TGF-beta inhibitors and their use for treating resistant or unresponsive cancer in patients

Abstract

The present disclosure provides TGFβ inhibitor therapy for treating immunosuppressive conditions such as cancer, either as monotherapy or as combination / adjunctive therapy. Selection of suitable therapy and patients likely to benefit from such therapy are also disclosed, as are methods for treating cancer and methods for predicting and monitoring therapeutic response. Related compositions, methods, and therapeutic uses are also disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 488,953, filed March 7, 2023, and U.S. Provisional Application No. 63 / 590,186, filed October 13, 2023, the entire contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing The XML sequence listing attached to this application (created on February 29, 2024, named "15094-0056-00304SL.xml", size 555,151 bytes) is incorporated herein by reference in its entirety.

[0003] TECHNICAL FIELD This application relates generally to TGFβ inhibitors and their therapeutic uses, as well as related assays for diagnosing, monitoring, prognosing, and treating disorders, including cancer. [Background technology]

[0004] Transforming growth factor β1 (TGFβ1) is a member of the TGFβ superfamily of growth factors, along with two other structurally related isoforms, TGFβ2 and TGFβ3, each encoded by a separate gene. These TGFβ isoforms function as pleiotropic cytokines that regulate cell proliferation, differentiation, immunoregulation (e.g., adaptive immune response), and a variety of other biological processes in both homeostasis and disease settings. The three TGFβ isoforms signal through the same cell surface receptor and trigger similar canonical downstream signaling events, including the SMAD2 / 3 pathway.

[0005] Transforming growth factor beta-1 (TGFβ1) has been implicated in mediating the immunosuppressive phenotype of tumors, which is associated with resistance to cancer therapies such as immune checkpoint inhibitors (Martin et al., Science Translational Medicine, 2020, 12(536):eaay8456).

[0006] In immuno-oncology, the terms "immunodesert" and "immune exclusion" have been coined to describe tumors that lack cytotoxic (CD8+) T cells and tumors that suppress cytotoxic T cells in the tumor microenvironment, respectively, and "inflammatory" tumors refer to tumors infiltrated with cytotoxic T cells.

[0007] Applicant previously demonstrated that selective inhibition of TGFβ1 is sufficient to reverse tumor immunosuppression by promoting T cell infiltration into tumors, thereby rendering tumors responsive to checkpoint inhibitors. These results suggested that a lack of intratumoral CD8+ T cells may indicate patients who are likely to benefit from TGFβ1 inhibitor therapy (WO 2020 / 014460, WO 2021 / 142448, and WO 2022 / 256723). Summary of the Invention [Problem to be solved by the invention]

[0008] Despite advances, a significant proportion of cancer patients remain resistant or refractory to cancer therapy. There remains an unmet need for improved selection and treatment of cancer patients. [Means for solving the problem]

[0009] The present disclosure includes the surprising finding that, contrary to conventional wisdom that tumor-bearing or immune cell-infiltrated tumor transformation renders treatments such as checkpoint inhibitor therapy more amenable, some patients may require additional intervention, e.g., TGFβ1 inhibition may further enhance treatments such as checkpoint inhibitor therapy. For example, patients with elevated Treg levels in the TME or a high ratio of Treg:CD8+ T cells within the TME may benefit from TGFβ1 inhibition despite exhibiting an immune-infiltrating phenotype. Such patients may also exhibit platelet-rich tumors, optionally with platelets expressing nicotinamide adenine dinucleotide N-methyltransferase (NNMT). In some embodiments, tumors may contain cells undergoing or that have undergone epithelial-mesenchymal transition (EMT), which may optionally be characterized by increased expression of stem cell-like or mesenchymal markers and / or decreased expression of epithelial markers. In some embodiments, stem cell-like or mesenchymal markers include α-SMA, vimentin, N-cadherin, fibronectin, and / or TCF7. These patients who may benefit from TGFβ1 inhibition may have failed to respond or responded inadequately to previous lines of treatment, such as previous checkpoint inhibitors and / or genotoxic therapies. In some patients, increased levels of circulating MDSCs (e.g., gMDSCs) may also be detected. In some embodiments, the carcinoma is renal cell carcinoma, preferably clear cell renal carcinoma (ccRCC).

[0010] The present disclosure further provides the surprising finding that reactive oxygen species (ROS) can enhance or promote TGFβ activation. The TME is characterized by elevated levels of cancer-derived and intracellular ROS. Furthermore, cancer therapies that include genotoxic agents, such as radiation therapy and chemotherapy, can induce high levels of ROS. Therefore, TGFβ inhibitors, particularly TGFβ1 inhibitors, can provide a protective effect against ROS-induced damage. Therefore, TGFβ inhibitors can be used in cancer therapy for subjects undergoing genotoxic agent therapy (optionally, the genotoxic agent therapy is radiation therapy and / or chemotherapy). In a preferred embodiment, the TGFβ inhibitor is a TGFβ1-selective inhibitor. Most preferably, the TGFβ1-selective inhibitor is SRK-181.

[0011] The present disclosure encompasses, inter alia, the recognition that the absence of intratumoral CD8+ T cells alone is not a sufficient marker for predicting a patient population likely to benefit from TGFβ1 inhibitor therapy. This recognition is based on the observation that some cancer patients (e.g., renal cell carcinoma (RCC) patients) exhibit resistance or refractory to conventional cancer therapies, such as checkpoint inhibitor therapy, despite the infiltration of cytotoxic T cells into their tumors. The present disclosure provides, in part, a method for addressing this shortcoming in the treatment of immune-infiltrated tumors by treating patients, particularly patients with immune-infiltrated tumors, with a TGFβ1 inhibitor. The TGFβ1 inhibitor can be administered as monotherapy and / or together with additional agents in an amount effective to treat cancer. The additional agents can be administered in combination with the TGFβ1 inhibitor or as add-on / adjuvant therapy and can include checkpoint inhibitors and / or genotoxic agents, such as radiation therapy and chemotherapy. The patient may have undergone previous cancer therapy, for example, prior checkpoint inhibitor therapy or genotoxic therapy.

[0012] Previous cancer therapies include, but are not limited to, checkpoint inhibitor therapy, chemotherapy, and radiation therapy. In some embodiments, the patient has previously received multiple lines of cancer therapy aimed at treating the carcinoma. In some embodiments, the carcinoma is resistant or unresponsive to the previous cancer therapy. In some embodiments, the disease has progressed during the previous therapy. In some embodiments, the patient experienced an adverse event in response to the previous cancer therapy, which led to the discontinuation of treatment.

[0013] Examples of previous cancer therapies include, but are not limited to, the following: anti-PD-(L)1 antibodies (e.g., pembrolizumab, nivolumab, cemiplimab, atezolizumab, dostallimab, darvalumab, avelumab), anti-CTLA4 antibodies (e.g., ipilimumab, tremelimumab), tyrosine kinase inhibitors (e.g., sunitinib, cabozantinib, imatinib, gefitinib, sorafenib, thiazol-1 ... phenib, erlotinib, lapatinib, canertinib, semaxinib, vatalanib, leflunomide, etc.), phosphoinositide 3 kinase (PI3K) inhibitors, paclitaxel, carboplatin, topotecan, doxil, gemcitabine, altretamine, bevacizumab, letrozole, carboplatin / taxol, leuprorelin, carboplatin / docetaxel, bevacizumab, etc. In some embodiments, one or more of the listed cancer therapies may be continued along with the treatment disclosed herein.

[0014] In some embodiments, the cancer is renal cell carcinoma, preferably renal clear cell carcinoma (ccRCC). In some embodiments, the renal cell carcinoma contains tumor-infiltrating CD8+ T cells (e.g., a "CD8+ T cell-infiltrated tumor") that are resistant or poorly responsive to cancer therapies such as checkpoint inhibitors and genotoxic agents (e.g., radiation therapy and chemotherapy). In some embodiments, the renal cell carcinoma has an elevated concentration of Tregs. In some embodiments, the renal cell carcinoma has an elevated concentration of platelets, optionally, the platelets express nicotinamide N-methyltransferase (NNMT). In some embodiments, the renal cell carcinoma contains cells undergoing or that have undergone epithelial-mesenchymal transition (EMT), where the EMT may optionally be characterized by increased expression of stem cell-like or mesenchymal markers and / or decreased expression of epithelial markers. In some embodiments, the stem cell-like or mesenchymal markers include α-SMA, vimentin, N-cadherin, fibronectin, and / or TCF7. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is urothelial carcinoma (UC). In some embodiments, the cancer is head and neck cancer, e.g., head and neck squamous cell carcinoma (HNSCC). In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is invasive ductal carcinoma, optionally triple-negative breast cancer. In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is squamous cell skin cancer.

[0015] In some embodiments, the patient has metastasis (i.e., the primary cancer has metastasized) at the time of screening (prior to initiation of TGFβ1 inhibitor treatment). In some cases, the cancer has metastasized to multiple sites.

[0016] The TGFβ1 inhibitor can be administered to a patient in an amount effective to treat cancer. In some embodiments, a therapeutically effective amount is an amount that achieves stable disease (SD), where SD indicates no disease progression for a defined period of time, such as 16 weeks or more (e.g., 6 months, 7 months, 8 months, 9 months, 10 months or more) on / while on treatment. In some embodiments, a therapeutically effective amount is an amount that achieves a partial response (PR, e.g., a 30% or greater shrinkage of the tumor. In some embodiments, tumor shrinkage is measured by the percent change in sum of target lesion diameters (SOD) from baseline. In some embodiments, a therapeutically effective amount achieves a 50% or greater reduction in SOD from baseline. In some embodiments, the response rate, defined as the percentage of patients achieving either a complete response (CR) or PR, is 20% or greater, e.g., 25%, 30%, 35%, 40%, 45%, 50%, or greater. In some embodiments, the duration of response is at least 6 months, e.g., 6, 7, 8, 9, 10, 11, 12 months, or greater.

[0017] According to the present disclosure, TGFβ1 inhibitors include any agent that is intended to reduce the TGFβ1 signaling pathway and can reduce it. Such agents include, for example, inhibitors of TGFβ1 activators, such as integrins that bind to the RGD motif in the LAP domain of latent TGFβ1; inhibitors of TGFβ1 activation, such as antibodies that bind to latent TGFβ1 and thereby inhibit the release of growth factors from latent complexes; inhibitors of mature (soluble) TGFβ1 ligands, such as neutralizing antibodies, ligand traps incorporating the ligand-binding module of TGFβ receptors, and nucleic acid-based inhibitors, such as siRNA and antisense oligonucleotides; and TGFβ receptor antagonists, such as Alk5 inhibitors.

[0018] In some embodiments, the TGFβ1 inhibitor preferentially inhibits TGFβ1 over TGFβ2 and / or TGFβ3. In some embodiments, the TGFβ1 inhibitor preferentially inhibits TGFβ1 and TGFβ2 over TGFβ3. In preferred embodiments, the TGFβ1 inhibitor is a TGFβ1 selective inhibitor.

[0019] Non-limiting examples of TGFβ1 inhibitors that can be used in the methods disclosed herein include: SRK-181 (from Scholar Rock), RG6440 (SOF10) (from Roche / Chugai), ABBV-151 (ribmoniplimab) (from AbbVie), NIS793 (XOMA-089) (from Novartis), PLN-10195 (from Pliant), ES014 (from Elpiscience), Cotsiranib (STP705) (from Sirnaomics), Bintrafusp alpha (M7824), Dalutrafusp alpha (AGEN14423), BMS-986416 (AVID200), MK-2225 (from MERCK), PM8001 (from Biotheus), Vactosertib (from Medpacto), BCA101 (from Bicara), TU2218 (NCE401) (from TiUM), ATB-301 (from Autotelic Bio / Clinigen), AdAPT-001 (AIM-001) (from EpicentRx), CART-PSMA-TGF-bRDN (from Tmunity Therapeutics), HCW9218 (from HCW Biologics), SH3051 (from Sanhome), TST005 (from Transcenta), GS19 (GT90008) (from Kintor (Gensun)), BJ-005 (from BJ Bioscience), QLS31901 (from Qilu Pharmaceutical), TQB2858 (from Chia Tal-tianqing), Y101D (YZY Biopharma), Charis 1000 (C1K) (Ensol Biosciences), and Fresolimumab (GC1008). In some embodiments, the TGFβ1 inhibitor is SRK-181 (Scholar Rock), RG6440 (SOF10) (Roche / Chugai), ABBV-151 (ribmoniplimab) (AbbVie), or Bintrafusp alpha (M7824). In some embodiments, the TGFβ1 inhibitor is a TGFβ1 selective inhibitor such as SRK-181, RG6440 (SOF10), or ABBV-151 (ribmoniplimab).In a preferred embodiment, the TGFβ1 selective inhibitor is SRK-181.

[0020] Further examples of TGFβ1 inhibitors include antibodies and antigen-binding fragments thereof disclosed in the following publications, as well as those that compete or cross-compete for antigen binding with such antibodies (e.g., share overlapping epitopes): WO 2020 / 104460, WO 2020 / 014473, WO 2019 / 163927, WO 2021 / 039945, WO 2015 / 015003, WO 2018 / 013939, WO 2021 / 142427, WO 2016 / 161410, WO 2019 / 075090, and WO 2020 / 160291.

[0021] In various embodiments, the TGFβ1-selective inhibitor is SRK-181 (also referred to herein as Ab6) or an antibody or engineered construct comprising an antigen-binding fragment (e.g., six CDRs) of Ab6. The CDR sequences of Ab6 are shown in Table 7, and the variable regions are shown in Table 8. In some embodiments, the TGFβ1-selective inhibitor comprises the heavy chain CDRs of Ab6 comprising the amino acid sequences of SEQ ID NO: 1001 (H-CDR1), SEQ ID NO: 1002 (H-CDR2), and SEQ ID NO: 1003 (H-CDR3), and the light chain CDRs of Ab6 comprising the 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. In some embodiments, the TGFβ1-selective inhibitor comprises the heavy chain variable region of Ab6 comprising SEQ ID NO: 1007 and the light chain variable region of Ab6 comprising SEQ ID NO: 1008. In some embodiments, the TGFβ1 selective inhibitor comprises a heavy chain of Ab6 comprising SEQ ID NO: 1009 and a light chain derived from Ab6 comprising SEQ ID NO: 1011. In some embodiments, the TGFβ1 selective inhibitor comprises an IgG4 constant region.

[0022] According to the present disclosure, a patient can be treated for cancer using an effective amount of a TGFβ1-selective inhibitor, such as SRK-181. For example, SRK-181 is administered to a patient at a dose of 240 to 3000 mg per dose every two or three weeks as monotherapy or combination therapy (e.g., with a checkpoint inhibitor) to reduce or delay tumor growth. For example, the dosing regimen can be coordinated with the dosing schedule of another therapy used in the combination therapy, such as checkpoint inhibitor therapy. For example, when a TGFβ1 inhibitor is used in combination with a PD-1 antibody administered every three weeks, a Q3W dosing schedule can be conveniently selected. Similarly, when used in combination with a PD-L1 antibody therapy administered every two weeks, a Q2W dosing schedule can be selected. In some embodiments, an effective amount of a TGFβ1-selective inhibitor, such as SRK-181, is sufficient to achieve stable disease (SD). In some embodiments, an effective amount of a TGFβ1-selective inhibitor, such as SRK-181, is sufficient to achieve a partial response (PR).

[0023] In some embodiments, the cancer treated with a TGFβ1 selective inhibitor, either as monotherapy, in combination, or as an adjunctive therapy, is characterized by increased alternative end-joining DNA repair or impaired double-strand break repair.

[0024] In some embodiments, cancers treated with a TGFβ1 selective inhibitor, either as monotherapy, in combination, or as adjunctive therapy, include solid tumors that are resistant or non-responsive to checkpoint inhibitor therapy, chemotherapy, radiation therapy, or any combination thereof.

[0025] In some embodiments, the cancer treated with a TGFβ1 selective inhibitor, either as monotherapy, in combination, or as adjunctive therapy, is ovarian cancer, renal cell carcinoma, breast cancer (e.g., triple-negative breast cancer), prostate cancer, or esophageal cancer.

[0026] In various embodiments, the cancer treated with a TGFβ1 selective inhibitor, either as monotherapy, in combination, or as adjunctive therapy, can be a carcinoma, and optionally the carcinoma is a basal cell carcinoma, a squamous cell carcinoma, a transitional cell carcinoma, a renal cell carcinoma, or an adenocarcinoma. In some embodiments, the basal cell carcinoma is a basal cell carcinoma of the skin. In some embodiments, the squamous cell carcinoma (SCC) is a squamous cell carcinoma of the skin (cutaneous SCC), a lung SCC, an esophageal SCC, or a head and neck SCC. In some embodiments, the transitional cell carcinoma is a renal transitional cell carcinoma. In some embodiments, the adenocarcinoma is a breast adenocarcinoma, a colon adenocarcinoma, a lung adenocarcinoma, a pancreatic adenocarcinoma, or a prostate adenocarcinoma.

[0027] In various embodiments, the cancer treated with a TGFβ1 selective inhibitor, either as monotherapy, in combination, or as an adjuvant therapy, is: uterine endometrial carcinoma (UCEC), thyroid carcinoma (THCA), testicular germ cell tumor (TGCT), cutaneous melanoma (SKCM), prostate adenocarcinoma (PRAD), ovarian serous cystadenocarcinoma (OV), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), hepatocellular carcinoma (LIHC), renal clear cell carcinoma (KIRC), renal clear cell carcinoma (ccRCC), head and neck squamous cell carcinoma (HNSCC), glioblastoma multiforme (GMB), esophageal carcinoma (ESCA), colon adenocarcinoma (COAD), invasive breast cancer (BRCA), or bladder urothelial carcinoma (BLCA).

[0028] In some embodiments, a TGFβ1 selective inhibitor (e.g., SRK-181) is used to treat cancer in a subject who is undergoing or has undergone background therapy including checkpoint inhibitors, chemotherapy and / or radiation therapy.

[0029] In some embodiments, genotoxic therapy (eg, chemotherapy and / or radiation therapy) is used to treat cancer in a subject receiving treatment with a TGFβ1 selective inhibitor (eg, SRK-181).

[0030] In some embodiments, a TGFβ1 selective inhibitor and a genotoxic therapy are used as a combination therapy to treat cancer in a subject, where the genotoxic therapy comprises chemotherapy and / or radiation therapy.

[0031] In some embodiments, a TGFβ1-selective inhibitor is used as monotherapy to treat cancer in a subject, where optionally the TGFβ1-selective inhibitor is SRK-181 (also referred to as Ab6), an antibody comprising an antigen-binding fragment of Ab6, a variant thereof, or an engineered construct comprising same. In some embodiments, the subject has a cancer for which no checkpoint inhibitors have been approved by regulatory authorities such as the FDA, EMA, and MHLW. In some embodiments, the subject has cancer. Optionally, the cancer is basal cell carcinoma, squamous cell carcinoma, transitional cell carcinoma, renal cell carcinoma, or adenocarcinoma. In some embodiments, the basal cell carcinoma is basal cell carcinoma of the skin. In some embodiments, the squamous cell carcinoma (SCC) is cutaneous squamous cell carcinoma (cutaneous SCC), lung SCC, esophageal SCC, or head and neck SCC. In some embodiments, the transitional cell carcinoma is renal transitional cell carcinoma. In some embodiments, the adenocarcinoma is breast adenocarcinoma, colorectal adenocarcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, or prostate adenocarcinoma. In some embodiments, the subject has ovarian cancer, eg, a malignant ovarian tumor.

[0032] In various embodiments, a subject or patient (e.g., a candidate) to be administered a cancer therapy, genotoxic agent, chemotherapy, radiation therapy, TGFβ inhibitor and / or TGFβ1 selective inhibitor in accordance with the present disclosure has not been treated with checkpoint inhibitor therapy, chemotherapy and / or radiation therapy.

[0033] In various embodiments, a subject or patient (e.g., a candidate) to be administered a cancer therapy, genotoxic agent, chemotherapy, radiation therapy, TGFβ inhibitor and / or TGFβ1 selective inhibitor in accordance with the present disclosure is a non-responder to checkpoint inhibitor therapy, chemotherapy and / or radiation therapy.

[0034] The cancer therapy, genotoxic agent, chemotherapy, radiation therapy, TGFβ inhibitor and / or TGFβ1 selective inhibitor is used to treat the cancer in the subject, and the subject may further receive checkpoint inhibitor therapy, where optionally the checkpoint inhibitor therapy includes an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0035] In some embodiments, the present disclosure further provides i) improved methods of image analysis aimed at providing better characterization of cellular structures within and surrounding tumors, ii) improved methods for determining circulating TGFβ levels aimed at achieving greater accuracy, and / or iii) LRRC33 as a promising blood-based biomarker indicative of immune suppression, and / or treatments, e.g., cancer treatments, incorporating i), ii), and / or iii). Accordingly, one or more of these features may be used as part of a diagnostic and / or therapeutic regimen for a subject (e.g., patient), either as a monotherapy or in combination / adjuvant therapy to treat cancer.

[0036] The present disclosure also relates to compositions comprising TGFβ inhibitors, methods for selecting appropriate TGFβ inhibitors for treating specific patient populations, and related therapies using TGFβ inhibitors. The present disclosure provides improved, more targeted therapies and treatment methods, including improved methods for identifying potential treatment targets (e.g., patients or patient populations likely to benefit from TGFβ inhibitor therapy) and / or monitoring treatment effectiveness. Related methods, such as treatment regimens, and methods for producing such inhibitors are also included herein. The selection of a particular TGFβ inhibitor for use in therapy aims to achieve in vivo efficacy while controlling potential risks (e.g., toxicities known to be associated with pan-TGFβ inhibition).

[0037] The present disclosure encompasses, in some embodiments, methods comprising selecting and / or administering a TGFβ inhibitor that does not target TGFβ3 signaling for use in therapy. In some embodiments, the TGFβ inhibitor does not inhibit TGFβ2 signaling at a therapeutically effective dose. In some embodiments, the TGFβ inhibitor does not inhibit TGFβ3 signaling at a therapeutically effective dose. In some embodiments, the TGFβ inhibitor does not inhibit TGFβ2 signaling and TGFβ3 signaling at a therapeutically effective dose. In preferred embodiments, such inhibitors are TGFβ1 selective.

[0038] Related embodiments include methods of manufacturing the medicament, comprising selecting a TGFβ inhibitor that does not inhibit TGFβ3 and / or TGFβ2. In some embodiments, the medicament may be used in cancer therapy. In preferred embodiments, the inhibitor is selective for TGFβ1.

[0039] According to the present disclosure, selection of a TGFβ inhibitor for therapeutic use may include testing the candidate TGFβ inhibitor for immune safety. Such testing may include cytokine release assays and may also include platelet assays.

[0040] In some embodiments, candidate TGFβ inhibitors selected for large-scale production and use, e.g., in cancer therapy, do not induce cytokine release (as described herein) or platelet aggregation (as described herein). In preferred embodiments, such inhibitors are selective for TGFβ1. In some embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising a TGFβ inhibitor, the method comprising the following steps: i) selecting a TGFβ inhibitor that meets immune safety criteria characterized by: no significant cytokine release is triggered compared to a control (e.g., IgG) in an in vitro cytokine release assay and / or in vivo testing, in which serum concentrations of cytokines are measured in response to administration of the TGFβ inhibitor; and / or no significant binding to, aggregation / activation of, human platelets, wherein the TGFβ inhibitor is effective in one or more preclinical animal models at doses below the MTD or NOAEL determined in preclinical toxicity testing; ii) producing the TGFβ inhibitor, e.g., an inhibitor selected as described herein, in a culture medium (e.g., a bioreactor) with a volume of 250 L or more; and optionally, further comprising: iii) formulating the TGFβ inhibitor and an excipient into a pharmaceutical composition.

[0041] In some embodiments, the pharmaceutical compositions and / or treatment regimens disclosed herein may further comprise a checkpoint inhibitor (e.g., a cancer therapeutic, e.g., a PD-1 antibody, a PD-L1 antibody, or a CTLA-4 antibody), either as separate molecular entities administered separately, as a single formulation (e.g., a mixture), or as part of a single molecular entity, e.g., an engineered multifunctional construct that functions as both a checkpoint inhibitor and a TGFβ inhibitor. When referring to cancer therapeutics (e.g., checkpoint inhibitors) and TGFβ inhibitors in the methods and treatment regimens described herein, these components may be provided as a single molecular entity.

[0042] In various embodiments, the disclosure provided herein includes the use of circulating MDSC levels as a predictive biomarker to improve diagnosis, monitoring, patient selection, prognosis, and / or ongoing treatment of subjects receiving a TGFβ inhibitor (e.g., a TGFβ1 inhibitor, e.g., a TGFβ1-selective inhibitor such as Ab6) by monitoring circulating MDSC levels. In some embodiments, the disclosure also encompasses methods of determining therapeutic efficacy and therapeutic agents (e.g., compositions) or dosing regimens for use in subjects with cancer by measuring circulating MDSC levels. Without being bound by theory, the inventors have discovered that reversal or overcoming of the immunosuppressive phenotype, e.g., in cancers or related conditions expressing ECM dysregulation, by administration of a TGFβ inhibitor can be demonstrated by analyzing circulating MDSC levels in a sample obtained from the subject, e.g., in blood or blood components, e.g., prior to a time point where a reduction in tumor volume or other biomarkers can be used to confirm therapeutic efficacy. In some embodiments, the circulating MDSCs are g-MDSCs. In some embodiments, the circulating MDSCs are m-MDSCs. In some embodiments, the circulating MDSCs are g-MDSCs and m-MDSCs. In some embodiments, the circulating MDSCs are characterized by cell surface expression of LRRC33. The terms "circulating" and "circulating" (e.g., "circulating MDSCs" and "circulating MDSCs") may be used interchangeably.

[0043] Tumor-associated MDSC cells may contribute to TGFβ1-mediated immunosuppression in the tumor microenvironment. Previously, the present applicants have shown that MDSCs are indeed enriched in solid tumors, and that inhibition of TGFβ1 in combination with 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 / US Patent Application Publication No. 2019 / 041373). In these efficacy studies, the efficacy of such combination therapy was observed over weeks to months (e.g., 6-12 weeks) by monitoring tumor growth. While tumor biopsies can reveal the immune profile of the tumor microenvironment (TME), in addition to being invasive, information based on biopsies can be inaccurate or biased because tumor-infiltrating lymphocytes (TILs) may not be uniformly present throughout the tumor, and therefore results may vary depending on the portion of the tumor biopsied. To overcome the limitations (e.g., drawbacks) of biopsy-based analyses, the data presented herein establish a correlation between tumor-associated (e.g., intratumoral) and circulating MDSC levels, raising the possibility that MDSCs measured in blood samples (e.g., whole blood or blood components, e.g., PBMCs) may serve as a surrogate for more accurately predicting patient populations likely to benefit from specific treatment regimens. MDSCs can be measured in blood samples by flow cytometry. Furthermore, it has been suggested that the degree of tumor burden (e.g., tumor size) correlates with the relative levels of circulating MDSCs in tumor-bearing subjects. Therefore, monitoring circulating MDSC levels in treated subjects can assess response to treatment (e.g., therapeutic efficacy) earlier than conventional methods, without the need for painful biopsies. Furthermore, more recent findings presented herein identify LRRC33 as a novel cell surface marker for circulating MDSCs (e.g., blood samples). This observation suggests the potential use of LRRC33 as a blood-based predictive biomarker.

[0044] In various embodiments, the inventors have identified circulating MDSCs, particularly gMDSCs, as an early biomarker for predicting the efficacy of combination therapy including a TGFβ inhibitor. The data disclosed herein demonstrate that following TGFβ inhibitor treatment, there is a significant decrease in circulating MDSC levels, e.g., as measured in blood or blood components, which can be detected well before an anti-tumor efficacy outcome is readily achieved, in some cases shortening the timeline by several weeks. Accordingly, the present disclosure provides for the use of circulating MDSCs as a predictive biomarker of patient responsiveness to cancer therapy, e.g., combination therapy. In related aspects of the present disclosure provided herein, the level of circulating MDSC cells can be determined within 1 to 10 weeks, e.g., within 3 to 6 weeks, after administration of a dose of a TGFβ inhibitor, optionally within 3 weeks or about 3 weeks after administration of a dose of a TGFβ inhibitor. In some embodiments, the level of circulating MDSC cells can be determined within 2 weeks after administration of a dose of a TGFβ inhibitor. In some embodiments, the level of circulating MDSC cells can be determined about 10 days after administration of a dose of a TGFβ inhibitor.

[0045] Cancer immunotherapy can harness or enhance the body's immunity to combat cancer. Without being bound by theory, it is believed that low levels of circulating MDSCs in cancer-affected subjects indicate that the body has maintained 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. Therefore, reduced levels of circulating MDSCs, particularly gMDSCs, after TGFβ inhibitor treatment indicate the pharmacodynamic effects of TGFβ inhibition (e.g., TGFβ1 inhibition) and can serve as an early predictive biomarker of therapeutic efficacy when treated with cancer therapies such as checkpoint inhibitors.

[0046] Advantageously, a patient's potential responsiveness to cancer immunotherapy can be assessed by measuring circulating MDSCs (e.g., in blood or blood components) as an indicator of TGFβ (e.g., TGFβ1)-mediated immunosuppression. In some embodiments, 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 / - In some embodiments, the circulating MDSCs are G-MDSCs.

[0047] When a cancer patient receives a combination therapy including a cancer therapy (such as a checkpoint inhibitor) and a TGFβ inhibitor that is not selective for TGFβ1 (a non-selective TGFβ inhibitor), there may be a higher risk of toxicity. To mitigate or manage such risks, the non-selective TGFβ inhibitor may be administered less frequently or intermittently, for example, on an "as needed" basis. For example, circulating MDSC levels may be monitored periodically to determine whether the effect of overcoming immunosuppression is sufficiently maintained to ensure the antitumor effect of the cancer therapy. If MDSC levels increase during the course of cancer treatment, this may indicate that the patient may benefit from additional doses of the TGFβ inhibitor. Such an approach may help reduce unnecessary risks and adverse events associated with overexposure to TGFβ inhibitors, particularly non-TGFβ1-selective inhibitors. In some embodiments, the TGFβ inhibitor targets TGFβ1 / 2 signaling. In some embodiments, the TGFβ inhibitor targets TGFβ1 / 3 signaling. In some embodiments, the TGFβ inhibitor targets TGFβ1 / 2 / 3 signaling. In some embodiments, the TGFβ inhibitor selectively targets TGFβ1 signaling. In some embodiments, a second TGFβ1-selective inhibitor is used to further reduce the frequency of exposure to the non-TGFβ1-selective inhibitor.

[0048] In some embodiments, methods of treating cancer are disclosed herein (and described herein in relation to compositions for treating or using in treating cancer). Additionally, methods of predicting, determining, or monitoring the efficacy of treatment in a subject with cancer are disclosed, e.g., methods of monitoring a patient's responsiveness to treatment and / or determining continued treatment based on monitored parameters. In some embodiments, the cancer is an immune-exclusion cancer and / or a myeloproliferative disorder, and the myeloproliferative disorder may be myelofibrosis. The cancer may have an immunosuppressive phenotype. In some embodiments, the cancer has an immune-exclusion, immunosuppressive phenotype. In some embodiments, the cancer has an immune-desert, immunosuppressive phenotype. In other specific embodiments, the cancer is not an immune-exclusion or immune-desert cancer. In certain embodiments, the cancer is an immune-infiltrative cancer. In certain embodiments, the cancer has an immune-infiltrative, immunosuppressive phenotype. In some cases, a cancer with an immune-exclusion phenotype comprises <5% CD8+ cells in the tumor and ≥5% CD8+ cells in the margin and / or stroma. In some embodiments, a cancer with an immune desert phenotype contains <5% CD8+ cells in all tumor compartments. In some embodiments, a cancer exhibiting an immune infiltration phenotype contains ≥5% CD8+ cells in the tumor. In some embodiments, a cancer has an immune infiltration phenotype, but the infiltrated CD8+ cells have reduced cytotoxic function, for example, the CD8+ cells express reduced levels of cytotoxic enzymes such as perforin and / or granzyme B. In some embodiments, the cancer is resistant or refractory to checkpoint inhibitor therapy, such as anti-PD(L)1 therapy. In some embodiments, the cancer is an immune-exclusion cancer and is resistant or refractory to checkpoint inhibitor therapy, such as anti-PD(L)1 therapy. Alternatively, in some embodiments, the cancer is an immune infiltration cancer and is resistant or refractory to checkpoint inhibitor therapy, such as anti-PD(L)1 therapy. In some embodiments, the cancer is a TGFβ1-positive cancer. A TGFβ1-positive cancer may co-express TGFβ1, TGFβ2, and / or TGFβ3. The TGFβ1 positive cancer may be a TGFβ1 dominant tumor. The TGFβ1 positive cancer may be a TGFβ1 dominant tumor and may co-express TGFβ1, TGFβ2 and / or TGFβ3.For example, a TGFβ1-positive cancer may be a TGFβ1-dominant tumor and may co-express TGFβ1 and TGFβ2. As another example, a TGFβ1-positive cancer may be a TGFβ1-dominant tumor and may co-express TGFβ1 and TGFβ3. Such cancers include advanced cancers, such as cancers with metastatic cancers (e.g., metastatic solid tumors) and locally advanced tumors (e.g., locally advanced solid tumors). In some embodiments, the treatment comprises administering to the subject a TGFβ inhibitor in an amount sufficient to reduce circulating MDSC levels, particularly circulating gMDSC levels. The circulating MDSC levels are reduced compared to the circulating MDSC levels before treatment with the TGFβ inhibitor. In some embodiments, the TGFβ inhibitor is a TGFβ1-selective inhibitor.

[0049] In some embodiments, the cancer is a solid tumor, e.g., an advanced solid tumor, and the solid tumor may be resistant or refractory to checkpoint inhibitor therapy, such as anti-PD(L)1 therapy. For example, the cancer may be a carcinoma. In some embodiments, the carcinoma comprises cells that have undergone epithelial-mesenchymal transition (EMT). The cancer may additionally or alternatively be non-small cell lung cancer (NSCLC), urothelial carcinoma, melanoma, renal cell carcinoma (e.g., renal clear cell carcinoma (ccRCC)), or head and neck cancer. The cancer may be or suspected to be an immune-exclusion cancer, and optionally have or suspected to have an immunosuppressive phenotype. In some cases, such as when the cancer is ccRCC, the cancer may be or suspected to be an immune-infiltrative cancer, and optionally have or suspected to have an immunosuppressive phenotype. If the cancer is renal cell carcinoma, particularly ccRCC, the cancer may contain cells that have undergone epithelial-mesenchymal transition (EMT).

[0050] In some embodiments, the cancer is a solid tumor, e.g., an advanced solid tumor, and the solid tumor may be resistant or refractory to checkpoint inhibitor therapy, such as anti-PD(L)1 therapy, and the patient is administered a TGFβ1 inhibitor, such as a TGFβ1-selective inhibitor, in combination with checkpoint inhibitor therapy, such as a PD-1 antagonist, a PDL1 antagonist, or a CTLA4 antagonist. For example, the cancer may be non-small cell lung cancer (NSCLC), urothelial carcinoma, melanoma, clear cell renal carcinoma (ccRCC), or head and neck cancer. The cancer may be or be suspected of being an immune-exclusion cancer, and optionally may have or be suspected of having an immunosuppressive phenotype. In some cases, for example, when the cancer is ccRCC, the patient is administered a TGFβ1 inhibitor, such as a TGFβ1-selective inhibitor, in combination with a PD-1 antagonist, a PDL1 antagonist, or a CTLA4 antagonist. The checkpoint inhibitor therapy can be an anti-PD-1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody. In some cases, for example, when the cancer is ccRCC, the patient is administered a TGFβ1 inhibitor, such as a TGFβ1-selective inhibitor, in combination with a CTLA4 antagonist (e.g., an anti-CTLA4 antibody).

[0051] In some embodiments, the subject has previously received at least one line of therapy (e.g., at least two, three, four, or five prior lines of therapy). The prior lines of therapy may include checkpoint inhibitor therapy (e.g., a PD-1 antagonist, a PD-L1 antagonist, and / or a CTLA4 antagonist), chemotherapy, and / or radiation therapy. In some embodiments, the subject has previously received one or more lines of therapy, where at least one of the prior lines of therapy is a checkpoint inhibitor therapy (e.g., a PD-1 antagonist, a PD-L1 antagonist, and / or a CTLA4 antagonist). In some embodiments, the subject has previously received at least one, two, three, four, or five lines of therapy, where at least one of the prior lines of therapy is a checkpoint inhibitor therapy (e.g., a PD-1 antagonist, a PD-L1 antagonist, and / or a CTLA4 antagonist). In some embodiments, the subject has previously received at least two lines of therapy. In some embodiments, the subject has received at least three prior lines of therapy, hi some embodiments, the subject has received at least four prior lines of therapy.

[0052] In some embodiments, the present disclosure includes a method of predicting or determining therapeutic efficacy in a subject with cancer, comprising determining circulating MDSC levels (e.g., circulating gMDSC levels) in the subject prior to administering a TGFβ inhibitor (alone or in combination with a cancer therapy), administering a therapeutically effective amount of a TGFβ inhibitor (alone or in combination with a cancer therapy) to the subject, and measuring circulating MDSC levels in the subject after administration, wherein a decrease in circulating MDSC levels after administration compared to circulating MDSC levels before administration predicts therapeutic efficacy. In one embodiment, circulating MDSC levels are determined by measuring MDSCs in a blood sample by flow cytometry.

[0053] In some embodiments, the disclosure includes a method of determining the therapeutic efficacy of a cancer therapy in a subject, the treatment comprising administering to the subject a combination therapy comprising a predetermined dose of a TGFβ inhibitor and a cancer therapy, the method comprising: (i) measuring circulating MDSC levels (e.g., circulating gMDSC levels) in a sample taken from the subject before administration of the TGFβ inhibitor, (ii) measuring circulating MDSC levels in a sample taken from the subject after administration of the TGFβ inhibitor, and (iii) determining whether the level measured in step (ii) is reduced compared to the level measured in step (i), wherein such a reduction indicates a therapeutic efficacy of the cancer therapy. In some embodiments, the doses of the TGFβ inhibitor and the cancer therapy in the combination therapy are for concurrent (e.g., simultaneous), separate, or sequential administration. In some embodiments, the TGFβ inhibitor is a TGFβ1 selective inhibitor, such as Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, and Ab46. In a preferred embodiment, the TGFβ inhibitor is Ab6.

[0054] In some embodiments, the present disclosure includes a method of treating cancer in a subject, comprising measuring circulating MDSC levels in the subject (e.g., circulating gMDSC levels) before administration of a TGFβ inhibitor, administering a first therapeutically effective dose of a TGFβ inhibitor to the subject, measuring circulating MDSC levels in the subject after administration of the TGFβ inhibitor, and, if the circulating MDSC levels measured after administration of the first therapeutically effective dose of the TGFβ inhibitor are reduced compared to the circulating MDSC levels measured before administration of the first therapeutically effective dose of the TGFβ inhibitor, administering a second therapeutically effective dose of a TGFβ1 inhibitor or combination therapy to the subject. In some embodiments, a combination therapy including a second cancer therapy (e.g., a checkpoint inhibitor therapy) is administered concurrently, sequentially, or simultaneously with the first therapeutically effective dose of the TGFβ inhibitor, and then continuing the combination therapy if the circulating MDSC levels measured after administration of the first therapeutically effective dose of the combination therapy are reduced compared to the circulating MDSC levels measured before administration of the first therapeutically effective dose.

[0055] In some embodiments, the present disclosure includes a cancer therapy for use in treating cancer in a subject, wherein the subject has received a dose of a TGFβ inhibitor, and it has been determined that the level of circulating MDSCs (e.g., circulating gMDSCs) measured in the subject after administration of the TGFβ inhibitor is reduced compared to the level of circulating MDSCs measured in the subject before administration of the dose of the TGFβ inhibitor. In some embodiments, the TGFβ inhibitor is a TGFβ1-selective inhibitor, such as Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, and Ab46. In a preferred embodiment, the TGFβ inhibitor is Ab6.

[0056] In some embodiments, the present disclosure encompasses a combination therapy comprising a predetermined dose of a TGFβ inhibitor and a cancer therapeutic agent for use in treating cancer, wherein the treatment comprises concurrent (e.g., simultaneous), separate, or sequential administration of the predetermined doses of the TGFβ inhibitor and the cancer therapeutic agent to a subject, wherein the level of circulating MDSCs (e.g., circulating gMDSCs) measured in the subject after administration of the TGFβ inhibitor is confirmed to be reduced compared to the level of circulating MDSCs measured in the subject before administration of the dose of the TGFβ inhibitor. In some embodiments, the TGFβ inhibitor is a TGFβ1-selective inhibitor, such as Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, and Ab46. In a preferred embodiment, the TGFβ inhibitor is Ab6.

[0057] In some embodiments, the present disclosure includes a TGFβ inhibitor for use in treating cancer in a subject, wherein the subject has received at least a first dose of a TGFβ inhibitor, and the treatment comprises administering an additional dose of the TGFβ inhibitor only if the circulating MDSC level (e.g., circulating gMDSC level) in the subject measured after administration of at least a first dose of the TGFβ inhibitor is reduced compared to the circulating MDSC level measured in the subject before administration of the given dose of the TGFβ inhibitor. In some embodiments, the TGFβ inhibitor is a TGFβ1-selective inhibitor, such as Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, and Ab46. In a preferred embodiment, the TGFβ inhibitor is Ab6.

[0058] In some embodiments, the present disclosure includes a TGFβ inhibitor used to treat cancer in a subject, wherein the subject is administered a dose of the TGFβ inhibitor, and the TGFβ inhibitor reduces or reverses immunosuppression in the cancer, wherein the reduced or reversed immunosuppression is determined by a decrease in circulating MDSC levels (e.g., circulating gMDSC levels) in the subject measured after administration of the TGFβ inhibitor compared to the circulating MDSC levels measured in the subject before administration of the dose of the TGFβ inhibitor. In some embodiments, the TGFβ inhibitor is a TGFβ1-selective inhibitor, such as Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, and Ab46. In a preferred embodiment, the TGFβ inhibitor is Ab6.

[0059] In some embodiments, the present disclosure includes a method of treating advanced cancer in a human subject, the method comprising selecting a subject with an advanced cancer (including a locally advanced tumor and / or a metastatic cancer) that exhibits primary resistance to checkpoint inhibitor therapy, administering a TGFβ inhibitor, and administering checkpoint inhibitor therapy to the subject. In the methods and compositions used for cancer treatment described herein, the cancer may be an advanced cancer. The cancer may include a locally advanced tumor and / or a metastatic cancer that exhibits primary resistance to checkpoint inhibitor therapy. The cancer therapy may include checkpoint inhibitor therapy. The subject may be a human subject. In some embodiments, the subject has elevated circulating MDSC levels (e.g., circulating gMDSC levels), e.g., compared to a healthy control subject or compared to a control subject with a cancer that is not resistant (e.g., responsive) to checkpoint inhibitor therapy. In some embodiments, a subject has elevated circulating MDSC levels (e.g., circulating gMDSC levels) when circulating MDSCs (e.g., circulating gMDSCs) are detectable in a sample, e.g., greater than 1% of the white blood cell / PBMC component or greater than 0.1% of whole blood. In some embodiments, a subject has elevated circulating MDSC levels (e.g., circulating gMDSC levels) when circulating MDSCs (e.g., circulating gMDSCs) represent 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the white blood cell / PBMC component of a blood sample. In some embodiments, a subject has elevated circulating MDSC levels (e.g., circulating gMDSC levels) when circulating MDSCs (e.g., circulating gMDSCs) represent greater than 10% of the white blood cell / PBMC component of a blood sample. In some embodiments, the treatment reduces circulating MDSC levels, hi some embodiments, continued treatment is contingent on an observed reduction in circulating MDSCs.

[0060] In some embodiments, the present disclosure encompasses methods of treating, predicting, determining, and / or monitoring the therapeutic efficacy of a cancer treatment in a subject administered a TGFβ inhibitor, alone or in combination with other cancer therapies (e.g., a checkpoint inhibitor), the method comprising determining the level of tumor-associated immune cells (e.g., CD8+ T cells and tumor-associated macrophages) in the subject prior to administration of the treatment, administering the treatment to the subject, and determining the level of tumor-associated immune cells in the subject after administration of the treatment, wherein a change in the level of one or more tumor-associated immune cell populations after administration of the inhibitor compared to the level of tumor-associated immune cells prior to administration indicates therapeutic efficacy. In some embodiments, the treatment results in a change in the level of tumor-associated immune cells. In some embodiments, continued treatment is contingent on an observed change in tumor-associated immune cells. In some embodiments, tumor-associated immune cell levels are monitored in combination with monitoring circulating MDSC levels (e.g., circulating gMDSC levels), and therapeutic efficacy and / or continued treatment is contingent on an observed change in both sets of biomarkers (e.g., a decrease in circulating MDSC levels and an increase in tumor-associated CD8+ T cells).

[0061] In some embodiments, the disclosure provides a checkpoint inhibitor and a TGFβ1 inhibitor for use in treating cancer in a subject in need thereof, wherein the treatment comprises administration of a checkpoint inhibitor and a TGFβ1 inhibitor in amounts effective to treat the cancer, optionally wherein the checkpoint inhibitor is a PD-(L)1 inhibitor, and further optionally wherein the PD-(L)1 inhibitor is budigalimab; optionally wherein the TGFβ1 inhibitor is a TGFβ1-selective inhibitor, and further optionally wherein the TGFβ1-selective inhibitor is SRK-181 (also referred to herein as Ab6); and optionally wherein the cancer comprises a solid tumor with an immunosuppressive phenotype.

[0062] In some embodiments, the disclosure provides a checkpoint inhibitor for use in treating cancer in a subject in need thereof, wherein the treatment comprises administering a checkpoint inhibitor to a subject treated with a TGFβ1 inhibitor in an amount effective to treat the cancer, wherein optionally, the checkpoint inhibitor is a PD-(L)1 inhibitor, and further optionally, the PD-(L)1 inhibitor is budigalimab; optionally, the TGFβ1 inhibitor is a TGFβ1-selective inhibitor, and further optionally, the TGFβ1-selective inhibitor is SRK-181 (also referred to herein as Ab6); and optionally, the cancer comprises a solid tumor with an immunosuppressive phenotype.

[0063] In some embodiments, the disclosure provides a TGFβ1 inhibitor for use in treating cancer in a subject in need thereof, wherein the treatment comprises administering a TGFβ1 inhibitor to a subject treated with a checkpoint inhibitor in an amount effective to treat the cancer, wherein optionally the checkpoint inhibitor is a PD-(L)1 inhibitor, and further optionally, the PD-(L)1 inhibitor is budigalimab; optionally, the TGFβ1 inhibitor is a TGFβ1-selective inhibitor, and further optionally, the TGFβ1-selective inhibitor is SRK-181 (also referred to herein as Ab6); and optionally, the cancer comprises a solid tumor with an immunosuppressive phenotype.

[0064] In some embodiments, the present disclosure includes methods of treating cancer in a subject and predicting, determining, and / or monitoring the therapeutic efficacy of a cancer treatment in a subject. In some embodiments, the methods include measuring the level of CD8+ cells within the tumor (or within one or more tumor nests within the tumor) and the surrounding stromal and / or marginal compartments in one or more tumor samples obtained from the subject. In some embodiments, the methods include identifying the immunophenotype of the subject's cancer based on the level of CD8+ cells within the tumor or tumor nests compared to the level of CD8+ cells outside the tumor or tumor nests (e.g., the surrounding stromal and / or marginal compartments). In certain embodiments, the cancer treatment includes a TGFβ inhibitor, e.g., a TGFβ1 inhibitor, e.g., Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, or Ab46. In certain embodiments, the cancer therapy comprises Ab6. In certain embodiments, the cancer therapy comprises an immune checkpoint inhibitor. In certain embodiments, the cancer therapy comprises a TGFβ1 inhibitor (e.g., Ab6) and an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, or a CTLA-4 antibody).

[0065] In some embodiments, the present disclosure provides methods of treating, predicting, and / or monitoring therapeutic efficacy of a cancer therapy in a subject administered a TGFβ inhibitor alone or in combination with another cancer therapy (e.g., a checkpoint inhibitor). The methods include measuring the level of circulating latent TGFβ in the subject prior to administration of the therapy, administering the therapy to the subject, and measuring the level of circulating latent TGFβ in the subject after administration of the therapy, wherein a change (e.g., an increase) in the level of circulating latent TGFβ after administration of the inhibitor compared to the level of circulating latent TGFβ before administration indicates therapeutic efficacy. In some embodiments, the treatment results in a change in the level of circulating latent TGFβ. In some embodiments, continued treatment is contingent on an observed change (e.g., an increase) in circulating latent TGFβ. In some embodiments, circulating latent TGFβ is monitored in combination with monitoring circulating MDSC levels (e.g., circulating gMDSC levels) and / or tumor-associated immune cell levels. In some embodiments, therapeutic efficacy and / or continued treatment is contingent on an observed change in two or more sets of biomarkers. In various embodiments, methods and compositions disclosed herein for use in treating cancer involving determining circulating MDSC levels (and optionally assessing changes in the levels of one or more tumor-associated immune cell populations) can further include assessing levels of circulating latent TGFβ, as described herein. Also disclosed are compositions for use in treating cancer comprising a therapeutically effective dose of a TGFβ inhibitor, which is administered when a decrease in circulating MDSC levels (alone or in combination with a change in circulating latent TGFβ) is determined after administration of a previous dose of the TGFβ inhibitor. In some embodiments, the TGFβ inhibitor is a TGFβ1-selective inhibitor, e.g., Ab6. In some embodiments, continued treatment is contingent on an observed change in circulating latent TGFβ. In some embodiments, circulating latent TGFβ is monitored in combination with monitoring circulating MDSC levels and / or tumor-associated immune cell levels. In some embodiments, therapeutic efficacy and / or continued treatment is contingent on observed changes in two or more sets of biomarkers (e.g., a decrease in circulating MDSC levels and / or an increase in tumor-associated CD8+ T cells and / or a decrease in circulating latent TGFβ).

[0066] In some embodiments, the present disclosure provides methods comprising administering to a subject a therapeutically effective amount of a TGFβ inhibitor (e.g., a TGFβ1 inhibitor) that does not cause significant release of one or more cytokines selected from interferon gamma (IFNγ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNFα), interleukin 1β (IL-1β), and chemokine CC motif ligand 2 (CCL2) / monocyte chemoattractant protein 1 (MCP-1). In some embodiments, the method does not induce a significant increase in platelet binding, activation, and / or aggregation. In some embodiments, the cancer has increased circulating MDSC levels (e.g., circulating gMDSC levels), e.g., compared to healthy control subjects or compared to control subjects with cancer that is not resistant (e.g., responsive) to checkpoint inhibitor therapy. In some embodiments, a subject has increased circulating MDSC levels (e.g., circulating gMDSC levels) when circulating MDSCs (e.g., circulating gMDSCs) are detectable in a sample (e.g., greater than 1% of the white blood cell / PBMC component or greater than 0.1% of whole blood). In some embodiments, a subject has increased circulating MDSC levels (e.g., circulating gMDSCs) when circulating MDSCs (e.g., circulating gMDSCs) are greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the white blood cell / PBMC component of a blood sample. In some embodiments, a subject has increased circulating MDSC levels (e.g., circulating gMDSCs) when circulating MDSCs (e.g., circulating gMDSCs) are greater than 10% of the white blood cell / PBMC component in a blood sample. In some embodiments, treatment with a therapeutically effective amount of a TGFβ inhibitor (e.g., a TGFβ1 inhibitor) reduces the level of circulating MDSCs. In some embodiments, continued treatment is contingent on an observed reduction in circulating MDSCs.

[0067] In some embodiments, the present disclosure provides a method for determining whether a TGFβ inhibitor (e.g., a TGFβ1 inhibitor) is tolerated in a patient, comprising contacting a cell culture or fluid sample with the TGFβ inhibitor and determining whether it causes a significant release of one or more cytokines selected from interferon gamma (IFNγ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNFα), interleukin 1β (IL-1β), and chemokine CC motif ligand 2 (CCL2) / monocyte chemoattractant protein 1 (MCP-1), where significant release indicates that the TGFβ inhibitor will not be well tolerated. The method may include monitoring cytokine release in an in vitro cytokine release assay. In some embodiments, the assay is performed on peripheral blood mononuclear cells (PBMCs) or whole blood, optionally obtained from the subject prior to administering the TGFβ inhibitor therapy. In some embodiments, the disclosure encompasses a TGFβ inhibitor (e.g., a TGFβ1-selective inhibitor) for use in treating cancer by administering to a subject a predetermined dose of the TGFβ inhibitor, wherein the TGFβ inhibitor does not cause significant release of one or more cytokines selected from interferon gamma (IFNγ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNFα), interleukin 1β (IL-1β), and chemokine C-C motif ligand 2 (CCL2) / monocyte chemoattractant protein 1 (MCP-1). In some embodiments, the disclosure encompasses combination therapies including a dose of a TGFβ inhibitor (e.g., a TGFβ1 inhibitor) and a cancer therapy (e.g., a checkpoint inhibitor therapy) used to treat cancer, wherein the treatment involves simultaneous, contemporaneous, or sequential administration of the doses of the TGFβ inhibitor and the cancer therapy to a subject, and wherein the TGFβ inhibitor does not cause significant release of one or more cytokines selected from interferon gamma (IFNγ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNFα), interleukin 1β (IL-1β), and chemokine C-C motif ligand 2 (CCL2) / monocyte chemoattractant protein 1 (MCP-1).In some embodiments, the TGFβ inhibitor used to treat cancer is administered in a therapeutically effective amount sufficient to reduce circulating MDSCs (e.g., circulating gMDSCs), the level of circulating MDSCs being reduced compared to the level of circulating MDSCs before treatment with the TGFβ inhibitor, i.e., the baseline circulating MDSC level.

[0068] In some embodiments, the present disclosure provides methods for determining whether a TGFβ inhibitor (e.g., a TGFβ1 inhibitor) causes a significant increase in platelet binding, activation, and / or aggregation after exposing the sample to the TGFβ inhibitor, the method comprising measuring platelet binding, activation, and / or aggregation in a plasma or whole blood sample. In some embodiments, the present disclosure includes a TGFβ inhibitor (e.g., a TGFβ1 inhibitor) for use in treating cancer by administering to a subject a predetermined dose of the TGFβ inhibitor, wherein the TGFβ inhibitor does not cause a significant increase in platelet binding, activation, and / or aggregation. In some embodiments, the present disclosure encompasses combination therapy for treating cancer, comprising a predetermined dose of a TGFβ inhibitor (e.g., a TGFβ1 inhibitor) and a cancer therapeutic agent (e.g., checkpoint inhibitor therapy), wherein the treatment comprises contemporaneous (e.g., simultaneous), separate, or sequential administration of predetermined doses of a TGFβ inhibitor and a cancer therapeutic agent to a subject, and wherein the TGFβ inhibitor does not cause a significant increase in platelet binding, activation, and / or aggregation. In some embodiments, the TGFβ inhibitor used is administered in a therapeutically effective amount sufficient to reduce circulating MDSCs (e.g., circulating gMDSCs), the circulating MDSC levels being reduced compared to the circulating MDSC levels before treatment with the TGFβ inhibitor, i.e., the baseline circulating MDSC levels.

[0069] In various embodiments of the methods and compositions disclosed herein in which a subject's circulating MDSC levels (e.g., circulating gMDSC levels) are assessed, the subject may have cancer, e.g., a highly metastatic cancer. In some embodiments, 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, gastric cancer (e.g., gastric cancer), or thyroid cancer.

[0070] In some embodiments, the present disclosure provides methods of making a TGFβ inhibitor for treating a pair of cancers, which meet the following criteria: a) the TGFβ inhibitor is effective in one or more preclinical models; b) the TGFβ inhibitor does not cause valvular disease or epithelial hyperplasia in toxicity studies in one or more animal species at doses at least greater than the minimum effective dose; c) the TGFβ inhibitor does not induce significant cytokine release from human PBMCs or whole blood in an in vitro cytokine release assay at the minimum effective dose determined in the one or more preclinical models of (a). The method further comprises selecting a TGFβ inhibitor that satisfies one or more, e.g., all, of the following criteria: (a) the TGFβ inhibitor does not induce platelet release; (b) the TGFβ inhibitor does not induce a significant increase in platelet binding, activation, and / or aggregation at a minimally effective dose determined in one or more preclinical models of (a); and (c) the TGFβ inhibitor reduces circulating MDSCs (e.g., circulating gMDSCs) at a minimally effective dose determined in one or more preclinical models of (a), wherein the method further comprises preparing a pharmaceutical composition comprising the TGFβ inhibitor and a pharmaceutically acceptable excipient. In some embodiments, the selected TGFβ inhibitor is a TGFβ1-selective inhibitor. In some embodiments, the TGFβ inhibitor is selective for proTGFβ1 and / or latent TGFβ1.

[0071] In some embodiments, the methods of the present disclosure can be used to select and treat patients who exhibit resistance to immunotherapy (e.g., checkpoint inhibitor therapy). The patients or subjects referred to in the methods of use and compositions disclosed herein may have resistance to immunotherapy (e.g., checkpoint inhibitor therapy). Patient populations included in the present disclosure may be treatment-naive (e.g., have not previously received cancer therapy), have primary resistance (i.e., present before treatment begins), or have acquired resistance to immunotherapy (e.g., checkpoint inhibitor therapy).

[0072] In some embodiments, the disclosure encompasses a TGFβ1-selective inhibitor for use in treating cancer, the treatment comprising selecting a subject whose cancer is highly metastatic and administering an isoform-selective TGFβ1 inhibitor to the subject. In some embodiments, 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, gastric cancer (e.g., gastric carcinoma), or thyroid cancer.

[0073] In some embodiments, the present disclosure encompasses a TGFβ1 selective inhibitor for use in treating cancer in a subject, the treatment comprising selecting a subject having myelofibrosis or at risk of developing myelofibrosis, and administering to the subject an amount of a TGFβ1 selective inhibitor effective to treat the cancer.

[0074] In some embodiments, the disclosure includes methods of treating cancer, wherein the subject has previously been treated, is currently being treated, or will be treated with a TGFβ inhibitor that inhibits TGFβ3, e.g., in combination with a checkpoint inhibitor. These patients may have their dosage or treatment frequency reduced by monitoring circulating MDSC levels (e.g., circulating gMDSC levels) and administering treatment only if MDSC levels increase. The above patients may also have their dosage or treatment frequency reduced by adding one or more doses of a TGFβ1 or TGFβ1 / 2 inhibitor. In some embodiments, the patient has previously been treated with a TGFβ inhibitor that inhibits TGFβ3 in combination with a checkpoint inhibitor. In some embodiments, a TGFβ1 or TGFβ1 / 2 inhibitor is provided for use in treating cancer in a subject, wherein the subject has previously been treated, is currently being treated, or will be treated with a TGFβ inhibitor that inhibits TGFβ3, e.g., in combination with a checkpoint inhibitor. In some embodiments, the cancer is metastatic cancer, desmoplastic tumor, or myelofibrosis. In some embodiments, the TGFβ inhibitor is a TGFβ1-selective inhibitor, such as Ab6 or a variant thereof, such as Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, and Ab46. In a preferred embodiment, the TGFβ inhibitor is Ab6. In some embodiments, the TGFβ inhibitor is isoform-nonselective and inhibits TGFβ1 / 2 / 3 or TGFβ1 / 3.

[0075] In some embodiments, the disclosure encompasses an isoform-nonselective TGFβ inhibitor for the treatment of cancer, the method comprising selecting a subject who has not been diagnosed with or is not at high risk for developing a fibrotic disease (e.g., a subject who does not exhibit increased MDSC levels compared to a control sample) and administering to the subject an amount of an isoform-nonselective TGFβ inhibitor effective to treat the cancer. In some embodiments, the isoform-nonselective TGFβ inhibitor is an antibody (or agent) that inhibits TGFβ1 / 2 / 3 or TGFβ1 / 3. In some embodiments, the isoform-nonselective TGFβ inhibitor is an engineered construct comprising a TGFβ receptor ligand binding site.

[0076] In some embodiments, the present disclosure provides a TGFβ inhibitor for use in an intermittent dosing regimen for cancer immunotherapy in a patient, the intermittent dosing regimen comprising the steps of: measuring circulating MDSCs (e.g., circulating gMDSCs) in a first sample collected from the patient prior to TGFβ inhibitor treatment; administering a TGFβ inhibitor to the patient treated with a cancer therapy (which cancer therapy is optionally checkpoint inhibitor treatment); measuring circulating MDSCs in a second sample collected from the patient after TGFβ inhibitor treatment; continuing the cancer therapy if the second sample shows a decrease in circulating MDSC levels compared to the first sample; measuring circulating MDSCs in a third sample; and administering an additional dose of a TGFβ inhibitor to the patient if the third sample shows an increased level of circulating MDSC levels compared to the second sample. The TGFβ inhibitor is an isoform-nonselective inhibitor. In some embodiments, the isoform-nonselective inhibitor inhibits TGFβ1 / 2 / 3, TGFβ1 / 2, or TGFβ1 / 3. In some embodiments, the sample is a blood sample or a blood component.

[0077] In some embodiments, the present disclosure provides a TGFβ inhibitor for use in treating cancer, including solid tumors in a patient, e.g., solid tumors that are CD8+ T cell infiltrated tumors, the treatment comprising administering the TGFβ inhibitor in combination with a checkpoint inhibitor (CPI) to treat the cancer, wherein the solid tumor has an immune infiltrate phenotype and the solid tumor is resistant or refractory to CPI therapy. In some embodiments, the TGFβ inhibitor is a TGFβ1 inhibitor. In some embodiments, the solid tumor is a carcinoma. In some embodiments, the solid tumor is a cancer comprising cells that have undergone epithelial-mesenchymal transition (EMT). In some embodiments, the cancer is renal cell carcinoma (RCC), particularly renal clear cell carcinoma (ccCC). In some embodiments, the CPI is a PD-1 antagonist, a PD-L1 antagonist, or a CTLA4 antagonist.

[0078] In any of the embodiments described herein, the TGFβ inhibitor, such as a TGFβ inhibitor, used in cancer therapy can be a TGFβ1-selective inhibitor, e.g., an anti-TGFβ1 antibody described herein, or an antibody having a sequence disclosed below, e.g., Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, and Ab46. For example, the TGFβ1-selective inhibitor can be as defined in any of embodiments 1-35 of paragraph

[1034] of the present specification. The TGFβ1-selective inhibitor may be an antibody comprising the CDRs and / or heavy chain variable region (VH) and / or light chain variable region (VL) of any one of Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, or Ab46. For example, the TGFβ1-selective inhibitor may be an antibody comprising the CDRs and / or VH and / or VL of Ab6. In a preferred embodiment, the TGFβ inhibitor is Ab6.

[0079] The TGFβ1-selective inhibitor may be a GARP-selective inhibitor (e.g., a GARP-TGFβ1 complex-selective inhibitor). The TGFβ1-selective inhibitor may be capable of binding to the GARP-TGFβ1 complex and the LRRC33-TGFβ1 complex (and not be able to bind to the LTBP1-TGFβ1 complex or the LTBP3-TGFβ1 complex). Alternatively, the TGFβ1-selective inhibitor may be a context-independent TGFβ1 inhibitor capable of binding to the following pro / latent complexes (GARP-TGFβ1, LRRC33-TGFβ1, LTBP1-TGFβ1, and LTBP3-TGFβ1). In some embodiments, the TGFβ1-selective inhibitor is capable of binding to the following pro / latent complexes: GARP-TGFβ1, LRRC33-TGFβ1, LTBP1-TGFβ1, and LTBP3-TGFβ1, and inhibits the release of mature TGFβ1 growth factor from the pro / latent complex.

[0080] The TGFβ inhibitor can inhibit integrin-dependent TGFβ1 activation. For example, the TGFβ inhibitor can be a TGFβ1-selective inhibitor that inhibits integrin-dependent TGFβ1 activation. The TGFβ inhibitor can be a TGFβ1-selective inhibitor that inhibits integrin-dependent activation of TGFβ1, which is a context-independent TGFβ1 inhibitor that can bind to the following pro / latent complexes (GARP-TGFβ1, LRRC33-TGFβ1, LTBP1-TGFβ1, and LTBP3-TGFβ1).

[0081] Alternatively or additionally, the TGFβ inhibitor may inhibit protease-dependent or protease-induced TGFβ1 activation. For example, the TGFβ inhibitor may be a TGFβ1-selective inhibitor that inhibits protease-dependent or protease-induced TGFβ1 activation. The TGFβ inhibitor is a TGFβ1-selective inhibitor that inhibits protease-dependent or protease-induced TGFβ1 activation and is a context-independent TGFβ1 inhibitor that can bind to the following pro / latent complexes: GARP-TGFβ1, LRRC33-TGFβ1, LTBP1-TGFβ1, and LTBP3-TGFβ1.

[0082] In some embodiments, the TGFβ inhibitors disclosed herein are well tolerated in preclinical safety / toxicity studies at doses of up to 100, 200, or 300 mg / kg when administered once weekly for at least four weeks. Such studies can be performed in animal models known to be sensitive to TGFβ inhibition, such as rats and non-human primates. In some embodiments, the TGFβ inhibitors disclosed herein do not cause the observable toxicities associated with general TGFβ inhibition. Observable toxicities can include cardiovascular toxicity (e.g., valvular disease). Other observable toxicities include epithelial hyperplasia. Additional observable toxicities are known in the art. In some embodiments, the TGFβ inhibitors disclosed herein do not induce significant cytokine release, platelet aggregation, binding, or activation. The TGFβ inhibitors are believed to not induce significant cytokine release (e.g., as determined by the methods described herein). The TGFβ inhibitors are believed to not cause a significant increase in platelet binding, activation, and / or aggregation (e.g., as determined by the methods described herein). The TGFβ inhibitor is determined, or is believed to be determined, by the methods described herein to not induce significant cytokine release and not cause a significant increase in platelet binding, activation and / or aggregation.

[0083] In some embodiments, the TGFβ inhibitors disclosed herein achieve a sufficient therapeutic window in that the effective amount of the inhibitor, as demonstrated by in vivo efficacy testing, is sufficiently lower (e.g., at least 3-fold, at least 6-fold, or at least 10-fold lower) than the amount or concentration that causes observable toxicity. In some embodiments, the therapeutically effective amount of the inhibitor is about 1 mg / kg to about 30 mg / kg per week. In some embodiments, the therapeutically effective amount of the inhibitor is about 1 mg / kg to about 10 mg / kg administered every three weeks. In some embodiments, the therapeutically effective amount of the inhibitor is about 2 mg / kg to about 7 mg / kg administered every three weeks.

[0084] In some embodiments, the TGFβ inhibitors disclosed herein achieve a sufficient therapeutic window in that the effective amount of the inhibitor, as demonstrated by in vivo efficacy testing, is well below (e.g., at least 3-fold, at least 6-fold, or at least 10-fold) the amount or concentration that causes dose-limiting toxicity (DLT). DLT is generally defined by the occurrence of serious toxicity during treatment (e.g., during the first cycle of cancer therapy). Such toxicity is evaluated based on the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) classification and typically includes all toxicities of grade 3 or higher, but excludes grade 3 non-febrile neutropenia and alopecia. In some embodiments, DLT may also include certain a priori untreatable or irreversible grade 2 toxicities (e.g., neurotoxicity, ocular toxicity, or cardiac toxicity), prolonged grade 2 toxicities (e.g., grade 2 toxicities that persist beyond a certain period of time), and / or prolonged DLT duration. Typically, the definition of DLT excludes toxicities clearly related to the disease itself (e.g., disease progression or intercurrent illness). In some embodiments, the therapeutically effective amount of the inhibitor is about 1 mg / kg to about 30 mg / kg per week. In some embodiments, the therapeutically effective amount of the inhibitor is about 1 mg / kg to about 10 mg / kg every three weeks. In some embodiments, the therapeutically effective amount of the inhibitor is about 2 mg / kg to about 7 mg / kg administered every three weeks.

[0085] In various embodiments, a TGFβ inhibitor disclosed herein (e.g., a TGFβ1-selective inhibitor, e.g., Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, or Ab46) is used in combination with at least one additional therapy. In some embodiments, the at least one additional therapy is a cancer therapy, such as immunotherapy, chemotherapy, radiation therapy (including radiotherapy), recombinant immune cell therapy (e.g., CAR-T therapy), cancer vaccine therapy, and / or oncolytic virus therapy. The cancer therapy can include, for example, a cancer therapeutic agent (e.g., an immunotherapy agent, a chemotherapy agent, a radiation therapy agent, a recombinant immune cell (e.g., CAR-T cell)), a cancer vaccine, and / or a therapeutic oncolytic virus (including any combination thereof). In some embodiments, the cancer therapy is immunotherapy, including checkpoint inhibitor therapy. The checkpoint inhibitor can include an agent that targets programmed cell death protein 1 (PD-1) or programmed cell death protein 1 ligand (PD-L1). For example, the checkpoint inhibitor can include an anti-PD-1 antibody or an anti-PD-L1 antibody.In some embodiments, a TGFβ inhibitor disclosed herein (e.g., a TGFβ1 selective inhibitor, e.g., Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, or Ab46) can be used in combination with at least one additional therapy selected from the following: 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, or an anti-B7H5 antibody. H4 antibody, anti-TIM3 antibody, anti-LAG3 antibody, anti-OX40 antibody (OX40 agonist), anti-CD27 antibody, anti-CD70 antibody, anti-CD47 antibody, anti-41BB antibody, anti-PD-1 antibody, anti-CD20 antibody, anti-CD3 antibody, anti-PD-1 / anti-PDL1 bispecific or multispecific antibody, anti-CD3 / anti-CD20 bispecific or multispecific antibody, anti-HER2 antibody, anti-CD79b antibody, anti-CD47 antibody, antibody that binds to T-cell immunoglobulin and ITIM domain protein (TIGIT), anti-ST2 antibody, anti-β7 integrin (e.g., anti-α4β7 integrin and / or αEβ7 integrin), CDK inhibitor, oncolytic virus, indoleamine 2,3-dioxygenase (IDO) inhibitor, and / or PARP inhibitor.

[0086] In the methods and compositions used in accordance with the present disclosure, including those relating to measuring circulating MDSC levels (e.g., circulating gMDSC levels) after administration of a TGFβ inhibitor (e.g., a TGFβ1-selective inhibitor or a non-isotype-selective TGFβ inhibitor), the subject may have not received prior cancer therapy, e.g., be untreated, have received prior cancer therapy, or be currently receiving cancer therapy. The prior cancer therapy may be the same cancer therapy administered in accordance with the present invention, or may be a portion thereof. The cancer therapy may be checkpoint inhibitor (CPI) therapy. The cancer may be an advanced cancer. The cancer may include locally advanced tumors and / or metastatic cancer. Furthermore, the subject may have a cancer that exhibits or is suspected of exhibiting immunosuppression (e.g., a tumor with an immune exclusion and / or immune suppressive phenotype). Alternatively, the subject may have a cancer that does not exhibit or is not suspected of immunoexclusion, e.g., a cancer that exhibits or is suspected of exhibiting an immune infiltration phenotype. For example, the cancer exhibiting or suspected of exhibiting an immune exclusion phenotype may be ccRCC, NSCLC, melanoma, urothelial carcinoma, or head and neck cancer. The cancer exhibiting or suspected of exhibiting an immune infiltration phenotype may be ccRCC. Subjects receiving or who have received a TGFβ inhibitor may have a cancer that has a high response rate to checkpoint inhibitor therapy (e.g., an overall response rate of greater than 30%, greater than 40%, greater than 50%, or higher) and may be resistant to checkpoint inhibitor therapy. Examples of cancers that exhibit high response rates to checkpoint inhibitor therapy include, but are not limited to, microsatellite unstable colorectal cancer (MSI-CRC), renal cell carcinoma (RCC), melanoma (e.g., metastatic melanoma), Hodgkin's lymphoma, NSCLC, cancers with microsatellite instability-high (MSI-H), cancers 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).In some embodiments, the subject may have a low response rate to checkpoint inhibitor therapy (e.g., an overall response rate of 30% or less, 20% or less, or 10% or less) and may be untreated. In some embodiments, the subject may have a low response rate to checkpoint inhibitor therapy (e.g., an overall response rate of 30% or less, 20% or less, or 10% or less) and may be resistant to checkpoint inhibitor therapy. Examples of cancers with low response rates to checkpoint inhibitor therapy include, but are not limited to, ovarian cancer, gastric cancer, and triple-negative breast cancer.

[0087] In some embodiments, a TGFβ inhibitor of the present disclosure (e.g., a TGFβ1-selective inhibitor, e.g., Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, or Ab46) can be used to improve the rate or ratio of complete to partial responses among responders to cancer therapy. Typically, even in cancer types that have relatively high response rates (e.g., ≧30% responders) to cancer therapy (e.g., checkpoint inhibitor therapy), the complete response rate is low. Thus, a TGFβ inhibitor of the present disclosure can be used to increase the proportion of complete responders among a responder population. In a preferred embodiment, the TGFβ inhibitor is Ab6.

[0088] In some embodiments, the TGFβ inhibitor does not inhibit TGFβ2 signaling at a therapeutically effective dose. In some embodiments, the TGFβ inhibitor does not inhibit TGFβ3 signaling at a therapeutically effective dose. In some embodiments, the TGFβ inhibitor does not inhibit TGFβ2 signaling or TGFβ3 signaling at a therapeutically effective dose. In some embodiments, the TGFβ inhibitor is a TGFβ1 selective inhibitor, such as Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, and Ab46. In a preferred embodiment, the TGFβ1 selective inhibitor is Ab6.

[0089] The present disclosure provides a method of treating fibrosis in a subject, the method comprising administering a therapeutically effective amount of a TGFβ inhibitor to the subject as a loading dose / maintenance dose regimen, wherein the TGFβ inhibitor inhibits TGFβ1 but does not inhibit either or both of TGFβ2 and / or TGFβ3, thereby treating fibrosis in the subject.

[0090] According to another aspect, the present disclosure provides a method of preventing fibrosis in a subject at risk of developing fibrosis, the method comprising administering a therapeutically effective amount of a TGFβ inhibitor to the subject as a loading / maintenance dose regimen, wherein the TGFβ inhibitor inhibits TGFβ1 but not either or both of TGFβ2 and / or TGFβ3, thereby preventing fibrosis in the subject at risk of developing fibrosis. According to some embodiments of the above aspects and embodiments, the method further comprises: (i) measuring the level of collagen, the level of new collagen synthesis, and / or the level of phosphorylated Smad2 present in fibrotic tissue of the subject before administration of the TGFβ inhibitor; and (ii) measuring the level of collagen, the level of new collagen synthesis, and / or the level of phosphorylated Smad2 present in fibrotic tissue of the subject after administration of the TGFβ 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 of the subject after administration compared to before administration indicates a therapeutic effect.

[0091] According to another aspect, the present disclosure provides a method of treating fibrosis in a subject, the method comprising the steps of: administering to a subject a TGFβ inhibitor in an amount effective to: reduce the amount of collagen present in the subject's fibrotic tissue after administration compared to the amount of collagen present in the subject's fibrotic tissue before administration; reduce the amount of new collagen synthesis present in the subject's fibrotic tissue after administration compared to the amount of new collagen synthesis present in the subject's fibrotic tissue before administration; and / or reduce the amount of phosphorylated Smad2 present in the subject's fibrotic tissue after administration compared to the amount of phosphorylated Smad2 present in the subject's fibrotic tissue before administration, wherein the TGFβ inhibitor inhibits TGFβ1 but does not inhibit either or both of TGFβ2 and / or TGFβ3, thereby treating fibrosis in the subject. According to one embodiment, the method further comprises: (a) determining the level of collagen, the level of new collagen synthesis, and / or the level of phosphorylated Smad2 present in the subject's fibrotic tissue before administering the TGFβ inhibitor; and (b) measuring the level of collagen, the level of new collagen synthesis, and / or the level of phosphorylated Smad2 present in the subject's fibrotic tissue after administering the TGFβ inhibitor. According to some embodiments of the above aspects and embodiments, the reduction in the amount of collagen in the fibrotic tissue, the reduction in the amount of new collagen synthesis, and / or the reduction in the amount of phosphorylated Smad2 in the fibrotic tissue is determined 24 hours, 48 ​​hours, 72 hours, or 96 hours after administering the TGFβ inhibitor. According to some embodiments of the above aspects and embodiments, the method further comprises selecting a subject who would benefit from a reduction in the level of collagen, the level of new collagen synthesis, and / or the level of phosphorylated Smad2 in the fibrotic tissue. According to some embodiments of the above aspects and embodiments, the TGFβ inhibitor is administered as a single dose regimen or as a loading dose / maintenance dose regimen. The presence of phosphorylated Smad2 in the subject's fibrotic tissue after administering the TGFβ inhibitor. In some embodiments, the single dose regimen comprises a single administration of about 1 mg / kg to about 100 mg / kg of the TGFβ inhibitor. In some embodiments, the single dose is about 3 mg / kg, about 10 mg / kg, or about 30 mg / kg.According to some embodiments of the above aspects and embodiments, a single dose is administered to a subject weekly, biweekly, or monthly. According to some embodiments of the above aspects and embodiments, the loading dose / maintenance dose regimen includes a loading dose of about 30 mg / kg to about 90 mg / kg and a maintenance dose of about 10 mg / kg to about 30 mg / kg. According to some embodiments of the above aspects and embodiments, the loading dose is about 30 mg / kg and the maintenance dose is about 10 mg / kg. According to some embodiments of the above aspects and embodiments, the loading dose is about 90 mg / kg and the maintenance dose is about 30 mg / kg. According to some embodiments of the above aspects and embodiments, the loading dose is administered intravenously and the maintenance dose is administered subcutaneously. According to some embodiments of the above aspects and embodiments, a single loading dose is administered, followed by maintenance doses administered weekly, biweekly, or monthly. According to some embodiments of the above aspects and embodiments, the fibrosis is pulmonary fibrosis or renal fibrosis. According to some embodiments, the pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF). According to some embodiments of the above aspects and embodiments, the administration is effective to alleviate symptoms of fibrosis in the subject. According to some embodiments, the symptoms of fibrosis are any one or more of pulmonary hypertension, right heart failure, respiratory failure, hypoxia, cough, thrombus formation, pneumonia, and / or lung cancer in the subject. According to some embodiments of the above aspects and embodiments, the subject has been diagnosed with a lung disease. According to some embodiments, the lung disease is an autoimmune lung disease, a viral lung infection, or a bacterial lung infection. According to some embodiments of the above aspects and embodiments, the subject has undergone radiation therapy. According to some embodiments, the radiation therapy is for lung cancer.

[0092] According to some embodiments of the above aspects and embodiments, the subject has one or more risk factors for fibrosis selected from the group consisting of smoking, environmental factors, and a genetic predisposition to pulmonary fibrosis. According to some embodiments of the above aspects and embodiments, the method further comprises selecting a TGFβ inhibitor that inhibits TGFβ1 but does not inhibit either or both of TGFβ2 and / or TGFβ3.

[0093] The present disclosure includes the selection of subjects or patients likely to respond to or benefit from TGFβ1 inhibitory therapy. Related diagnostic methods, as well as methods for monitoring or determining therapeutic response to TGFβ1 inhibitory therapy, are also encompassed by the present disclosure.

[0094] The present disclosure encompasses processes and methods for identifying or selecting TGFβ1-selective inhibitors suitable for therapeutic use. In preferred embodiments, the selection involves one or more antibodies or antigen-binding fragments with particularly favorable kinetic criteria, characterized by: i) subnanomolar affinity (e.g., K ) for each of the human LTBP1 / 3-proTGFβ1 complexes; D <1 nM), and ii) a low dissociation rate (k ) as measured by a suitable in vitro binding / kinetics assay (e.g., surface plasmon resonance (SPR), e.g., a BIACORE®-based system). OFF ), e.g., ≦5.00E-4. The selected antibody or antibodies are evaluated in preclinical studies, including efficacy studies and toxicity / safety studies, using a suitable preclinical model. The effective amount of the antibody or antibodies, as determined in the efficacy study, is below the level that causes undesirable toxicity, as determined in the toxicity / safety study. Preferably, an antibody or antibodies having a therapeutic window of at least 3-fold, 6-fold, or more preferably 10-fold is selected. The effective amount of an antibody according to the present disclosure can be from about 0.1 mg / kg to about 30 mg / kg when administered weekly. In a preferred embodiment, the maximum tolerated dose (MTD) of an antibody of the present disclosure is >100 mg / kg when administered weekly for at least 4 weeks. [Brief explanation of the drawings]

[0095] [Figure 1] FIG. 1 shows a schematic diagram illustrating an example of pathological analysis of tumor tissue samples. [Figure 2] FIG. 1 shows a schematic diagram illustrating an example of pathological analysis of tumor tissue samples. [Figure 3A] P-Smad2 immunohistochemistry (IHC) analysis of melanoma samples is shown. [Figure 3B]1 shows pSmad-2 signaling in MBT2 tumors after Ab6-mIgG1 treatment. [Figure 4A] Circulating gMDSC and mMDSC levels in whole blood of MBT2 tumor-bearing mice are shown. [Figure 4B] Figure 1 shows intratumoral gMDSC and mMDSC levels in mice bearing MBT2 tumors. [Figure 5] The mean pharmacokinetic (PK) profiles of SRK-181 by dose are shown. [Figure 6] Preliminary efficacy by treatment duration is shown. [Figure 7] Best target lesion response in Part A1 and Part A2 is shown. [Figure 8A-C] 1 shows an exemplary analysis of MDSCs by signal filtering. [Figure 9A-C] Figure 1 shows the identification of tumor MDSC populations in various solid tumor samples. [Figure 10A-C] 1 shows an analysis of gMDSC and mMDSC populations in various solid tumor samples. [Figure 11A-C] Figure 11A shows the relative gene expression of LTBP1, LRRC33, and COL3A1 in the adenine model, Figure 11B shows the relative gene expression of COL3A1 at 24, 48, and 96 hours, and Figure 11C shows the relative gene expression of LRRC33 at 24, 48, and 96 hours. [Figure 12] The left panel provides updated information on dose, cancer type, and treatment duration from DRAGON Part A. The right panel shows a summary of treatment response in three ovarian cancer patients who achieved SD for ≥6 months in response to SRK-181 monotherapy. [Figure 13] Figure 1 shows an overview of ccRCC patients who achieved PR in response to combination treatment with SRK-181 and anti-PD-1 antibody. Two sets of images are collected from patient 1 and patient 2, showing pre- and post-treatment images. [Figures 14A-E]Representative pre- and post-treatment paired biopsy images from patients with UC (Figure 14A), melanoma (Figure 14B), NSCLC (Figures 14C and 14D), and ccRCC (Figure 14E) are shown. Biopsies were stained for CD8, demonstrating CD8+ cells within the tumor compartment. Figures 14A, B, C, and D show an increase in intratumoral CD8+ T cells after treatment with SRK-181 and anti-PD-1. Figure 14E also shows a slight increase in CD8+ infiltration. [Figures 15A-E] Major compartment analysis of %CD8+ T cells by tumor compartment is shown for each patient with UC (Figure 15A), melanoma (Figure 15B), NSCLC (Figures 15C and 15D), and ccRCC (Figure 15E). Corresponding biopsy pairs are shown in Figures 14A-E. Figures 15A, B, C, and D show the increase in intratumoral CD8+ T cells after SRK-181 and anti-PD-1 antibody treatment. [Figures 16A-E] Tumor nest analysis is shown for each patient with UC (Figure 16A), melanoma (Figure 16B), NSCLC (Figures 16C and 16D), and ccRCC (Figure 16E). Corresponding biopsy pairs are shown in Figures 14A-E. Figures 16A, B, C, and D show the CD8+ cells (%) plotted against the size of each tumor nest. Figure 16E shows the percentage of tumors (by cell count) displaying each phenotype (exclusion, desert, and infiltration) before and after treatment (tumor % = nest cell count / total cell count in tumor compartment × 100). Figures 16A, B, C, and D show an increase in infiltrating tumor nests after SRK-181 and anti-PD1 treatment. Figure 16E also shows a slight increase in CD8+ infiltration. [Figure 17] This table shows the correlation between CD8+ T-cell infiltration and tumor shrinkage observed in some patients. In the table, "++" indicates a significant increase, "+" indicates an increase, "+-" indicates no change, "-" indicates a decrease, and "--" indicates a significant decrease. [Figure 18]

[0023] Figure 1 shows baseline CD8+ cell levels in 11 ccRCC patients prior to treatment with SRK-181. Eight patients were found to have tumors exhibiting an invasive phenotype despite being non-responsive to anti-PD(L)1 therapy. [Figures 19A-19B]19A and 19B are two graphs showing the change in circulating gMDSC levels after treatment with SRK-181. Figure 19A shows the mean change across all patients, stratified according to response (PR = partial response, SD = stable disease, PD = progressive disease). Figure 19B shows the change in circulating gMDSC levels for individual patients who showed a PR. For patients who showed a PR or SD response, a decrease in circulating gMDSCs can be seen. [Figure 20] Figure 20 is another graph showing the change in circulating gMDSC levels after SRK-181 treatment in patients with ccRCC. Figure 20 shows the mean change in all patients with ccRCC stratified according to response (PR, SD, PD). [Figure 21A-C] Summary of ccRCC patient response to SRK-181 and anti-PD-1 therapy. Patients with PR, SD, or SD responses are highlighted, and a graph showing duration of treatment (FIG. 21A), a waterfall graph showing best response in target lesions as % change from baseline (FIG. 21B), and a spider graph showing change in tumor volume over time as % change from baseline (FIG. 21C) are provided. [Figure 22] Data from a CAGA reporter assay are shown, demonstrating that TGFβ1-selective antibodies can block ascorbic acid / Fe(III)Cl-mediated growth factor release from TGFβ C4S small latent complexes (SLCs). The TGFβ1-selective antibodies tested included Ab46 (Ab 37021), SKR-181 (Ab 36993), Ab42 (Ab 49247), and Ab 36956. The assay was repeated twice, and the corresponding data are shown in two bar graphs. TGFb1: TGFβ1 growth factor; 1D11: pan-inhibitor of TGFβ (used as a positive control); Asc: ascorbic acid + Fe(III)Cl + EDTA treatment; HuNEG: human IgG (used as a negative control). [Figures 23A-23B]Figure 23 shows kallikrein digestion of the latent-associated peptide (LAP) in the TGFβ1 C4S small latent complex (SLC) with or without preincubation with TGFβ-selective antibodies. As shown in Figure 23A, LAP can be cleaved by kallikrein to generate the R58LAP-D and L59LAP-D fragments. Figure 23B shows that none of the TGFβ1-selective antibodies tested, including Ab46 (Ab 37021), SKR-181 (Ab 36993), Ab42 (Ab 49247), and Ab 36956, were able to block kallikrein-mediated cleavage of LAP. M: marker; C4S: TGFβ1 C4S SLC; HuNEG: human IgG used as a negative control. DETAILED DESCRIPTION OF THE INVENTION

[0096] definition In order to make this disclosure more readily understandable, certain terms are first defined. These definitions should be interpreted in light of the remainder of this disclosure and as understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Additional definitions are set forth throughout the detailed description.

[0097] Advanced Cancer, Advanced Malignancy: As used herein, the term "advanced cancer" or "advanced malignancy" has the meaning understood in the relevant art, e.g., by an oncologist in the context of diagnosing or treating a subject / patient with cancer. Advanced malignancies involving solid tumors can be locally advanced or metastatic. The term "locally advanced cancer" is used to describe a cancer (e.g., a tumor) that has grown outside the organ where it began but has not yet metastasized to distant parts of the body. Thus, this term includes cancer that has metastasized from the place where it began to nearby tissues or lymph nodes. In contrast, a "metastatic cancer" is a cancer that has metastasized from the part of the body where it began (the primary site) to other parts of the body (e.g., distant sites).

[0098] Affinity: Affinity is the strength of binding between a molecule (such as an antibody) and its ligand (such as an antigen). It is typically measured by the equilibrium dissociation constant (K D ) is measured and recorded. In relation to antibody-antigen interactions, K D is the dissociation rate ("off rate" or K off ), i.e., the rate at which an antibody dissociates from its antigen, and the antibody association rate (the "on rate" or K on ), i.e., the ratio of how quickly an antibody binds to its antigen. For example, an antibody with an affinity of ≦5 nM has a K of 5 nM or less as determined by a suitable in vitro binding assay. D Suitable in vitro assays include biolayer interferometry (BLI) and solution equilibrium titration (e.g., MSD-SET) to measure the K of an antibody against its antigen. D In a preferred embodiment, affinity is measured by surface plasmon resonance (e.g., Biacore®). Antibodies with suitable affinity in a surface plasmon resonance assay have, for example, a K of up to about 1 nM, e.g., up to about 0.5 nM, e.g., up to about 0.5, 0.4, 0.3, 0.2, 0.15 nM or less. D may have:

[0099] Antibody: The term "antibody" includes any naturally occurring, recombinant, modified, or engineered immunoglobulin or immunoglobulin-like structure, or antigen-binding fragment or portion thereof, or derivatives thereof, as described in more detail elsewhere herein. Thus, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, including, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and multispecific antibodies (such as bispecific antibodies). Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, but in some cases may also include fewer chains, such as naturally occurring antibodies in camelids, which may contain only heavy chains. Antibodies may be derived from only a single source or may be "chimeric" (i.e., different portions of the antibody may be derived from two different antibodies). Antibodies, or antigen-binding portions thereof, may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. The term antibody, as used herein, includes each of 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, and antibody fusions (sometimes referred to herein as "antibody conjugates"). In some embodiments, the term also encompasses peptibodies.

[0100] Antigen: The term "antigen" broadly encompasses any molecule containing an antigenic determinant within the binding region to which an antibody or fragment specifically binds. An antigen can be a single unit molecule (such as a protein monomer or fragment) or a complex composed of multiple components. An antigen provides an epitope, a molecule or portion of a molecule, or a complex of molecules, that can be bound by a selective binding agent, such as an antigen-binding protein (including, for example, an antibody). Thus, a selective binding agent can specifically bind to an antigen formed by two or more components in a complex. In some embodiments, an antigen can be used in an animal to generate antibodies capable of binding to that antigen. An antigen can have one or more epitopes that can interact with different antigen-binding proteins, such as antibodies. In the context of the present disclosure, a suitable antigen is a complex (e.g., a multimeric complex composed of associated components) comprising a proTGF dimer associated with a presentation molecule. Each monomer of the proTGF dimer contains a prodomain and a growth factor domain, separated by a furin cleavage sequence. Two such monomers form a proTGF dimeric complex. This is then covalently linked to a presentation molecule via a disulfide bond, which contains a cysteine ​​residue present near the N-terminus of each proTGF monomer. This multimeric complex formed by proTGF dimers bound to a presentation molecule is commonly referred to as a large latent complex. Antigen complexes suitable for screening antibodies or antigen-binding fragments include, for example, the presentation molecule component of the large latent complex. Such presentation molecule components can be full-length presentation molecules or fragments thereof. The minimum required portion of a presentation molecule typically contains at least 50 amino acids, but more preferably at least 100 amino acids, of a presentation molecule polypeptide containing two cysteine ​​residues capable of forming covalent bonds with a proTGFβ1 dimer.

[0101] Antigen-binding portion / fragment: As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TGFβ1). Antigen-binding portions include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. In some embodiments, an antigen-binding portion of an antibody can be derived from an intact antibody molecule using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Non-limiting examples of antigen-binding portions include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a single-chain Fv (scFv) molecule (see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Nat'l. Acad. Sci. USA 85:5879-5883); (vi) a dAb fragment (see, e.g., Ward et al., (1989) Nature 341:544-546); and (vii) minimal recognition units composed of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs)). Other forms of single-chain antibodies, such as diabodies, are also encompassed.The term antigen-binding portion of an antibody includes a "single-chain Fab fragment," also known as "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 the antibody domains and the linker have one of the following sequences from N-terminal to C-terminal: 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; wherein the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids.

[0102] Bias: In the context of the present disclosure, the term "bias" (as in "biased binding") refers to an antibody's skewed or heterogeneous affinity toward or for a subset of antigens to which it can specifically bind. For example, an antibody is said to be biased if its affinity for one antigen complex is not equivalent to its affinity for another antigen complex (e.g., the affinity differs by more than five-fold). Context-independent antibodies according to the present disclosure have equivalent affinities (i.e., non-biased or homogeneous) for such antigen complexes. Preferred biased antibodies of the present disclosure include "matrix-biased" (or "LTBP-biased" antibodies), which preferentially bind to EMC-associated complexes (LTBP1-proTGFβ1 and LTBP3-proTGFβ) and have a relative affinity between at least one matrix-associated complex and at least one cell-associated complex (GARP-proTGFβ1 and / or LRRC33-proTGFβ1 complex) that is greater than five-fold. In contrast, antibodies characterized as "non-biased" have roughly equivalent affinities for such antigen complexes (e.g., less than a five-fold difference in affinity).

[0103] Binding region: As used herein, a "binding region" is a portion of an antigen (e.g., an antigen complex) that, when bound to an antibody or fragment thereof, can form the interface of the antibody-antigen interaction. Upon antibody binding, the binding region is protected from surface exposure and can be detected by a suitable technique, such as HDX-MS. An antibody-antigen interaction can be mediated through multiple (e.g., two or more) binding regions. A binding region can comprise an antigenic determinant, or epitope.

[0104] Biolayer Interferometry (BLI): BLI is a label-free technique for optically measuring biomolecular interactions, for example, between a ligand immobilized on the surface of a biosensor chip and an analyte in solution. BLI offers the ability to accurately and precisely monitor binding specificity, association and dissociation rates, or concentration. BLI platform instruments are commercially available, for example, from ForteBio, and are commonly referred to as the Octet® System.

[0105] Cancer: As used herein, the term "cancer" refers to a physiological condition in multicellular eukaryotic organisms that is typically characterized by abnormal cell growth and malignant tumors. The term broadly encompasses solid and liquid malignancies, including tumors, blood cancers (e.g., leukemia, lymphoma, and myeloma), and myelofibrosis.

[0106] Cancer-associated fibroblasts (CAFs): The term "cancer-associated fibroblasts (CAFs)," also known as tumor-associated fibroblasts, oncogenesis-associated fibroblasts, and activated fibroblasts, refers to a cell type within the tumor microenvironment that promotes tumorigenic properties by initiating extracellular matrix remodeling or secreting cytokines. CAFs express smooth muscle actin α (actin α), platelet-derived growth factor receptor α (PDGFRα / CD140a), platelet-derived growth factor receptor β (PDGFRβ / CD140b), fibroblast-specific protein 1 (FSP-1 / S100A4), fibroblast activation protein (FAP), and nicotinamide N-methyltransferase (NNMT), all of which have been used as markers to identify CAFs.

[0107] Cell-associated TGFβ1 / proTGFβ1: This term refers to membrane-bound (e.g., cell surface-tethered) TGFβ1 or its signaling complex (e.g., pro / latent TGFβ1). Typically, such cells are immune cells. TGFβ1 presented by GARP or LRRC33 is cell-associated TGFβ1. GARP and LRRC33 are transmembrane presentation molecules expressed on the cell surface of certain cells. GARP-proTGFβ1 and LRRC33-proTGFβ1 are sometimes collectively referred to as "cell-associated" (or "cell surface") proTGFβ1 complexes, which mediate cell-associated (e.g., immune cell-associated) TGFβ1 activation / signaling. This term also includes recombinant, purified GARP-proTGFβ1 and LRRC33-proTGFβ1 complexes in solution that are not physically associated with the cell membrane (e.g., in vitro assays). The average KD values ​​of antibodies (or fragments thereof) for the GARP-proTGFβ1 complex and the LRRC33-proTGFβ1 complex can be calculated to collectively represent their affinity for cell-associated (e.g., immune cell-associated) proTGFβ1 complexes. Human counterparts of the presenting molecule or presenting molecule complex can be indicated by an "h" preceding the protein or protein complex, e.g., "hGARP," "hGARP-proTGFβ1," "hLRRC33," and "hLRRC33-proTGFβ1." In addition to blocking the release of active TGFβ1 growth factor from cell-associated complexes, cell-associated proTGFβ1 can be targeted for internalization (e.g., endocytosis) and / or cell killing, such as ADCC-, ADCP-, or ADC-mediated depletion, of target cells expressing such cell surface complexes.

[0108] Checkpoint inhibitor: In the context of this disclosure, checkpoint inhibitor refers to an immune checkpoint inhibitor and has the meaning as understood in the art. "Checkpoint inhibitor therapy" or "checkpoint blockade therapy" targets a checkpoint molecule to partially or completely alter its function. Typically, checkpoints are receptor molecules on T cells or NK cells or corresponding cell surface ligands on antigen-presenting cells (APCs) or tumor cells. Without being bound by theory, immune checkpoints are activated in immune cells to prevent the development of inflammatory immunity against "self." Thus, shifting the balance of the immune system through checkpoint blockade may enable the immune system to fully activate and detect and eliminate cancer. The best-known inhibitory receptors involved in regulating immune responses are cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed cell death receptor ligand 1 (PD-L1), T-cell immunoglobulin domain and mucin domain-3 (TIM3), lymphocyte activation gene 3 (LAG3), killer cell immunoglobulin-like receptor (KIR), glucocorticoid-induced tumor necrosis factor receptor (GITR), and V-domain immunoglobulin (Ig)-containing inhibitor of T-cell activation (VISTA). Non-limiting examples of checkpoint inhibitors include nivolumab, pembrolizumab, cemiplimab, BMS-936559, atezolizumab, avelumab, darvalumab, ipilimumab, tremelimumab, IMP-321 (eftiragimod alfa or ImmuFact®), BMS-986016 (relatimab), budigalimab (ABBV-181, an anti-PD-1 antibody), and lirilumab. Keytruda® is an example of an anti-PD-1 antibody. Budigalimab is a humanized recombinant IgG1 monoclonal antibody that targets PD-1 and has been shown to be equally safe and well-tolerated in patients with HNSCC and NSCLC in a phase I trial (Italiano et al., Cancer Immunology, Immunotherapy (2022) 71:417-431).Opdivo® is an example of an anti-PD-1 antibody. Therapies or treatment regimens that use one or more immune checkpoint inhibitors are sometimes referred to as checkpoint blockade therapy (CBT) or checkpoint inhibitor therapy (CPI).

[0109] Clinical benefit: As used herein, the term "clinical benefit" is intended to include both the efficacy and safety of a treatment. Thus, a therapeutic treatment that achieves a desired clinical benefit is effective (e.g., achieves a therapeutically beneficial effect) and safe (e.g., with an acceptable or tolerable level of toxicity or adverse events).

[0110] Clinical benefit rate: The term "clinical benefit rate" refers to the percentage of patients who experience a complete response, a partial response, or stable disease for at least several months as a result of treatment. This term can be used to characterize the ability of a treatment to promote tumor stasis and / or stable disease.

[0111] Combination therapy: "Combination therapy" refers to a therapeutic regimen for a clinical indication that includes two or more therapeutic agents. Accordingly, the term refers to a therapeutic regimen in which a first therapeutic agent, comprising a first composition (e.g., active ingredient(s)), is administered to a patient together with at least one second therapeutic agent, comprising a second composition (e.g., active ingredient(s)), for the purpose of treating the same or overlapping diseases or clinical conditions. The term can further encompass therapeutic regimens in which a first therapeutic agent, comprising a first composition (e.g., active ingredient(s)), is administered together with a second therapeutic agent, comprising a second composition (e.g., active ingredient(s) such as a checkpoint inhibitor), a third therapeutic agent, comprising a third composition (e.g., active ingredient(s) such as a chemotherapeutic agent), or more (e.g., additional, different active ingredients). The first, second, and (optionally additional) compositions may act on the same cellular target or distinct cellular targets. The phrase "together with" in reference to combination therapy means that the therapeutic effect of the first therapeutic agent overlaps in time and / or space with the therapeutic effect of the second and additional therapeutic agents in a subject receiving combination therapy. The first, second, and / or additional compositions can be administered contemporaneously (e.g., simultaneously), separately, or sequentially. Thus, a combination therapy can be formulated as a single formulation for simultaneous administration or as separate formulations for sequential, contemporaneous, or simultaneous administration of the therapeutic agents. When a subject treated with a first therapy to treat a disease is administered a second and additional therapeutic agent to treat the same disease, the second and additional therapeutic agents may also be referred to as add-on or adjunctive therapy.

[0112] Combinatorial or combinatorial epitope: A combinatorial epitope is an epitope recognized and bound by a combinatorial antibody at a site (i.e., antigenic determinant) formed by non-contiguous portions of one or more components of an antigen, which are closely related in a three-dimensional structure to form an epitope. Thus, the antibodies of the present disclosure can bind to an epitope formed by two or more components (e.g., portions or segments) of the pro / latent TGFβ1 complex. A combinatorial epitope can include amino acid residues from a first component of the complex and amino acid residues from a second component of the complex, etc. Each component can be either a single protein or two or more proteins of the antigen complex. A combinatorial epitope is formed by structural contributions from two or more components (e.g., portions or segments, such as amino acid residues) of an antigen or antigen complex.

[0113] Competition or cross-competition; cross-blocking: When used in reference to antigen-binding proteins (e.g., antibodies or antigen-binding portions thereof) that compete for the same epitope, the term "competition" refers to competition between antigen-binding proteins as determined by an assay in which the antigen-binding protein under test prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein to a common antigen (e.g., TGFβ1 or a fragment thereof). Various types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another antigen-binding protein, including, for example, solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays; solid-phase direct biotin-avidin EIAs; solid-phase direct label assays, and solid-phase direct label sandwich assays. Typically, when a competing antigen-binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of the reference antigen-binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, when the competing antibody is present in excess, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more. In some embodiments, competition is determined using an SPR (e.g., Biacore) assay. In some embodiments, competition is determined using a BLI (e.g., Octet®) assay.

[0114] In some embodiments, a first antibody or antigen-binding portion thereof and a second antibody or antigen-binding portion thereof "cross-block" each other for the same antigen, as determined by, for example, biolayer interferometry (e.g., Octet®) or surface plasmon resonance (e.g., a Biacore system) using standard test conditions and following the manufacturer's instructions (e.g., binding is assayed at room temperature, approximately 20-25°C). In some embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have the same epitope. In other embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have overlapping, but not identical, epitopes. In yet other embodiments, the first antibody or fragment thereof and the second antibody or fragment thereof may have distinct (different) epitopes that are close together in three-dimensional space, such that antibody binding is cross-blocked through steric hindrance. "Cross-blocking" means that the binding of a first antibody to an antigen prevents the binding of a second antibody to the same antigen, and similarly, the binding of a second antibody to an antigen prevents the binding of the first antibody to the same antigen.

[0115] Antibody binning (sometimes called epitope binning or epitope mapping) can be performed to characterize and classify a set (e.g., a "library") of monoclonal antibodies generated against a target protein or protein complex (i.e., an antigen). These antibodies against the same target are tested pairwise against all other antibodies in the library to assess whether they block each other's binding to the antigen. Closely related binning profiles indicate that the antibodies have the same or closely related (e.g., overlapping) epitopes and are "binned" together. Because biological activity (e.g., intervention; efficacy) generated by antibody binding to a target is likely to be inherited by other antibodies in the same bin, binning provides a useful structure-function profile of antibodies that share similar binding regions within the same antigen. Thus, antibodies with higher affinity (lower KD) within the same epitope bin generally have greater efficacy.

[0116] In some embodiments, an antibody that binds to the same epitope as Ab6 binds to the proTGFβ1 complex such that the epitope of the antibody includes one or more amino acid residues in Region 1, Region 2, and Region 3, which are identified as the binding regions of Ab6.

[0117] Complementarity-Determining Region (CDR): As used herein, the term "CDR" refers to a complementarity-determining region within an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, which are designated CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single variable region that is capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to various systems. The system described by Kabat (Kabat et al., (1987; 1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) not only provides an unambiguous residue numbering system applicable to any antibody variable region, but also provides precise residue boundaries defining the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Chothia et al. (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; and Chothia et al., (1989) Nature 342:877-883) have used the Kabat CDRs to It has been discovered that certain subportions within the CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. These subportions are designated L1, L2, and L3, or H1, H2, and H3, or L-CDR1, L-CDR2, and L-CDR3, or H-CDR1, H-CDR2, and H-CDR3, where "L" and "H" indicate the light chain and heavy chain regions, respectively. These regions are sometimes called Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described by Padlan (1995) FASEB J. 9:133-139 and MacCallum (1996) J. Mol. Biol. 262(5):732-45.Yet other CDR boundary definitions may not strictly follow one of the systems herein, but will still overlap with the Kabat CDRs (although they may be shortened or lengthened to accommodate predictions or experimental findings that particular residues or groups of residues, or entire CDRs, do not significantly affect antigen binding) (see, e.g., 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 CDRs defined by Kabat or Chothia.

[0118] Conformational epitope: A conformational epitope is an epitope that is recognized and bound by a conformational antibody in a three-dimensional conformation, but not in an unfolded peptide of the same amino acid sequence. A conformational epitope may be referred to as a conformation-specific epitope, a conformation-dependent epitope, or a conformation-sensitive epitope. The corresponding antibody or fragment thereof that specifically binds to such an epitope may be referred to as a conformation-specific antibody, a conformation-selective antibody, or a conformation-dependent antibody. The binding of an antigen to a conformational epitope depends on the three-dimensional structure (conformation) of the antigen or antigen complex.

[0119] Constant region / domain: Immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy and light chain constant domain amino acid sequences are known in the art.

[0120] Context bias: As used herein, a "context-biased antibody" refers to a type of conformational antibody that binds to an antigen with differential affinity when the antigen is associated with (i.e., bound or attached to) an interacting protein or fragment thereof. Thus, a context-biased antibody that specifically binds to an epitope within proTGFβ1 may bind to LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1 with different affinities. For example, if an antibody has higher affinity for matrix-bound proTGFβ1 complexes (e.g., LTBP1-proTGFβ1 and LTBP3-proTGFβ1) than for cell-associated proTGFβ1 complexes (e.g., GARP-proTGFβ1 and LRRC33-proTGFβ1), the antibody is said to be "matrix-biased." The relative affinity of [matrix-bound complex]:[cell-associated complex] is determined by the average K of the former, as exemplified herein. D Obtain the average K value of the latter D After obtaining the values, the ratio of the two can be calculated. A context-biased antibody may preferentially bind to or not bind to one presentation molecule-proTGFβ1 complex compared to other presentation molecule-proTGFβ1 complexes, such that the affinity of the former (measured by the dissociation constant KD) is more than 10-fold weaker or stronger, respectively, than the average affinity of the latter.

[0121] Context-independent: According to the present disclosure, a "context-independent antibody" that binds to proTGFβ1 has equivalent affinity for the four known presentation molecule-proTGFβ1 complexes, i.e., LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. Context-independent antibodies disclosed herein may also be characterized as unbiased or balanced. Typically, context-independent antibodies exhibit equivalent (i.e., 5-fold or less biased) affinities such that the relative ratio of measured KD values ​​between matrix-bound and cell-associated complexes is 5 or less, 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). In a preferred embodiment, surface plasmon resonance is used.

[0122] Dissociation rate: As used herein, the term "dissociation rate" has the meaning understood by those skilled in the relevant art (e.g., antibody technology) and refers to a kinetic parameter measured by how fast / slow a ligand (e.g., antibody or fragment) dissociates from its binding target (e.g., antigen). The dissociation rate can also be expressed as the "off rate" ("k OFF The relative on / off rates (i.e., k ON and k OFF ) determines the overall strength of the interaction, or affinity, and is typically measured as the dissociation constant (K D ) and therefore fast binding (high k ON ), slow dissociation (low k OFF ) or the contribution of both factors results in equivalent affinity (e.g., K D It is possible to achieve dissociation rates of 0.05 to 0.15 (values). Monovalent interactions can be measured using monovalent antigen-binding molecules / fragments such as fAb (Fab), and bivalent interactions can be measured using bivalent antigen-binding molecules such as whole immunoglobulins (e.g., IgG). Dissociation rates can be measured experimentally in suitable in vitro binding assays, such as OCTET® and BIACORE®-based systems.

[0123] ECM-associated TGFβ1 / proTGFβ1: This term refers to TGFβ1 or its signaling complexes (e.g., pro / latent TGFβ1) that are components of the extracellular matrix (e.g., deposited in the cell matrix). TGFβ1 presented by LTBP1 or LTBP3 is ECM-associated TGFβ1, i.e., LTBP1-proTGFβ1 and LTBP3-proTGFβ1, respectively. LTBPs are important for the correct deposition and subsequent bioavailability of TGFβ in the ECM, and fibrillin (Fbn) and fibronectin (FN) are thought 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 and certain disease-related tissues, such as tumor stroma and fibrotic tissues. Human counterparts of presentation molecules or presentation molecule complexes can be indicated by an "h" preceding the protein or protein complex, e.g., "hLTBP1," "hLTBP1-proTGFβ1," "hLTBP3," and "hLTBP3-proTGFβ1." The average dissociation constants (KD values) of antibodies (or fragments thereof) against the LTBP1-proTGFβ1 complex and the LTBP3-proTGFβ1 complex can be calculated to comprehensively represent their affinity for the ECM-associated (or matrix-associated) proTGFβ1 complex.

[0124] Effective amount: The terms "effective" and "therapeutically effective" refer to the ability or amount sufficient to produce a detectable change in a disease parameter, e.g., a slowing, cessation, reversal, reduction, or improvement in the symptoms or downstream effects of the disease. The term encompasses, but does not require, the use of an amount that completely cures the disease. An "effective amount" (or therapeutically effective amount or therapeutic dose) can be a dose or dosing regimen that achieves a statistically significant clinical benefit (e.g., efficacy) in a patient population. For example, for an antibody that has been shown to be effective in preclinical models at doses of 3 mg / kg to 30 mg / kg, the effective amount can be said to be about 3 to 30 mg / kg. For example, Ab6 has been shown to be effective in preclinical models at doses as low as 3 mg / kg and as high as 30 mg / kg. The terms "minimum effective dose" or "minimum effective amount" refer to the lowest amount, dose, or dosing regimen that achieves a detectable change in a disease parameter, e.g., a statistically significant clinical benefit. As used herein, when referring to a dose of a drug (e.g., a dose of a TGFβ1 inhibitor), it may be a therapeutically effective dose, as described herein. In clinical settings, such as human clinical trials, the term "pharmacologically active dose (PAD)" may be used to refer to an effective dose. An effective amount may be expressed in terms of the dose administered or the exposure level (e.g., serum concentration) achieved as a result of administration.

[0125] Effective tumor control: The term "effective tumor control" can be used to refer to the degree of tumor regression achieved in response to treatment, e.g., the tumor regresses by a defined percentage (e.g., <25%) of the endpoint tumor volume. For example, in a particular model, the endpoint tumor volume is 2,000 mm 3 If the threshold is set to <25%, the tumor will be 500 mm 3Effective tumor control is achieved when the tumor shrinks to less than 100%. Thus, effective tumor control encompasses complete regression. Clinically, effective tumor control can be measured by objective response, including partial response (PR) and complete response (CR), as determined by art-accepted criteria such as RECIST v1.1 and the corresponding iRECIST (iRECIST v1.1). In some embodiments, effective tumor control in clinical settings also includes stable disease, in which a tumor that is generally expected to grow at a steady rate is prevented from growing by treatment, even if shrinkage is not achieved.

[0126] Effector T cell: As used herein, effector T cells are T lymphocytes that respond readily and actively to stimuli, such as costimulation, including, but not limited to, CD4+ T cells (also called T helper or Th cells) and CD8+ T cells (also called cytotoxic T cells). Th cells assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or support active immune responses. These cells can differentiate into one of several subtypes, including Th1, Th2, Th3, Th17, Th9, or Tfh, which secrete various cytokines to promote different types of immune responses. Signaling from APCs directs T cells toward specific subtypes: cytotoxic (killer). In contrast, cytotoxic T cells (TC cells, CTLs, T killer cells, killer T cells) destroy virus-infected and cancer cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells. Markers of cytotoxic effector cells (e.g., CD8+ cells) include, for example, perforin and granzyme B.

[0127] Endpoint: In studies aimed at assessing the effectiveness of a treatment (e.g., clinical benefit or improvement), such as clinical trials of cancer therapy, an endpoint represents a measure of a predetermined parameter indicative of treatment effect. 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 assessments) and biomarker assessments such as blood- or fluid-based assessments.

[0128] Epithelial hyperplasia: The term "epithelial hyperplasia" refers to increased tissue proliferation resulting from the proliferation of epithelial cells. As used herein, epithelial hyperplasia refers to unwanted toxicity caused by TGFβ inhibition, including, but not limited to, abnormal proliferation of epithelial cells in the oral cavity, esophagus, breast, and ovaries.

[0129] Epitope: The term "epitope," sometimes referred to as an antigenic determinant, is a molecular determinant (e.g., a polypeptide determinant) capable of specific binding 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 groups, and in certain embodiments, may have specific three-dimensional structural and / or charge characteristics. The epitope recognized by an antibody or antigen-binding fragment of an antibody is a structural element of the antigen that interacts with the CDR (e.g., complementary site) of the antibody or fragment. An epitope may be formed by contributions from several amino acid residues, which interact with the CDR of the antibody to provide specificity. An antigenic fragment may contain more than one epitope. In certain embodiments, an antibody may specifically bind to 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 they cross-compete (one prevents the binding or regulatory action of the other).

[0130] Equivalent Affinity: In the context of the present disclosure, the term "equivalent affinity" is intended to mean: i) the antibody binds to matrix-associated and cell-associated proTGFβ1 complexes with an affinity bias of less than 5-fold, as measured by a suitable in vitro binding assay, such as solution equilibrium titration (e.g., MSD-SET), biolayer interferometry (e.g., Octet®), or surface plasmon resonance (e.g., Biacore system); and / or ii) the antibody's relative affinity for the four complexes is uniform, either by having the lowest affinity (highest KD value) among the four antigen complexes be no more than 5-fold the average calculated from the affinities of the remaining three; or by having the highest affinity (lowest KD value) among the four antigen complexes be no more than 5-fold the average calculated from the affinities of the remaining three. Antibodies with equivalent affinities may achieve a more uniform inhibitory effect, regardless of the specific presentation molecule associated with the proTGFβ1 complex (hence "context-independent"). In some embodiments, the observed bias in the average affinity between the matrix-associated complex and the cell-associated complex is 3-fold or less. In a preferred embodiment, affinity is measured by surface plasmon resonance (e.g., a Biacore system). Such methods are performed using standard test conditions, e.g., according to the manufacturer's instructions.

[0131] Extended Latent Lasso: As used herein, the term "extended latent lasso" refers to a portion of a prodomain that includes a latent lasso and an alpha-2 helix, e.g., LASPPSQGEVPPGPLPEAVLALYNSTR (SEQ ID NO: 1127). In some embodiments, the extended latent lasso further includes a portion of an alpha-1 helix, e.g., LVKRKRIEA (SEQ ID NO: 1132) or a portion thereof.

[0132] Fibrosis: The term "fibrosis" or "fibrotic condition / disorder" refers to a process or condition characterized by the pathological accumulation of extracellular matrix (ECM) components, such as collagen, in a tissue or organ. Indeed, collagen accumulation is a hallmark of fibrosis. According to some embodiments, the fibrosis is pulmonary (also called pulmonary fibrosis) fibrosis.

[0133] Pulmonary fibrosis: The term "pulmonary fibrosis" or "pulmonary fibrosis," as used in connection with this disclosure, refers to the formation of excess fibrous connective tissue in the lungs. According to some embodiments, pulmonary fibrosis can be a secondary effect of other lung diseases. Examples of such diseases include autoimmune diseases, viral infections, and bacterial infections (such as tuberculosis). Pulmonary fibrosis can be idiopathic, with smoking, environmental factors (e.g., occupational exposure to gases, smoke, chemicals, or dust), or genetic predisposition being considered risk factors.

[0134] Fibrotic microenvironment: The term "fibrotic microenvironment" refers to the localized disease niche within tissues where fibrosis occurs in vivo. The fibrotic microenvironment may contain disease-associated molecular signatures (a collection of chemokines, cytokines, etc.), disease-associated cell populations (activated macrophages, MDSCs, etc.), and disease-associated ECM environments (alterations in ECM components and / or structure). The fibrotic microenvironment is thought to support the transition of fibroblasts to α-smooth muscle actin-positive myofibroblasts in a TGFβ-dependent manner. The fibrotic microenvironment may further be characterized by the infiltration of specific immune cells (such as macrophages and MDSCs).

[0135] Finger-1 (of TGFβ1 growth factor): As used herein, "finger-1" is a domain within the TGFβ1 growth factor domain. In its unmutated form, finger-1 of human proTGFβ1 contains the following amino acid sequence: CVRQLYIDFRKDLGWKWIHEPKGYHANFC (SEQ ID NO: 1124). In the 3D structure, the finger-1 domain is adjacent to the latent lasso.

[0136] Finger-2 (of TGFβ1 growth factor): As used herein, "finger-2" is a domain within the TGFβ1 growth factor domain. In its unmutated form, finger-2 of human proTGFβ1 contains the following amino acid sequence: CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 1125). Finger-2 contains "binding region 6," which is spatially located adjacent to the latent lasso.

[0137] Gamma delta (γδ) T cells: As used herein, the term "gamma delta (γδ) T cells" refers to a subpopulation of atypical T cells with functions not limited to MHC-mediated antigen presentation. γδ T cells are actively recruited to the tumor microenvironment (TME) and are generally considered cytotoxic and antitumor lymphocytes; however, some γδ T cell subsets, particularly those expressing IL-17, CD39, or FOXP3, are immunosuppressive or tumor-promoting cells (Park et al., Exp Mol Med, 2021 Mar, 53(3):318-327). TGFβ can also suppress the antitumor cytotoxicity of human Vγ9Vδ2 T cells (Rafia et al., Front Immunol, 2023 Jan 19, 13:1066-336).

[0138] GARP-TGFβ1 / GARP-proTGFβ1 complex: As used herein, the term "GARP-TGFβ1 complex" (or "GARP-proTGFβ1 complex") refers to a protein complex comprising a proprotein or latent form of transforming growth factor-β1 (TGFβ1) protein and glycoprotein-A repeat dominant protein (GARP) or a fragment or variant thereof. In some embodiments, the proprotein or latent form of the TGFβ1 protein may also be referred to as a "pro / latent TGFβ1 protein." In some embodiments, a GARP-TGFβ1 complex comprises GARP covalently linked to pro / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed with a cysteine ​​residue present near the N-terminus (e.g., amino acid position 4) of the proTGFβ1 dimeric complex. In other embodiments, a GARP-TGFβ1 complex comprises GARP non-covalently linked to pro / latent TGFβ1. In some embodiments, the GARP-TGFβ1 complex is a naturally occurring complex, for example, an intracellular GARP-TGFβ1 complex. The term "hGARP" refers to human GARP.

[0139] High affinity: As used herein, the term "high affinity," as in "high affinity proTGFβ1 antibody," refers to a K D Thus, high affinity, context-independent proTGFβ1 antibodies encompassed by the present disclosure have a K of ≦5 nM, more preferably ≦1 nM, for each of the following antigen complexes: LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. D It has a value.

[0140] Human antibody: As used herein, the term "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, for example, amino acid residues in the CDRs, and particularly CDR3, that are 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). However, 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.

[0141] Humanized antibody: The term "humanized antibody" refers to an antibody containing heavy and light chain variable region sequences derived from a species other than human (e.g., mouse), but in which at least a portion of the VH and / or VL sequences have been modified to be more "human-like," i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. A "humanized antibody" also refers to an antibody or variant, derivative, analog, or fragment thereof that immunospecifically binds to an antigen of interest and comprises FR regions having substantially the amino acid sequence of a human antibody and CDR regions having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in reference to 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 the 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 of human immunoglobulin consensus sequences. In embodiments, a humanized antibody also comprises at least a portion of an immunoglobulin Fc region, typically at least a portion of a human immunoglobulin. In some embodiments, a humanized antibody contains at least the variable domains of a light chain and a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In certain embodiments, a humanized antibody contains only humanized variable domains of a light chain and / or a humanized heavy chain.

[0142] Immune-exclusion or immune-exclusion tumor: As used herein, a tumor characterized by "immune exclusion" is deficient or substantially deficient in intratumoral antitumor lymphocytes. For example, a tumor with poor T cell infiltration may have T cells surrounding the tumor, e.g., around the periphery of the tumor mass and / or near the tumor's vasculature ("perivascular"), but the T cells are nevertheless unable to effectively recruit within the tumor and exert cytotoxic function against cancer cells. In other situations, tumors are unable to mount a strong immune response (so-called "immune desert" tumors), resulting in the absence of many T cells near and within the tumor environment. In contrast to immune-exclusion tumors, tumors infiltrated with anti-tumor lymphocytes (such as CD8+ T cells) ("immune-infiltrated tumors") are sometimes characterized as "highly immunogenic" or "inflammatory" tumors; these tumors tend to be more responsive to immune checkpoint blockade therapy (CBT) and can therefore be targeted, although some immune-infiltrated tumors are still resistant or refractory to checkpoint blockade therapy. However, typically, only a small proportion of patients respond to CBT due to immune exclusion, which renders the tumor resistant to CBT.

[0143] Immune safety (assessment): As used herein, this term refers to a safety assessment related to immune response (immune activation), and acceptable immune safety criteria include the following: no significant cytokine release as determined by in vitro or in vivo cytokine release testing (e.g., assays); and no significant platelet aggregation or activation as determined using human platelets. Statistical significance in these tests can be determined relative to an appropriate reference control. For example, in the case of a test molecule that is a human monoclonal antibody, an appropriate control can be an immunoglobulin of the same subtype, e.g., an antibody of the same subtype known to have a good safety profile in humans.

[0144] Immunosuppression, immune suppression, immunosuppressive: These terms refer to the ability to suppress immune cells such as T cells, NK cells, and B cells. The gold standard for assessing immunosuppressive function is inhibition of T cell activity, which can include antigen-specific and non-specific suppression. Regulatory T cells (Tregs), subsets of gamma delta (γδ) T cells, and MDSCs can be considered immunosuppressive cells. M2-polarized macrophages (e.g., disease-limiting macrophages such as TAMs and FAMs) can also be characterized as immunosuppressive. Thus, cancers (e.g., solid tumors) with an immunosuppressive phenotype can contain infiltrating Tregs, MDSCs, and / or macrophages (e.g., high levels of infiltrating Tregs, MDSCs, and / or macrophages). Cancers with an immunosuppressive phenotype are also thought to be associated with elevated levels of circulating MDSCs, for example, compared to healthy controls, or when circulating MDSCs (e.g., circulating gMDSCs) are detectable (e.g., ≥1% of the leukocyte / peripheral blood mononuclear cell component).

[0145] Immunological memory refers to the immune system's ability to rapidly and specifically recognize previously encountered antigens and mount a corresponding immune response. Generally, these are secondary, tertiary, and subsequent immune responses to the same antigen. Immunological memory is responsible for adaptive components of the immune system, specialized T cells and B cells (so-called memory T and B cells). After antigen-naive T cells encounter their cognate antigen in the context of MHC molecules on the surface of professional antigen-presenting cells (e.g., dendritic cells), they expand and differentiate into memory and effector T cells. A single unifying theme for all memory T cell subtypes is that they are long-lived and can rapidly expand into large numbers of effector T cells upon re-exposure to their cognate antigen. Through this mechanism, they provide the immune system with "memory" against previously encountered pathogens. Memory T cells can be either CD4+ or CD8+ and typically express CD45RO. In a preclinical setting, immunological memory can be tested in a tumor rechallenge paradigm.

[0146] Inhibit or Inhibition of: As used herein, the terms "inhibit" or "inhibition of" mean to reduce by a measurable amount and can include, but does not require, complete prevention or inhibition.

[0147] Isoform-nonspecific / isoform-nonselective: The terms "isoform-nonspecific" or "isoform-nonselective" refer to the ability of an agent to bind to two or more structurally related isoforms. Isoform-nonspecific TGFβ inhibitors exert inhibitory activity against two or more isoforms of TGFβ, such as TGFβ1 / 3, TGFβ1 / 2, TGFβ2 / 3, and TGFβ1 / 2 / 3.

[0148] Isoform-specific / selective: The terms "isoform specificity" or "isoform selection" refer to the ability of an agent to distinguish one isoform from other structurally related isoforms (i.e., isoform selectivity). An isoform-specific TGFβ inhibitor exerts its inhibitory activity against one isoform of TGFβ but not against other isoforms of TGFβ at a given concentration. For example, an isoform-specific TGFβ1 antibody selectively binds to TGFβ1. A TGFβ1-specific inhibitor (antibody) preferentially targets (binds to and thereby inhibits) the TGFβ1 isoform with substantially higher affinity than TGFβ2 or TGFβ3. For example, selectivity in this context may exhibit at least a 10-fold, 100-fold, 500-fold, 1000-fold, or more difference in respective affinity as measured by an in vitro binding assay such as BLI (Octet®) or, preferably, SPR (Biacore®). In some embodiments, selectivity is such that the inhibitor, when used at a dose effective to inhibit TGFβ1 in vivo, does not inhibit TGFβ2 and TGFβ3. For example, an antibody may preferentially bind to TGFβ1 with an affinity of about 1 pM, while the same antibody may bind to TGFβ2 and / or TGFβ3 with an affinity of about 0.5-50 nM. For such inhibitors to be useful as therapeutic agents, the dosage to achieve the desired effect (e.g., a therapeutically effective amount) must fall within a therapeutic window in which the inhibitor can effectively inhibit the TGFβ1 isoform without inhibiting TGFβ2 or TGFβ3. In some embodiments, a TGFβ1-selective inhibitor is an agent that interferes with the function or activity of TGFβ1 but not with the function or activity of TGFβ2 and / or TGFβ3, regardless of mechanism of action. The terms "isoform-specific" and "isoform-selective" are used interchangeably herein.

[0149] Isolated: As used herein, an "isolated" antibody refers to an antibody that is substantially free of other antibodies having different antigen specificities. In some embodiments, an isolated antibody is substantially free of other unintended cellular material and / or chemicals.

[0150] Large latent complex: In the context of the present disclosure, the term "large latent complex" ("LLC") refers to a complex consisting of a proTGFβ1 dimer bound to a so-called presentation molecule. Thus, large latent complexes are presentation molecule-proTGFβ1 complexes such as LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. Such complexes can be formed in vitro using recombinant purified components capable of complex formation. For screening purposes, the presentation molecule used to form such an LLC does not need to be a full-length polypeptide. However, typically, a portion of a protein capable of forming a disulfide bond with the proTGFβ1 dimer complex via a cysteine ​​residue near its N-terminal region is required.

[0151] Latency-associated peptide (LAP): LAP is the so-called "prodomain" of proTGFβ1. As described in more detail herein, LAP is composed of a "straight jacket" domain and an "arm" domain. The straight jacket itself is further subdivided into an alpha-1 helix domain and a latency lasso domain.

[0152] Latent Lasso: As used herein, the "latent lasso," sometimes referred to as the cryptic loop, is a domain flanked by the α-1 helix and arms within the prodomain of proTGFβ1. In its unmutated form, the latent lasso of human proTGFβ1 comprises the amino acid sequence: LASPPSQGEVPPGPL (SEQ ID NO: 1126), from which region 1 extends. As used herein, the term "extended latent lasso region" refers to the latent lasso including its adjacent C-terminal motif, called the alpha-2 helix (α2-helix) of the prodomain. A proline residue at the C-terminus of the latent lasso provides an "elbow"-like vertical "bend" that connects the lasso loop with the α2-helix. Particular high-affinity TGFβ1 activation inhibitors bind, at least in part, to a latent Lasso or a portion thereof to confer inhibitory capability (e.g., the ability to block activation), optionally wherein the portion of the latent Lasso is ASPPSQGEVPPGPL (SEQ ID NO: 1170). In some embodiments, the antibodies of the present disclosure bind the proTGFβ1 complex to ASPPSQGEVPPGPL (SEQ ID NO: 1170) or a portion thereof. Particular high-affinity TGFβ1 activation inhibitors bind, at least in part, to an extended latent Lasso or a portion thereof to confer inhibitory capability (e.g., the ability to block activation), optionally wherein the portion of the extended latent Lasso is KLRLASPPSQGEVPPGPLPEAVL (SEQ ID NO: 1142) or LASPPSQGEVPPGPLPEAVLALYNSTR (SEQ ID NO: 271).

[0153] Localized: In the context of this disclosure, the term "localized" (as in "localized tumor," "disease-localized," etc.) refers to an anatomically isolated or isolatable abnormality, such as a solid malignant tumor, as opposed to a systemic disease. A particular leukemia, for example, may have both a localized (e.g., bone marrow) and a systemic (e.g., circulating blood cells) component to the disease.

[0154] LRRC33-TGFβ1 / LRRC33-proTGFβ1 complex: As used herein, the term "LRRC33-TGFβ1 complex" (or "LRRC33-proTGFβ1 complex") refers to a complex of the proprotein or latent form of transforming growth factor-β1 (TGFβ1) protein with leucine-rich repeat-containing protein 33 (LRRC33; also known as negative regulator of reactive oxygen species or NRROS) or a fragment or variant thereof. In some embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 covalently linked to the pro / latent form of TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed with a cysteine ​​residue present near the N-terminus of the proTGFβ1 dimeric complex (e.g., amino acid position 4). In other embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 non-covalently linked to the pro / latent form of TGFβ1. In some embodiments, the LRRC33-TGFβ1 complex is a naturally occurring complex, for example, an intracellular LRRC33-TGFβ1 complex. The term "hLRRC33" refers to human LRRC33. LRRC33 on the cell surface and LRRC33-containing complexes can be internalized in vivo. LRRC33 is expressed on a subset of myeloid cells, including M2-polarized macrophages (such as TAMs) and MDSCs. MDSCs that express LRRC33 on their cell surface include tumor-associated MDSCs and circulating MDSCs. LRRC33-expressing tumor-associated MDSCs may include g-MDSCs. Circulating LRRC33-expressing MDSCs may include g-MDSCs.

[0155] LTBP1-TGFβ1 / LTBP1-proTGFβ1 complex: As used herein, the term "LTBP1-TGFβ1 complex" (or "LTBP1-proTGFβ1 complex") refers to a protein complex comprising a proprotein or latent form of transforming growth factor-β1 (TGFβ1) protein and latent TGF-β binding protein 1 (LTBP1) or a fragment or variant thereof. In some embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 covalently linked to pro / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed with a cysteine ​​residue present near the N-terminus (e.g., amino acid position 4) of the proTGFβ1 dimeric complex. In other embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 non-covalently linked to pro / latent TGFβ1. In some embodiments, the LTBP1-TGFβ1 complex is a naturally occurring complex, e.g., an intracellular LTBP1-TGFβ1 complex. The term "hLTBP1" means human LTBP1.

[0156] LTBP3-TGFβ1 / LTBP3-proTGFβ1 complex: As used herein, the term "LTBP3-TGFβ1 complex" (or "LTBP3-proTGFβ1 complex") refers to a protein complex comprising a proprotein or latent form of transforming growth factor-β1 (TGFβ1) protein and latent TGF-β binding protein 3 (LTBP3) or a fragment or variant thereof. In some embodiments, the LTBP3-TGFβ1 complex comprises LTBP3 covalently linked to pro / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed with a cysteine ​​residue present near the N-terminus (e.g., amino acid position 4) of the proTGFβ1 dimeric complex. In other embodiments, the LTBP3-TGFβ1 complex comprises LTBP1 non-covalently linked to pro / latent TGFβ1. In some embodiments, the LTBP3-TGFβ1 complex is a naturally occurring complex, e.g., an intracellular LTBP3-TGFβ1 complex. The term "hLTBP3" means human LTBP3.

[0157] M2 or M2-like macrophages: M2 macrophages represent a subset of activated or polarized macrophages and include disease-associated macrophages in both fibrotic and tumor microenvironments. Cell surface markers of M2-polarized macrophages typically include CD206 and CD163 (i.e., CD206+ / CD163+). Applicant recently discovered that M2-polarized macrophages can also express cell surface LRRC33. M2 macrophage activation is primarily driven by IL-4, IL-13, IL-10, and TGFβ; M2 macrophages secrete the same cytokines (IL-4, IL-13, IL-10, and TGFβ) that activate them. These cells have high phagocytic capacity and produce ECM components, angiogenic, and chemotactic factors. Release of TGFβ by macrophages can perpetuate myofibroblast activation, EMT, and EndMT induction in diseased tissues, such as fibrotic tissues and tumor stroma. For example, M2 macrophages play a specific role in TGFβ-driven pulmonary fibrosis and are enriched in many tumors.

[0158] Matrix-associated proTGFβ1: LTBP1 and LTBP3 represent molecules that are components of the extracellular matrix (ECM). LTBP1-proTGFβ1 and LTBP3-proTGFβ1 can be collectively referred to as "ECM-associated" (or "matrix-associated") proTGFβ1 complexes that mediate ECM-associated TGFβ1 activation / signaling. This term also includes recombinant, purified LTBP1-proTGFβ1 and LTBP3-proTGFβ1 complexes in solution that are not physically bound to a matrix or substrate (e.g., in vitro assays).

[0159] Maximum Tolerated Dose (MTD): The term MTD generally refers to the highest dose of a test substance (such as a TGFβ1 inhibitor) evaluated at the no-observed-adverse-effect level (NOAEL) in relation to safety / toxicology considerations. For example, based on a 4-week toxicity study, the NOAEL for Ab6 in rats was the highest dose evaluated (100 mg / kg), suggesting that the MTD for Ab6 is >100 mg / kg. The NOAEL for Ab6 in non-human primates was the highest dose evaluated (300 mg / kg), based on a 4-week toxicity study, suggesting that the MTD for Ab6 in non-human primates is >300 mg / kg.

[0160] Mesoscale Discovery: "Mesoscale Discovery" or "MSD" is a type of immunoassay that uses electrochemiluminescence (ECL) as a detection technique. Typically, a high-binding carbon electrode is used to capture a protein (e.g., an antibody). The antibody can be incubated with a specific antigen, and its binding can be detected using a secondary antibody conjugated to an electrochemiluminescent label. Once an electrical signal is obtained, the light intensity can be measured to quantify the analyte in the sample.

[0161] Myelofibrosis: "Myelofibrosis," also known as osteomyelofibrosis, is a relatively rare myeloproliferative disorder (e.g., cancer) that belongs to a group of diseases called myeloproliferative disorders, including primary myelofibrosis and secondary myelofibrosis. Myelofibrosis is generally characterized by abnormal clonal proliferation of hematopoietic stem cells in the bone marrow and other sites, leading to fibrosis, or replacement of the bone marrow with scar tissue. The term myelofibrosis encompasses primary myelofibrosis (PMF), also called chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary in this case mean that the disease is of unknown or spontaneous origin), as well as secondary types of myelofibrosis, such as myelofibrosis occurring following polycythemia vera (PV) or essential thrombocytopenia (ET). Myelofibrosis is a form of myeloid metaplasia, which refers to a change in cell type in the hematopoietic tissue of the bone marrow; the two terms are often used interchangeably. The terms myeloid metaplasia of unknown etiology and myelofibrosis with myeloid metaplasia (MMM) are also used to refer to primary myelofibrosis, which is characterized by mutations that lead to upregulation or hyperactivation of the downstream JAK pathway.

[0162] Myeloid cells: In hematopoiesis, myeloid cells are blood cells that arise from precursors of granulocytes, monocytes, erythrocytes, or platelets (common myeloid progenitors, i.e., CMPs or CFU-GEMMs), or are often used in the narrower sense to distinguish them from lymphoid cells, i.e., lymphocytes derived from common lymphoid progenitors that give rise to B and T cells, particularly blood cells from the myeloblast lineage (myelocytes, monocytes, and their daughter types). Specific myeloid cell types, their general morphology, typical cell surface markers, and their immunosuppressive capabilities in both mice and humans are summarized below. In some embodiments, human neutrophils express the cell surface marker CD11b + , CD14 - , CD15 + , and CD66b + In some embodiments, the human neutrophils are LOX-1-. In some embodiments, the human neutrophils are HLA-DR - / medIn some embodiments, classical human monocytes express the cell surface marker CD14 + CD15 - CD16 - HLA-DR + In some embodiments, classical human monocytes can be identified by at least one (e.g., all) of the following: CD33 + and / or CD11b + In some embodiments, the classical human monocyte is CD16 - In some embodiments, the intermediate human monocytes express the cell surface marker CD14 + CD15 - CD16 + HLA-DR + In some embodiments, non-classical human monocytes can be identified by at least one (e.g., all) of the cell surface markers CD14 - CD15 - CD16 + HLA-DR + In some embodiments, human M1 macrophages can be identified by at least one (e.g., all) of the cell surface markers CD15 - CD16 + CD80 + HLA-DR + / 高 CD33 + In some embodiments, human M1 macrophages can be identified by at least one (e.g., all) of the following: - In some embodiments, human M1 macrophages are CD11b + In some embodiments, the human M1 macrophages are CD14 - In some embodiments, human M2 macrophages express the cell surface marker CD11b + and CD15 - In some embodiments, human M2 macrophages can be identified by at least one (e.g., all) of the following: + In some embodiments, human M2 macrophages are CD163 +In some embodiments, human M2 macrophages are HLA-DR + In some embodiments, the human M2 macrophages are CD14 - In some embodiments, human M2 macrophages are CD33 + In some embodiments, human M2 macrophages are CD66b - is.

[0163] [Table 1]

[0164] Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells produced during various pathological conditions and are thought to represent pathological states of monocyte and relatively immature neutrophil activation. MDSCs include at least two categories of cells: i) "granulocytic" (G-MDSCs) or polymorphonuclear (PMN-MDSCs), which are phenotypically and morphologically similar to neutrophils; and ii) monocytic (M-MDSCs), which are phenotypically and morphologically similar to monocytes. MDSCs are characterized by a set of distinct genomic and biochemical features and can be distinguished by specific surface molecules. In certain embodiments, suitable cell surface markers for identifying MDSCs may include one or more of CD11b, CD33, CD14, CD15, HLA-DR, and CD66b. For example, human G-MDSCs / PMN-MDSCs typically express the cell surface markers CD11b, CD33, CD15, and CD66b. In some embodiments, human G-MDSCs may express low levels of the CD33 cell surface marker. Human G-MDSCs / PMN-MDSCs may express LOX-1 and / or arginase. In contrast, human M-MDSCs typically express the cell surface markers CD11b, CD33, and CD14. Furthermore, both human G-MDSCs / PMN-MDSCs and M-MDSCs may exhibit low or undetectable levels of HLA-DR. In certain embodiments, G-MDSCs can be differentiated from M-MDSCs based on the presence or absence of specific cell surface markers (e.g., CD14, CD15, and / or CD66b). MDSCs may also express CD39 and CD73 to mediate adenosine signaling, which is involved in organ fibrosis (such as liver fibrosis and pulmonary fibrosis), cancer, and bone marrow fibrosis. Furthermore, human M-MDSCs may express HLA-DR. In some embodiments, human G-MDSC / PMN-MDSC may express LOX-1 and / or arginase. In contrast, human M-MDSC typically express the cell surface markers CD11b, CD33, and CD14. Furthermore, both human G-MDSC / PMN-MDSC and M-MDSC may exhibit low or undetectable levels of HLA-DR. In some embodiments, human G-MDSC may express HLA-DR.- In certain embodiments, G-MDSCs can be differentiated from M-MDSCs based on the presence or absence of specific cell surface markers (e.g., CD14, CD15, and / or CD66b). In some embodiments, G-MDSCs can be identified by the presence or increased expression of surface markers CD11b, CD33, CD15, CD66b, and / or LOX-1 and the absence of CD14, while M-MDSCs can be identified by the presence or increased expression of surface markers CD11b, CD33, and / or CD14 and the absence of CD15. In some embodiments, M-MDSCs can be identified by the presence or increased expression of surface markers CD66b - In addition to these cell surface markers, MDSCs may be characterized by their ability to suppress immune cells such as T cells, NK cells, and B cells. The immunosuppressive function of MDSCs may include inhibition of antigen-nonspecific functions and inhibition of antigen-specific functions. MDSCs, including tumor-associated MDSCs and circulating MDSCs, can express cell surface LRRC33 and / or LRRC33-proTGFβ1. In some embodiments, the signal intensity of cell surface markers may be classified or binned as "low," "medium," or "high" based on normalization of signal intensity to reduce background and bleed-through signals. In some embodiments, the signal intensity of cell surface markers may be classified based on the cutoff thresholds provided in Table 14A. In some embodiments, the signal intensity of cell surface markers may be determined by binary intensity selection. In some embodiments, binary intensity selection involves classifying the signal intensity measured for a particular cell surface marker as "positive" or "negative." In some embodiments, the signal intensity of cell surface markers may be classified based on the cutoff thresholds provided in Table 14B. In some embodiments, the signal intensities of a set of surface markers may be determined by sequential application of signal filtering, where a signal intensity threshold for one or more surface markers is determined before a threshold for one or more further surface markers is determined.

[0165] Myofibroblasts: Myofibroblasts are cells with a specific phenotype of fibroblasts and smooth muscle cells and generally express vimentin, α-smooth muscle actin (α-SMA; human gene ACTA2), and palladin. In many disease states involving extracellular matrix dysregulation (e.g., increased matrix stiffness), normal fibroblasts dedifferentiate into myofibroblasts in a TGFβ-dependent manner. Abnormal overexpression of TGFβ is common among myofibroblast-driven pathologies. TGFβ is known to promote myofibroblast differentiation, cell proliferation, and matrix production. Myofibroblasts or myofibroblast-like cells within a fibrotic microenvironment are sometimes referred to as fibrosis-associated fibroblasts (or "FAFs"), and myofibroblasts or myofibroblast-like cells within a tumor microenvironment are sometimes referred to as cancer-associated fibroblasts (or "CAFs").

[0166] Pan-TGFβ inhibitor / pan-TGFβ inhibitor: The term "pan-TGFβ inhibitor" or "pan-TGFβ inhibitor" refers to any agent capable of inhibiting or antagonizing all three isoforms of TGFβ. Such inhibitors may be small molecule inhibitors of TGFβ isoforms, such as those known in the art. This term includes pan-TGFβ antibodies, which refer to any antibody that can bind to each of the TGFβ isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3. In some embodiments, the pan-TGFβ antibody binds to and neutralizes the activity of all three isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3. The antibody 1D11 (or the human analog fresolimumab (GC1008)) is a well-known example of a pan-TGFβ antibody that neutralizes all three isoforms of TGFβ. An example of a small molecule pan-TGFβ inhibitor is galunisertib (LY2157299 monohydrate, CAS number 700874-72-2), an antagonist of TGFβ receptor I kinase / ALK5, which mediates signaling of all three TGFβ isoforms.

[0167] Perivascular (infiltration): The prefix "perivascular" means "around," "surrounding," or "near," so "perivascular" is taken literally as surrounding blood vessels. As used herein in reference to tumor cell infiltration, the term "perivascular infiltration" refers to the mode of entry for tumor-infiltrating immune cells (e.g., lymphocytes) through the vasculature of solid tumors.

[0168] Potency: As used herein, the term "potency" refers to the activity of a drug, such as an inhibitory antibody (or fragment) with inhibitory activity, in terms of the concentration or amount of drug that produces a defined effect. For example, an antibody that can produce a specific effect at a given dose is more potent than another antibody that requires twice the amount (dosage) to produce the same effect. Potency can be measured in a cell-based assay, such as a TGFβ activation / inhibition assay, whereby the extent of TGFβ activation, such as activation caused by integrin binding, can be measured in a cell-based system in the presence or absence of a test substance (e.g., an inhibitory antibody). Typically, among those that can bind to the same or overlapping binding region of an antigen (e.g., a cross-blocking antibody), those with higher affinity (lower K D An antibody with a lower affinity (higher K D These antibodies tend to exhibit higher potency than antibodies with higher IgG values.

[0169] Preclinical Model: The term "preclinical model" refers to a cell line or animal that exhibits certain characteristics of a human disease that is used to test the mechanism of action, efficacy, pharmacology, and toxicity of a drug, treatment, or therapy before it is tested on humans. Typically, cell-based preclinical studies are referred to as "in vitro" studies, and animal-based preclinical studies are referred to as "in vivo" studies. For example, in vivo mouse preclinical models encompassed by the present disclosure include the MBT2 bladder cancer model, the Cloudman S91 melanoma model, and the EMT6 breast cancer model.

[0170] Predictive Biomarkers: Predictive biomarkers provide information regarding the probability or likelihood of response to a particular therapy. Typically, predictive biomarkers are measured before and after treatment, and changes or relative levels of the marker in samples taken from a subject indicate or predict therapeutic benefit.

[0171] Presentation molecule: In the context of this disclosure, a presentation molecule refers to a protein that forms a covalent bond with a latent proprotein (e.g., proTGFβ1) to tether ("present") the inactive complex to an extracellular niche (e.g., ECM or immune cell surface, etc.), thereby maintaining its latency until an activation event occurs. Known presentation molecules for proTGFβ1 include LTBP1, LTBP3, GARP (also known as LRRC32), and LRRC33, each of which can form a presentation molecule-proTGFβ1 complex (i.e., LLC), i.e., LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1, respectively. In nature, LTBP1 and LTBP3 are components of the extracellular matrix (ECM); therefore, LTBP1-proTGFβ1 and LTBP3-proTGFβ1 can be collectively referred to as "ECM-associated" (or "matrix-associated") proTGFβ1 complexes that mediate ECM-associated TGFβ1 signaling / activity. On the other hand, GARP and LRRC33 are transmembrane proteins expressed on the cell surface of certain cells; therefore, GARP-proTGFβ1 and LRRC33-proTGFβ1 can be collectively referred to as "cell-associated" (or "cell surface") proTGFβ1 complexes that mediate cell-associated (e.g., immune cell-associated) TGFβ1 signaling / activity.

[0172] Protection (from solvent exposure): In the context of assessing protein-protein interactions, such as antibody-antigen binding, by HDX-MS, the degree to which a protein (e.g., a region of a protein containing an epitope) is exposed to solvent (whereby proton exchange occurs) is inversely correlated with the degree of binding / interaction. Thus, when an antibody described herein binds to a region of an antigen, that binding region is "protected" from solvent exposure because the protein-protein interaction makes the surrounding solvent inaccessible to the binding region. Thus, the protected region represents the site of interaction. Typically, a suitable solvent is a physiological buffer.

[0173] ProTGFβ1: As used herein, the term "proTGFβ1" is intended to encompass the precursor form of the inactive TGFβ1 complex, which contains the prodomain sequence of TGFβ1 within the complex. Thus, this term can include the proform as well as the latent form of TGFβ1. The terms "pro / latent TGFβ1" can be used interchangeably. The "pro" form of TGFβ1 exists prior to proteolytic cleavage at the furin site. Upon cleavage, the resulting form is said to be the "latent" form of TGFβ1. The "latent" complex remains noncovalently associated until a further activation trigger, such as an integrin-driven activation event. The proTGFβ1 complex consists of dimeric TGFβ1 proprotein polypeptides linked by disulfide bonds. The latent dimeric complex is covalently linked to a single presentation molecule via the cysteine ​​residue at position 4 (Cys4) of each proTGFβ1 polypeptide. The adjective "latent" can be used generally / broadly to describe the "inactive" state of TGFβ1 prior to an integrin-mediated or other activation event. The proTGFβ1 polypeptide contains a prodomain (LAP) and a growth factor domain (SEQ ID NO: 1119).

[0174] Regression: Tumor regression or tumor growth can be used as a measure of in vivo efficacy. For example, in preclinical settings, median tumor volume (MTV) and regression response criteria for treatment efficacy can be determined from the tumor volume of animals remaining in the study on the final day. Treatment efficacy can also be determined from the incidence and extent of regression responses observed during the study. Treatment may result in partial regression (PR) or complete regression (CR) of tumors in animals. Complete regression achieved in response to a therapy (e.g., administration of a drug) can be referred to as a "complete response," and subjects who achieve a complete response can be referred to as "complete responders." Thus, complete response excludes spontaneous complete regression. In some embodiments of preclinical tumor models, a PR response is defined as three consecutive measurements during the study period that are 50% or less of their Day 1 volume, and one or more of these three measurements are 13.5 mm or less. 3 In some embodiments, a CR response is defined as a tumor volume of 13.5 mm or greater on three consecutive measurements over the course of the study. 3 In preclinical models, animals with a CR response at the end of the study can be separately classified as tumor-free survivors (TFS). The term "effective tumor control" can be used to refer to the degree of tumor regression achieved in response to treatment, where, for example, tumor volume is reduced to <25% of the endpoint tumor volume in response to treatment. For example, in certain models, an endpoint tumor volume of 2,000 mm 3 If the tumor is 500 mm 3 Effective tumor control is achieved when the tumor shrinks to less than 100%. Therefore, effective tumor control encompasses complete regression as well as partial regression that reaches a threshold shrinkage. Similarly, the regression of fibrosis can be used as a measure of the in vivo efficacy of therapies such as TGFβ1 inhibitors. The regression of fibrotic status can be determined based on standard criteria for assessing the severity of fibrotic symptoms by stage.

[0175] Regulatory T cells: "Regulatory T cells" or Tregs are a type of immune cell characterized by the expression of the biomarkers CD4, FOXP3, and CD25. Tregs, also called suppressor T cells, are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells (Teff). Tregs originate in the thymus (so-called CD4+Foxp3+ "natural" Tregs) or differentiate from naive CD4+ T cells in the periphery, for example, after exposure to TGFβ or retinoic acid. Tregs can express cell surface GARP-proTGFβ1.

[0176] Resistance (to a treatment): Resistance to a particular treatment (e.g., CBT) can be due to an innate feature of a disease, such as cancer ("primary resistance," i.e., present before treatment begins) or an acquired phenotype that develops over time after treatment ("acquired resistance"). Patients who do not respond to a treatment (e.g., patients who are non-responders or patients who respond poorly to a treatment) are said to have primary or acquired resistance to that treatment and may be characterized as primary non-responders. Patients who have not previously received a treatment and do not respond to a treatment are said to have primary resistance. Patients who initially respond to a treatment but later lose response (e.g., progress or relapse despite continued therapy) are said to have acquired resistance to that treatment. In the context of immunotherapy, such resistance may represent immune escape.

[0177] Response Evaluation Criteria in Solid Tumors (RECIST) and iRECIST: RECIST is a set of published rules that define when a cancer patient's tumor improves ("responds"), stays the same ("stabilizes"), or worsens ("progresses") during treatment. The criteria were published in February 2000 by an international collaboration including the European Organization for Research and Treatment of Cancer (EORTC), the U.S. National Cancer Institute, and the National Cancer Institute of Canada Clinical Trials Group. A revised version of the RECIST guidelines (RECIST v 1.1) has since been widely adopted (see Eisenhauera et al., (2009), "New response evaluation criteria in solid tumor: Revised RECIST guideline (version 1.1)" Eur J Cancer 45:228-247; which are incorporated herein).

[0178] The criteria for determining treatment response are as follows: complete response (CR): disappearance of all target lesions; partial response (PR): at least a 30% reduction in the sum of the LD of target lesions, based on baseline total LD; stable disease (SD): neither a sufficient reduction to qualify for PR nor a sufficient increase to qualify for PD, based on the smallest total LD ​​since the start of treatment; progressive disease (PD): at least a 20% increase in the sum of the LD of target lesions, based on the smallest total LD ​​recorded since the start of treatment or the appearance of one or more new lesions.

[0179] On the other hand, iRECIST provides a modified set of criteria that takes immune-related responses into account (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). RECIST and iRECIST criteria are standardized, may be revised from time to time as more data becomes available, and are well understood in the art.

[0180] Response rate: As used herein, the term response rate (such as "low response rate") has the ordinary meaning understood by those skilled in the art of medicine, such as oncologists. Response rate is the proportion (e.g., fraction or percentage) of subjects in a patient population who show clinical improvement upon receiving treatment (e.g., pharmacological intervention), and may include complete and partial responses. In oncology, clinical improvement may include tumor shrinkage (e.g., partial response) or disappearance (e.g., complete response). When used as a clinical endpoint in clinical trials of cancer treatments, this is typically expressed as the objective response rate (ORR). The FDA defines ORR as the proportion of patients who have a reduction in tumor size by a predetermined amount in a minimum period of time. Reference: "Clinical Trial Endpoints for the Approval of Candidate Drugs and Biologics—Guidance for Industry," published by the USDaPt of Health and Human Services, Food and Drug Administration, Oncology Center of Excellence, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER), the contents of which are incorporated herein by reference.

[0181] Solid tumor: The term "solid tumor" refers to a proliferative disorder that results in an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Solid tumors include locally advanced solid tumors and tumors of advanced malignancies, such as metastatic cancer. Solid tumors typically consist of multiple cell types, including, but not limited to, cancerous (malignant) cells, stromal cells such as CAFs, and infiltrating leukocytes such as macrophages, MDSCs, and lymphocytes. Solid tumors to be treated with TGFβ1 isoform-selective inhibitors are typically TGFβ1-positive (TGFβ1+) tumors, such as those described herein, which may contain multiple cell types that produce TGFβ1. In certain embodiments, TGFβ1+ tumors can also co-express TGFβ3 (i.e., TGFβ3-positive). For example, certain tumors are TGFβ1 / 3-codominant. In some embodiments, such tumors are caused by cancer of epithelial cells, such as carcinomas. In certain embodiments, such tumors include ovarian cancer, breast cancer, bladder cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), prostate cancer (e.g., prostate adenocarcinoma), melanoma (e.g., cutaneous melanoma), lung cancer (e.g., lung squamous cell carcinoma and lung adenocarcinoma), liver cancer (e.g., hepatocellular carcinoma), uterine cancer (e.g., uterine endometrial cancer), 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 cancer, and tenosynovial giant cell tumor (TGCT). In some embodiments, the solid tumors treated herein, such as one or more of those listed above, exhibit elevated TGFβ1 expression compared to other tumor types and exhibit reduced responsiveness to mainstream therapies, such as genotoxic therapy. Therefore, TGFβ inhibitors (e.g., Ab6) can be used in combination with one or more genotoxic therapies (e.g., chemotherapy and / or radiation therapy, including radiation therapy) to treat such cancers in subjects.

[0182] Solution equilibrium titration (SET): SET is an assay method that can measure the binding between two molecules (e.g., an antigen and an antibody that binds to the antigen) at equilibrium in solution. For example, SET based on Meso-Scale Discovery ("MSD"), i.e., MSD-SET, is a useful method for determining the dissociation constant at equilibrium, particularly for high-affinity protein-protein interactions, such as picomolar affinity antibodies that bind to antigens (e.g., Ducata et al., (2015) J. Biomolecular Screening 20(10):1256-1267). SET-based assays are particularly useful for determining the KD values ​​of antibodies with subnanomolar (e.g., picomolar) affinity.

[0183] Specific binding: As used herein, the terms "specific binding" or "specifically binds" refer to the binding of an antibody or antigen-binding portion thereof to a particular structure (e.g., antigenic determinant or epitope) in an antigen with a particular affinity (e.g., K as measured by Biacore®). D For example, an antibody or antigen-binding portion thereof may bind to a specific protein rather than proteins in general. In some embodiments, an antibody or antigen-binding portion thereof may be used to demonstrate that the antibody binds to a target with at least about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less K D In some embodiments, the term "specifically binding to an epitope of proTGFβ1," "specifically binds to an epitope of proTGFβ1," "specifically binds to proTGFβ1," or "specifically binds to proTGFβ1," as used herein, refers to a molecule that binds to proTGFβ1 and has a binding affinity of 1.0×10 or more, as determined by a suitable in vitro binding assay, such as surface plasmon resonance and biolayer interferometry (BLI), to a target, such as TGFβ1. -8 The dissociation constant (K D In a preferred embodiment, the term "antibody" refers to an antibody or antigen-binding portion thereof having a kinetic rate constant (e.g., KD ) is determined by surface plasmon resonance (e.g., a Biacore system). In one embodiment, the antibody or antigen-binding portion thereof can specifically bind to both human and non-human (e.g., murine) orthologs of proTGFβ1. In some embodiments, an antibody may also "selectively" (i.e., "preferentially") bind to a target antigen if it binds to that target with a relative affinity that is relatively greater than the strength of binding exhibited for other antigens, for example, a 10-fold, 100-fold, 1000-fold, or more relative affinity for the target antigen (e.g., TGFβ1) compared to non-target antigens (e.g., TGFβ2 and / or TGFβ3). In preferred embodiments, an isoform-selective inhibitor shows no detectable binding or efficacy for other isoforms or counterparts. In some embodiments, an antibody that specifically binds to a set of antigens may have high affinity for the antigens but may not be able to distinguish between the antigens (i.e., the antibody is specific but not selective). In some embodiments, an antibody that binds to an antigen with particularly high affinity relative to other antigens may be considered selective for the antigen. For example, an antibody that binds to antigen X with 1000 times greater affinity than antigen Y can be considered an antibody that is selective for antigen X over antigen Y. In the context of this disclosure, an "antibody that specifically binds to an antigen with high affinity" generally has an affinity of 1.0 x 10 -8 Refers to KD below M.

[0184] Subject: In the context of therapeutic applications, the term "subject" refers to an individual receiving or in need of clinical care or intervention, such as treatment or diagnosis. Suitable subjects include, but are not limited to, vertebrates, including mammals (e.g., humans and non-human mammals). When the subject is a human subject, the term "patient" may be used interchangeably. In the clinical context, the term "patient population" or "patient subpopulation" is used to refer to a group of individuals who meet a set of criteria, such as clinical criteria (e.g., symptoms, stage of disease, susceptibility to a particular condition, responsiveness to treatment, etc.), medical history, health status, sex, age group, genetic criteria (e.g., carriers of particular mutations, polymorphisms, gene duplications, DNA sequence repeats, etc.), and lifestyle factors (e.g., smoking, alcohol consumption, exercise, etc.).

[0185] Surface Plasmon Resonance (SPR): Surface plasmon resonance is an optical phenomenon that allows unlabeled reactants to be detected in real time. SPR-based biosensors, such as those commercially available from Biacore, can be used to measure biomolecular interactions, including protein-protein interactions such as antigen-antibody binding. This technique is widely known in the art and is useful for measuring parameters such as binding affinity, reaction rate constants, and thermodynamics.

[0186] Target binding: As used herein, the term target binding refers to the ability of a molecule (e.g., a TGFβ inhibitor) to bind to its intended target (e.g., endogenous TGFβ) in vivo. In the case of an activated inhibitor, the intended target may be the large latent complex.

[0187] TGFβ1-related indication: "TGFβ1-related indication" refers to any disease or disorder and / or condition that is a TGFβ1-related disorder and whose etiology and / or progression are at least in part attributable to TGFβ1 signaling or dysregulation thereof. Certain TGFβ1-related disorders are primarily driven by TGFβ1 isoforms. Subjects with TGFβ1-related indications can benefit from inhibiting TGFβ1 activity and / or levels. Certain TGFβ1-related indications are primarily driven by TGFβ1 isoforms. TGFβ1-related indications include, but are not limited to, fibrosis (e.g., organ fibrosis and tissue fibrosis associated with chronic inflammation), proliferative disorders (cancer, such as solid tumors and myelofibrosis), diseases associated with ECM dysregulation (e.g., conditions involving matrix stiffening and remodeling), diseases involving mesenchymal transition (e.g., EndMT and / or EMT), diseases involving proteases, and diseases with aberrant gene expression of certain markers described herein. These disease categories are not intended to be mutually exclusive. According to some embodiments, the TGFβ1-related indication is fibrosis, for example, pulmonary fibrosis.

[0188] TGFβ inhibitor: The term "TGFβ inhibitor" refers to any agent capable of antagonizing the biological activity, signaling, or function of a TGFβ growth factor (e.g., TGFβ1, TGFβ2, and / or TGFβ3). This term is not intended to be limiting in terms of its mechanism of action and includes, for example, neutralizing inhibitors, receptor antagonists, soluble ligand traps, TGFβ activation inhibitors, and integrin inhibitors (e.g., antibodies that bind to αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibit downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3). This term encompasses TGFβ inhibitors that are isoform-selective and non-selective inhibitors. The latter include, for example, small molecule receptor kinase inhibitors (e.g., ALK5 inhibitors), antibodies that preferentially bind to two or more isoforms (such as neutralizing antibodies), and engineered constructs (e.g., fusion proteins) that contain ligand-binding moieties. In certain embodiments, these antibodies may contain or be engineered to contain mutations or modifications that result in an increased half-life of the antibody. In some embodiments, such mutations or modifications may be present in the Fc domain of the antibody (e.g., an Fc-modified antibody). In some embodiments, the mutation is a so-called YTE mutation. TGFβ inhibitors also include antibodies that can reduce the availability of latent proTGFβ that can be activated in the niche, e.g., by inducing antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADPC), as well as antibodies that can cause internalization of cell surface complexes containing latent proTGFβ, thereby removing precursors from the plasma membrane without depleting the cells themselves. Internalization may be a preferred mechanism of action of LRRC33-containing protein complexes (e.g., human LRRC33-proTGFβ1), resulting in a reduction in the levels of cells expressing the LRRC33-containing protein complex on the cell surface.

[0189] The "TGFβ family" is a class within the TGFβ superfamily, which in humans includes three structurally similar members: TGFβ1, TGFβ2, and TGFβ3. These three growth factors are known to signal through the same receptor.

[0190] TGFβ1-positive cancer / tumor: As used herein, this term refers to a cancer / tumor with aberrant TGFβ1 expression (overexpression). Many human cancer / tumor types exhibit predominant expression of the TGFβ1 (note that "TGFB" is sometimes used to refer to the gene as opposed to the protein) isoform. In some cases, such cancers / tumors may exhibit co-dominant expression of another isoform, such as TGFβ3. Some epithelial cancers (e.g., carcinomas) may co-express TGFβ1 and TGFβ3. Within the tumor environment of a TGFβ1-positive tumor, TGFβ1 can 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). In the context of the present disclosure, a preclinical cancer / tumor model that recapitulates the human condition is a TGFβ1-positive cancer / tumor.

[0191] Therapeutic window: The term "therapeutic window" refers to the dose / concentration range that produces a therapeutic response without causing significant / observable / unacceptable adverse effects in a subject (e.g., within the range of tolerable or acceptable adverse effects). The therapeutic window can be calculated as the ratio of the minimum effective concentration (MEC) to the minimum toxic concentration (MTC). For example, a TGFβ1 inhibitor that achieves in vivo efficacy at a dose of 10 mg / kg and exhibits tolerable or acceptable toxicity at 100 mg / kg provides a therapeutic window of at least 10 times (e.g., 10×). In contrast, a pan-inhibitor of TGFβ that is effective at 10 mg / kg but causes adverse effects at less than the effective dose (e.g., 5 mg / kg) is said to have "dose-limiting toxicity." In general, the maximum tolerated dose (MTD) can set the upper limit of the therapeutic window. For example, Ab6 has been found to be effective at doses ranging from about 3 to 30 mg / kg / week, and has also demonstrated no observable toxicity associated with pan-TGFβ inhibition in rats or non-human primates at doses of at least 100 or 300 mg / kg / week for 4 weeks. Based on this, Ab6 exhibits a therapeutic window of at least 3.3-fold and up to 100-fold. In some embodiments, the concept of a therapeutic window can be expressed in terms of a safety factor.

[0192] Toxicity: As used herein, the term "toxicity" or "toxicity" refers to undesirable in vivo effects in a subject (e.g., patient), e.g., unwanted side effects and adverse events, associated with a therapy administered to the subject (e.g., patient). "Tolerability" refers to the level of toxicity associated with a treatment or therapeutic regimen that can be reasonably tolerated by a patient without discontinuing treatment due to toxicity. Typically, toxicity / toxicology testing is conducted in one or more preclinical models prior to clinical development to evaluate the safety profile of a drug candidate (e.g., monoclonal antibody therapy). Toxicity / toxicology testing helps determine the "no observed adverse effect level (NOAEL)" and "maximum tolerated dose (MTD)" of a test substance, based on which a therapeutic window can be estimated. Preferably, a species shown to be sensitive to a particular intervention should be selected as the preclinical animal model in which safety / toxicity testing is performed. In the case of TGFβ inhibition, suitable species include rats, dogs, and cynomolgus monkeys. Although mice have been reported to be less sensitive to pharmacological inhibition of TGFβ and may not exhibit potentially dangerous toxicities in other species, including humans, certain studies have reported toxicities observed with pan-inhibition of TGFβ in mice. To illustrate in the context of this disclosure, the NOAEL for Ab6 in rats was the highest dose evaluated (100 mg / kg) based on a 4-week toxicity study, suggesting that the MTD is >100 mg / kg. The MTD for Ab6 in non-human primates is >300 mg / kg based on a 4-week toxicity study.

[0193] To determine the NOAEL and MTD, preferably, a species that has proven sensitive to a particular intervention should be selected as the preclinical animal model in which safety / toxicity testing is conducted. In the case of TGFβ inhibition, suitable species include, but are not limited to, rats, dogs, and cynomolgus monkeys. Mice have been reported to be less sensitive to pharmacological inhibition of TGFβ and may not reveal potentially serious or dangerous toxicities in other species, including humans.

[0194] Translatability: In the context of drug discovery and clinical development, the term "translatability" or "translatable" refers to a particular quality or characteristic of a preclinical model or data that recapitulates a human pathology. As used herein, preclinical models that recapitulate TGFβ1 indications typically exhibit predominant expression of TGFβ1 (or TGFβ1) compared to TGFβ2 (or TGFβ2) and TGFβ3 (or TGFβ3). For example, in a combination therapy paradigm, translatability may require the same underlying mechanism of action that the combination of active agents in the model aims to achieve. As an example, many human tumors are immune-excluded TGFβ1-positive tumors that exhibit primary resistance to checkpoint blockade therapy (CBT). To overcome resistance to CBT, a second therapy (such as a TGFβ1 inhibitor) can be used in combination. In this scenario, an appropriate translatable preclinical model includes a TGFβ1-positive tumor that exhibits primary resistance to checkpoint blockade therapy (CBT).

[0195] Treat / Treatment: The term "treat" or "treatment" includes therapeutic treatments, prophylactic treatments, and applications that reduce a subject's risk of developing a disorder or other risk factor. Thus, the term is intended to be broadly interpreted: providing a therapeutic benefit to a patient by, for example, slowing disease progression, reversing certain disease characteristics, normalizing gene expression, or enhancing the body's immunity; reducing or reversing immunosuppression; reducing, removing, or eradicating harmful cells or substances from the body; reducing disease burden (e.g., fibrosis and tumor burden); preventing recurrence or relapse; extending refractory periods, and / or otherwise improving survival. The term includes therapeutic treatments, prophylactic treatments, and applications that reduce a subject's risk of developing a disorder or other risk factor. Treatment does not require a complete cure of a disorder, but encompasses embodiments that reduce symptoms or underlying risk factors. In the context of combination therapy, the term may also refer to: i) the ability of the second therapeutic agent to reduce the effective dose of the first therapeutic agent, thereby reducing side effects and increasing tolerability; ii) the ability of the second therapeutic agent to make the patient more responsive to the first therapeutic agent; and / or iii) the ability to provide additive or synergistic clinical benefit.

[0196] Tumor-associated macrophages (TAMs): TAMs are polarized / activated macrophages with a tumor-promoting phenotype (M2-like macrophages). TAMs can be either monocytes / macrophages of bone marrow origin recruited to tumor sites or tissue-resident macrophages derived from erythroid-myeloid progenitor cells. The differentiation of monocytes / macrophages into TAMs is influenced by many factors, including local chemical signals such as cytokines, chemokines, growth factors, and other molecules acting as ligands, as well as cell-cell interactions between monocytes / macrophages present in the niche (tumor microenvironment). Generally, monocytes / macrophages can polarize into so-called "M1" or "M2" subtypes, the latter associated with a more tumor-promoting phenotype. In solid tumors, up to 50% of the tumor mass can correspond to macrophages that are predominantly M2-polarized. Among tumor-associated monocytic and myeloid cell populations, M1 macrophages typically express cell surface HLA-DR, CD68, and CD86, whereas 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-proTGFβ1. M2-like macrophages can also be enriched in fibrotic microenvironments.

[0197] Tumor microenvironment: The term "tumor microenvironment (TME)" refers to the local disease niche in which a tumor (e.g., a solid tumor) resides in vivo. The TME can include disease-associated molecular signatures (collections of chemokines, cytokines, etc.), disease-associated cell populations (TAMs, CAFs, MDSCs, etc.), and disease-associated ECM milieu (alterations in ECM components and / or structure).

[0198] Valvular disease: The term "valvular disease" refers to a disease, disorder, or condition that affects one or more of the four valves of the heart and is often characterized by pathology of the heart valves. It is also commonly known as valvular heart disease or valvular heart disease. Types of valvular disease include, but are not limited to, aortic valve disease (e.g., aortic stenosis), mitral valve disease, tricuspid valve disease, and pulmonary valve disease.

[0199] Variable region: The term "variable region" or "variable domain" refers to a portion of an antibody light and / or heavy chain, typically comprising about 120-130 amino acids in the heavy chain and about 100-110 amino-terminal amino acids in the light chain. In certain embodiments, the variable regions of different antibodies vary significantly in amino acid sequence, even among antibodies of the same species. The variable regions of an antibody typically determine the specificity of a particular antibody for its target.

[0200] Except in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood in all instances as modified by the term "about," which means ±10% of the stated value.

[0201] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0202] As used herein in the specification and claims, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements present conjunctively in some cases and disjunctively in other cases. Other elements, whether related or unrelated to the elements specifically identified, may optionally be present other than the elements specifically identified, unless clearly indicated to the contrary. Thus, as a non-limiting example, when a reference to "A and / or B" is used in combination with an open-ended term such as "comprising," this can indicate, in one embodiment, A without B (optionally including elements other than B); in another embodiment, B without A (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements), etc.

[0203] As used herein in the specification and claims, 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 in the list of elements, and not excluding all combinations of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to the elements specifically set forth. Thus, as a non-limiting example, "at least one of A and B" (or, similarly, "at least one of A or B" or, similarly, "at least one of A and / or B") can refer to, in one embodiment, no B and at least one (optionally including more than one) A (and optionally including elements other than B); in another embodiment, no A and at least one (optionally including more than one) B (and optionally including elements other than A); in yet another embodiment, 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.

[0204] The use of ordinal terms such as "first," "second," and "third" in the claims to modify claim elements does not, by itself, imply any priority, precedence, or order of one claim element over another, or the chronological order in which acts of a method are performed, but is merely used as a label to distinguish one claim element having a particular name from another element having the same name for purposes of distinguishing claim elements (other than the use of ordinal terms).

[0205] Ranges provided herein are understood to be shorthand for all values ​​within that range, for example, a range of 1 to 50 is understood to encompass any and all values, combinations of values, or subranges 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.

[0206] Transforming growth factor beta (TGFβ) Transforming growth factor beta (TGFβ) activity and the subsequent partial purification of the soluble growth factor were first described in the late 1970s and early 1980s, thus initiating the field of TGFβ approximately 40 years ago. To date, 33 gene products that constitute the large TGFβ superfamily have been identified. The TGFβ superfamily can be divided into at least three subclasses based on structural similarity: TGFβ, growth differentiation factors (GDFs), and bone morphogenetic proteins (BMPs). The TGFβ subclass consists of three highly conserved isoforms, TGFβ1, TGFβ2, and TGFβ3, which are encoded by three distinct genes in humans.

[0207] [Table 2]

[0208] TGF-β is thought to play a key role in diverse processes, including cell proliferation inhibition, extracellular matrix (ECM) remodeling, and immune homeostasis. The importance of TGF-β1 for T cell homeostasis is demonstrated by the observation that TGF-β1- / - mice survive only 3-4 weeks and then die due to multiple organ failure caused by intense immune activation (Kulkarni, AB, et al., Proc Natl Acad Sci USA, 1993. 90(2):770-4; Shull, MM, et al., Nature, 1992. 359(6397):693-9). The roles of TGF-β2 and TGF-β3 are less clear. Although the three TGFβ isoforms have distinct temporal and spatial expression patterns, they signal through the same receptors, TGFβRI and TGFβRII, although in some cases, they also require type III receptors, such as betaglycan, for TGFβ2 signaling (Feng, XH and R. Derynck, Annu Rev Cell Dev Biol, 2005.21:659-93; Massague, J., Annu Rev Biochem, 998.67:753-91). Ligand-induced oligomerization of TGFβRI / II triggers phosphorylation of SMAD transcription factors, resulting in the transcription of target genes such as Col1a1, Col3a1, ACTA2, and SERPINE1 (Massague, J., J. Seoane, and D. Wotton, Genes Dev, 2005.19(23):2783-810). SMAD-independent TGFβ signaling pathways have also been described, for example, in cancer and in aortic lesions in Marfan syndrome mice (Derynck, R. and YE Zhang, Nature, 2003. 425(6958): p. 577-84; Holm, TM, et al., Science, 2011. 332(6027): p. 358-61).

[0209] The biological importance of the TGF-β pathway in humans is validated by genetic disorders. Camurati-Engelman disease results from an autosomal dominant mutation in the TGFB1 gene, resulting in dysplastic bone formation and constitutive activation of TGF-β1 signaling (Janssens, K., et al., J Med Genet, 2006, 43(1):pp. 1-11). Patients with Loeys-Dietz syndrome have autosomal dominant mutations in components of the TGF-β signaling pathway, which cause aortic aneurysms, sequestration, and a bifid uvula (Van Laer, L., H. Dietz, and B. Loeys, Adv Exp Med Biol, 2014, 802:pp. 95-105). Because TGF-β pathway dysregulation has been implicated in multiple diseases, several drugs targeting the TGF-β pathway have been developed and tested in patients, but with limited success.

[0210] Dysregulation of TGFβ signaling is associated with a wide variety of human diseases. Indeed, in many disease states, such dysregulation may involve multiple aspects of TGFβ function. Affected tissues, such as fibrotic and / or inflamed tissues and tumors, may form local environments in which TGFβ activation can lead to disease exacerbation or progression, which may be mediated at least in part by the interaction of multiple TGFβ-responsive cells that are activated in an autocrine and / or paracrine manner, along with several other cytokines, chemokines, and growth factors that play a role in specific disease settings.

[0211] For example, the tumor microenvironment (TME) contains multiple cell types that express TGFβ1, including cancer (i.e., malignant) cells as well as activated myofibroblast-like fibroblasts, stromal cells, infiltrating macrophages, MDSCs, and other immune cells. Thus, the TME represents a heterogeneous population of cells within the niche that express and / or respond to TGFβ1, but that are associated with multiple types of presentation molecules, such as LTBP1, LTBP3, LRRC33, and GARP.

[0212] Advances in immunotherapy have transformed the treatment landscape available to an increasing number of cancer patients. Most notably, checkpoint blockade therapy (CBT) has become part of standard treatment regimens for an ever-increasing number of cancers. While robust and durable responses to CBT have been observed in an increasing number of cancer types, it is clear that a significant fraction of tumors appear unresponsive to CBT even at the onset of treatment. Thus, primary resistance presents a major challenge to enabling many patients' immune systems to target and eliminate tumor cells. Efforts to understand and address the underlying mechanisms conferring primary resistance to CBT have been undertaken to extend therapeutic efficacy to more patients. However, this enthusiasm has been limited by lackluster clinical trial results and failures when CBT is combined with drugs known to affect the same tumor type or to modulate seemingly related components of the immune system. A possible reason is that a clear mechanistic rationale for a given combination is often not rooted in clinically obtained data, thus leading to uncertain and confounding outcomes in trials intended to enhance approved monotherapies. It has become clear that the design of combination immunotherapies must be rooted in scientific evidence of links to basic tumor and immune system biology.

[0213] Recently, the phenomenon known as "immune exclusion" has been coined to describe the tumor environment in which antitumor effector T cells (e.g., CD8+ T cells) are kept away (and thus "eliminated") by immunosuppressive local cues. More recently, numerous retrospective analyses of clinically derived tumors have implicated TGFβ pathway activation in mediating primary resistance to CBT. For example, transcriptional profiling and analysis of pretreatment melanoma biopsies revealed enrichment of TGFβ-related pathways and biological processes in tumors unresponsive to anti-PD-1 CBT. In immune-excluded tumors, effector cells that would otherwise be able to attack cancer cells by recognizing cell-surface tumor antigens are prevented from accessing the cancer site. In this way, cancer cells evade host immunity and evade immuno-oncological therapeutics, such as checkpoint inhibitors, that exploit and rely on such immunity. Indeed, such tumors are resistant to checkpoint blockade, such as anti-PD-1 and anti-PD-L1 antibodies, likely because targeted T cells are prevented from entering the tumor and therefore are unable to exert their anticancer effects.

[0214] Several retrospective analyses of clinically derived tumors have pointed to TGFβ pathway activation in mediating primary resistance to CBT. For example, transcriptional profiling and analysis of pretreatment melanoma biopsies revealed enrichment of TGFβ-related pathways and biological processes in tumors unresponsive to anti-PD-1 CBT. More recently, a similar analysis of tumors from patients with metastatic urothelial carcinoma found that the lack of response to PD-L1 blockade with atezolizumab was associated with a transcriptional signature of TGFβ signaling, particularly in tumors where CD8+ T cells appear to be excluded from tumor entry. The critical role of TGFβ signaling in mediating immune exclusion leading to anti-PD-(L)1 resistance has been confirmed in the EMT-6 syngeneic mouse model of breast cancer. EMT-6 tumors respond poorly to treatment with anti-PD-L1 antibodies, but combining this checkpoint inhibitor with 1D11, an antibody that blocks the activity of all TGFβ isoforms, significantly increased the frequency of complete responses compared with treatment with individual inhibitors. The synergistic antitumor activity is suggested to be due to the alteration of the cancer-associated fibroblast (CAF) phenotype and the resolution of the immune exclusion phenotype, leading to the infiltration of activated CD8+ T cells into the tumor. Similar results were found in a mouse model of colorectal cancer and metastasis using a combination of an anti-PD-L1 antibody and galunisertib, a small molecule inhibitor of the type I TGFβ receptor ALK5 kinase. Collectively, these findings suggest that inhibiting the TGFβ pathway in CBT-resistant tumors may be a promising approach to improve or increase the number of clinical responses to CBT. While recent studies suggest a relationship between TGFβ pathway activation and primary CBT resistance, TGFβ signaling has long been associated with hallmarks of cancer pathogenesis. As a potent immunosuppressant, TGFβ blocks antitumor T cell activity and promotes immunosuppressive macrophages. Malignant cells often become resistant to TGFβ signaling and its tumor-suppressing effects as a mechanism to avoid their proliferation. TGFβ activates CAFs, inducing extracellular matrix production and promoting tumor progression. Finally, TGFβ induces EMT, thus supporting tissue invasion and tumor metastasis.

[0215] Mammals have distinct genes encoding and expressing the three TGFβ growth factors TGFβ1, TGFβ2, and TGFβ3, all of which signal through the same heteromeric TGFβ receptor complex. Despite a common signaling pathway, each TGFβ isoform appears to have distinct biological functions, as evidenced by the phenotypes of non-overlapping TGFβ knockout mice. All three TGFβ isoforms are expressed as inactive prodomain-growth factor complexes, and the TGFβ prodomain, also known as latency-associated peptide (LAP), envelops the growth factor and maintains it in a latent, non-signaling state. Furthermore, latent TGFβ is coexpressed with latent TGFβ binding protein, forming a large latent complex (LLC) via disulfide bonds. Binding of latent TGFβ to latent TGFβ binding protein-1 (LTBP1) or LTBP3 allows tethering to the extracellular matrix, whereas binding to the transmembrane proteins GARP and LRRC33 allows assimilation onto the surface of Tregs or macrophages, respectively. In vivo, latent TGF-β1 and latent TGF-β3 are activated by a subset of αV integrins, which bind to a consensus RGD sequence on LAP and trigger a conformational change that releases growth factors. The mechanism by which latent TGF-β2 is activated is less clear due to the lack of a consensus RGD motif. Proteolytic cleavage of LAP to release TGF-β1 has also been implicated as an activation mechanism, but its biological relevance is less clear.

[0216] While the pathogenic role of TGFβ activation is evident in several disease states, it is equally clear that therapeutic targeting of the TGFβ pathway has been challenging due to the pleiotropic effects resulting from broad and sustained pathway inhibition. For example, several studies have demonstrated that small-molecule-mediated inhibition of the TGFβ type I receptor kinase ALK5 (TGFβR1) or blockade of all three highly related TGFβ growth factors with high-affinity antibodies caused severe valvular heart disease in mice, rats, and dogs. Thus, these "pan" TGFβ approaches that block all TGFβ signaling have a very narrow therapeutic window, which has proven to be an obstacle to treating numerous disease-related processes with significant unmet medical need. To date, no TGFβ-targeted therapies have been approved, and the results of clinical trials using such modalities have been largely disappointing, likely due to the use of ineffective dosing regimens required to address safety concerns.

[0217] All references cited herein are incorporated by reference for any purpose. In the event of a conflict between a reference and the present specification, the present specification shall control. While certain features of the disclosed compositions and methods are described herein in the context of separate embodiments for clarity, it will be understood that they may also be provided in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods are described in the context of a single embodiment for brevity, but they may also be provided individually or in any subcombination.

[0218] Highly active TGFβ1 selective inhibitor The present disclosure provides a method for the production of thrombin-containing ... D ) and a low dissociation rate (i.e., low k OFFThe present invention provides monoclonal antibodies and antigen-binding fragments thereof capable of binding to TGFβ1 at the TGFβ1 isoform (SEQ ID NO: 276), which can be used to treat fibrotic diseases and disorders, particularly pulmonary fibrosis. These antibodies and antigen-binding fragments include isoform-selective inhibitors of TGFβ1. One example of such an antibody or antigen-binding fragment thereof comprises H-CDR1, H-CDR2, and H-CDR3, L-CDR1, L-CDR2, and L-CFR3, wherein H-CDR1 comprises GFTFADYA (SEQ ID NO: 276); H-CDR2 comprises a sequence represented by the formula ISGSGX1AT (wherein, optionally, X1 is A or K) (SEQ ID NO: 277); and H-CDR3 comprises a sequence represented by the formula VSSGX1WDX2D (wherein, optionally, X1 is H, D, or Q, and further optionally, X2 is F or and X4 is S or T) (SEQ ID NO: 280); and L-CDR3 comprises a sequence represented by the formula QQTYX1VPLT (wherein, optionally, X1 is T or G) (SEQ ID NO: 281). In a preferred embodiment, H-CDR2 comprises ISGSGAAT (SEQ ID NO: 282), H-CDR3 comprises VSSGHWDYD (SEQ ID NO: 287), and L-CDR2 comprises AASGLES (SEQ ID NO: 284); and L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285). In some embodiments, the antibody or fragment thereof binds to an epitope that includes one or more of the following amino acid residues of the proTGFβ1 polypeptide sequence: S35, G37, E38, V39, P40, P41, G42, P43, R274, K280, H283, and K309.In some embodiments, H-CDR1 may comprise the sequence GFTFADYA (SEQ ID NO: 276); H-CDR2 may comprise the sequence ISGSGAAT (SEQ ID NO: 282); H-CDR3 may comprise the sequence represented by the formula VSSGX1WDX2D (wherein, optionally, X1 is H or Q, and further optionally, X2 is Y or F) (SEQ ID NO: 283); L-CDR1 may comprise the sequence QSISSY (SEQ ID NO: 279); L-CDR2 may comprise the sequence AASGLES (SEQ ID NO: 284); L-CDR3 may comprise the sequence QQTYGVPLT (SEQ ID NO: 285). In a preferred embodiment, H-CDR3 is VSSGHWDYD (SEQ ID NO: 287). In some embodiments, the antibody or fragment thereof binds to an epitope that includes one or more of the following amino acid residues of the proTGFβ1 polypeptide sequence: S35, G37, E38, V39, P40, P41, G42, P43, R274, K280, H283, and K309.

[0219] The table below shows the CDR sequences of useful variants.

[0220] [Table 3]

[0221] Optionally, one or more of the six CDRs may contain one or more (eg, one or two) amino acid changes.

[0222] In some embodiments, an antibody or antigen-binding fragment thereof selected for use or production in accordance with the present disclosure comprises H-CDR1, H-CDR2, and H-CDR3, L-CDR1, L-CDR2, and L-CFR3, wherein H-CDR1 comprises GFTFADYA (SEQ ID NO: 276); H-CDR2 comprises a sequence represented by the formula ISGSGX1AT, where optionally, X1 is A or K (SEQ ID NO: 277); and H-CDR3 comprises a sequence represented by the formula VSSGX1WDX2D, where optionally, X1 is H, D, or Q, and optionally and optionally, X2 is F or Y (SEQ ID NO: 278); L-CDR1 comprises QSISSY (SEQ ID NO: 279); L-CDR2 comprises a sequence represented by the formula AASX1X2X3X4 (wherein, optionally, X1 is N, G, or V; further optionally, X2 is L and N is E; further optionally, X3 is Q or E; and further optionally, X4 is S or T) (SEQ ID NO: 280); and L-CDR3 comprises a sequence represented by the formula QQTYX1VPLT (wherein, optionally, X1 is T or G) (SEQ ID NO: 281). In preferred embodiments, H-CDR2 comprises ISGSGAAT (SEQ ID NO: 282); H-CDR3 comprises VSSGHWDYD (SEQ ID NO: 287); L-CDR2 comprises AASGLES (SEQ ID NO: 284); and L-CDR3 comprises QQTYGVPLT (SEQ ID NO: 285). In some embodiments, the antibody or fragment thereof binds to an epitope comprising one or more of the following amino acid residues of the proTGFβ1 polypeptide sequence: S35, G37, E38, V39, P40, P41, G42, P43, R274, K280, H283, and K309.

[0223] The table below provides further CDR sequences of useful variants.

[0224] [Table 4]

[0225] In some embodiments, one or more of the six CDRs may contain one or more (eg, one or two) amino acid changes.

[0226] Non-limiting examples of preferred inhibitors of TGFβ1 activation are shown in the table below, and are referred to herein as Ab37, Ab38, Ab39, Ab40, Ab41, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, and Ab52. Each of these antibodies may be in the form of a whole immunoglobulin (e.g., IgG) or an antigen-binding fragment thereof, such as a Fab fragment. The antigen-binding fragment can be used to generate engineered constructs comprising the fragment or derivative thereof (e.g., bispecific antibodies and other fusion proteins that function as TGFβ1 inhibitors). The six CDRs of each exemplary antibody are listed in the table below. In some embodiments, the inhibitor of TGFβ1 activation is AB46.

[0227] [Table 5]

[0228] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), where V H is the following: EVQLLESGGGLVQPGGSLRLSCAASGFTFADYAMTWVRQAPGKGLEWVSAISGSGAATYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDYDYWGQGTLVTVSS (SEQ ID NO: 297) and V 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 with L is the following: DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASGLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGVPLTFGGGTKVEIK (SEQ ID NO: 298) In some embodiments, the antibody or fragment thereof binds to an epitope comprising one or more of the following amino acid residues of the proTGFβ1 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.

[0229] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), where V H is the following: EVQLLESGGGLVQPGGSLRLSCAASGFTFADYAMTWVRQAPGKGLEWVSAISGSGAATYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDYDYWGQGTLVTVSS (SEQ ID NO: 297) Including V L is the following: DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASGLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGVPLTFGGGTKVEIK (SEQ ID NO: 298) Includes:

[0230] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), where V H is the following: EVQLLESGGGLVQPGGSLRLSCAASGFTFADYAMTWVRQAPGKGLEWVSAISGSGAATYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDFDYWGQGTLVTVSS (Sequence number 299) Including V Lis the following: DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYTVPLTFGGGTKVEIK (SEQ ID NO: 300) Includes:

[0231] The present disclosure includes nucleic acid sequences encoding any one of the aforementioned amino acid sequences. Included herein are vectors (e.g., DNA plasmids and related nucleic acid formulations, such as mammalian expression vectors) comprising the nucleic acid sequences; cells transfected with the vectors; cell lines with stable expression of the nucleic acids; and cell cultures comprising the cells, optionally wherein the cell cultures comprise mammalian cells capable of large-scale production of protein constructs comprising antibodies or antigen-binding fragments of antibodies.

[0232] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof that selectively inhibits activation of TGFβ1 comprises: a heavy chain complementarity 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 complementarity 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 complementarity 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 complementarity 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 complementarity determining region 2 (CDRL2) having an amino acid sequence at least 95% identical to the sequence set forth in AASGLES (SEQ ID NO: 284); and a light chain complementarity determining region 3 (CDRL3) having an amino acid sequence at least 95% identical to the sequence set forth in QQTYGVPLT (SEQ ID NO: 285).

[0233] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof that selectively inhibits activation of TGFβ1 comprises: a heavy chain complementarity 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 complementarity 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 complementarity 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 complementarity 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 complementarity determining region 2 (CDRL2) having an amino acid sequence at least 96% identical to the sequence set forth in AASGLES (SEQ ID NO: 284); and a light chain complementarity determining region 3 (CDRL3) having an amino acid sequence at least 96% identical to the sequence set forth in QQTYGVPLT (SEQ ID NO: 285).

[0234] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof that selectively inhibits activation of TGFβ1 comprises: a heavy chain complementarity 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 complementarity 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 complementarity 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 complementarity 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 complementarity determining region 2 (CDRL2) having an amino acid sequence at least 98% identical to the sequence set forth in AASGLES (SEQ ID NO: 284); and a light chain complementarity determining region 3 (CDRL3) having an amino acid sequence at least 98% identical to the sequence set forth in QQTYGVPLT (SEQ ID NO: 285).

[0235] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof that selectively inhibits activation of TGFβ1 comprises: a heavy chain complementarity 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 complementarity 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 complementarity 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 complementarity 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 complementarity determining region 2 (CDRL2) having an amino acid sequence at least 99% identical to the sequence set forth in AASGLES (SEQ ID NO: 284); and a light chain complementarity determining region 3 (CDRL3) having an amino acid sequence at least 99% identical to the sequence set forth in QQTYGVPLT (SEQ ID NO: 285).

[0236] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof that selectively inhibits activation of TGFβ1 has the following: heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in GFTFADYA (SEQ ID NO: 276); heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in ISGSGAAT (SEQ ID NO: 282); heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in VSSGHWDYD (SEQ ID NO: 287); light chain complementarity determining region 1 (CDRL1) having the amino acid sequence set forth in QSISSY (SEQ ID NO: 279); light chain complementarity determining region 2 (CDRL2) having the amino acid sequence set forth in AASGLES (SEQ ID NO: 284); and light chain complementarity determining region 3 (CDRL3) having the amino acid sequence set forth in QQTYGVPLT (SEQ ID NO: 285). In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V) comprising a sequence having, comprising, or consisting of at least 95% identity, 96% identity, 97% identity, 98% identity, 99% identity to SEQ ID NO: 297. H) and a light chain variable region (V) comprising a sequence having, comprising, or consisting of at least 95% identity, 96% identity, 97% identity, 98% identity, 99% identity to SEQ ID NO: 298. L ) and

[0237] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: EVQLVQSGGVVVQPGGSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSLISWDGGSTYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKDADDSTFDIWGQGTMVTVSS (SEQ ID NO: 1173) and the VL has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%) sequence identity with: ETTLTQSPATLSVSPGERVTLSCRASQSVSRNLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSVPYTFGQGTKLEIK (SEQ ID NO: 1174) In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH is selected from the group consisting of: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPMFGTTNYAQKFQGRVTIIADESTSTAYMELRSLRSDDTAVYYCARDREWEPAYGMDVWGQGTTVTVSS (SEQ ID NO: 1175) and the VL has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity with: QSALTQPASVSGSPGQSITISCIGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEAMYYCSAYTVSSTWVFGGGTKVTVL (SEQ ID NO: 1176) and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%) sequence identity with

[0238] Antibody binding kinetics The antibodies and antigen-binding fragments thereof (e.g., Fabs) disclosed herein are characterized by enhanced binding properties. These antibodies and antigen-binding fragments can specifically bind to presentation molecule-proTGFβ1 complexes (sometimes referred to as "large latent complexes" or LLCs, which are trimeric complexes consisting of proTGFβ1 dimers bound to a single presentation molecule), i.e., LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. Recombinantly produced and purified protein complexes can be used as antigens (e.g., antigen complexes) to screen, evaluate, or confirm the ability of antibodies to bind to the antigen complex in appropriate in vitro binding assays. Such assays are well known in the art and include assays using biolayer interferometry (BLI) (e.g., OCTET®) and surface plasmon resonance (SPR) (e.g., BIACORE®).

[0239] To date, we have reported that ATP possesses high affinity (e.g., subnanomolar K) for LLC. D ) have been identified. Advantageously, in the present disclosure, antibodies and fragments have been specifically selected that have a particularly slow dissociation rate, with the aim of achieving a particularly long-lasting inhibitory effect.

[0240] Treatment methods and biomarkers of treatment efficacy Circulating / circulating MDSCs as biomarkers MDSCs are a heterogeneous population of cells named for their myeloid origin and their primary immunosuppressive function (Gabrilovich. Cancer Immunol Res. 2017 Jan;5(1):3-8). MDSCs generally exhibit high plasticity and a potent ability to attenuate the cytotoxic function of T cells and natural killer (NK) cells, including the ability to promote T regulatory cell (Treg) proliferation and thus suppress T effector cell function (Gabrilovich et al., Nat Rev Immunol. (2012) 12:253-68). MDSCs are typically classified into two subsets based on the expression of surface markers: monocytic (m-MDSCs) and granulocytic (G-MDSCs or PMN-MDSCs) (Consonni et al., Front Immunol. 2019 May 3;10:949). Suppressive G-MDSCs can be characterized by the production of reactive oxygen species (ROS) as a primary mechanism of immunosuppression, whereas M-MDSCs mediate immunosuppression primarily by upregulating the inducible nitric oxide synthase gene (iNOS), producing nitric oxide (NO) and an array of immunosuppressive cytokines (Youn and Garilovich, Eur J Immunol. 2010 Nov;40(11):2969-2975).

[0241] MDSCs have been implicated in a variety of diseases, including chronic inflammation, infection, autoimmune diseases, and graft-versus-host disease. In recent years, MDSCs have become a notable immune population in cancer due to their role in inducing T cell tolerance through checkpoint inhibitors such as programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA4) (Trovato et al., J Immunother Cancer. 2019 Sep 18;7(1):255). Furthermore, MDSCs have generally been characterized as supporting tumor progression through mechanisms in addition to immunosuppression, including promoting tumor angiogenesis. Previous studies have focused on MDSCs present in tumor biopsies due to their tendency to be enriched around inflammatory tissues (Passro et al., Clin Transl Oncol. 2019 Jun 28; Ai et al., BMC Cancer. 2018 Dec 5;18(1):1220; Nakamura. Front Med (Lausanne). 2019;6:119). However, no such studies have reported in the literature that elucidated a clear relationship between MDSC levels and treatment response. For example, low baseline monocytic MDSC frequencies demonstrated a poor correlation with treatment benefit (Pico de Coana et al., Oncotarget. 2017 Mar 28;8(13):21539-21553).

[0242] Many human cancers (e.g., solid tumors) are known to exhibit elevated levels of MDSCs in biopsies from patients compared to healthy controls (e.g., reviewed in Elliott et al., (2017) Frontiers in Immunology, Vol. 8, Article 86). These human cancers include, but are not limited to, bladder cancer, colorectal cancer, prostate cancer, breast cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, lung cancer, melanoma, NSCLC, ovarian cancer, pancreatic cancer, and renal cell carcinoma. The compositions and methods according to the present disclosure may be applied to one or more of these cancers.

[0243] Previously, we demonstrated that immunosuppressive tumors contain elevated levels of tumor-infiltrating or intratumor MDSCs, also referred to as tumor-associated MDSCs, and this evidence clearly indicates that this is inversely correlated with antitumor immunity in a TGFβ1-dependent manner. For example, in MBT2 tumors, mice treated with a combination of Ab6 (a TGFβ1-selective inhibitor) and a PD-1 antibody triggered a robust influx of cytotoxic CD8+ T cells, with a corresponding decrease in the tumor-associated MDSC population (e.g., from approximately 11% to 1.4% of CD45+ cells). These data suggested that closely examining tumor-associated immune cells (e.g., MDSCs and / or CD8+ T cells), e.g., by biopsy, may be useful for characterizing antitumor effects in cancer patients. Furthermore, applicants have made the surprising discovery that a relatively simple and non-invasive blood test can provide equivalent information, which led to the realization that the pharmacological effect of TGFβ1 inhibition in overcoming the immunosuppressive phenotype can be determined by measuring circulating MDSC levels (e.g., circulating gMDSC levels).

[0244] The present disclosure includes the discovery that circulating MDSC levels (including gMDSCs and / or mMDSCs) can be determined by detecting or measuring LRRC33-positive cells in a blood sample, thereby recognizing LRRC33 as a novel blood-based biomarker for circulating MDSCs. See Figures 11A and 11B. For example, LRRC33-positive cells in a blood sample collected from a patient (e.g., a cancer patient) can be detected or measured by a FACS-based assay using an antibody that binds to cell surface LRRC33. In some embodiments, LRRC33-expressing cells in a blood sample collected from a subject with cancer are G-MDSCs. Although MDSCs are derived from bone marrow-derived monocytes, cell surface expression of LRRC33 appears to be narrowly restricted to MDSCs, not monocytes, in the circulation. This realization has raised new possibilities for using LRRC33 as a blood-based marker for circulating MDSCs. LRRC33 expression can be determined using an LRRC33-specific antibody or a TGFβ1-LRRC33 complex-specific antibody. In some embodiments, LRRC33 expression can be determined using any of the antibodies disclosed in International Publication Nos. 2018 / 208888 and 2018 / 081287, the entire contents of which are incorporated herein. Here, applicants have established a correlation between circulating and tumor-associated MDSC levels. Combined with the finding that circulating MDSCs appear to exhibit strong and uniform cell surface LRRC33 expression, determining LRRC33 levels measured in blood samples can serve as an effective alternative for assessing tumor immune phenotypes, such as immunosuppression, without the need for more invasive procedures such as tumor biopsies.

[0245] In various embodiments, the disclosure provides a method for treating TGFβ2-related leukemia (TGFβ2-related leukemia) by administering to a patient a TGFβ1-selective inhibitor (e.g., a selective pro- or latent TGFβ1 inhibitor, such as Ab6), a non-isoform-selective TGFβ2 inhibitor (e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, such as GC1008 and variants, an antibody that binds to TGFβ1 / 3, a ligand trap, such as a TGFβ1 / 3 inhibitor), and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, such as selective inhibition of TGFβ1 and / or TGFβ3). The present invention provides methods for treating cancer, predicting or determining efficacy, and / or confirming pharmacological responses by monitoring levels of circulating MDSCs (e.g., circulating gMDSCs) in a sample obtained (e.g., in blood or in a patient's blood components). Exemplary integrin inhibitors include the anti-αVβ8 integrin antibodies provided in WO2020051333, the disclosure of which is incorporated by reference. In various embodiments disclosed herein, circulating MDSCs can 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) after administration of a therapeutic agent to a subject, e.g., administration of a therapeutic dose of a TGFβ inhibitor.

[0246] In certain embodiments, the TGFβ therapeutic agent may be administered alone or in combination with an additional cancer therapy. The therapeutic agent may be administered to a subject with an immunosuppressive cancer or myeloproliferative disorder. In some embodiments, the TGFβ inhibitor is a TGFβ1-selective antibody or antigen-binding fragment thereof (e.g., Ab6) encompassed by the present disclosure. In some embodiments, the TGFβ1-selective antibody or antigen-binding fragment does not inhibit TGFβ2 and TGFβ3 at therapeutically effective doses. In some embodiments, the TGFβ inhibitor is an isoform-nonselective TGFβ inhibitor (e.g., a low-molecular-weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants, an antibody and ligand trap that binds to TGFβ1 / 3, e.g., a TGFβ1 / 3 inhibitor). In some embodiments, the TGFβ inhibitor is an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3). Exemplary integrin inhibitors include the anti-αVβ8 integrin antibodies provided in WO2020051333, the disclosure of which is incorporated by reference. In some embodiments, the additional cancer therapy may include chemotherapy, radiation therapy (including radiotherapeutic agents), cancer vaccines, or immunotherapy, including checkpoint inhibitor therapy such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies. In some embodiments, 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®). In a preferred embodiment, the combination cancer therapy comprises Ab6 and at least one checkpoint inhibitor (such as those listed above).Thus, in some embodiments, the combination of Ab6 and a checkpoint inhibitor is used to treat cancer in a human patient in an amount effective to treat the cancer. In some embodiments, the TGFβ treatment may additionally or alternatively include a second checkpoint inhibitor and / or chemotherapy.

[0247] For example, without being bound by theory, evidence suggests that an overactive TGFβ pathway may correlate with tumor unresponsiveness 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., epithelial cancers, e.g., carcinomas. In certain embodiments, 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., cutaneous melanoma, lung cancer, e.g., lung squamous cell carcinoma and lung adenocarcinoma, liver cancer, e.g., hepatocellular carcinoma, uterine cancer, e.g., endometrial cancer, 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 cancer, and tenosynovial giant cell tumor (TGCT). Thus, TGFβ inhibitors (e.g., Ab6) can be used in combination with one or more genotoxic therapies (e.g., chemotherapy and / or radiation therapy, including radiotherapeutic agents) to treat such cancers in a subject. In certain embodiments, such cancers can have elevated TGFβ levels, e.g., elevated TGFβ activity, as indicated by direct measurement and / or alterations in one or more downstream gene regulation (e.g., one or more genes involved in DNA repair). For example, a cancer, such as one of the cancers listed above, can have elevated TGFβ signaling, as indicated by upregulation of one or more genes associated with non-homologous end joining (NHEJ), e.g., cyclin-dependent kinase inhibitor 1A (CDKN1A), or downregulation of one or more genes associated with alternative end joining, e.g., LIG1 (DNA ligase 1), PARP1, and / or POLQ. In some embodiments, the cancer is a cancer with elevated TGFβ1 levels associated with ROS (e.g., elevated ROS). Without being bound by theory, ROS may induce an increase in TGFβ levels (e.g., TGFβ1 levels), which can be reduced by the TGFβ inhibitors (e.g., TGFβ1 inhibitors) disclosed herein.

[0248] The present disclosure also provides methods for utilizing measurements of circulating MDSCs in cancer therapy in subjects administered TGFβ inhibitors alone or in combination with immunotherapy. Furthermore, the description provided herein also provides support for circulating MDSC populations (e.g., circulating gMDSC populations) as an early predictive marker of efficacy, particularly in cancer subjects treated with TGFβ inhibitor and checkpoint inhibitor combination therapy, at time points before other markers of therapeutic efficacy, such as reduction in tumor volume, can be detected.

[0249] In certain embodiments, a TGFβ inhibitor, e.g., a TGFβ1-selective inhibitor such as Ab6, a non-isoform-selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants, an antibody that binds to TGFβ1 / 3, a ligand trap, e.g., a TGFβ1 / 3 inhibitor, and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3), is administered contemporaneously (e.g., simultaneously), separately, or sequentially with checkpoint inhibitor therapy such that the amount (e.g., dose) of TGFβ1 inhibitor administered is sufficient to reduce circulating MDSC levels by at least 10%, at least 15%, at least 20%, at least 25% or more compared to baseline MDSC levels. In some preferred embodiments, the circulating MDSC level is the circulating gMDSC level. Circulating MDSC levels can be measured before or after each treatment or each dose of TGFβ inhibitor, such that a decrease in circulating MDSC levels of at least 10%, at least 15%, at least 20%, at least 25% or more can indicate or predict therapeutic efficacy. In some embodiments, the circulating MDSC level can be used to determine disease burden (e.g., measured by the change in relative tumor volume before and after a treatment regimen). In certain embodiments, a decrease in circulating MDSC levels can indicate a decrease in disease burden (e.g., a decrease in relative tumor volume).For example, circulating MDSC levels can be measured before and after administration of a dose of a TGF inhibitor (an isoform-selective inhibitor, e.g., Ab6; a non-isoform-selective TGFβ inhibitor, e.g., a low molecular weight ALK5 antagonist; a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants; an antibody that binds to TGFβ1 / 3; a ligand trap, e.g., a TGFβ1 / 3 inhibitor; and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3), and a decrease in circulating MDSC levels indicates a pharmacological effect, e.g., a reduction in disease burden. In certain embodiments, circulating MDSC levels may be indicative of or predictive of a progression of the disease (e.g., a reduction in relative tumor size). In certain embodiments, circulating MDSC levels may be measured before and after administration of a first dose of a TGFβ inhibitor, e.g., a TGFβ1-selective inhibitor, e.g., Ab6, a non-isoform-selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants, an antibody that binds to TGFβ1 / 3, a ligand trap, e.g., a TGFβ1 / 3 inhibitor, and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3).In some embodiments, administration of a first dose of a TGFβ inhibitor, e.g., Ab6, a non-isoform-selective TGFβ inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants, an antibody that binds to TGFβ1 / 3, a ligand trap, e.g., a TGFβ1 / 3 inhibitor, and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3) can be used to reduce tumor volume, such that administration of the TGFβ inhibitor reduces circulating MDSC levels by at least 10%, at least 20%, at least 25% or more compared to circulating MDSC levels before administration. In some embodiments, a decrease in circulating MDSC levels may be indicative of or predictive of a pharmacological effect and may also warrant the administration of a second or subsequent dose of a TGFβ inhibitor. In some embodiments, the first dose of a TGFβ inhibitor is the very first dose of a TGFβ inhibitor a patient receives. In some embodiments, the first dose of a TGFβ inhibitor is the first dose of a given treatment regimen comprising two or more doses of a TGFβ inhibitor. In another embodiment, circulating MDSC levels may be measured before and after a combination therapy comprising a TGFβ inhibitor (e.g., Ab6) and checkpoint inhibitor therapy administered contemporaneously (e.g., simultaneously), separately, or sequentially, and a decrease in circulating MDSC levels is indicative of or predictive of therapeutic efficacy.In some embodiments, a decrease in circulating MDSC levels following combined treatment with a TGFβ inhibitor, e.g., a TGFβ1 inhibitor, e.g., a TGFβ1 selective inhibitor, e.g., Ab6, a non-isoform selective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants, an antibody that binds to TGFβ1 / 3, a ligand trap, e.g., a TGFβ1 / 3 inhibitor, and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3) and checkpoint inhibitor therapy may warrant continuation of treatment.

[0250] In certain embodiments of the present disclosure, levels of circulating MDSCs can be used to predict, determine, and monitor the pharmacological effect of therapeutic agents, including a dose of a TGFβ inhibitor, e.g., a TGFβ1-selective inhibitor, e.g., Ab6; a non-isoform-selective inhibitor, e.g., a low-molecular-weight ALK5 antagonist; a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants; an antibody that binds to TGFβ1 / 3; a ligand trap, e.g., a TGFβ1 / 3 inhibitor; and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3), administered alone or in combination with another cancer therapy, such as a checkpoint inhibitor. In certain embodiments, circulating MDSCs can be measured within six weeks of administration of the initial treatment (e.g., a (first) dose of a TGFβ inhibitor). In certain embodiments, circulating MDSC levels can be measured within 30 days of administration of the first dose of the TGFβ inhibitor. In some embodiments, MDSC levels can be measured within 3 weeks or about 3 weeks after administration of the first dose of the TGFβ inhibitor. In some embodiments, MDSC levels can be measured within 2 weeks or about 2 weeks after administration of the first dose of the TGFβ inhibitor. In some embodiments, MDSC levels can be measured within 10 days or about 10 days after administration of the first dose of the TGFβ inhibitor.

[0251] In certain embodiments, circulating MDSC levels (e.g., circulating gMDSC levels) can be used to select, inform, and / or predict treatment for patients who have not previously received checkpoint inhibitor therapy. Patients who have not previously received checkpoint inhibitor therapy and who have been diagnosed with a cancer type for which high response rates to checkpoint inhibitor therapy have been reported (e.g., overall response rates of greater than 30%, greater than 40%, greater than 50%, or greater, as reported in the art) can be tested to first determine whether their tumor exhibits an immune exclusion, immune infiltration, or immunosuppressive phenotype. In some embodiments, circulating MDSCs can be used in combination with immunohistochemistry, flow cytometry, and / or in vivo imaging methods known in the art to determine the immunophenotype of the tumor. Patients with cancers that exhibit an immune exclusion and / or immunosuppressive phenotype can be selected to receive a TGFβ inhibitor. Cancer patients exhibiting an immune infiltrative phenotype may also be selected to receive a TGFβ1 inhibitor if the patient's circulating MDSC levels (e.g., circulating gMDSC levels) indicate that the cancer exhibits an immunosuppressive phenotype (e.g., if the circulating MDSC or gMDSC levels are above a threshold level and are higher than, for example, the circulating MDSC or gMDSC levels of a control patient, or higher than the circulating MDSC or gMDSC levels of a control patient whose cancer is not immunosuppressive). In some embodiments, if the circulating MDSC or gMDSC levels are detectable, the circulating MDSC or gMDSC levels are higher than the threshold level. In some embodiments, the threshold circulating MDSC or gMDSC level is greater than 1%, e.g., greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the leukocyte / PBMC component in a blood sample.The TGFβ inhibitor may be a TGFβ1-selective inhibitor, such as Ab6, an isoform-nonselective inhibitor, such as a low-molecular-weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, such as GC1008 and its variants, an antibody that binds to TGFβ1 / 3, a ligand trap, such as a TGFβ1 / 3 inhibitor, and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, such as selective inhibition of TGFβ1 and / or TGFβ3), and combination therapy with a checkpoint inhibitor (e.g., an anti-PD1 or anti-PD-L1 antibody). Circulating MDSC levels (e.g., gMDSC levels) may further be monitored as an early predictor of therapeutic response. In certain embodiments, patients who have not previously received checkpoint inhibitor treatment and who have been diagnosed with a cancer type for which low response rates to checkpoint inhibitor therapy have been reported (e.g., overall response rates of 30% or less, 20% or less, or 10% or less, as reported in the art) may be treated with a TGFβ inhibitor, e.g., a TGFβ1 selective inhibitor such as Ab6, a non-isoform selective inhibitor such as a low molecular weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3 such as GC1008 and variants, an antibody that binds to TGFβ1 / 3, a ligand trap such as a TGFβ1 / 3 inhibitor, and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3), in combination with checkpoint inhibitor therapy. In some embodiments, therapeutic response in these patients can be predicted by monitoring circulating MDSC levels (eg, circulating gMDSC levels).

[0252] In certain embodiments, circulating MDSC levels (e.g., circulating gMDSC levels) can be used to select, inform treatment, and predict response in patients who are resistant to or who cannot tolerate checkpoint inhibitor therapy (e.g., due to adverse effects). These patients may have primary resistance (i.e., never responded to checkpoint inhibitor therapy) or acquired resistance (i.e., initially responded to checkpoint inhibitor therapy but developed resistance over time). In some embodiments, resistance to checkpoint inhibitor therapy in patients indicates immune suppression and / or exclusion, and therefore, these patients may be selected as candidates for TGFβ inhibitor therapy, such as a TGFβ1-selective inhibitor, e.g., Ab6, an isoform-nonselective inhibitor, e.g., a low molecular weight ALK5 antagonist, a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants, an antibody and ligand trap that binds to TGFβ1 / 3, e.g., a TGFβ1 / 3 inhibitor, and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3). In certain embodiments, patients with either primary or acquired resistance to checkpoint inhibitors can be administered TGFβ inhibitors such as TGFβ1 selective inhibitors, e.g., Ab6, isoform non-selective inhibitors, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind to two or more of TGFβ1 / 2 / 3, e.g., GC1008 and variants, antibodies that bind to TGFβ1 / 3, ligand traps, e.g., TGFβ1 / 3 inhibitors, and / or integrin inhibitors (e.g., antibodies that bind to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibit downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3), and their response to treatment can be monitored and / or predicted by circulating MDSC levels.In some embodiments, a decrease in circulating MDSC levels of at least 10%, at least 15%, at least 20%, at least 25% or more may indicate a response to TGFβ inhibitor therapy. In some embodiments, a decrease in circulating MDSC levels of at least 10%, at least 15%, at least 20%, at least 25% or more may indicate, for example, a pharmacological effect of treatment with a TGFβ inhibitor. In certain embodiments, a decrease in circulating MDSC levels may indicate a decrease in tumor size.

[0253] Most TGFβ inhibitors currently under development are not isoform-selective. These include pan-TGFβ inhibitors and inhibitors targeting TGFβ1 / 2 and TGFβ1 / 3. Approaches taken to manage potential toxicities associated with such inhibitors include careful dosing regimens to seize the small window of opportunity to achieve both efficacy and an acceptable safety profile. This may include reducing the dose of non-isoform-selective inhibitors, which may involve less frequent administration and / or a smaller dose per administration. For example, monthly administration of biologic TGFβ inhibitors may be considered instead of weekly administration. Another example is administering them only in the early stages of combination immunotherapy to avoid or minimize toxicity associated with TGFβ inhibition.

[0254] Because combination therapy including a cancer therapy (e.g., checkpoint inhibitor therapy) and a non-isoform-selective TGFβ inhibitor may result in higher toxicity compared to a TGFβ1-selective inhibitor (e.g., Ab6), to mitigate or manage such risk, the non-isoform-selective TGFβ inhibitor may be administered less frequently or intermittently, for example, on an "as needed" basis. In such a treatment paradigm, circulating MDSC levels (e.g., circulating gMDSC levels) may be monitored periodically to determine whether the effectiveness of overcoming immunosuppression is sufficiently maintained to ensure the anti-tumor efficacy of the cancer treatment. If MDSCs increase during the course of cancer treatment, this indicates that the patient would benefit from additional administration of a TGFβ inhibitor. Such an approach may help reduce unnecessary risks and adverse events associated with TGFβ inhibition, particularly with non-isoform-selective inhibitors. In some embodiments, the TGFβ inhibitor targets TGFβ1 / 2. In some embodiments, the TGFβ inhibitor targets TGFβ1 / 3. In some embodiments, the TGFβ inhibitor targets TGFβ1 / 2 / 3. In some embodiments, the TGFβ inhibitor selectively targets TGFβ1.

[0255] Thus, the present disclosure provides a TGFβ inhibitor for use in an intermittent dosing regimen for cancer immunotherapy in a patient, the intermittent dosing regimen further comprising the steps of: measuring circulating MDSCs (e.g., circulating gMDSCs) in a first sample collected from the patient before TGFβ inhibitor treatment; administering the TGFβ inhibitor to the patient treated with cancer therapy, where the cancer therapy is optionally checkpoint inhibitor therapy; measuring circulating MDSCs in a second sample collected from the patient after TGFβ inhibitor treatment; continuing the cancer therapy if the second sample shows a decreased level of circulating MDSCs compared to the first sample; measuring circulating MDSCs in a third sample; and administering an additional dose of a TGFβ inhibitor to the patient if the third sample shows an elevated level of circulating MDSCs compared to the second sample. In some embodiments, the TGFβ inhibitor is a non-isoform-selective inhibitor. In some embodiments, the sample is a blood or blood component sample. In some embodiments, the non-isoform-selective inhibitor inhibits TGFβ1 / 2 / 3, TGFβ1 / 2, or TGFβ1 / 3. Baseline circulating MDSC levels are likely elevated in cancer patients compared to healthy individuals, and subjects with immunosuppressive cancers may have even higher circulating MDSC levels. Thus, reduced levels of circulating MDSCs in patients treated with TGFβ inhibitor therapy, such as a TGFβ1-selective inhibitor (e.g., Ab6), an isoform-nonselective inhibitor (e.g., a low molecular weight ALK5 antagonist), a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3 (e.g., GC1008 and variants), an antibody that binds to TGFβ1 / 3, a ligand trap (e.g., a TGFβ1 / 3 inhibitor), and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3), alone or in combination with checkpoint inhibitor therapy, may indicate a reduction or reversal of immune suppression in cancer.In certain embodiments, a TGFβ inhibitor, such as a TGFβ1-selective inhibitor (e.g., Ab6), an isoform-nonselective inhibitor (e.g., a low molecular weight ALK5 antagonist), a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3 (e.g., GC1008 and variants), an antibody that binds to TGFβ1 / 3, a ligand trap (e.g., a TGFβ1 / 3 inhibitor), and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3), is administered to a subject with cancer such that the dose of the TGFβ inhibitor is sufficient to reduce or reverse immune suppression of the cancer, as indicated by a decrease in circulating MDSC levels and / or a change in levels of tumor-associated immune cells measured after administration of the TGFβ inhibitor treatment compared to levels measured before administration. In some embodiments, the levels of circulating MDSCs and / or tumor-associated immune cells are measured before and after administration of a TGFβ inhibitor therapy, such as a TGFβ1-selective inhibitor (e.g., Ab6), a non-isoform-selective inhibitor (e.g., a low molecular weight ALK5 antagonist), a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3 (e.g., GC1008 and variants), an antibody that binds to TGFβ1 / 3, a ligand trap (e.g., a TGFβ1 / 3 inhibitor), and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3), in combination with checkpoint inhibitor therapy; and a decrease in circulating MDSC levels and / or a change in the levels of tumor-associated immune cells measured after treatment compared to levels measured before treatment indicates a reduction or reversal of cancer immunosuppression.

[0256] Circulating MDSC levels can be determined in samples such as whole blood samples or blood components (e.g., PBMCs). In some embodiments, the sample is fresh whole blood or a blood component of a sample that has not been previously frozen. In certain embodiments, circulating MDSCs can be collected by drawing peripheral blood into heparinized tubes. For example, peripheral blood mononuclear cells can be isolated from peripheral blood using art-known methods such as elutriation, magnetic bead separation, or density gradient centrifugation (e.g., Ficoll-Paque®). In some embodiments, MDSCs can be separated from peripheral blood mononuclear cells by CD11b+ marker selection (e.g., using CD11b+ microbeads or antibodies). G-MDSCs and M-MDSCs can also be further distinguished from CD11b+ cells by flow cytometry / FACS analysis based on surface marker expression. For example, human G-MDSCs can be identified by the expression of cell surface markers CD11b, CD33, CD15, and CD66b. In some embodiments, human G-MDSCs may also express LOX-1, arginase, and / or low levels of HLA-DR. Human M-MDSCs can be identified by expression of the cell surface markers CD11b, CD33, and CD14, and in some embodiments, low levels of HLA-DR expression. Quantification of circulating MDSCs can be expressed as a percentage of total CD45+ cells.

[0257] Tumor-associated immune cell markers Immune cell markers can be used to determine whether a cancer has an immune exclusion phenotype and / or can be used alone or in combination with other circulating biomarkers, such as circulating MDSCs, to determine therapeutic efficacy or treatment regimens. If a tumor is determined to have an immune exclusion phenotype, cancer treatment (such as CBT) alone may not be effective. Without being bound by theory, the tumor may lack sufficient cytotoxic cells in the tumor environment for effective CBT treatment alone. Therefore, alternative and / or add-on therapy with a TGFβ inhibitor (such as those described herein) can reduce immunosuppression, thereby providing improved treatment alone or making resistant tumors more responsive to cancer treatment. In some embodiments, immune cell markers are measured in a biopsy (e.g., core needle biopsy). In some embodiments, a patient with an immune exclusion tumor is administered a therapeutic agent including one or more TGFβ inhibitors (e.g., a TGFβ1 inhibitor, e.g., Ab6). In some embodiments, patients with immune-negative tumors are administered a therapeutic agent comprising one or more TGFβ inhibitors (e.g., TGFβ1 inhibitors, e.g., Ab6) and monitored for improvement in their condition (e.g., increased immune cell infiltration into the tumor, decreased tumor volume, etc.). In some embodiments, patients who show improvement in their condition after a first round of treatment are administered one or more additional rounds of the therapeutic agent. In some embodiments, subjects are administered one or more additional therapeutic agents in combination with one or more TGFβ inhibitors (e.g., TGFβ1 inhibitors, e.g., Ab6).

[0258] Tumor-associated immune cells that can be used to indicate the immune context of the tumor / cancer microenvironment include, but are not limited to, cytotoxic T cells and tumor-associated macrophages (TAMs), as well as tumor-associated MDSCs. Biomarkers for detecting cytotoxic T cell levels include, but are not limited to, CD8 glycoprotein, granzyme B, perforin, and IFNγ, the latter three of which may also indicate activated cytotoxic T cells. Protein markers, such as HLA-DR, CD68, CD163, CD206, and other biomarkers, and any method known in the art, can be used to measure TAM levels. In certain embodiments, increased levels of cytotoxic T cells, e.g., activated cytotoxic T cells, detected within the tumor microenvironment may indicate a reduction or reversal of immunosuppression. For example, increased CD8 expression and perforin, granzyme B, and / or IFNγ expression by tumor-associated immune cells may indicate a reduction or reversal of immunosuppression in cancer. In certain embodiments, decreased levels of TAMs or tumor-associated MDSCs detected within the tumor microenvironment may indicate a reduction or reversal of immunosuppression. For example, reduced expression of HLA-DR, CD68, CD163, and CD206 by tumor-associated immune cells may indicate a reduction or reversal of immunosuppression in cancer. In certain embodiments, tumor-associated immune cells, such as CD8+ T cells, may be used in combination with one or more additional biomarkers to indicate the immune architecture of the tumor / cancer microenvironment. In certain embodiments, the immune architecture of a tumor may be characterized by the density, location, organization, and / or functional orientation of tumor-infiltrating immune cells. In certain embodiments, such markers may be used to determine the immune phenotype of a tumor, for example, to determine whether the tumor is immune-negative, immune-inflammatory, or immune-desert.

[0259] In various embodiments, cytotoxic T cells can be used, for example, to determine whether a cancer in a patient sample has an immune-exclusion phenotype and / or can be used alone or in combination with other biomarkers, such as circulating MDSCs, to determine therapeutic efficacy or treatment regimens. For example, CD8 expression and / or the distribution of CD8 expression in a tumor sample can be used. For example, CD8 expression can be examined in a sample to determine its distribution in the tumor (i.e., tumor compartment), stroma (i.e., stromal compartment), and margin (i.e., margin compartment; e.g., determined by evaluating an area of ​​approximately 10-100 μm, 25-75 μm, or 30-60 μm, e.g., 50 μm, between the tumor and stroma). In certain embodiments, the tumor, intratumoral stroma, and / or margin compartment can be determined using histological methods (e.g., pathologist evaluation, pathologist-trained machine learning algorithms, and / or immunohistochemistry). In certain embodiments, CD8+ T cells in the tumor compartment may be referred to as "tumor-associated CD8+ cells." In certain embodiments, CD8+ T cells in the stromal compartment may be referred to as "stroma-associated CD8+ cells." In certain embodiments, CD8+ T cells in the marginal compartment may be referred to as "marginal-associated CD8+ cells." In some embodiments, the CD8 distribution is determined by the presence of tumor nests (e.g., clusters of cells extending from a common center found in cancerous growths; in one embodiment, tumor nests contain at least 250 cells and at least 500 μm 2The stroma may be determined in the stroma surrounding the tumor nest, and in the margin between the tumor nest and its surrounding stroma (e.g., by evaluating an area of ​​approximately 10-100 μm, or 25-75 μm, or 30-60 μm, e.g., 50 μm, between the tumor nest and the surrounding stroma). In certain embodiments, tumor nests may be identified using histological methods (e.g., pathologist evaluation, pathologist-trained machine learning algorithms, and / or immunohistochemistry). In certain embodiments, one or more tumor nests may be found within the tumor compartment. In certain embodiments, a tumor may contain multiple (e.g., at least 5, at least 10, at least 20, at least 25, at least 50, or more) tumor nests. By default, unless the context dictates otherwise, the terms "stroma" or "stromal compartment" refer to the stroma surrounding the tumor, and "margin" or "margin compartment" refer to the margin between the tumor and the stroma surrounding the tumor. In some embodiments, the structural interface between the tumor / tumor nest and the surrounding stroma is determined by imaging analysis. Thus, the margin can be defined as the region surrounding the interface in either direction by a predetermined distance, e.g., 10-100 μm. In some embodiments, prior to administration of a TGFβ inhibitor, such as a TGFβ1 inhibitor (e.g., Ab6), this distribution can be used to select patients for treatment and / or predict and / or determine the likelihood of a therapeutic response (e.g., anti-tumor response) to an anti-cancer therapy including an anti-TGFβ inhibitor. For example, if no or few cytotoxic T cells (e.g., less than 5% CD8+ T cells) are found in a tumor sample, including the stroma and margin, this may indicate a patient who will not benefit from TGFβ inhibitor therapy (without being bound by theory, this is thought to be because few immune cells are recruited to the tumor). Similarly, if a high density of cytotoxic T cells (e.g., more than 5% CD8+ T cells) is observed in the tumor as well as the stroma and margin, this patient may also receive limited benefit from TGFβ inhibitor therapy (without being bound by theory, this is thought to be because immune cells have already infiltrated the tumor).In contrast, in certain embodiments, the subject's cancer may exhibit an immune exclusion phenotype, in which cytotoxic T cells (e.g., CD8+ T cells) are observed clustered primarily within or near the margin, e.g., at the margin-tumor border, and do not significantly infiltrate the tumor itself (e.g., less than 5% CD8+ T cells within the tumor compartment and more than 10% CD8+ T cells in the margin and / or stromal compartment). In certain embodiments, the subject's cancer may exhibit an immune exclusion phenotype, in which cytotoxic T cells (e.g., CD8+ T cells) are observed clustered primarily within or near the margin, e.g., at the margin-tumor border (or peri-vasculature), and do not significantly infiltrate the tumor core itself (e.g., less than 5% CD8+ T cells within the tumor compartment and more than 5% CD8+ T cells in the margin and / or stromal compartment). In certain embodiments, the subject's cancer may exhibit an immune exclusion phenotype, in which cytotoxic T cells (e.g., CD8+ T cells) are observed clustered primarily within or near the margin, e.g., at the margin-tumor border, and do not significantly infiltrate the tumor itself (e.g., less than 5%, less than 10%, less than 15%, or fewer CD8+ T cells within the tumor compartment and more than 5%, more than 10%, more than 15%, or more CD8+ T cells in the margin and / or stromal compartment). In some embodiments, the CD8+ content in the tumor compartment can be 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 US A. 2007 Mar 6;104(10):3967-72), or Echarti et al. (Cancers (Basel). 2019 Sep;11(9):1398), the contents of which are incorporated herein in their entirety. Any of these methods can be used to determine the immunophenotype of a tumor.Tumor samples from patients with this pattern may indicate patients who are likely to benefit from TGF inhibitor therapy (without being bound by theory, this is because the tumor is actively suppressing the immune response and preventing sufficient infiltration of cytotoxic T cells, which can be partially or completely reversed by TGF inhibitors).

[0260] In some embodiments, the immune exclusion phenotype is characterized by determining the cluster score of cytotoxic T cells (e.g., CD8+ T cells) within a tumor-associated compartment, e.g., within the tumor, within the periphery of the tumor mass, and / or near the tumor vasculature. In some embodiments, the cluster score of cytotoxic T cells (e.g., CD8+ T cells) can be determined based on the homogeneity of immune cells in a particular tumor-associated compartment, such that compartments containing a highly uniform distribution of cytotoxic T cells (e.g., CD8+ T cells) obtain a high cluster score. In certain embodiments, tumors exhibiting an immune exclusion phenotype may be characterized by a lower density of cytotoxic T cells (e.g., CD8+ T cells) within the tumor compared to the density outside the tumor (e.g., at the periphery of the tumor mass and / or near the tumor vasculature). In some embodiments, the immune exclusion phenotype is characterized by cytotoxic T cells (e.g., CD8+ T cells) in the tumor stroma located in close proximity (e.g., less than 100 μm) to the tumor. In some embodiments, the immune exclusion phenotype is characterized by cytotoxic T cells (e.g., CD8+ T cells) that can infiltrate tumor nests and locate within close distance (e.g., less than 100 μm) from the tumor. In some embodiments, CD8+ T cells can be observed in clusters within the tumor near intratumoral blood vessels, as determined, for example, by endothelial markers. In comparison, upon reversal of immune suppression with a TGF-beta inhibitor, a more uniform distribution of CD8+ T cells within the tumor can be observed, likely due to CD8+ cells being able to infiltrate from perivascular regions and proliferate within the tumor.

[0261] In certain embodiments, the level of tumor-infiltrating cytotoxic T cells (e.g., CD8+ T cells) and their activation state can be determined from a tumor biopsy sample obtained from the subject. In some embodiments, a tumor biopsy sample, e.g., a core needle biopsy, can be obtained at least 28 days before and at least 100 days after administration of the therapeutic agent. In some embodiments, a tumor biopsy sample, e.g., a core needle biopsy, can be obtained from about 21 days to about 45 days after administration of the therapeutic agent. In some embodiments, a tumor biopsy sample can be obtained via core needle biopsy. In some embodiments, if an increase is detected, treatment is continued.

[0262] In certain embodiments, the immunophenotype of a subject's cancer may be determined by measuring the cell density of cytotoxic T cells in a tumor biopsy sample (e.g., the percent of CD8+ T cells per square millimeter or other defined distance). In certain embodiments, the immunophenotype of a subject's cancer may be determined by comparing the density of cytotoxic T cells (e.g., CD8+ T cells) within the tumor with the density outside the tumor (e.g., cells within the margin, e.g., cells on the periphery of the tumor mass and / or near the tumor vasculature). In some embodiments, the immunophenotype of a subject's cancer may be determined by comparing the proportion of CD8+ lymphocytes within the tumor with that outside the tumor. In certain embodiments, the immunophenotype of a subject's cancer may be determined by comparing the clustering or distribution of cytotoxic T cells in the tumor, stroma, or margin (e.g., the average number of CD8+ T cells surrounding other CD8+ T cells). In certain embodiments, the immunophenotype of a subject's cancer may be determined by measuring the average distance from cytotoxic T cells (e.g., CD8+ T cells) in the stroma to the tumor. In certain embodiments, the immunophenotype of a subject's cancer may be determined by measuring the mean depth of cytotoxic T cell (e.g., CD8+ T cell) infiltration into tumor nests. Cell number and density may be determined using immunostaining and computerized or manual measurement protocols. In certain embodiments, the level of cytotoxic T cells (e.g., CD8+ T cells) may be measured using immunohistochemical analysis of tumor biopsy samples. In certain embodiments, the level of cytotoxic T cells (e.g., CD8+ T cells) may be determined at least 28 days before and / or at least 100 days after administration of TGFβ therapy. In certain embodiments, the level of cytotoxic T cells (e.g., CD8+ T cells) may be determined up to about 45 days (e.g., about 21 days to about 45 days) after administration of TGFβ therapy. In some embodiments, the level of cytotoxic T cells (e.g., CD8+ T cells) is determined 5, 10, 15, 20, 25, 30 or more days before and / or at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 days after (or any time in between) administration of the TGFβ therapeutic agent.

[0263] In some embodiments, tumors that have low levels of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor compared to levels of cytotoxic T cells (e.g., CD8+ T cells) outside the tumor (e.g., near the tumor periphery and / or tumor vasculature) can be identified as immune-exclusion tumors. In some embodiments, immune-exclusion tumors can also have higher levels of cytotoxic T cells (e.g., CD8+ T cells) in the tumor stroma compared to the tumor interior. In certain embodiments, immune-exclusion tumors can be identified by determining the ratio of cytotoxic T cell density (e.g., CD8+ T cells) inside the tumor to the tumor exterior, where the ratio is less than 1. In certain embodiments, immune-exclusion tumors can be identified by determining the ratio of cytotoxic T cell density inside the tumor to the density within the tumor margin, where the ratio is less than 1. In certain embodiments, immune-exclusion tumors can be identified by determining the ratio of cell density inside the tumor to the density in the tumor stroma, where the ratio is less than 1. In certain embodiments, immune-exclusion tumors can be identified by comparing the absolute number, percentage, and / or density of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor to the outside of the tumor (e.g., the margin and / or stroma). In some embodiments, in immune-exclusion tumors, 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 higher than inside the tumor. In some embodiments, immune-exclusion tumors contain less than 5% CD8+ T cells inside the tumor and more than 10% CD8+ T cells in the tumor margin and / or stroma. In some embodiments, immune-exclusion tumors can be identified by comparing the ratio of compartmentalized cytotoxic T cell density (e.g., density of CD8+ cells inside the tumor relative to density at the tumor margin and / or in the stroma) to the ratio of total tissue cytotoxic T cell density (e.g., CD8+ cells inside the tumor relative to CD8+ cells in the entire tumor tissue or biopsy), where the compartmental ratio is greater than the total tissue ratio. In some embodiments, tumors with increased cell density of cytotoxic T cells (e.g., CD8+ T cells) at an average distance of about 100 μm or less outside the tumor can be identified as immune-exclusion tumors.In some embodiments, cytotoxic T cell density (e.g., CD8+ T cells) may be used in combination with one or more parameters, such as mean CD8+ cluster score. In some embodiments, a mean CD8+ cluster score of 50% or less in the tumor indicates immune exclusion.

[0264] In some embodiments, tumors with low levels of CD8+ T cells within the tumor (e.g., core) compared to CD8+ T cells outside the tumor (e.g., at the periphery of the tumor, e.g., the periphery of the tumor and / or near the tumor vasculature, e.g., tumor margin and / or within the stroma) can be identified as immune-excluded tumors. In some embodiments, immune-excluded tumors comprise less than 5%, less than 10%, or less than 15% of CD8+ T cells within the tumor and / or one or more tumor nests, and more than 5%, more than 10%, or more than 15% of CD8+ T cells outside the tumor and / or one or more tumor nests. In some embodiments, immune-excluded tumors comprise less than 5% of CD8+ T cells within the tumor and / or one or more tumor nests, and more than 5% of CD8+ T cells outside the tumor and / or one or more tumor nests. In some embodiments, immune-excluded tumors comprise less than 10% of CD8+ T cells within the tumor and / or one or more tumor nests, and more than 10% of CD8+ T cells outside the tumor and / or one or more tumor nests. In some embodiments, an immune-excluded tumor contains less than 15% CD8+ T cells within the tumor and / or one or more tumor nests, and more than 15% CD8+ T cells outside the tumor and / or one or more tumor nests.

[0265] In some embodiments, tumors with higher levels of CD8+ T cells within the tumor compared to CD8+ T cells outside the tumor (e.g., the periphery of the tumor and / or near the tumor vasculature, e.g., the tumor margin and / or stroma) can be identified as immunoinflammatory (or immune-infiltrated) tumors. In some embodiments, immunoinflammatory (or immune-infiltrated) tumors contain greater than 5% CD8+ T cells within the tumor. In some embodiments, immunoinflammatory (or immune-infiltrated) tumors contain greater than 10% CD8+ T cells within the tumor and / or within one or more tumor nests. In some embodiments, immunoinflammatory (or immune-infiltrated) tumors contain greater than 15% CD8+ T cells within the tumor and / or within one or more tumor nests. Although immune-infiltrated tumors may sometimes have limited benefit from TGFβ inhibitor therapy, some immune-infiltrated tumors may benefit from TGFβ inhibitor therapy, especially if the tumor is also resistant or refractory to checkpoint inhibitor therapy. Such tumors may exhibit an immunosuppressive phenotype. Additionally or alternatively, the cells may contain infiltrating CD8+ cells with reduced cytotoxic function, for example, CD8+ cells that express low levels of cytotoxic enzymes such as perforin and / or granzyme B.

[0266] In some embodiments, tumors with low levels of CD8+ T cells both inside and outside the tumor can be identified as immune desert tumors. In some embodiments, immune desert tumors contain less than 5% CD8+ T cells within the tumor and less than 10% CD8+ T cells at the tumor margin and / or stroma. In some embodiments, immune desert tumors contain less than 5% CD8+ T cells within the tumor (and / or within one or more tumor nests) and less than 5% CD8+ T cells at the tumor margin and / or stroma.

[0267] In some embodiments, the CD8+ content in the tumor compartment can 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 incorporated herein in their entirety. In some embodiments, any of these methods can be used to determine the immunophenotype of a tumor.

[0268] In certain embodiments, the immunophenotype of a subject's cancer can be determined by the average percent CD8 positivity (i.e., the percentage of CD8+ lymphocytes) measured for multiple (e.g., at least 5, at least 15, at least 25, at least 50, or more) tumor nests in a tumor (e.g., in one or more tumor biopsy samples). In certain embodiments, the immunophenotype of a given tumor nest can be determined by comparing the CD8 positivity within the tumor nest with the CD8 positivity outside the tumor nest (e.g., at the tumor nest edge and / or within the tumor nest stroma). In certain embodiments, a tumor nest can be identified as immunoinflammatory if the CD8 positivity within the tumor nest is less than 5% and the CD8 positivity within the tumor nest edge is greater than 5%. In certain embodiments, a tumor nest can be identified as immune-deleted if the CD8 positivity within the tumor nest is less than 5% and the CD8 positivity within the tumor nest edge is less than 5%. In certain embodiments, a tumor nest can be identified as immune-desert if the CD8 positivity within the tumor nest is less than 5% and the CD8 positivity within the tumor nest edge is less than 5%. In certain embodiments, a subject's cancer can be identified as immunoinflammatory if more than 50% of the total tumor area analyzed contains tumor nests exhibiting an immunoinflammatory phenotype. In certain embodiments, a subject's cancer can be identified as immunoexclusion if more than 50% of the total tumor area analyzed contains tumor nests exhibiting an immune exclusion phenotype. In certain embodiments, a subject's cancer can be identified as immune desert if more than 50% of the total tumor area analyzed contains tumor nests exhibiting an immune desert phenotype. In certain embodiments, a subject's cancer can be identified based on determining the CD8 positivity rate from two or more samples (e.g., at least three samples, e.g., four samples) taken from the same tumor.

[0269] In certain embodiments, a patient treated with a TGFβ inhibitor (e.g., a TGFβ inhibitor disclosed herein), e.g., concurrently with a second therapy, has an immune infiltrate phenotype. In certain embodiments, the immune phenotype of the subject's cancer is assessed to determine the ratio of Treg / CD8+ T cells in the TME. In certain embodiments, the patient has an immune infiltrate phenotype. In certain embodiments, the patient has a high Treg / CD8+ T cell ratio in the TME and / or infiltrating CD8+ T cells expressing reduced levels of cytotoxic enzymes (e.g., perforin and granzyme A / B) and / or inflammatory cytokines (e.g., IFNγ). In certain embodiments, the level of circulating MDSCs is determined in the patient (instead of, or in addition to, measuring the Treg / CD8+ T cell ratio in the TME). In certain embodiments, a patient treated with a TGFβ inhibitor has elevated numbers of circulating MDSCs. Such patients may have failed to respond to previous treatments, e.g., previous checkpoint inhibitor treatment, and may be selected for combination therapy (e.g., a therapy comprising a checkpoint inhibitor) that includes administration of one or more TGFβ inhibitors.

[0270] In certain embodiments, tumor biopsy samples can be obtained by core needle biopsy. In certain embodiments, three to five samples (e.g., four samples) can be collected from the same tumor. In certain embodiments, the needle can be inserted along a single trajectory, and multiple samples (e.g., three to five samples, e.g., four samples) can be collected at different tumor penetration depths along the same needle trajectory. In certain embodiments, samples collected at different tumor penetration depths can be used to analyze the total CD8 positivity rate for multiple tumor nests. In certain embodiments, the total CD8 positivity rate determined for these samples can represent the CD8 positivity rate in the remainder of the tumor. In certain embodiments, the total CD8 positivity rate determined for these samples can be used to identify the immunophenotype of the subject's cancer.

[0271] In certain embodiments, the immunophenotype of a subject's tumor may be determined by a combined analysis of the absolute number, percentage, ratio, and / or density of CD8+ cells in the tumor and the total CD8 positivity (i.e., percentage of CD8+ lymphocytes) for tumor nests throughout the tumor.

[0272] In certain embodiments, tumor compartments may be identified, determined, and / or analyzed for markers such as CD8 content manually, e.g., by pathologist examination of tumor samples. In some embodiments, tumor compartments may be identified, determined, and / or analyzed for markers such as CD8 content by digital analysis, e.g., by using software or computer programs for automatic identification. In certain embodiments, one skilled in the art may use such software or computer programs for automatic identification of tumor nests and the boundaries between tumor nests, stromal compartments, and / or tumor margin compartments. In certain embodiments, software or computer programs may be used to assess the distribution of appropriate markers, such as CD8+ T cells, in recognized tumor nests, stromal compartments, and / or tumor margin compartments. In certain embodiments, the software or computer program may be based on one or more machine learning algorithms. In certain embodiments, the one or more machine learning algorithms may initially be based on manual classification of reference samples, e.g., by a trained pathologist. In some embodiments, the software or computer program may use a neural network approach with machine learning based on reference samples manually classified, e.g., by a pathologist. Exemplary software or computer programs include any software or computer program capable of capturing images (e.g., microscopic images of tumor samples containing immunostaining), processing and analyzing the images, and segmenting tumor compartments within the images based on specific parameters (e.g., nuclear staining, fibroblast staining, CD8+ staining, other biomarkers). In certain embodiments, the software or computer program may be any of those provided by Visiopharm, HALO (Indica Labs), CellProfiler Analyst, Aperio Image Analysis, Zeiss ZEN Intellesis, or ImageJ.Such programs may advantageously achieve sufficient resolution to visualize specific features of individual tumor nests within a solid tumor (e.g., boundaries of tumor nests, stroma, and / or marginal compartments) rather than substantially analyzing the entire tumor as a whole.

[0273] In certain embodiments, subjects whose cancer exhibits an immunoinflammatory phenotype but is unresponsive to checkpoint inhibitor therapy may be more responsive to therapy involving administration of a TGFβ inhibitor (e.g., Ab6). In some embodiments, such subjects are identified for treatment. In some embodiments, such subjects may have a high intratumoral Treg / CD8+ T cell ratio and / or CD8+ T cells expressing reduced levels of cytotoxic enzymes and / or inflammatory cytokines. In some embodiments, such subjects may have an increased number of circulating MDSCs. In some embodiments, such subjects have RCC, such as ccRCC. In some embodiments, such subjects are administered a treatment comprising a TGF inhibitor such as a TGFβ1-selective inhibitor (e.g., Ab6), an isoform-nonselective inhibitor (e.g., a low molecular weight ALK5 antagonist), a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3 (e.g., GC1008 and variants), an antibody that binds to TGFβ1 / 3, a ligand trap (e.g., a TGFβ1 / 3 inhibitor), and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3).

[0274] In certain embodiments, subjects whose cancer exhibits an immunoinflammatory phenotype but is unresponsive to checkpoint inhibitor therapy may be more responsive to a combination therapy comprising a TGFβ inhibitor, such as a TGFβ1-selective inhibitor (e.g., Ab6), an isoform-nonselective inhibitor (e.g., a low molecular weight ALK5 antagonist), a neutralizing antibody that binds to two or more of TGFβ1 / 2 / 3 (e.g., GC1008 and variants), an antibody that binds to TGFβ1 / 3, a ligand trap (e.g., a TGFβ1 / 3 inhibitor), and / or an integrin inhibitor (e.g., an antibody that binds to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, e.g., selective inhibition of TGFβ1 and / or TGFβ3), and an additional cancer therapy, e.g., a checkpoint inhibitor. In some embodiments, the additional cancer therapy may include chemotherapy, radiation therapy (including radiotherapeutic agents), a cancer vaccine, or immunotherapy including a checkpoint inhibitor such as an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody. In some embodiments, 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®). In certain embodiments, subjects whose cancer exhibits an immune exclusion phenotype are administered a combination therapy including a TGFβ inhibitor, such as a TGFβ1-selective inhibitor (e.g., Ab6), and an additional cancer therapy, such as a checkpoint inhibitor. In some embodiments, such subjects may have a high Treg / CD8+ T cell ratio in the tumor and / or CD8+ T cells expressing reduced levels of cytotoxic enzymes and / or inflammatory cytokines. In some embodiments, such subjects have RCC, such as ccRCC.

[0275] In certain embodiments, subjects whose cancer exhibits an immunoinflammatory phenotype but is unresponsive to checkpoint inhibitor therapy may be amenable to combination therapy including a TGFβ inhibitor, such as a TGFβ1-selective inhibitor (e.g., Ab6), and checkpoint inhibitor therapy (e.g., a PD1 or PDL1 antibody). In some embodiments, such subjects are identified for combination therapy. In some embodiments, such subjects are identified for combination therapy before receiving checkpoint inhibitor therapy alone. In some embodiments, such subjects are identified for combination therapy before receiving either checkpoint inhibitor therapy or a TGFβ inhibitor alone. In some embodiments, such subjects are treatment-naive. In some embodiments, such subjects have previously received checkpoint inhibitor therapy and are unresponsive to checkpoint inhibitor therapy. In some embodiments, such subjects have cancer that exhibits an immune-exclusion phenotype. In some embodiments, such subjects have previously received checkpoint inhibitor therapy and are directly administered combination therapy (e.g., avoiding the need to first attempt treatment with a checkpoint inhibitor alone). In some embodiments, such subjects receive a ...

Claims

1. 1. A TGFβ1 inhibitor for use in treating cancer in a patient who has previously received therapy for said cancer, wherein said treatment comprises administering a TGFβ1 inhibitor in an amount sufficient to inhibit the growth of or reduce the volume of a solid tumor, wherein said cancer is renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC), ovarian cancer, melanoma, testicular cancer, colorectal cancer, pancreatic cancer, squamous cell skin cancer, triple-negative breast cancer (TNBC), or liver cancer.

2. 2. The TGFβ1 inhibitor for use according to claim 1, wherein the previous cancer therapy comprises checkpoint inhibitor therapy, chemotherapy, and / or radiation therapy.

3. The previous cancer therapy may be an anti-PD-(L)1 antibody (e.g., pembrolizumab, nivolumab, cemiplimab, atezolizumab, dostallimab, darvalumab, avelumab), an anti-CTLA4 antibody (e.g., ipilimumab, tremelimumab), a tyrosine kinase inhibitor (e.g., sunitinib, cabozantinib, imatinib, gefitinib, sorafenib, erlotinib, lapatinib, canertinib, semaphorinib, semaphorinib), or a combination of these.

3. The TGFβ1 inhibitor for use according to claim 1 or 2, comprising an inhibitor such as rifapril, sirolimus, sirolimus, sirolimus (sirolimus), ...

4. The TGFβ1 inhibitor for use according to any one of claims 1 to 3, wherein said patient has undergone multiple lines of previous cancer therapy aimed at treating said cancer.

5. The TGFβ1 inhibitor for use according to any one of claims 1 to 4, wherein the cancer has been resistant or unresponsive to previous cancer therapies.

6. The TGFβ1 inhibitor for use according to any one of claims 1 to 5, wherein the disease has progressed during the previous therapy.

7. The TGFβ1 inhibitor for use according to any one of claims 1 to 6, wherein said patient experienced an adverse event in response to said previous cancer therapy, which resulted in said therapy being discontinued.

8. The TGFβ1 inhibitor for use according to any one of claims 1 to 7, wherein the cancer is from RCC, HNSCC, ovarian cancer, TNBC, pancreatic adenocarcinoma, colorectal cancer, or squamous cell skin cancer.

9. The TGFβ1 inhibitor for use according to any one of claims 1 to 8, wherein the patient has metastases (i.e. the primary cancer has metastasized) at the time of screening (before treatment with the TGFβ1 inhibitor is started), and optionally the cancer has metastasized to multiple sites.

10. A therapeutically effective amount of the TGFβ1 inhibitor is administered to the patient, and optionally, the therapeutically effective amount is one of the following: (i) achieving stable disease (SD), e.g., an amount showing no disease progression for 16 weeks or more (e.g., 6 months, 7 months, 8 months, 9 months, 10 months or more) on / while on treatment; (ii) a partial response (PR), e.g., an amount that achieves 30% or more tumor shrinkage; The TGFβ1 inhibitor for use according to any one of claims 1 to 9, wherein In some embodiments, tumor shrinkage is measured by percent change in sum of diameters of target lesions (SOD) from baseline, and optionally, said therapeutically effective amount achieves a 50% or greater reduction in SOD from baseline.

11. The TGFβ1 inhibitor comprises an agent that targets and is capable of reducing the TGFβ1 signaling pathway, and optionally the agent is one of the following: (i) inhibitors of TGFβ1 activators, such as integrins that bind to the RGD motif within the LAP domain of latent TGFβ1; (ii) inhibitors of TGFβ1 activation, such as antibodies that bind to latent TGFβ1 and thereby inhibit the release of the growth factor from the latent complex; (iii) inhibitors of mature (soluble) TGFβ1 ligands, such as neutralizing antibodies, ligand traps incorporating the ligand-binding module of the TGFβ receptor, and nucleic acid-based inhibitors, such as siRNA and antisense oligonucleotides; and (iv) TGFβ receptor antagonists, e.g., Alk5 inhibitors The TGFβ1 inhibitor for use according to any one of claims 1 to 10, selected from:

12. The TGFβ1 inhibitor for use according to any one of claims 1 to 11, wherein said TGFβ1 inhibitor preferentially inhibits TGFβ1 over TGFβ2 and / or TGFβ3.

13. The TGFβ1 inhibitor for use according to any one of claims 1 to 11, wherein said TGFβ1 inhibitor preferentially inhibits TGFβ1 and TGFβ2 over TGFβ3.

14. 12. The TGFβ1 inhibitor for use according to any one of claims 1 to 11, wherein said TGFβ1 inhibitor is a TGFβ1 selective inhibitor, optionally wherein said TGFβ1 selective inhibitor is SRK-181.

15. The TGFβ1 inhibitor is selected from the group consisting of SRK-181 (from Scholar Rock), RG6440 (SOF10) (from Roche / Chugai), ABBV-151 (ribmoniplimab) (from AbbVie), NIS793 (XOMA-089) (from Novartis), PLN-10195 (from Pliant), ES014 (from Elpiscience), Cotsiranib (STP705) (from Sirnaomics), Bintrafusp alpha (M7824), Dalutrafusp alpha (AGEN14423), BMS-986416 (AVID200), MK-2225 (from MERCK), PM8001 (from Biotheus), Vactosertib (from Medpacto), BCA101 (from Bicara), TU2218 (NCE401) (from TiUM), ATB-301 (from Autotelic Bio / Clinigen), AdAPT-001 (AIM-001) (from EpicentRx), CART-PSMA-TGF-bRDN (from Tmunity Therapeutics), HCW9218 (HCW 12. The TGFβ1 inhibitor for use according to any one of claims 1 to 11, which is or comprises: EGFR-1 (from EGFR Biologics), SH3051 (from Sanhome), TST005 (from Transcenta), GS19 (GT90008) (from Kintor (Gensun)), BJ-005 (from BJ Bioscience), QLS31901 (from Qilu Pharmaceuticals), TQB2858 (from Chia Tal-tianqing), Y101D (from YZY Biopharma), Charis 1000 (C1K) (from Ensol Biosciences), and / or Fresolimumab (GC1008).

16. 16. The TGFβ1 inhibitor for use according to any one of claims 1 to 15, wherein the TGFβ1 inhibitor is selected from antibodies and antigen-binding fragments thereof disclosed in the following publications: WO 2020 / 104460, WO 2020 / 014473, WO 2019 / 163927, WO 2021 / 039945, WO 2015 / 015003, WO 2018 / 013939, WO 2021 / 142427, WO 2016 / 161410, WO 2019 / 075090, and WO 2020 / 160291, and those that compete or cross-compete for antigen binding with such antibodies (e.g., share overlapping epitopes).

17. 1. A TGFβ1 selective inhibitor for use in treating ovarian cancer in a patient, wherein the TGFβ1 selective inhibitor is an antibody that binds to latent TGFβ1 and thereby inhibits its activation, the treatment comprising administering the TGFβ1 selective inhibitor to achieve stable disease (SD) for six months or more, and optionally the TGFβ1 selective inhibitor is administered at a dose of 240 to 2400 mg. SRK-181 administered Q3W or Q2W as monotherapy, wherein the patient has received a prior cancer therapy, and optionally, the prior cancer therapy is a TGFβ1 selective inhibitor comprising paclitaxel / carboplatin, topotecan, doxil, gemcitabine, altretamine, bevacizumab / gemcitabine / carboplatin, letrozole, carboplatin / taxol, leuprorelin, carboplatin / docetaxel, carboplatin / docetaxel / bevacizumab, or any combination thereof.

18. 1. A TGFβ1-selective inhibitor for use in the treatment of RCC, HNSCC, melanoma, or squamous cell skin cancer in patients whose disease has progressed on a prior anti-PD-(L)1 therapy, wherein the TGFβ1-selective inhibitor is an antibody that inhibits activation of the latent TGFβ1 complex by binding to it, and wherein the treatment comprises administration of the TGFβ1-selective inhibitor in combination with anti-PD-(L)1 therapy, and optionally the TGFβ1-selective inhibitor is SRK-181 administered at 240-2400 mg Q3W or Q2W (e.g., 1500 mg Q3W or 1000 mg Q2W).

19. The TGFβ1 inhibitor for use according to any one of claims 1 to 18, wherein the cancer comprises a solid tumor, and the solid tumor is a CD4+ T cell infiltrated tumor.

20. The TGFβ1 inhibitor for use according to any one of claims 1 to 19, wherein the tumor microenvironment (TME) of said CD8+ T cell infiltrated tumor has a high Treg / CD8+ T cell ratio before said treatment.

21. The TGFβ1 inhibitor for use according to any one of claims 1 to 20, wherein CD8+ T cells in the TME express low levels of inflammatory cytokines and / or cytotoxic enzymes before the treatment.

22. 22. The TGFβ1 inhibitor for use according to claim 21, wherein the inflammatory cytokines include IFNγ and the cytotoxic enzymes include perforin and granzyme A / B.

23. The TGFβ1 inhibitor for use according to any one of claims 1 to 22, wherein the patient has elevated levels of circulating MDSCs, preferably gMDSCs, before treatment.

24. The TGFβ1 inhibitor for use according to any one of claims 1 to 23, wherein the patient has an elevated number of platelets in the cancer prior to said treatment.

25. The TGFβ1 inhibitor for use according to any one of claims 1 to 24, wherein TGFβ1 is overexpressed in the cancer before treatment.

26. The TGFβ1 inhibitor for use according to any one of claims 1 to 25, wherein increased levels of TGFβ1 activators are detected in the cancer before treatment.

27. The TGFβ1 inhibitor for use according to claim 26, wherein the TGFβ1 activator comprises an integrin capable of binding to an RGD motif, including alpha-v, alpha-5, alpha-11, beta-6, and / or beta-8.

28. 27. The TGFβ1 inhibitor for use according to claim 26, wherein the TGFβ1 activator comprises kallikrein, chemotrypsin, trypsin, elastase, plasmin, thrombin, zinc metalloproteases (MMPs), and / or ADAM proteases.

29. 29. The TGFβ1 inhibitor for use according to any one of claims 1 to 28, wherein an increase in ROS markers is present and / or is expected to be present in said patient before and / or during said treatment, said ROS markers comprising isoprostanes (IsoPs), malondialdehyde (MDA), nitrotyrosine, S-glutathionylation, myeloperoxidase (MPO), oxidized low density lipoprotein (OxLDL), and / or antioxidant enzymes.

30. 30. The TGFβ1 inhibitor for use according to any one of claims 1 to 29, wherein increased deposition of extracellular matrix (ECM) is detected in the cancer prior to treatment, and wherein increased deposition of ECM is indicated by levels of ECM markers including collagen, fibronectin, and / or fibrillin, and / or by levels of cancer-associated fibroblast (CAF) markers including actin α, platelet-derived growth factor receptor α (PDGFRα / CD140a), platelet-derived growth factor receptor β (PDGFRβ / CD140b), fibroblast-specific protein 1 (FSP-1 / S100A4), fibroblast activation protein (FAP), and / or nicotinamide N-methyltransferase (NNMT).

31. The TGFβ1 inhibitor for use according to any one of claims 1 to 30, wherein a decreased level of epithelial markers and / or an increased level of mesenchymal markers are detected in the cancer before treatment, the epithelial markers including E-cadherin, α-catenin, γ-catenin, and / or cytokeratin, and the mesenchymal markers including fibronectin, vimentin, and / or N-cadherin.

32. The TGFβ1 inhibitor for use according to any one of claims 1 to 31, wherein an immunosuppressive marker is detected in the cancer before treatment, the immunosuppressive marker comprising a Treg marker and / or LRRC33, and the Treg marker comprising CD4, FOXP3, and / or CD25.

33. The TGFβ1 inhibitor for use according to any one of claims 1 to 32, wherein the cancer is SMAD4 deficient.

34. The TGFβ1 inhibitor for use according to any one of claims 1 to 33, wherein the cancer is methylthioadenosine phosphorylase (MTAP) deficient.

35. (A) the cancer has a deletion at the 9p21 locus, and optionally, further comprises a further deletion of a tumor suppressor gene; (B) the cancer has reduced expression of MTAP; and / or (C) a mutant MTAP with reduced activity is produced in the cancer; A TGFβ1 inhibitor for use according to claim 34.

36. The TGFβ1 inhibitor for use according to any one of claims 1 to 35, wherein low levels of interferon gamma (IFNγ) are detected in the cancer before treatment.

37. The treatment comprises: (i) measuring biomarkers in the patient; (ii) administering to the patient an amount of said TGFβ1 inhibitor sufficient to inhibit the growth of or reduce the volume of a solid tumor; (iii) measuring the biomarker after administration of the TGFβ1 inhibitor; and (iv) continuing administration of the treatment if a change in the biomarker is detected.

37. A TGFβ1 inhibitor for use according to any one of claims 1 to 36, comprising:

38. The biomarker is selected from the group consisting of: (A) TGFβ1 transcript and / or TGFβ1 protein; (B) Integrins capable of binding to an RGD motif, including α-v, α-5, α-11, β-6, and / or β-8 integrins; (C) a protease capable of activating TGFβ1; (D) ROS marker, (E) ECM markers, (F) CAF marker, (G) epithelial markers; (H) Mesenchymal markers; (I) immunosuppressive markers, (J) the SMAD4 gene, SMAD4 transcript, and / or SMAD4 protein; (K) MTAP gene, MTAP transcript, and / or MTAP protein; (L) IFNγ, (M) Circulating MDSC, (N) Platelets, (O) an increase in the Treg / CD8+ T cell ratio in CD8+ T cell-infiltrated solid tumors; and / or (P) CD8+ T cells expressing low levels of inflammatory cytokines and / or cytotoxic enzymes in CD8+ T cell-infiltrated solid tumors.

38. The TGFβ1 inhibitor for use according to claim 37, comprising:

39. 38. The TGFβ1 inhibitor for use according to claim 37, wherein step (iii) of said treatment comprises detecting a deletion of the 9p21 locus in said cancer, and optionally further detecting a further deletion of a tumor suppressor gene.

40. 1. A TGFβ1 inhibitor for use in treating cancer in a patient, said treatment comprising: (i) administering a TGFβ1 inhibitor in an amount sufficient to inhibit the growth of or reduce the volume of a solid tumor; (ii) administering a genotoxic therapy prior to and / or concurrently with the administration of the TGFβ1 inhibitor, or administering a genotoxic therapy concurrently and / or after the administration of the TGFβ1 inhibitor. A TGFβ1 inhibitor comprising:

41. 41. The TGFβ1 inhibitor for use according to claim 40, wherein the administration of said genotoxic therapy begins at least 1 hour after the start of administration of said TGFβ1 inhibitor.

42. 41. The TGFβ1 inhibitor for use according to claim 40, wherein the administration of said genotoxic therapy begins at least one day after the start of administration of said TGFβ1 inhibitor.

43. 41. The TGFβ1 inhibitor for use according to claim 40, wherein the administration of said genotoxic therapy begins at least one week after the start of administration of the previous TGFβ1 inhibitor.

44. 44. The TGFβ1 inhibitor for use according to any one of claims 40 to 43, wherein said treatment further comprises continuing administration of said TGFβ1 inhibitor after termination of said genotoxic therapy.

45. The TGFβ1 inhibitor for use according to any one of claims 40 to 44, wherein said genotoxic therapy is chemotherapy and / or radiotherapy.

46. The TGFβ1 inhibitor for use according to any one of claims 40 to 45, wherein said chemotherapy comprises administering to the patient 5-FU, paclitaxel, cisplatin, and / or bleomycin.

47. The TGFβ1 inhibitor for use according to any one of claims 40 to 46, wherein the patient further receives a checkpoint inhibitor treatment.

48. 48. The TGFβ1 inhibitor for use according to any one of claims 40 to 47, wherein the patient has undergone checkpoint inhibitor therapy, chemotherapy, and / or radiotherapy for the cancer prior to receiving the TGFβ1 inhibitor.

49. 49. The TGFβ1 inhibitor for use according to any one of claims 40 to 48, wherein said TGFβ1 inhibitor is a TGFβ1 selective inhibitor, optionally wherein the TGFβ1 selective inhibitor is SRK-181.

50. 50. The TGFβ1 inhibitor for use according to any one of claims 40 to 49, wherein the cancer is RCC, HNSCC, ovarian cancer, melanoma, testicular cancer, colorectal cancer, pancreatic cancer, cutaneous squamous cell carcinoma, TNBC, or liver cancer.

51. The TGFβ1 inhibitor for use according to any one of claims 1 to 50, wherein the cancer is ccRCC.

52. 1. A TGFβ1 inhibitor for use in treating cancer in a subject, said treatment comprising administering to said subject a TGFβ1 inhibitor in an amount effective to treat said cancer, wherein CD8+ T cells are infiltrating the tumor microenvironment (TME) of said cancer, and said cancer is resistant or refractory to cancer therapy, optionally wherein said cancer therapy comprises a checkpoint inhibitor and / or genotoxic agent therapy, and further optionally wherein said genotoxic agent therapy is radiation therapy or chemotherapy.

53. 53. The TGFβ1 inhibitor for use according to claim 52, wherein the TME has a high Treg / CD8+ T cell ratio before the treatment.

54. 54. The TGFβ1 inhibitor for use according to claim 52 or 53, wherein the CD8+ T cells in the TME express low levels of inflammatory cytokines and / or cytotoxic enzymes prior to the treatment.

55. 55. The TGFβ1 inhibitor for use according to claim 54, wherein the inflammatory cytokines include IFNγ and the cytotoxic enzymes include perforin and granzyme A / B.

56. The TGFβ1 inhibitor for use according to any one of claims 52 to 55, wherein the patient has elevated levels of circulating MDSCs, preferably gMDSCs, before treatment.

57. The TGFβ1 inhibitor for use according to any one of claims 52 to 56, wherein the patient has an increased number of platelets in the cancer prior to the treatment.

58. 58. The TGFβ1 inhibitor for use according to any one of claims 52 to 57, wherein the subject is further treated with a cancer therapy selected from a checkpoint inhibitor and a genotoxic agent therapy.

59. 59. The TGFβ1 inhibitor for use according to any one of claims 52 to 58, wherein the cancer is undergoing or has undergone epithelial-mesenchymal transition (EMT).

60. The TGFβ1 inhibitor for use according to any one of claims 52 to 59, wherein the cancer comprises a higher number of Treg cells than non-cancerous / normal tissue.

61. 61. The TGFβ1 inhibitor for use according to any one of claims 52 to 60, wherein a biological sample taken from the subject exhibits elevated levels of TGFB1 and / or TGFβ1 transcripts, integrins capable of binding to RGD motifs, proteases capable of activating TGFβ1, ROS markers, mesenchymal markers; CAF markers; immunosuppressive markers, Treg cells, platelets, circulating MDSCs, and / or the Treg / CD8+ T cell ratio.

62. 61. The TGFβ1 inhibitor for use according to any one of claims 52 to 60, wherein a biological sample taken from the subject exhibits reduced levels of epithelial markers, SMAD4 transcripts and / or proteins, methylthioadenosine phosphorylase (MTAP) transcripts and / or proteins, pro-inflammatory cytokines, and / or cytotoxic enzymes.

63. 63. The TGFβ1 inhibitor for use according to any one of claims 52 to 62, wherein the cancer is selected from renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC), ovarian cancer, melanoma, testicular cancer, colorectal cancer, pancreatic cancer, squamous cell skin cancer, triple-negative breast cancer (TNBC), and liver cancer.

64. The TGFβ1 inhibitor for use according to any one of claims 52 to 63, wherein the cancer is ccRCC.

65. The TGFβ1 inhibitor for use according to any one of claims 52 to 64, wherein the TGFβ1 inhibitor is SRK-181.

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