of the inhibitors shown by in vivo efficacy studies are well below (such as at least 3-fold, at least 6-fold, or at least 10-fold) the amounts or concentrations that cause observable toxicities.
  • the therapeutically effective amounts of the inhibitors are between about 1 mg/kg and about 30 mg/kg per week.
  • therapeutically effective amounts of the inhibitors are between about 1 mg/kg and about 10 mg/kg dosed every three weeks.
  • therapeutically effective amounts of the inhibitors are between about 2 mg/kg and about 7 mg/kg dosed every three weeks.
  • the TGF ⁇ inhibitors disclosed herein achieve a sufficient therapeutic window in that effective amounts of the inhibitors shown by in vivo efficacy studies are well below (such as at least 3-fold, at least 6-fold, or at least 10-fold) the amounts or concentrations that cause dose-limiting toxicities (DLTs).
  • DLTs are generally defined by the occurrence of severe toxicities during therapy (e.g., during first cycle of cancer therapy). Such toxicities may be assessed according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE) classification, and usually encompass all grade 3 or higher toxicities with the exception of grade 3 nonfebrile neutropenia and alopecia.
  • CCAE Common Terminology Criteria for Adverse Events
  • DLTs may also include certain a priori unbeatable or irreversible grade 2 toxicities (e.g., neurotoxicities, ocular toxicities, or cardiac toxicities), prolonged grade 2 toxicities (e.g., grade 2 toxicities lasting longer than a certain period), and/or the prolongation of the DLT period.
  • the definition of DLTs exclude toxicities that are clearly related to the disease itself (e.g., disease progression or intercurrent illness).
  • the therapeutically effective amounts of the inhibitors are between about 1 mg/kg and about 30 mg/kg per week. In some embodiments, therapeutically effective amounts of the inhibitors are between about 1 mg/kg and about 10 mg/kg dosed every three weeks. In some embodiments, therapeutically effective amounts of the inhibitors are between about 2 mg/kg and about 7 mg/kg dosed every three weeks.
  • the TGF ⁇ inhibitors disclosed herein e.g., a TGF ⁇ 1-selective inhibitor, e.g., Ab4, Ab5, Ab6, Ab21 , Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31 , Ab32, Ab33, or Ab34
  • the at least one additional therapy is a cancer therapy, such as immunotherapy, chemotherapy, radiation therapy (including radiotherapeutic agents), engineered immune cell therapy (e.g., CAR-T therapy), cancer vaccine therapy, and/or oncolytic viral therapy.
  • a cancer therapy may, for example, comprise a cancer therapy agent (e.g., an immunotherapeutic agent, a chemotherapeutic agent, a radiotherapeutic agent, engineered immune cells (e.g., CAR-T cells)), a cancer vaccine and/or a therapeutic oncolytic virus (including any combination thereof).
  • the cancer therapy is immunotherapy comprising 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 (0X40 ).
  • 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 e.g., a CTLA4 antagonist
  • a GITR agonist e.
  • compositions for use according to the present disclosure including those referring to the determination of circulating MDSC levels following administration of a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1-selective inhibitor or an isotype-non-selective TGF ⁇ inhibitor), the subject may not have received previous cancer therapy, e.g., may be treatment-naive, may have received previous cancer therapy, or may be 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.
  • the cancer may be advanced cancer.
  • the cancer may comprise a locally advanced tumor and/or metastatic cancer.
  • the subject may have cancer which exhibits or is suspected of exhibiting immune suppression (e.g., a tumor with an immune-excluded or immunosuppressive phenotype).
  • the subject who receives or has received the TGF ⁇ inhibitor may have 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) and may be resistant to checkpoint inhibitor therapy.
  • 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 subject may have 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) and may be treatment-naive.
  • the subject may have cancer with low response rates to checkpoint inhibitor therapy (e.g., overall response rate of 30% or less, 20% or less, or 10%, or less) and 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.
  • a TGF ⁇ inhibitor (e.g., a TGF ⁇ 1 -selective inhibitor, e.g., Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31 , Ab32, Ab33, or Ab34) 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 (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 does not inhibit TGF ⁇ 2 signaling at a therapeutically effective dose. In some embodiments, 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.
  • a TGF ⁇ inhibitor is a TGF ⁇ 1-selective inhibitor, e.g., Ab4, Ab5, Ab6, Ab21 , Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31 , Ab32, Ab33, and Ab34. In preferred embodiments, the TGF ⁇ 1 -selective inhibitor is Ab6.
  • the disclosure provides a method of treating fibrosis in a subject, the method comprising steps of administering a therapeutically effective amount of a TGF ⁇ inhibitor to the subject as a loading dose / maintenance dose regimen, wherein the TGF ⁇ inhibitor inhibits TGF ⁇ 1 but does not inhibit one or both of TGF ⁇ 2 and/or TGF ⁇ 3, thereby treating fibrosis in the subject.
  • the disclosure provides a method of preventing fibrosis in a subject at risk of developing fibrosis, the method comprising the steps of administering a therapeutically effective amount of a TGF ⁇ inhibitor to the subject as a loading dose / maintenance dose regimen, wherein the TGF ⁇ inhibitor inhibits TGF ⁇ 1 but does not inhibit one or both of TGF ⁇ 2 and/or TGF ⁇ 3, thereby preventing fibrosis in the subject at risk of developing fibrosis.
  • the method further comprises the steps of: (i) determining a level of collagen, a level of new collagen synthesis, and/or a level of phosphorylated Smad2, present in a fibrotic tissue in the subject prior to administering the TGF ⁇ inhibitor; and (ii) determining a level of collagen, a level of new collagen synthesis, and/or a level of phosphorylated Smad2, present in a fibrotic tissue in the subject after administering the TGF ⁇ inhibitor, wherein a decrease in the level of collagen, the level of new collagen synthesis and/or the level of phosphorylated Smad2 present in the fibrotic tissue in the subject after administration, as compared to prior to administration, indicates therapeutic efficacy.
  • the disclosure provides a method of treating fibrosis in a subject, the method comprising steps of administering to the subject a TGF ⁇ inhibitor, wherein the TGF ⁇ inhibitor inhibits TGF ⁇ 1 but does not inhibit one or both of TGF ⁇ 2 and/or TGF ⁇ 3, in an amount effective to reduce the amount of collagen present in a fibrotic tissue in the subject after administration, as compared to the amount of collagen present in the fibrotic tissue in the subject prior to administration; reduce the amount of new collagen synthesis in a fibrotic tissue in the subject after administration, as compared to the amount of new collagen synthesis present in the fibrotic tissue in the subject prior to administration; and/or reduce the amount of phosphorylated Smad2 in a fibrotic tissue in the subject after administration, as compared to the amount of phosphorylated Smad2 present in the fibrotic tissue in the subject prior to administration; thereby treating fibrosis in the subject.
  • the method further comprises the steps of (a) determining a level of collagen, a level of new collagen synthesis, and/or a level of phosphorylated Smad2, present in the fibrotic tissue in the subject prior to administering the TGF ⁇ inhibitor; and (b) determining a level of collagen, a level of new collagen synthesis, and/or a level of phosphorylated Smad2, present in the fibrotic tissue in the subject after administering the TGF ⁇ inhibitor.
  • reduction in the amount of collagen present in the fibrotic tissue, reduction in the amount of new collagen synthesis, and/or reduction in the amount of phosphorylated Smad2 in the fibrotic tissue is determined 24 hours, 48 hours, 72 hours, or 96 hours after administration of the TGF ⁇ inhibitor.
  • the method further comprises a step of selecting a subject who would benefit from a reduction in a level of collagen, a level of new collagen synthesis, and/or a level of phosphorylated Smad2 in a fibrotic tissue.
  • the TGF ⁇ inhibitor is administered as a single dose regimen, or as a loading dose / maintenance dose regimen.
  • the single dose regimen comprises administration of a single dosage of between about 1 mg/kg to about 100 mg/kg of the TGF ⁇ inhibitor.
  • the single dosage is about 3 mg/kg, about 10 mg/kg, or about 30 mg/kg.
  • the single dosage is administered to the subject weekly, biweekly, or monthly.
  • the loading dose /maintenance dose regimen comprises a loading dosage of between about 30 mg/kg and about 90 mg/kg and a maintenance dosage of between about 10 mg/kg and about 30 mg/kg.
  • the loading dosage is about 30 mg/kg and the maintenance dosage is about 10 mg/kg. According to some embodiments of the above aspects and embodiments, the loading dosage is about 90 mg/kg and the maintenance dosage is about 30 mg/kg. According to some embodiments of the above aspects and embodiments, the loading dosage is administered intravenously, and wherein the maintenance dosage is administered subcutaneously. According to some embodiments of the above aspects and embodiments, the loading dosage is administered once, and the maintenance dosage is administered weekly, biweekly, or monthly thereafter. According to some embodiments of the above aspects and embodiments, the fibrosis is pulmonary fibrosis or kidney fibrosis.
  • the pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF).
  • the administration is effective to reduce symptoms of fibrosis in the subject.
  • the symptoms of fibrosis are one or more of pulmonary hypertension, right-sided heart failure, respiratory failure, hypoxia, cough, formation of blood clots, pneumonia, and/or lung cancer in the subject.
  • the subject has been diagnosed with a pulmonary disease.
  • the pulmonary disease is an autoimmune disorder of the lung, a viral infection of the lung, or a bacterial infection of the lung.
  • the subject has received radiation therapy.
  • the radiation therapy is for lung cancer.
  • the subject has one or more risk factors for fibrosis selected from the group consisting of cigarette smoking, environmental factors and genetic predisposition for lung fibrosis.
  • the method further comprises a step of selecting a TGF ⁇ inhibitor that inhibits TGF ⁇ 1 but does not inhibit one or both of TGF ⁇ 2 and/or TGF ⁇ 3.
  • the present disclosure includes selection of subjects or patients who are likely to respond to or benefit from a TGF ⁇ 1 inhibition therapy.
  • Related diagnostic methods, as well as methods for monitoring or determining therapeutic response to the TGF ⁇ 1 inhibition therapy, are encompassed herein.
  • selection includes one or more antibodies or antigen-binding fragments with particularly advantageous kinetics criteria characterized by: i) sub-nanomolar affinities to each of human LTBP1/3-proTGF ⁇ 1 complexes (e.g., K D ⁇ 1 nM), and, ii) low dissociation rates (k OFF ), e.g., ⁇ 5.00E-4, as measured by a suitable in vitro binding/kinetics assay, such as by surface plasmon resonance (SPR), e.g., BIACORE®-based systems.
  • SPR surface plasmon resonance
  • the selected antibody or the plurality of antibodies are evaluated in preclinical studies comprising an efficacy study and a toxicology/safety study, employing suitable preclinical models. Effective amounts of the antibody or the antibodies determined in the efficacy study are below the level that results in undesirable toxicities determined in the toxicology/safety study. Preferably, the antibody or antibodies are selected which has/have at least 3-fold, 6-fold, and more preferably 10-fold therapeutic window. Effective amounts of the antibodies according to the present disclosure may be between about 0.1 mg/kg and about 30 mg/kg when administered weekly. In preferred embodiments, the maximally tolerated dose (MTD) of the antibodies according to the present disclosure is >100 mg/kg when dosed weekly for at least 4 weeks.
  • MTD maximally tolerated dose
  • the present disclosure includes a surprising finding that, contrary to the general belief that inhibition of multiple isoforms is needed for antifibrotic effects, concurrent inhibition of TGF ⁇ 3 produced pro-fibrotic effects in mice.
  • This observation raises the possibility that non-selective TGF ⁇ inhibitors (such as pan-inhibitors and TGF ⁇ 1/3 inhibitors) may in fact exacerbate fibrosis.
  • the antibodies disclosed herein are isoform- selective in that they specifically target the latent TGF ⁇ 1 complex and do so with low dissociation rates.
  • the disclosure includes the recognition that when selecting a particular TGF ⁇ inhibitor for patients with a fibrotic condition (e.g., disease involving ECM dysregulation, such as cardiovascular diseases), isoform selectivity should be carefully considered so as to avoid risk of exacerbating ECM dysregulation.
  • a fibrotic condition e.g., disease involving ECM dysregulation, such as cardiovascular diseases
  • the present disclosure includes therapeutic methods comprising selecting a TGF ⁇ inhibitor that does not inhibit TGF ⁇ 3 to treat a subject with a fibrotic condition, wherein optionally the subject has organ fibrosis or cancer, wherein further optionally the cancer is myelofibrosis.
  • the subject has, or is at risk of developing a cardiovascular disease.
  • the organ fibrosis is liver fibrosis, kidney fibrosis or lung fibrosis (e.g., IPF).
  • the liver fibrosis is associated with NASH.
  • Patients at risk of developing fibrosis or a condition with ECM dysregulation may include those suffering from a metabolic condition, such as diabetes, obesity and NASH.
  • FIG. 1A provides graphs showing cytokine release from the plate-bound assay format.
  • FIG. 1B provides graphs showing cytokine release from the soluble assay format.
  • FIG. 2A shows amplitude of platelet aggregation in human PRP with ADP agonist.
  • FIG. 2B shows area under the curve of platelet aggregation in human PRP with ADP agonist.
  • FIG. 3A shows tumor MDSC levels measured in MBT-2 tumors.
  • FIG. 3B shows tumor volume and percent circulating G-MDSC and M-MDSC measured in MBT-2 mice.
  • FIG. 4 shows a schematic of an exemplary TGF ⁇ inhibitor treatment regimen.
  • FIG. 5 shows circulating TGF ⁇ 1 levels (pg/mL) in MBT-2 mice.
  • FIG. 6A shows plasma levels of Ab6 ( ⁇ g/mL, left) and TGF ⁇ 1 (pg/mL, right).
  • FIG. 6B 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. 7A shows plasma platelet factor 4 levels (ng/mL) in MBT-2 mice.
  • FIG. 7B shows sample outliers as determined by interquartile range.
  • FIG. 7C shows identified sample outliers (left) and outlier-corrected levels (pg/mL) of circulatory TGF ⁇ 1 (right).
  • FIG. 8A shows tissue compartment data of bladder cancer samples.
  • FIG. 8B shows tissue compartment data of melanoma samples.
  • FIG. 9A shows representative CD8+ staining in bladder cancer samples.
  • FIG. 9B shows subdivision of CD8+ staining in the tumor margin compartment.
  • FIG. 9C shows subdivision of CD8+ staining in the tumor margin compartment of a bladder sample.
  • FIG. 10 shows comparison of compartment CD8+ ratio and absolute percent CD8 positivity.
  • FIG. 11 shows comparison of CD8+ cell density and absolute percent CD8 positivity.
  • FIG. 12 shows tumor volume in MBT-2 mice across treatment groups.
  • FIG. 13 shows baseline level of circulating MDSCs in non-tumor bearing mice.
  • FIG. 14 shows levels of circulating MDSCs in tumor-bearing mice.
  • FIG. 15 shows a comparison of circulating MDSC levels in non-tumor bearing mice and tumor-bearing mice.
  • FIG. 16A shows a comparison of circulating M-MDSC and G-MDSC levels on days 3-10.
  • FIG. 16B shows time-course of changes in circulating M-MDSC and G-MDSC levels from day 3 to day 10.
  • FIG. 17 is a plot of circulating MDSC level and tumor volume on day 10 across treatment groups.
  • FIG. 18 shows tumor MDSC levels in different treatment groups.
  • FIG. 19 shows a comparison of circulating G-MDSC levels and tumor MDSC levels on day 10 across treatment groups.
  • FIG. 20 shows correlation of tumor MDSC levels to circulating MDSC levels.
  • FIG. 21 is a plot of levels of tumor G-MDSC and tumor CD8+ cells across all treatment groups.
  • FIG. 22 shows circulatory TGF ⁇ levels in NHP following a single dose of Ab6.
  • FIG. 23 shows circulatory TGF ⁇ levels in rats following a single dose of Ab6.
  • FIG. 24 shows tumor depth of bladder samples.
  • FIG. 25 shows CD8 density in a melanoma sample.
  • FIG. 26 shows a schematic of an exemplary pathology analysis of tumor tissue sample.
  • FIG. 27 shows a schematic of an exemplary pathology analysis of tumor tissue sample.
  • FIG. 28 shows binding affinity of Ab6 to latent TGF ⁇ from human, rat, and cynomolgus monkey.
  • FIG. 29 shows mean Ab6 serum concentration time profiles following single doses to C57BL/6 mice, Sprague Dawley rats, and cynomolgus monkeys.
  • FIG. 30 shows serum concentration time profiles following multiple doses to Sprague Dawley rats and cynomolgus monkeys.
  • FIG. 31 shows density of CD8+ cells in bladder cancer samples as analyzed based on tumor nest.
  • FIG. 32 shows immune phenotype analysis of a single bladder cancer sample based on density of CD8+ cells measured in tumor nests.
  • FIG. 33A 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).
  • FIG. 33B 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).
  • FIG. 33C shows tumor nest data and immune phenotyping for individual tumor nests identified from bladder cancer samples.
  • FIG. 33D shows tumor nest CD8+ data and immune phenotyping for bladder cancer and melanoma samples.
  • FIG. 33E shows percent CD8+ cells in tumor, tumor margin, and stroma compartments of commercially available bladder cancer samples.
  • FIG. 34A shows a P-Smad2 IHC analysis of melanoma samples.
  • FIG. 34B shows pSmad-2 signaling in MBT2 tumors following treat with Ab6-mlgG1 .
  • FIG. 35A shows an exemplary sample collection and processing method for evaluating circulating TGF ⁇ 1 levels in blood.
  • FIG. 35B shows circulating TGF ⁇ 1 levels in blood samples as evaluated under various sample processing conditions.
  • FIG. 35C shows platelet factor 4 (PF4) levels in blood samples as evaluated under various sample processing conditions.
  • FIG. 35D shows correlation of circulating TGF ⁇ 1 levels and PF4 levels in blood samples as evaluated under various sample processing conditions.
  • FIG. 35E shows PF4 levels in blood samples as evaluated under various sample processing conditions.
  • FIG. 35F shows exemplary outlier analysis based on measurement of PF4 levels.
  • FIG. 35G shows exemplary outlier analysis based on measurement of PF4 levels.
  • FIG. 36A shows circulating gMDSC and mMDSC levels in whole blood of mice bearing MBT2 tumors.
  • FIG. 36B shows intratumoral gMDSC and mMDSC levels in mice bearing MBT2 tumors.
  • FIG. 37 demonstrates mean pharmacokinetic (PK) profiles of SRK-181 by dose.
  • FIG. 38 depicts the preliminary efficacy by duration of treatment.
  • FIG. 39 depicts the best response in target lesions in Part A1 and Part A2.
  • FIGs. 40A-C show exemplary analysis of MDSC by signal filtering.
  • FIGs. 41A-C shows identification of tumor MDSC populations in various solid cancer samples.
  • FIGs. 42A-C shows analysis of gMDSC and mMDSC populations in various solid cancer samples.
  • FIG. 43 shows representative OCTET® binding curves showing association and dissociation of AB2 to six different antigen complexes.
  • AB2 specifically binds TGF ⁇ 1 small latent complexes (SLCs), but not mature growth factors, in isoform-selective manner.
  • SLCs small latent complexes
  • FIG. 44 shows representative Biacore binding curves showing association and dissociation of Ab46 and reference antibody. Summary of binding kinetics is provided.
  • FIG. 45 provides 4 graphs that shows dose-dependent binding by ELISA of 5 antibodies (hlgG4) to the LLCs shown.
  • FIG. 46 provides 4 graphs that shows dose-dependent binding by ELISA of 5 antibodies (hlgG4) to the LLCs.
  • FIG. 47 provides 2 graphs showing picosirius red area (%) in liver sections of CDHFD mice treated with Ab2 or control.
  • FIG. 48 provides 3 representative PSR-stained images from CDHFD mice treated with AB2 or control.
  • FIG. 49 provides two graphs showing hydroxyproline content ( ⁇ g/mg tissue) in liver of CDHFD mice treated with AB2 or control.
  • FIG. 50 provides two graphs showing type 1 collagen-positive area of liver sections of CDHFD mice treated with AB2 or control.
  • FIG. 51 provides a graph showing picosirius red area (%) in kidney sections of adenine-induced rat kidney fibrosis model.
  • FIG. 52 provides 5 representative PSR-stained images from controls, CDHFD mice treated with AB2, a TGF ⁇ 3 inhibitor, or both (left); A graph showing picosirius red area (%) in liver sections of CDHFD mice treated with AB2, a TGF ⁇ 3 inhibitor, or both, as compared to control, is also provided (right).
  • FIG. 53 provides immunocytochemistry images of mouse liver cells visualized with 2 fluorescent labels: green depicts TGF ⁇ 1 and red depicts TGF ⁇ 3.
  • FIG. 54 provides a graph showing cell-based potency assay data by reporter cells. TGF ⁇ activities are inhibited by increasing concentrations of Ab2 or Ab46.
  • FIG. 55 provides a graph showing ratios of phosphorylated vs. total SMAD2/3 in the medial lobe of CDHFD mice liver. Significant reduction in pSMAD2/3 was seen at all doses tested of Ab46 in the medial lobe (p ⁇ 0.05 (t test)).
  • FIG. 56 provides a graph showing percent (%) positive phospho-SMAD2 (pSMAD2) nuclei in the liver of mice after 10 weeks of a choline-deficient high fat diet (CDHFD). Significant reduction in % positive pSMAD2 nuclei was seen in mice treated with 30 mg/kg dose of Ab46.
  • FIG. 57 provides a graph showing percent (%) positive phospho-SMAD2 (pSMAD2) nuclei in the liver of rats (eft lateral lobe) after 12 weeks of a CDHF diet.
  • a human IgG (HuNeg; negative control) or Ab46 were administered on Day 1 and Day 3 after 12 weeks of CDHFD.
  • An ALK5 inhibitor (ALK5i; positive control) was given to a control group of rats two hours before rats were harvested. T reatment with all doses of Ab46 (3mg/kg, 10mg/kg, and 30 mg/kg) resulted in suppression of SMAD2 phosphorylation, as did treatment with the positive control (ALK5i).
  • FIG. 58 provides a graph showing percent (%) positive phospho-SMAD2 (pSMAD2) nuclei in the liver of rats after 12 weeks of a CDHF diet.
  • a human IgG Human IgG (HuNeg; negative control) or Ab46 at doses of 0.3mg/kg, 1 mg/kg, 3mg/kg, 10mg/kg, and 30mg/kg were administered on Day 1 after 12 weeks of CDHFD.
  • An ALK5 inhibitor (ALK5i; positive control) was given to a control group of rats two hours before rats were harvested. Treatment with doses of Ab46 10/mg/kg and 30 mg/kg appeared to fully suppress SMAD2 phosphorylation, as did treatment with the positive control (ALK5i).
  • FIG. 59A is a graph that shows quantitation of picosirius red (PSR) staining of collagen fibers (by percent (%)) in fibrotic kidneys.
  • FIG. 59B is a graph that shows quantitation of hydroxyproline (HYP) content as a measurement of collagen levels (by percent (%)) in fibrotic kidneys.
  • FIG. 59C is a graph that depicts the results of immunohistochemical (IHC) analysis to determine the amount of phosphorylated Smad2 (active Smad2) in fibrotic kidney samples after treatment with Ab46.
  • IHC immunohistochemical
  • FIG. 60A is a graph that shows the average serum exposure for Ab46 based on the loading dose approach described in Example 23.
  • FIG. 60B is a graph that shows the PSR/serum exposure correlation for Ab46.
  • the serum exposure/HYP correlation of Ab46 in the adenine efficacy study described in the examples is shown in FIG. 60C.
  • the PSR and HYP correlation of Ab46 in the adenine efficacy study described in Example 23 is shown in FIG. 60D.
  • FIG. 61 is a graph that shows that exposure with Ab46 was dose-proportional and maintained across all time points in the PK/PD study shown in Example 24.
  • FIGs. 62A, 62B, and FIG. 62C are graphs that depict the quantitation of the results of immunohistochemical (IHC) analysis to determine the amount of phosphorylated Smad2 (active Smad2) in fibrotic kidney samples after treatment with Ab46.
  • IHC immunohistochemical
  • pSmad2-positive nuclei were assayed at 24 hours (FIG. 62A) and 48 hours (FIG. 62B) after antibody treatment.
  • FIG. 62C shows that there was continued pSmad2 reduction after 96 hours (FIG. 62C).
  • FIG. 62D shows that at 48hrs, Ab46 antibody serum exposure of ⁇ 80ug/mL was sufficient for target engagement, i.e., elicited reduction in pSmad2.
  • FIG. 63 is a graphs that shows that target engagement with Ab46 at doses of 10mg/kg and 30mg/kg remained at 96 hours post single dose. As shown in FIG. 22, there was full suppression of pSmad2 by ALK5i (10 mg/kg).
  • FIG. 64 depicts a panel of graphs showing that the average body weight (BW) in the treatment groups did not significantly change during the course of the study (24, 48 and 96 hour time points shown).
  • FIG. 65 depicts a series of graphs showing that all animals had a similar course of disease progression by assessment of hydroxyproline levels (normalized to protein levels) during the course of the study (24, 48 and 96 hour time points shown).
  • FIG. 66 is a graph that shows treatment with Ab46 at all doses tested (30 mg/kg, 10 mg/kg, 3 mg/kg and 1 mg/kg) showed a significant reduction in relative phosphorylation of SMAD2/3, as compared to isotype treated control knockout (Col4a3-/-) mice.
  • FIG. 67A is four graphs depicting relative gene expression of Serpine 1, COL1A1, LOXL2, and FN1 in an adenine model.
  • FIG. 67B depicts relative gene expression of FN1 at 24 hours, 48 hours, and 96 hours.
  • FIG. 67C depicts LOXL2 relative gene expression at 24, 48 and 96 hours.
  • FIG. 68 depicts relative gene expression of MMP2, MMP9, THBS1 , and CTGF in the adenine model.
  • FIG. 69A depicts relative gene expression of HAVcr-1/KIM1 and LCN2/NGAL in an adenine model.
  • FIG. 69B depicts relative gene epression of KIM1 at 24, 48 and 96 hours, and
  • FIG. 69C depicts relative gene expression of NGAL at 24, 48 and 96 hours.
  • FIG. 70A depicts relative gene expression of IL-1beta, CD68, IL-6 and CCL2/MCP1 in an adenine model.
  • FIG. 70B depicts relative gene expression of IL-6 at 24, 48 and 96 hours, and 70C depicts IL-1 beta expression at 24, 48, and 96 hours.
  • FIG. 71A depicts relative gene expression of TGFbetal, TGFbeta3, LTBP3, and GARP in an adenine model.
  • FIG. 71B depicts GARP expression at 24, 48 and 96 hours.
  • FIG. 72 depicts relative gene expression of TNFa, TGFb1 , TGFb2, and TGFb3 in an adenine model.
  • FIG. 73A depicts LTBP1 , LRRC33, and COL3A1 relative gene expression in an adenine model
  • FIG. 73B depicts COL3A1 relative gene expression at 24, 48, and 96 hours
  • FIG. 73C depicts LRRC33 relative gene expression at 24, 48 and 96 hours.
  • Advanced cancer advanced malignancy
  • advanced malignancy 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.
  • tumor includes cancer that has spread from where it started to nearby tissue or lymph nodes.
  • metalastatic cancer is a cancer that has spread from the part of the body where it started (the primary site) to other parts (e.g., distant parts) of the body.
  • 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.
  • Antibody encompasses any naturally-occurring, recombinant, modified or engineered immunoglobulin or immunoglobulin-like structure or antigen-binding fragment or portion thereof, or derivative thereof, as further described elsewhere herein.
  • 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.
  • the term antibodies, as used herein, 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.
  • 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.
  • Such 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 refers to skewed or uneven affinity towards or against a subset of antigens to which an antibody is capable of specifically binding.
  • an antibody is said to have bias when the affinity for one antigen complex and the affinity for another antigen complex are not equivalent (e.g., more than five-fold difference in affinity).
  • Context-independent antibodies according to the present disclosure have equivalent affinities towards such antigen complexes (i.e., unbiased or uniform).
  • Preferred biased antibodies of the present disclosure include “matrix-biased” (or “LTBP-biased') antibodies, which preferentially bind EMC-associated complexes (LTBP1-proTGF ⁇ 1 and LTBP3-proTGF ⁇ ), such that relative affinities between at least one of the matrix-associated complexes and at least one of the cell- associated complexes (GARP-proTGF ⁇ 1 and/or LRRC33-proTGF ⁇ 1 complexes) is greater than five-fold.
  • antibodies characterized as “unbiased” have approximately equivalent affinities towards such antigen complexes (e.g., less than five-fold difference in affinity).
  • Binding region is a portion of an antigen (e.g., an antigen complex) that, when bound to an antibody or a fragment thereof, can form an interface of the antibody-antigen interaction. Upon antibody binding, a binding region becomes protected from surface exposure, which can be detected by suitable techniques, such as HDX-MS. Antibody-antigen interaction may be mediated via multiple (e.g., two or more) binding regions. A binding region can comprise an antigenic determinant, or epitope.
  • an antigen e.g., an antigen complex
  • BLI Biolayer Interferometry
  • BLI is a label-free technology for optically measuring biomolecular interactions, e.g., between a ligand immobilized on the biosensor tip surface and an analyte in solution.
  • BLI provides the ability to monitor binding specificity, rates of association and dissociation, or concentration, with precision and accuracy.
  • BLI platform instruments are commercially available, for example, from ForteBio and are commonly referred to as the Octet® System.
  • 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 TGF ⁇ 1/proTGF ⁇ 1 refers to TGF ⁇ 1 or its signaling complex (e.g., pro/latent TGF ⁇ 1) that is membrane-bound (e.g., tethered to cell surface). Typically, such cell is an immune cell.
  • 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- 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 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 (ARC) or tumor cell.
  • ARC 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 , anti-PD-1 antibody), and Lirilumab. Keytruda® is an example of anti-PD-1 antibodies.
  • Budigalimab is a humanized, recombinant IgG 1 monoclonal antibody targeting PD-1 , that has been shown to be equally safe and well-tolerated in patients with HNSCC and NSCLC in a phase I study (Italiano et al. , Cancer Immunology, Immunotherapy (2022) 71 :417-431 ).
  • Opdivo® is one example of an anti-PD-1 antibody.
  • Therapies or therapeutic regimens that employ one or more of immune checkpoint inhibitors may be referred to as checkpoint blockade therapy (CBT) or checkpoint inhibitor therapy (CPI).
  • CBT checkpoint blockade therapy
  • CPI checkpoint inhibitor therapy
  • Clinical benefit is intended to include both efficacy and safety of a therapy.
  • 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 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).
  • 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
  • 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.
  • the second and additional therapies may be referred to as an add-on therapy or adjunct therapy.
  • 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.
  • 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
  • a BLI e.g., Octet®
  • 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.
  • the 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.
  • Binning provides useful structure-function profiles of antibodies that share similar binding regions within the same antigen because biological activities (e.g., intervention; potency) effectuated by binding of an antibody to its target is likely to be carried over to another antibody in the same bin.
  • biological activities e.g., intervention; potency
  • those with higher affinities lower KD typically have greater potency.
  • 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
  • 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 Radian (1995) FASEB J. 9: 133-139 and MacCallum (1996) J. Mol. Biol. 262(5): 732-45.
  • CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding (see, for example: Lu X et al., MAbs. 2019 Jan;11(1):45-57).
  • the methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs.
  • 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/domain 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 K D values of the former, taking average K D values of the latter, and calculating the ratio of the two, as exemplified herein.
  • a context-biased antibody may also be biased for or against one presenting molecule-proTGF ⁇ 1 complex relative to the other presenting molecule-proTGF ⁇ 1 complexes, such that the affinity (as measured by KD) for the former is more than 10-fold weaker or greater than the average of the latter, respectively.
  • 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 or balanced.
  • 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).
  • BLI Biolayer Interferometry
  • MSD-SET solution equilibrium titration
  • Dissociation rate The term dissociation rate as used herein has the meaning understood by the skilled artisan in the pertinent art (e.g., antibody technology) as refers to a kinetics parameter measured by how fast/slow a ligand (e.g., antibody or fragment) dissociates from its binding target (e.g., antigen). Dissociation rate is also referred to as the “off' rate (“k OFF "). Relative on/off rates between an antibody and its antigen (i.e., k ON and k OFF ) determine the overall strength of the interaction, or affinity, typically expressed as a dissociation constant, or K D .
  • equivalent affinities may be achieved by having fast association (high k ON ), slow dissociation (low k OFF ), or contribution from both factors.
  • Monovalent interactions may be measured by the use of monovalent antigen-binding molecules/fragments, such as fAb (Fab), whilst divalent interactions may be measured by the use of divalent antigen-binding molecules such as whole immunoglobulins (e.g., IgGs).
  • Dissociation rates can be experimentally measured in suitable in vitro binding assays, such as OCTET®- and BIACORE®-based systems.
  • ECM-associated TGF ⁇ 1/proTGF ⁇ 1 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.
  • Such matrix-associated latent complexes are enriched in connective tissues, as well as certain disease-associated tissues, such as tumor stroma and fibrotic tissues.
  • Human counterpart of a presenting molecule or presenting molecule complex may be indicated by an “h” preceding the protein or protein complex, e.g., “hLTBP1 ,” “hLTBPI- proTGF ⁇ 1 ,” hLTBP3” and “hLTBP3-proTGF ⁇ 1 .”
  • Average KD values of an antibody (or its fragment) to an LTBP1 - proTGF ⁇ 1 complex and an LTBP3-proTGF ⁇ 1 complex may be calculated to collectively represent affinities for ECM-associated (or matrix-associated) proTGF ⁇ 1 complexes.
  • 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” (or therapeutically effective amount, or therapeutic dose) may be a dosage or dosing regimen that achieves a statistically significant clinical benefit (e.g., efficacy) in a patient population.
  • the effective amount can be said to be between about 3-30 mg/kg.
  • 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, concentration, 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. For instance, in a particular model, if the endpoint tumor volume is set at 2,000 mm 3 , 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. Clinically, 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 ). In some embodiments, 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.
  • PR partial response
  • CR complete response
  • effective tumor control in clinical settings also includes stable disease, where tumors that are
  • 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 ARC 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
  • markers include, e.g., perforin and granzyme B.
  • Endpoints In studies aimed to assess effectiveness (e.g., clinical benefit or improvements) of a therapy, such as in clinical trials for a cancer therapy, endpoints represent the measures of predetermined parameters indicative of treatment effects. In oncology, 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 an increase in tissue growth resulting from proliferation of epithelial cells. As used herein, 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).
  • Equivalent affinity is intended to mean: i) the antibody binds matrix-associated proTGF ⁇ 1 complexes and cell-associated proTGF ⁇ 1 complexes with less than five-fold bias in affinity, as measured by suitable in vitro binding assays, such as solution equilibrium titration (such as MSD-SET), Biolayer Interferometry (such as Octet®) or surface plasmon resonance (such as Biacore System; and/or, ii) relative affinities of the antibody for the four complexes are uniform in that: either, the lowest affinity (highest KD numerical value) that the antibody shows among the four antigen complexes is no more than five-fold less than the average value calculated from the remaining three affinities; or, the highest affinity (lowest KD numerical value) that the antibody shows among the four antigen complexes is no more than five-fold greater than the average calculated from the remaining three affinities.
  • Antibodies with equivalent affinities may achieve more uniform inhibitory effects, irrespective of the particular presenting molecule associated with the proTGF ⁇ 1 complex (hence “context-independent”).
  • bias observed in average affinities between matrix-associated complexes and cell-associated complexes is no more than three-fold.
  • affinities are measured by surface plasmon resonance (e.g., a Biacore system). Such methods are to be carried out using standard test conditions, e.g., according to the manufacturer’s instructions.
  • Extended Latency Lasso refers to a portion of the prodomain that comprises Latency Lasso and Alpha-2 Helix, e.g., LASPPSQGEVPPGPLPEAVLALYNSTR (SEQ ID NO: 1127). In some embodiments, Extended Latency Lasso further comprises a portion of Alpha-1 Helix, e.g., LVKRKRIEA (SEQ ID NO: 1132) or a portion thereof.
  • Fibrosis refers to the process or manifestation characterized by the pathological accumulation of extracellular matrix (ECM) components, such as collagens, within a tissue or organ. Indeed, collagen accumulation is a hallmark of fibrosis. According to some embodiments, the fibrosis is lung (also referred to as pulmonary) fibrosis.
  • ECM extracellular matrix
  • Pulmonary fibrosis The term "pulmonary fibrosis” or “lung fibrosis” as used in the context of the present disclosure refers to the formation of excess fibrous connective tissue in the lung. According to some embodiments, pulmonary fibrosis may be a secondary effect of other lung diseases. Examples of such diseases include autoimmune disorders, viral infections and bacterial infections (such as tuberculosis). Pulmonary fibrosis may also be idiopathic, with cigarette smoking, environmental factors (e.g. occupational exposure to gases, smoke, chemicals or dusts) or genetic predisposition thought to be risk factors.
  • environmental factors e.g. occupational exposure to gases, smoke, chemicals or dusts
  • Fibrotic microenvironment refers to a local disease niche within a tissue, in which fibrosis occurs in vivo.
  • the fibrotic microenvironment may comprise disease-associated molecular signature (a set of chemokines, cytokines, etc.), disease-associated cell populations (such as activated macrophages, MDSCs, etc.) as well as disease-associated ECM environments (alterations in ECM components and/or structure).
  • Fibrotic microenvironment is thought to support the transition of fibroblast to ⁇ -smooth muscle actin-positive myofibroblast in a TGF ⁇ -dependent manner.
  • Fibrotic microenvironment may be further characterized by the infiltration of certain immune cells (such as macrophages and MDSCs).
  • Finger-1 (of TGF ⁇ 1 Growth Factor): As used herein, “Finger-1” is a domain within the TGF ⁇ 1 growth factor domain. In its unmutated form, Finger-1 of human proTGF ⁇ 1 contains the following amino acid sequence: CVRQLYIDFRKDLGWKWIHEPKGYHANFC (SEQ ID NO: 1124). 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: 1125). Finger-2 includes the “binding region 6", which spatially lies in close proximity to Latency Lasso.
  • GARP-TGF ⁇ 1/GARP-proTGF ⁇ 1 complex refers to a protein complex comprising a pro-protein form or latent form of a transforming growth factor-01 (TGF ⁇ 1) protein and a glycoprotein-A repetitions predominant protein (GARP) or fragment or variant thereof.
  • TGF ⁇ 1 protein transforming growth factor-01
  • 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 K D 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
  • human 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 96%, at least 97%, 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.
  • HDX-MS Hydrogen/deuterium exchange mass spectrometry
  • 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
  • immune-excluded tumors 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 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
  • TAMs and FAMs may also be characterized as immunosuppressive.
  • 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. Generally, these are secondary, tertiary, and other subsequent immune responses to the same antigen. 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-naive 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
  • 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/isoform-non-selective 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/selective The term “isoform specificity” or “isoform selectivity” 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.
  • an antibody may preferentially bind TGF ⁇ 1 at affinity of ⁇ 1 pM, while the same antibody may bind TGF ⁇ 2 and/or TGF ⁇ 3 at ⁇ 0.5-50 nM.
  • 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.
  • the terms “isoform-specific” and “isoform-selective” are used interchangeably herein.
  • 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.
  • presenting molecules used for forming such LLCs need not be full length polypeptides; however, the portion of the protein capable of forming disulfide bonds with the proTGF ⁇ 1 dimer complex via the cysteine residues near its N-terminal regions is typically required.
  • 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: 1126) which is spanned by Region 1 . 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: 1170).
  • the antibodies of the present disclosure bind a proTGF ⁇ 1 complex at ASPPSQGEVPPGPL (SEQ ID NO: 1170) 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: 1142) or LASPPSQGEVPPGPLPEAVLALYNSTR (SEQ ID NO: 271 ).
  • localized refers to anatomically isolated or isolatable abnormalities, such as solid malignancies, as opposed to systemic disease.
  • Certain leukemia 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.
  • LRRC33-TGF ⁇ 1/LRRC33-proTGF ⁇ 1 complex refers to a complex between a pro-protein form or latent form of transforming growth factor-01 (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.
  • LRRC33-TGF ⁇ 1 complex comprises LRRC33 covalently linked with pro/latent TGF ⁇ 1 via one or more disulfide bonds.
  • 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. In vivo, 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-TGF ⁇ 1/LTBP1-proTGF ⁇ 1 complex refers to a protein complex comprising a pro-protein form or latent form of transforming growth factor-01 (TGF ⁇ 1) protein and a latent TGF-beta binding protein 1 (LTBP1 ) or fragment or variant thereof.
  • LTBP1 -TGF ⁇ 1 complex comprises LTBP1 covalently linked with pro/latent TGF ⁇ 1 via one or more disulfide bonds.
  • 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 "hLTBPT" denotes human LTBP1.
  • LTBP3-TGF ⁇ 1/LTBP3-proTGF ⁇ 1 complex refers to a protein complex comprising a pro-protein form or latent form of transforming growth factor-01 (TGF ⁇ 1) protein and a latent TGF-beta binding protein 3 (LTBP3) or fragment or variant thereof.
  • LTBP3-TGF ⁇ 1 complex comprises LTBP3 covalently linked with pro/latent TGF ⁇ 1 via one or more disulfide bonds.
  • 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+).
  • CD206+/CD163+ CD206+/CD163+.
  • Applicant recently discovered that the 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 ⁇ transforming growth factor beta ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ fibroblasts fibroblasts.
  • M2 macrophages play a role in TGF ⁇ -driven lung fibrosis and are also enriched in a number of tumors.
  • Matrix-associated proTGF ⁇ 1 LTBP1 and LTBP3 are presenting molecules that are components of the extracellular matrix (ECM). 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 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), which belongs to a group of diseases called myeloproliferative disorders and includes primary myelofibrosis and secondary myelofibrosis.
  • 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 CD 16-.
  • 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-. In some embodiments, a human M1 macrophage is CD11b + . In some embodiments, a human M1 macrophage is CD14-. In some embodiments, a human M2 macrophage can be identified by at least one (e.g., all) of the cell surface markers CD11b + and CD15-. In some embodiments, a human M2 macrophage is CD206 + . In some embodiments, a human M2 macrophage is CD 163 + . In some embodiments, a human M2 macrophage is HLA-DR + . In some embodiments, a human M2 macrophage is CD 14-. In some embodiments, a human M2 macrophage is CD33 + . In some embodiments, a human M2 macrophage is CD66b-.
  • Myeloid-derived suppressor cell Myeloid-derived suppressor cells
  • MDSCs are a heterogeneous population of cells generated during various pathologic conditions and thought to represent a pathologic state of activation of monocytes and relatively immature neutrophils.
  • 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
  • MDSCs are characterized by a distinct set of genomic and biochemical features, and can be distinguished by specific surface molecules.
  • 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 also 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.
  • 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).
  • the MDSCs may also express CD39 and CD73 to mediate adenosine signaling involved in organ fibrosis (such as liver fibrosis, and lung fibrosis), cancer and myelofibrosis).
  • human M-MDSCs may also express HLA-DR.
  • 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 45A.
  • 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 T able 45B.
  • 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.
  • Myofibroblast 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
  • Myofibroblasts or myofibroblast-like cells within the fibrotic microenvironment may be referred to as fibrosis-associated fibroblasts (or “FAFs”), and myofibroblasts or myofibroblast-like cells within the tumor microenvironment may be referred to as cancer- associated fibroblasts (or “CAFs”).
  • FAFs fibrosis-associated fibroblasts
  • CAFs cancer- associated fibroblasts
  • 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 ⁇ .
  • small molecule pan-TGF ⁇ inhibitors include galunisertib (LY2157299 monohydrate, , CAS No. 700874-72-2), which is an antagonist for the TGF ⁇ receptor I kinase/ALK5 that mediates signaling of all three TGF ⁇ isoforms.
  • Perivascular (infiltration) The prefix “peri-”” means “around” “surrounding” or “near,” hence “perivascular” literally translates to around the blood vessels.
  • 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.
  • antibodies with higher affinities tend to show higher potency than antibodies with lower affinities (greater K D values).
  • 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
  • animal-based preclinical studies are referred to as “in vivo” studies.
  • in vivo mouse preclinical models encompassed by the current disclosure include the MBT2 bladder cancer model, the Cloudman S91 melanoma model, and the EMT6 breast cancer model.
  • Predictive biomarker 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.
  • latent pro-proteins e.g., proTGF ⁇ 1
  • tether present
  • ECM extracellular niche
  • presenting molecules for proTGF ⁇ 1 include: LTBP1, LTBP3, GARP (also known as LRRC32) 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 “mafr/x-associated”) proTGF ⁇ 1 complexes, that mediate ECM- associated TGF ⁇ 1 signaling/activities.
  • ECM-associated or “mafr/x-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.
  • 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 .
  • the expression “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: 1119).
  • Regression of tumor or tumor growth can be used as an in vivo efficacy measure. For example, in precl i ni cal settings, median tumor volume (MTV) and Criteria for Regression Responses T reatment 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. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. Complete regression achieved in response to therapy (e.g., administration of a drug) may be referred to as "complete response” and the subject that achieves complete response may be referred to as a “complete responder”. Thus, complete response excludes spontaneous complete regression.
  • MTV median tumor volume
  • Criteria for Regression Responses T reatment 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. Treatment may cause partial regression (PR) or complete regression
  • 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.
  • fibrosis can be used as an in vivo efficacy measure of a therapy such as a TGF ⁇ 1 inhibitor. The regression of fibrotic conditions may be determined based on the standard criteria to assess the severity of fibrotic manifestation by disease stage.
  • Tregs are a type of immune cells characterized by the expression of the biomarkers CD4, FOXP3, and CD25. T regs are sometimes referred to as suppressor T cells and represent a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T (Teff) cells. Tregs can develop in the thymus (so-called CD4+ Foxp3+ “natural” Tregs) or differentiate from naive CD4+ T cells in the periphery, for example, following exposure to TGF ⁇ or retinoic acid. Tregs can express cell surface GARP-proTGF ⁇ 1.
  • 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
  • acquired resistance e.g., acquired phenotypes that develop over time following the treatment.
  • 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
  • such resistance can indicate immune escape.
  • 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.
  • EORTC European Organisation for Research and Treatment of Cancer
  • National Cancer Institute of the United States 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.
  • iRECIST provides a modified set of criteria that takes into account immune-related response (see: ncbi.nlm.nih.gov/pmc/articles/PMC5648544/, Seymour et al., iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics, Lancet Oncol. , 2017, the 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 (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 patients with tumor size reduction of a predefined amount and for a minimum time period.
  • 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).
  • TGF ⁇ 3-positive tumors are TGF ⁇ 1/3-co-dominant.
  • 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 e.g., pancreatic adenocarcinoma
  • prostate cancer e.g
  • 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
  • the 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 Meso-Scale Discovery
  • MSD-SET is a useful mode of determining dissociation constants for particularly high-affinity protein-protein interactions at equilibrium, such as picomolar-affinity antibodies binding to their antigens (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.
  • 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 K D measured by Biacore®).
  • an antigen e.g., a K D measured by Biacore®.
  • the antibody, or antigen- binding portion thereof binds to a specific protein rather than to proteins generally.
  • 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 proTG ⁇ 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.
  • 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. In a clinical context, the term “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.
  • lifestyle factors e.g., smoking, alcohol consumption, exercise, etc.
  • SPR Surface plasmon resonance
  • the SPR-based biosensors such as those commercially available from Biacore, can be employed to measure biomolecular interactions, including protein-protein interactions, such as antigen-antibody binding.
  • the technology is widely known in the art and is useful for the determination of parameters such as binding affinities, kinetic rate constants and thermodynamics.
  • Target engagement refers to the ability of a molecule (e.g., TGF ⁇ inhibitor) to bind to its intended target in vivo ⁇ e.g., endogenous TGF ⁇ ).
  • a molecule e.g., TGF ⁇ inhibitor
  • the intended target can be a large latent complex.
  • 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.
  • the TGF ⁇ 1 -related indication is fibrosis, e.g., lung fibrosis.
  • 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 , ⁇ llb ⁇ 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 , ⁇
  • TGF ⁇ inhibitors that are isoform-selective and non-selective inhibitors.
  • the latter commonly referred to as “pan-inhibitors" of TGF ⁇ , 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.
  • 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.
  • Internalization may be a suitable mechanism of action for LRRC33-containing protein complexes (such as human LRRC33-proTGF ⁇ 1) which results in reduced levels of cells expressing LRRC33-containing protein complexes on cell surface.
  • TGF ⁇ family is a class within the TGF ⁇ superfamily and in human contains three members: TGF ⁇ 1 , TGF ⁇ 2, and TGF ⁇ 3, which are structurally similar. The three growth factors are known to signal via the same receptors.
  • TGF ⁇ 1-positive cancer/tumor refers to a cancer/tumor with aberrant TGF ⁇ 1 expression (overexpression). Many human cancer/tumor types show predominant expression of the TGF ⁇ 1 (note that “TGFB” is sometimes used to refer to the gene as opposed to protein) isoform. In some cases, such cancer/tumor may show co-dominant expression of another isoform, such as TGF ⁇ 3. A number of epithelial cancers (e.g., carcinoma) may co-express TGF ⁇ 1 and TGF ⁇ 3.
  • TGF ⁇ 1 may arise from multiple sources, including, for example, cancer cells, tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and the surrounding extracellular matrix (ECM).
  • TAMs tumor-associated macrophages
  • CAFs cancer-associated fibroblasts
  • Regs regulatory T cells
  • MDSCs myeloid-derived suppressor cells
  • ECM extracellular matrix
  • preclinical cancer/tumor models that recapitulate human conditions are TGF ⁇ 1 -positive cancer/tumor.
  • Therapeutic window refers to a dosage/concentration 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 (e.g., at 5 mg/kg) 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 (see, for example, Example 26 herein).
  • 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. Mice are reported to be less sensitive to pharmacological inhibition of TGF ⁇ and may not reveal toxicities that are potentially serious or dangerous in other species, including human.
  • translatability In the context of drug discovery and clinical development, the term “translatability” or “translatable” refers to certain quality or property of preclinical models or data that recapitulate human conditions. As used herein, a preclinical model that recapitulates a TGF ⁇ 1 indication typically shows predominant expression of TGFB1 (or TGF ⁇ 1), relative to TGFB2 (or TGF ⁇ 2) and TGFB3 (or TGF ⁇ 3). In combination therapy paradigms, for example, translatability may require the same underlining mechanisms of action that the combination of actives is aimed to effectuate in the model. As an example, many human tumors are immune excluded, TGF ⁇ 1 -positive tumors that show primary resistance to a checkpoint blockade therapy (CBT).
  • CBT checkpoint blockade therapy
  • a second therapy (such as TGF ⁇ 1 inhibitors) may be used in combination to overcome the resistance to CBT.
  • suitable translatable preclinical models include TGF ⁇ 1 -positive tumors that show primary resistance to a checkpoint blockade therapy (CBT).
  • CBT checkpoint blockade therapy
  • 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, slowing disease progression, reversing certain disease features, normalizing gene expression, 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., fibrosis and tumor burden); preventing recurrence or relapse; prolonging a refractory period, and/or otherwise improving survival.
  • disease burden e.g., fibrosis and tumor burden
  • 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. T reatment 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 (such as M2c and M2d subtypes) can express cell surface LRRC33 and/or LRRC33-proTGF ⁇ 1.
  • M2-like macrophages may be also enriched in fibrotic microenvironment.
  • 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).
  • Valvulopathy refers to a disease, disorder, or condition affecting one or more of the four valves of the heart, often characterized by lesions on the valve(s) of the heart. It is also generally known as valvular heart disease, or cardiac valvulopathy. Types of valvulopathies include, but are not limited to, aortic valvulopathies (e.g., aortic stenosis), mitral valvulopathies, tricuspid valvulopathies, and pulmonary valvulopathies.
  • aortic valvulopathies e.g., aortic stenosis
  • mitral valvulopathies e.g., tricuspid valvulopathies
  • pulmonary valvulopathies e.g., pulmonary valvulopathies.
  • 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.
  • 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.
  • 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.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one" refers, whether related or unrelated to those elements specifically identified.
  • “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.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • 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 ⁇ Transforming Growth Factor-beta
  • GDFs Growth- Differentiation Factors
  • BMPs Bone-Morphogenetic Proteins
  • 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/ll Ligand-induced oligomerization of TGF ⁇ RI/ll triggers the phosphorylation of SMAD transcription factors, resulting in the transcription of target genes, such as Coll a1 , Col3a1 , ACTA2, and SERPINE1 (Massague, J., J. Seoane, and D. Wotton, Genes Dev, 2005. 19(23): p. 2783- 810).
  • target genes such as Coll a1 , 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. Zhang, Nature, 2003. 425(6958): p. 577-84; Holm, T
  • 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
  • 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.
  • a number of retrospective analyses of clinically-derived tumors points to 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.
  • 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.
  • 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. While the EMT-6 tumors are weakly responsive to treatment with an anti-PD-L1 antibody, combining this checkpoint inhibitor with 1 D11 , an antibody that blocks the activity of all TGF ⁇ isoforms, resulted in a profound increase in the frequency of complete responses when compared to treatment with individual inhibitors.
  • the synergistic antitumor activity is proposed to be due to a change in cancer-associated fibroblast (CAF) phenotype and a breakdown of the immune excluded phenotype, resulting in infiltration of activated CD8+ T cells into the tumors.
  • CAF cancer-associated fibroblast
  • TGF ⁇ Malignant cells often become resistant to TGF ⁇ signaling as a mechanism to evade its growth and tumor-suppressive effects.
  • TGF ⁇ activates CAFs, inducing extracellular matrix production and promotion of tumor progression.
  • TGF ⁇ induces EMT, thus supporting tissue invasion and tumor metastases.
  • TGF ⁇ 1 , TGF ⁇ 2, and TGF ⁇ 3 are 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.
  • TGF ⁇ prodomain also called latency-associated peptide (LAP)
  • LAP latency-associated peptide
  • latent TGF ⁇ is co-expressed with latent TGF ⁇ - binding proteins and forms large latent complexes (LLCs) through disulfide linkage.
  • LLCs latent complexes
  • 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.
  • 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
  • TGFBR1 TGFBR1
  • TGFBR1 TGFBR1
  • TGFBR1 TGFBR1
  • TGFBR1 TGFBR1
  • the present disclosure provides monoclonal antibodies and antigen-binding fragments thereof capable of binding each of the four known human LLCs (hLTBP1-proTGF ⁇ 1 , hLTBP3-proTGF ⁇ 1 , hGARP-proTGF ⁇ 1 and hLRRC33-proTGF ⁇ 1) with high affinity (e.g., below 1 nM K D ) and with slow dissociation rates (i.e., low k OFF values), as measured for example by surface plasmon resonance (SPR), that are can be used in the treatment of fibrotic diseases and disorders, in particular in the treatment of pulmonary fibrosis.
  • SPR surface plasmon resonance
  • the antibodies and the antigen-binding fragments include isoform-selective inhibitors of TGF ⁇ 1 .
  • An example of such an antibody or the antigen-binding fragment thereof comprises an H-CDR1 , an H-CDR2, and H-CDR3, an L-CDR1 , an L-CDR2 and an L-CFR3, wherein: the H-CDR1 comprises GFTFADYA (SEQ ID NO: 276); the H-CDR2 comprises a sequence represented by the formula ISGSGX 1 AT, wherein optionally the X 1 is an A or K (SEQ ID NO: 277); the H-CDR3 comprises a sequence represented by the formula VSSGX 1 WDX 2 D, wherein optionally the X 1 is an H, D or Q, and wherein further optionally the X 2 is an F or Y (SEQ ID NO: 278); the L-CDR1 comprises QSISSY (SEQ ID NO: 279); the L- CDR2 comprises a sequence represented by the
  • the H- CDR2 comprises ISGSGAAT (SEQ ID NO: 282); the H-CDR3 comprises VSSGHWDYD (SEQ ID NO: 287); the L- CDR2 comprises AASGLES (SEQ ID NO: 284); and, the L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285).
  • the antibody or the fragment binds an epitope that comprises one or more of the following amino acid residues of the proTGF ⁇ 1 polypeptide sequence: S35, G37, E38, V39, P40, P41 , G42, P43, R274, K280, H283 and K309.
  • the H-CDR1 may comprise the sequence GFTFADYA (SEQ ID NO: 276); the H-CDR2 may comprise the sequence ISGSGAAT (SEQ ID NO: 282); the H-CDR3 may comprise a sequence represented by the formula VSSGX 1 WDX 2 D, wherein optionally the X 1 is an H or Q, and wherein further optionally the X 2 is a Y or F (SEQ ID NO: 283); the L-CDR1 may comprise the sequence QSISSY (SEQ ID NO: 279); the L-CDR2 may comprise the sequence AASGLES (SEQ ID NO: 284); and, the L-CDR3 may comprise the sequence QQTYGVPLT (SEQ ID NO: 285).
  • the H-CDR3 is VSSGHWDYD (SEQ ID NO: 287).
  • the antibody or the fragment binds an epitope that comprises one or more of the following amino acid residues of the proTGF ⁇ 1 polypeptide sequence: S35, G37, E38, V39, P40, P41 , G42, P43, R274, K280, H283 and K309.
  • one or more of the six CDRs may include one or more (e.g., 1 or 2) amino acid change(s).
  • an antibody or an antigen-binding fragment thereof selected for use or manufacture according to the present disclosure comprises an H-CDR1 , an H-CDR2, and H-CDR3, an L-CDR1, an L-CDR2 and an L-CFR3, wherein: the H-CDR1 comprises GFTFADYA (SEQ ID NO: 276); the H-CDR2 comprises a sequence represented by the formula ISGSGX 1 AT, wherein optionally the X 1 is an A or K (SEQ ID NO: 277); the H-CDR3 comprises a sequence represented by the formula VSSGX 1 WDX 2 D, wherein optionally the X 1 is an H, D or Q, and wherein further optionally the X 2 is an F or Y (SEQ ID NO: 278); the L-CDR1 comprises QSISSY (SEQ ID NO: 279); the L-CDR2 comprises a sequence represented by the formula AASX 1 X 2 X 3 X 4 wherein optionally the
  • the L-CDR2 comprises AASGLES (SEQ ID NO: 284); and, the L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285).
  • the antibody or the fragment binds an epitope that comprises one or more of the following amino acid residues of the proTGF ⁇ 1 polypeptide sequence: S35, G37, E38, V39, P40, P41 ,
  • one or more of the six CDRs may include one or more (e.g., 1 or 2) amino acid change.
  • Non-limiting examples of preferred activation inhibitors of TGF ⁇ 1 are provided in the table below, herein referred to as: Ab37, Ab38, Ab39, Ab40, Ab41 , Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51 and
  • Each of these antibodies may be in the form of whole immunoglobulin (such as IgG) or an antigen-binding fragment thereof, such as the Fab fragment.
  • the antigen-binding fragment may be used to make an engineered construct that comprises the fragment or a derivative thereof, such as bispecific antibodies and other fusion proteins that functions as a TGF ⁇ 1 inhibitor.
  • the six CDRs of each of the exemplary antibodies are listed in the table below.
  • the activation inhibitors of TGF ⁇ 1 is AB46.
  • the antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein, the V H comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%) sequence identity to:
  • V L comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
  • the antibody or the fragment binds an epitope that comprises one or more of the following amino acid residues of the proTGF ⁇ 1 polypeptide sequence: S35, G37, E38, V39, P40, P41 , G42, P43, R274, K280, H283 and K309.
  • Ab46 comprises the VH amino acid sequence of SEQ ID NO: 297 and the VL amino acid sequence of SEQ ID NO: 298.
  • the antibody or the antigen-binding fragment comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein, the V H comprises
  • V L comprises
  • the antibody or the antigen-binding fragment comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein, the V H comprises
  • V L comprises
  • the disclosure includes nucleic acid sequences that encode any one of the amino acid sequences provided above.
  • vectors e.g., DNA plasmids, such as mammalian expression vectors, and related nucleic acid preparations
  • cells transfected with the vector(s); a cell line with stable expression of the nucleic acids; a cell culture comprising the cell, wherein optionally the cell culture comprises mammalian cells capable of large-scale production of the antibody or a protein construct comprising an antigen-binding fragment of the antibody.
  • the monoclonal antibody or an antigen-binding fragment thereof, which selectively inhibits TGF ⁇ 1 activation comprises a heavy chain complementary determining region 1 (CDRH1 ) having an amino acid sequence at least 95% identical to the sequence set forth in GFTFADYA (SEQ ID NO: 276); a heavy chain complementary determining region 2 (CDRH2) having an amino acid sequence at least 95% identical to the sequence set forth in ISGSGAAT (SEQ ID NO: 282); a heavy chain complementary determining region 3 (CDRH3) having an amino acid sequence at least 95% identical to the sequence set forth in VSSGHWDYD (SEQ ID NO: 287); a light chain complementary determining region 1 (CDRL1 ) having an amino acid sequence at least 95% identical to the sequence set forth in QSISSY (SEQ ID NO: 279); a light chain complementary determining region 2 (CDRL2) having an amino acid sequence at least 95% identical to the sequence set forth in AASGLES (SEQ ID NO:
  • the monoclonal antibody or an antigen-binding fragment thereof, which selectively inhibits TGF ⁇ 1 activation comprises a heavy chain complementary determining region 1 (CDRH1 ) having an amino acid sequence at least 96% identical to the sequence set forth in GFTFADYA (SEQ ID NO: 276); a heavy chain complementary determining region 2 (CDRH2) having an amino acid sequence at least 96% identical to the sequence set forth in ISGSGAAT (SEQ ID NO: 282); a heavy chain complementary determining region 3 (CDRH3) having an amino acid sequence at least 96% identical to the sequence set forth in VSSGHWDYD (SEQ ID NO: 287); a light chain complementary determining region 1 (CDRL1 ) having an amino acid sequence at least 96% identical to the sequence set forth in QSISSY (SEQ ID NO: 279); a light chain complementary determining region 2 (CDRL2) having an amino acid sequence at least 96% identical to the sequence set forth in AASGLES (SEQ ID NO:
  • the monoclonal antibody or an antigen-binding fragment thereof, which selectively inhibits TGF ⁇ 1 activation comprises a heavy chain complementary determining region 1 (CDRH1 ) having an amino acid sequence at least 98% identical to the sequence set forth in GFTFADYA (SEQ ID NO: 276); a heavy chain complementary determining region 2 (CDRH2) having an amino acid sequence at least 98% identical to the sequence set forth in ISGSGAAT (SEQ ID NO: 282); a heavy chain complementary determining region 3 (CDRH3) having an amino acid sequence at least 98% identical to the sequence set forth in VSSGHWDYD (SEQ ID NO: 287); a light chain complementary determining region 1 (CDRL1 ) having an amino acid sequence at least 98% identical to the sequence set forth in QSISSY (SEQ ID NO: 279); a light chain complementary determining region 2 (CDRL2) having an amino acid sequence at least 98% identical to the sequence set forth in AASGLES (SEQ ID NO:
  • the monoclonal antibody or an antigen-binding fragment thereof, which selectively inhibits TGF ⁇ 1 activation comprises a heavy chain complementary determining region 1 (CDRH1 ) having an amino acid sequence at least 99% identical to the sequence set forth in GFTFADYA (SEQ ID NO: 276); a heavy chain complementary determining region 2 (CDRH2) having an amino acid sequence at least 99% identical to the sequence set forth in ISGSGAAT (SEQ ID NO: 282); a heavy chain complementary determining region 3 (CDRH3) having an amino acid sequence at least 99% identical to the sequence set forth in VSSGHWDYD (SEQ ID NO: 287); a light chain complementary determining region 1 (CDRL1 ) having an amino acid sequence at least 99% identical to the sequence set forth in QSISSY (SEQ ID NO: 279); a light chain complementary determining region 2 (CDRL2) having an amino acid sequence at least 99% identical to the sequence set forth in AASGLES (SEQ ID NO:
  • the monoclonal antibody or an antigen-binding fragment thereof, which selectively inhibits TGF ⁇ 1 activation comprises a heavy chain complementary determining region 1 (CDRH1 ) having an amino acid sequence set forth in GFTFADYA (SEQ ID NO: 276); a heavy chain complementary determining region 2 (CDRH2) having an amino acid sequence set forth in ISGSGAAT (SEQ ID NO: 282); a heavy chain complementary determining region 3 (CDRH3) having an amino acid sequence set forth in VSSGHWDYD (SEQ ID NO: 287); a light chain complementary determining region 1 (CDRL1 ) having an amino acid sequence set forth in QSISSY (SEQ ID NO: 279); a light chain complementary determining region 2 (CDRL2) having an amino acid sequence set forth in AASGLES (SEQ ID NO: 284); and, a light chain complementary determining region 3 (CDRL3) having an amino acid sequence set forth in QQTYGVPLT (SEQ ID NO: 285)
  • CDRH1 heavy chain complementary
  • the antibody or antigen- binding fragment thereof comprises a heavy chain variable domain (V H ) comprising a sequence having at least 95% identity, 96% identity, 97% identity, 98% identity, 99% identity to, comprises, or consists of SEQ ID NO:297; and a light chain variable domain (V L ) comprising a sequence having at least 95% identity, 96% identity, 97% identity, 98% identity, 99% identity to, comprises, or consists of SEQ ID NO:298.
  • V H heavy chain variable domain
  • V L light chain variable domain
  • the antibodies and antigen-binding fragments thereof are characterized by enhanced binding properties.
  • the antibodies and the antigen-binding fragments are capable of specifically binding to each of the presenting molecule-proTGF ⁇ 1 complexes (sometimes referred to as “Large Latency Complex” or LLC, 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 may be used as antigens (e.g., antigen complexes) to screen, evaluate or confirm the ability of an antibody to bind the antigen complexes in suitable in vitro binding assays.
  • antigens e.g., antigen complexes
  • assays include but are not limited to: Bio-Layer Interferometry (BLI)-based assays (such as OCTET®) and surface plasmon resonance (SPR)-based assays (such as BIACORE®).
  • TGF ⁇ inhibitors for carrying out the methods and therapeutic use in accordance with the present disclosure may include carrying out in vitro binding assays to measure binding kinetics.
  • the antibody or the antigen-binding fragment binds each of the following large latent complexes with a sub-nanomolar affinity, e.g., with K D of 1.0 nM or less, and with k OFF of 10E-4 (1/s) or lower: hLTBP1-proTc, hLTBP3-proTGF ⁇ 1, hGARP-proTGF ⁇ 1 and hLRRC33-proTGF ⁇ 1.
  • the antibody or the fragment further binds each of the murine LLC counterparts, namely, mLTBP1-proTGF ⁇ 1, mLTBP3-proTGF ⁇ 1, mGARP-proTGF ⁇ 1 and mLRRC33-proTGF ⁇ 1, with equivalent affinities as human LLCs.
  • In vitro binding kinetics may be readily determined by measuring interactions of test antibodies (such as antigen-binding fragments) and suitable antigen, such as large latent complexes (LLCs) and small latent complexes (SLCs).
  • suitable methods for in vitro binding assays to determine the parameters of binding kinetics include BLI-based assays such as OCTET®, and surface plasmon resonance-based assays, such as BIACORE® systems.
  • FIG. 43 An example of an Octet-based in vitro binding assays is provided in FIG. 43.
  • FIG. 44 An example of SPR-based in vitro binding assays is provided in FIG. 44.
  • Fab fragments of Ab46 and a reference antibody, which are both activation inhibitors of TGF ⁇ 1 were used in this experiment. As illustrated in FIG. 44, the two Fabs have similar “ON" rates (k ON ) indicating that they engage (i.e., associate) with antigen at similar rates.
  • the disclosure includes a method of selecting a TGF ⁇ activation inhibitor for therapeutic use, wherein the method comprises selection of an antibody or antigen-binding fragment thereof that has a dissociation rate of 10.0e-4 (s -1 ) or less as measured by SPR.
  • the antibody or the fragment binds antigen with an affinity of less than 1 nM (i.e., sub-nanomolar), e.g., less than 500 pM, 400 pM, 300 pM, 200 pM,
  • the table below exemplifies binding kinetics of the listed antibodies (e.g., Fabs) obtained by OCTET®-based binding assays.
  • the experiments were conducted with immobilized, biotinylated antigen and Fab fragments (e.g., test antibodies) in solution.
  • Table 10 mGARP-proTGF ⁇ 1
  • Table 11 hLRRC33-proTGF ⁇ 1
  • Circuiating/circuiatory 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. 2010 Nov; 40(11 ): 2969-2975).
  • MDSCs have been implicated in various 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.
  • human cancers e.g., solid tumors
  • 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.
  • tumor-associated MDSCs also referred to as tumor-associated MDSCs
  • TGF ⁇ 1 -dependent manner mice treated with a combination of Ab6 (TGF ⁇ 1 -selective inhibitor) and a PD-1 antibody triggered a robust influx of cytotoxic CD8+ T cells and a corresponding reduction in the tumor-associated MDSC population (e.g., from about 11 % to 1 .4% of CD45+ cells).
  • 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. See, Example 35 and FIGs 73A and 73B.
  • 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.
  • 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 tummor-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,
  • 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • integrin inhibitors include the anti- ⁇ V ⁇ 8 integrin antibodies provided in W02020051333, 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®); budigalimab (ABBV-181); pembrolizumab (e.g., Keytruda®); avelumab (e.g., Bavencio®); cemiplimab (e.g., Libtayo®); atezolizumab (e.g., Tecentriq®)); and durvalumab (e.g., Imfinzi®).
  • 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 and/or chemotherapy.
  • TGF ⁇ pathways may correlate with unresponsiveness of a tumor to genotoxic therapies, such as chemotherapy and radiation therapy (Liu et al., Sci Transl Med. 2021 Feb 10;13(580):eabc4465). This is observed across multiple cancer types, e.g., cancers of the epithelia, e.g., carcinoma.
  • such 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, esophageal carcinoma, and tenosynovial giant cell tumor (TGCT).
  • pancreatic cancer e.g., pancreatic adenocarcinoma
  • prostate cancer e.g
  • TGF ⁇ inhibitors may be used in conjunction with one or more genotoxic therapies (e.g., chemotherapy and/or radiation therapy, including radiotherapeutic agents) to treat such a cancer in a subject.
  • 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 1 A (CDKN1 A), or downregulation of one or more genes relating to alternative end joining, e.g., LIG1 (DNA ligase 1 ), PARP1 , and/or POLQ.
  • 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.
  • 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 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 , ⁇ llb ⁇ 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 A
  • 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).
  • a decrease in circulating MDSC 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 after the administration of a dose of TGF inhibitor (such as isoform-selective 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ , e.g., selective inhibition of TGF ⁇ 1 and/or TGF ⁇ 3) and a reduction in circulating MDSC levels may be indicative or predictive TGF
  • 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1 -selective inhibitor, e.g., Ab
  • TGF ⁇ inhibitor 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • TGF ⁇ inhibitor 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
  • the TGF ⁇ inhibitor 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 prior to administration. In some embodiments, 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.
  • the first dose of the TGF ⁇ inhibitor is the very first dose of TGF ⁇ inhibitor received by the patient. In some embodiments, 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 circulating MDSC levels is indicative or predictive of therapeutic efficacy.
  • a TGF ⁇ inhibitor e.g., Ab6
  • a checkpoint inhibitor therapy administered concurrently (e.g., simultaneously), separately, or sequentially, and a reduction in 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ . e.g., selective inhibition of TGF ⁇ 1 and/or TGF ⁇ 3), and
  • 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • TGF ⁇ inhibitor such as a TGF ⁇ 1-selective inhibitor,
  • circulating MDSCs may be measured within six weeks following administration of the initial treatment (e.g., the (first) dose of TGF ⁇ inhibitor). 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.
  • circulating MDSC levels may be used to select, inform treatment in, and/or predicting 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ 1 -selective inhibitor e.g., Ab6, an
  • Circulating MDSC levels may be further monitored as an early predictor of treatment response.
  • checkpoint inhibitor therapy e.g., an anti-PD1 or anti-PD-L1 antibody.
  • 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). These patients may have primary resistance (i.e. , have never shown response to checkpoint inhibitor therapy) or have acquired resistance (i.e., have responded checkpoint inhibitor therapy initially and developed resistance over time).
  • resistance to checkpoint inhibitor therapy in patients is indicative of immune suppression or exclusion, thus these patients may be selected as candidates for receiving 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, 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ 1 -selective inhibitor e.g., Ab6, an
  • 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ 1 -selective inhibitor e.g., Ab6
  • 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.
  • 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. Ab6)
  • 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.
  • the TGF ⁇ inhibitor targets TGF ⁇ 1/2. In some embodiments, 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 present disclosure provides a TGF ⁇ inhibitor for use in an intermittent dosing regimen for cancer immunotherapy in a patient, wherein the intermittent dosing regimen comprises the following steps: measuring circulating MDSCs 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; measuring circulating MDSCs 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 to the first sample; measuring circulating MDSCs 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.
  • 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 , ⁇ llb ⁇ 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
  • 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 , ⁇ llb ⁇ 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),
  • 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 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.
  • 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ inhibitor treatment such as a TGF ⁇ 1 -selective inhibitor (e.g., Ab6), an is
  • TGF ⁇ 1 and/or TGF ⁇ 3 selective inhibition of TGF ⁇ 1 and/or TGF ⁇ 3 in combination with a checkpoint inhibitor therapy, and 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.
  • Circulating MDSC levels may be determined in a sample such as a whole blood sample or a blood component (e.g., PBMCs).
  • 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. From peripheral blood, 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 CD11b+ marker selection (e.g., using CD11 b+ microbeads or antibodies).
  • G-MDSCs and M- MDSCs may be further distinguished from CD11b+ cells via e.g., flow cytometry/FACS analysis based on surface marker expression.
  • human G-MDSCs may be identified by expression of the cell-surface markers CD11b, CD33, CD15 and CD66b.
  • human G-MDSCs may also express LOX-1 , Arginase, and/or low levels of HLA-DR.
  • Human M-MDSCs may be identified by expression of the cell surface markers CD11b, CD33 and CD14, as well as low levels of HLA-DR in some embodiments. Quantification of circulating MDSCs may be represented as percentage of total CD45+ cells.
  • 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.
  • subjects are administered one or more additional treatment in combination with the one or more TGF ⁇ inhibitor (e.g., TGF ⁇ 1 inhibitor, e.g., Ab6).
  • 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.
  • 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.
  • protein markers such as HLA-DR, CD68, CD163, CD206, and other biomarkers, any method known in the art may be used.
  • 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.
  • cytotoxic T cells e.g., activated cytotoxic T cells
  • 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 (see Example 30).
  • 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
  • Tumor samples with this pattern from a patient may indicate a patient likely to benefit from TGF inhibitor therapy (without being bound by theory, this may be because the tumor is actively suppressing the immune response, preventing sufficient ingress of cytotoxic T cells, which could be partially or completely reversed by the TGF inhibitor).
  • 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.
  • the cluster score of cytotoxic T cells e.g. , CD8+ T cells
  • 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. By comparison, upon overcoming immunosuppression by TGF beta inhibitors, more uniform distribution of CD8+ T cells within the tumor can be observed, presumably as a result of the CD8+ cells being able to infiltrate from the perivascular regions and possibly proliferate in the tumor.
  • 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
  • tumor biopsy samples 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. In certain embodiments, 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).
  • cytotoxic T cells e.g., CD8+ T cells
  • 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. In certain embodiments, 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.
  • cytotoxic T cells e.g., CD8+ T cells
  • 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. In certain embodiments, levels of cytotoxic T cells (e.g., CD8+ T cells) may be measured using immunohistochemical analysis of tumor biopsy samples. In certain embodiments, levels of cytotoxic T cells (e.g., CD8+ T cells) may be determined at least 28 days prior to and/or at least 100 days following administering a TGF ⁇ therapy.
  • cytotoxic T cell e.g., CD8+ T cell
  • 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) inside the tumor as compared to cytotoxic T cell levels (e.g., CD8+ T cells) outside the tumor may be identified as an immune-excluded 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 . In certain embodiments, 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 . In certain embodiments, 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 .
  • cytotoxic T cell density e.g., CD8+ T cells
  • 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
  • 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 may be identified as an immune-excluded tumor.
  • 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.
  • tumor biopsy samples may be obtained by core needle biopsy.
  • three to five samples e.g., four samples
  • 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.
  • samples taken at different tumor depths may be used to analyze combined CD8 positivity over multiple tumor nests.
  • the combined CD8 positivity determined in these samples may be representative of CD8 positivity in the rest of the tumor.
  • 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 Imaged.
  • 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 TGF ⁇ inhibitor e.g., Ab6
  • such 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.
  • TGF ⁇ 1/3, ligand traps e.g., TGF ⁇ 1/3 inhibitors
  • an integrin inhibitor e.g., an antibodies that bind to ⁇ V ⁇ 1 , ⁇ V ⁇ 3, ⁇ V ⁇ 5, ⁇ V ⁇ 6, ⁇ V ⁇ 8, ⁇ 5 ⁇ 1 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibit downstream activation of TGF ⁇ . e.g., selective inhibition of TGF ⁇ 1 and/or TGF ⁇ 3).
  • 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • a TGF ⁇ inhibitor such as a TGF ⁇ 1-selective inhibitor (e.
  • 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 (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
  • 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 TGF ⁇ inhibitor such as a TGF ⁇ 1 -selective inhibitor (e.g., Ab6)
  • 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 TGF ⁇ inhibitor such as a TGF ⁇ 1 -selective inhibitor (e.g., Ab6)
  • a checkpoint inhibitor therapy e.g., a PD1 or PDL1 antibody
  • such 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.
  • such a subject is identified for receiving the combination therapy prior to receiving either the checkpoint inhibitor therapy or the TGF ⁇ inhibitor alone.
  • such a subject is treatment-naive.
  • such a subject has previously received a checkpoint inhibitor therapy and is non-responsive to the checkpoint inhibitor therapy.
  • such a subject has cancer that exhibits an immune-excluded phenotype.
  • 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 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).
  • 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).
  • 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 proinfiammatory 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.
  • a subject whose cancer exhibits an immune-infiamed 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 (ABBV-181); and durvalumab (e.g., Imfinzi®).
  • a subject whose cancer exhibits an immune-inflamed phenotype is administered a checkpoint inhibitor.
  • 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.
  • tissue sections prepared from needle biopsy samples may not remain intact during sample processing, and the possibility that a needle may be inserted in the portion of the tumor tissue that does not accurately represent the tumor phenotype, collecting four samples may help mitigate such limitations and provides more representative tumor phenotyping for improved accuracy.
  • 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 e.g., CD8+ 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%
  • a low percentage e.g., less than 5%
  • cytotoxic T cells e.g., CD8+ T cells
  • a low percentage of cytotoxic T cells (e.g., CD8+ T cells) in both the tumor and the stroma may be indicative of 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. In certain embodiments, a tumor-to-stroma CD8 ratio of greater than 1 may be indicative of an inflamed tumor phenotype. In certain embodiments, a tumor-to-stroma CD8 ratio of less than 1 may be indicative of an immune-excluded tumor. In certain embodiments, 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 pm may be measured in increments of 100 pm.
  • 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).
  • 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. 26, 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 such as a TGF ⁇ 1-selective inhibitor (e.g., 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 e.g. , the proportion of necrotic area inside the tumor margin
  • a therapeutic response may be determined according to any part of the exemplary flow chart shown in FIG. 27.
  • an increased level of tumor-infiltrating cytotoxic T cells (e.g., CD8+ T cells), especially 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.
  • TGF ⁇ inhibitor therapy e.g., a TGF ⁇ 1 inhibitor such as Ab6
  • TGF ⁇ inhibitor therapy e.g., a TGF ⁇ 1 inhibitor such as Ab6
  • conversion of an immune-excluded tumor microenvironment toward an immune-infiltrated or “inflamed” microenvironment for instance, 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.
  • 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.
  • treatment with the TGF ⁇ inhibitor therapy e.g., a TGF ⁇ 1 inhibitor such as Ab6 is continued if such a reduction or reversal of immune suppression in the cancer is detected.
  • Immunosuppressive lymphocytes associated with TMEs include TAMs and MDSCs.
  • TAMs and MDSCs A significant fraction of tumor-associated macrophages is of so-called “M2" type, which has an immunosuppressive phenotype. 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. In some embodiments, 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.
  • Tumor immune contexture examines the TME from the perspective of tumor-infiltrating lymphocytes (i.e., tumor immune microenvironment or TIME).
  • Tumor immune contexture refers to the localization (e.g., spatial organization) and/or density of the immune infiltrate in the TME.
  • 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. In some embodiments, TILs are analyzed by immunohistochemical (IHC) methods. In some embodiments, TILs are analyzed by so-called digital pathology (see, for example, Saltz et al., (2016) Cell Reports 23, 181-193. “Spatial organization and molecular correlation of tumor- infiltrating lymphocytes using deep learning on pathology images.”); (Scientific Reports 9: 13341 (2019) “A novel digital score for abundance of tumor infiltrating lymphocytes predicts disease free survival in oral squamous cell carcinoma”). In some embodiments, 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.
  • TGF ⁇ inhibitor alone (e.g., Ab6) or in conjunction with a checkpoint inhibitor therapy.
  • circulating 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 latent TGF beta before and after administration.
  • circulating TGF ⁇ 1 in a blood sample e.g., plasma and/or serum
  • a blood sample e.g., plasma and/or serum
  • comprises both latent and mature forms the former of which representing vast majority of circulatory TGF ⁇ 1.
  • total circulating TGF ⁇ (e.g., total circulating TGF ⁇ 1) may be measured, i.e., comprising both latent and mature TGF ⁇ , for example by using an acid treatment step to liberate the mature growth factor (e.g. TGF ⁇ 1) from its latent complex and detecting with an enzyme-linked immunosorbent assay (ELISA) assay.
  • 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.
  • only circulating latent TGF ⁇ (e.g., circulating latent TGF ⁇ 1) is detectable.
  • circulating latent TGF ⁇ (e.g., circulating latent circulating TGF ⁇ 1) is measured.
  • circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1) 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.
  • 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 0.11 M 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.
  • circulating TGF ⁇ 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.
  • the collection tube is coated with a solution comprising 0.1-0.25 M dipyridamole.
  • the collection tube is coated with a solution having a pH of 4.0-6.0.
  • 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.
  • CTAD citrate-theophylline-adenosine-dipyridamole
  • ACD acid-citrate-dextrose
  • EDTA ethylenediaminetetraacetic acid
  • the collection tube is coated with CTAD.
  • the collection tube is coated with a CTAD solution comprising about 0.11 M 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 HemogardTM 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 VacutainerTM 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 (PPP).
  • PPP platelet-poor plasma
  • a PPP fraction may be prepared from a blood sample for the measurement of circulatory TGF ⁇ 1 levels.
  • the term PPP typically refers to blood plasma that contains less than 10,000 platelets per microliter (i.e., ⁇ 10 x 10 3 / ⁇ L).
  • 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 than5 ⁇ 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. In some embodiments, processing the blood sample comprises one or more incubation steps as described in Example 34.
  • 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. In some embodiments, processing the blood sample comprises a centrifugation step at a speed of about 2500xg. In some embodiments, 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.
  • processing the blood sample comprises a first centrifugation step at a speed below 2500xg, and a second centrifugation step at a speed above 10000xg.
  • 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the supernatant portion of the sample following the second centrifugation step is used for measuring circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1) levels. In some embodiments, TGF ⁇ (e.g., circulating latent TGF ⁇ 1) levels may be determined using Bio-Plex ProTM TGF- ⁇ Assays (Strauss eta/. Clin Cancer Res. 2018 Mar 15;24(6): 1287- 1295). [396] In some embodiments, collection, processing, and/or determination of circulating TGF ⁇ (e.g., circulating latent TGF ⁇ 1) levels are conducted at about 4 degrees C.
  • TGF ⁇ e.
  • 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 degrees 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 degrees 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 degrees 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 degrees 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 degrees 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 normalizing to control markers of platelet activation during collection, e.g., PF4 levels.
  • the resulting samples may be et al. used to carry out one or more measuring steps for circulatory TGF ⁇ .
  • the present disclosure provides, in various embodiments, a method for measuring circulating 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
  • 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.
  • the 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 , WO 2021/039945).
  • the TGF ⁇ inhibitors include neutralizing antibodies and engineered constructs that incorporate an antigen-binding fragment thereof. Examples of neutralizing antibodies include GC1008 and its variants, and NIS-793 (XOMA089).
  • the TGF ⁇ inhibitors also include so-called ligand traps, which comprise the ligand binding fragments) of the TGF ⁇ receptor(s).
  • 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.
  • 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 naive to a cancer therapy, e.g., a checkpoint inhibitor (i.e., a checipoint inhibitor-naive patient).
  • a checkpoint inhibitor i.e., a checipoint inhibitor-naive patient.
  • the checkpoint inhibitor-naive 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 latent TGF ⁇ may be monitored alone or in conjunction with one or more of the biomarkers disclosed herein (e.g., MDSCs).
  • 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ .
  • 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.
  • 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 latent 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 latent 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 latent 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 ⁇ 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.
  • an increase in circulating TGF ⁇ levels e.g., circulating latent TGF ⁇ 1 levels
  • after administration as compared to before administration indicates target engagement of the TGF ⁇ inhibitor.
  • an increase in circulating latent TGF ⁇ (e.g., circulating latent TGF ⁇ 1) of 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, after administration of the TGF inhibitor indicates target engagement.
  • 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 latent 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 latent TGF ⁇ 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
  • an additional cancer therapy e.g., a checkpoint inhibitor therapy
  • the amount of TGF ⁇ 1 inhibition administered is sufficient to increase the levels of circulating latent-TGF ⁇ (e.g., circulating latent TGF ⁇ 1) as compared to baseline circulating latent-TGF ⁇ levels.
  • Circulating latent-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 latent-TGF ⁇ levels e.g., circulating latent TGF ⁇ 1
  • a TGF ⁇ inhibitor e.g., Ab6
  • an increase in circulating latent-TGF ⁇ levels e.g., latent TGF ⁇ 1 following the treatment predicts therapeutic efficacy.
  • treatment is continued if an increase is detected.
  • circulating latent-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 latent- 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 latent- 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 latent TGF ⁇ (e.g., TGF ⁇ 1) of 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, after administration of the TGF inhibitor indicates therapeutic efficacy.
  • the increase in circulating latent-TGF ⁇ levels 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 TGFb is elevated.
  • the level of 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 the level of circulating TGF ⁇ before the first dose of the TGF ⁇ inhibitor.
  • 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 latent 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 latent 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
  • the TGF ⁇ inhibitor 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 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.
  • circulating TGF ⁇ levels are measured from a blood sample (e.g., a plasma sample, serum sample, etc.).
  • circulating latent 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 latent TGF ⁇ levels e.g., latent TGF ⁇ 1 levels
  • a first dose of TGF ⁇ inhibitor e.g., Ab6
  • circulating latent 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 latent TGF ⁇ levels (e.g., latent TGF ⁇ 1) following the administration indicates therapeutic efficacy.
  • TGF ⁇ inhibitor e.g., Ab6
  • treatment is continued if an increase in circulating latent-TGF ⁇ levels (e.g., latent TGF ⁇ 1) following administration of a TGF ⁇ inhibitor (e.g., Ab6) is detected.
  • circulating TGF ⁇ levels are measured from a blood sample (e.g., a plasma sample, serum sample, etc.).
  • circulating latent-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 latent-TGF ⁇ levels (e.g., latent TGF ⁇ 1) after the administration indicates target engagement and/or treatment response, and/or further warrants administration of a second or more dose(s) of the TGF ⁇ inhibitor.
  • a TGF ⁇ inhibitor e.g., Ab6
  • an increase in circulating latent-TGF ⁇ levels e.g., latent TGF ⁇ 1 after the administration indicates target engagement and/or treatment response, and/or further warrants administration of a second or more dose(s) of the TGF ⁇ inhibitor.
  • circulating latent-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 latent-TGF ⁇ levels after the administration indicates target engagement and/or 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 comprising 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ . e.g., selective inhibition of TGF ⁇ 1 and/orTGF ⁇ 3).
  • circulating TGF ⁇ levels are measured from a blood (e.g., plasma sample, serum sample, etc.).
  • circulating TGF ⁇ is circulating TGF ⁇ 1 .
  • the circulating 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.
  • 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.
  • cytokine storm a Phase 1 Trial of the anti-CD28 monoclonal antibody TGN1412 in healthy volunteers led to a life-threatening “cytokine storm” response resulted from an unexpected systemic and rapid induction of proinflammatory cytokines (Suntharalingam G et al., N Engl J Med. 2006 Sep 7;355(10):1018-28). This incident prompted heightened awareness of the potential danger associated with pharmacologic stimulation of T cells.
  • 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 such assays are described in Example 23 herein.
  • selection of a TGF ⁇ inhibitor for therapeutic use and/or large-scale production thereof includes an assessment of the ability for the TGF ⁇ inhibitor to trigger cytokine release from cytokine- producing cells.
  • one or more of the cytokines e.g., inflammatory cytokines
  • MCP-1 peripheral blood mononuclear cell
  • 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, or any other suitable control depending on the TGF ⁇ inhibitor being tested.
  • Cytokine activation may be assessed in plate-bound (e.g., immobilized) and/or soluble assay formats.
  • Levels of IFN ⁇ , IL-2, IL-1 ⁇ , TNF ⁇ , IL-6, and CCL2 (MCP-1 ) 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 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.
  • a negative control e.g., saline solution
  • a reference sample e.g., a buffered solution.
  • the candidate drug should be evaluated to ensure that it does not trigger spontaneous or agonist-induced activation.
  • the drug should not interfere with the normal function of platelets (e.g., aggregation or clotting).
  • platelet aggregation and binding do not exceed 10% above the aggregation in the negative control.
  • 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 an immune-directed safety study in non-human primates, 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.
  • such preclinical models comprise TGF ⁇ 1 -positive fibrosis.
  • the preclinical models are selected from liver fibrosis model, kidney fibrosis model, lung fibrosis model, heart (cardiac) fibrosis model, skin fibrosis model.
  • 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 or a fibrotic disorder, such as organ fibrosis.
  • the organ fibrosis can be pulmonary (lung) fibrosis.
  • 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.
  • Such 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.
  • TGF ⁇ inhibitors useful for carrying out various embodiments of the disclosure are aimed to pharmacologically interfere with one or more aspects of TGF ⁇ 1 function in vivo.
  • the TGF ⁇ inhibitor may be a TGF ⁇ 1 inhibitor, such as a TGF ⁇ 1 -isoform selective inhibitor, or an isoform-non-selective inhibitor.
  • Isoform-non- selective inhibitors include, without limitation, 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.
  • TGF ⁇ inhibitors that target more than one isoform.
  • ALK5 antagonists such as Galunisertib (LY2157299 monohydrate, Eli Lilly
  • monoclonal antibodies such as neutralizing antibodies
  • 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 ⁇ llb ⁇ 3 , or ⁇ 8 ⁇ 1 integrins and inhibit downstream activation of TGF ⁇ , e.g., selective inhibition of TGF ⁇ 1 and/or TGF ⁇ 3 (e.g.,
  • inhibitors of integrins such as also block integrin-dependent activation of both TGF ⁇ 1 and TGF ⁇ 3 and therefore may be considered as isoform- non-selective inhibitors of TGF ⁇ signaling.
  • TGF ⁇ 1 -selective inhibitors are shown to mitigate fibrosis in preclinical models, including mouse liver fibrosis model where both of the TGF ⁇ 1/3 isoforms are co- expressed in the fibrotic tissue, albeit in discrete cell types (herein).
  • TGF ⁇ 3 promoted pro-fibrotic phenotypes. The exacerbation of fibrosis is observed when the TGF ⁇ 3 inhibitor is used alone.
  • the TGF ⁇ 3 inhibitor when used in combination with a TGF ⁇ 1 -selective inhibitor, attenuated the anti-fibrotic effect of the TGF ⁇ 1 -selective inhibitor, as evidenced by increased collagen accumulation in the fibrotic liver.
  • a TGF ⁇ inhibitor that does not specifically target TGF ⁇ 3 is selected.
  • such inhibitor is an isoform-selective inhibitor of TGF ⁇ 1 .
  • Related methods include a method for selecting a TGF ⁇ inhibitor for use in the treatment of a fibrotic disorder in a subject, wherein the method includes the steps of: testing potency of one or more candidate inhibitors for the ability to inhibit TGF ⁇ 1 , TGF ⁇ 2 and TGF ⁇ 3, and selecting an inhibitor that inhibits TGF ⁇ 1 but does not inhibit TGF ⁇ 3, for therapeutic use.
  • Related treatment methods can further comprise a step of administering to the subject the inhibitor that inhibits TGF ⁇ 1 but does not inhibit TGF ⁇ 3 in an amount sufficient to treat the fibrotic disorder or treat a subject having or at risk of developing a fibrotic disorder and/or a cardiovascular disease.
  • the subject has pulmonary fibrosis, which may be a secondary effect of other lung diseases, such as autoimmune disorders, viral infections and bacterial infections (such as tuberculosis) of the lung.
  • the subject has pulmonary fibrosis which is a secondary effect of radiation therapy received as a treatment for lung or breast cancer.
  • the pulmonary fibrosis in the subject is idiopathic, with cigarette smoking, environmental factors (e.g.
  • subjects at risk of developing a fibrotic disorder may suffer from a metabolic disorder, such as diabetes, obesity and NASH.
  • a metabolic disorder such as diabetes, obesity and NASH.
  • the proposed exclusion of the subpopulation of patients is aimed to reduce risk of triggering, facilitating or exacerbating a pro-fibrotic effect.
  • a TGF ⁇ inhibitor for use in the treatment of a fibrotic disorder is an isoform- selective activation inhibitor of TGF ⁇ 1 (such as the novel antibodies with low k OFF disclosed herein) capable of targeting TGF ⁇ 1 -containing latent complexes in vivo.
  • the inhibitor is selected from the group consisting of: Ab37, Ab38, Ab39, Ab40, Ab41 , Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51 and Ab52.
  • the isoform-selective activation inhibitor of TGF ⁇ 1 is Ab2, Ab46, Ab50, or derivatives thereof.
  • the isoform-selective activation inhibitor of TGF ⁇ 1 is Ab46 or an engineered molecule comprising an antigen-binding fragment thereof.
  • the isoform-selective activation inhibitor of TGF ⁇ 1 is Ab2, Ab46, Ab50, or derivatives thereof.
  • the isoform-selective activation inhibitor of TGF ⁇ 1 is Ab46 or an engineered molecule comprising an antigen-binding fragment thereof.
  • the antibody of the disclosure is aimed to target the following complexes in a disease site (e.g., TME or fibrotic tissue) where it preemptively binds the latent complex thereby preventing the growth factor from being released: i) proTGF ⁇ 1 presented by GARR; ii) proTGF ⁇ 1 presented by LRRC33; iii) proTGF ⁇ 1 presented by LTBP1 ; and iv) proTGF ⁇ 1 presented by LTBP3.
  • a disease site e.g., TME or fibrotic tissue
  • complexes (i) and (ii) above are present on cell surface because both GARP and LRRC33 are transmembrane proteins capable of presenting or tethering latent proTGF ⁇ 1 on the extracellular face of the cell expressing GARP or LRRC33, whilst complexes (iii) and (iv) are components of the extracellular matrix.
  • the inhibitors embodied herein do away with having to complete binding with endogenous high affinity receptors for exerting inhibitory effects.
  • targeting upstream of the ligand/receptor interaction may enable more durable effects since the window of target accessibility is longer and more localized to relevant tissues than conventional inhibitors that target active, soluble growth factors only after it has been released from the latent complex.
  • ⁇ V integrins bind the RGD sequence present in TGF ⁇ 1 and TGF ⁇ 1 LAPs with high affinity (Dong, X., et al., Nat Struct Mol Biol, 2014. 21(12): p. 1091-6).
  • mice that lack both ⁇ 6 and ⁇ 8 integrins recapitulate all essential phenotypes of TGF ⁇ 1 and TGF ⁇ 3 knockout mice, including multiorgan inflammation and cleft palate, confirming the essential role of these two integrins for TGF ⁇ 1/3 activation in development and homeostasis (Aluwihare, P., et al. , J Cell Sci, 2009. 122(Pt 2): p. 227-32).
  • latent TGF ⁇ 1 illuminates how integrins enable release of active TGF ⁇ 1 from the latent complex: the covalent link of latent TGF ⁇ 1 to its presenting molecule anchors latent TGF ⁇ 1 , either to the ECM through LTBPs, or to the cytoskeleton through GARP or LRRC33. Integrin binding to the RGD sequence results in a force-dependent change in the structure of LAP, allowing active TGF ⁇ 1 to be released and bind nearby receptors (Shi, M., et al., Nature, 2011. 474(7351 ): p. 343-9). The importance of integrin-dependent TGF ⁇ 1 activation in disease has also been well validated.
  • a small molecular inhibitor of ⁇ V ⁇ 1 protects against bleomycin-induced lung fibrosis and carbon tetrachloride-induced liver fibrosis (Reed, N.L, et al., Sci Transl Med, 2015. 7(288): p. 288ra79), and ⁇ v ⁇ 6 blockade with an antibody or loss of integrin ⁇ 6 expression suppresses bleomycin-induced lung fibrosis and radiation-induced fibrosis (Munger, J.S., et al. , Cell, 1999. 96(3): p. 319-28); Horan, G.S., et al., Am J Respir Crit Care Med, 2008. 177(1): p. 56-65).
  • thrombospondin- 1 In addition to integrins, other mechanisms of TGF ⁇ 1 activation have been implicated, including thrombospondin- 1 and activation by proteases such as thrombin, Plasmin, matrix metalloproteinases (MMPs, e.g., MMP2, MMP9 and MMP12), cathepsin D and kallikrein. Knockout of thrombospondin- 1 recapitulates some aspects of the TGF ⁇ 1-/- phenotype in some tissues, but is not protective in bleomycin-induced lung fibrosis, known to be TGF ⁇ -dependent (Ezzie, M.E., et al., Am J Respir Cell Mol Biol, 2011. 44(4): p. 556-61).
  • Various antibodies of the present disclosure work by preventing the step of TGF ⁇ 1 activation.
  • such inhibitors can inhibit integrin-dependent (e.g., mechanical or force-driven) activation of TGF ⁇ 1.
  • such inhibitors can inhibit protease-dependent or protease-induced activation of TGF ⁇ 1.
  • the latter includes inhibitors that inhibit the TGF ⁇ 1 activation step in an integrin-independent manner.
  • such inhibitors can inhibit TGF ⁇ 1 activation irrespective of the mode of activation, e.g., inhibit both integrin-dependent activation and protease-dependent activation of TGF ⁇ 1.
  • Non-limiting examples of proteases which may activate TGF ⁇ 1 include serine proteases, such as Kallikreins, Chemotrypsin, Trypsin, Elastases, Plasmin, thrombin, as well as zinc metalloproteases (MMP family) such as MMP-2, MMP-9, MMP-12, MMP-13 and ADAM proteases (e.g., ADAM10 and ADAM17).
  • Kallikreins include plasma-Kallikreins and tissue Kallikreins, such as KLK1 , KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11 , KLK12, KLK13, KLK14 and KLK15.
  • inhibitors of the present disclosure prevent release or dissociation of active (mature) TGF ⁇ 1 growth factor from the latent complex.
  • the antibodies according to the present disclosure may induce internalization of the complex comprising proTGF ⁇ 1 bound to LRRC33 or GARR on cell surface.
  • the antibodies are inhibitors of cell-associated TGF ⁇ 1 (e.g., GARP-presented proTGF ⁇ 1 and LRRC33-presented proTGF ⁇ 1).
  • the disclosure includes antibodies or fragments thereof that specifically bind such complex (e.g., GARP-pro/latent TGF ⁇ 1 and LRRC33-pro/latent TGF ⁇ 1), thereby triggering internalization of the complex (e.g., endocytosis). This mode of action causes removal or depletion of the inactive TGF ⁇ 1 complexes from the cell surface (e.g., Treg, macrophages, MDSCs, etc.), hence reducing latent TGF ⁇ 1 available for activation.
  • the inactive TGF ⁇ 1 complexes e.g., Treg, macrophages, MDSCs, etc.
  • TGF ⁇ activities e.g., TGF ⁇ 1 activities
  • TGF ⁇ 1 activities 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.
  • cancers 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. More particular examples of such 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
  • 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 (
  • TGF ⁇ 1 -positive 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. Typically, 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.
  • Cancers that may be treated according to the present disclosure are TGF ⁇ 1 -positive and 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.
  • 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.
  • TGF ⁇ activation can be triggered by ROS (Jobling el al., 2006. Radial Res. 166: 839-848).
  • a TGF ⁇ 1 inhibitors can be used to block TGF ⁇ activation by ROS.
  • 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, may be treated with an isoform-selective inhibitor of TGF ⁇ 1 in accordance with the present disclosure.
  • TGF ⁇ (e.g., TGF ⁇ 1) may be either growth promoting or growth inhibitory.
  • TGF ⁇ e.g., TGF ⁇ 1
  • 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.
  • SMAB4- null pancreatic cancers there are SMAB4- 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
  • 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 into AML; AML and myelodysplastic syndrome (MDS), therapy- related, which category includes patients who have had prior chemotherapy and/or radiation and subsequently develop A
  • 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, 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.
  • 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 chondros
  • 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).
  • cancer or cancer-related conditions 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).
  • Tregs 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.
  • the number of Tregs is markedly increased. While 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.
  • Tregs can suppress the proliferation of effector T cells.
  • Tregs exert contact-dependent inhibition of immune cells (e.g., naive CD4+ T cells) through the production of TGF ⁇ 1 . To combat a tumor, therefore, it is advantageous to inhibit Tregs so sufficient effector T cells can be available to exert anti-tumor effects.
  • TGF ⁇ activation especially TGF ⁇ 1 activation
  • Bone marrow-derived monocytes e.g., CD11b+
  • tumor-derived cytokines/chemokines such as CCL2, CCL3 and CCL4
  • monocytes undergo differentiation and polarization to acquire pro-cancer phenotype (e.g., M2-biased or M2-like macrophages, TAMs).
  • 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
  • a majority of TAMs in many tumors are M2-biased.
  • M2c and M2d subtypes, but not M1 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.
  • 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.
  • LRRC33 As disclosed herein, a majority of tumor-infiltrating M2 macrophages and MDSCs express cell-surface LRRC33 and/or LRRC33-proTGF ⁇ 1 complex. Interestingly, cell- surface expression of LRRC33 (or LRRC33-proTGF ⁇ 1 complex) appears to be highly regulated.
  • the TGF ⁇ inhibitors described herein, e.g., Ab6 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.
  • 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). At the disease loci, this may reduce the availability of activatable latent LRRC33-proTGF ⁇ 1 levels.
  • 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. Phenotypically, these are immunosuppressive cells, contributing to the immunosuppressive tumor microenvironment, which is at least in part mediated by the TGF ⁇ 1 pathway. Given that many tumors are enriched with these cells, the antibodies that are capable of targeting multiple arms of TGF ⁇ 1 function, such as those described herein, should provide a particular functional advantage.
  • 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).
  • 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, NSCL, ovarian cancer, pancreatic cancer, and renal cell carcinoma.
  • Elevated levels of MDSCs may be detected in biological samples such as peripheral blood mononuclear cell (PBMC) and tissue samples (e.g., tumor biopsy).
  • 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 CD 14+ cells, percent (%) of CD45+ cells; percent (%) of mononuclear cells, percent (%) of total cells, percent (%) of CD 11 b+ 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.
  • tumors effectively penetrated by effector T cells (e.g., CD8+ T cells) following the treatment with a combination of a checkpoint inhibitor and a context-independent TGF ⁇ 1 inhibitor.
  • 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.
  • F4/80-positive macrophages extensive infiltration/expansion of the tumor by F4/80-positive macrophages is observed. This may be indicative of M1 (anti-tumor) macrophages clearing cancer cell debris generated by cytotoxic cells and is presumably a direct consequence of TGF ⁇ 1 inhibition.
  • these tumor-infiltrating macrophages are identified predominantly as non-M2 macrophages for their lack of CD163 expression, indicating that circulating monocytes are recruited to the tumor site upon checkpoint inhibitor and TGF ⁇ 1 inhibitor treatment and differentiate into M1 macrophages, and this observation is accompanied by a marked influx of CD8+ T cells into the tumor site.
  • the TGF ⁇ 1 inhibitors of the present disclosure may be used to increase non-M2 macrophages associated with tumor.
  • CBT checkpoint blockade therapy
  • urothelial cancer and melanoma tumors have 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 ⁇ may be a primary player in creating and/or maintaining immunosuppression in disease tissues, including the immune-excluded tumor environment. Therefore, TGF ⁇ inhibition may unblock the immunosuppression and enable effector T cells (particularly cytotoxic CD8+ T cells) to access and kill target cancer cells. In addition to tumor infiltration, TGF ⁇ inhibition may also promote CD8+ T cell expansion. Such expansion may occur in the lymph nodes and/or in the tumor (intratumorally). While the exact mechanism underlining this process has yet to be elucidated, it is contemplated that immunosuppression is at least in part mediated by immune cell-associated TGF ⁇ 1 activation involving regulatory T cells and activated macrophages.
  • Treg a regulatory (immunosuppressive) phenotype
  • 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).
  • LRRC33 which is a presenting molecule for TGF ⁇ 1
  • TAMs tumor-associated macrophages
  • a number of solid tumors are characterized by having tumor stroma enriched with myofibroblasts or myofibroblast-like cells. These cells produce collagenous matrix that surrounds or encases the tumor (such as desmoplasia), which at least in part may be caused by overactive TGF ⁇ 1 signaling. It is contemplated that the 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.
  • TGF ⁇ activation such as TGF ⁇ 1 inhibition
  • 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.
  • the present disclosure teaches that selective inhibition of 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.
  • additional isoform such as TGF ⁇ 3.
  • TGF ⁇ 3 may include certain types of carcinoma, such as breast cancer.
  • suitable phenotypes of human tumors include: i) a subsets) 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
  • ii evidence of immune exclusion
  • iii) evidence of 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, budigalimab and pembrolizumab
  • PD-1 antibodies 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.
  • T cells e.g., T cell depletion
  • CD4 and/or CD8 T cells may at least in part underline the observed anti-PD-1 resistance in certain patient populations.
  • 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 ).
  • 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.
  • 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. The 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 Biologies, 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.
  • the isoform-selective activation inhibitor of TGF ⁇ 1 is Ab46, Ab50, a derivative thereof, or an engineered molecule comprising an antigen-binding fragment thereof.
  • 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.
  • 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.
  • the isoform-selective activation inhibitor of TGF ⁇ 1 is Ab46, Ab50, a derivative thereof, or an engineered molecule comprising an antigen-binding fragment thereof.
  • 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.
  • the LRRC33 axis may play a crucial role in recruitment of circulating monocytes to the tumor microenvironment, subsequent differentiation into tumor- associated macrophages (TAMs), infiltration into the tumor tissue and exacerbation of the disease.
  • TAMs tumor- associated macrophages
  • 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
  • Evidence suggest that 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.
  • MDSCs may induce differentiation of Tregs by secreting TGF ⁇ and IL-10, further adding to the immunosuppressive effects.
  • 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
  • this may further enhance (e.g., restore or potentiate) the body’s responsiveness or sensitivity to another therapy, such as cancer therapy.
  • 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.
  • checkpoint blockade therapies such as PD-1 antagonists and PD-L1 antagonists.
  • resistance to PD-1 checkpoint blockade in inflamed head and neck carcinoma (HNC) associates with expression of GM-CSF and Myeloid Derived Suppressor Cell (MDSC) markers.
  • HNC inflamed head and neck carcinoma
  • 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.
  • the invention therefore provides the use of an isoform-specific, TGF ⁇ 1 inhibitor described herein for the treatment of cancer that comprises a solid tumor.
  • Such treatment comprises administration of the isoform-specific, TGF ⁇ 1 inhibitor to a subject diagnosed with cancer that includes at least one localized tumor (solid tumor) in an amount effective to treat the cancer.
  • the isoform-selective activation inhibitor of TGF ⁇ 1 is Ab46, Ab50, a derivative thereof, or an engineered molecule comprising an antigen-binding fragment thereof.
  • the disclosure therefore provides the use of 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).
  • 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. In some embodiments, 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. In some embodiments, 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 (0X40 agonist), an anti-CD27 antibody, an anti-CD70 antibody, an anti- CD47 antibody, an anti-41 BB 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.
  • 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.
  • checkpoint inhibitory therapy include, but are not limited to: 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.
  • bladder urothelial carcinoma such as metastatic urothelial carcinoma
  • squamous cell carcinoma such as head & neck
  • kidney clear cell carcinoma such as head & neck
  • kidney papillary cell carcinoma such as liver hepatocellular carcinoma
  • lung adenocarcinoma adenocarcinoma
  • skin cutaneous melanoma skin cutaneous melanoma
  • stomach adenocarcinoma a
  • 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.
  • the isoform-selective activation inhibitor of TGF ⁇ 1 is Ab46, Ab50, a derivative thereof, or an engineered molecule comprising an antigen-binding fragment thereof.
  • 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- ⁇ -lnduced 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.
  • 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
  • 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 may alleviate symptoms and/or complications related to anemia through their hematopoiesis-promoting effects and that BMP inhibitors (antagonists) (e.g., a BMP6 inhibitor, e.g., a RGMc inhibitor) may improve iron-deficiency anemia (e.g., chemotherapy-induced anemia).
  • BMP inhibitors antagonists
  • 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.
  • BMP6 examples include anti-BMP6 antibodies (e.g., WO 2016/098079, Novartis; and, KY-1070, KyMab).
  • the BMP inhibitor is an inhibitor of a BMP6 co-receptor, such as RGMc.
  • such inhibitor may include an antibody that binds RGMa/c. (Boser 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).
  • Therapeutic Indications and/or Subjects Likely to Benefit from a Therapy Comprising a TGF ⁇ -lnhibitor
  • the current disclosure encompasses methods of treating cancer and predicting or monitoring therapeutic efficacy using a TGF ⁇ inhibitor, e.g., Ab6.
  • a 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.
  • murine syngeneic cancer models e.g., EMT-6 and 4T1
  • numerous other cancer models e.g., S91 , B16 and MBT-2
  • TGF ⁇ 1 appears to be more frequently the dominant isoform over TGF ⁇ 2/3.
  • the TGF ⁇ isoform(s) predominantly expressed under homeostatic conditions may not be the disease-associated isoform(s).
  • TGF ⁇ 1 appears to become markedly upregulated in disease conditions, such as lung fibrosis.
  • determination of relative isoform expression may be made post- treatment.
  • patients’ responsiveness e.g., clinical response/benefit
  • overexpression of the TGF ⁇ 1 isoform shown ex post facto correlates with greater responsiveness to the treatment.
  • 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. These observations suggest that the pharmacologic inhibition of 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 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 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.
  • 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).
  • the TGF ⁇ -related indication is cancer, wherein optionally the cancer comprises a solid tumor, such as locally advanced cancer and metastatic cancer, n some embodiments, the TGF ⁇ -related indication is myelofibrosis.
  • the TGF ⁇ -related indication is an immune disorder.
  • the TGF ⁇ -related indication is fibrosis.
  • 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 i) 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).
  • 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 fibrotic disorder such as organ fibrosis
  • 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.
  • 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ . e.g., selective inhibition of TGF ⁇ 1 and/or TGF ⁇ 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., referred to as TGF ⁇ 1 -dominant.
  • TGFB1 TGF ⁇ 1 expression
  • T aken together 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.
  • TGF ⁇ 1 inhibitor for promoting tumor regression, where the tumor is TGF ⁇ 1 +/TGF ⁇ 3+.
  • 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.
  • Certain tumors such as various carcinomas, may be characterized as low mutational burden tumors (MBTs). Such tumors are often poorly immunogenic and fail to elicit sufficient T cell response.
  • Cancer therapies that include chemotherapy, radiation therapy (such as a radiotherapeutic agent), cancer vaccines and/or oncolytic virus, may be helpful to elicit T cell immunity in such tumors. Therefore, TGF ⁇ 1 inhibition therapy detailed herein 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.
  • Examples of such tumors include breast cancer, ovarian cancer, and pancreatic cancer, e.g., pancreatic ductal adenocarcinoma (PDAC).
  • PDAC pancreatic ductal adenocarcinoma
  • cancers are characterized by increased alternative end-joining DNA repair.
  • cancer types are of epithelial origin, e.g., carcinomas.
  • 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, such as chemotherapy, radiation therapy cancer vaccines and oncolytic virus.
  • the present disclosure provides combination or adjunct (add-on) cancer therapy comprising a TGF ⁇ inhibitor (e.g., TGF ⁇ 1 inhibitor such as Ab6) and a genotoxic agent (e.g., chemotherapeutic agent, radiation therapy, etc.).
  • a TGF ⁇ inhibitor e.g., TGF ⁇ 1 inhibitor such as Ab6
  • a genotoxic agent e.g., chemotherapeutic agent, radiation therapy, etc.
  • a TGF ⁇ inhibitor is used in the treatment of cancer in a subject, wherein the cancer is characterized by increased alternative end-joining DNA repair or impaired double-strand break repair, and wherein the subject receives cancer therapty comprising a genotoxic agent, wherein optionally the cancer therapy comprises chemotherapy and/or radiation therapy.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1 -selective inhibitor.
  • the presend disclosure also provides a genotoxic agent for use in the treatment of cancer in a subject, wherein the cancer is characterized by increased alternative end-joining DNA repair or impaired double-strand break repair, and wherein the subject is treated with a TGF ⁇ inhibitor, wherein optionally the TGF ⁇ inhibitor is a TGF ⁇ 1 -selective inhibitor.
  • the genotoxic agent is a chemotherapeutic agent.
  • the presend disclosure also provides radiation therapy for use in the treatment of cancer in a subject, wherein the cancer is characterized by increased alternative end-joining DNA repair or impaired double-strand break repair, and wherein the subject is treated with a TGF ⁇ inhibitor, wherein optionally the TGF ⁇ inhibitor is a TGF ⁇ 1 -selective inhibitor.
  • the present disclosure further provides a TGF ⁇ inhibitor and a cancer therapy comprising chemotherapy or radiation therapy, is used in the treatment of cancer in a subject, wherein the cancer is characterized by increased alternative end-joining DNA repair or impaired double-strand break repair,
  • the subject may be naive to checkpoint inhibitor therapy.
  • the cancer may be uterine corpus endometrial carcinoma (UCEC), thyroid carcinoma (THCA), testicular germ cell tumors (TGCT), skin cutaneous melanoma (SKCM), prostate adenocarcinoma (PRAD), ovarian serous cystadenocarcinoma (OV), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), liver hepatocellular carcinoma (LIHC), kidney renal clear cell carcinoma (KIRC), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme (GMB), esophageal carcinoma (ESCA), colon adenocarcinoma (COAD), breast invasive carcinoma (BRCA), or bladder urothelial carcinoma (BLCA).
  • the subject may further receive a checkpoint inhibitor therapy.
  • the immunosuppressive tumor environment may be mediated in a TGF ⁇ 1 -dependent fashion.
  • TGF ⁇ 1 tumors that are typically immunogenic; however, T cells cannot sufficiently infiltrate, proliferate, and elicit their cytotoxic effects due to the immune-suppressed environment.
  • tumors are poorly responsive to cancer therapies such as CBTs.
  • 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. More recently, non-invasive imaging methods are being developed which will allow the detection of cells of interest (e.g., cytotoxic T cells) in vivo. See for example, imaginab.com/technology/; Tavare et al., (2014) PNAS, 111(3): 1108-1 113; Tavare et al.., (2015) J Nucl Med 56(8): 1258-1264; Rashidian etal. , (2017) J Exp. Med 214(8): 2243-2255; Beckford Vera (2016) PLoS ONE 13(3): et al.
  • cells of interest e.g., cytotoxic T cells
  • 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+). In this way, it is possible to determine whether the tumor has an immune-excluded phenotype. If the tumor is determined to have an immune-excluded phenotype, cancer therapy (such as CBT) alone may not be efficacious because the tumor lacks sufficient cytotoxic cells within the tumor environment. 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.
  • a detection moiety e.g., radiolabel
  • 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, CDS, 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrin
  • 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 , ⁇ llb ⁇ 3, or
  • a TGF ⁇ 1 inhibitor such as a TGF ⁇ 1 -selective
  • 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)).
  • 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.
  • 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.
  • 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).
  • 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. For example, 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.
  • TGF ⁇ inhibition such as TGF ⁇ 1 inhibition
  • 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits
  • 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 , ⁇ llb ⁇ 3, or ⁇ 8 ⁇ 1 integrins, and inhibits downstream activation of TGF ⁇ . e.g., selective inhibition of TGF ⁇ 1 and/or TGF ⁇ 3).
  • a TGF ⁇ 1- selective inhibitor e.g., Ab
  • 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 .
  • 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.
  • TNBC breast cancer
  • prostate cancer such as Castration resistant prostate cancer (CRPC)
  • pancreatic cancer such as pancreatic adenocarcinoma (PDAC)
  • TGF ⁇ 1 of the present disclosure can inhibit Plasmin-induced activation of TGF ⁇ 1 .
  • 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.
  • 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).
  • 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.
  • such 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 (such as biopsies) 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 such as context-independent 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.
  • LTBP1 and LTBP3 may be highly expressed in CAFs within the tumor stroma, while GARP and LRRC33 may be highly expressed by tumor-associated immune cells, such as Tregs and leukocyte infiltrate, respectively.
  • LTBP1 and LTBP3 may be highly expressed in FAFs (e.g., myofibroblasts) within the fibrotic microenvironment, while LRRC33 may be highly expressed by fibrosis-associated immune cells, such as M2 macrophages and MDSCs.
  • 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.
  • 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.
  • 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.
  • the present disclosure provides therapeutic use and related treatment methods comprising an immune checkpoint inhibitor, e.g., a PD-(L)1 antibody.
  • an immune checkpoint inhibitor e.g., a PD-(L)1 antibody.
  • 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, 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).
  • an effective amount of TGF ⁇ inhibitor is used to treat cancer (e.g., carcinoma) in a patient, wherein no checkpoint inhibitor is approved for the treatment of the cancer.
  • the TGF ⁇ inhibitor is a TGF ⁇ 1-selective inhibitor, wherein optionally the TGF ⁇ 1 -selective inhibitor is Ab6 or a variant thereof.
  • the TGF ⁇ inhibitor may be used as a monotherapy or used in conjunction with an approved cancer therapy, such as chemotherapy and radiation therapy.
  • Ab6 may be used as monotherapy to treat cancr such as ovarian cancer, colorectal cancer, and prostate cancer, in a patient.
  • the effective amount of Ab6 may be an amount sufficient to stabilize disease (SD), e.g., the observed change in tumor size is below the progressive disease (PD) and above the partial response (PR) levels according to the RECIST response evaluation criteria in solid tumors.
  • the effective amount is between 240-3000 mg per dose, administered every 2 weeks or every 3 weeks.
  • the patient has ovarian cancer and is dosed at 240 mg per dose every 3 weeks, at 800 mg per dose every 3 weeks, 1600 mg per dose every 3 weeks, or 2400 mg per dose every 3 weeks.
  • the patient is a candidate for further receiving a genotoxic therapy such as chemotherapy and radiation therapy.
  • Ab6 may be used as combination therapy or adjunct therapy to treat cancer such as renal cell carcinoma, liver cancer and oropharynx cancer, in a patient
  • the effevtive amount of Ab6 may be an amount sufficient to achieve partial response (PR) or disease stabilization (SD) according to the RECIST response evaluation criteria in solid tumors.
  • the effective amount of Ab6 is between 240-3000 mg per dose, administered every 2 weeks or every 3 weeks, in conjunction with a checkpoint inhibitor therapy, such as anti-PD-1 antibody and anti-PD-L1 antibody.
  • a checkpoint inhibitor therapy such as anti-PD-1 antibody and anti-PD-L1 antibody.
  • the patient is a CPI-naive patient (who has never received a CPI).
  • the patient has a renal cell carcinoma and is dosed at 800 mg of Ab6 every 3 weeks inconjunction with anti-PD-1 (e.g., Pembro at 200 mg every 3 weeks).
  • the poartial response (PR) may comprise 50% or greater reduction in tumor size (volume) relative to baseline.
  • the patient has a history of primary nonresponse to the checkpoint inhibitor therapy (alone or in combination with other therapy).
  • the patient may have had disease progression (DP) on prior checkpoint inhibitor therapy, such as anti-PD-(L)1.
  • 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.
  • such subjects may receive 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 , ⁇ llb ⁇ 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).
  • the therapies may be given more than about 1 minute, about
  • 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) 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 (CTLA-4),
  • 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 may be administered alone at 3000 mg once every four weeks. In certain embodiments, 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. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) 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.
  • the TGF ⁇ inhibitor 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. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) 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.
  • the TGF ⁇ inhibitor 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. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) 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.
  • the TGF ⁇ inhibitor 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. In certain embodiments, the TGF ⁇ inhibitor (e.g., Ab6) 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.
  • 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.
  • theTGF ⁇ 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 may be administered alone at 3000 mg once every two weeks. In certain embodiments, 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. In certain embodiments, 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 may be administered alone at 2400 mg once every two weeks. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, 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 with checkpoint inhibitor therapy, e.g., an anti-PD-(L)1 therapy), to a subject who is a non-responder to checkpoint inhibitor therapy, e.g., an anti-PD-(L)1 therapy).
  • 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 (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.
  • 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.
EP22733495.0A 2021-06-03 2022-06-03 Tgf-beta inhibitoren und deren therapeutische verwendung Pending EP4348260A2 (de)

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US202163202260P 2021-06-03 2021-06-03
US202163221843P 2021-07-14 2021-07-14
US202163221588P 2021-07-14 2021-07-14
US202163256927P 2021-10-18 2021-10-18
US202263298128P 2022-01-10 2022-01-10
US202263302999P 2022-01-25 2022-01-25
US202263313355P 2022-02-24 2022-02-24
US202263313386P 2022-02-24 2022-02-24
PCT/US2022/022063 WO2022204581A2 (en) 2021-03-26 2022-03-25 Tgf-beta inhibitors and use thereof
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