JP2024511029A - TGF-β inhibitors and their uses - Google Patents

Abstract

The present disclosure provides TGFp inhibitor therapy for treating immunosuppressive conditions such as cancer. A method of predicting and monitoring therapeutic response is disclosed. Related compositions, methods and therapeutic uses are also disclosed.

Description

RELATED APPLICATIONS This application is filed in U.S. Provisional Patent Application No. 63/166,824, filed March 26, 2021, entitled “TGF-BETA INHIBITORS AND USE THEREOF,” June 3, 2021, respectively. Specification No. 63/202,260 filed on January 25, 2022; Specification No. 63/302,999 filed on January 25, 2022; and No. 63 filed on February 24, 2022 No. 313,386, the contents of which are hereby expressly incorporated by reference in their entirety.

FIELD OF THE INVENTION This application relates generally to TGFβ inhibitors and their therapeutic uses and related assays for diagnosing, monitoring, prognosing and treating disorders, including cancer.

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 of which is encoded by a separate gene. These TGFβ isoforms function as pleiotropic cytokines that regulate cell proliferation, differentiation, immune regulation (eg, adaptive immune responses), and a variety of other biological processes in both homeostasis and disease settings. The three TGFβ isoforms signal through the same cell surface receptors and trigger similar canonical downstream signaling events, including the SMAD2/3 pathway.

TGFβ has been implicated in the pathogenesis and progression of various disease states such as cancer, fibrosis and immune diseases. Often these conditions are associated with dysregulation of the extracellular matrix (ECM). For these and other reasons, TGFβ has been an attractive therapeutic target for immune disorders, various proliferative disorders, and fibrosis. However, observations from preclinical studies, including those in rats and dogs, have revealed significant toxicity associated with systemic inhibition of TGFβ in vivo, and to date, no TGFβ therapeutics that are considered safe and effective are commercially available. do not have.

Dose-limiting toxicity noted by inhibition of the TGFβ pathway remains a major concern in the development of anti-TGFβ therapies. These include cardiovascular abnormalities, skin lesions, oral epithelial hyperplasia and gingival bleeding (Vitsky 2009; Lonning 2011; Stauber 2014; Mitra 2020). Although many of these toxicities are either reversible or manageable, cardiovascular pathologies such as inflammation, hemorrhage, or hyperplasia in the heart valves, aortic arch, and associated arteries are not reversible and therefore remain associated with TGFβ. It is an important safety issue when developing inhibitors (Stauber 2014; Anderton 2011; Mitra 2020).

Previously, the applicant has described monoclonal antibodies with a novel mechanism of action for modulating growth factor signaling (see, e.g., WO 2014/182676; the contents of which are incorporated herein by reference in its entirety). (incorporated herein by reference). These antibodies take advantage of the fact that TGFβ1 is expressed as a latent proprotein complex consisting of a prodomain and a growth factor, which requires an activation step to release the growth factor from the latent complex. designed for. Rather than taking traditional approaches that directly target the mature growth factor itself after activation (e.g., neutralizing antibodies), a new class of inhibitory antibodies targets the activation step upstream of the ligand-receptor interaction. specifically targeted the inactive pro-proprotein complex itself to pre-emptively block it. Without being bound by theory, this unique mechanism of action suggests that they act at the source, i.e., by targeting latent proTGFβ1 complexes within the disease microenvironment before activation occurs. It was reasoned that it would offer advantages for achieving both spatial and temporal benefits.

Using this approach, additional monoclonal antibodies were generated that specifically bind to TGFβ1 and inhibit its activation step (i.e., release of the mature growth factor from the latent complex) in an isoform-selective manner (International See Publication No. 2017/156500; the contents of which are incorporated herein by reference in their entirety). The data presented for such antibodies support the notion that isoform-specific inhibition (as opposed to pan-inhibition) of TGFβ may result in an improved safety profile for antagonizing TGFβ in vivo. In view of this, we proposed a therapeutic agent for conditions driven by pleiotropic TGFβ1 actions and their dysregulation that is i) isoform-specific; and ii) sensitive to different presenting molecules. Efforts have been made to develop TGFβ1 inhibitors that can broadly target multiple relevant TGFβ1 signaling complexes. Non-limiting examples of such isoform-specific inhibitors include TGFβ1 selective antibodies, such as Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, as disclosed herein. Ab28, Ab29, Ab30, Ab31, Ab32, Ab33 or Ab34.

Examples of such antibodies are later described in WO 2018/129329 and PCT/US 2019/041373, the contents of each of which are incorporated herein by reference in their entirety. ). These isoform-specific inhibitors have demonstrated both efficacy and safety in vivo.

For example, PCT/U.S. Patent Application Publication No. 2019/041373 discloses that isoform-selective high-affinity antibodies that can target the large latent complex (LLC) of TGFβ1 for TGFβ1-related indications. For example, diseases involving aberrant gene expression (e.g. TGFB1, Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, Loxl2, CCL2 and Mmp2), diseases associated with ECM dysregulation (e.g. fibrosis, myelofibrosis and solid tumors), diseases characterized by an increase in immunosuppressive cells (e.g. Tregs, MDSCs and/or M2 macrophages), diseases involving mesenchymal transition, diseases involving proteases, aberrant stem cell proliferation and/or differentiation. Discloses that it may be effective in treating the associated diseases.

In multiple preclinical tumor models, these TGFβ1 inhibitors have been shown to eliminate primary tumor resistance (i.e., present before treatment initiation) to immunotherapies (e.g., checkpoint inhibitors); are infiltrated with immunosuppressive cell types such as regulatory T cells, M2-type macrophages and/or myeloid-derived suppressor cells (tumor-associated MDSCs). Upon treatment, a decrease in the number of tumor-associated immunosuppressive cells (eg, MDSCs) and a corresponding increase in the number of anti-tumor effector T cells was observed. In multiple preclinical models (including tumors co-expressing TGFβ1/3 isoforms), significant and durable antitumor effects were achieved with survival benefits when used in conjunction with checkpoint blockade therapy; This suggests that inhibitors of TGFβ1 alone were sufficient to sensitize immunosuppressed tumors to cancer immunotherapy drugs such as checkpoint inhibitors. Martin et al. See Science Translational Medicine (2020), 12 (536): eaay8456.

As of the filing date of this application, the general consensus in the art is that it is necessary to inhibit multiple isoforms of TGFβ to achieve therapeutic efficacy while managing toxicity through careful dosing regimens. or considered advantageous. Consistent with this premise, numerous groups are developing TGFβ1 inhibitors that target more than one isoform. These include: low molecular weight antagonists of the TGFβ receptor, such as ALK5 antagonists, such as galunisertib (LY2157299 monohydrate); monoclonal antibodies that inhibit all three isoforms (such as neutralizing antibodies). (“pan-inhibitor” antibodies) (see e.g. WO 2018/134681); monoclonal antibodies that preferentially inhibit two of the three isoforms (e.g. antibodies against TGFβ1/2 (e.g. , WO 2016/161410 pamphlet) and antibodies against TGFβ1/3 (e.g., WO 2006/116002 pamphlet and WO 2020/051333 pamphlet); integrin inhibitors, such as αVβ3, αVβ5, αVβ6, αVβ8 , antibodies that bind to α5β1, αIIbβ3 or α8β1 integrin and inhibit downstream activation of TGFβ, such as selective inhibition of TGFβ1 and/or TGFβ3 (e.g. PLN-74809), as well as engineered molecules such as ligand traps (e.g. fusion protein) (for example, International Publication No. 2018/029367 pamphlet; International Publication No. 2018/129331 and International Publication No. 2018/158727 pamphlet).

Immune checkpoint inhibitors have become one of the most successful cancer treatments in recent years, but these treatments are only effective in a small proportion of the patient population (Hedge et al., Immunity .2020 Jan 14;52(1):17-35). As single agents, many immune checkpoint inhibitors typically exhibit response rates of only about 10-35%. An unmet need in cancer immunotherapy has been the limited availability of reliable and predictive biomarkers (see, for example: Zhang et al., Front. Med. 2019, 13(1): 32-44, “Monitoring checkpoint inhibitors: predictive biomarkers in immunotherapy” and Arora et al., Adv. Ther. 2019, 36:2638-2678, “Ex isting and emerging biomarkers for immune checkpoint immunotherapy in solid tumors). Although biopsies provide useful information about the disease, possible limitations of biopsies include that they are invasive, sample collection/access is not always feasible, and that they are potentially representative of the entire tumor area. Alternatives to biopsy are being actively explored, including gene expression profiling and non-invasive imaging techniques. Certain serum markers are useful for diagnostic purposes. However, they are not very useful for prognostic purposes (see, e.g., Zhang et al.). This makes blood-based assessments difficult to determine what is happening in the tumor microenvironment (TME). (Galon & Bruni, Nature Reviews Drug Discovery, 2019 Mar; 18 (3): 197-218 “Approaches to treat immunity hot, altered and cold tumors with combination immunotherapies”). There remains a need for better guidance regarding both the selection of suitable TGFβ inhibitors tailored to this patient population and related treatment regimens that may provide improved cancer therapy.

The present disclosure relates to compositions containing TGFβ inhibitors and methods of selecting subjects suitable for treatment with TGFβ inhibitors, as well as related treatment methods and methods of monitoring therapeutic parameters such as efficacy and target binding. The present disclosure provides improved and more targeted methods for identifying treatment candidates, such as patients or patient populations likely to benefit from TGFβ inhibitor therapy, and/or monitoring treatment efficacy. Provide standardized treatments and treatment methods. Related methods, including therapeutic regimens, and methods for manufacturing such inhibitors are encompassed herein. The selection of specific targets and TGFβ inhibitors for therapeutic use is aimed at achieving in vivo efficacy.

More specifically, the present disclosure provides, inter alia, i) improved analytical methods aimed at providing better characterization of intra- and peritumoral cellular structures; ii) achieving higher accuracy; an improved method for measuring circulating TGFβ levels, with the aim of improved methods for assessing circulating MDSC levels, including; and iv) additional biomarkers, including p-Smad2, that are useful in predicting and monitoring therapeutic efficacy and determining target binding. Accordingly, one or more of these characteristics may be used as part of a diagnostic and/or therapeutic regimen for a subject (eg, a patient) with a condition associated with TGFβ1 dysregulation, such as cancer.

In some embodiments, the pharmaceutical compositions and/or treatment regimens disclosed herein combine a TGFβ inhibitor and one or more additional therapies, such as genotoxic therapy and/or checkpoint inhibitor therapy. include. In some embodiments, the one or more additional therapies are used in combination with the TGFβ inhibitor, as separate molecular entities administered separately, as a single formulation (e.g., a mixture), or as a single It can be administered as part of a recombinant multifunctional construct that functions as a molecular entity, eg, both a checkpoint inhibitor and a TGFβ inhibitor. In some embodiments, the checkpoint inhibitor therapy is an antibody or antigen-binding fragment that targets PD-1, PD-L1, CTLA-4, or LAG3. In some embodiments, the genotoxic therapy is chemotherapy or radiation therapy. In some embodiments, the TGFβ inhibitor and one or more additional therapies are administered contemporaneously, separately, or sequentially. In some embodiments, the TGFβ inhibitor is a TGFβ1 inhibitor, such as apitegromab or Ab6. In some embodiments, the TGFβ inhibitor is any one of the antibodies or antigen-binding fragments disclosed in PCT/JP2015/006323, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the TGFβ inhibitor is GYM329. In some embodiments, the TGFβ inhibitor is an inhibitor of TGFβ1 and TGFβ2, such as NIS793/XOMA-089 or GC1008. In some embodiments, the TGFβ inhibitor is an inhibitor of TGFβ1 and TGFβ3, such as M7824 (bintrafusp alpha) or AVID200. In some embodiments, the TGFβ inhibitor is a pan-TGFβ inhibitor (i.e., an agent that inhibits TGFβ1, TGFβ2, and TGFβ3), and is an agent that inhibits GC1008 or a derivative thereof, SAR439459, LY3022859, or the ligand binding domain of a TGFβ receptor. Contains drugs that block In some embodiments, the TGFβ inhibitor is an agent that binds to the RGD motif present in latent TGFβ1 and/or TGFβ3, such as a low molecular weight (small molecule) compound or an antibody or antigen binding fragment. In some embodiments, the TGFβ inhibitor is an RNA-based inhibitor of TGFβ1 expression. In some embodiments, the TGFβ inhibitor is a soluble ligand trap, such as M7824 (vintrafsp alpha) or AVID200.

In various embodiments, the present disclosure provides methods for treating, predicting, predicting the therapeutic effects of TGFβ inhibitor treatment in a subject by measuring or monitoring circulating TGFβ levels (e.g., circulating TGFβ1 levels, e.g., circulating latent TGFβ1 levels). and/or provide a method for monitoring. Without wishing to be bound by theory, we believe that administering a TGFβ inhibitor to a subject increases TGFβ levels in the subject's blood, presumably due to accumulation of latent TGFβ as a result of inhibition of the TGFβ activation pathway. was found to increase. Accordingly, the present disclosure contemplates the use of circulating TGFβ, eg, from a blood sample obtained from a subject, as a biomarker to determine and monitor therapeutic efficacy and/or target binding and guide decisions regarding therapy. The terms circulating and blood (such as "circulating TGFβ" and "blood TGFβ") may be used interchangeably.

In some embodiments, the present disclosure provides a method of treating, predicting, and/or monitoring the therapeutic effect of TGFβ inhibitor treatment in a subject, the method comprising: (i) prior to administering the TGFβ inhibitor; determining the level of circulating TGFβ in the subject; (ii) administering to the subject a therapeutically effective dose of a TGFβ inhibitor; and (iii) determining the level of circulating TGFβ in the subject after administration, , an increase in circulating TGFβ after administration compared to pre-administration indicates a therapeutic effect. In some embodiments, the increase is at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, or more. In some embodiments, the treatment alters the level of circulating TGFβ. In some embodiments, continued treatment is conditional on an observed increase in circulating TGFβ.

In some embodiments, the present disclosure encompasses a method of determining target binding and/or therapeutic efficacy of TGFβ inhibitor treatment in a subject, wherein the treatment comprises: (i) prior to administering the TGFβ inhibitor; , determining circulating TGFβ levels in a sample obtained from the subject; (ii) administering a first dose of a TGFβ inhibitor to the subject; and (iii) determining, after administration, a circulating TGFβ level in a sample obtained from the subject; determining circulating TGFβ levels, where an increase in circulating TGFβ levels after administration compared to pre-administration is indicative of target binding and/or therapeutic effect. In some embodiments, the method comprises administering to the subject a second dose of the TGFβ inhibitor if an increase in circulating TGFβ levels is observed after administration compared to pre-administration. In some embodiments, treatment is continued if an increase in circulating TGFβ levels is observed after administration compared to pre-administration. In some embodiments, the increase is at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, or more.

In any one of the foregoing embodiments, the treatment may further include administering to the subject one or more additional therapies, such as genotoxic therapy and/or immunotherapy, where one or more Additional therapies may be administered contemporaneously (eg, simultaneously), separately, or sequentially. For example, additional therapy may include checkpoint inhibitor therapy.

In some embodiments, the present disclosure provides an improved method for measuring circulating TGFβ levels from a blood sample or blood-derived sample, the method comprising: measuring the sample in a sample tube containing an anticoagulant; It involves processing at a temperature of 2-8°C. In some embodiments, the anticoagulant is citrate-theophylline-adenosine-dipyridamole (CTAD). In some embodiments, the tube contains citric acid-theophylline-adenosine-dipyridamole (CTAD) solution, 0.11 M buffered trisodium citrate solution, 15 M theophylline, 3.7 M adenosine, and 0.198 M dipyridamole. where the solution has a pH of 5.0. In some embodiments, sample processing includes one or more centrifugation steps at speeds greater than 100×g, and/or one or more centrifugation steps at speeds less than 15000×g. In some embodiments, sample processing includes i) a first step of 10 minutes at 150 x g and a second step of 20 minutes at 2500 x g; or ii) a first step of 10 minutes at 2500 x g. and a second step of 2500 x g for 20 min; or iii) a first step of 1500 x g for 10 min and a second step of 12000 x g for 5 min. include. In some embodiments, the method includes analyzing plasma factor 4 (PF4) levels in the same sample in which circulating TGFβ levels are determined, such that PF4 levels provide quality control of the sample. In some embodiments, a sample is only used to determine circulating TGFβ levels if the PF4 level in the same sample is below a concentration indicative of plasma activation. In some embodiments, a PF4 level greater than 500 ng/ml indicates plasma activation. Accordingly, in some embodiments, a sample is used to assess circulating TGFβ levels only if the PF4 level in the sample is 500 ng/ml or less.

In some embodiments, the present disclosure provides a method of treating, predicting, and/or monitoring the therapeutic effect of TGFβ inhibitor treatment in a subject, the method comprising: (i) prior to administering the TGFβ inhibitor; determining the level of phosphorylated Smad2 (P-Smad2) nuclear translocation in a tumor sample obtained from the subject (pre-treatment tumor sample); (ii) administering to the subject one or more doses of a TGFβ inhibitor; and (iii) determining the level of P-Smad2 nuclear translocation in a tumor sample obtained from the subject (post-treatment tumor sample) after administration; A reduction in migration indicates therapeutic efficacy. In some embodiments, treatment is continued if a decrease in P-Smad2 nuclear translocation is observed in the tumor sample after treatment. In some embodiments, the reduction is at least 1.3 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times. In some embodiments, the level of nuclear translocation of P-Smad2 is determined by nuclear masking.

In various embodiments, the present disclosure provided herein is useful for diagnosing, monitoring subjects receiving TGFβ inhibitors (e.g., TGFβ1 selective inhibitors such as Ab6) by monitoring circulating MDSC levels. , including the use of circulating MDSC levels as a predictive biomarker to improve patient selection, prognosis and/or ongoing treatment. In some embodiments, the present disclosure also provides methods for determining therapeutic efficacy and therapeutic agents (e.g., compositions) or dosage regimens for use in a subject with cancer by measuring levels of circulating MDSCs. include. Without being bound by theory, the present inventors believe that amelioration or overcoming of the immunosuppressive phenotype in cancers or related pathologies that express ECM dysregulation, such as by administration of TGFβ inhibitors, may improve the immune system, e.g. e.g., by analyzing circulating MDSC levels in a sample obtained from the subject, e.g., in blood or blood components, before a reduction in volume or other biomarkers can be used to confirm treatment efficacy. I found that I can get it. In some embodiments, circulating MDSCs are characterized by cell surface expression of LRRC33. In some embodiments, subpopulations of circulating MDSCs, such as m-MDSCs and/or g-MDSCs, are measured. The terms circulating and blood (such as "circulating MDSC" and "blood MDSC") may be used interchangeably.

Tumor-associated MDSC cells may contribute to TGFβ1-mediated immunosuppression in the tumor microenvironment. Previously, the applicant demonstrated that MDSCs were indeed enriched in solid tumors and that inhibition of TGFβ1 in combination with checkpoint inhibitor treatment significantly reduced intratumoral MDSCs, which was slowed down. It has been shown to correlate with tumor growth and, in some cases, achieve complete regression in multiple preclinical tumor models (PCT/US Patent Application Publication No. 2019/041373). In these efficacy studies, the effectiveness of such combination therapy was monitored over weeks to months (eg, 6-12 weeks) by monitoring tumor growth. Tumor biopsies can reveal the immune profile of the tumor microenvironment (TME); in addition to being invasive, tumor-infiltrating lymphocytes (TILs) may not be uniformly present throughout the tumor. Biopsy-based information may therefore be inaccurate or biased, as results may vary depending on where the tumor is biopsied. To overcome the limitations of biopsy-based analyses, the data presented herein establish a correlation between tumor-associated (e.g., intratumoral) and circulating MDSC levels and It is possible that MDSCs measured in whole blood or blood components (e.g., PBMCs) may serve as a surrogate to more accurately predict which patient populations are likely to benefit from a particular treatment regimen. It brings up sexuality. Furthermore, evidence suggests that the extent of tumor burden (eg, tumor size) correlates with the relative level of circulating MDSCs in tumor-bearing subjects. Thus, by monitoring circulating MDSC levels in a subject after receiving therapy, response to therapy (e.g., treatment efficacy) can be assessed without the need for invasive biopsy, and results can be obtained faster than traditional methods. obtain. Furthermore, more recent discoveries presented herein identify LRRC33 as a novel cell surface marker for MDSCs in circulation (eg, blood samples), among others. This observation raises the possibility that surface LRRC33 expression can be used as a blood-based predictive biomarker.

In various embodiments, the methods disclosed herein utilize circulating MDSCs as an early biomarker for predicting the effectiveness of combination therapy including a TGFβ inhibitor. The data disclosed herein show that after TGFβ1 inhibitor treatment, there is a significant reduction in circulating MDSC levels compared to baseline, which may be a reduction in mMDSC levels and/or gMDSC levels. Such circulating MDSC levels can be measured in the blood or blood components and can be detected long before antitumor efficacy outcomes are readily available, in some cases weeks ahead of schedule. Shorten. Accordingly, the present disclosure provides the use of circulating MDSCs as predictive biomarkers of patient responsiveness to cancer therapy, such as combination therapy. In a related correspondence of the disclosure provided herein, the level of circulating MDSC cells is increased within 1 to 10 weeks, such as within 3 to 6 weeks, after administration of a dose of a TGFβ inhibitor, optionally within 3 to 6 weeks of TGFβ inhibition. The determination can be made within or about 3 weeks after administration of the dose of agent. In some embodiments, the level of circulating MDSC cells can be determined within two weeks after administration of a dose of TGFβ inhibitor. In some embodiments, the level of circulating MDSC cells can be determined at about 10 days after administration of a dose of TGFβ inhibitor.

Cancer immunotherapy may 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 subjects with cancer may indicate that the body uses disease-fighting immunity (e.g., anti-tumor activity), more specifically lymphocytes such as CD8+ T cells. It is thought that the cells retain or recover the cells, which may be mobilized to attack malignant cells. Therefore, reduced levels of circulating MDSCs after TGFβ inhibitor treatment may indicate a pharmacodynamic effect of TGFβ inhibition (e.g., TGFβ1 inhibition) and an early onset of therapeutic effects when treated with cancer therapies such as checkpoint inhibitors. It can serve as a predictive biomarker.

When cancer patients receive a combination therapy that includes cancer therapy (such as a checkpoint inhibitor) and a TGFβ inhibitor that is not selective for TGFβ1 (non-selective TGFβ inhibitor), there may be a higher risk of toxicity. To reduce or manage such risks, non-selective TGFβ inhibitors can be administered, for example, infrequently or intermittently on an “as needed” basis. For example, circulating MDSC levels can be monitored periodically to determine that the efficacy to overcome immunosuppression is maintained sufficiently to ensure the antitumor efficacy of cancer therapy. If MDSC levels increase during the course of cancer treatment, this may indicate that the patient may benefit from additional doses of TGFβ inhibitors. 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, methods of treating cancer (also described herein in connection with treating cancer or compositions for use in treating cancer) are disclosed herein. Also disclosed are methods of predicting, determining, or monitoring treatment efficacy in a subject suffering from cancer, such as monitoring patient responsiveness to treatment and/or determining continued treatment based on monitored parameters. Ru. In some embodiments, the cancer is an immunodepleted cancer and/or a myeloproliferative disease, and the myeloproliferative disease can be myelofibrosis. In some embodiments, the cancer is a TGFβ1 positive cancer. TGFβ1-positive cancers may co-express TGFβ1, TGFβ2 and/or TGFβ3. A TGFβ1-positive cancer can be a TGFβ1-dominant tumor. A TGFβ1 positive cancer may be a TGFβ1 predominant 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 predominant tumor and may co-express TGFβ1 and TGFβ3. Such cancers include advanced cancers, such as metastatic cancers (eg, metastatic solid tumors) and cancers associated with locally advanced tumors (eg, 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.

In some embodiments, the present disclosure provides a method for predicting or determining therapeutic efficacy in a subject with cancer, the method comprising: administering a TGFβ inhibitor (alone or in combination with cancer therapy), determining the MDSC level; administering to the subject a therapeutically effective amount of a TGFβ inhibitor (alone or in combination with cancer therapy); and determining the circulating MDSC level in the subject after administration; A decrease in circulating MDSC levels after administration compared to circulating MDSC levels of is predictive of therapeutic efficacy.

In some embodiments, the present disclosure encompasses a method of determining the therapeutic effect of TGFβ inhibitor treatment in a subject, wherein treatment is obtained from the subject prior to (i) administering the TGFβ inhibitor. (ii) administering to the subject a first dose of a TGFβ inhibitor; and (iii) determining the level of circulating MDSC in a sample obtained from the subject after administration; wherein a decrease in circulating MDSC levels after administration compared to pre-administration indicates a therapeutic effect. In some embodiments, the method comprises administering to the subject a second dose of the TGFβ inhibitor if a decrease in circulating MDSC levels is observed after administration compared to pre-administration. In some embodiments, treatment is continued if a decrease in circulating MDSC levels is observed after administration compared to pre-administration.

In some embodiments, the present disclosure encompasses combination therapy comprising administration of a TGFβ inhibitor and checkpoint inhibitor therapy and/or genotoxic therapy for use in the treatment of cancer, wherein the treatment includes combined (eg, simultaneous), separate or sequential administration of doses of TGFβ inhibitors, and a reduction in circulating MDSC levels following administration compared to pre-administration has been determined.

In some embodiments, the present disclosure provides a TGFβ inhibitor for use in treating cancer in a subject, wherein the subject is administered a dose of a TGFβ inhibitor, and the TGFβ inhibitor provides immunosuppression in the cancer. said reduced or reversed immunosuppression measured after administration of the TGFβ inhibitor compared to circulating MDSC levels measured in the subject before administering the dose of the TGFβ inhibitor. Includes TGFβ inhibitors, as determined by a reduction in circulating MDSC levels in a subject.

In some embodiments, the present disclosure provides a method for treating advanced cancer in a human subject comprising selecting a subject with advanced cancer and administering a combination therapy comprising a TGFβ inhibitor and checkpoint inhibitor therapy. wherein the advanced cancer comprises a locally advanced tumor and/or metastatic cancer with primary resistance to checkpoint inhibitor therapy, and the subject has elevated circulating MDSC levels. In some embodiments, the combination therapy reduces circulating MDSC levels in the subject. In some embodiments, continued treatment is conditional on an observed decrease in the subject's circulating MDSC levels.

In various embodiments, the circulating MDSC level can be that of mMDSC and/or gMDSC. In some embodiments, the mMDSCs are identified by cell surface markers of CD11b+, HLA-DR-/low, CD14+, CD15-, CD33+/high, and CD66b-. In some embodiments, gMDSCs are identified by cell surface markers of CD11b+, HLA-DR-, CD14-, CD15+, CD33+/low, and CD66b+. In some embodiments, the reduction in circulating MDSC levels (eg, circulating mMDSCs and/or circulating gmMDSCs) can be at least a 10% reduction.

In some embodiments, the present disclosure provides cancer treatment in a subject who has been administered a TGFβ inhibitor alone or in combination with one or more additional cancer therapies (e.g., checkpoint inhibitor therapy and/or genotoxic therapy). The method includes the steps of: (i) obtaining a pre-treatment biopsy sample from a subject; (ii) a tumor-associated tumor in the pre-treatment biopsy sample; determining the level of CD8+ cells; (iii) administering a treatment to the subject; (iv) obtaining a post-treatment biopsy sample from the subject; and (v) tumor-associated CD8+ in the pre-treatment biopsy sample. determining the level of tumor-associated CD8+ cells in the biopsy sample, wherein the level of tumor-associated CD8+ cells in the biopsy sample is determined by immunohistochemical analysis of individual tumor foci within the tumor.

In some embodiments, a subject is selected for treatment who has an immunoinflammatory tumor characterized by a pre-treatment biopsy sample having greater than 5% CD8+ cells in individual tumor foci. In some embodiments, the immunoinflammatory tumor is characterized by having greater than 5% CD8+ cells in greater than 50% of individual tumor foci detected.

In some embodiments, a subject with an immune excluded tumor characterized by a pre-treatment biopsy sample having less than 5% intratumoral CD8+ cells and more than 5% marginal CD8+ cells is selected for treatment. selected. In some embodiments, the immunocompromised tumor is characterized by having less than 5% CD8+ cells in individual tumor foci. In some embodiments, an immunocompromised tumor is characterized by having less than 5% CD8+ cells in more than 50% of individual tumor foci detected.

In some embodiments, the therapeutic effect is determined by the difference between the pre-treatment biopsy sample and the post-treatment biopsy sample, such that a change in the CD8+ level in the post-treatment biopsy sample compared to the pre-treatment CD8+ level is indicative of the treatment effect. It can be monitored by comparing the level of CD8+ cells in the test sample. In some embodiments, an increase in intratumoral CD8+ cells (eg, an increase in CD8+ cells within tumor nests) in a post-treatment biopsy sample compared to a pre-treatment biopsy sample is indicative of a therapeutic effect.

In some embodiments, any one of the biomarkers disclosed herein may be used in combination with one or more other biomarkers provided herein. For example, circulating TGFβ is monitored in combination with one or more of the biomarkers disclosed herein (e.g., circulating MDSCs, tumor-associated CD8+ cells, intratumoral or circulating cytokines, and/or p-Smad2 nuclear translocation). obtain. In some embodiments, treatment efficacy and/or continued treatment may be conditional on observed changes in two or more sets of biomarkers.

In various embodiments, methods and compositions for use of the present disclosure include treating or selecting for treatment a subject with cancer, wherein the cancer is a highly metastatic cancer and a /or may be a solid cancer. In some embodiments, the subject has melanoma, triple negative breast cancer, HER2 positive breast 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), uterus have cancer, prostate cancer, gastric cancer (eg gastric cancer) or thyroid cancer.

In some embodiments, methods and compositions for use of the present disclosure include treating, or selecting for treatment, a subject with a cancer that is resistant to immunotherapy. The subject may be treatment naive (e.g., have not previously received cancer therapy) or may have primary resistance to the immunotherapy (i.e., resistance exists prior to initiation of treatment); or may have acquired resistance to immunotherapy (ie, resistance as a result of at least one dose of treatment). In some embodiments, the immunotherapy is checkpoint inhibitor therapy, eg, an anti-PD-1 antibody or an anti-PD-L1 antibody.

In some embodiments, methods and compositions for use according to the present disclosure include providing treatment to a treatment-naive subject. In some embodiments, methods and compositions for use according to the present disclosure include providing treatment to a subject who has previously undergone cancer therapy or is currently undergoing cancer therapy. . The previous cancer therapy may be the same cancer therapy administered according to the present invention. Cancer therapy can be checkpoint inhibitor (CPI) therapy. In some embodiments, methods and compositions for use according to the present disclosure include providing treatment to a subject with cancer, wherein the cancer is or is immunosuppressive. Suspected (e.g., having a tumor with an immunocompromised or immunosuppressive phenotype).

In some embodiments, methods and compositions for use according to the present disclosure provide a high response rate (e.g., greater than 30%, greater than 40%, greater than 50%, or higher overall response rate) to checkpoint inhibitor therapy. treatment to a subject with a cancer that has a high incidence of cancer. Examples of cancers with 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). cancer), Hodgkin lymphoma, NSCLC, cancer with frequent microsatellite instability (MSI-H), cancer with mismatch repair deficiency (dMMR), primary mediastinal large B-cell lymphoma (PMBCL), and Merkel cell cancer (eg, as reported in Haslam et al., JAMA Network Open. 2019;2(5):e192535).

In some embodiments, methods and compositions for use according to the present disclosure provide low response rates to checkpoint inhibitor therapy (e.g., 30% or less, 20% or less, or 10% or less overall response). treatment to a subject with a cancer that has a high incidence of cancer. In some embodiments, the subject may be treatment naive. In some embodiments, the subject may be refractory 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.

In various embodiments, methods and compositions for use according to the present disclosure include providing treatment to a subject with a solid cancer. In some embodiments, the solid cancer is melanoma (e.g., metastatic melanoma), renal cell carcinoma, breast cancer, e.g., triple-negative breast cancer, HER2-positive breast cancer, colorectal cancer, e.g., microsatellite stable colorectal cancer. cancer and colon adenocarcinoma, lung cancer (e.g. metastatic non-small cell lung cancer, small cell lung cancer), esophageal cancer, pancreatic cancer, bladder cancer, kidney cancer, e.g. transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC) (including clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC), uterine cancer, e.g. endometrial cancer, prostate cancer, gastric cancer (e.g. gastric cancer), head and neck cancer. The cancer is selected from, for example, head and neck squamous cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, thyroid carcinoma, or tenosynovial giant cell tumor (TGCT).

In some embodiments, the TGFβ inhibitors of the present disclosure can be used to treat cancer therapy, including improving the rate or ratio of complete response to partial response among responders. Typically, even cancer types with relatively high response rates (eg, ≧30% responders) to cancer therapy (eg, checkpoint inhibitor therapy) have low complete response rates. Accordingly, the TGFβ inhibitors of the present disclosure can be used to increase the proportion of complete responders in a responder population. In a preferred embodiment, the TGFβ inhibitor is Ab6.

In various embodiments, the TGFβ inhibitors of the present disclosure do not inhibit TGFβ2 signaling at therapeutically effective doses. 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 various embodiments, the TGFβ inhibitor is a TGFβ1 selective inhibitor. In some embodiments, the TGFβ inhibitor has an affinity of 0.5 nM or greater (K < 0.5 nM) with a dissociation rate of 10.0E-4 (1/s) or less as measured by SPR. Can bind to TGFβ1. More preferably, the TGFβ inhibitor is an activation inhibitor of TGFβ1. For example, the activation inhibitor can be a monoclonal antibody or antigen-binding fragment thereof that binds to the latent lasso region of the latent TGFβ1 complex. In some embodiments, the TGFβ inhibitor is Ab4, Ab5, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, or Ab34. Most preferably, the TGFβ inhibitor is Ab6 or a variant thereof (e.g., a variant of Ab6 as used herein means at least 80%, 90%, 95% or more sequence relative to Ab6). one that retains similarity and/or one or more binding and/or therapeutic properties of Ab6 so as to achieve the desired therapeutic effect).

In various embodiments, the methods and compositions for use disclosed herein can be used in combination with (e.g., in combination with) checkpoint inhibitors and/or genotoxic therapy. comprising the use of a TGFβ inhibitor, wherein the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG3 antibody, or an antigen-binding fragment thereof; and/ or the genotoxic therapy is chemotherapy or radiotherapy; optionally the chemotherapy is PARP inhibitor therapy.

In various embodiments, the methods and compositions for use disclosed herein include the use of a TGFβ inhibitor disclosed herein in combination with at least one additional therapy. In some embodiments, the at least one additional therapy is cancer therapy, e.g., immunotherapy, genotoxic therapy, including chemotherapy and radiation therapy (including radiotherapeutic agents), recombinant immune cell therapy (e.g., CAR-T therapy). ), cancer vaccine therapy and/or oncolytic virus therapy. In some embodiments, the at least one additional therapy is chemotherapy or radiation therapy (including radiotherapeutic agents). In some embodiments, the at least one additional cancer therapy is checkpoint inhibitor therapy. In some embodiments, checkpoint inhibitors can include agents that target programmed cell death protein 1 (PD-1) or programmed cell death protein 1 ligand (PD-L1). For example, checkpoint inhibitors can include anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the TGFβ inhibitors disclosed herein are PD-1 antagonists (e.g., PD-1 antibodies), PDL1 antagonists (e.g., PDL1 antibodies), PD-L1 or PDL2 fusion proteins, CTLA4 Antagonist (e.g. CTLA4 antibody), GITR agonist (e.g. GITR antibody), anti-ICOS antibody, anti-ICOSL antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-TIM3 antibody, anti-LAG3 antibody, anti-OX40 antibody (OX40 agonist), anti-CD27 Antibodies, anti-CD70 antibodies, anti-CD47 antibodies, anti-41BB antibodies, anti-PD-1 antibodies, anti-CD20 antibodies, anti-CD3 antibodies, anti-PD-1/anti-PDL1 bispecific or multispecific antibodies, anti-CD3/anti-CD20 Bispecific or multispecific antibodies, anti-HER2 antibodies, anti-CD79b antibodies, anti-CD47 antibodies, antibodies that bind T cell immunoglobulin and ITIM domain protein (TIGIT), anti-ST2 antibodies, anti-β7 integrins (e.g. anti-α4 - β7 integrin and/or αEβ7 integrin), CDK inhibitors, oncolytic viruses, indoleamine 2,3-dioxygenase (IDO) inhibitors and/or PARP inhibitors. obtain.

Shows tumor MDSC levels measured in MBT-2 tumors. Tumor volume and circulating G-MDSC and M-MDSC levels in MBT-2 mice are shown. Tumor volumes in MBT-2 mice across treatment groups are shown. Baseline levels of circulating MDSC in non-tumor-bearing mice are shown. Levels of circulating MDSC in tumor-bearing mice are shown. Comparison of circulating MDSC levels in non-tumor-bearing and tumor-bearing mice is shown. Figure 7A shows a comparison of circulating M-MDSC and G-MDSC levels from days 3 to 10. Figure 7B shows the time course of circulating M-MDSC and G-MDSC levels from days 3 to 10. Figure 2 is a plot of circulating MDSC levels and tumor volume at day 10 across treatment groups. Figure 9A shows tumor MDSC levels in different treatment groups. Figure 9B shows a comparison of circulating G-MDSC and tumor MDSC levels at day 10 across treatment groups. Correlation between circulating and tumor MDSC levels is shown. Tumor G-MDSC and tumor CD8+ cells across all treatment groups are shown. Figure 12A shows circulating gMDSC and mMDSC levels in whole blood of mice bearing MBT2 tumors. Figure 12B shows intratumoral gMDSC and mMDSC levels in mice bearing MBT2 tumors. Figure 13A shows circulating TGFβ1 levels (pg/mL) in MBT-2 mice. Figure 13B shows plasma levels of Ab6 (μg/mL, left) and TGFβ1 (pg/mL, right). Figure 13C shows the correlation between plasma levels of Ab6 (μg/mL) and TGFβ1 (pg/mL) in MBT-2 mice treated with AB6 alone or in combination with anti-PD1 antibody. Plasma platelet factor 4 levels (ng/mL) in MBT-2 mice (right) and sample outliers determined by interquartile range (left) are shown. Identified sample outliers (left) and outlier correction levels (pg/mL) (right) of circulating TGFβ1 are shown. Circulating TGFβ levels in NHPs after a single administration of Ab6. Circulating TGFβ levels in rats after a single administration of Ab6. 1 illustrates an exemplary sample collection and processing method for assessing circulating TGFβ1 levels in blood. Circulating TGFβ1 levels in blood samples evaluated under various sample processing conditions. Figure 2 shows platelet factor 4 (PF4) levels in blood samples evaluated under various sample processing conditions. Figure 2 shows the correlation between circulating TGFβ1 and PF4 levels in blood samples evaluated under various sample processing conditions. FIG. 21A shows PF4 levels in blood samples evaluated under various sample processing conditions. 21B and 21C show an exemplary outlier analysis based on measurements of PF4 levels. PF4 versus TGFβ1 levels are shown before and 1 hour after administration. Figure 23A shows the fold change in TGFβ levels over time in subjects treated with 80-240 mg of Ab6. FIG. 23B shows the fold change in TGFβ levels over time in subjects treated with 800 mg of Ab6. Figure 23C shows the fold change in TGFβ levels over time in subjects treated with 1600 mg of Ab6. P-Smad2 IHC analysis of melanoma samples is shown. pSmad-2 signaling in MBT2 tumors after treatment with Ab6-mIgG1. FIG. 26A shows tissue compartmentalization data for bladder cancer samples. FIG. 26B shows tissue compartment data for melanoma samples. Figure 3 shows the density of CD8+ cells in bladder cancer samples analyzed based on tumor foci. Immunophenotypic analysis of a single bladder cancer sample based on the density of CD8+ cells measured within tumor nests. Figure 29A shows mean percentages of CD8+ cells and immunophenotypes in bladder cancer and melanoma samples analyzed by tumor compartment (left) and tumor foci (right). Underlined phenotypes indicate differences between assays. Figure 29B shows the average percentage of CD8+ cells and immunophenotypes in bladder cancer and melanoma samples analyzed by tumor compartment (left) and tumor focus (right). Underlined phenotypes indicate differences between assays. Figure 29C shows tumor nest data and immunophenotypes of individual tumor foci identified from bladder cancer samples. Figure 29D shows tumor focal CD8+ data and immunophenotypes of bladder cancer and melanoma samples. Figure 29E shows the percentage of CD8+ cells in the tumor, tumor margin, and stromal compartment of commercially available bladder cancer samples. Figure 30A shows representative CD8+ staining in bladder cancer samples. FIG. 30B shows subdivision of CD8+ staining in tumor margin compartments. Figure 30C shows subdivision of CD8+ staining in the tumor margin compartment of bladder samples. Comparison of compartment CD8+ ratio and absolute percent CD8 positivity is shown. Comparison of CD8+ cell density and absolute percentage of CD8 positivity rate is shown. The tumor invasion depth of the bladder sample is shown. CD8 density in melanoma samples is shown. Figures 35A-C show exemplary analysis of MDSCs by signal filtering. Figures 36A-C show the identification of tumor MDSC populations in various solid tumor samples. Figures 37A-C show analysis of gMDSC and mMDSC populations in various solid tumor samples. FIG. 3 depicts a schematic of an exemplary pathological analysis of a tumor tissue sample. FIG. 3 depicts a schematic of an exemplary pathological analysis of a tumor tissue sample. 1 shows a schematic diagram of an exemplary TGFβ inhibitor treatment regimen. Identification of three binding regions (region 1, region 2, and region 3) after statistical analysis is shown. Region 1 overlaps with a region called "latent lasso" within the prodomain of proTGFβ1, whereas regions 2 and 3 are within the growth factor domain. Various domains and motifs of proTGFβ1 are shown with respect to the three binding regions involved in Ab6 binding. A sequence alignment between the three isoforms is also provided.

Definitions In order that this disclosure may be more easily understood, certain terms will first be defined. These definitions should be construed 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 one of ordinary skill in the art. Additional definitions are provided throughout the detailed description.

Advanced cancer, advanced malignancy: As used herein, the term "advanced cancer" or "advanced malignancy" has the meaning as understood in the relevant art, e.g. or as understood by the oncologist in the context of treatment. Advanced malignancies associated with solid tumors may be locally advanced or metastatic. The term "locally advanced cancer" is used to describe cancer (eg, a tumor) that has grown outside the organ in which it started, but has not yet spread to distal parts of the body. Thus, the term includes cancer that has spread to nearby tissues or lymph nodes from where it started. In contrast, a "metastatic cancer" is a cancer that has spread from the part of the body where it started (the primary site) to other parts of the body (eg, distal parts).

Affinity: Affinity is the strength of binding between a molecule (such as an antibody) and its ligand (such as an antigen). This is typically measured and recorded by the equilibrium dissociation constant (K _D ). In the context of antibody-antigen interactions, the K _D is the rate of antibody dissociation (the "off rate" or K _off ), ie, the rate at which the antibody dissociates from its antigen, and the rate of association of the antibody (the "on rate" or K off ). K _on ), the ratio of how quickly an antibody binds to its antigen. For example, an antibody with an affinity of ≦5 nM has a K _D value of 5 nM or less (ie, an affinity of 5 nM or greater) as determined by a suitable in vitro binding assay. Suitable in vitro assays can be used to measure the K _D value of an antibody against its antigen, such as biolayer interferometry (BLI) and solution equilibrium titration (eg, MSD-SET). In a preferred embodiment, affinity is measured by surface plasmon resonance (eg, Biacore®). Antibodies with suitable affinities in surface plasmon resonance assays include, for example, up to about 1 nM, such as up to about 0.5 nM, such as up to about 0.5, 0.4, 0.3, 0.2, 0. It may have a K _D of 15 nM or less.

Antibody: The term "antibody" refers to any naturally occurring, recombinant, modified or engineered immunoglobulin or immunoglobulin-like structure or antigen, as described in more detail elsewhere herein. It includes binding fragments, parts thereof, and derivatives thereof. The term thus refers to immunoglobulin molecules that specifically bind to a target antigen and includes, 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 contain fewer chains, e.g. only heavy chains, as naturally occurring in camelids. Antibodies and the like may also be included. An antibody may be derived from only a single source, or may be "chimeric" (ie, different portions of the antibody may be derived from two different antibodies). Antibodies or antigen-binding portions thereof can be produced in hybridomas, by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. As used herein, the term antibody refers to monoclonal antibodies, multispecific antibodies such as bispecific antibodies, minibodies, domain antibodies, and 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"), respectively. In some embodiments, the term also encompasses peptibodies.

Antigen: The term "antigen" broadly encompasses any molecule that contains an antigenic determinant within the binding region to which an antibody or fragment specifically binds. Antigens can be single unit molecules (such as protein monomers or fragments) or complexes composed of multiple components. An antigen provides an epitope, eg, a molecule or portion of a molecule or a molecule or complex of molecules, that can be bound by a selective binding agent such as an antigen binding protein (including, eg, 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 produce antibodies that can bind to the 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 comprising a proTGF dimer associated with a presentation molecule (eg, a multimeric complex of associated components). 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 the presentation molecule via a disulfide bond, which includes a cysteine residue present near the N-terminus of each proTGF monomer. This multiple complex formed by proTGF dimers bound to presentation molecules is commonly referred to as the large latent complex. Antigen complexes suitable for screening antibodies or antigen-binding fragments include, for example, displaying molecular components of large latent complexes. Such display molecule components can be full-length display molecules or fragments thereof. The minimum necessary portion of the display molecule typically comprises at least 50 amino acids of the display molecule polypeptide, including two cysteine residues capable of forming a covalent bond with the proTGFβ1 dimer; More preferably it contains at least 100 amino acids.

Antigen-binding portion/fragment: As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to an antibody that retains the ability to specifically bind an antigen (e.g., TGFβ1). Refers to the above fragments. Antigen-binding moieties include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to the antigen. bind to form a complex. In some embodiments, the antigen-binding portion of an antibody is produced by any suitable method, such as proteolytic digestion or recombinant genetic engineering techniques, including, for example, manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. can be derived from complete antibody molecules using standard techniques. Non-limiting examples of antigen binding moieties include: (i) Fab fragments, monovalent fragments composed of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of a VH domain and a CH1 domain; (iv) a VL domain and a VH domain of a single arm of the antibody. (v) single-chain Fv (scFv) molecules (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) dAb fragments (see, eg, Ward et al., (1989) Nature 341:544-546); and (vii) antibodies. A minimal recognition unit composed of amino acid residues that mimic the hypervariable regions of (e.g., isolated complementarity determining regions (CDRs)). Also included are other forms of single chain antibodies such as diabodies. The term antigen-binding portion of an antibody includes the antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and linker. ”, wherein the antibody domain and the linker have one of the following sequences in the direction from the N-terminus to the C-terminus: 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; in this case said linker comprises at least 30 amino acids, Preferably it is a polypeptide of 32 to 50 amino acids.

Bias: In the context of this disclosure, the term "bias" (as in "biased binding") refers to bias toward or against a subset of antigens to which an antibody can specifically bind. or refer to heterogeneous affinities. For example, an antibody is said to be biased if its affinity for one antigen complex is not equal to its affinity for another antigen complex. Context-independent antibodies according to the present disclosure have comparable affinities (ie, unbiased or homogeneous) for such antigen complexes.

Binding region: As used herein, a "binding region" is a portion of an antigen that, when bound to an antibody or fragment thereof, is capable of forming an interface for antibody-antigen interaction. Once the antibody binds, the binding region is protected from surface exposure and can be detected by a suitable technique such as HDX-MS. Antibody-antigen interactions can be mediated through multiple (eg, two or more) binding regions. A binding region can include an antigenic determinant, or epitope.

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

Cancer: As used herein, the term "cancer" refers to a physiological condition in multicellular eukaryotes that is typically characterized by abnormal cell growth and malignancy. The term broadly encompasses solid and liquid malignancies, including tumors, hematological cancers (eg, leukemia, lymphoma, and myeloma), and myelofibrosis.

Cell-associated proTGFβ1: This term refers to membrane-bound (eg, tethered to the cell surface) TGFβ1 or its signaling complex (eg, pro/latent TGFβ1). Typically such cells are immune cells. TGFβ1 presented by GARP or LRRC33 is cell-associated TGFβ1. GARP and LRRC33 are transmembrane display molecules expressed on the cell surface of certain cells. GARP-proTGFβ1 and LRRC33- may also be collectively referred to as "cell-associated" (or "cell surface") proTGFβ1 complexes, which are responsible for cellular proTGFβ1 binding (e.g., immune cell-associated) TGFβ1 activation/signaling. mediate. The term also includes recombinant, purified GARP-proTGFβ1 and LRRC33-proTGFβ1 complexes in solution and not physically associated with cell membranes (eg, in vitro assays). The average K value of antibodies (or fragments thereof) against the GARP-proTGFβ1 complex and the LRRC33-proTGFβ1 complex can be calculated to collectively represent the affinity for cell-associated (e.g., immune cell-associated) proTGFβ1 complexes. . See, for example, column (G) of Table 5. The human counterpart of a presentation molecule or presentation molecule complex may be designated by an "h" preceding the protein or protein complex, such as "hGARP", "hGARP-proTGFβ1", "hLRRC33" and "hLRRC33-proTGFβ1". . In addition to blocking the release of active TGFβ1 growth factor from cell-tethered complexes, cell-associated proTGFβ1 inhibits internalization (e.g., endocytosis) and/or ADCC, ADCP, or may be a target for cell killing such as ADC-mediated depletion.

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 inhibition therapy" targets checkpoint molecules and partially or completely alters their function. Typically, the checkpoint is a receptor molecule on a T cell or NK cell or a corresponding cell surface ligand on an antigen presenting cell (APC) or tumor cell. Without wishing to be bound by theory, immune checkpoints are activated within immune cells to prevent the development of inflammatory immunity against "self." Therefore, changing the balance of the immune system through checkpoint inhibition may allow the immune system to be fully activated to detect and eliminate cancer. The most well-known inhibitory receptors involved in the control of immune responses are cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), and 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, davalumab, ipilimumab, tremelimumab, IMP-321 (eftiragimod alfa or ImmuFact®), BMS-986016 (leratimab), budigalimab (ABBV-181) and rililumab. Keytruda® is an example of an anti-PD-1 antibody, and Opdivo® is an example of an anti-PD-L1 antibody. Therapy using one or more immune checkpoint inhibitors is sometimes referred to as checkpoint blockade therapy (CBT) or checkpoint inhibitor therapy (CPI).

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

Combination therapy: "Combination therapy" refers to a treatment regimen for a clinical indication that includes two or more therapeutic agents. Accordingly, the term refers to the use of a first therapeutic agent comprising a first composition (e.g., active ingredient) to treat a second composition (active ingredient) for the purpose of treating the same or overlapping disease or clinical condition. Refers to a therapeutic regimen that is administered to a patient together with at least one second therapeutic agent. This term means that a first therapeutic agent comprising a first composition (e.g. an active ingredient) is a second therapeutic agent comprising a second composition (e.g. an active ingredient such as a checkpoint inhibitor). The therapeutic regimens may further include therapeutic regimens administered together with a third therapeutic agent or more (eg, additional different active ingredients) comprising three compositions (eg, active ingredients such as chemotherapeutic agents). The first, second and (optionally additional) compositions may act on the same cellular target or on separate cellular targets. The phrase "in conjunction with" in the context of combination therapy means that the therapeutic effect of the first therapeutic agent is temporally and/or spatially similar to the therapeutic effect of the second and additional therapeutic agent in the subject receiving the combination therapy. It means overlapping. The first, second and/or additional compositions may be administered contemporaneously (eg, simultaneously), individually or sequentially. Accordingly, combination therapy may 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 agent is an add-on. Sometimes referred to as (add-on) therapy or adjunctive therapy.

Combinatorial or combinatorial epitope: A combinatorial epitope is an epitope that is recognized and bound by a combinatorial antibody at a site (i.e., an antigenic determinant) formed by noncontiguous portions of one or more components of an antigen; structures that come together in close proximity to form epitopes. Thus, antibodies of the present disclosure can bind to epitopes formed by two or more components (eg, portions or segments) of the pro/latent TGFβ1 complex. A combinatorial epitope can include an amino acid residue from a first component of a complex, an amino acid residue from a second component of a complex, and the like. Each component can be either a single protein or two or more proteins of the antigen complex. Combinatorial epitopes are formed by structural contributions from two or more components (eg, portions or segments such as amino acid residues) of an antigen or antigen complex.

competition or cross-competition; cross-blocking: When used in reference to antigen-binding proteins (e.g., antibodies or antigen-binding portions thereof) competing for the same epitope, the term "competition" means that the antigen-binding protein being tested is Refers to competition between antigen binding proteins as determined by an assay that prevents or inhibits (eg, reduces) the specific binding of a standard antigen binding protein to a common antigen (eg, TGFβ1 or a fragment thereof). Various types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, such as solid-phase direct or indirect radioimmunoassays (RIA). ), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay; solid phase direct biotin-avidin EIA; solid phase direct labeling assay, and solid phase direct labeling sandwich assay. Typically, the presence of an excess of competing antigen binding proteins inhibits the specific binding of a standard antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%. , 65-70%, 70-75% or more than 75%. 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 (eg, Biacore) assay. In some embodiments, competition is determined using a BLI (eg, Octet®) assay.

In some embodiments, biolayer interferometry (Octet®, registered The first antibody or antigen-binding portion thereof and the second antibody or antigen-binding portion thereof “cross-block” each other against the same antigen, as assayed by surface plasmon resonance (such as the Biacore system) or by surface plasmon resonance (such as the Biacore system). . 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 another embodiment, the first antibody or fragment thereof and the second antibody or fragment thereof are separate ( different) epitopes. "Cross-blocking" means that the binding of a first antibody to one antigen prevents the binding of a second antibody to the same antigen; similarly, the binding of a second antibody to one antigen interferes with the binding of a first antibody to the same antigen. means to prevent the combination of

Perform antibody binning (sometimes referred to as epitope binning or epitope mapping) to characterize a set (e.g., a "library") of monoclonal antibodies raised against a target protein or protein complex (i.e., antigen). , it can be classified. These antibodies against the same target are tested in a pairwise fashion against all other antibodies in the library to assess whether the antibodies block each other's binding to the antigen. A closely related binning profile indicates that the antibodies have the same or closely related (eg, overlapping) epitopes and are "binned" together. Binning combines similar binding regions within the same antigen because the biological activity (e.g., intervention; potency) generated by the binding of an antibody to a target is likely to be inherited by another antibody within the same bin. Provides useful structure-function profiles of shared antibodies. Thus, among antibodies within the same epitope bin, antibodies with higher affinity (lower KD) generally have greater potency.

In some embodiments, an antibody that binds to the same epitope as Ab6 is such that the epitope of the antibody includes one or more amino acid residues of region 1, region 2, and region 3 identified as the binding region of Ab6. Binds to proTGFβ1 complex.

Complementarity Determining Region: 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, referred to as CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs that occur in a single variable region that is capable of binding an antigen. The exact boundaries of these CDRs are defined differently according to different systems. Kabat (Kabat et al., (1987; 1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD. The system described in ) is applicable to any variable region of an antibody with well-defined residues. In addition to providing a numbering scheme, it also provides the precise residue boundaries that define 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), although certain subparts within the Kabat CDRs have great diversity at the amino acid sequence level. We found that the peptides adopt nearly identical backbone conformations. Referred to as H-CDR2 and H-CDR3, where "L" and "H" indicate the light chain and heavy chain regions, respectively. These regions are also referred to as Chothia CDRs, which are Kabat CDRs. Other boundaries delimiting CDRs that overlap with Kabat CDRs include Padlan (1995) FASEB J.9:133-139 and MacCallum (1996) J. Mol. Biol. 262(5):732. Further CDR boundary definitions may not strictly follow one of the systems herein, but would still overlap with the Kabat CDRs (although certain In view of predictions or experimental findings that a residue or group of residues or an entire CDR does not significantly affect antigen binding, they may be shortened or lengthened (e.g., Lu X et al., MAbs 2019 Jan; 11(1):45-57). Although the methods used herein may utilize CDRs defined according to any of these systems, in certain embodiments , Kabat or Chothia.

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. Conformational epitopes may be referred to as conformation-specific epitopes, conformation-dependent epitopes, or conformation-sensitive epitopes. Corresponding antibodies or fragments thereof that specifically bind to such epitopes may be referred to as conformation-specific antibodies, conformation-selective antibodies or conformation-dependent antibodies. The binding of an antigen to a conformational epitope depends on the three-dimensional structure (conformation) of the antigen or antigen complex.

Constant region: 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.

Context Bias: As used herein, a "context biased antibody" refers to an antigen with differential affinity when the antigen is associated with (i.e., binds to or attached to) an interacting protein or fragment thereof. Refers to one type of conformational antibody that binds to. Thus, context-biased antibodies that specifically bind to epitopes within proTGFβ1 can bind to LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1 and LRRC33-proTGFβ1 with different affinities. For example, if the antibody has a 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). , antibodies can be said to be "matrix biased." [Matrix-bound complex]: The relative affinity of [cell-associated complex] is determined by obtaining the average K _D value of the former and obtaining the average K _D value of the latter, as exemplified herein. It can be obtained by calculating the ratio of both.

Context-independent: According to the present disclosure, "context-independent antibodies" that bind to proTGFβ1 include four known presenting molecule-proTGFβ1 complexes, namely LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1 and LRRC33-proTGFβ1. have the same affinity. Context-independent antibodies disclosed in this application can also be characterized as non-biased. Typically, context-independent antibodies are used such that the relative ratio of measured KD values between matrix-bound and cell-associated complexes is determined by surface plasmon resonance, biolayer interferometry (BLI), and/or solution equilibrium. It exhibits comparable (ie, 5-fold bias or less) affinity of 5 or less as determined by a suitable in vitro binding assay such as titration (eg, MSD-SET). In a preferred embodiment, surface plasmon resonance is used.

ECM-associated TGFβ1/proTGFβ1: This term refers to TGFβ1 or its signaling complex (eg, pro/latent TGFβ1) that is a component of the extracellular matrix (eg, deposited in the cellular matrix). TGFβ1 presented by LTBP1 or LTBP3 is ECM-bound TGFβ1, namely LTBP1-proTGFβ1 and LTBP3-proTGFβ1, respectively. LTBP is important for the correct deposition and subsequent bioavailability of TGFβ in the ECM, and fibrillin (Fbn) and fibronectin (FN) are thought to be the major matrix proteins responsible for the association of LTBP with the ECM. Such matrix-bound latent complexes are enriched in connective tissue and certain disease-related tissues such as tumor stroma and fibrotic tissue. The human counterpart of a presentation molecule or presentation molecule complex may be designated by an "h" preceding the protein or protein complex, such as "hLTBP1", "hLTBP1-proTGFβ1", "hLTBP3" and "hLTBP3-proTGFβ1". .

Effective amount: The terms "effective" and "therapeutically effective" refer to an amount sufficient to cause a detectable change in a parameter of the disease, such as slowing, halting, reversing, reducing, or ameliorating symptoms or downstream effects of the disease. Refers to the ability or amount to bring about. This term encompasses, but does not require, the use of amounts that completely cure the disease. An "effective amount" (or therapeutically effective amount or dose) can be a dose or dosing regimen that achieves a statistically significant clinical benefit (eg, efficacy) in a patient population. For example, Ab6 has been shown to be effective at doses as low as 3 mg/kg and as high as 30 mg/kg in preclinical models. The term "minimum effective dose" or "minimum effective amount" refers to the lowest amount, dosage, or dosing regimen that achieves a detectable change in a parameter of a disease, such as a statistically significant clinical benefit. When referring to a dose of a drug (eg, a dose of a TGFβ1 inhibitor) herein, it can 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 can be expressed in terms of the dose administered or the level of exposure (eg, serum concentration) achieved as a result of administration.

Effective tumor control: The term "effective tumor control" can be used to refer to the extent of tumor regression achieved in response to treatment, e.g. regress by a percentage (eg <25%). For example, in a particular model, if the endpoint tumor volume is set at 2,000 ^mm , effective tumor control will be achieved if the tumor shrinks below 500 ^mm , assuming a threshold of <25%. . Effective tumor control therefore includes complete regression. Clinically, effective tumor control is defined as partial response (PR) and complete response (CR) as determined by art-recognized criteria such as RECIST v1.1 and corresponding iRECIST (iRECIST v1.1). can be measured by objective response including In some embodiments, effective tumor control in a clinical setting also includes stable disease, where a tumor that is generally expected to grow at a constant rate, even if no shrinkage is achieved. Treatment prevents such growth.

Effector T cell: As used herein, an effector T cell is a T lymphocyte that immediately and actively responds to stimuli such as costimulation, including, but not limited to, CD4+ T cells ( CD8+ T cells (also called T helper or Th cells) and CD8+ T cells (also called cytotoxic T cells). Th cells assist other leukocytes 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). When activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist in active immune responses. These cells can differentiate into one of several subtypes, including Th1, Th2, Th3, Th17, Th9, or TFh, which secrete various cytokines and stimulate different types of immune responses. promote. Signaling from APCs directs T cells toward specific subtypes. Cytotoxic (killer). On the other hand, cytotoxic T cells (TC cells, CTLs, T killer cells, killer T cells) destroy virus-infected cells 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 present on the surface of all nucleated cells. Cytotoxic effector cell (eg, CD8+ cell) markers include, for example, perforin and granzyme B.

Endpoint: In studies aimed at evaluating 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 given parameter indicative of treatment efficacy. . In oncology, relevant endpoints are overall survival, disease-free survival (DFS), event-free survival (EFS), progression-free survival (PFS), objective response rate (ORR), and complete response (CR). , partial response (PR), time to progression (TTP), and biomarker assessments such as patient-reported outcomes (eg, symptom ratings) and blood- or body fluid-based assessments.

Epithelial hyperplasia: The term "epithelial hyperplasia" refers to increased tissue proliferation resulting from proliferation of epithelial cells. As used herein, epithelial hyperplasia refers to the undesirable toxicity caused by TGFβ inhibition, including, but not limited to, the abnormal proliferation of epithelial cells in the oral cavity, esophagus, breast, and ovary. can be mentioned.

Epitope: The term "epitope," sometimes referred to as an antigenic determinant, is a molecular determinant (e.g., a polypeptide determinant) that can be specifically bound by a binding agent, immunoglobulin, or T-cell receptor. be. Epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and, in certain embodiments, possess particular three-dimensional structural characteristics, and/or particular charge characteristics. may have. An epitope recognized by an antibody or antigen-binding fragment of an antibody is a structural element of the antigen that interacts with the CDRs (eg, complementary sites) of the antibody or fragment. An epitope can be formed by contributions from several amino acid residues, which interact with the CDRs of an antibody to provide specificity. Antigenic fragments 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 the same epitope" if they cross-compete (one interferes with the binding or modulatory effect of the other).

Elongated latent lasso: As used herein, the term "extended latent lasso" refers to the portion of the prodomain that includes the latent lasso and the α-2 helix, such as LASPPSQGEVPPGPLPEAVLALYNSTR (SEQ ID NO: 127). In some embodiments, the elongated latent lasso further comprises a portion of the α-1 helix, such as LVKRKRIEA (SEQ ID NO: 132) or a portion thereof.

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, within a tissue or organ. Point.

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: 124). In the 3D structure, the finger-1 domain is in close proximity to the latent lasso.

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: 125). Finger-2 includes a "binding region 6", which is located spatially close to the latent lasso.

GARP-proTGFβ1 complex: As used herein, the term “GARP-TGFβ1 complex” refers to transforming growth factor-β1 (TGFβ1) protein and glycoprotein-A repeat-predominant protein (GARP) or its Refers to protein complexes that include proprotein or latent forms of fragments or variants. In some embodiments, the pro-protein or latent form of TGFβ1 protein may also be referred to as "pro/latent TGFβ1 protein." In some embodiments, the 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 (eg, amino acid position 4) of the proTGFβ1 dimeric complex. In other embodiments, the GARP-TGFβ1 complex comprises GARP non-covalently linked to pro/latent TGFβ1. In some embodiments, the GARP-TGFβ1 complex is a naturally occurring complex, such as an intracellular GARP-TGFβ1 complex. The term "hGARP" means human GARP.

High affinity: As used herein, the term "high affinity" as in "high affinity proTGFβ1 antibody" refers to an in vitro binding activity with a K _D value of ≦5 nM, more preferably ≦1 nM. Point. Accordingly, high affinity, context-independent proTGFβ1 antibodies encompassed by the present disclosure are directed against each of the following antigen complexes: LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1 and LRRC33-proTGFβ1. , ≦5 nM, more preferably ≦1 _nM .

Human antibody: As used herein, the term "human antibody" is intended to include antibodies that have variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure contain amino acid residues not encoded by human germline immunoglobulin sequences, e.g., in the CDRs and particularly CDR3, such as by random or site-directed mutagenesis in vitro or by somatic mutagenesis in vivo. mutations introduced by mutation). 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. .

Humanized antibody: The term "humanized antibody" includes heavy chain variable region sequences 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 are Refers to antibodies that are more "human-like", ie, have been modified to more closely resemble 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. Furthermore, a "humanized antibody" immunospecifically binds to a target antigen and includes an FR region that substantially has the amino acid sequence of a human antibody and a CDR region that substantially has the amino acid sequence of a non-human antibody. An antibody or a variant, derivative, analog or fragment thereof. As used herein, the term "substantially" in the context of CDRs means at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or Refers to CDRs that have at least 99% identical amino acid sequences. A humanized antibody is one in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., the donor antibody) and all or substantially all of the FR regions are of human immunoglobulin consensus sequences. Contains substantially all of one, typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv). In embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin Fc region, typically a human immunoglobulin. In some embodiments, a humanized antibody contains at least the variable domain of a light chain as well as a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, humanized antibodies contain only humanized light chains. In some embodiments, humanized antibodies contain only humanized heavy chains. In certain embodiments, a humanized antibody comprises only the humanized variable domains of a light chain and/or a humanized heavy chain.

Immune- or immuno-cleared tumor: As used herein, a tumor characterized by "immune clearance" is deficient or substantially free of intratumoral anti-tumor lymphocytes. It is missing. For example, a tumor with poorly infiltrated T cells may have T cells surrounding the tumor, e.g., around the periphery of the tumor mass and/or in close proximity to the tumor's vasculature (“perivascular”); First, T cells cannot effectively gather within tumors and exert cytotoxic functions against cancer cells. In other situations, the tumor is unable to elicit a strong immune response (so-called "immune desert" tumors) and, as a result, there are few T cells near and within the tumor environment. In contrast to immune-cleared tumors, tumors infiltrated by anti-tumor lymphocytes are sometimes characterized as "hyperimmunogenic" or "inflammatory" tumors; such tumors can be treated with immune checkpoint blockade therapy. (“CBT”), making it a target because it tends to be more responsive to CBT. However, typically only a small proportion of patients respond to CBT due to immune clearance that renders the tumor resistant to CBT.

Immune safety (assessment): As used herein, this term refers to safety assessment with respect to immune response (immune activation), and acceptable immunosafety standards include: in vitro or no significant cytokine release, as determined by an in vivo cytokine release test (eg, assay); and no significant platelet aggregation, activation, as determined using human platelets. Statistical significance in these tests can be determined against appropriate reference controls. For example, in the case of a test molecule that is a human monoclonal antibody, a suitable control may be an immunoglobulin of the same subtype, eg, an antibody of the same subtype known to have a good safety profile in humans.

Immunosuppression, immunosuppression, 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 inhibition. Regulatory T cells (Tregs) and MDSCs can be considered immunosuppressive cells. M2-polarized macrophages (eg, disease-localized macrophages such as TAM and FAM) can also be characterized as immunosuppressive.

Immunological memory: Immunological memory refers to the ability of the immune system to rapidly and specifically recognize antigens that the body has encountered in the past and mount a corresponding immune response. Generally, these are secondary, tertiary and other subsequent immune responses to the same antigen. Immune memory is responsible for the adaptive components of the immune system, specialized T and B cells (so-called memory T and B cells). Antigen-naive T cells expand and differentiate into memory and effector T cells after encountering cognate antigen within the environment of MHC molecules on the surface of professional antigen presenting cells (eg dendritic cells). The single unifying theme for all memory T cell subtypes is that they are long-lived and can rapidly expand to large numbers of effector T cells upon re-exposure to their cognate antigen. By this mechanism, they provide the immune system with "memory" for previously encountered pathogens. Memory T cells can be either CD4+ or CD8+ and usually express CD45RO. In a preclinical setting, immunological memory can be tested in a tumor rechallenge paradigm.

Inhibition or inhibition of: As used herein, the term "inhibition" or "inhibition of" means to reduce by a measurable amount, and may include complete prevention or inhibition, but I'm not asking for that.

Isoform non-specific: The term "isoform non-specific" refers 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.

Isoform specific: The term "isoform specificity" refers to the ability of a drug to discriminate one isoform from other structurally related isoforms (ie, isoform selectivity). An isoform-specific TGFβ inhibitor exerts its inhibitory activity against one isoform of TGFβ, but not against the other isoform of TGFβ, at a given concentration. For example, isoform-specific TGFβ1 antibodies selectively bind TGFβ1. TGFβ1-specific inhibitors (antibodies) preferentially target (bind to and thereby inhibit) TGFβ1 isoforms with substantially higher affinity than TGFβ2 or TGFβ3. For example, selectivity in this situation is at least 10-fold, 100-fold higher than the respective affinity, as measured by an in vitro binding assay such as BLI (Octet®) or preferably SPR (Biacore®). , 500-fold, 1000-fold or more. In some embodiments, the selectivity is such that the inhibitor does not inhibit TGFβ2 and TGFβ3 when used at a dose effective to inhibit TGFβ1 in vivo. For such an inhibitor to be useful as a therapeutic agent, a dosage (e.g., a therapeutically effective amount) to achieve the desired effect is such that the inhibitor inhibits TGFβ1 isoforms without inhibiting TGFβ2 or TGFβ3. must be within a therapeutic range that can effectively inhibit In some embodiments, a TGFβ1 selective inhibitor is an agent that interferes with the function or activity of TGFβ1, but not the function or activity of TGFβ2 and/or TGFβ3, regardless of the mechanism of action.

Isolation: As used herein, an "isolated" antibody refers to an antibody that is substantially free of other antibodies with different antigen specificities. In some embodiments, isolated antibodies are substantially free of other unintended cellular materials and/or chemicals.

Large latent complex: In the context of this disclosure, the term "large latent complex" ("LLC") refers to a complex consisting of a proTGFβ1 dimer bound to a so-called presentation molecule. Therefore, 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 forming complexes. For screening purposes, the display molecules used to form such LLCs need not be full-length polypeptides. However, typically a portion of the protein is required that is capable of forming disulfide bonds with the proTGFβ1 dimeric complex via cysteine residues near its N-terminal region.

Latent-associated peptide (LAP): LAP is the so-called "prodomain" of proTGFβ1. As described in more detail herein, a LAP is composed of a "straight jacket" domain and an "arm" domain. The straight jacket itself is further divided into alpha-1 helical domains and latent lasso domains.

Latent Lasso: As used herein, the “latent lasso”, also referred to as the latent loop, is the domain flanked by the α-1 helix and arms within the prodomain of proTGFb1. In its non-mutant form, the latent lasso of human proTGFβ1 contains the amino acid sequence: LASPPSQGEVPPGPL (SEQ ID NO: 126), into which region 1, identified in FIG. 41, extends. As used herein, the term "extended cryptic lasso region" refers to a cryptic lasso that includes 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. Certain high-affinity TGFβ1 activation inhibitors bind at least in part to latent Lasso or a portion thereof to confer inhibitory ability (e.g., the ability to block activation) and optionally bind to latent Lasso or a portion thereof. The above part of the type lasso is ASPPSQGEVPPGPL (SEQ ID NO: 170). In some embodiments, the antibodies of the present disclosure bind the proTGFβ1 complex to ASPPSQGEVPPGPL (SEQ ID NO: 170) or a portion thereof. Certain high-affinity TGFβ1 activation inhibitors bind, at least in part, to an elongated latent lasso or a portion thereof, conferring an inhibitory ability (e.g., the ability to block activation), in which case an optional Optionally, the portion of the elongated latent lasso is KLRLASPPSQGEVPPGPLPEAVL (SEQ ID NO: 142).

Localized: In the context of this disclosure, the term “localized” (as in “localized tumor,” “disease localized,” etc.) refers to an anatomical disease, such as a solid malignant tumor, as opposed to a systemic disease. Refers to a chemically isolated or isolable abnormality. Certain leukemias, for example, may have both localized (eg, bone marrow) and systemic (eg, circulating blood cell) components to the disease.

LRRC33-proTGFβ1 complex: As used herein, the term "LRRC33-TGFβ1 complex" refers to the proprotein or latent form of the transforming growth factor-β1 (TGFβ1) protein and the 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 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 (eg, amino acid position 4) of the proTGFβ1 dimeric complex. In other embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 non-covalently linked to pro/latent TGFβ1. In some embodiments, the LRRC33-TGFβ1 complex is a naturally occurring complex, eg, an intracellular LRRC33-TGFβ1 complex. The term "hLRRC33" means human LRRC33. In vivo, LRRC33 and LRRC33-containing complexes on the cell surface can be internalized. LRRC33 is expressed on a subset of myeloid cells, including M2-polarized macrophages (such as TAMs) and MDSCs.
MDSCs that express LRRC33 on the cell surface include tumor-associated MDSCs and circulating MDSCs. LRRC33-expressing tumor-associated MDSCs can include gMDSCs. LRRC33-expressing MDSCs in circulation may include g-MDSCs.

LTBP1-proTGFβ1 complex: As used herein, the term “LTBP1-TGFβ1 complex” refers to transforming growth factor-β1 (TGFβ1) protein and latent TGF-β binding protein 1 (LTBP1) or its Refers to protein complexes that include proprotein forms or cryptic forms of fragments or variants. 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 (eg, 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, such as an intracellular LTBP1-TGFβ1 complex. The term "hLTBP1" means human LTBP1.

LTBP3-proTGFβ1 complex: As used herein, the term “LTBP3-TGFβ1 complex” refers to transforming growth factor-β1 (TGFβ1) protein and latent TGF-β binding protein 3 (LTBP3) or its Refers to protein complexes that include proprotein forms or latent forms of fragments or variants. 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 (eg, 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, such as an intracellular LTBP3-TGFβ1 complex. The term "hLTBP3" means human LTBP3.

M2 or M2-like macrophages: M2 macrophages represent a subset of activated or polarized macrophages and include disease-associated macrophages in both fibrotic and neoplastic microenvironments. Cell surface markers for M2-polarized macrophages typically include CD206 and CD163 (ie, CD206+/CD163+). M2-polarized macrophages can also express cell surface LRRC33. Activation of M2 macrophages is primarily promoted by IL-4, IL-13, IL-10 and TGFβ; 10 and TGFβ). 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 tissue and tumor stroma. For example, M2 macrophages play a specific role in TGFβ-driven pulmonary fibrosis and are enriched in many tumors.

Matrix-associated proTGFβ1: LTBP1 and LTBP3 present molecules that are components of the extracellular matrix (ECM). LTBP1-proTGFβ1 and LTBP3-proTGFβ1 may be collectively referred to as the “ECM-associated” (or “matrix-associated”) proTGFβ1 complex that mediates ECM-associated TGFβ1 activation/signaling. The term also includes recombinant, purified LTBP1-proTGFβ1 and LTBP3-proTGFβ1 complexes in solution that are not physically bound to a matrix or substrate (eg, in vitro assays).

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 Adverse Effect Level (NOAEL) in relation to safety/toxicological considerations. refers to 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 of Ab6 is >100 mg/kg. There is. The NOAEL for Ab6 in non-human primates was the highest dose evaluated (300 mg/kg) based on a 4-week toxicity study, which indicates that the MTD of Ab6 in non-human primates is >300 mg/kg. It suggests something.

Mesoscale Discovery: "Mesoscale Discovery" or "MSD" is a type of immunoassay that uses electrochemiluminescence (ECL) as the detection technique. Typically, high binding carbon electrodes are used to capture proteins (eg, antibodies). Antibodies can be incubated with a particular antigen, and binding can be detected using a second antibody conjugated to an electrochemiluminescent label. Once the electrical signal is obtained, the light intensity can be measured to quantify the analyte in the sample.

Myelofibrosis: "Myelofibrosis", also known as osteomyelofibrosis, is a relatively rare bone marrow proliferative disorder (e.g., cancer); It is characterized by abnormal clonal proliferation of hematopoietic stem cells in the brain, leading to fibrosis, or replacement of bone marrow by scar tissue. The term myelofibrosis refers to primary myelofibrosis (PMF), also called chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary above refer to cases in which the disease is of unknown or spontaneous origin). ) as well as secondary types of myelofibrosis, such as myelofibrosis following polycythemia vera (PV) or essential thrombocytopenia (ET). Myelofibrosis is a form of myeloid metaplasia, which refers to changes in cell types in the hematopoietic tissue of the bone marrow, and the two terms are often used interchangeably. The terms unexplained myeloid metaplasia and myelofibrosis with myeloid metaplasia (MMM) are also used to refer to primary myelofibrosis. Myelofibrosis is characterized by mutations that cause upregulation or overactivation of the downstream JAK pathway.

Myeloid cells: In hematopoiesis, myeloid cells are blood cells that arise from granulocyte, monocyte, red blood cell or platelet progenitor cells (common myeloid progenitor cells, i.e. CMP or CFU-GEMM), or from lymphocytes. lineage cells, i.e. lymphocytes derived from the common lymphoid progenitors that give rise to B cells and T cells, are also often used in the narrow sense, especially the myeloblast lineage (myeloid cells, monocytes and blood cells derived from their daughter types). Specific myeloid cell types, their general morphology, typical cell surface markers and their immunosuppressive potential in both mice and humans are summarized below. In some embodiments, human neutrophils can be identified by at least one (eg, all) of the cell surface markers CD11b ⁺ , CD14 ⁻ , CD15 ⁺ , and CD66b ⁺ . In some embodiments, the human neutrophil is LOX-1-. In some embodiments, the human neutrophil is HLA-DR ^−/med . In some embodiments, classical human monocytes can be identified by at least one (eg, all) of the cell surface markers CD14 ⁺ CD15 ⁻ CD16 ⁻ HLA-DR ⁺ . In some embodiments, the classical human monocytes are CD33 ⁺ and/or CD11b ⁺ . In some embodiments, the classical human monocytes are CD16 ⁻ . In some embodiments, intermediate human monocytes can be identified by at least one (eg, all) of the cell surface markers CD14 ⁺ CD15 ⁻ CD16 ⁺ HLA-DR ⁺ . In some embodiments, non-classical human monocytes can be identified by at least one (eg, all) of the cell surface markers CD14 ⁻ CD15 ⁻ CD16 ⁺ HLA-DR ⁺ . In some embodiments, human M1 macrophages can be identified by at least one (eg, all) of the cell surface markers CD15 ⁻ CD16 ⁺ CD80 ⁺ HLA-DR ^{+ /} CD33 ⁺ high. In some embodiments, the human M1 macrophage is CD66b ⁻ . In some embodiments, the human M1 macrophage is CD11b ⁺ . In some embodiments, the human M1 macrophage is CD14 ⁻ . In some embodiments, human M2 macrophages can be identified by at least one (eg, all) of the cell surface markers CD11b ⁺ and CD15 ⁻ . In some embodiments, the human M2 macrophage is CD206 ⁺ . In some embodiments, the human M2 macrophage is CD163 ⁺ . In some embodiments, the human M2 macrophage is HLA-DR ⁺ . In some embodiments, the human M2 macrophage is CD14 ⁻ . In some embodiments, the human M2 macrophage is CD33 ⁺ . In some embodiments, the human M2 macrophage is CD66b ⁻ .

Myeloid-derived suppressor cells: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells produced during the course of various pathological conditions. MDSCs are i) "granulocytic" (G-MDSCs) or polymorphonuclear (PMN-MDSCs), which are phenotypically and morphologically similar to neutrophils; and ii) phenotypically and morphologically. Contains at least two categories of cells called monocytes (M-MDSCs), which are chemically similar to monocytes. MDSCs are characterized by a distinct set of genomic and biochemical features and can be identified 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-MDSC/PMN-MDSC typically express cell surface markers CD11b, CD33, CD15 and CD66b. In some embodiments, human G-MDSCs may express low levels of the CD33 cell surface marker. In some embodiments, human G-MDSC/PMN-MDSC 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-MDSC/PMN-MDSC and M-MDSC may exhibit low or undetectable levels of HLA-DR. In some embodiments, human G-MDSCs can be HLA-DR ^- . In certain embodiments, G-MDSCs can be differentiated from M-MDSCs based on the presence or absence of certain cell surface markers (eg, CD14, CD15, and/or CD66b). In some embodiments, G-MDSCs may be identified by the presence or increased expression of surface markers CD11b, CD33, CD15, CD66b and/or LOX-1 and the absence of CD14, whereas M-MDSCs may be identified by surface markers It can be identified by the presence or increased expression of CD11b, CD33 and/or CD14 and the absence of CD15. In some embodiments, M-MDSCs can be ^CD66b- . In addition to these cell surface markers, MDSCs can be characterized by the ability to suppress immune cells such as T cells, NK cells and B cells. Immunosuppressive functions of MDSCs can 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 the cell surface marker is classified as "low,""medium," or "high" based on normalization of the signal intensity to reduce background and bleed-through signals. Can be binned. In some embodiments, the signal intensities of cell surface markers can be classified based on the cutoff thresholds provided in Table 38A. In some embodiments, the signal intensity of a cell surface marker can 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 intensities of cell surface markers can be classified based on the cutoff thresholds provided in Table 38B. In some embodiments, the signal strength of a set of surface markers may be determined by successive applications of signal filtering, where one or more A signal intensity threshold for the surface marker is determined.

Myofibroblasts: Myofibroblasts are cells with specific phenotypes of fibroblasts and smooth muscle cells, and commonly express vimentin, α-smooth muscle actin (α-SMA; human gene ACTA2), and palladin. do. In many disease states with extracellular matrix dysregulation (such as increased matrix stiffness), normal fibroblasts dedifferentiate into myofibroblasts in a TGFβ-dependent manner. Aberrant 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 the fibrotic microenvironment are sometimes referred to as fibrosis-associated fibroblasts (or "FAFs"), and myofibroblasts or myofibroblasts within the tumor microenvironment. Fibroblast-like cells are sometimes referred to as cancer-associated fibroblasts (or "CAFs").

Pan-TGFβ inhibitor/pan-inhibition of TGFβ: The term “pan-TGFβ inhibitor” refers to any agent that can inhibit or antagonize all three isoforms of TGFβ. Such inhibitors can be small molecule inhibitors of TGFβ isoforms such as those known in the art. The term includes pan-TGFβ antibodies, which refer to any antibody capable of binding to each of the TGFβ isoforms, namely TGFβ1, TGFβ2 and TGFβ3. In some embodiments, the pan-TGFβ antibody binds to and neutralizes the activity of all three isoforms: TGFβ1, TGFβ2, and TGFβ3. 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 includes galunisertib (LY2157299 monohydrate), an antagonist of TGFβ receptor I kinase/ALK5 that mediates signaling of all three TGFβ isoforms.

Perivascular (infiltration): The prefix "perivascular" means "around", "around" or "near", so "perivascular" is interpreted literally as around a blood vessel. As used herein in the context of tumor cell invasion, the term "perivascular invasion" refers to a mode of entry for tumor-infiltrating immune cells (e.g., lymphocytes) through the vasculature of a solid tumor. .

Efficacy: As used herein, the term "efficacy" refers to the activity of a drug, such as an inhibitory antibody (or fragment) that has inhibitory activity, with respect to the concentration or amount of drug that produces a defined effect. For example, an antibody that can produce a particular effect at a given dose is more potent than another antibody that requires twice that amount (dose) to produce the same effect. Efficacy can be measured in cell-based assays, such as TGFβ activation/inhibition assays, whereby the extent of TGFβ activation, such as activation caused by integrin binding, is determined by the test substance (e.g. , inhibitory antibodies). Typically, antibodies with higher affinity (lower K _D value) that are able to bind to the same or overlapping binding regions of an antigen (e.g., cross-blocking antibodies) have lower affinity (higher K _D values) tend to exhibit higher efficacy.

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

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 the subject indicate or predict therapeutic benefit.

Display molecules: Presentation molecules related to the present disclosure form covalent bonds with latent proproteins (e.g., proTGFβ1) to tether an inactive complex to an extracellular niche (e.g., the ECM or immune cell surface). (“present”) and thereby maintain its latency until an activation event occurs. As for Protgfβ111, LTBP1, LTBP3, GARP and LRC33 are listed, and each of them are presented molecules -PROTGFβ1 complex (that is, that is, LTBP1 -PROTGFGFβ1, LTBP3 -PROTGFβ1, GARP -PROTGFβ1 and LRC33 - proTGFβ1 can be formed respectively. In nature, LTBP1 and LTBP3 are components of the extracellular matrix (ECM); therefore, LTBP1-proTGFβ1 and LTBP3-proTGFβ1 are considered “ECM-associated” (or “matrix-associated”) that mediate ECM-associated TGFβ1 signaling/activity. ) may be collectively referred to as the proTGFβ1 complex. On the other hand, GARP and LRRC33 are transmembrane proteins expressed on the cell surface of certain cells; therefore, GARP-proTGFβ1 and LRRC33-proTGFβ1 are involved in cell-associated (e.g., immune cell-associated) TGFβ1 signaling/activation. may be collectively referred to as "cell-associated" (or "cell surface") proTGFβ1 complexes that mediate

proTGFβ1: As used herein, the term “proTGFβ1” is intended to encompass the precursor form of the inactive TGFβ1 complex that includes the prodomain sequence of TGFβ1 within the complex. Thus, this term may include the pro-form as well as the latent form of TGFβ1. The expression "pro/latent TGFβ1" may be used interchangeably. The "pro" form of TGFβ1 exists prior to proteolytic cleavage at the furin site. Once cleaved, the resulting form is said to be the "latent" form of TGFβ1. A "latent" complex remains non-covalently 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 display molecule via the cysteine residue at position 4 (Cys4) of each proTGFβ1 polypeptide. The adjective "latent" may be used generally/broadly to describe the "inactive" state of TGFβ1 prior to integrin-mediated or other activation events. The proTGFβ1 polypeptide contains a prodomain (LAP) and a growth factor domain (SEQ ID NO: 119).

Regression (tumor regression): Tumor regression or tumor growth can be used as a measure of in vivo efficacy. For example, in a preclinical setting, median tumor volume (MTV) and regression response treatment efficacy criteria can be determined from the tumor volume of animals remaining in the study at 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 the tumor in the animal. Complete regression achieved in response to a therapy (eg, administration of a drug) can be referred to as a "complete response," and a subject who achieves a complete response can be referred to as a "complete responder." Complete response therefore excludes spontaneous complete regression. In some embodiments of the preclinical tumor model, a PR response is defined as 50% or less of its Day 1 volume in three consecutive measurements during the study period, and one of these three measurements. It is defined as a tumor volume that is 13.5 mm ³ or more. In some embodiments, a CR response is defined as a tumor volume that is ^less than 13.5 mm for 3 consecutive measurements during the study period. In preclinical models, animals with a CR response at the end of the study can be classified separately 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, in which case, for example, tumor volume is defined as the endpoint tumor volume in response to treatment. <25% of For example, in a particular model, if the endpoint tumor volume is ^2,000 mm, effective tumor control is achieved if the tumor shrinks to less than 500 ^mm . Effective tumor control therefore includes complete regression as well as partial regression that reaches a threshold reduction.

Resistance (to a treatment): Resistance to a particular treatment (such as CBT) can be an innate characteristic of a disease such as cancer (“primary resistance”, i.e., present before treatment begins) or an acquired expression that develops over time after treatment. ('acquired resistance'). A patient who does not show a therapeutic response to a treatment (eg, a patient who is a non-responder or a patient who has a poor response to a treatment) is said to have primary or acquired resistance to that treatment. Patients who have not previously received treatment and who do not show a therapeutic response to treatment are said to have primary resistance. Patients who initially exhibit a therapeutic response to a treatment, but whose response subsequently disappears (eg, progress or relapse despite continued treatment) are said to have acquired resistance to that treatment. In the context of immunotherapy, such resistance may indicate immune escape.

Response Criteria for Solid Tumors (RECIST) and iRECIST: RECIST determines whether a cancer patient's tumor improves (“responses”), remains the same (“stabilizes”), or worsens during treatment. A set of published rules that define when (to "proceed"). The standards were published in February 2000 by an international collaboration including the European Organization for Research and Treatment of Cancer (EORTC), the US National Cancer Institute, and the National Cancer Institute Clinical Trials Group of Canada. Subsequently, a revised version of the RECIST guidelines (RECIST v 1.1) has been widely adapted (see: Eisenhauera et al., (2009), “New response evaluation criteria in solid tumor: Revised RE CIST guideline (version 1.1)” Eur J Cancer 45:228-247; incorporated herein).

Treatment response criteria are as follows: complete response (CR): disappearance of all target lesions; partial response (PR): at least 30% of the total LD of target lesions, relative to the baseline total LD. Decrease; Stable (SD): Based on the lowest total LD since the start of treatment, there is neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD; progressive disease (PD) : An increase of at least 20% in the total LD of the target lesions, based on the minimum total LD recorded since the start of treatment or the appearance of one or more new lesions.

In contrast, iRECIST provides a modified set of criteria that takes into account immune-related responses (see: www.ncbi.nlm.nih.gov/pmc/articles/PMC5648544/; (the contents of which are incorporated herein by reference). The RECIST and iRECIST standards are standardized, subject to revision from time to time as more data becomes available, and are well understood in the art.

Response rate: As used herein, the term response rate (such as "low response rate") has its ordinary meaning as 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 demonstrate clinical improvement upon receiving treatment (e.g., a pharmacological intervention) and can include complete responses and partial responses. In oncology, clinical improvement can include tumor shrinkage (eg, partial response) or disappearance (eg, complete response). When used as a clinical endpoint in clinical trials of cancer therapy, it is typically expressed as the objective response rate (ORR). The FDA defines ORR as the percentage of patients who have a given amount of reduction in tumor size over a minimal period of time. Reference: U.S., the contents of which are incorporated herein by reference. S. Department of Health and Human Services, Food and Drug Administration, Oncology Center of Excellence, Center for Drug Evaluation “Clinical Trial Endpoints for the Approval of Can” published by Center for Biologics Evaluation and Research (CBER), der Drugs and Biologics-Guidance for Industry”.

Solid tumor: The term "solid tumor" refers to a proliferative disorder that usually results in abnormal growth or mass of tissue without cysts or areas of fluid. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Solid tumors include tumors of progressive malignancies such as locally advanced solid tumors and metastatic cancers. Solid tumors are typically composed of multiple cell types including, but not limited to, cancerous (malignant) cells, stromal cells such as CAFs, and infiltrating leukocytes such as macrophages, MDSCs, lymphocytes. Solid tumors to be treated with isoform-selective inhibitors of TGFβ1, such as those described herein, are typically TGFβ1-positive (TGFβ1+) tumors, which contain multiple cells that produce TGFβ1. May contain types. In certain embodiments, TGFβ1+ tumors may also co-express TGFβ3 (ie, TGFβ3 positive). For example, certain tumors are TGFβ1/3-codominant. In some embodiments, such tumors are caused by cancers 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 adenocarcinoma, e.g. prostatic 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 of the uterine body, renal cancer, e.g., kidney clear cell carcinoma, head and neck cancer, e.g. Epithelial cancers include colon cancers, such as 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 are less susceptible to mainstream therapy. , for example, exhibits decreased responsiveness to genotoxic therapy. Accordingly, a TGFβ inhibitor (e.g., Ab6) may be used in combination with one or more genotoxic therapies (e.g., chemotherapy and/or radiotherapy, including radiotherapy agents) to treat such cancer in a subject. It can be used as

Specific binding: As used herein, the term "specific binding" or "specifically binds" means that an antibody or antigen-binding portion thereof binds to a particular structure in an antigen (e.g., an antigenic determinant). or epitope) exhibiting a particular affinity (eg, K _D as measured by Biacore®). In some embodiments, the antibody or antigen-binding portion thereof is such that the antibody has a molecular weight of at least about 10 ⁻⁸ M, 10 ⁻⁹ M, 10 ⁻¹⁰ M, 10 ⁻¹¹ M, 10 ⁻¹² M, or less to the target. specifically binds to a target, eg, TGFβ1, if it has a K _D below. In some embodiments, "specifically binds to an epitope of proTGFβ1,""specifically binds to an epitope of proTGFβ1,""specifically binds to proTGFβ1," or "specifically binds to proTGFβ1." As used herein, the term "binds to proTGFβ1 and binds to 1.0 x 10" as determined by a suitable in vitro binding assay such as surface plasmon resonance and biolayer interferometry (BLI) ^. Refers to an antibody or antigen-binding portion thereof that has a dissociation constant (K _D ) of ⁸ M or less. In preferred embodiments, the kinetic rate constant (eg, K _D ) is determined by surface plasmon resonance (eg, Biacore system). In one embodiment, the antibody or antigen-binding portion thereof is capable of specifically binding both human and non-human (eg, murine) orthologs of proTGFβ1. In some embodiments, the antibody also binds to the target antigen with a relatively greater strength than that shown to other antigens, e.g., compared to non-target antigens (e.g., TGFβ2 and/or TGFβ3). may "selectively" (i.e., "preferentially") bind to a target antigen if it binds to that target with a relative affinity of 10-fold, 100-fold, 1000-fold or more to (e.g., TGFβ1) . In preferred embodiments, isoform-selective inhibitors exhibit no detectable binding or potency toward other isoforms or counterparts. In some embodiments, antibodies that specifically bind to a set of antigens may have high affinity for said antigens, but may not be able to distinguish said antigens from each other (i.e., antibodies that specifically bind (but not selective). In some embodiments, antibodies that bind an antigen with particularly high affinity compared to other antigens may be considered selective for said antigen. For example, an antibody that binds antigen X with 1000 times higher 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 an antigen with high affinity" generally refers to a KD of 1.0×10 ⁻⁸ M or less.

Subject: The term "subject" in the context of therapeutic applications refers to an individual receiving or in need of clinical care or intervention, such as treatment, diagnosis. Suitable subjects include vertebrates, including, but not limited to, mammals (eg, 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" refers to clinical criteria (e.g., symptoms, stage of disease, susceptibility to a particular condition, responsiveness to treatment, etc.), medical history, health status, gender, Individuals who fit a set of criteria, such as age group, genetic criteria (e.g., carriers of certain mutations, polymorphisms, gene duplications, DNA sequence repeats, etc.), and lifestyle factors (e.g., smoking, alcohol consumption, exercise, etc.) used to indicate a group of

Target binding: As used herein, the term target binding refers to the ability of a molecule (eg, a TGFβ inhibitor) to bind to its intended target (eg, endogenous TGFβ) in vivo. In the case of activation inhibitors, the intended target may be a large latent complex.

TGFβ1-related indication: “TGFβ1-related indication” is a TGFβ1-related disorder, including any disease or disorder and/or condition whose pathogenesis and/or progression is at least in part due to TGFβ1 signaling or its dysregulation. means. Certain TGFβ1-related disorders are primarily driven by TGFβ1 isoforms. Subjects with TGFβ1-related indications may benefit from inhibition of TGFβ1 activity and/or levels. Certain TGFβ1-related indications are primarily driven by the TGFβ1 isoform. TGFβ1-related indications include, but are not limited to: fibrosis (e.g. organ fibrosis and tissue fibrosis with chronic inflammation), proliferative disorders (cancer, e.g. 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, as described herein. A disease with abnormal gene expression of specific markers of. These disease categories are not intended to be mutually exclusive.

TGFβ inhibitor: The term “TGFβ inhibitor” refers to any agent that can antagonize the biological activity, signaling, or function of a TGFβ growth factor (eg, TGFβ1, TGFβ2, and/or TGFβ3). This term is not intended to limit the mechanism of action and includes, for example, neutralizing inhibitors, receptor antagonists, soluble ligand traps, TGFβ activation inhibitors as well as integrin inhibitors (e.g. αVβ1, αVβ3, Antibodies that bind to αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 or α8β1 integrin and inhibit downstream activation of TGFβ, such as selective inhibition of TGFβ1 and/or TGFβ3). The 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 more than one isoform (such as neutralizing antibodies) and engineered constructs containing a ligand binding moiety. (eg, fusion proteins). In certain embodiments, these antibodies may include, or be engineered to include, mutations or modifications that increase the half-life of the antibody. In some embodiments, such mutations or modifications may be within the Fc domain of the antibody (eg, an Fc-modified antibody). In some embodiments, the mutation is a so-called YTE mutation. TGFβ inhibitors 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). It also includes antibodies that are capable of causing internalization of cell surface complexes containing latent proTGFβ, thereby removing the precursor from the plasma membrane without depleting the cell itself. Internalization may be a preferred mechanism of action of LRRC33-containing protein complexes (such as human LRRC33-proTGFβ1) resulting in a reduction in the level of cells expressing the LRRC33-containing protein complex on the cell surface.

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.

TGFβ1-positive cancer/tumor: As used herein, this term refers to a cancer/tumor that has aberrant TGFβ1 expression (overexpression). Many human cancer/tumor types exhibit predominant expression of the TGFβ1 (note that "TGFB" is sometimes used to refer to a gene as opposed to a protein) isoform. In some cases, such cancers/tumors may exhibit co-dominant expression of another isoform such as TGFβ3. Some epithelial cancers (eg, carcinomas) can co-express TGFβ1 and TGFβ3. Within the tumor environment of TGFβ1-positive tumors, TGFβ1 is associated with e.g. cancer cells, tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and surrounding cells. can originate from multiple sources, including extracellular matrix (ECM). In the context of this disclosure, a preclinical cancer/tumor model that reproduces the human condition is a TGFβ1 positive cancer/tumor.

Therapeutic Window: The term "therapeutic window" refers to a dosage range that produces a therapeutic response without causing significant/observable/unacceptable adverse effects (e.g., within a range of tolerable or tolerable adverse effects) in a subject. refers to The therapeutic window can be calculated as the ratio of minimum effective concentration (MEC) to minimum toxic concentration (MTC). To illustrate, 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×) do. In contrast, a pan-inhibitor of TGFβ that is effective at 10 mg/kg but causes adverse effects at less than effective doses is said to have "dose-limiting toxicity." Generally, the maximum tolerated dose (MTD) may 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 been shown to be effective against TGFβ at doses of at least 100 or 300 mg/kg/week for 4 weeks in rats or non-human primates. It was also demonstrated that there is no observable toxicity associated with pan-inhibition of . On this basis, Ab6 exhibits at least a 3.3-fold and up to a 100-fold therapeutic window. In some embodiments, the concept of therapeutic window may be expressed in terms of a safety factor.

Toxicity: As used herein, the term "toxicity" refers to one or more of the undesirable in vivo effects in a subject (e.g., patient) associated with the therapy administered to the subject (e.g., patient); For example, it refers to undesirable side effects and adverse events. "Tolerability" refers to the level of toxicity associated with a treatment or treatment regimen that can be reasonably tolerated by a patient without discontinuing treatment due to toxicity. Typically, toxicity/toxicology testing is performed in one or more preclinical models prior to clinical development to evaluate the safety profile of a drug candidate (eg, monoclonal antibody therapy). Toxicity/toxicology studies help 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 that has been shown to be sensitive to a particular intervention should be selected as the preclinical animal model in which safety/toxicity testing is performed. For TGFβ inhibition, suitable species include rat, dog and cynomolgus monkey. Although it has been reported that mice are less sensitive to pharmacological inhibition of TGFβ and may not reveal potentially dangerous toxicity in other species, including humans, certain studies have shown that Toxicity observed with pan-inhibition of TGFβ has been reported. 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, which was /kg. The MTD of Ab6 in non-human primates is >300 mg/kg based on a 4 week toxicity study.

In order to determine the NOAEL and MTD, preferably a species that has proven to be sensitive to a particular intervention should be selected as the preclinical animal model in which safety/toxicity testing is performed. For TGFβ inhibition, suitable species include, but are not limited to, rat, dog, and cynomolgus monkey. 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.

Treating/Treatment: The term "treating" or "therapy" includes therapeutic treatment, prophylactic treatment, and applications that reduce the risk of a subject developing the disorder or other risk factor. Accordingly, this term is intended to mean broadly: for example, enhancing or increasing the body's immunity; reducing or reversing immunosuppression; reducing, eliminating or eliminating harmful cells or substances from the body. provide a therapeutic benefit to the patient by eradicating; reducing disease burden (e.g., tumor burden); preventing relapse or relapse; prolonging the refractory period; and/or otherwise improving survival. thing. The term includes therapeutic treatment, prophylactic treatment, and applications that reduce a subject's risk of developing a disorder or other risk factor. Treatment does not require complete cure of the disorder, but includes embodiments that reduce symptoms or underlying risk factors. In the context of combination therapy, the term may also refer to: i) reducing the effective dose of the first therapeutic agent such that the second therapeutic agent reduces side effects and improves tolerability; ii) the ability of a second therapeutic agent to make a patient more responsive to the first therapeutic agent; and/or iii) the ability to provide an additive or synergistic clinical benefit.

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 the tumor site 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 that act as ligands, as well as the niche (tumor microenvironment). Cell-to-cell interactions between monocytes/macrophages that occur in In general, monocytes/macrophages can be polarized into so-called "M1" or "M2" subtypes, the latter being associated with a more tumor-promoting phenotype. In solid tumors, up to 50% of the tumor mass can correspond to macrophages that are preferentially M2 polarized. Among tumor-associated monocyte and myeloid cell populations, M1 macrophages typically express cell surface HLA-DR, CD68 and CD86, whereas M2 macrophages typically express cell surface HLA-DR, Expresses CD68, CD163 and CD206. Tumor-associated, M2-like macrophages (such as M2c and M2d subtypes) can express cell surface LRRC33 and/or LRRC33-proTGFβ1.

Tumor microenvironment: The term "tumor microenvironment (TME)" refers to the local disease niche in which tumors (eg, solid tumors) reside in vivo. The TME may include disease-associated molecular signatures (collections of chemokines, cytokines, etc.), disease-associated cell populations (TAMs, CAFs, MDSCs, etc.) and disease-associated ECM environments (alterations in ECM components and/or structure).

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

Except as indicated in the examples or otherwise, any numerical value expressing an amount of a component or reaction condition used herein is to be understood as modified by the term "about" in all instances. Should. The term "about" means ±10% of the stated value.

As used herein in the specification and claims, the indefinite articles "a" and "an" refer to "at least one," unless the contrary clearly indicates. should be understood to mean.

As used herein in the specification and claims, the phrase "and/or" refers to "one or both" of the elements so conjoined, i.e., in some cases present conjunctively. However, in other cases it should be understood to mean elements that exist disjunctively. Any other elements other than those specifically identified by the "and/or" clause, whether related or unrelated to the elements specifically identified, are optional, unless there is a clear indication to the contrary. Can exist by choice. Thus, by way of non-limiting example, when a reference to "A and/or B" is used in conjunction with an open-ended term such as "comprising", this means, in one embodiment, that A ( In another embodiment, B without A (optionally containing an element other than A); in yet another embodiment, both A and B (optionally containing an element other than A); in another embodiment, B without A (optionally containing an element other than A); optionally including other elements), etc.

As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements refers to the phrase "at least one" selected from any one or more of the elements in the list of elements. but does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not necessarily include at least one of each and every element specifically listed in the list of elements. does not exclude any combination of This definition means that within the list of elements referred to by the phrase "at least one," any element other than the specifically identified element is optional, whether or not related to the specifically identified element. It also makes it possible to exist by choice. Thus, as a non-limiting example, "at least one of A and B" (or equally "at least one of A or B" or similarly "at least one of A and/or B") refers to one implementation. In the form, B is absent and at least one (and optionally more than one) A (and optionally includes elements other than B); in another embodiment, A is absent and at least one (optionally including two or more) of B (and optionally including an element other than A); in yet another embodiment, at least one (optionally including two or more) of A; and at least one (optionally including two or more) B (and optionally including other elements), etc.

The use of ordinal terms such as "first," "second," "third," etc. in a claim to modify a claim element, by itself, indicates the priority of one claim element relative to another; The use of one claim element with a particular name from another with the same name does not imply precedence or order or temporal order in which acts of a method are performed, and for purposes of distinguishing claim elements. It is only used as a label to differentiate (excluding the use of ordinal terms).

Ranges provided herein are understood to be shorthand for all values within that range. For example, the range 1 to 50 is 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, such as from 10 to 20, from 1 to 10, from 30 to 40, etc.

Transforming growth factor β (TGFβ)
Transforming growth factor β (TGFβ) activity and subsequent partial purification of soluble growth factors were first described in the late 1970s to early 1980s, with which the field of TGFβ began approximately 40 years ago. To date, 33 gene products have been identified that make up the large TGFβ superfamily. The TGFβ superfamily can be divided into at least three subclasses by 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 in humans are encoded by three separate genes.

TGFβ is thought to play important roles in diverse processes such as inhibition of cell proliferation, extracellular matrix (ECM) remodeling, and immune homeostasis. The importance of TGFβ1 for T cell homeostasis is demonstrated by the observation that TGFβ1−/− mice survive only 3–4 weeks and die due to multiple organ failure due to intense immune activation ( Kulkarni, A.B., et al., Proc Natl Acad Sci USA, 1993.90(2): p.770-4; Shull, M.M., et al., Nature, 1992.359 (6397 ): p.693-9). The roles of TGFβ2 and TGFβ3 are less clear. The three TGFβ isoforms have different temporal and spatial expression patterns, although they signal through the same receptors TGFβRI and TGFβRII, in some cases, e.g., for TGFβ2 signaling, β-glycans, etc. It also requires type III receptors (Feng, X.H. and R. Deryck, Annu Rev Cell Dev Biol, 2005.21: p. 659-93; Massague, J., Annu Rev Biochem, 998.67: p.753-91). Ligand-induced oligomerization of TGFβRI/II triggers phosphorylation of SMAD transcription factors, resulting in transcription of target genes such as Col1a1, Col3a1, ACTA2 and SERPINE1 (Massague, J., J. Seoane, and D. .Wotton, Genes Dev, 2005.19(23): p.2783-810). SMAD-independent TGFβ signaling pathways have also been described, for example, in cancer or in aortic lesions of Marfan syndrome mice (Derynk, R. and Y.E. Zhang, Nature, 2003.425 (6958): p.577-84; Holm, T. M., et al., Science, 2011.332(6027): p.358-61).

The biological importance of the TGFβ pathway in humans has been verified by genetic diseases. Camurati-Engelman disease results in bone dysplasia due to an autosomal dominant mutation in the TGFB1 gene, leading to constitutive activation of TGFβ1 signaling (Janssens, K., et al., J Med Genet, 2006.43(1): p.1-11). Patients with Loey-Dietz syndrome have autosomal dominant mutations in components of the TGFβ signaling pathway, which cause aortic aneurysms, sequestra, and bifid uvula (Van Laer, L., H. Dietz, and B. Loeys, Adv Exp Med Biol, 2014.802: p.95-105). As TGFβ pathway dysregulation has been implicated in multiple diseases, several drugs targeting the TGFβ pathway have been developed and tested in patients, with limited success.

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. Diseased tissues, such as fibrotic and/or inflamed tissues and tumors, can form a local environment in which TGFβ activation can cause disease aggravation or progression, which may play a role in specific disease settings. may be mediated, at least in part, by interactions between multiple TGFβ-responsive cells that are activated in an autocrine and/or paracrine manner, along with several other cytokines, chemokines, and growth factors. .

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

Advances in immunotherapy have changed the treatment landscape for a growing number of cancer patients. The most prominent is checkpoint blockade therapy (CBT), which is now part of standard treatment regimens for a growing number of cancers. Although strong and durable responses to CBT are being observed in an increasing number of cancer types, it is clear that a significant proportion of tumors appear to be unresponsive to CBT even at the beginning of treatment. , and thus primary resistance presents as a major challenge to enable the immune system of many patients to target and eliminate tumor cells. Efforts have been made to understand and address the underlying mechanisms that confer primary resistance to CBT in order to extend therapeutic efficacy to a larger number of patients. However, this enthusiasm has led to lackluster clinical trials when CBT is combined with drugs known to affect the same tumor type or to modulate seemingly related components of the immune system. It has been limited by results and failures. A possible reason is that the clear mechanistic basis for a given combination is often not rooted in clinically available data and is therefore uncertain in trials intended to enhance approved monotherapies. This is because it has led to confusing outcomes. It has become clear that the design of combination immunotherapy should be rooted in scientific evidence of the relationship with basic tumor and immune system biology.

Recently, a phenomenon called "immune exclusion" has been coined to describe a 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, many retrospective analyzes of tumors of clinical origin have implicated TGFβ pathway activation in mediating primary resistance to CBT. For example, transcriptional profiling and analysis of pre-treatment melanoma biopsies revealed enrichment of TGFβ-related pathways and biological processes in tumors unresponsive to anti-PD-1 CBT. In immunocompromised tumors, effector cells that would otherwise attack cancer cells by recognizing cell surface tumor antigens are prevented from accessing the site of the cancer cells. In this way, cancer cells evade host immunity as well as immuno-oncological therapeutics such as checkpoint inhibitors that utilize and depend on such immunity. Indeed, such tumors are resistant to checkpoint blockade, such as anti-PD-1 and anti-PD-L1 antibodies, presumably because target T cells are blocked from entering the tumor and This is because it is therefore unable to exhibit anticancer effects.

Several retrospective analyzes of clinically derived tumors point to TGFβ pathway activation in mediating primary resistance to CBT. For example, transcriptional profiling and analysis of pre-treatment 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 showed that the lack of response to PD-L1 blockade with atezolizumab was particularly important in tumors where CD8+ T cells appeared to be excluded from invading the tumor. It was found to be associated with a transcriptional signature of TGFβ signaling. The important role of TGFβ signaling in mediating immune clearance leading to anti-PD-(L)1 tolerance has been confirmed in the EMT-6 syngeneic mouse model of breast cancer. EMT-6 tumors are poorly responsive to treatment with anti-PD-L1 antibodies, but combining this checkpoint inhibitor with 1D11, an antibody that blocks the activity of all TGFβ isoforms, Compared to treatment, the frequency of complete responses was significantly increased. It is suggested that the synergistic anti-tumor activity is due to changes in the cancer-associated fibroblast (CAF) phenotype and degradation of the immune exclusion phenotype, resulting in 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 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. Although recent studies have suggested a relationship between TGFβ pathway activation and primary CBT resistance, TGFβ signaling has long been associated with hallmarks of cancer pathogenesis. As a potent immunosuppressive factor, TGFβ blocks antitumor T cell activity and promotes immunosuppressive macrophages. Malignant cells often become resistant to TGFβ signaling and tumor suppressive effects as a mechanism to evade their proliferation. TGFβ activates CAF and induces extracellular matrix production and promotion of tumor progression. Finally, TGFβ induces EMT and thus supports tissue invasion and tumor metastasis.

Mammals have distinct genes encoding and expressing the three TGFβ growth factors TGFβ1, TGFβ2 and TGFβ3, which all signal through the same heteromeric TGFβ receptor complex. Despite common signaling pathways, each TGFβ isoform appears to have distinct biological functions, as evidenced by non-redundant TGFβ knockout mouse phenotypes. All three TGFβ isoforms are expressed as inactive prodomain-growth factor complexes; the TGFβ prodomain, also called the latent-associated peptide (LAP), envelops the growth factor and renders it a latent, non-signaling complex. Hold in transmission state. Furthermore, latent TGFβ is co-expressed with latent TGFβ binding proteins to form large latent complexes (LLC) through disulfide bonds. Binding of latent TGFβ to latent TGFβ binding protein-1 (LTBP1) or LTBP3 enables tethering to the extracellular matrix, whereas binding to the transmembrane proteins GARP or LRRC33 inhibits Treg or LTBP3 binding, respectively. Allows assimilation on the surface of macrophages. In vivo, latent TGFβ1 and latent TGFβ3 are activated by a subset of αV integrins and bind to the consensus RGD sequence on LAP, triggering a conformational change to release growth factors. The mechanism by which latent TGFβ2 is activated is less clear due to the lack of a consensus RGD motif. TGFβ1 release by proteolytic cleavage of LAP has also been implicated as an activation mechanism, but its biological relevance is less clear.

Although the pathogenic role of TGFβ activation is clear in several disease states, therapeutic targeting of the TGFβ pathway has likewise been difficult due to the pleiotropic effects resulting from widespread and sustained pathway inhibition. It is clear that For example, 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, induces severe valvular heart disease in mice, rats, and dogs. Several studies have shown that this is the cause. Therefore, these "pan" TGFβ approaches that block all TGFβ signaling have very narrow therapeutic windows, which is an obstacle to the treatment of numerous disease-related processes with very high unmet medical needs. It has been proven that there is. Although no TGFβ-targeted therapy has been approved to date and the results of clinical trials using such modalities have been largely disappointing, this is perhaps necessary to address safety concerns. This may be due to the use of a dosing regimen that has proven to be ineffective.

All references cited herein are incorporated by reference for any purpose. In case of conflict between a reference and the present specification, the present specification will control. Although certain features of the disclosed compositions and methods are described herein, for clarity, in the context of separate embodiments, it is understood that they may be provided in combination in a single embodiment. I want to be Conversely, while the various features of the disclosed compositions and methods are, for simplicity, described in the context of a single embodiment, they can also be provided individually or in any subcombination.

Treatment Methods and Biomarkers of Treatment Efficacy The subject matter of the present disclosure relates generally to the disclosure of PCT/2021/012969, filed on January 11, 2021, the entire contents of which are hereby incorporated by reference. Incorporated.

Circulating/circulating MDSC as a biomarker
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 strong ability to reduce the cytotoxic function of T cells and natural killer (NK) cells, which promotes T regulatory cell (Treg) proliferation and thus T effector cell function. (Gabrilovich et al., Nat Rev Immunol. (2012) 12:253-68). MDSCs are typically classified into two subsets, monocytic (m-MDSC) and granulocytic (G-MDSC or PMN-MDSC), based on the expression of surface markers (Consoni 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 major mechanism of immunosuppression. In contrast, 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).

MDSCs have been implicated in various diseases such as chronic inflammation, infection, autoimmune diseases, and graft-versus-host disease. In recent years, MDSCs have attracted attention in cancer due to their role in inducing T cell tolerance through checkpoint inhibitory molecules such as programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA4). It has become an immune population (Trovato et al., J Immunother Cancer. 2019 Sep 18; 7(1): 255). Furthermore, MDSCs have generally been characterized as supporting tumor progression by mechanisms in addition to immunosuppression, including promotion of tumor angiogenesis. Previous studies have focused on MDSCs present in tumor biopsies, as they tend to be enriched around inflamed tissue. (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 they have elucidated a clear relationship between MDSC levels and treatment response. For example, low baseline monocytic MDSC frequency has been shown to correlate poorly with treatment benefit (Pico de Coana et al., Oncotarget. 2017 Mar 28;8(13):21539-21553).

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., 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, and pancreatic cancer. cancer, and renal cell carcinoma. Compositions and methods according to the present disclosure may be applied to one or more of these cancers.

Previously, it was demonstrated by the applicant that immunosuppressive tumors contain increased levels of tumor-infiltrating or intratumoral MDSCs, also called tumor-associated MDSCs, and this evidence suggests that this may lead to antitumor immunity in a TGFβ1-dependent manner. It is clearly shown that there is an inverse correlation between The data is provided in PCT/US2019/041373, which is incorporated herein in its entirety. These data suggested that probing tumor-associated immune cells, eg, by biopsy, may be useful in characterizing anti-tumor effects in cancer patients. Additionally, Applicants have made the surprising discovery that a relatively simple and non-invasive blood test can provide comparable information, which suggests that TGFβ1 in overcoming the immunosuppressive phenotype This led to the realization that the pharmacological effects of inhibition can be determined by measuring circulating MDSC levels.

The present disclosure includes the finding that circulating MDSC levels (including gMDSCs and/or mMDSCs) can be determined by detecting or measuring LRRC33-positive cells in a blood sample, whereby LRRC33 is a novel relative to circulating MDSCs. recognized as a blood-based biomarker. For example, LRRC33-positive cells in a blood sample taken from a patient (such as a cancer patient) can be detected or measured by a FACS-based assay using antibodies that bind to cell surface LRRC33. In some embodiments, the LRRC33-expressing cells in a blood sample taken 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 rather than monocytes in the circulation. This recognition has raised new possibilities for using LRRC33 as a blood-based marker for circulating MDSCs. In some embodiments, LRRC33 expression may be determined by any of the antibodies disclosed in WO 2018/208888 and WO 2018/081287, the contents of which are incorporated herein in their entirety. . 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, determination of LRRC33 levels measured in blood samples may not require more invasive means such as tumor biopsy. can serve as an effective alternative for assessing tumor immunophenotypes, such as immunosuppression.

In various embodiments, the present disclosure describes TGFβ inhibitors, such as TGFβ1 selective inhibitors (e.g., selective pro- or latent TGFβ1 inhibitors, such as Ab6), isoform non-selective TGFβ inhibitors (e.g., low Molecular weight ALK5 antagonists, neutralizing antibodies that bind to two or more of TGFβ1/2/3, such as GC1008 and variants, antibodies that bind to TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors), and/or integrin inhibitors. (and integrin inhibitors (e.g., antibodies that bind to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibit downstream activation of TGFβ, such as selective inhibition of TGFβ1 and/or TGFβ3) Treat cancer, predict or determine efficacy, and/or treat cancer by monitoring the levels of circulating MDSC in a sample obtained from a patient receiving treatment (e.g., in the blood or in a blood component of the patient). Provides a method for confirming the reaction. Exemplary integrin inhibitors include anti-αVβ8 integrin antibodies provided in WO 2020051333, the disclosure of which is incorporated by reference. In various disclosed embodiments, circulating MDSCs are collected within 1, 2, 3, 4, 5, 6, or 7 days or 1 day after administration of a therapeutic agent to a subject, e.g., administration of a therapeutic dose of a TGFβ inhibitor. , 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks (eg, preferably less than 6 weeks).

In certain embodiments, TGFβ therapeutics may be administered alone or in combination with 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 (eg, Ab6) that is encompassed by this disclosure. In some embodiments, the TGFβ1 selective antibody or antigen binding fragment does not inhibit TGFβ2 and TGFβ3 at a therapeutically effective dose. 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 mutants, antibodies that bind to TGFβ1/3 and ligand traps, such as TGFβ1/3 inhibitors). 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 anti-αVβ8 integrin antibodies provided in WO2020051333, the disclosure of which is incorporated by reference. In some embodiments, additional cancer therapies include chemotherapy, radiation therapy (including radiotherapy drugs), cancer vaccines, or checkpoints such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies. Can include immunotherapy, including inhibitor therapy. In some embodiments, checkpoint inhibitor therapy includes 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®) selected from the group. In a preferred embodiment, the combination cancer therapy comprises Ab6 and at least one checkpoint inhibitor (such as those listed above). Accordingly, in some embodiments, a combination of Ab6 and a checkpoint inhibitor is used to treat cancer in a human patient in an amount effective to treat cancer. In some embodiments, TGFβ treatment may additionally or alternatively include a second checkpoint inhibitor. In some embodiments, TGFβ treatment may additionally or alternatively include chemotherapy (eg, genotoxic therapy or radiation therapy).

For example, without wishing to be bound by theory, evidence suggests that an overactive TGFβ pathway may correlate with tumor unresponsiveness to genotoxic therapies such as chemotherapy and radiotherapy (Liu et al. ., Sci Transl Med. 2021 Feb 10; 13 (580): eabc4465). This is observed across multiple cancer types, including epithelial cancers such as carcinomas. In certain embodiments, such cancer types include ovarian cancer, breast cancer, bladder cancer, pancreatic cancer, e.g., pancreatic adenocarcinoma, prostate cancer, e.g., prostatic adenocarcinoma, melanoma, e.g., cutaneous melanoma, lung cancer, For example, lung squamous cell carcinoma and lung adenocarcinoma, liver cancer, for example, hepatocellular carcinoma, uterine cancer, for example, uterine endometrial cancer, kidney cancer, for example, kidney clear cell carcinoma, head and neck cancer, for example, head and neck cancer. Includes squamous cell carcinoma, colon cancer, such as colon adenocarcinoma, esophageal carcinoma, and tenosynovial giant cell tumor (TGCT). In some embodiments, the cancer is a cancer that has elevated TGFβ1 levels associated with ROS (eg, elevated ROS). Without wishing to be bound by theory, ROS can induce an increase in TGFβ levels (e.g., TGFβ1 levels) that can be reduced by the TGFβ inhibitors (e.g., TGFβ1 inhibitors) disclosed herein.

Accordingly, a TGFβ inhibitor (e.g., Ab6) may be used in combination with one or more genotoxic therapies (e.g., chemotherapy and/or radiotherapy, including radiotherapy agents) to treat such cancer in a subject. It can be used as In certain embodiments, such cancers have elevated levels of TGFβ, as indicated by direct measurement and/or alterations in one or more downstream gene regulations (e.g., one or more genes involved in DNA repair). , for example, may have an increase in TGFβ activity. For example, a cancer, such as one of the cancers listed above, may be associated with upregulation of one or more genes associated with non-homologous end joining (NHEJ), e.g., cyclin-dependent kinase inhibitor 1A (CDKN1A), or alternative end joining. There may be an increase in TGFβ signaling as indicated by downregulation of one or more genes associated with binding, eg, LIG1 (DNA ligase 1), PARP1, and/or POLQ.

The present disclosure also provides methods of using measurement of circulating MDSCs in treating cancer in a subject who is administered a TGFβ inhibitor alone or in conjunction with immunotherapy. Furthermore, the description presented herein specifically provides that patients treated with TGFβ inhibitor and checkpoint inhibitor combination therapy at a time point before other markers of treatment efficacy, such as, for example, reduction in tumor volume, can be detected. We also provide support for circulating MDSC population as an early predictive marker of efficacy in cancer subjects.

In certain embodiments, TGFβ inhibitors, e.g., TGFβ1 selective inhibitors such as Ab6, isoform non-selective inhibitors, e.g., low molecular weight ALK5 antagonists, neutralizing antibodies that bind to two or more of TGFβ1/2/3; For example, GC1008 and variants, antibodies that bind to TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors and/or integrin inhibitors (e.g., bind to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 or α8β1 integrins) Antibodies that inhibit downstream activation of TGFβ (e.g., selective inhibition of TGFβ1 and/or TGFβ3) may be used to determine whether the amount (e.g., dose) of TGFβ1 inhibition administered increases the contemporaneously (e.g., at the same time) with checkpoint inhibitor therapy and individually sufficient to reduce MDSC levels by at least 10%, at least 15%, at least 20%, at least 25% or more. or administered sequentially. Circulating MDSC levels may be measured before or after each treatment or each dose of a TGFβ inhibitor such that a reduction in circulating MDSC levels by at least 10%, at least 15%, at least 20%, at least 25% or more may indicate or predict therapeutic efficacy. In some embodiments, the level of circulating MDSCs can be used to determine disease burden (eg, as measured by the change in relative tumor volume before and after a treatment regimen). In some embodiments, the level of circulating mMDSC can be used to determine disease burden (eg, as measured by the change in relative tumor volume before and after a treatment regimen).

In certain embodiments, a decrease in circulating MDSC levels (eg, mMDSC levels) can be indicative of a decrease in disease burden (eg, a decrease in relative tumor volume). For example, circulating MDSC levels (e.g., mMDSC levels) may be affected by a dose of TGF inhibitor (isoform-selective inhibitors, e.g. Ab6, isoform-nonselective TGFβ inhibitors, e.g. low molecular weight ALK5 antagonists, TGFβ1/2/3 neutralizing antibodies, such as GC1008 and variants, antibodies that bind TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors, and/or integrin inhibitors (e.g., 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). The reduction may indicate or be predictive of a pharmacological effect, eg, a reduction in disease burden (eg, a reduction in relative tumor size).

In certain embodiments, circulating MDSC levels (e.g., circulating mMDSC levels) are determined by the first dose of a TGFβ inhibitor, e.g., a TGFβ1 selective inhibitor, e.g., Ab6, an isoform non-selective inhibitor, e.g., a low molecular weight ALK5 antagonist, Neutralizing antibodies that bind to two or more of TGFβ1/2/3, such as GC1008 and variants, antibodies that bind to TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors and/or integrin inhibitors (e.g., αVβ1, can be measured before and after administration of an antibody that binds to 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, a first dose of a TGFβ inhibitor, e.g., Ab6, 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; For example, GC1008 and variants, antibodies that bind to TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors and/or integrin inhibitors, such as αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 or α8β1 integrins. Thus, administration of antibodies that inhibit downstream activation of TGFβ, such as selective inhibition of TGFβ1 and/or TGFβ3, can be used to reduce tumor volume, such that administration of TGFβ inhibitors Decreasing circulating MDSC levels (eg, circulating mMDSC levels) by at least 10%, at least 20%, at least 25% or more compared to previous circulating MDSC levels.

In some embodiments, a reduction in circulating MDSC levels (e.g., circulating mMDSC levels) is indicative of or may be predictive of a pharmacological effect, and further, a decrease in circulating MDSC levels (e.g., circulating mMDSC levels) is indicative of, or may be predictive of, a pharmacological effect, and in addition, a second or subsequent dose of the TGFβ inhibitor. It also serves as the basis for the administration of In some embodiments, a reduction in circulating mMDSC levels is indicative or predictive of a pharmacological effect and also provides a basis for administration of a second or subsequent dose of a TGFβ inhibitor. . In some embodiments, the first dose of TGFβ inhibitor is the very first dose of TGFβ inhibitor that the patient receives. In some embodiments, the first dose of the TGFβ inhibitor is the first dose of a given treatment regimen that includes two or more doses of the TGFβ inhibitor. In another embodiment, circulating MDSC levels (e.g., circulating mMDSC levels) are lower than those of a TGFβ inhibitor (e.g., Ab6) and checkpoint inhibitor therapy, administered contemporaneously (e.g., simultaneously), separately or sequentially. A reduction in circulating MDSC levels is indicative of or predictive of treatment efficacy, which can be measured before and after combination therapy including. In some embodiments, two or more of a TGFβ inhibitor, e.g. a TGFβ1 inhibitor, e.g. a TGFβ1 selective inhibitor, e.g. Ab6, an isoform non-selective inhibitor, e.g. a low molecular weight ALK5 antagonist, TGFβ1/2/3. Neutralizing antibodies that bind, such as GC1008 and variants, antibodies that bind TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors and/or integrin inhibitors (e.g., αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, The reduction in circulating MDSC levels after combined treatment with an antibody that binds αIIbβ3 or α8β1 integrin and inhibits downstream activation of TGFβ (e.g. selective inhibition of TGFβ1 and/or TGFβ3) and checkpoint inhibitor therapy This may be the basis for continued treatment. In some embodiments, checkpoint inhibitor therapy is combined with a TGFβ inhibitor, such as a TGFβ1 inhibitor, such as a TGFβ1 selective inhibitor, such as Ab6, or an isoform non-selective inhibitor, such as a low molecular weight ALK5 antagonist, TGFβ1/ Neutralizing antibodies that bind two or more of the two thirds, such as GC1008 and variants, antibodies that bind TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors and/or integrin inhibitors (e.g. αVβ1, αVβ3, Reduction in circulating mMDSC levels following combination treatment with antibodies that bind to αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 or α8β1 integrin and inhibit downstream activation of TGFβ (e.g. selective inhibition of TGFβ1 and/or TGFβ3) , may be the basis for continued treatment. In some embodiments, checkpoint inhibitor therapy is combined with a TGFβ inhibitor, such as a TGFβ1 inhibitor, such as a TGFβ1 selective inhibitor, such as Ab6, or an isoform non-selective inhibitor, such as a low molecular weight ALK5 antagonist, TGFβ1/ Neutralizing antibodies that bind two or more of the two thirds, such as GC1008 and variants, antibodies that bind TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors and/or integrin inhibitors (e.g. αVβ1, αVβ3, Reduction in circulating gMDSC levels after combination treatment with antibodies that bind to αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 or α8β1 integrin and inhibit downstream activation of TGFβ (e.g. selective inhibition of TGFβ1 and/or TGFβ3) , may be the basis for continued treatment.

In certain embodiments of the present disclosure, levels of circulating MDSCs are used to administer a dose of a TGFβ inhibitor, e.g., a TGFβ1 selective inhibitor, administered alone or in combination with another cancer therapy, such as a checkpoint inhibitor. , e.g. Ab6, isoform non-selective inhibitors, e.g. low molecular weight ALK5 antagonists, neutralizing antibodies that bind to more than one of TGFβ1/2/3, e.g. GC1008 and variants, antibodies that bind 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 integrins and inhibit downstream activation of TGFβ, such as TGFβ1 and or selective inhibition of TGFβ3) can be predicted, determined and monitored. In certain embodiments, baseline circulating MDSC levels can be measured prior to administering the initial treatment (eg, the first dose of the TGFβ inhibitor). In certain embodiments, lower baseline circulating MDSC levels predict a better response to TGFβ inhibitor treatment. In certain embodiments, lower baseline circulating mMDSC levels predict a better response to TGFβ inhibitor treatment. In certain embodiments, lower baseline circulating mMDSC levels predict a better response to TGFβ inhibitor treatment. In certain embodiments, patients receiving TGFβ inhibitor treatment have low baseline circulating mMDSC levels. In certain embodiments, circulating MDSCs can be measured within 6 weeks of administration of the initial treatment. In certain embodiments, circulating MDSC levels can be measured within 30 days of administration of the initial dose of the TGFβ inhibitor. In some embodiments, MDSC levels can be measured within or about 3 weeks after administration of the initial dose of the TGFβ inhibitor. In some embodiments, MDSC levels can be measured within or about 2 weeks after administration of the initial dose of TGFβ inhibitor. In some embodiments, MDSC levels can be measured within or about 10 days after administration of the initial dose of the TGFβ inhibitor. In certain embodiments, the MDSCs are circulating mMDSCs. In certain embodiments, the MDSCs are circulating gMDSCs.

In certain embodiments, circulating MDSC levels can be used to select treatment, inform treatment, and/or predict response in patients who have not previously received checkpoint inhibitor therapy. High response rates to checkpoint inhibitor therapy (e.g., greater than 30%, greater than 40%, greater than 50%, or less, as reported in the art) in patients who have not received prior checkpoint inhibitor therapy. Patients diagnosed with cancer types for which an overall response rate of greater than or equal to 10% has been reported can be tested to initially determine whether the tumor exhibits an immune-eliminated 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 exhibiting an immunocompromised or immunosuppressive phenotype may be treated with TGFβ inhibitors, such as TGFβ1 selective inhibitors, such as Ab6, isoform non-selective inhibitors, such as low molecular weight ALK5 antagonists, TGFβ1/2/3 Neutralizing antibodies that bind two or more, such as GC1008 and variants, antibodies that bind TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors, and/or integrin inhibitors (e.g., αVβ1, αVβ3, αVβ5, αVβ6, Antibodies that bind to αVβ8, α5β1, αIIbβ3 or α8β1 integrin and inhibit downstream activation of TGFβ, e.g. selective inhibition of TGFβ1 and/or TGFβ3) and checkpoint inhibitors (e.g. anti-PD1 or anti- PD-L1 antibody). In some embodiments, the circulating MDSCs are circulating mMDSCs. In some embodiments, the circulating MDSCs are circulating gMDSCs. In some embodiments, patients exhibiting an immunodeprived or immunosuppressive phenotype are treated with a TGFβ inhibitor.

In some embodiments, circulating MDSC levels (eg, circulating mMDSC levels) can be further monitored as an early predictor of treatment response. In certain embodiments, low response rates to checkpoint inhibitor therapy (e.g., 30% or less, 20% or less, as reported in the art) in patients who have not received prior checkpoint inhibitor therapy. Patients diagnosed with cancer types for which an overall response rate of 10% or less has been reported may be treated with TGFβ1 selective inhibitors, e.g. Ab6, isoform non-selective inhibitors, e.g. low molecular weight ALK5 antagonists, TGFβ1/2/3 neutralizing antibodies, such as GC1008 and variants, antibodies that bind TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors, and/or integrin inhibitors (e.g., αVβ1, αVβ3, αVβ5, αVβ6). , antibodies that bind to αVβ8, α5β1, αIIbβ3 or α8β1 integrin and inhibit downstream activation of TGFβ, e.g. selective inhibition of TGFβ1 and/or TGFβ3) in combination with checkpoint inhibitor therapy. can be treated with. In some embodiments, therapeutic response in these patients can be predicted by monitoring circulating MDSC levels. In some embodiments, therapeutic response in these patients can be predicted by monitoring circulating mMDSC levels. In some embodiments, therapeutic response in these patients can be predicted by monitoring circulating gMDSC levels. In some embodiments, treatment is continued based on circulating MDSC levels.

In certain embodiments, circulating MDSC levels are used to select and inform treatment in patients who are resistant to or do not tolerate checkpoint inhibitor therapy (e.g., due to adverse effects). and its response can be predicted. These patients either have primary resistance (i.e., have never responded to checkpoint inhibitor therapy) or have acquired resistance (i.e., initially respond to checkpoint inhibitor therapy and develop resistance over time). formed). In some embodiments, resistance to checkpoint inhibitor therapy in patients is indicative of immunosuppression or clearance, and these patients are therefore treated with 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, such as GC1008 and variants, antibodies that bind to TGFβ1/3 and ligand traps, such as 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). May be selected as a candidate for therapy. In certain embodiments, patients with either primary or acquired resistance to checkpoint inhibitors are treated with TGFβ1 selective inhibitors, such as Ab6, isoform non-selective inhibitors, such as low molecular weight ALK5 antagonists, TGFβ1/2 Neutralizing antibodies that bind to two or more of TGFβ1/3, such as GC1008 and variants, antibodies that bind to TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors, and/or integrin inhibitors (e.g., αVβ1, αVβ3, αVβ5 , αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin, and inhibits downstream activation of TGFβ, an antibody that binds to αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, such as selective inhibition of TGFβ1 and/or TGFβ3) can be administered and treated. Its response to can be monitored and/or predicted by circulating MDSC levels. In some embodiments, a reduction in circulating MDSC levels by at least 10%, at least 15%, at least 20%, at least 25% or more can indicate a response to TGFβ inhibitor therapy. In some embodiments, a reduction in circulating MDSC levels by at least 10%, at least 15%, at least 20%, at least 25% or more may indicate a pharmacological effect of treatment with, eg, a TGFβ inhibitor. In certain embodiments, a reduction in circulating MDSC levels may be indicative of a reduction in tumor size. In certain embodiments, a decrease in circulating mMDSC levels may be indicative of a decrease in tumor size. In certain embodiments, a decrease in circulating gMDSC levels may be indicative of a decrease in tumor size. A chart summarizing an exemplary treatment regimen is displayed in FIG.

Most of the TGFβ inhibitors currently in development are not isoform selective. These include pan-inhibitors of TGFβ as well as inhibitors targeting TGFβ1/2 and TGFβ1/3. The approach taken to manage the potential toxicity associated with such inhibitors is to use careful dosing to seize the small window of opportunity that can achieve both efficacy and an acceptable safety profile. Including regimen. These can include reducing the dose of the isoform non-selective inhibitor, which can include less frequent administration and/or lower doses per administration. For example, rather than weekly administration of a biologic TGFβ inhibitor, monthly administration can be considered. Another example is administering only in the early stages of combination immunotherapy to avoid or minimize toxicity associated with TGFβ inhibition.

Combination therapies involving cancer therapy (such as checkpoint inhibitor therapy) and isoform-nonselective TGFβ inhibitors may result in higher toxicity compared to TGFβ1-selective inhibitors (e.g., Ab6) and therefore pose such a risk. In order to reduce or manage the isoform non-selective TGFβ inhibitor can be administered, for example, on an “as needed” basis, infrequently or intermittently. In such treatment paradigms, circulating MDSC levels are regularly monitored for the purpose of determining that the efficacy to overcome immunosuppression is maintained sufficiently to ensure the antitumor efficacy of cancer treatments. It is possible. If, during the course of cancer treatment, MDSC increases, it indicates that the patient would benefit from additional administration of TGFβ inhibitors. Such an approach may help reduce unnecessary risks and adverse events associated with TGFβ inhibition, particularly 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.

Accordingly, 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 following steps: collecting the TGFβ inhibitor from the patient prior to treatment. measuring circulating MDSCs (e.g., circulating mMDSCs and/or circulating gMDSCs) in a first sample that has been treated; administering a TGFβ inhibitor to a patient treated with cancer therapy, the cancer therapy comprising: optionally checkpoint inhibitor therapy and/or chemotherapy; measuring circulating MDSCs (e.g., circulating mMDSCs and/or circulating gMDSCs) in a second sample taken 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. In some embodiments, the intermittent dosing regimen comprises: measuring circulating MDSCs (e.g., circulating mMDSCs and/or circulating gMDSCs) in a third sample; The method further comprises administering an additional dose of the TGFβ inhibitor to the patient if the patient exhibits a relatively elevated level of circulating MDSC levels. In some embodiments, the TGFβ inhibitor is an isoform non-selective inhibitor. In some embodiments, the sample is a blood or blood component sample. In some embodiments, the isoform non-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 are likely to have even higher circulating MDSC levels. Thus, TGFβ1-selective inhibitors (e.g., Ab6), isoform-nonselective inhibitors (e.g., low molecular weight ALK5 antagonists), neutralizing antibodies that bind to two or more of TGFβ1/2/3 (e.g., GC1008 and mutants), antibodies that bind TGFβ1/3, ligand traps (e.g., TGFβ1/3 inhibitors) and/or integrin inhibitors (e.g., αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 or α8β1 integrins). of patients treated with TGFβ inhibitor therapy, such as antibodies that bind and inhibit downstream activation of TGFβ, such as selective inhibition of TGFβ1 and/or TGFβ3, alone or in combination with checkpoint inhibitor therapy. Reduced levels of circulating MDSC (eg, reduced levels of circulating mMDSC) may indicate a reduction or reversal of immunosuppression in cancer.

In certain embodiments, 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 mutants), antibodies that bind TGFβ1/3, ligand traps (e.g. TGFβ1/3 inhibitors) and/or integrin inhibitors (e.g. αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 or α8β1 TGFβ inhibitors, such as antibodies that bind to integrins and inhibit downstream activation of TGFβ, e.g. Doses of TGFβ inhibitors reduce cancer immunosuppression, as shown by reduced circulating MDSC levels (e.g., reduced circulating mMDSC levels) and/or changes in the levels of tumor-associated immune cells, as measured after administration of or administered to a subject with cancer so as to be sufficient to reverse it. In some embodiments, the level of circulating MDSCs and/or tumor-associated immune cells is reduced by a TGFβ1 selective inhibitor (e.g., Ab6), an isoform non-selective inhibitor (e.g., a low molecular weight ALK5 antagonist), a TGFβ1/2 neutralizing antibodies that bind to two or more of TGFβ1/3 (e.g., GC1008 and mutants), antibodies that bind to TGFβ1/3, ligand traps (e.g., TGFβ1/3 inhibitors), and/or integrin inhibitors (e.g., αVβ1 Checkpoint TGFβ inhibitor therapy, such as 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) A decrease in circulating MDSC levels and/or a change in the level of tumor-associated immune cells measured after treatment compared to the levels measured before treatment, measured before and after administration in combination with inhibitor therapy, Demonstrates reduction or reversal of cancer immunosuppression. In some embodiments, a reduction in circulating mMDSC levels and/or a change in the level of tumor-associated immune cells measured after treatment compared to levels measured before treatment indicates a reduction or reversal of immunosuppression in cancer. show. In some embodiments, a decrease in circulating gMDSC levels and/or a change in the level of tumor-associated immune cells measured after treatment compared to levels measured before treatment indicates a reduction or reversal of immunosuppression in cancer. show. In some embodiments, treatment is continued in patients who show reversal of immunosuppression.

Circulating MDSC levels can be determined in samples such as whole blood samples or blood components (eg, 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. Isolating peripheral blood mononuclear cells from peripheral blood using, for example, elutriation methods, magnetic bead separation methods, or density gradient centrifugation methods (e.g., Ficoll-Paque®) known in the art. Can be done. In some embodiments, MDSCs can be separated from peripheral blood mononuclear cells by surface marker selection (eg, using immunofluorescence, eg, flow cytometry/FACS analysis or immunohistochemistry). G-MDSC and M-MDSC can be further differentiated using the surface markers provided herein.

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 for treatment. It can also be used to determine efficacy or treatment regimens. If a tumor is determined to have an immune exclusion phenotype, cancer treatments alone (such as CBT) may not be effective. Without wishing to be bound by theory, it is possible that the tumor does not have enough cytotoxic cells within the tumor environment for effective CBT treatment alone. Therefore, alternative and/or add-on therapies using TGFβ inhibitors (such as those described herein) may reduce immunosuppression and thereby provide improved treatment alone or treat resistant tumors. It can be made more responsive to cancer treatment. In some embodiments, immune cell markers are measured on a biopsy (eg, core needle biopsy). In some embodiments, a patient with an immunocompromised tumor is administered a therapeutic agent that includes one or more TGFβ inhibitors (eg, a TGFβ1 inhibitor, eg, Ab6). In some embodiments, a patient with an immunocompromised tumor is administered a therapeutic agent that includes one or more TGFβ inhibitors (e.g., TGFβ1 inhibitors, e.g., Ab6) to improve the condition (e.g., reduce the tumor burden). (increased immune cell penetration, decreased tumor volume, etc.). In some embodiments, patients who show improvement in their medical condition after a first round of treatment are administered one or more additional rounds of treatment. In some embodiments, the subject is administered one or more additional therapeutic agents in combination with one or more TGFβ inhibitors (eg, TGFβ1 inhibitors, eg, Ab6).

Tumor-associated immune cells that can be used to represent the immune context of the tumor/cancer microenvironment include, but are not limited to, cytotoxic T cells and tumor-associated macrophages (TAMs) and 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 are markers of activated cytotoxicity. Sexual T cells may also be present. To measure the levels of TAMs, protein markers such as HLA-DR, CD68, CD163, CD206, and other biomarkers, any method known in the art can be used. In certain embodiments, increased levels of cytotoxic T cells, such as 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, reduced levels of TAMs or tumor-associated MDSCs detected within the tumor microenvironment may indicate a reduction or reversal of immunosuppression. For example, reduction of HLA-DR, CD68, CD163 and CD206 expression by tumor-associated immune cells may indicate reduction or reversal of immunosuppression in cancer. In certain embodiments, tumor-associated immune cells, such as CD8+ T cells, can be used in combination with one or more additional biomarkers to indicate the immune structure of the tumor/cancer microenvironment. In certain embodiments, the immune architecture of a tumor can be characterized by the density, location, organization, and/or functional orientation of tumor-infiltrating immune cells. In certain embodiments, such markers are used to determine the immunophenotype of a tumor, e.g., to determine whether the tumor is immunocompromised, immunoinflammatory, or immune desert. can be used.

In various embodiments, cytotoxic T cells can be used, for example, to determine whether a cancer has an immune exclusion phenotype in a patient sample, and/or alone or in combination with circulating MDSCs, etc. It can be used in combination with other biomarkers to determine treatment 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 may be distributed within the tumor (i.e., tumor compartment), in the stroma (i.e., stromal compartment), and in the margins (i.e., marginal compartment; e.g., approximately 10-100 μm between tumor and stroma, or 25 75 μm, or 30-60 μm, such as 50 μm). In certain embodiments, the tumor, intratumoral stroma, and/or marginal compartments are determined using histological methods (e.g., pathologist assessment, pathologist-trained machine learning algorithms, and/or immunohistochemistry). can be determined. In certain embodiments, CD8+ T cells within a tumor compartment may be referred to as "tumor-associated CD8+ cells." In certain embodiments, CD8+ T cells within the stromal compartment may be referred to as "stroma-associated CD8+ cells." In certain embodiments, CD8+ T cells within the limbal compartment may be referred to as "limb-associated CD8+ cells." In some embodiments, CD8 distribution is distributed between tumor foci (e.g., clumps of cells extending from a common center found in cancerous growths), the stroma surrounding the tumor foci, and between the tumor foci and the surrounding stroma. (e.g., by assessing an area of about 10-100 μm, or 25-75 μm, or 30-60 μm, such as 50 μm, between the tumor nest and the surrounding stroma).

In certain embodiments, tumor foci may be identified using histological methods (eg, pathologist assessment, pathologist-trained machine learning algorithms, and/or immunohistochemistry). In certain embodiments, one or more tumor foci may be found within a tumor compartment. In certain embodiments, a tumor can include a plurality (eg, at least 5, at least 10, at least 20, at least 25, at least 50 or more) tumor foci.

By default, unless the context dictates otherwise, the term "stroma" or "stromal compartment" refers to the stroma surrounding the tumor, and the term "margin" or "marginal compartment" refers to the tumor and the stroma surrounding the tumor. Refers to the edge between the surrounding stroma. In some embodiments, the structural interface between the tumor/tumor nest and the surrounding stroma is determined by imaging analysis. Thus, an edge may be defined as a region surrounding the interface in either direction by a predetermined distance, eg 10-100 μm. In some embodiments, this distribution is used to select patients for treatment and/or to administer an anti-TGFβ inhibitor prior to administering a TGFβ inhibitor, such as a TGFβ1 inhibitor (e.g., Ab6). The likelihood of a therapeutic response (eg, an anti-tumor response) to an anti-cancer therapy comprising the anti-cancer therapy can be predicted and/or determined. For example, if no or few cytotoxic T cells are found in a tumor sample that includes the stroma and margins (e.g., less than 5% CD8+ T cells), this indicates that patients will not benefit from TGF inhibitor therapy. (While not being bound by theory, this is thought to be due to few immune cells being recruited to the tumor). Similarly, if high densities of cytotoxic T cells (e.g. >5% CD8+ T cells) are observed in the tumor as well as in the stroma and margins, this patient may also have limited benefit from TGF inhibitor therapy. Possibly (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 cancer may exhibit an immune exclusion phenotype, in which cytotoxic T cells (e.g., CD8+ T cells) are primarily located in or near the margin. , for example, observed clustered at the margin and tumor border and not significantly infiltrating the tumor itself (e.g., <5% CD8+ T cells within the tumor compartment and 10% in the margin and/or stromal compartment). CD8+ T cells). In certain embodiments, the cancer of interest may exhibit an immune exclusion phenotype, in which cytotoxic T cells (e.g., CD8+ T cells) are primarily located in or near the margin, e.g. CD8+ T cells are observed clustered at the peri-vasculature (or peri-vasculature) and do not significantly infiltrate the tumor core itself (e.g. <5% CD8+ T cells within the tumor compartment and the peri-vasculature). or >5% CD8+ T cells in the stromal compartment). In certain embodiments, the cancer of interest may exhibit an immune exclusion phenotype, in which cytotoxic T cells (e.g., CD8+ T cells) are primarily located in or near the margin, e.g. CD8+ T cells are observed clustered at the border of the tumor compartment and do not significantly infiltrate the tumor itself (e.g., less than 5%, less than 10%, less than 15%, or less CD8+ T cells within the tumor compartment and at the margins and/or or >5%, >10%, >15%, or more CD8+ T cells in the stromal compartment). In some embodiments, CD8+ content in tumor compartments is determined as described by Ziai et al., incorporated herein in its entirety. (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 USA. 2007 Mar 6; 104(10):3967-72), or Echarti et al. (Cancers (Basel). 2019 Sep; 11(9): 1398). 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 may indicate that the tumor is actively suppressing the immune response). and prevent sufficient entry of cytotoxic T cells, which can be partially or completely reversed by TGF inhibitors).

In some embodiments, the immune exclusion phenotype includes cytotoxic T cells (e.g., CD8+ T cells) within tumor-associated compartments, e.g., within the tumor, within the margins near the outer periphery of the tumor mass, and/or in the vicinity of the tumor vasculature. characterized by determining the cluster score of In some embodiments, a cluster score for cytotoxic T cells (e.g., CD8+ T cells) can be determined based on the homogeneity of immune cells in a particular tumor-associated compartment, resulting in cytotoxic T cells. Compartments containing a highly uniform distribution of cells (eg, CD8+ T cells) obtain high cluster scores. In certain embodiments, a tumor exhibiting an immune exclusion phenotype has a lower density of cytotoxicity within the tumor compared to the density outside the tumor (e.g., around the periphery of the tumor mass and/or near the tumor vasculature). may be characterized as sexual T cells (eg, CD8+ T cells). In some embodiments, the immune exclusion phenotype is characterized by cytotoxic T cells (eg, CD8+ T cells) in the tumor stroma located in close proximity (eg, 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 foci and be located at 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, for example, as determined by endothelial markers. In comparison, when immunosuppression is reversed with TGFbeta inhibitors, a more uniform distribution of CD8+ T cells within the tumor is observed, as CD8+ cells are able to infiltrate from the perivascular region and proliferate within the tumor. obtain.

In certain embodiments, the levels of tumor-infiltrating cytotoxic T cells (eg, CD8+ T cells) and their activation status can be determined from a tumor biopsy sample obtained from the subject. In some embodiments, a tumor biopsy sample, such as a core needle biopsy, may 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, such as a core needle biopsy, may be obtained from about 21 days to about 45 days after administration of the therapeutic agent. In some embodiments, tumor biopsy samples may be obtained via core needle biopsy. In some embodiments, treatment is continued if an increase is detected.

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

In some embodiments, the level of cytotoxic T cells (e.g., CD8+ T cells) outside the tumor (e.g., around the periphery of the tumor and/or in the vicinity of the tumor vasculature) Tumors with low levels of sexual T cells (eg, CD8+ T cells) can be identified as immunocompromised tumors. In some embodiments, an immunocompromised tumor may also have higher levels of cytotoxic T cells (eg, CD8+ T cells) in the tumor stroma compared to inside the tumor. In certain embodiments, an immunocompromised tumor can be identified by determining the ratio of cytotoxic T cell density (e.g., CD8+ T cells) inside the tumor to outside the tumor; is less than 1. In certain embodiments, an immunocompromised tumor can be identified by determining the ratio of cytotoxic T cell density within the tumor to the density within the tumor margin, where the ratio is less than 1. . In certain embodiments, an immunocompromised tumor can be identified by determining the ratio of cell density within the tumor to density in the tumor stroma, where the ratio is less than one. In certain embodiments, an immunocompromised tumor reduces the absolute number, percentage, and/or density of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor compared to the outside of the tumor (e.g., margin and/or stroma). It can be identified by comparing with In some embodiments, in immunocompromised tumors, the absolute number, percentage, and/or density of cytotoxic T cells (e.g., CD8+ T cells) outside the tumor is at least 2 times, 3 times, 4 times that inside the tumor. times, 5 times, 7 times or 10 times. In some embodiments, the immunocompromised tumor contains less than 5% CD8+ T cells within the tumor and more than 10% CD8+ T cells in the tumor margin and/or stroma. In some embodiments, an immunocompromised tumor is determined by the ratio of compartmental cytotoxic T cell density (e.g., the density of CD8+ cells inside the tumor relative to the density in the tumor margin and/or stroma) and the tissue can be identified by comparing the proportion of overall cytotoxic T cell density (e.g., CD8+ cells inside the tumor to CD8+ cells in the whole tumor tissue or in a biopsy), where the compartmental proportions , which is larger than the overall proportion of the organization. In some embodiments, a tumor that has an increased cell density of cytotoxic T cells (eg, CD8+ T cells) at an average distance of about 100 μm or less outside the tumor can be identified as an immunocompromised tumor. In some embodiments, cytotoxic T cell density (eg, CD8+ T cells) may be used in combination with one or more parameters such as average CD8+ cluster score. In some embodiments, a mean CD8+ cluster score of 50% or less in a tumor indicates immune clearance.

In some embodiments, compared to CD8+ T cells external to the tumor (e.g., at the periphery of the tumor, e.g., at the periphery of the tumor and/or near the vasculature of the tumor, e.g., within the tumor margin and/or stroma) , tumors with low levels of CD8+ T cells within the tumor (eg, core) can be identified as immunocompromised tumors. In some embodiments, the immunocompromised tumor has less than 5%, less than 10%, or less than 15% CD8+ T cells within the tumor and/or one or more tumor foci, and less than 5%, less than 10%, or less than 15% CD8+ T cells outside the tumor and/or one or more tumor foci. contains >5%, >10%, or >15% of CD8+ T cells outside the tumor focus. In some embodiments, the immunocompromised tumor has less than 5% CD8+ T cells within the tumor and/or one or more tumor foci and more than 5% outside the tumor and/or one or more tumor foci. Contains CD8+ T cells. In some embodiments, an immunocompromised tumor has less than 10% CD8+ T cells within the tumor and/or one or more tumor foci and more than 10% outside the tumor and/or one or more tumor foci. Contains CD8+ T cells. In some embodiments, an immunocompromised tumor has less than 15% CD8+ T cells within the tumor and/or one or more tumor foci and more than 15% outside the tumor and/or one or more tumor foci. Contains CD8+ T cells.

In some embodiments, there is a high level of CD8+ T cells inside the tumor compared to outside the tumor (e.g., around the periphery of the tumor and/or near the tumor vasculature, e.g. within the tumor margin and/or stroma). Tumors with high levels of CD8+ T cells can be identified as immunoinflammatory tumors. In some embodiments, the immunoinflammatory tumor contains greater than 5% CD8+ T cells within the tumor. In some embodiments, the immunoinflammatory tumor comprises greater than 10% CD8+ T cells within the tumor and/or within one or more tumor foci. In some embodiments, the immunoinflammatory tumor comprises greater than 15% CD8+ T cells within the tumor and/or within one or more tumor foci.

In some embodiments, tumors with low levels of CD8+ T cells both inside and outside the tumor may be identified as immune desert tumors. In some embodiments, the immune desert tumor contains 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, the immune desert tumor has less than 5% CD8+ T cells within the tumor (and/or within one or more tumor foci) and less than 5% CD8+ T cells at the tumor margin and/or stroma. Contains cells.

In some embodiments, CD8+ content in tumor compartments is determined as described by Ziai et al., incorporated herein in its entirety. (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 USA. 2007 Mar 6;104(10):3967-72), or Echarti et al. (Cancers (Basel). 2019 Sep; 11(9): 1398). In some embodiments, any of these methods may be used to determine the immunophenotype of a tumor.

In certain embodiments, the immunophenotype of the subject's cancer is determined by a plurality (e.g., at least 5, at least 15, at least 25, at least 50) of the tumor (e.g., in one or more tumor biopsy samples). The mean percent CD8 positivity (ie, the percentage of CD8+ lymphocytes) measured for tumor foci (or greater than or equal to 100%) can be determined by the mean percent CD8 positivity (ie, the percentage of CD8+ lymphocytes). In certain embodiments, the immunophenotype of a given tumor nest determines the CD8 positivity rate within the tumor nest compared to the CD8 positivity rate outside the tumor nest (e.g., within the tumor nest margin and/or the tumor nest stroma). It can be determined by comparing with In certain embodiments, a tumor nest can be identified as immunoinflammatory if the CD8 positivity rate within the tumor nest is greater than 5%. In certain embodiments, a tumor nest can be identified as immunocompromised if the CD8 positivity rate within the tumor nest is less than 5% and the CD8 positivity rate within the tumor nest edge is greater than 5%. In certain embodiments, a tumor nest can be identified as an immune desert if the CD8 positivity rate within the tumor nest is less than 5% and the CD8 positivity rate in the tumor nest edge is less than 5%. . In certain embodiments, a subject's cancer may be identified as immunoinflammatory if more than 50% of the total tumor area analyzed contains tumor foci exhibiting an immunoinflammatory phenotype. In certain embodiments, a subject's cancer may be identified as immunocompromised if more than 50% of the total tumor area analyzed contains tumor foci exhibiting an immunoexclusion phenotype. In certain embodiments, a subject's cancer may be identified as an immune desert if more than 50% of the total tumor area analyzed contains tumor foci exhibiting an immune desert phenotype. In certain embodiments, the cancer of interest can be identified based on determining CD8 positivity from two or more samples (e.g., at least three samples, e.g., four samples) taken from the same tumor. can.

In certain embodiments, tumor biopsy samples can be obtained by core needle biopsy. In certain embodiments, three to five samples (eg, four samples) may be taken from the same tumor. In certain embodiments, the needle may be inserted along a single trajectory, and multiple samples (e.g., 3-5 samples, eg, 4 samples) may be inserted at different tumor depths along the same needle trajectory. It can be collected in In certain embodiments, samples taken at different tumor depths can be used to analyze the total CD8 positivity rate for multiple tumor foci. In certain embodiments, the total CD8 positivity determined in these samples may represent the CD8 positivity in the remainder of the tumor. In certain embodiments, the total CD8 positivity determined in these samples can be used to identify the immunophenotype of the subject's cancer.

In certain embodiments, the immunophenotype of the tumor of interest includes the absolute number, percentage, ratio, and/or density of CD8+ cells in the tumor and the total CD8 positivity rate (i.e., CD8+ lymphocytes) for tumor foci throughout the tumor. (percentage of).

In certain embodiments, tumor compartments may be identified, determined, and/or analyzed for markers, such as CD8 content, manually, eg, 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, eg, by use of 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 foci and boundaries between tumor foci, 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 foci, 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, one or more machine learning algorithms may initially be based on manual classification of a reference sample, eg, by a trained pathologist. In some embodiments, the software or computer program may use a machine learning neural network approach based on manually classified reference samples, such as by a pathologist. Exemplary software or computer programs include image capture (e.g., microscopic images of tumor samples, including immunostaining), image processing and analysis, and specific parameters (e.g., nuclear staining, fibroblast staining, CD8+ staining). Any software or computer program capable of segmenting tumor compartments within an image based on tumor compartments (e.g., tumor markers, other biomarkers) is included. In certain embodiments, the software or computer program is one of those provided by Visiopharm, HALO (Indica Labs), CellProfiler Analyst, Aperio Image Analysis, Zeiss ZEN Intelligence, or ImageJ. It's possible. Such programs do not essentially analyze the entire tumor as a whole, but instead analyze specific features of individual tumor foci within a solid tumor (e.g., boundaries of tumor foci, stroma, and/or marginal compartments). Advantageously, sufficient resolution can be achieved to visualize.

In certain embodiments, subjects whose cancer exhibits an immune exclusion phenotype may be more responsive to treatment comprising administration of a TGFβ inhibitor (eg, Ab6). In some embodiments, such subjects are identified for treatment. In some embodiments, such subjects are treated with two or more of a TGFβ1 selective inhibitor (e.g., Ab6), an isoform non-selective inhibitor (e.g., a low molecular weight ALK5 antagonist), a TGFβ1/2/3. Neutralizing antibodies that bind (e.g., GC1008 and variants), antibodies that bind to TGFβ1/3, ligand traps (e.g., TGFβ1/3 inhibitors), and/or integrin inhibitors (e.g., αVβ1, αVβ3, αVβ5, αVβ6, A therapeutic agent comprising a TGF inhibitor, such as an antibody that binds to αVβ8, α5β1, αIIbβ3, or α8β1 integrin and inhibits downstream activation of TGFβ, such as selective inhibition of TGFβ1 and/or TGFβ3, is administered.

In certain embodiments, a subject whose cancer exhibits an immune exclusion phenotype is treated with a TGFβ1 selective inhibitor (e.g., Ab6), an isoform non-selective inhibitor (e.g., a low molecular weight ALK5 antagonist), a TGFβ1/2/3 Neutralizing antibodies that bind to two or more (e.g., GC1008 and variants), antibodies that bind to TGFβ1/3, ligand traps (e.g., TGFβ1/3 inhibitors), and/or integrin inhibitors (e.g., αVβ1, αVβ3, TGFβ inhibitors, such as antibodies that bind to αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 or α8β1 integrin and inhibit downstream activation of TGFβ, e.g. selective inhibition of TGFβ1 and/or TGFβ3) and additional cancer therapy; For example, they may be more responsive to combination therapy including checkpoint inhibitors. In some embodiments, additional cancer therapies include chemotherapy, radiation therapy (including radiotherapeutic agents), cancer vaccines, or checkpoint inhibitors, such as anti-PD-1, anti-PD-L1 or anti-CTLA-4 antibodies. immunotherapy including. In some embodiments, checkpoint inhibitor therapy includes 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®) ) selected from the group consisting of In certain embodiments, a subject whose cancer exhibits an immune exclusion phenotype receives combination therapy comprising a TGFβ inhibitor, such as a TGFβ1 selective inhibitor (e.g., Ab6), and an additional cancer therapy, e.g., a checkpoint inhibitor. administered.

In certain embodiments, subjects whose cancer exhibits an immune exclusion phenotype receive a combination comprising a TGFβ inhibitor, such as a TGFβ1 selective inhibitor (e.g., Ab6), and a checkpoint inhibitor therapy (e.g., a PD1 or PDL1 antibody). May be more responsive to therapy. In some embodiments, such subjects are identified for treatment of combination therapy. In some embodiments, such subjects are identified for treatment of combination therapy prior to receiving checkpoint inhibitor therapy alone. In some embodiments, such subjects are identified for combination therapy prior to receiving either checkpoint inhibitor therapy or TGFβ inhibitor alone. In some embodiments, such subjects are treatment naive. In some embodiments, such subject has previously received checkpoint inhibitor therapy and is unresponsive to checkpoint inhibitor therapy. In some embodiments, such a subject has a cancer that exhibits an immune exclusion phenotype. In some embodiments, such subjects have previously received checkpoint inhibitor therapy and are administered combination therapy directly (e.g., eliminating the need to first try treatment with a checkpoint inhibitor alone). To avoid). In some embodiments, such subjects receive a combination therapy comprising a TGFβ inhibitor, such as a TGFβ1 selective inhibitor (eg, Ab6), and an additional cancer therapy, eg, a PD1 or PDL1 antibody.

In some embodiments, a subject whose cancer exhibits an immune exclusion phenotype is selected for treatment that includes a TGFβ inhibitor, such as a TGFβ1 selective inhibitor (e.g., Ab6), and/or receives a therapeutic agent that includes the same. It may be monitored during and/or after administration. In some embodiments, patient selection and/or treatment efficacy determines the level of cytotoxic T cells (e.g., CD8+ T cells) within the tumor compared to the level of cytotoxic T cells (e.g., CD8+ T cells) outside the tumor (e.g., within the margins). Determined by measuring the level of cytotoxic T cells (eg, CD8+ T cells). In certain embodiments, the external part of the tumor (e.g., margin and/or stromal ) may indicate a therapeutic response (eg, an anti-tumor response). For example, at least 10%, 15%, 20% of the tumor-infiltrating cytotoxic T cell level after TGFβ inhibitor treatment (e.g., Ab6) compared to the tumor-infiltrating cytotoxic T cell level before treatment; An increase of 25% or more may indicate a therapeutic response (eg, an anti-tumor response). In some embodiments, an increase in total tumor area including immunoinflammatory tumor foci of at least 10%, 15%, 20%, 25% or more may indicate a therapeutic response. In some embodiments, levels of cytolytic proteins, such as perforin or granzyme B, or proinflammatory cytokines, such as IFNγ, expressed by tumor-infiltrating cytotoxic T cells are measured to The activation state of cells can also be determined. In some embodiments, an increase of at least 1.5-fold, or 2-fold, or 5-fold or more in cytolytic protein levels can indicate a therapeutic response (eg, an anti-tumor response). In some embodiments, a change in IFNγ levels of at least 1.5-fold, 2-fold, 5-fold, or 10-fold or more increase can be indicative of a therapeutic response (eg, an anti-tumor response). In some embodiments, treatment is continued if an increase in tumor-infiltrating cytotoxic T cells (eg, CD8+ T cells) is detected.

In certain embodiments, a subject whose cancer exhibits an immunoinflammatory phenotype may be more responsive to a treatment that includes a checkpoint inhibitor that does not include a TGFβ inhibitor than a subject that has an immune exclusion phenotype. . In some embodiments, checkpoint inhibitor therapy includes ipilimumab (e.g., Yervoy®); nivolumab (e.g., Opdivo®); pembrolizumab (e.g., Keytruda®); avelumab (e.g., , Bavencio®); cemiplimab (e.g., Libtayo®); atezolizumab (e.g., Tecentriq®); budigalimab (e.g., ABBV-181); and durvalumab (e.g., Imfinzi®); trademark))). In certain embodiments, a checkpoint inhibitor is administered to a subject whose cancer exhibits an immunoinflammatory phenotype.

In certain embodiments, the immunophenotype of the tumor of interest is determined, for example, histologically, from a tumor biopsy sample (e.g., core needle biopsy sample), including but not limited to cytotoxic T cells (e.g., CD8+ T cells). one or more parameters, such as the distribution of cytotoxic T cells (e.g., CD8+ T cells) in the tumor compartment relative to the stromal compartment, and the percentage of cytotoxic T cells (e.g., CD8+ T cells) within the tumor margin. can be determined using

Recognizing that samples taken by traditional needle biopsy protocols carry the risk of inadvertent bias due to the location within the tumor where the needle is inserted, this disclosure uses needle biopsy for tumor analysis. It also provides an improved method for doing so. According to the present disclosure, the risk of bias inherent in needle biopsies is reduced by collecting adjacent tumor samples, e.g., at least three, preferably four, samples taken from adjacent tumor tissue (e.g., from the same tumor). This can be significantly reduced by This can be done from a single needle insertion point, for example by changing the angle and/or depth of insertion. Some tissue sections prepared from needle biopsy samples may not remain intact during sample processing, and the needle may be inserted into parts of tumor tissue that do not accurately represent the tumor phenotype. Considering the nature of the tumor, collection of four samples would help alleviate such constraints and provide a more representative tumor phenotype for improved accuracy.

In certain embodiments, a sample may be analyzed for its distribution of cytotoxic T cells (eg, CD8+ T cells) using methods such as CD8 immunostaining. In certain embodiments, the distribution of cytotoxic T cells (e.g., CD8+ T cells) can be relatively uniform (e.g., the distribution is homogeneous across the sample, e.g., the CD8 density across the tumor foci is with a variance of 10% or less). In some embodiments, a tumor focus (or cancer focus) refers to a mass of cells extending from a common center of cancerous growth. In some embodiments, the tumor nest may include cells interspersed with the stroma. In certain embodiments, a sample, such as a sample with a uniform distribution of cytotoxic T cells (e.g., CD8 T cells), is analyzed to determine the number of cytotoxic T cells (e.g., CD8+ T cells) within the tumor and stroma. A percentage can be determined. In certain embodiments, a high percentage (e.g., greater than 5%) of cytotoxic T cells (e.g., CD8+ T cells) within the tumor and a lower percentage (e.g., less than 5%) of cytotoxic T cells within the stroma. (eg, CD8+ T cells) may exhibit an inflammatory tumor phenotype. In certain embodiments, a low percentage of cytotoxic T cells (e.g., CD8+ T cells) in both the tumor and stroma (e.g., total CD8 percentage of tumor and stroma less than 5%) is associated with a low immunogenic tumor. may exhibit a phenotype (eg, an immune desert phenotype). In certain embodiments, a low percentage (e.g., less than 5%) of cytotoxic T cells (e.g., CD8+ T cells) within the tumor and a high percentage (e.g., greater than 5%) of cytotoxic T cells within the stroma. (e.g., CD8+ T cells) may exhibit an immune-excluded tumor phenotype. In certain embodiments, the tumor:stromal CD8 ratio can be determined by dividing the percentage of CD8 within the tumor by the percentage within the stroma. In certain embodiments, a tumor:stromal CD8 ratio of greater than 1 may indicate an inflammatory tumor phenotype. In certain embodiments, a tumor:stromal CD8 ratio of less than 1 may indicate an immunocompromised tumor. In certain embodiments, the percentage of cytotoxic T cells can be determined by immunohistochemical analysis of CD8 immunostaining.

In certain embodiments, a sample, e.g., a sample with a heterogeneous distribution of cytotoxic T cells (e.g., CD8 density across tumor foci has a variance of >10%), is analyzed to determine whether the margin: quality CD8 ratio can be determined. In certain embodiments, such a ratio may be calculated by dividing the CD8 density within the tumor margin by the CD8 density within the tumor stroma. In certain embodiments, the immunocompromised tumor exhibits a margin:stromal CD8 ratio of greater than 0.5 and less than 1.5.

In certain embodiments, samples with a margin:stromal CD8 ratio greater than 1.5 are further analyzed to determine and/or confirm the immunophenotype by assessing tumor depth (e.g., determining and/or confirming whether a tumor has an immune exclusion phenotype). In certain embodiments, tumor invasion depth can be measured in units of 20 μm to 200 μm (eg, 100 μm). In certain embodiments, tumor depth may be determined by pathological analysis and/or digital image analysis. In certain embodiments, significant tumor depth of invasion may be indicated by a distance of about twice the depth of invasion of the tumor margin or greater. In certain embodiments, the tumor sample may have a tumor margin depth of 100 μm and a tumor depth measurement of greater than 200 μm, such sample having a tumor depth invasion score of greater than 2. , thus will have significant tumor depth. In certain embodiments, significant tumor depth can be indicated by a ratio of two or more determined by dividing the tumor depth by the tumor margin depth. In certain embodiments, tumor depth can be measured in unit increments corresponding to the depth of tumor margin invasion. For example, tumor depth for a tumor nest with a 100 μm tumor margin can be measured in 100 μm increments. In certain embodiments, a tumor sample with significant tumor depth may exhibit shallow infiltration by cytotoxic T cells (e.g., a tumor sample with more than 5% CD8 T cells may have one tumor depth unit). does not show tumor penetration beyond increments of ). In certain embodiments, a tumor sample with significant tumor depth that exhibits shallow CD8 penetration may be indicative of an immunocompromised tumor.

In certain embodiments, tumor phenotyping may be performed according to any portion of the exemplary flowchart shown in FIG. 38, eg, using all steps shown in the figure.

In certain embodiments, subjects whose cancer exhibits an immune exclusion phenotype can be selected for TGFβ inhibitor therapy (eg, a TGFβ1 inhibitor such as Ab6). In certain embodiments, subjects whose cancer exhibits an immune exclusion phenotype may be more responsive to TGFβ inhibitor therapy (eg, a TGFβ1 inhibitor such as Ab6). In certain embodiments, a subject whose cancer exhibits an immune exclusion phenotype is treated with a TGFβ inhibitor, such as a TGFβ1 selective inhibitor (e.g., Ab6), and a second cancer therapy, e.g., checkpoint inhibitor therapy (e.g., PD1 or PDL1 antibodies) may be more responsive to combination therapy.

In certain embodiments, response to TGFβ inhibitor therapy (eg, a TGFβ1 inhibitor such as Ab6) can be monitored and/or determined using parameters such as any of those described above. In certain embodiments, a change in the distribution of cytotoxic T cells (e.g., CD8+ T cells) in a pre-treatment tumor sample compared to a corresponding post-treatment sample from a corresponding tumor can be indicative of a therapeutic response to the treatment. In certain embodiments, the tumor:stromal CD8 density ratio between pre- and post-treatment tumor samples is at least 1 times (e.g., 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1 A change (eg, an increase) of .5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold or more) may indicate a therapeutic response. In certain embodiments, a 1.5-fold or greater change (eg, increase) in tumor:stromal CD8 density ratio between pre- and post-treatment tumor samples may indicate a therapeutic response. In certain embodiments, the tumor:stromal CD8 density ratio can be determined by dividing the CD8 cell density within the tumor nest by the CD8 cell density within the tumor stroma. In certain embodiments, a 1.5-fold or greater change (e.g., increase) in the density of cytotoxic T cells (e.g., CD8+ T cells) within the tumor margin between pre- and post-treatment tumor samples indicates a treatment response. can be shown. In certain embodiments, a 1.5-fold or greater change (eg, increase) in tumor depth score of a tumor sample from pre-treatment to post-treatment may indicate a therapeutic response. In some embodiments, TGFβ inhibitor therapy (e.g., a TGFβ1 inhibitor such as Ab6), when used in combination with a checkpoint inhibitor such as, e.g., a PD-(L)1 antibody, increases the , achieving an increase in the number of intratumoral T cells by at least 2-fold, such as 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold or more. do. In certain embodiments, once a therapeutic response is observed, treatment with TGFβ inhibitor therapy (e.g., a TGFβ1 inhibitor such as Ab6), e.g., alone or in combination with one or more additional cancer therapy agents, is Can be continued.

In certain embodiments, the pre-treatment and post-treatment samples have comparable tumor depth scores (e.g., a variance of less than 0.25 in the tumor depth scores of the pre-treatment and post-treatment tumor samples). , the sample can be analyzed to determine a therapeutic response according to one or more of the parameters described above. In certain embodiments, the pre-treatment and post-treatment samples have comparable total and compartment areas (e.g., a variance of less than 0.25 in the analyzable total and compartment areas of the pre- and post-treatment tumor samples); The sample can be analyzed to determine therapeutic response according to one or more of the parameters described above.

In some embodiments, the percent necrosis of a tumor sample can be assessed by histology and/or digital image analysis, which may represent the presence or activity of cytotoxic cells in the tumor. There is. In some embodiments, percent necrosis of a tumor sample can be compared in pre- and post-treatment tumor samples taken from subjects who have been administered a TGFβ inhibitor (eg, Ab6). In some embodiments, a greater than 10% increase in percent necrosis (e.g., the ratio of necrotic area to total tissue area in a tumor sample) between pre- and post-treatment samples is due to TGFβ inhibitor therapy, e.g., Ab6, etc. may exhibit a therapeutic response to TGFβ1 inhibitors. In some embodiments, an increase of 10% or more in percent necrosis at or near the center of the tumor (eg, percentage of necrotic area within the tumor margin) may indicate a therapeutic response.

In certain embodiments, therapeutic response can be determined according to any portion of the exemplary flowchart shown in FIG. 39.

In some embodiments, increased levels of tumor-infiltrating cytotoxic T cells (e.g., CD8+ T cells), particularly activated cytotoxic T cells (e.g., TGFβ1 inhibitors such as Ab6) after TGFβ inhibitor therapy may indicate an immune infiltration or conversion of the immune-excluded tumor microenvironment into an "inflammatory" microenvironment. For example, at least 1%, 2%, 3%, 4% of the tumor-associated cytotoxic T cell level after TGFβ inhibitor treatment (e.g., Ab6) compared to the tumor-associated cytotoxic T cell level before treatment. , 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more may indicate a reduction or reversal of immunosuppression in cancer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% of the tumor area includes immunoinflammatory tumor foci. , 20%, 25% or more may indicate a reduction or reversal of immunosuppression in cancer. In some embodiments, the levels of cytolytic proteins, such as perforin or granzyme B, or proinflammatory cytokines, such as IFNγ, expressed by tumor-associated cytotoxic T cells are measured to The activation state can be determined. In some embodiments, the increase in cell lytic protein levels by at least 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold or 2-fold or 5-fold or more is , may be shown to reduce or reverse immunosuppression in cancer. In some embodiments, a change in IFNγ levels of at least 1.5-fold, 2-fold, 5-fold, or 10-fold or more increase can indicate a reduction or reversal of immunosuppression in cancer. In some embodiments, treatment with TGFβ inhibitor therapy (eg, a TGFβ1 inhibitor such as Ab6) is continued once a reduction or reversal of such immunosuppression is detected in the cancer.

Immunosuppressive lymphocytes associated with the TME include TAMs and MDSCs. A significant proportion of tumor-associated macrophages are of the so-called "M2" type and have an immunosuppressive phenotype. Most of these cells are monocyte-derived cells that originate from the bone marrow. Levels of immunosuppressive cells such as TAMs and MDSCs within a tumor (eg, tumor-associated) can also be measured to determine the state of immunosuppression in cancer. In some embodiments, a reduction in the level of TAM by at least 10%, 15%, 20%, 25% or more can indicate a reduction or reversal of immunosuppression. In certain embodiments, tumor-associated immune cells can be measured from a biopsy sample from a subject before and after TGFβ inhibitor treatment (eg, Ab6). In certain embodiments, biopsy samples may be obtained 28 to 130 days after treatment administration.

The concept of "immune context" considers the TME from the perspective of tumor-infiltrating lymphocytes (ie, the tumor immune microenvironment or TIME). Tumor immune context refers to the localization (eg, spatial organization) and/or density of the immune infiltrate in the TME. TIME is commonly associated with clinical outcomes in cancer patients and has been used to estimate cancer prognosis (e.g., Fridman et al., (2017) Nat Rev Clin Oncol. 14(12):717 -734) “The immune context in cancer prognosis and treatment”). Typically, tissue samples from the tumor are collected for TIL analysis (eg, a biopsy, such as a core needle biopsy). In some embodiments, TILs are analyzed by FACS-based methods. In some embodiments, TILs are analyzed by immunohistochemical (IHC) methods. In some embodiments, TILs are analyzed by so-called digital pathology (see, e.g., Saltz et al., (2018) Cell Reports 23, 181-193. “Spatial organization and molecular correlation o f (Scientific Reports 9:13341 (2019) “A novel digital score for abundance of tumor infiltrating lymphocytes predictors disease free survival in oral squamous cell carcinoma”). In some embodiments, tumor biopsy samples can be used in various DNA and/or RNA-based assays (eg, RNAseq or Nanostring) to assess tumor immune context. While not wishing to be bound by theory, reduction or reversal of immunosuppression in cancers/tumors, as demonstrated by increased cytotoxic T cells and decreased TAMs, has been demonstrated by the use of TGFβ inhibitors (e.g., Ab6) alone. or in combination with checkpoint inhibitor therapy can be predicted.

Circulating/circulating latent TGFβ
According to the present disclosure, circulating latent TGFβ can be used as a target binding biomarker. If an activation inhibitor is selected as a therapeutic candidate, such biomarkers can be used to assess in vivo target binding, e.g., by monitoring the levels of circulating TGFβ (circulating TGFβ) before and after administration. Or you can check. In some embodiments, a target binding marker comprising circulating latent TGFβ (eg, circulating latent TGFβ1) is measured in the sample. In some embodiments, circulating TGFβ1 in a blood sample (eg, plasma and/or serum) includes both latent and mature forms, with the former representing the majority of circulating TGFβ1. In some embodiments, total circulating TGFβ (e.g., total circulating TGFβ1), including both latent TGFβ and mature TGFβ, can be measured, such as by using an acid treatment step to This is accomplished by releasing the mature growth factor (eg, TGFβ1) from the complex and detecting it using an enzyme-linked immunosorbent assay (ELISA) assay. In some embodiments, circulating latent TGFβ1 can be specifically measured using reagents such as antibodies that specifically bind to the latent form of TGFβ1 (eg, TGFβ1). In some embodiments, the majority of the measured circulating TGFβ (eg, circulating TGFβ1) is released from the latent complex. In some embodiments, the total circulating TGFβ (e.g., circulating TGFβ1) that is measured includes any free TGFβ (e.g., TGFβ1) that is present before acid treatment, plus any dissociated latent TGFβ (e.g., latent TGFβ1). ), which is known to be only a small fraction of circulating TGFβ1. In some embodiments, only circulating latent TGFβ (eg, circulating latent TGFβ1) is detectable. In some embodiments, circulating latent TGFβ (eg, circulating latent TGFβ1) is measured.

In some embodiments, circulating TGFβ (e.g., circulating latent TGFβ1) is described by Mussbacher et al., the entire contents of which are incorporated herein by reference. , PLos One. 2017 Dec 8;12(12):e0188921 and Mancini et al. Transl Res. 2018 Feb; 192:15-29, or adapted from a blood sample.

The challenges associated with accurately measuring blood/serum TGFβ levels are well recognized. Platelets are the main source of TGFβ1 in the circulation, and even moderate handling of blood samples, such as drawing blood, pipetting blood samples, and mechanical agitation, can cause the release of TGFβ1 from the platelets in the sample. As a result, readings are known to be distorted.

Aspects of the present disclosure include improved assays for measuring circulating TGFβ levels. Such assays include sample collection steps, sample processing steps, and measurement steps.

Sample collection involves placing a blood sample obtained from a subject (eg, a cancer patient) into a container (eg, a collection tube). Preferably, the collection tube is a sterile, evacuated glass or plastic tube containing an anticoagulant. In some embodiments, such tubes are approximately 13 mm x 75 mm in size and have a capacity of approximately 2.7 mL. In some embodiments, the collection tube contains an anticoagulant solution that includes a form of sodium citrate. In a preferred embodiment, the anticoagulant solution is a so-called CTAD. CTAD contains buffered trisodium citrate solution, theophylline, adenosine, and dipyrdamole. For example, a CTAD may include 0.11 M buffered trisodium citrate solution (pH approximately 5.0), 15 M theophylline, 3.7 M adenosine, and 0.198 M dipyridamole. Such collection tubes may contain an internal silicone coating to minimize contact activation. Such tubes may be equipped with a closure (eg, a cap or stopper) intended to protect the user from blood that may be splattered when the tube is opened. Such a closure is a rubber stopper, which may be placed inside a plastic shield to prevent exposure to blood present on the stopper. Examples of commercially available collection tubes include BD Vacutainer™ CTAD blood collection tubes with Hemogard™ closures. The manufacturer's product instructions suggest that when the blood is drawn into the tube, the sample is centrifuged at 1500g for 15 minutes at room/ambient temperature (18-25°C). Surprisingly, however, applicants have discovered that, contrary to the manufacturer's recommendations, sample collection and processing performed at 2-8°C (e.g., about 4°C) is superior for the purpose of measuring circulating TGFβ1 levels. found that it produces results.

Accordingly, in some embodiments, circulating TGFβ (e.g., circulating latent TGFβ1) in a blood sample is determined by collecting the blood sample into a collection tube that includes (contains or is coated with) an anticoagulant. Measured by In some embodiments, the collection tube includes a citrate coating. In some embodiments, the collection tube is coated with a solution containing 0.1-0.5M buffered trisodium citrate. In some embodiments, the collection tube is coated with a solution containing 10-20M theophylline. In some embodiments, the collection tube is coated with a solution containing 2-5M adenosine. In some embodiments, the collection tube is coated with a solution containing 0.1-0.25M dipyridamole. In some embodiments, the collection tube is coated with a solution having a pH of 4.0-6.0. In some embodiments, the collection tube contains citric acid-theophylline-adenosine-dipyridamole (CTAD), citric acid (e.g., sodium citrate), acid-citric acid-dextrose (ACD), ethylenediaminetetraacetic acid (EDTA), and heparin. In some embodiments, the collection tube is coated with CTAD. In some embodiments, the collection tube is coated with a CTAD solution comprising about 0.11 M buffered trisodium citrate solution, about 15 M theophylline, about 3.7 M adenosine, and about 0.198 M dipyridamole. Ru. In some embodiments, the CTAD solution has a pH of about 5.0. In some embodiments, the collection tube is glass. In some embodiments, the collection tube has a silicone coating. In some embodiments, the collection tube has a Hemogard™ closure. In some embodiments, the collection tube has a volume capacity of 2-3 mL (eg, 2.7 mL). In some embodiments, the collection tube is sterile. In some embodiments, the collection tube is a BD Vacutainer™ CTAD blood collection tube (Macey et al. Clin Chem. 2002 Jun; 48(6 Pt 1):891-9).

Sample processing refers to any handling or processing of a biological sample (eg, a blood sample) following the sample collection steps described above. Sample processing steps can include, for example, centrifugation, sample fractionation or separation, pipetting, mechanical agitation (eg, shaking or mixing), and the like. In some embodiments, sample processing is performed to prepare platelet poor plasma (PPP). PPP fractions may be prepared from blood samples for measurement of circulating TGFβ1 levels. The term PPP typically refers to plasma containing fewer than 10,000 platelets per microliter (ie, <10 x ¹⁰ /μL).

In some embodiments, processing a blood sample includes incubation and/or centrifugation at a temperature below room temperature. In some embodiments, processing the blood sample includes incubation and/or centrifugation at a temperature of less than 20°C, less than 15°C, less than 10°C, less than 5°C, or lower. In some embodiments, processing the blood sample includes incubation at 2-8°C and/or centrifugation. In some embodiments, processing the blood sample includes incubation at about 4°C and/or centrifugation. In some embodiments, processing the blood sample includes one or more incubation steps as described in Example 4.

In some embodiments, processing the blood sample includes one or more centrifugation steps. In some embodiments, processing the blood sample includes one or more centrifugation steps performed at about 4°C. In some embodiments, processing the blood sample at a rate of less than 1500 x g, less than 1000 x g, less than 800 x g, less than 400 x g, less than 250 x g, less than 200 x g, or less. including a centrifugation step. In some embodiments, processing the blood sample includes a centrifugation step at a speed of about 150 xg. In some embodiments, processing the blood sample comprises: greater than 1500×g, greater than 2000×g, greater than 2500×g, greater than 5000×g, greater than 7500×g, greater than 10000×g, greater than 12000×g, or a centrifugation step at higher speeds. In some embodiments, processing the blood sample includes a centrifugation step at a speed of about 2500 xg. In some embodiments, processing the blood sample includes a centrifugation step at a speed of about 12,000 xg.

In some embodiments, processing the blood sample includes a first centrifugation step at a speed less than 1000 x g and a second centrifugation step at a speed greater than 2000 x g, optionally and one or both steps are performed at about 4°C. In some embodiments, processing the blood sample includes a first centrifugation step at a speed of about 150×g and a second centrifugation step at a speed of about 2000×g. In some embodiments, processing the blood sample includes a first centrifugation step at a speed less than 2500 xg and a second centrifugation step at a speed greater than 10000 xg. In some embodiments, processing the blood sample includes a first centrifugation step at a speed of about 1500×g and a second centrifugation step at a speed of about 12000×g. In some embodiments, processing the blood sample includes a first centrifugation step at a speed of 1000 x g to 5000 x g, and a second centrifugation step at a speed of 1000 x g to 5000 x g. Including process. In some embodiments, processing a blood sample includes a first step and a second centrifugation step, where the two centrifugation steps are performed at the same speed. In some embodiments, processing the blood sample includes a first centrifugation step at a speed of about 2500 xg and a second centrifugation step at a speed of about 2500 xg.

In some embodiments, processing the blood sample is performed for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, or more. This includes a centrifugation step. In some embodiments, the blood sample is subjected to a first centrifugation step for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, or more; a second centrifugation step for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, or more. In some embodiments, processing the blood sample includes a first centrifugation step for about 10 minutes and a second centrifugation step for about 20 minutes. In some embodiments, processing the blood sample includes a first centrifugation step for about 10 minutes and a second centrifugation step for about 5 minutes. In some embodiments, processing the blood sample includes transferring a supernatant portion of the sample to another tube after a first centrifugation step and further processing the supernatant in a second centrifugation step. . In some embodiments, the supernatant portion of the sample after the second centrifugation step is used to measure circulating TGFβ (eg, circulating latent TGFβ1) levels. In some embodiments, TGFβ (e.g., circulating latent TGFβ1) levels can be determined using the Bio-Plex Pro™ TGF-β assay (Strauss et al. Clin Cancer Res. 2018 Mar 15; 24(6):1287-1295).

In some embodiments, the collection, processing, and/or determination of circulating TGFβ (eg, circulating latent TGFβ1) levels is performed at about 4°C.

In some embodiments, processing the blood sample includes a first centrifugation step at 100-500 x g for 5-25 minutes, and a second centrifugation step at 1000-3000 x g for 10-40 minutes. steps, each step optionally performed at about 4°C. In some embodiments, processing the blood sample includes a first centrifugation step at 1000-3000 x g for 5-25 minutes, and a second centrifugation step at 1000-3000 x g for 10-40 minutes. steps, each step optionally performed at about 4°C. In some embodiments, processing the blood sample includes a first centrifugation step at 1000-3000 x g for 5-25 minutes, and a second centrifugation step at 5000-15000 x g for 2-10 minutes. steps, each step optionally performed at about 4°C.

In some embodiments, processing the blood sample includes a first centrifugation step at 1500 x g for 10 minutes and a second centrifugation step at 12000 x g for 5 minutes, optionally, One or both steps are performed at about 4°C. In some embodiments, the blood sample is processed by a first centrifugation step at 2500 x g for 10 minutes, followed by a second centrifugation step at 2500 x g for 10 minutes, optionally with one or Both steps are carried out at approximately 4°C. In some embodiments, one or more additional centrifugation steps are applied.

In various embodiments, the present disclosure provides methods for determining the level of circulating latent TGFβ in a sample obtained from a patient such that undesired or inadvertent TGFβ activation associated with sample processing and preparation is reduced. and provide a method for monitoring. In certain embodiments, the methods disclosed herein detect markers of platelet activation, e.g., PF4 levels, during collection, e.g., by using the sample collection methods disclosed herein. By normalizing to control, it can be used to determine or monitor the level of circulating latent TGFβ1.

Following the sample processing steps described above, one or more measurement steps can be performed on circulating TGFβ using the obtained sample (eg, PPP, etc.). Accordingly, the present disclosure provides, in various embodiments, a method of measuring circulating TGFβ levels in a blood sample, wherein the method includes a collection step and a processing step, each of which includes a CTAD collection tube. It is carried out at 2-8°C using The processing step may include two centrifugation steps as described above to generate the PPP fraction from the blood sample. The obtained PPP is used to measure TGFβ levels. In some embodiments, total TGFβ levels are measured, including both active and latent TGFβ forms. In some embodiments, active TGFβ (mature growth factor) levels are measured. In some embodiments, latent TGFβ levels are measured. In some embodiments, the majority of TGFβ measured in the acidified sample is derived from circulating latent TGFβ. In some embodiments, levels of TGFβ1 isoforms are selectively measured. In some embodiments, the measuring step can include acidifying the sample to release TGFβ (ie, mature growth factor) from the latent complex (ie, proTGFβ, such as proTGFβ1). An ELISA-based method may then be used to capture and detect/quantitate TGFβ present in the sample.

Such assay steps may be incorporated into a patient's treatment regimen. For example, such assays can be used to provide information to assist in prognosis, diagnosis, target binding, monitoring therapeutic response, and the like.

In some embodiments, circulating TGFβ levels can serve as a predictive biomarker.

In some embodiments, circulating TGFβ levels can serve as a predictive biomarker of therapeutic response to checkpoint inhibitor therapy. In some embodiments, high baseline levels of circulating TGFβ levels (e.g., in plasma) can predict poor therapeutic response to checkpoint inhibitor therapy (e.g., pembrolizumab) (Feun et al. Cancer.2019 Oct 15;125(20):3603-3614).

Accordingly, in various embodiments, a treatment regimen can include the administration of a therapy that includes a TGFβ inhibitor, such as a TGFβ1 inhibitor. TGFβ inhibitors include, for example, monoclonal antibodies that bind to the latent form of TGFβ (i.e., proTGFβ, such as proTGFβ1), thereby preventing the release of growth factors such as Ab6, and other antibodies that act by the same mechanism of action. Included (for example, see WO 2000/014460 pamphlet, WO 2000/041390 pamphlet, PCT/2021/012930, WO 2018/013939 pamphlet, WO 2020/160291 pamphlet) . TGFβ inhibitors include neutralizing antibodies and engineered constructs incorporating antigen-binding fragments thereof. Examples of neutralizing antibodies include GC1008 and its variants, and NIS-793 (XOMA089). TGFβ inhibitors also include so-called ligand traps, which contain ligand-binding fragments of TGFβ receptors. Examples of ligand traps include M7824 (vintrafsp alpha) and AVID200. TGFβ inhibitors also include low molecular weight receptor kinase inhibitors such as ALK5 inhibitors.

In various embodiments, the patient receiving the treatment regimen has been diagnosed with, or is at risk for developing, a TGFβ-related disease, such as cancer, myeloproliferative disorders (such as myelofibrosis), fibrosis, and immune disorders. There is, or there is a suspicion that there is. Thus, in some embodiments, the present disclosure provides a TGFβ inhibitor for use in treating a TGFβ-related disease in a subject, wherein the treatment comprises inhibiting TGFβ in an amount sufficient to treat the disease. The treatment further comprises measuring circulating TGFβ levels according to the disclosure herein. In some embodiments, the treatment further includes determining circulating MDSCs. In some embodiments, circulating MDSC levels are determined by measuring cell surface markers. In some embodiments, the cell surface marker is LRRC33. In some embodiments, the patient is a cancer patient, and optionally the cancer comprises a solid tumor, such as a locally advanced tumor or a metastatic tumor. In some embodiments, the patient has previously undergone cancer therapy, and the cancer therapy is a checkpoint inhibitor, radiation therapy, and/or chemotherapy. In some embodiments, the subject is refractory or refractory to cancer therapy, and optionally, the cancer therapy includes a checkpoint inhibitor (eg, checkpoint inhibitor resistance). In some embodiments, the tumor is resistant to cancer therapy. In some embodiments, the patient has never received cancer therapy, eg, a checkpoint inhibitor (ie, checkpoint inhibitor naive patient). In some embodiments, checkpoint inhibitor-naive patients have a low response rate to checkpoint inhibitors, such as anti-PD-(L)1 (e.g., less than 30%, less than 25%, less than 20%, less than 15%). be diagnosed with a type of cancer that is statistically shown to occur (e.g., less than %). In some embodiments, the solid tumor has an immune exclusion phenotype. In some embodiments, the solid tumor has low expression of PD-L1.

In various embodiments, the present disclosure provides a method of treating a TGFβ-related disorder, wherein the present disclosure provides a method for treating a TGFβ-related disorder in which a TGFβ inhibitor is including monitoring levels of circulating TGFβ, such as levels of circulating latent TGFβ (eg, TGFβ1). In certain embodiments, circulating TGFβ, such as circulating latent TGFβ (eg, TGFβ1), can be measured in a plasma sample obtained from a subject. In certain embodiments, measuring TGFβ from plasma, such as circulating latent TGFβ (e.g., TGFβ1), reduces the risk of inadvertently activating TGFβ as observed during serum preparation and/or processing. can be reduced. Accordingly, the present disclosure includes TGFβ inhibitors for use in treating diseases such as cancer, myelofibrosis and fibrosis in a subject, the treatment comprising measuring circulating TGFβ levels from a plasma sample taken from the subject. This includes the step of Such samples may be taken before and/or after administration of a TGFβ inhibitor to treat such diseases.

Levels of circulating TGFβ (eg, circulating latent TGFβ1) can be monitored alone or in conjunction with one or more biomarkers disclosed herein (eg, MDSC). In certain embodiments, circulating TGFβ (eg, circulating latent TGFβ1) may be monitored alone or in combination with one or more of total platelet count, phosphorylated Smad2 levels, and/or treatment duration. In certain embodiments, TGFβ inhibitors can be administered alone or in conjunction with additional cancer therapy. In some embodiments, a therapeutic agent can be administered to a subject suffering from a TGFβ-related cancer or myeloproliferative disorder. In some embodiments, the TGFβ inhibitor is a TGFβ1 selective antibody or antigen-binding fragment thereof (eg, Ab6) that is encompassed by this disclosure. 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 mutants, antibodies and ligand traps that bind to TGFβ1/3, such as TGFβ1/3 inhibitors). 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). In some embodiments, the additional cancer therapy includes chemotherapy, radiation therapy (including radiotherapeutic agents), cancer vaccines or checkpoint inhibitor therapy, such as anti-PD-1, anti-PD-L1 or anti-CTLA- 4 antibodies may be included. In some embodiments, checkpoint inhibitor therapy includes 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®) selected from the group.

In various embodiments, circulating TGFβ (eg, circulating latent TGFβ1) can be measured in a sample (eg, whole blood or blood component) obtained from a subject. In various embodiments, the circulating latent TGFβ level (e.g., latent TGFβ1) is 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14 after administration of the TGFβ inhibitor to the subject. , 16, 18, 21, 22, 25, 28, 30, 35, 40, 45, 48, 50 or 56 days, eg, up to 56 days after administration of a therapeutic dose of the TGFβ inhibitor. In various embodiments, circulating latent TGFβ levels (eg, circulating latent TGFβ1) can be measured from about 8 to about 672 hours after administration of a therapeutic dose of a TGFβ inhibitor. In various embodiments, circulating latent TGFβ levels (e.g., circulating latent TGFβ1) range from about 72 to about 240 hours (e.g., about 72 to about 168 hours, about 84 to about 156 hours, about 96 hours to about 144 hours, about 108 hours to about 132 hours). In various embodiments, circulating latent TGFβ levels (eg, circulating latent TGFβ1) can be measured about 120 hours after administration of a therapeutic dose of a TGFβ inhibitor. In some embodiments, circulating latent TGFβ levels (eg, circulating latent TGFβ1) can be measured by any method known in the art (eg, ELISA). In a preferred embodiment, circulating TGFβ levels are measured from a blood sample (eg, a plasma sample).

In various embodiments, the present disclosure encompasses a method of treating cancer in a subject, wherein the treatment comprises determining the level of circulating TGFβ in the subject prior to administering the TGFβ inhibitor, administering a therapeutically effective dose. administering a TGFβ inhibitor to the subject, and determining the level of circulating TGFβ in the subject after administration. In some embodiments, circulating TGFβ levels are or have been determined by processing a blood sample from a subject at below room temperature in a sample tube coated with an anticoagulant.

In various embodiments, a method of treating cancer or other TGF-related disorders comprises administering a TGFβ inhibitor (e.g., an anti-TGFβ1 antibody) to a patient in need thereof; and controlling the level of target binding by the inhibitor. and a step of confirming. In some embodiments, determining the level of target binding comprises determining the level of circulating TGFβ (e.g., circulating latent TGFβ1). In some embodiments, an increase in circulating TGFβ (eg, circulating latent TGFβ1) after administration of a TGF inhibitor is indicative of target binding. In some embodiments, the present disclosure provides a method of determining target binding in a subject with cancer, the method comprising determining the level of circulating TGFβ (e.g., circulating latent TGFβ1) in the subject prior to administering a TGFβ inhibitor. A method is provided that includes determining, administering to a subject a therapeutically effective dose of a TGFβ inhibitor, and, following administration, determining the level of circulating TGFβ in the subject. In some embodiments, an increase in circulating TGFβ levels (eg, circulating latent TGFβ1 levels) after administration of the inhibitor compared to before administration is indicative of targeted binding of the TGFβ inhibitor. In some embodiments, an increase in circulating TGFβ (e.g., circulating latent TGFβ1) after administration of a TGFβ inhibitor is indicative of target binding, wherein the increase is at least 1.5-fold from baseline levels, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times or more. In some embodiments, circulating TGFβ levels are or have been determined by processing a blood sample from the subject below room temperature in a sample tube coated with an anticoagulant. In some embodiments, if target binding is detected, an additional therapeutically effective amount of TGFβ inhibitor is administered.

In various embodiments, the present disclosure uses circulating TGFβ levels (e.g., circulating latent TGFβ1 levels) to predict treatment response and to inform further treatment decisions (e.g., reducing treatment if an increase is observed). (by continuation of ). In some embodiments, if target binding is detected, an additional dose of TGFβ inhibitor (eg, anti-TGFβ1 antibody) is administered. In some embodiments, a method of determining therapeutic efficacy comprises determining the level of circulating TGFβ in a subject prior to administering the TGFβ inhibitor, administering to the subject a therapeutically effective dose of the TGFβ inhibitor, and After administration, the method includes determining the level of circulating TGFβ in the subject. In a preferred embodiment, circulating TGFβ levels are measured from a blood sample. In some embodiments, circulating TGFβ levels are or have been determined by processing a blood sample from a subject at below room temperature in a sample tube coated with an anticoagulant. In some embodiments, if efficacy is detected, an additional therapeutically effective amount of TGFβ inhibitor is administered.

In one aspect of the disclosure, levels of circulating TGFβ (e.g., circulating latent TGFβ1) are determined to inform treatment in a subject administered a TGFβ inhibitor, such as a TGFβ1 selective inhibitor described herein. and also predict treatment efficacy. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is such that the amount of TGFβ1 inhibition administered increases the level of circulating TGFβ (e.g., circulating latent TGFβ1) as compared to baseline levels. Administered alone or contemporaneously (e.g., simultaneously) with an additional cancer therapy, e.g., checkpoint inhibitor therapy, separately or sequentially, as may be sufficient. Circulating TGFβ levels (eg, circulating latent TGFβ1) can be measured before or after each treatment such that an increase in circulating latent TGFβ levels (eg, latent TGFβ1) after treatment is indicative of treatment efficacy. For example, circulating TGFβ levels (e.g., circulating latent TGFβ1) can be measured before and after administration of a TGFβ inhibitor (e.g., Ab6), and increases in circulating TGFβ levels (e.g., latent TGFβ1) after treatment , predicting treatment effectiveness. In some embodiments, treatment is continued if an increase in circulating TGFβ is detected. In certain embodiments, circulating TGFβ levels (e.g., circulating latent TGFβ1) are measured before and after administration of a first dose of a TGFβ inhibitor, such as a TGFβ1 inhibitor (e.g., Ab6) described herein. An increase in circulating TGFβ levels (eg, circulating latent TGFβ1) after administration can be predictive of therapeutic efficacy and provide the basis for administration of a second or subsequent dose of a TGFβ inhibitor. In some embodiments, circulating TGFβ levels (e.g., circulating latent TGFβ1) are reduced by TGFβ inhibition, such as a TGFβ1 selective inhibitor (e.g., Ab6), administered contemporaneously (e.g., simultaneously), individually or sequentially. Changes in circulating latent TGFβ levels after treatment are predictive of treatment efficacy, which can be measured before and after combination treatment with an agent and further treatment (eg, checkpoint inhibitor therapy). In some embodiments, treatment is continued if an increase is detected. In some embodiments, an increase in circulating TGFβ (e.g., circulating latent TGFβ1) after administration of a TGFβ inhibitor is indicative of a therapeutic effect, wherein the increase is at least 1.5-fold compared to baseline. , at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times or more. In some embodiments, an increase in circulating TGFβ levels (eg, circulating latent TGFβ1 levels) following combination treatment may be the basis for continued treatment. In a preferred embodiment, circulating TGFβ levels are measured from a blood sample, which is optionally processed in an anticoagulant-coated sample tube at below room temperature.

In certain embodiments, the present disclosure provides a method of treating cancer in a subject, comprising administering a second dose of a TGFβ inhibitor to a subject who has an elevated level of circulating TGFβ after receiving a first dose of a TGFβ inhibitor. A method is provided comprising administering a TGFβ inhibitor, wherein the level of TGFβ is measured by processing a blood sample from a subject at below room temperature in a sample tube coated with an anticoagulant.

In certain embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising: determining the level of circulating TGFβ in the subject prior to administering the TGFβ inhibitor; administering a first dose of the TGFβ inhibitor; administering to a subject; after administration, determining the level of circulating TGFβ in the subject; and if the circulating TGFβ level is elevated, administering to the subject a second dose of a TGFβ inhibitor. provide. In some embodiments, measuring the level of circulating TGFβ comprises processing a blood sample from the subject at below room temperature in a sample tube coated with an anticoagulant. In some embodiments, the circulating TGFβ level after the first dose of the TGFβ inhibitor is at least 1.5 compared to baseline (the level of circulating TGFβ before the first dose of the TGFβ inhibitor). increase by at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, or more. In some embodiments, the circulating TGFβ is latent TGFβ. In some embodiments, the circulating TGFβ is circulating TGFβ1.

In some embodiments, the cancer comprises a solid tumor, and optionally, the solid tumor comprises melanoma (e.g., metastatic melanoma), triple-negative breast cancer, HER2-positive breast cancer, colorectal cancer (e.g., microsatellite stable colorectal cancer), lung cancer (e.g. metastatic non-small cell lung cancer, small cell lung cancer), esophageal cancer, pancreatic cancer, bladder cancer, kidney cancer (e.g. transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC) ) (including clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC), uterine cancer, prostate cancer, gastric cancer (e.g., gastric cancer), head and neck squamous cell carcinoma, urothelial cancer (eg, metastatic urothelial carcinoma), hepatocellular carcinoma or thyroid carcinoma.

In various embodiments, the present disclosure encompasses a method of treating a TGFβ-related disorder comprising administering to a subject having a TGFβ-related disorder a therapeutically effective amount of a TGFβ inhibitor, wherein the therapeutically effective amount comprises circulating TGFβ (e.g. , in an amount sufficient to increase the level of circulating latent TGFβ1). In certain embodiments, the TGFβ inhibitor is a TGFβ activation inhibitor. In certain embodiments, the TGFβ inhibitor is a TGFβ1 inhibitor (eg, Ab6). In certain embodiments, the circulating TGFβ is latent TGFβ1. In some embodiments, a therapeutically effective amount of a TGFβ inhibitor (eg, Ab6) is 0.1-30 mg/kg per dose. In some embodiments, a therapeutically effective amount of a TGFβ inhibitor (eg, Ab6) is 1-30 mg/kg per dose. In some embodiments, a therapeutically effective amount of a TGFβ inhibitor (eg, Ab6) is 5-20 mg/kg per dose. In some embodiments, a therapeutically effective amount of a TGFβ inhibitor (eg, Ab6) is 3-10 mg/kg per dose. In some embodiments, a therapeutically effective amount of a TGFβ inhibitor (eg, Ab6) is 1-10 mg/kg per dose. In some embodiments, a therapeutically effective amount of a TGFβ inhibitor (eg, Ab6) is 2-7 mg/kg per dose. In some embodiments, a therapeutically effective amount of a TGFβ inhibitor (eg, Ab6) is about 2-6 mg/kg per dose. In some embodiments, a therapeutically effective amount of a TGFβ inhibitor (eg, Ab6) is about 1 mg/kg per dose. In some embodiments, doses are administered about every three weeks. In some embodiments, the TGFβ inhibitor (e.g., Ab6) is administered weekly, every 2 weeks, every 3 weeks, every 4 weeks, every month, every 6 weeks, every 8 weeks, every other month, every 10 weeks, every 12 weeks. , every 3 months, every 4 months, every 6 months, every 8 months, every 10 months, or once a year. In a preferred embodiment, circulating TGFβ levels are measured from a blood sample (eg, plasma sample, serum sample, etc.).

In various embodiments, total circulating TGFβ1 (e.g., circulating latent TGFβ1) in a blood sample drawn from a patient can range from about 2 to 200 ng/mL at baseline; Varies depending on health status and specific assay employed. In certain embodiments, total circulating TGFβ1 (e.g., circulating latent TGFβ1) in a blood sample drawn from a patient ranges from about 1 ng/mL to about 10 ng (e.g., about 1000 pg/mL to about 7000 pg/mL). It can be. In certain embodiments, the level of circulating latent TGFβ (e.g., latent TGFβ1) after administration of the TGFβ inhibitor (e.g., Ab6) is compared to the level of circulating latent TGFβ (e.g., baseline) prior to administration. at least 1.5 times (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) or more). In a preferred embodiment, circulating TGFβ levels are measured from a blood sample (eg, plasma sample, serum sample, etc.).

In certain embodiments, circulating TGFβ levels (e.g., circulating latent TGFβ1) are used to target the binding and binding of TGFβ inhibitors in subjects receiving TGFβ inhibitor therapy (e.g., TGFβ activation inhibitors, e.g., Ab6). Pharmacological activity can be monitored. In certain embodiments, circulating TGFβ levels (e.g., circulating latent TGFβ1 levels) can be measured before and after administration of a first dose of a TGFβ inhibitor (e.g., Ab6), such that circulating latent TGFβ1 levels after administration type TGFβ levels from baseline at least 1.5 times (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, an increase of at least 9-fold, at least 10-fold or more) is indicative of target binding (eg, binding of the TGFβ inhibitor to the human large latent proTGFβ1 complex). In certain embodiments, circulating TGFβ levels (e.g., circulating latent TGFβ1) can be measured before and after administration of a first dose of a TGFβ inhibitor (e.g., Ab6), such that post-administration circulating TGFβ levels (e.g., , an increase in circulating latent TGFβ1) indicates therapeutic efficacy. In certain embodiments, treatment is continued if an increase in circulating TGFβ levels (eg, circulating latent TGFβ1) is detected after administration of a TGFβ inhibitor (eg, Ab6). In a preferred embodiment, circulating TGFβ levels (eg, circulating latent TGFβ1) are measured from a blood sample (eg, plasma sample, serum sample, etc.).

In some embodiments, circulating TGFβ levels (e.g., circulating latent TGFβ1) can be measured before and after administration of a first dose of a TGFβ inhibitor (e.g., Ab6), and post-administration circulating TGFβ levels (e.g., Increases in circulating latent TGFβ1) indicate target binding, therapeutic response, and/or further justify administration of a second or subsequent dose of TGFβ inhibitor. In another embodiment, circulating TGFβ levels (e.g., circulating latent TGFβ1) are measured prior to administration of the first dose of a combination therapy comprising checkpoint inhibitor therapy and a TGFβ inhibitor, such as a TGFβ1 selective inhibitor (e.g., Ab6). An increase in circulating TGFβ levels (eg, circulating latent TGFβ1) after administration indicates target binding, therapeutic response, and/or provides further evidence for continued treatment. In various embodiments, the combination therapy includes checkpoint inhibitor therapy and a TGFβ1 selective inhibitor (e.g., Ab6), an isoform non-selective inhibitor (e.g., a low molecular weight ALK5 antagonist), a TGFβ1/2/3 Neutralizing antibodies that bind to two or more (e.g., GC1008 and variants), antibodies that bind to TGFβ1/3, and/or integrin inhibitors (e.g., αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, or α8β1 integrin TGFβ inhibitors, such as antibodies that bind to and inhibit downstream activation of TGFβ, such as selective inhibition of TGFβ1 and/or TGFβ3. In a preferred embodiment, circulating TGFβ levels are measured from blood (eg, plasma sample, serum sample, etc.).

In any of the foregoing embodiments, the circulating TGFβ1 may be circulating TGFβ1 or circulating latent TGFβ1. In various embodiments, circulating TGFβ1 or circulating latent TGFβ1 is measured from a blood sample taken from the subject. In various embodiments, the blood sample is processed below room temperature in a sample tube containing or coated with an anticoagulant.

Smad2 Phosphorylation According to the present disclosure, activation of Smad2 can serve as a marker of target binding and/or therapeutic efficacy. In certain embodiments, Smad2 activation is detected by measuring the level of Smad2 phosphorylation (p-Smad2) and/or p-Smad2 nuclear translocation. In certain embodiments, p-Smad2 levels and/or p-Smad2 nuclear translocation is measured by immunohistochemistry. In certain embodiments, P-Smad2 nuclear translocation is determined by nuclear masking analysis.

In certain embodiments, a method of determining therapeutic efficacy in a subject undergoing treatment for cancer comprises determining p-Smad2 nuclear translocation levels in a tumor sample obtained from the subject prior to administering a therapy comprising a TGFβ inhibitor. administering to the subject one or more doses of a TGFβ inhibitor; and determining the level of p-Smad2 nuclear translocation in a tumor sample obtained from the subject after administration; A decrease in p-Smad2 nuclear translocation after administration compared to before administration indicates a therapeutic effect. In certain embodiments, p-Smad2 nuclear translocation after administration is at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least as compared to p-Smad2 nuclear translocation before administration. 4 times, at least 5 times, or more. In certain embodiments, one or more additional doses of treatment are administered if a decrease in p-Smad2 nuclear translocation is observed.

In certain embodiments, a method of determining target binding in a subject with cancer comprises determining p-Smad2 nuclear translocation levels in a tumor sample obtained from the subject prior to administering a therapy comprising a TGFβ inhibitor. administering to the subject one or more doses of a TGFβ inhibitor; determining, after administration, the level of p-Smad2 nuclear translocation in a tumor sample obtained from the subject; A decrease in p-Smad2 nuclear translocation after administration indicates target binding of the TGFβ inhibitor. In certain embodiments, p-Smad2 nuclear translocation after administration is at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least as compared to p-Smad2 nuclear translocation before administration. 4 times, at least 5 times, or more. In certain embodiments, one or more additional doses of treatment are administered if a decrease in p-Smad2 nuclear translocation is observed.

In certain embodiments, a method of treating cancer in a subject, or a TGFβ inhibitor for use in the treatment of cancer, wherein the method or use of a TGFβ inhibitor comprises a therapy comprising a TGFβ inhibitor. determining the level of p-Smad2 nuclear translocation in a tumor sample obtained from the subject prior to administering a first dose of the TGFβ inhibitor to the subject; determining the level of p-Smad2 nuclear translocation in the sample; and if p-Smad2 nuclear translocation after administration of the first dose is decreased compared to p-Smad2 nuclear translocation before administration of the first dose. , comprising administering to the subject one or more additional doses of the TGFβ inhibitor. In certain embodiments, p-Smad2 nuclear translocation after administration is at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least as compared to p-Smad2 nuclear translocation before administration. 4 times, at least 5 times, or more. In certain embodiments, if a decrease in p-Smad2 nuclear translocation is observed, the treatment comprises administering one or more additional doses of the treatment.

In any of the foregoing embodiments, the TGFβ inhibitor is a TGFβ1 inhibitor, eg, Ab6.

Immune Safety Cytokines play an important role in normal immune responses, but when the immune system is triggered to become overactive, a positive feedback loop of cytokine production can lead to a “cytokine storm” or hypercytokinemia. However, this is a situation in which excessive cytokine production causes an immune response that damages organs, especially the lungs and kidneys, and even leads to death. Such conditions are characterized by markedly increased proinflammatory cytokines in the serum. In the past, a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 in healthy volunteers resulted in a life-threatening "cytokine storm" response due to unexpected systemic and rapid induction of pro-inflammatory cytokines (Suntharalingam G. et al., N Engl J Med. 2006 Sep 7;355(10):1018-28). This case raised awareness of the potential risks associated with pharmacological stimulation of T cells.

Although TGFβ-directed therapies do not target specific T-cell receptors or their ligands, Applicants of the present disclosure have developed, for example, in vitro cytokine release assays from plasma samples of non-human primates treated with TGFβ inhibitors. We deemed it prudent to perform an immunosafety assessment including in vivo cytokine measurements and platelet assays using human platelets. An exemplary assay was described, for example, in Example 23 of PCT/US Patent Application Publication No. 2021/012969.

In some embodiments, one or more of the cytokines IL-2, TNFα, IFNγ, IL-1β, CCL2 (MCP-1), and IL-6 are, for example, peripheral blood mononuclear cells (PBMCs) from a healthy donor. ) can be assayed by exposure to constituents. The cytokine response following exposure to an antibody disclosed herein, eg, Ab6, can be compared to the release following exposure to a control, eg, an IgG isotype negative control antibody. Cytokine activation can be assessed in plate binding and/or soluble assay formats. Levels of IFNγ, IL-2, IL-1β, TNFα, IL-6 and CCL2 (MCP-1) should not exceed 10 times, eg 8, 6, 4 or 2 times the activation in the negative control. In some embodiments, positive controls can also be used to confirm cytokine activation in a sample, eg, PBMC. In some embodiments, these in vitro cytokine release results are evaluated in vivo, e.g., in a monkey toxicology study, e.g., as described in Example 24 of PCT/U.S. Patent Application Publication No. 2021/012969. Further confirmation can be provided in animal models such as a 4 week GLP repeated dose monkey study.

Human platelets have been reported to express GARP, which can form TGFβ1 LLC (Tran et al., 2009. Proc Natl Acad Sci USA. 106(32):13445-13450 ). In some embodiments, the antibodies disclosed herein, eg, Ab6, do not significantly bind to and/or activate platelets. In some embodiments, platelet activation is assessed in vitro, as described in Example 23. In some embodiments, platelet aggregation, binding and activation can be assessed in human whole blood or platelet-rich plasma from healthy donors. Platelet aggregation and binding after exposure to an antibody disclosed herein, eg, Ab6, can be compared to exposure to a negative control, eg, saline, or a reference sample, eg, a buffer solution. In certain embodiments, platelet aggregation and binding is 10% or less of the aggregation in the negative control. In some embodiments, platelet activation following exposure to an antibody disclosed herein, eg, Ab6, can be compared to exposure to a positive control, eg, adenosine diphosphate (ADP). The activation state of platelets can be determined by the surface expression of activation markers such as CD62P (P-selectin) and GARP, which can be detected by flow cytometry. Platelet activation should be less than 10% of the activation in the negative control. In some embodiments, in vitro platelet response results can be further confirmed in vivo, eg, in an animal model, such as a monkey toxicology study, eg, a 4-week GLP repeated dose monkey study.

In some embodiments, selecting an antibody or antigen-binding fragment thereof for therapeutic use comprises: identifying an antibody or antigen-binding fragment that meets one or more of the criteria described herein; conducting an in vivo efficacy study in a suitable preclinical model to determine the effective amount; a safe or toxic amount of antibody (e.g., MTD, NOAEL, or skill in the art to assess safety/toxicity); performing in vivo safety/toxicity studies in a suitable model to determine the therapeutic concentration range (preferably 6-fold, more preferably 10-fold therapeutic); selecting an antibody or fragment that provides a concentration range, more preferably a 15-fold therapeutic concentration range). In certain embodiments, in vivo efficacy testing is performed in two or more suitable preclinical models that reproduce the human condition. In some embodiments, such preclinical models include TGFβ1 positive cancers, which may optionally include immunosuppressive tumors. Immunosuppressive tumors can be resistant to cancer therapies such as CBT, chemotherapy and radiotherapy (including radiotherapeutic agents). In some embodiments, the preclinical model is selected from MBT-2, Cloudman S91 and EMT6 tumor models.

Identification of antibodies or antigen-binding fragments thereof for therapeutic use may further include performing immunosafety assays, including, but not limited to, measurements of cytokine release and/or platelet binding, activation and/or or determining the effect of the antibody or antigen-binding fragment on aggregation. In certain embodiments, cytokine release can be measured in vitro using PBMCs or in vivo using preclinical models such as non-human primates. In certain embodiments, the antibody or antigen-binding fragment thereof induces the release of IL-6, IFNγ, and/or TNFα levels that are 10-fold or less compared to the levels in an IgG control sample in an immunosafety assessment. In certain embodiments, assessment of platelet binding, activation and aggregation may be performed in vitro using PBMC. In some embodiments, the antibody or antigen-binding fragment thereof induces no more than a 10% increase in platelet binding, activation, and/or aggregation compared to a buffer or isotype control in an immunosafety assessment.

Selected antibodies or fragments can be used in the manufacture of pharmaceutical compositions containing the antibodies or fragments. Such pharmaceutical compositions can be used to treat TGFβ indications in the subjects described herein. For example, the TGFβ indication can be a proliferative disorder, such as a TGFβ1 positive cancer. Accordingly, the present invention includes a method of manufacturing a pharmaceutical composition comprising a TGFβ inhibitor, the method comprising the step of selecting a TGFβ inhibitor, the TGFβ inhibitor being tested for immunosafety, which Immunosafety evaluation includes release assays and optionally further includes platelet assays. TGFβ inhibitors selected by this method do not trigger unacceptable levels of cytokine release (eg, no more than 10-fold, more preferably no more than 2.5-fold, compared to a control, such as an IgG control). Similarly, TGFβ inhibitors selected by this method do not cause unacceptable levels of platelet aggregation, platelet activation and/or platelet binding. These TGFβ inhibitors are then manufactured on a large scale, such as 250L or more, such as 1000L, 2000L, 3000L, 4000L or more, for commercial production of pharmaceutical compositions comprising the TGFβ inhibitor.

Cancer/Malignancy A variety of cancers involve TGFβ activity, such as TGFβ1 activity, and therefore can be treated with the antibodies, compositions, and methods of the present disclosure. As used herein, the term "cancer" includes any of a variety of malignant neoplasms, optionally those associated with TGFβ1 positive cells. Such malignant neoplasms are characterized by the proliferation of undifferentiated cells that tend to invade surrounding tissues and metastasize to new body sites, and also refer to pathological conditions characterized by the proliferation of such malignant neoplasms. . Sources of TGFβ1 are believed to be diverse and may include the malignant (cancer) cells themselves as well as their surrounding or supporting cells/tissues such as the extracellular matrix, various immune cells, and any combination thereof. .

Examples of cancers that can be treated according to the present disclosure include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, or lymphoid malignancies. More specific examples of such cancers include anal cancer; bile duct cancer; brain tumors (including glioblastoma); breast cancer, such as HER2+ breast cancer and triple negative breast cancer (TNBC); ductal carcinoma in situ ( DCIS); cervical cancer; colorectal cancer; endometrial or uterine cancer; esophageal cancer; gastric or gastrointestinal cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); head and neck cancer, e.g. liver cancer, such as hepatocellular carcinoma (HCC); lung cancer such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), metastatic NSCLC, lung adenocarcinoma, and lung squamous cell carcinoma; melanoma; Ovarian cancer; pancreatic cancer (e.g. pancreatic ductal adenocarcinoma (PDAC)); penile cancer; prostate cancer, e.g. castration-resistant prostate cancer (CRPC); renal cell carcinoma (RCC), e.g. clear cell RCC; peritoneal cancer; salivary gland cancer; Thyroid cancer; urothelial carcinoma (UC) of the bladder and urinary tract, including metastatic UC (mUC); urothelial bladder cancer, muscle invasive bladder cancer (MIBC), and non-muscle invasive bladder cancer (NMIBC) ); Myeloproliferative diseases such as chronic myeloid leukemia (CML), polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET), and chronic neutrophilic leukemia (CNL) Neoplasm (MPN); myelodysplastic syndrome (MDS); myeloproliferative neoplasm (MDS/MPN); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL) ; multiple myeloma; Hodgkin lymphoma; non-Hodgkin lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and hairy cell leukemia; mantle cell lymphoma; monoclonal gamma globulinemia of undetermined significance. plasma cell myeloma; Waldenström macroglobulinemia; and mature T and NK neoplasms. In certain embodiments, cancers that can be treated according to the present disclosure include those with high tumor mutational burden.

Definitive determination of cancer as "TGFβ1 positive" is not required to practice the therapeutic methods described herein, but is included in some embodiments. Typically, a particular cancer type is known or suspected to be associated with TGFβ1 signaling based on reliable evidence.

Cancers can be localized (eg, solid tumors) or systemic. In the context of this disclosure, the term "localized" (as in "localized tumor") refers to an anatomical disease such as a solid malignant tumor, as opposed to a systemic disease (e.g., a so-called liquid tumor or hematological cancer). Refers to a scientifically isolated or isolable abnormality/lesion. Certain cancers, such as certain leukemias (e.g., myelofibrosis) and multiple myeloma, have both localized (e.g., bone marrow) and systemic (e.g., circulating blood cells) components to the disease. obtain. In some embodiments, the cancer may be systemic, such as a hematological malignancy. Cancers that may be treated according to the present disclosure are TGFβ1 positive and include, but are not limited to, anus, bladder, bile duct, bone, brain, breast, cervix, color/rectum, endometrium, esophagus, eye, gallbladder, head and neck. Any cancer or tumor found in the body, liver, kidneys, larynx, lungs, mediastinum (chest), oral cavity, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, coccyx, testicles, thyroid, and uterus. Includes types of lymphoma/leukemia, carcinoma and sarcoma. In some embodiments, the cancer can be an advanced cancer, such as locally advanced solid tumors and metastatic cancer.

In some embodiments, the cancer can be a cancer that has elevated TGFβ1 levels associated with reactive oxygen species (ROS). In some embodiments, the cancer can be one that has elevated ROS levels and expresses high levels of TGFβ1.

Antibodies or antigen-binding fragments thereof encompassed by this disclosure can be used to treat cancers including, but not limited to: myelofibrosis, melanoma, adjuvant melanoma, renal cell carcinoma (RCC) including cellular RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC), bladder cancer, colorectal cancer (CRC) (e.g., microsatellite-stable CRC, mismatch repair-deficient colorectal cancer), colon Cancer, Rectal Cancer, Anal Cancer, Breast Cancer, Triple Negative Breast Cancer (TNBC), HER2 Negative Breast Cancer, HER2 Positive Breast Cancer, BRCA Mutated Breast Cancer, Hematologic Malignancy, Non-Small Cell Cancer, Non-Small Cell Lung Cancer/Carcinoma (NSCLC), Small Cell Lung cancer/carcinoma (SCLC), extensive small cell lung cancer (ES-SCLC), lymphoma (classic Hodgkin and non-Hodgkin), primary mediastinal large B-cell lymphoma (PMBCL), T-cell lymphoma, diffuse large cell Type B-cell lymphoma, histiocytic sarcoma, follicular dendritic cell sarcoma, digital intrusive dendritic cell sarcoma, myeloma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), small lymphocytic lymphoma ( SLL), head and neck cancer (e.g., head and neck squamous cell carcinoma), urothelial cancer (e.g., metastatic urothelial carcinoma), Merkel cell carcinoma (e.g., metastatic Merkel cell carcinoma), Merkel cell skin cancer, high Cancers with microsatellite instability (MSI-H), cancers with mismatch repair deficiency (dMMR), high tumor mutation burden cancers, mesothelioma (e.g. malignant pleural mesothelioma), gastric cancer, gastroesophageal junction cancer (GEJ), gastric adenocarcinoma, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), gastric cardia adenocarcinoma, renal cancer, biliary tract cancer, bile duct cancer, pancreatic cancer, prostate cancer, adenocarcinoma, squamous cell carcinoma, non-squamous cell carcinoma Cancer, cutaneous squamous cell carcinoma (CSCC), ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, peritoneal cancer, gastric cancer, brain tumor, malignant glioma, glioblastoma, gliosarcoma, neuroblastoma, Thyroid cancer, adrenocortical cancer, oral intraepithelial neoplasia, esophageal cancer, nasal and paranasal sinus squamous cell carcinoma, nasopharyngeal cancer, salivary gland cancer, liver cancer, basal cell carcinoma; and hepatocellular carcinoma (HCC). However, any cancer (e.g., a patient with such a cancer) in which TGFβ1 is overexpressed, or at least the predominant isoform, as determined by biopsy, for example, may be treated with an isoform of TGFβ1 according to the present disclosure. Can be treated with form-selective inhibitors.

In the case of cancer, TGFβ (eg, TGFβ1) can be either pro-proliferative or anti-proliferative. As an example, in the case of pancreatic cancer, SMAD4 wild-type tumors may experience inhibited proliferation in response to TGFβ, but as the disease progresses, they typically develop constitutively activated type II receptors. A body exists. In addition, there are also SMAD4-null pancreatic cancers. In some embodiments, the antibodies, antigen-binding portions thereof, and/or compositions of the present disclosure selectively target components of the TGFβ signaling pathway that function uniquely in one or more forms of cancer. Designed to. Leukemia, or cancer of the blood or bone marrow characterized by abnormal proliferation of white blood cells, or leukocytes, can be divided into four major categories, including: acute lymphoblastic leukemia ( ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia or acute myeloid leukemia (AML) (chromosomes 10 and 11 [t(10,11)], chromosomes 8 and 21 [t (8;21)], AML with translocation between chromosomes 15 and 17 [t(15;17)] and inversion in chromosome 16 [inv(16)]; multiphyletic dysplasia treatment-related AML (including patients with previous myelodysplastic syndromes (MDS) or myeloproliferative disorders converting to AML); d) AML not otherwise classified, including subtypes of AML that do not fall into the above categories; and e) acute leukemia of uncertain lineage, which occurs when leukemic cells cannot be classified as either myeloid or lymphoid cells, or when both cell types are present); as well as chronic myeloid leukemia (CML).

In some embodiments, any one of the TGFβ1 positive cancers listed above may also be TGFβ3 positive. In some embodiments, a tumor that is both TGFβ1 positive and TGFβ3 positive can be TGFβ1/TGFβ3 codominant. In some embodiments, such cancer is a carcinoma, including solid tumors. In some embodiments, such tumor is breast cancer. In some embodiments, the breast cancer may be of the triple negative genotype (triple negative breast cancer). In some embodiments, the subject with a TGFβ1 positive cancer has elevated levels of MDSC. For example, such tumors may contain MDSCs recruited to the tumor site resulting in increased numbers of MDSC infiltrates. In some embodiments, elevated levels of MDSCs can be detected in the blood (ie, circulating MDSCs). In some embodiments, subjects with breast cancer exhibit elevated levels of C-reactive protein (CRP), an inflammatory marker associated with recurrence and poor prognosis. In some embodiments, the subject with breast cancer exhibits elevated levels of IL-6.

The TGFβ inhibitors of the present disclosure are used to treat patients suffering from chronic myeloid leukemia, a stem cell disease in which the BCR/ABL oncoprotein is believed to be essential for the abnormal growth and accumulation of neoplastic cells. be able to. Imatinib is an approved therapy to treat this condition; however, a significant proportion of myeloid leukemia patients develop resistance to imatinib. TGFβ inhibition achieved by inhibitors as described herein enhances repopulation/expansion to counteract the BCR/ABL-driven aberrant proliferation and accumulation of neoplastic cells, thereby increasing clinical can bring benefits.

TGFβ inhibitors such as those described herein can be used to treat multiple myeloma. Multiple myeloma is a cancer of B lymphocytes (eg, plasma cells, plasmablasts, memory B cells) that originates and spreads within the bone marrow, causing destructive bone lesions (ie, osteolytic lesions). Typically, the disease manifests as enhanced osteoclast bone resorption, suppression of osteoblast differentiation (e.g., differentiation arrest), and impaired bone formation, including, in part, osteolytic lesions, bone loss, It is characterized by osteoporosis, hypercalcemia, and plasmacytoma, thrombocytopenia, neutropenia, and neurological disorders. TGFβ inhibitor therapy described herein can be effective in ameliorating one or more such clinical symptoms or symptoms in a patient. TGFβ1 inhibitors may be administered to patients undergoing additional treatments or therapies to treat multiple myeloma, including those listed elsewhere herein. In some embodiments, the multiple myeloma is treated with a myostatin inhibitor (e.g., an antibody disclosed in WO 2017/049011, e.g., apitegromab, also known as SRK-015) or an IL-6 inhibitor. can be treated with TGFβ inhibitors, such as isoform-specific context-independent inhibitors (eg, Ab6). In some embodiments, the TGFβ inhibitors include bortezomib, lenalidomide, carfilzomib, pomalidomide, thalidomide, doxorubicin, corticosteroids (e.g., dexamethasone and prednisone), chemotherapy (e.g., melphalan), radiation therapy (radiotherapy ), stem cell transplantation, plitidepsin, elotuzumab, ixazomib, masitinib and/or panobinostat.

Types of carcinomas that may be treated by the methods of the present disclosure include, but are not limited to, papillomas/carcinomas, choriocarcinomas, endodermal sinus tumors, teratomas, adenomas/adenocarcinomas, melanomas, fibromas, lipomas, leiomyomas. , rhabdomyoma, mesothelioma, hemangioma, osteoma, chondroma, glioma, lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, basal cell carcinoma, and cribriform nasal cavity carcinoma. .

Types of sarcoma include, but are not limited to, soft tissue sarcomas such as alveolar soft tissue sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small cell tumor, extraosseous chondrosarcoma, extraosseous osteosarcoma, fibrosarcoma, and vascular Exocytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (undifferentiated neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.

TGFβ inhibitors such as those described herein are considered suitable for treating malignancies involving cells of neural crest origin. Cancers of the neural crest lineage (i.e., neural crest-derived tumors) include, but are not limited to, melanoma (cancer of melanocytes), neuroblastoma (cancer of sympathoadrenal progenitor cells), and gangliocytoma (peripheral nervous system nerve cancer). medullary thyroid carcinoma (cancer of the thyroid C cells), pheochromocytoma (cancer of the chromaffin cells of the adrenal medulla), and MPNST (cancer of the Schwann cells). In some embodiments, the antibodies and methods of the present disclosure can be used to treat one or more types of cancer or cancer-related conditions, including, but not limited to, colon cancer, kidney cancer, breast cancer. , malignant melanoma, urothelial carcinoma, and glioblastoma (Schlingensiepen et al., 2008. Cancer Res. 177:137-50; Ouhtit et al., 2013. J Cancer. 4(7) :566-572.

Immunological Characteristics Under normal conditions, regulatory T cells (Tregs) represent a small subset of the overall CD4-positive lymphocyte population and play an important role in maintaining the immune system in homeostasis. However, in almost all cancers, the number of Tregs is significantly increased. Tregs play an important role in suppressing the immune response in healthy individuals, but increased numbers of Tregs in cancer are associated with poor prognosis. Increased Tregs in cancer can weaken host anti-cancer immunity and contribute to tumor progression, metastasis, tumor recurrence and/or treatment resistance. For example, human ovarian cancer ascites is infiltrated with Foxp3+GARP+Treg (Downs-Canner et al., Nat Commun. 2017, 8:14649). Similarly, Tregs were positively correlated with a more immunosuppressive and more aggressive phenotype in advanced hepatocellular carcinoma (Kalathil et al., Cancer Res. 2013, 73(8): 2435 -44). Tregs can suppress the proliferation of effector T cells. In addition, Tregs exert contact-dependent inhibition of immune cells (eg, naive CD4+ T cells) through the production of TGFβ1. Therefore, to treat tumors, it is advantageous to inhibit Tregs so that sufficient effector T cells are available to exert an antitumor effect.

A growing body of evidence suggests a role for macrophages in tumor/cancer progression. The present disclosure embraces the view that this is partially mediated by TGFβ activation, particularly TGFβ1 activation, in the tumor microenvironment. Bone marrow-derived monocytes (e.g., CD11b+) are recruited to tumor sites in response to tumor-derived cytokines/chemokines (e.g., CCL2, CCL3, and CCL4), where they undergo differentiation and polarization to develop a cancer-promoting phenotype. (e.g., M2-biased or M2-like macrophages, TAMs). As previously demonstrated (WO 2018/129329), monocytes isolated from human PBMCs can be divided into different subtypes of macrophages, e.g. M1 (pro-fibrotic, anti-cancer). and M2 (pro-cancer). The majority of TAMs in many tumors are M2 biased. Among M2-like macrophages, M2c and M2d subtypes have been found to express increased LRRC33 on the cell surface, whereas M1 does not. Furthermore, macrophages can be further skewed or activated by exposure to certain cytokines, such as M-CSF, resulting in a significant increase in LRRC33 expression concomitant with TGFβ1 expression. Increased levels of circulating M-CSF (ie, serum M-CSF concentrations) have also been observed in patients with myeloproliferative disorders (eg, myelofibrosis). Generally, tumors with high macrophage (TAM) and/or MDSC infiltration are associated with poor prognosis. Similarly, increased levels of M-CSF are also indicative of poor prognosis. Accordingly, in some embodiments, TGFβ inhibitors such as those encompassed herein can be used to treat cancers characterized by increased levels of cancer-promoting macrophages and/or MDSCs. In some embodiments, TGFβ inhibitors such as those encompassed herein can be used to treat cancers characterized by increased levels of MDSCs, regardless of levels of other macrophages. The LRRC33 arm of the inhibitor may at least partially mediate its inhibitory effects on disease-associated immunosuppressive myeloid cells, such as M2-macrophages and MDSCs.

The high prevalence of tumor-associated M2-like macrophages is reproduced in murine syngeneic tumor models such as those previously described by Applicants. Please refer to the data disclosed in PCT/US2019/041373. In the case of MBT-2 tumors, for example, approximately 40% of CD45-positive cells isolated from established tumors are M2 macrophages. This is halved in animals treated with a combination of isoform-selective TGFβ1 and anti-PD-1. In comparison, no significant changes in the number of tumor-associated M1 macrophages are observed in the same animals. Similar to M2 macrophages, tumor-associated MDSCs are also increased in established tumors (approximately 10-12% of CD45+ cells) and significantly reduced (to negligible levels) by inhibiting both PD-1 and TGFβ1 in treated animals. do. As previously disclosed by Applicants, the majority of tumor-infiltrating M2 macrophages and MDSCs express cell surface LRRC33 and/or LRRC33-proTGFβ1 complexes. Interestingly, cell surface expression of LRRC33 (or LRRC33-proTGFβ1 complex) appears to be highly regulated. The TGFβ inhibitors described herein, such as Ab6, can be rapidly internalized in cells expressing LRRC33 and proTGFβ1, and the rate of internalization achieved by TGFβ inhibitors is similar to that of cell surface LRRC33. significantly higher than that achieved with the reference antibody that recognizes. Similar results are obtained with primary human macrophages. These observations indicate that Ab6, upon binding to its target LRRC33-proTGFβ1, can promote internalization and thereby remove LRRC33-containing complexes from the cell surface. Accordingly, target binding by a TGFβ inhibitor of the present disclosure, such as Ab6, can induce antibody-dependent downregulation of the target protein (eg, cell-associated proTGFβ1 complex). At the disease locus, this may reduce the availability of activatable latent LRRC33-proTGFβ1 levels. Accordingly, the TGFβ inhibitors of the present disclosure may inhibit the LRRC33 arm of TGFβ1 through a dual mechanism of action: i) inhibition of the release of mature growth factors from the latent complex; and ii) Removal of LRRC33-proTGFβ1 complex from the cell surface by naturalization. In the tumor microenvironment, antibodies are thought to target cell-associated latent proTGFβ1 complexes and may enhance inhibitory effects on target cells such as M2 macrophages (eg, TAMs), MDSCs, and Tregs. Phenotypically, these are immunosuppressive cells and contribute to an immunosuppressive tumor microenvironment, which is mediated, at least in part, by the TGFβ1 pathway. Given that many tumors are enriched in these cells, antibodies capable of targeting multiple arms of TGFβ1 function, such as those described herein, may offer particular functional advantages. It should be provided.

Many human cancers are known to cause increased levels of MDSCs in patients compared to healthy controls (e.g., Elliott et al., (2017) “Human tumor-infiltrating myeloid cells: phenotypic and functional diversity” Frontiers in Immunology, Vol. 8, Article 86). These human cancers include, but are not limited to, bladder cancer, colon cancer, prostate cancer, breast cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, lung cancer, melanoma, NSCL, ovarian cancer, pancreatic cancer, and renal cancer. Examples include cell cancer. Increased levels of MDSCs can be detected in biological samples such as peripheral blood mononuclear cells (PBMCs) and tissue samples (eg, tumor biopsies). For example, changes in the frequency or number of MDSCs can be determined using appropriate cell surface markers (phenotypes): percent (%) total PBMC, percent (%) CD14+ cells, percent (%) CD45+ cells; mononuclear Percentage of cells (%), Percentage of total cells (%), Percentage of CD11b+ cells (%), Percentage of monocytes (%), Percentage of non-lymphocytic MNCs (%), Percentage of KLA-DR cells (%) ) can be measured as

On the other hand, macrophage infiltration into the tumor may also signify the effectiveness of the treatment. As exemplified herein, tumors are effectively infiltrated by effector T cells (eg, CD8+ T cells) after treatment with a combination of checkpoint inhibitors and context-independent TGFβ1 inhibitors. Intratumoral effector T cells can lead to the recruitment of phagocytic monocytes/macrophages that eliminate cellular debris.

It was observed that the combination of anti-PD-1 and TGFβ inhibitor resulted in robust CD8 T cell influx/expansion throughout the tumor compared to anti-PD-1 monotherapy. Correspondingly, robust increases in CD8 effector genes can be achieved with combination therapy. Accordingly, the TGFβ1 inhibitors of the present disclosure can be used to promote effector T cell infiltration into tumors.

Furthermore, significant tumor infiltration/expansion by F4/80 positive macrophages is observed. This is thought to represent M1 (anti-tumor) macrophages clearing cancer cell debris produced by cytotoxic cells, which is likely a direct result of TGFβ1 inhibition. As described in more detail in PCT/U.S. Patent Application Publication No. 2019/041373, these tumor-infiltrating macrophages are primarily classified as non-M2 macrophages because they lack CD163 expression. This observation indicates that circulating monocytes are recruited to tumor sites and differentiate into M1 macrophages upon checkpoint inhibitor and TGFβ1 inhibitor treatment; It is accompanied by a significant influx of CD8+ T cells. Accordingly, the TGFβ1 inhibitors of the present disclosure can be used to increase tumor-associated non-M2 macrophages.

In recent years, checkpoint blockade therapy (CBT) has become a standard therapy for treating many cancer types. Despite significant advances in cancer immunotherapy, primary resistance to CBT remains a major unmet need for patients. The majority of patient cancers remain unresponsive to PD-(L)1 inhibition. Recently, retrospective analyzes of urothelial carcinoma and melanoma tumors have implicated TGFβ activation as a potential promoter of primary resistance, which is associated with the elimination of cytotoxic T cells from tumors and tumor It is likely to be through multiple mechanisms, including their expansion within the microenvironment (immune exclusion). These observations and subsequent preclinical validation point to TGFβ pathway inhibition as a promising means to resolve primary resistance to CBT. However, therapeutic targeting of the TGFβ pathway is hampered by dose-limiting preclinical cardiotoxicity, likely due to inhibition of signaling from one or more TGFβ isoforms.

Many tumors lack a primary response to CBT. In this scenario, CD8+ T cells are generally excluded from the tumor parenchyma, suggesting that tumors may co-opt the immunomodulatory function of TGFβ signaling to generate an immunosuppressive microenvironment. These insights from retrospective clinical tumor sample analysis provided the rationale to examine the role of TGFβ signaling in primary resistance to CBT.

Regarding responses to TGFβ and CBT, we here observe a predominant expression of TGFβ1 in many human tumors, suggesting that this family member contributes to primary resistance in this pathway. This suggests that it may be an important promoting factor.

Increasing evidence suggests that TGFβ may be a major agent in producing and/or maintaining immunosuppression in diseased tissues, including immune-excluded tumor environments. Therefore, TGFβ inhibition may unblock immunosuppression and allow effector T cells (particularly cytotoxic CD8+ T cells) to reach and kill target cancer cells. In addition to tumor invasion, TGFβ inhibition can also promote CD8+ T cell proliferation. Such spread may occur in the lymph nodes and/or in the tumor (intratumoral). Although the precise mechanisms underlying this process remain to be elucidated, immunosuppression is thought to be mediated, at least in part, by immune cell-associated TGFβ1 activation, including regulatory T cells and activated macrophages. TGFβ has been reported to directly promote Foxp3 expression in CD4+ T cells, thereby converting them to a regulatory (immunosuppressive) phenotype (ie, Tregs). Furthermore, Tregs suppress effector T cell proliferation, thereby reducing immune responses. This process has been shown to be TGFβ1 dependent and likely involves GARP-associated TGFβ1 signaling. Observations in both humans and animal models have shown that increased Tregs in the TME are associated with poor prognosis in multiple cancer types. Furthermore, the applicant has previously shown that M2-polarized macrophages exposed to tumor-derived factors such as M-CSF significantly upregulate the cell surface expression of LRRC33, a presenting molecule for TGFβ1. (See, eg, PCT/US Patent Application Publication No. 2018/031759). These so-called tumor-associated macrophages (or TAMs) are thought to contribute to the TGFβ1-dependent immunosuppression observed in the TME and promote tumor growth.

Some solid tumors are characterized by having a tumor stroma enriched with myofibroblasts or myofibroblast-like cells. These cells produce a collagenous matrix that surrounds or envelops the tumor (such as fibrosis), which may be caused, at least in part, by overactive TGFβ1 signaling. TGFβ1 activation is thought to be mediated by ECM-related presentation molecules such as LTBP1 and LTBP3 in the tumor stroma.

Selective inhibition of TGFβ activation, such as TGFβ1 inhibition, may be sufficient to resolve primary resistance to CBT. By targeting the prodomain of latent TGFβ1, isoform-selective inhibitors of TGFβ1 can acquire isoform specificity and inhibit latent TGFβ1 activation.

Selective inhibition of the TGFβ pathway, such as the TGFβ1 pathway, may result in significantly improved preclinical safety versus broad inhibition of all isoform activities. The pleiotropic effects associated with broad TGFβ pathway inhibition have hindered therapeutic targeting of the TGFβ pathway. Most experimental therapeutics to date (e.g., galunisertib, LY3200882, fresolimumab) lack selectivity for a single TGFβ isoform, which may contribute to the dose-limiting toxicities observed in non-clinical and clinical trials. There is sex. Genetic data from knockout mouse and human loss-of-function mutations in the TGFβ2 or TGFβ3 genes suggest that cardiotoxicity observed with nonspecific TGFβ inhibitors may be due to inhibition of TGFβ2 or TGFβ3. are doing. The present disclosure shows that selective inhibition of TGFβ1 activation by such antibodies has an improved safety profile and is sufficient to induce robust antitumor responses when combined with PD-1 inhibition. , teachings that allow evaluation of the efficacy of TGFβ1 inhibitors at clinically manageable dose levels.

The preclinical studies and results presented herein and in PCT/U.S. Patent Application Publication No. 2019/041373 demonstrate that combination therapy with a TGFβ1 inhibitor (e.g., Ab6) and a checkpoint inhibitor may It has been demonstrated that this can have a significant effect on context (eg, increased levels of tumor-associated CD8+ T cells). These may include unexpected enrichment of Treg cells by combination treatment with anti-PD-1/TGFβ1 inhibitors.

In addition to the expected and observed effects on the distribution of cytotoxic T cells within tumors, TGFβ inhibitor/anti-PD-1 combination therapy may also have beneficial effects on the immunosuppressive myeloid compartment. Therefore, therapeutic strategies that involve targeting these important immunosuppressive cell types may have a greater effect in the tumor microenvironment than targeting a single immunosuppressive cell type (ie, Treg cells only). Accordingly, the TGFβ1 inhibitors of the present disclosure can be used to reduce tumor-associated immunosuppressive cells such as M2 macrophages and MDSCs.

Applicants' data also demonstrate that highly specific inhibition of TGFβ1 activation may enable the host immune system to overcome an important mechanism of primary resistance to checkpoint inhibition therapy and is a major limitation in clinical application. We demonstrate that this method avoids the previously recognized toxicity of broad-spectrum TGFβ inhibition.

Therefore, TGFβ inhibitors, such as selective TGFβ1 inhibitors, can be used to counter primary resistance to CBT, thereby making tumors/cancers more sensitive to CBT. Such effects may be applicable to the treatment of a wide variety of malignant tumor types where the cancer/tumor is TGFβ1 positive. In some embodiments, such tumors/cancers may further express additional isoforms such as TGFβ3. Non-limiting examples of the latter may include certain types of carcinoma, such as breast cancer.

Accordingly, the present disclosure, in some embodiments, provides selection criteria for identifying or selecting patients or patient populations/subpopulations for which a TGFβ1 inhibitor is likely to achieve clinical benefit. In some embodiments, preferred phenotypes of human tumors include: i) a subset has been shown to be responsive to CBT (e.g., PD-(L) system inhibition); ii) immune exclusion and/or iii) evidence of TGFB1 expression and/or TGFβ signaling. Various cancer types match the profile, including, for example, melanoma and bladder cancer.

As mentioned above, TGFβ inhibitors such as those described herein can be used in the treatment of melanoma. Types of melanoma that may be treated with such inhibitors include, but are not limited to, lentiginous malignancy, lentigo maligna, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, and nodular melanoma. Includes melanoma, polypoid melanoma, and desmoplastic melanoma. In some embodiments, the melanoma is metastatic melanoma. In some embodiments, the melanoma is a cutaneous melanoma.

More recently, immune checkpoint inhibitors have been used to effectively treat patients with advanced melanoma. In particular, anti-programmed death (PD)-1 antibodies (such as nivolumab and pembrolizumab) are now the standard of care for certain types of cancer, such as advanced melanoma, and have shown significant activity and efficacy with manageable toxicity profiles. Demonstrates sustained response. However, effective clinical application of PD-1 antagonists is hampered by a high rate of innate resistance (approximately 60-70%) (see Hugo et al., (2016) Cell 165:35-44). Perrot et al., (2013) Ann Dermatol 25 explains that ongoing challenges continue to include issues of patient selection and predictors of response and resistance as well as optimization of combination strategies (2):135-144). Additionally, studies suggest that approximately 25% of melanoma patients who initially respond to anti-PD-1 therapy eventually develop acquired resistance (Ribas et al., (2016) JAMA 315:1600 -9).

The number of tumor-infiltrating CD8+ T cells expressing PD-1 and/or CTLA-4 appears to be an important indicator of successful checkpoint inhibition, and both PD-1 and CTLA-4 inhibition can be increased. However, in patients with a higher presence of tumor-associated macrophages, the anti-cancer effect of CD8 cells may be suppressed.

LRRC33-expressing cells, such as myeloid cells including myeloid progenitors, MDSCs and TAMs, are exposed to immunosuppressive environments (TME and bone marrow) by inhibiting T cells such as CD4 and/or CD8 T cells (e.g., T cell depletion). fibrotic bone marrow, etc.), which may at least partially explain the observed anti-PD-1 resistance in certain patient populations. Indeed, evidence suggests that resistance to anti-PD-1 monotherapy is characterized by failure to accumulate CD8+ cytotoxic T cells and a reduced Teff/Treg ratio. In particular, we found that certain cancer patients, such as melanoma patient populations, are dichotomized with respect to LRRC33 expression levels (i.e., one group exhibits high LRRC33 expression (LRRC33 ^high ), whereas the other group The group was observed to exhibit relatively low LRRC33 expression (LRRC33 ^low ). Accordingly, the present disclosure includes the notion that the LRRC33 ^high patient population may represent those who are less responsive to or resistant to immune checkpoint inhibitor therapy. Therefore, agents that inhibit LRRC33, such as those described herein, are useful in cancers such as melanoma, lymphoma, and myeloproliferative disorders that are resistant to checkpoint inhibitor therapy (e.g., anti-PD-1). It can be particularly beneficial for treatment.

In some embodiments, the cancer/tumor is inherently resistant or refractory to immune checkpoint inhibitors (eg, primary resistance). While not intending to be bound by any particular theory, the inventors of the present disclosure believe that this is at least partially due to upregulation of the TGFβ1 signaling pathway, whereby checkpoint inhibitors are unable to exert their effects. We believe that an immunosuppressive microenvironment may be formed. TGFβ1 inhibition can make such cancers more responsive to checkpoint inhibitor therapy. Non-limiting examples of cancer types that can benefit from a combination of immune checkpoint inhibitors and TGFβ1 inhibitors include myelofibrosis, melanoma, renal cell carcinoma, bladder cancer, colon cancer, hematologic malignancies, Small cell carcinoma, non-small cell lung cancer/carcinoma (NSCLC), lymphoma (classical Hodgkin and non-Hodgkin), head and neck cancer, urothelial carcinoma (e.g., metastatic urothelial carcinoma), with high microsatellite instability Examples include cancer, cancer with mismatch repair function deficiency, gastric cancer, renal cancer, and hepatocellular carcinoma. However, any cancer in which TGFβ1 is overexpressed, coexpressed with TGFβ3, or is the dominant isoform for TGFβ2/3, as determined by biopsy, e.g. patients) can also be treated with TGFβ inhibitors according to the present disclosure.

In some embodiments, the cancer/tumor becomes resistant over time. This phenomenon is called acquired tolerance. Similar to primary resistance, in some embodiments acquired resistance is mediated at least in part by a TGFβ1-dependent pathway. The TGFβ inhibitors described herein may be effective in restoring anti-cancer immunity in these cases. TGFβ inhibitors of the present disclosure can be used to reduce tumor recurrence. The TGFβ inhibitors of the present disclosure can be used to increase the durability of cancer treatments such as CBT. The term "durability" as used in connection with treatment refers to the time between clinical effect (eg, tumor control) and tumor regrowth (eg, recurrence). Presumably, persistence and relapse are correlated with secondary or acquired resistance, where the treatment to which the patient originally responded no longer works. Accordingly, the TGFβ inhibitors of the present disclosure can be used to extend the period during which cancer treatment is effective. TGFβ inhibitors of the present disclosure can be used to reduce the probability of developing acquired resistance among responders to treatment. TGFβ inhibitors of the present disclosure can be used to prolong progression free survival in patients. In some embodiments, the TGFβ inhibitors described herein can be used to improve disease-free survival of patients. In some embodiments, the TGFβ inhibitors of the present disclosure improve patient-reported outcomes, reduce complications, reduce time to treatment completion, provide more sustained treatment, increase time to re-treatment, etc. It can be effective. In some embodiments, TGFβ inhibitors of the present disclosure can be used to improve overall survival in patients.

In some embodiments, the TGFβ inhibitors of the present disclosure can be used to improve the complete:partial response rate or ratio among responders to cancer treatment. Typically, CR rates are quite low, even for cancer types with relatively high response rates (eg, ≧35%) to cancer treatments (such as CBT). Therefore, the TGFβ inhibitors of the present disclosure are used to increase the proportion of complete responders within the responder population.

In addition, TGFβ inhibitors may also be effective in enhancing or increasing the degree of partial response among partial responders.

In some embodiments, the clinical endpoints of the TGFβ inhibitors described herein are as specified in the 2018 Food and Drug Administration Guidelines for Clinical Trial Endpoints for the Approval of Cancer. Including those listed in Drugs and Biologics, the contents of which are Incorporated herein in its entirety.

In some embodiments, combination therapies including immune checkpoint inhibitors and LRRC33 inhibitors (such as those described herein) may be used in conjunction with the methods disclosed herein, and such can be effective in treating cancer. Furthermore, high LRRC33-positive cell infiltration in tumors or sites/tissues with abnormal cell proliferation can serve as a biomarker of host immunosuppression and immune checkpoint resistance. Similarly, effector T cells can be excluded from immunosuppressive niches that limit the body's ability to fight cancer.

As disclosed in PCT/US Patent Application Publication No. 2019/041373, Tregs expressing GARP-presenting TGFβ1 suppress effector T cell proliferation. Collectively, TGFβ1 may be an important facilitator in the generation and maintenance of immune-inhibitory disease microenvironments (such as the TME), and multiple TGFβ1 presentation contexts are associated with tumors. In some embodiments, combination therapy may achieve a more favorable Teff/Treg ratio.

In some embodiments, an antibody that specifically binds to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex as described herein; The antigen-binding portion can be used in a method for treating cancer in a subject in need thereof, the method comprising administering the antibody or antigen-binding portion thereof to the subject, thereby treating the cancer. be treated. In certain embodiments, the cancer is colon cancer. In certain embodiments, the cancer is melanoma. In certain embodiments, the cancer is bladder cancer. In certain embodiments, the cancer is head and neck cancer. In certain embodiments, the cancer is lung cancer.

In some embodiments, an antibody that specifically binds to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex, as described herein. or antigen-binding portions thereof can be used in methods of treating solid tumors. In some embodiments, the solid tumor can be a desmoplastic tumor, which is typically dense and hard for penetration by therapeutic molecules. By targeting ECM components of such tumors, such antibodies can "loosen" and disrupt dense tumor tissue, facilitating therapeutic access to exert their anticancer effects. . Accordingly, additional therapeutic agents such as any known anti-tumor drug may be used in combination.

Additionally or alternatively, isoform-specific, context-independent antibodies or fragments thereof capable of inhibiting TGFβ1 activation, such as those disclosed herein, may be used in cancer immunotherapy, etc. to treat cancer. can be used in conjunction with chimeric antigen receptor T cell (“CAR-T”) technology as a cell-based immunotherapy.

In some embodiments, an antibody that specifically binds to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex, as described herein. or an antigen-binding portion thereof can be used in a method of inhibiting or reducing solid tumor growth in a subject having a solid tumor, the method comprising administering the antibody or antigen-binding portion thereof to the subject, thereby Inhibit or reduce solid tumor growth. In certain embodiments, the solid tumor is a colon cancer tumor. In some embodiments, antibodies or antigen-binding portions thereof useful for treating cancer are isoform-specific, context-independent inhibitors of TGFβ1 activation. In some embodiments, such antibodies target the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex. In some embodiments, such antibodies target the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, and the LTBP3-TGFβ1 complex. In some embodiments, such antibodies target the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex. In some embodiments, such antibodies target the GARP-TGFβ1 complex and the LRRC33-TGFβ1 complex.

The present disclosure includes the use of TGFβ inhibitors, such as context-independent, isoform-specific inhibitors of TGFβ1, in the treatment of cancer, including solid tumors in subjects. In some embodiments, such TGFβ inhibitors may inhibit activation of TGFβ1. In some embodiments, such TGFβ inhibitors include antibodies or antigen-binding portions thereof that bind to the proTGFβ1 complex. This binding occurs when the complex binds to any one of the presentation molecules, such as LTBP1, LTBP3, GARP or LRRC33, and may thereby inhibit the release of mature TGFβ1 growth factor from the complex. In some embodiments, the solid tumor is characterized by having a stroma enriched in CD8+ T cells that are in direct contact with CAFs and collagen fibers. Such tumors can create an immunosuppressive environment that prevents anti-tumor immune cells (eg, effector T cells) from effectively infiltrating the tumor and limits the body's ability to fight cancer. Instead, such cells may accumulate within or near the tumor stroma. These characteristics may render such tumors refractory to immune checkpoint inhibitor therapy. As discussed in more detail below, the TGFβ1 inhibitors disclosed herein can be used, for example, with immune checkpoint inhibitors to enable effector cells to reach and kill cancer cells. The suppression may be unblocked to allow.

TGFβ, particularly TGFβ1, is thought to play multifaceted roles in the tumor microenvironment, including tumor growth, host immunosuppression, malignant cell proliferation, vascularity, angiogenesis, migration, invasion, metastasis, and chemoresistance. Therefore, each “context” of TGFβ1 presentation in the environment may be involved in the regulation (or dysregulation) of disease progression. For example, the GARP system is particularly important in Treg responses that regulate effector T cell responses that mediate host immune responses to combat cancer cells. The LTBP1/3 system is thought to regulate the ECM, including the stroma, where cancer-associated fibroblasts (CAFs) play a role in cancer pathogenesis and progression. The LRRC33 system may play an important role in the recruitment of circulating monocytes to the tumor microenvironment, subsequent differentiation into tumor-associated macrophages (TAMs), infiltration into tumor tissues and disease progression.

In some embodiments, TGFβ1-expressing cells infiltrate the tumor and create or contribute to an immunosuppressive local environment. The degree to which such infiltration is observed may correlate with a worse prognosis. In some embodiments, higher infiltration indicates a poorer therapeutic response to another cancer therapy, such as an immune checkpoint inhibitor. In some embodiments, TGFβ1-expressing cells in the tumor microenvironment include immunosuppressive immune cells such as Tregs and/or myeloid cells. In some embodiments, myeloid cells include, but are not limited to, macrophages, monocytes (resident or bone marrow-derived tissue), and MDSCs.

In some embodiments, the LRRC33-expressing cells in the TME are myeloid-derived suppressor cells (MDSCs). MDSC infiltration (eg, solid tumor infiltration) may clearly represent at least one mechanism of immune escape by creating an immunosuppressive niche from which the host's anti-tumor immune cells are excluded. Evidence suggests that MDSCs are recruited by inflammation-related signals such as tumor-associated inflammatory factors, Opon recruitment, and that MDSCs exert immunosuppressive effects by impairing disease-fighting cells such as CD8+ T cells and NK cells. can have an impact on In addition, MDSCs can induce Treg differentiation by secreting TGFβ and IL-10, further increasing the immunosuppressive effect. Accordingly, TGFβ inhibitors, such as those described herein, may be administered to patients with immune escape (e.g., decreased immune surveillance) to restore or restore the body's ability to fight disease (such as cancer or tumors). Can be strengthened. As described in more detail herein, this may further enhance (eg, restore or improve) the body's responsiveness or sensitivity to another treatment, such as cancer treatment.

In some embodiments, an increase in the frequency (eg, number) of circulating MDSCs in a patient predicts decreased responsiveness to checkpoint blockade therapy, such as PD-1 antagonists and PD-L1 antagonists. For example, biomarker studies revealed that pre-treatment circulating HLA-DR ^lo /CD14+/CD11b+ myeloid-derived suppressor cells (MDSCs) were associated with progression and worse OS (p=0.0001 and 0.0009). In addition, resistance to PD-1 checkpoint inhibition in inflammatory head and neck cancers (HNC) is associated with the expression of GM-CSF and myeloid-derived suppressor cell (MDSC) markers. This observation suggested that strategies to deplete MDSCs, such as chemotherapy, should be considered in combination with anti-PD-1 (e.g., contemporaneously (e.g., simultaneously), individually or sequentially). . LRRC33 or the LRRC33-TGFβ complex represents a new target for cancer immunotherapy due to its selective expression on immunosuppressive myeloid cells. Therefore, without intending to be bound by any particular theory, targeting this complex may increase the effectiveness of standard-of-care checkpoint inhibitor therapy in patient populations.

Accordingly, the present disclosure provides the use of TGFβ inhibitors, such as the isoform-specific TGFβ1 inhibitors described herein, for the treatment of cancer, including solid tumors. Such treatment involves administering to a subject diagnosed with cancer, including at least one localized tumor (solid tumor), a TGFβ inhibitor encompassed by the present disclosure, such as Ab6, in an amount effective to treat the cancer. including administering. Preferably, the subject is further treated with cancer therapy such as CBT, chemotherapy and/or radiotherapy (such as a radiotherapeutic agent). In some embodiments, the TGFβ inhibitor increases the proportion/proportion of the patient population that are primary responders to cancer treatment. In some embodiments, the TGFβ inhibitor increases the degree of responsiveness of a primary responder to cancer treatment. In some embodiments, the TGF1 inhibitor increases the ratio of complete to partial responders to cancer treatment. In some embodiments, TGFβ inhibitors increase the durability of cancer treatment such that the period before recurrence and/or before cancer therapy becomes ineffective is extended. In some embodiments, the TGFβ inhibitor reduces the incidence or probability of acquired resistance to cancer treatment among primary responders.

In some embodiments, cancer progression (eg, tumor proliferation/growth, invasion, angiogenesis, and metastasis) can be driven at least in part by tumor-stromal interactions. In particular, CAFs may contribute to this process by secreting various cytokines and growth factors and ECM remodeling. Factors involved in this process include, but are not limited to, stromal cell-derived factor 1 (SCD-1), MMP2, MMP9, MMP3, MMP-13, TNF-α, TGFβ1, VEGF, IL-6, M - Includes CSF. Furthermore, CAFs can recruit TAMs by secreting factors such as CCL2/MCP-1 and SDF-1/CXCL12 to tumor sites; subsequently, the pro-TAM niche to which TAMs preferentially attach ( For example, interstitial regions rich in hyaluronic acid) are formed. Since TGFβ1 has been suggested to promote the activation of normal fibroblasts into myofibroblast-like CAFs, isoform-specific, context-independent TGFβ1 as described herein Administration of inhibitors can be effective in countering the cancer-promoting activity of CAF. Data disclosed in PCT/U.S. Patent Application Publication No. 2019/041373 show that isoform-specific context-independent antibodies that block activation of TGFβ1 are linked to CCL2/MCP, which is also involved in many cancers. -1, α-SMA, FN1 and Col1, suggesting that UUO-induced upregulation of marker genes such as α-SMA, FN1 and Col1 can be inhibited.

In certain embodiments, an antibody or its The antigen-binding portion is administered alone or in combination with an additional agent, such as an anti-PD-1 antibody (eg, an anti-PD-1 antagonist), to a subject having cancer or a tumor. Other combination therapies included in this disclosure are those in which the antibodies described herein, or antigen-binding portions thereof, are administered together with radiation (including radiotherapeutic agents, radiotherapy) or chemotherapeutic agents (chemotherapy). It is. Exemplary additional agents for use with anti-TGFβ inhibitors include, but are not limited to, PD-1 antagonists (e.g., PD-1 antibodies), PDL1 antagonists (e.g., PDL1 antibodies), PD-L1 or PDL2 fusions. protein, CTLA4 antagonist (e.g. CTLA4 antibody), GITR agonist (e.g. GITR antibody), anti-ICOS antibody, anti-ICOSL antibody, anti-B7H3 antibody, anti-B7H4 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-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-α4-β7 integrin and/or αE β7 integrin), CDK inhibition agents, oncolytic viruses, indoleamine 2,3-dioxygenase (IDO) inhibitors, and/or PARP inhibitors. Examples of useful oncolytic viruses include adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus, Seneca virus, enterovirus, and vaccinia. In certain embodiments, oncolytic viruses are engineered for tumor selectivity.

In some embodiments, determining or selecting a treatment approach for a combination therapy that is appropriate for a particular cancer type or patient population may include: a) consideration of cancer types for which standard treatments are available (e.g. b) considerations regarding treatment-resistant subpopulations (e.g., immune exclusion); and c) are “TGFβ1 pathway active” or are otherwise at least partially TGFβ1-dependent (e.g., Considerations regarding cancers/tumors that are (or are generally suspected to be) sensitive to TGFβ1 inhibition. For example, many cancer samples show that TGFβ1 is the predominant isoform, eg, by TCGA RNAseq. In some embodiments, DNA and/or RNA-based assays (e.g., RNAseq or Nanostring) can be used to assess the level of TGFβ signaling (e.g., TGFβ1 signaling) in a tumor sample. In some embodiments, more than 50% (eg, 50%, 60%, 70%, 80% and 90%) of samples from each tumor type are positive for TGFβ1 isoform expression. In some embodiments, cancers/tumors that are "TGFβ1 pathway active" or otherwise at least partially TGFβ1 dependent (e.g., sensitive to TGFβ1 inhibition) include K-ras, N-ras and and/or at least one Ras mutation, such as a H-ras mutation. In some embodiments, the cancer/tumor comprises at least one K-ras mutation.

Confirmation of TGFβ1 expression in clinical samples obtained from patients (e.g., biopsy samples) is not a prerequisite for TGFβ1 inhibition therapy, in which specific conditions are generally known to involve the TGFβ pathway. or suspected.

In some embodiments, TGFβ inhibitors, such as those described herein, have been diagnosed with a cancer for which one or more checkpoint inhibitor therapies have been approved or proven effective. It is administered along with checkpoint blockade therapy to patients who have These cancers include, but are not limited to, bladder urothelial carcinoma (such as metastatic urothelial carcinoma), squamous cell carcinoma (such as head and neck), kidney clear cell carcinoma, renal papillary cell carcinoma, liver hepatocellular carcinoma, and lung glandular carcinoma. Includes cancer, cutaneous melanoma, and gastric adenocarcinoma. In certain embodiments, such patients are poorly responsive or refractory to checkpoint inhibitor therapy. In some embodiments, this hyporesponsiveness is due to primary resistance. In some embodiments, cancers that are resistant to checkpoint inhibition exhibit downregulation of TCF7 expression. In some embodiments, TCF7 downregulation in checkpoint inhibition resistant tumors may correlate with low numbers of intratumoral CD8+ T cells.

TGFβ inhibitors, such as those described herein, can be used to treat chemotherapy- or radiotherapy-resistant cancers. Accordingly, in some embodiments, a TGFβ1 inhibitor, e.g., Ab6, is administered to a patient diagnosed with cancer who is receiving or has received chemotherapy and/or radiation therapy (such as a radiotherapeutic agent). can do. In particular, the use of TGFβ1 inhibitors is advantageous when the cancer (patient) is resistant to such treatment. In some embodiments, such cancers contain quiescent tumor-proliferating cancer cells (TPCs), and TGFβ signaling controls their reversible entry into a growth-arrested state, which is inhibited by chemotherapy or Protect TPC from radiation therapy (such as radiotherapeutic drugs). It is contemplated that pharmacological inhibition of TGFβ1 will result in impaired TPCs being unable to enter a quiescent state and thus becoming susceptible to chemotherapy and/or radiotherapy (such as radiotherapeutic agents). Such cancers include various carcinomas, such as squamous cell carcinoma. For example, Brown et al. , (2017) “TGF-β-Induced Quiescence Mediates Chemoresistance of Tumor-Propagating Cells in Squamous Cell Carcinoma.”Cell Ste. m Cell. 21(5):650-664.

In some embodiments, a TGFβ inhibitor, such as an isoform-selective TGFβ1 inhibitor (eg, Ab6), can be used to treat (eg, alleviate) anemia in a subject, eg, a cancer patient. In some embodiments, a TGFβ inhibitor (e.g., Ab6), such as an isoform-selective TGFβ1 inhibitor, is combined with a BMP inhibitor (e.g., a BMP6 inhibitor, e.g., an RGMc inhibitor, the entire contents of which are herein Any of the RGMc inhibitors disclosed in incorporated WO 2020/086736) can be used, for example, to treat (eg, alleviate) anemia in a subject. In some embodiments, the anemia is reduced or impaired red blood cell production (e.g., as a result of myelofibrosis or cancer), iron limitation (e.g., as a result of cancer or treatment-induced anemia, such as chemotherapy-induced anemia). ) or both. In some embodiments, the combination of a TGFβ inhibitor and a BMP inhibitor (antagonist) has sufficient therapeutic efficacy to alleviate one or more anemia-related symptoms and/or complications in a subject, e.g., a cancer patient. It may be administered in any quantity or amount. In some embodiments, a combination of a TGFβ inhibitor and a BMP inhibitor (antagonist) can be administered in a therapeutically effective amount sufficient to increase or normalize red blood cell production and/or reduce iron limitation. Without wishing to be bound by theory, TGFβ1 inhibitors (e.g., Ab6) may alleviate symptoms and/or complications associated with anemia through their pro-hematopoietic effects, and BMP inhibitors It is believed that (antagonists) (eg, BMP6 inhibitors, eg, RGMc inhibitors) may ameliorate iron deficiency anemia (eg, chemotherapy-induced anemia). In some embodiments, treating anemia further comprises administering one or more JAK inhibitors (eg, a Jak1/2 inhibitor, a Jak1 inhibitor, and/or a Jak2 inhibitor).

In some embodiments, the BMP inhibitor is an antagonist of a kinase associated with a BMP receptor (eg, a type I receptor and/or a type II receptor).

In some embodiments, the BMP inhibitor is a "ligand trap" that binds to (or sequesters) BMP growth factors, such as BMP6.

In some embodiments, the BMP inhibitor is an antibody that neutralizes a BMP growth factor, such as BMP6. Examples include anti-BMP6 antibodies (eg, WO 2016/098079, Novartis; and KY-1070, KyMab).

In some embodiments, the BMP inhibitor is an inhibitor of a BMP6 co-receptor, such as RGMc. For example, such inhibitors can include antibodies that bind RGMa/c (Boeser et al. AAPS J. 2015 Jul; 17(4):930-938). More preferably, such an inhibitor is an antibody that selectively binds to RGMc (see, eg, WO 2020/086736).

Therapeutic Indications and/or Subjects That May Benefit from Therapy Including TGFβ Inhibitors The present disclosure encompasses methods of treating cancer and predicting or monitoring treatment efficacy using TGFβ inhibitors, such as Ab6. do. In some embodiments, the identification/screening/selection of suitable indications and/or patient populations for which TGFβ inhibitors, such as those described herein, are likely to have advantageous therapeutic benefit. , i) whether the disease is driven by or primarily dependent (or at least co-dominant) on the TGFβ1 isoform compared to other isoforms in humans; ii) the condition ( iii) whether the disease involves both matrix-associated and cell-associated TGFβ1 function.

Differential expression of the three known TGFβ isoforms, TGFβ1, TGFβ2 and TGFβ3, has been observed in various tissues under normal (healthy; homeostatic) and disease conditions. Nevertheless, in traditional approaches supporting pan-inhibition of TGFβ across multiple isoforms, the concept of isoform selectivity has not been fully exploited or reliably achieved. Furthermore, the expression patterns of isoforms can be differentially regulated not only in normal (homeostatic) and abnormal (pathological) conditions, but also in different patient subpopulations. Most preclinical studies are conducted in a limited number of animal models, which may or may not mimic the human condition, so data obtained using such models can be biased. This can lead to erroneous interpretations of the data or erroneous conclusions regarding their translatability for therapeutic drug development.

Previous analyzes of human tumor samples have implicated TGFβ signaling as an important contributor to disease progression and primary resistance to treatment response, including checkpoint blockade therapy (“CBT”) for various types of malignancies. It was said that Studies reported in the literature have found that TGFB gene expression may be particularly relevant to treatment resistance, as the activity of this isoform may inhibit TGFβ signaling in these diseases. This suggests that it may be driven. As detailed in PCT/U.S. Patent Application Publication No. 2019/041373, TGFB1 expression appears to be most prevalent in the majority of human tumor types profiled in The Cancer Genome Atlas (TCGA); This suggests that selection of preclinical models that more faithfully reproduce human disease expression patterns of TGFβ isoforms may be beneficial.

Without wishing to be bound by theory, TGFβ1 and TGFβ3 are often co-dominant in certain murine syngeneic cancer models (e.g., EMT-6 and 4T1) that are widely used in preclinical studies ( (see, eg, FIG. 21B of PCT/US Patent Application Publication No. 2019/041373). In contrast, numerous other cancer models (e.g. S91, B16 and MBT-2) are observed in many human tumors in which TGFβ1 appears to be the predominant isoform more frequently than TGFβ2/3. (See Figures 20 and 21A of PCT/US Patent Application Publication No. 2019/041373). Furthermore, the TGFβ isoforms that are predominantly expressed under homeostatic conditions may not be disease-associated isoforms. For example, in normal lung tissue of healthy rats, sustained TGFβ signaling appears to be primarily mediated by TGFβ3. However, TGFβ1 appears to be significantly upregulated in disease states such as pulmonary fibrosis. Overall, although not a prerequisite, it may be beneficial to test or confirm the relative expression of TGFβ isoforms in clinical samples in order to select suitable therapeutic agents to which a patient is likely to respond. In some embodiments, determination of relative isoform expression may be performed post-treatment. Under such circumstances, a patient's responsiveness (eg, clinical response/benefit) to TGFβ1 inhibitory therapy may be correlated with the relative expression levels of TGFβ isoforms. In some embodiments, subsequent overexpression of TGFβ1 isoforms correlates with greater responsiveness to treatment.

Although inhibition of TGFβ1 alone appears to be sufficient to resolve primary resistance to cancer immunotherapy, as demonstrated in tumor models expressing both TGFβ1 and TGFβ3, the findings disclosed herein suggest that inhibition of TGFβ3 may actually be harmful. Surprisingly, in a murine liver fibrosis model, mice treated with isoform-selective inhibitors of TGFβ3 develop worsening of fibrosis. The significant increase in collagen deposition in liver sections of these animals suggests that inhibition of TGFβ3 may indeed result in greater dysregulation of the ECM. Without wishing to be bound by theory, this suggests that TGFβ3 inhibition may promote a profibrotic phenotype.

A hallmark of the profibrotic phenotype is increased collagen deposition and/or accumulation in the ECM, which is associated with increased stiffness of tissue ECM. This has been observed during the pathological progression of cancer, fibrosis and cardiovascular diseases. Consistent with this, Applicants have used primary fibroblasts grown on silicone-based substrates with defined stiffness (e.g. 5 kPa, 15 kPa or 100 kPa) to stimulate the integrin-dependent activation of TGFβ. We have previously demonstrated the role of matrix hardness (see WO 2018/129329 pamphlet). Matrices with greater stiffness enhanced TGFβ1 activation, which was suppressed by isoform-specific inhibitors of TGFβ1. These observations indicate that pharmacological inhibition of TGFβ3 may antagonize TGFβ1 inhibition by creating a tumor-promoting microenvironment where greater stiffness of the tissue matrix may support cancer progression. It suggests that.

Given the common pathways involved in the fibrotic phenotype and the many aspects of cancer progression such as increased invasiveness and metastasis (e.g. Chakravarthy et al., Nat Com (2018) 9:4692. “TGF-β -associated extracellular matrix genes link cancer-associated fibroblasts to immune evasion and immunotherapy failure) , the profibrotic effects of TGFβ3 inhibition observed in fibrosis models may be applicable to the cancer context.

Therefore, the aforementioned findings raise the possibility that TGFβ inhibitors with inhibitory potency against TGFβ3 are not only ineffective in the treatment of cancer, but may actually be harmful. In some embodiments, TGFβ3 inhibition is avoided in patients suffering from statistically highly metastatic cancer types. Cancer types that are typically considered highly metastatic include, but are not limited to, colorectal cancer, lung cancer, bladder cancer, kidney cancer (e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC) (clear cell RCC). , papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC), uterine cancer, prostate cancer, gastric cancer, and thyroid cancer. Additionally, TGFβ3 inhibition may be associated with fibrotic conditions and/or cardiovascular It is best avoided in patients who have or are at risk of developing the disease. Such patients who are at risk of developing fibrotic conditions and/or cardiovascular disease include metabolic disorders such as NAFLD and NASH, obesity, and Includes, but is not limited to, patients with type 2 diabetes. Similarly, TGFβ3 inhibition may be best avoided in patients diagnosed with or at risk of developing myelofibrosis. Patients at risk of developing myelofibrosis include those with one or more genetic mutations involved in the pathogenesis of myelofibrosis.

In addition to the concerns about potential inhibition of TGFβ3 mentioned above, Takahashi et al. (Nat Metab. 2019, 1(2):291-303) recently reported the beneficial role of TGFβ2 in regulating metabolism. The authors identified TGFβ2 as an exercise-induced adipokine, which stimulated glucose and fatty acid uptake in vitro and tissue glucose uptake in vivo. However, it improved the metabolism of obese mice; it also reduced high-fat diet-induced inflammation. Furthermore, the authors found that lactate, a metabolite released from muscle during exercise, stimulated TGFβ2 expression in human adipocytes and that lactate-lowering agents reduced circulating TGFβ2 levels, reducing exercise-stimulated glucose tolerance. It was observed that the improvement in Accordingly, in some embodiments, TGFβ inhibitors are used to treat a subject that lacks inhibitory activity against the TGFβ2 isoform, e.g., potentially deleterious effects on one or more metabolic functions of the subject being treated. can be avoided.

More recently, the potential link between cancer and various metabolic conditions has been recognized. For example, Braun et al. An increased risk of cancer mortality is associated with metabolic syndrome in men, as reviewed by (Braun et al. Int J Biol Sci. 2011;7(7):1003-1015). Similarly, the authors conclude that "metabolic dysregulation plays an important role in the pathogenesis and progression of certain cancer types and can worsen the outcome of some cancers. Obesity and diabetes mellitus are associated with breast cancer, intrauterine [Braun et al. Int J Biol Sci. 2011;7(7):1003-1015].

Thus, in various embodiments, a TGFβ inhibitor can be used to treat a TGFβ-related indication (e.g., cancer) in a subject, where the TGFβ inhibitor inhibits TGFβ1 at a therapeutically effective dose administered. , TGFβ2 is not inhibited. In some embodiments, the subject benefits from improved metabolism following such treatment, in which case the subject optionally suffers from a metabolic disease, such as obesity, high-fat diet-induced inflammation and glucose regulation. have or are at risk of developing a disorder (eg, diabetes). In some embodiments, the TGFβ-related indication is cancer, in which case the cancer optionally includes solid tumors, such as locally advanced cancers and metastatic cancers.

In some embodiments, the TGFβ inhibitor is TGFβ1 selective (eg, does not inhibit TGFβ2 and/or TGFβ3 signaling at therapeutically effective doses). In certain embodiments, TGFβ1 selective inhibitors are selected for use in treating cancer patients. In some embodiments, such treatment i) avoids TGFβ3 inhibition to reduce the risk of exacerbating ECM dysregulation (which may contribute to tumor growth and invasiveness) and ii) avoids TGFβ2 inhibition. to reduce the risk of increasing the patient's metabolic load. Related methods for selecting TGFβ inhibitors for therapeutic use are also encompassed herein.

The present disclosure includes methods for selecting TGFβ inhibitors for use in the treatment of cancer, wherein the TGFβ inhibitors have no or little inhibitory efficacy against TGFβ3 (e.g., the TGFβ inhibitors inhibit TGFβ3 (not targeted). In certain embodiments, the TGFβ inhibitor is a TGFβ1 selective inhibitor (eg, an antibody or antigen-binding fragment that does not inhibit TGFβ2 and/or TGFβ3 signaling at a therapeutically effective dose). This selection strategy is contemplated to provide the benefits of TGFβ1 inhibition to treat cancer while reducing the risk of exacerbating ECM dysregulation in cancer patients. In some embodiments, cancer patients also receive treatment with cancer therapies such as immune checkpoint inhibitors. In some embodiments, cancer patients are at risk of developing metabolic diseases, such as fatty liver, obesity, high fat diet-induced inflammation, and glucose or insulin dysregulation (eg, diabetes).

The present disclosure also includes related methods for selecting and/or treating appropriate patient populations who may be candidates to receive TGFβ inhibitors capable of inhibiting TGFβ3. Such methods may be used to treat subjects who have not been diagnosed with a fibrotic disorder (such as organ fibrosis), who have not been diagnosed with myelofibrosis, who have not been diagnosed with cardiovascular disease, and/or who have such conditions. Includes the use of TGFβ inhibitors capable of inhibiting TGFβ3 for the treatment of cancer in subjects who are not at risk of developing it. Similarly, such methods include the use of TGFβ inhibitors capable of inhibiting TGFβ3 for the treatment of cancer in a subject, where the cancer is not considered highly metastatic. TGFβ inhibitors capable of inhibiting TGFβ3 include pan-inhibitors of TGFβ (e.g., low molecular weight antagonists of TGFβ receptors, such as ALK5 inhibitors and neutralizing antibodies that bind to TGFβ1/2/3), Isoform non-selective inhibitors such as antibodies that bind and engineered fusion proteins capable of binding TGFβ1/3, such as ligand traps and integrin inhibitors (e.g., αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, Antibodies that bind to αIIbβ3 or α8β1 integrin and inhibit downstream activation of TGFβ, such as selective inhibition of TGFβ1 and/or TGFβ3, may be included.

The surprising finding that TGFβ3 inhibition can actually be disease-promoting is that patients who have previously received or are currently being treated with TGFβ inhibitors that have inhibitory activity against TGFβ3 may be treated with TGFβ3 inhibitors. suggest that they may benefit from additional treatment with TGFβ1 selective inhibitors to counteract the possible profibrotic effects of TGFβ1. Accordingly, the present disclosure includes TGFβ1 selective inhibitors for use in treating cancer in a subject, wherein the subject has been treated with a TGFβ inhibitor that inhibits TGFβ3 in conjunction with a checkpoint inhibitor. and the method comprises administering to said subject a TGFβ1 selective inhibitor, optionally, the cancer is metastatic cancer, desmoplastic tumor, myelofibrosis, and/or the subject has The subject has or is at risk of developing a fibrotic disorder and/or cardiovascular disease, and optionally the subject at risk of developing a fibrotic disorder or cardiovascular disease has a metabolic disease and is optionally at risk of developing a fibrotic disorder and/or cardiovascular disease. Optionally, the metabolic disease is NAFLD, NASH, obesity or diabetes.

As described herein, isoform-selective TGFβ1 inhibitors are particularly advantageous for treating diseases in which TGFβ1 isoforms are predominantly expressed over other isoforms (e.g., referred to as TGFβ1-dominant). be. As an example, a non-limiting list of human cancer clinical samples with relative expression levels of TGFB1 (left), TGFB2 (middle) and TGFB3 (right) is provided in PCT/U.S. Patent Application No. 2019/041373, for example It was described in FIG. 20 and FIG. 21A. Each horizontal line across the three isoforms represents one patient. As can be seen, global TGFβ1 expression (TGFB1) is significantly higher in most of these human tumors/cancers than the other two isoforms in many tumor/cancer types, suggesting that TGFβ1 selective inhibition is suggesting that it may be beneficial in these disease types. Taken together, these lines of evidence support the view that selective inhibition of TGFβ1 activity may resolve primary resistance to CBT. This is important because the generation of highly selective TGFβ1 inhibitors will also allow evaluation of whether such approaches address the major safety issues observed with pan-TGFβ inhibition. This will be important for evaluating usefulness.

It was previously thought that TGFβ1 inhibitors may not be effective, particularly in cancer types where TGFβ1 is co-dominant with another isoform or where TGFβ2 and/or TGFβ3 expression is significantly higher than TGFβ1. It was getting worse. However, more recently, the inventors of the present application have discovered that TGFβ1 inhibitors, such as TGFβ1 selective inhibitors (e.g., Ab6), are combined with checkpoint inhibitors (e.g., anti-PD-1 antibodies). We made the unexpected discovery that when used together, both TGFβ1 and TGFβ3 can cause significant tumor regression in the EMT-6 model, which is known to express similar levels. Codominance has been confirmed by both RNA measurements and ELISA assays (see Figure 35 of PCT/US Patent Application Publication No. 2019/041373). This observation suggests that in the context of checkpoint inhibition, both co-dominant isoforms must be specifically inhibited to achieve the requisite effect in tumors co-expressing TGFβ1 and TGFβ3. This was surprising since it had been assumed. Accordingly, the treatment methods disclosed herein include the use of a TGFβ1 inhibitor to promote tumor regression, where the tumor is TGFβ1+/TGFβ3+. Such tumors include, for example, cancers of epithelial origin, ie, carcinomas (e.g., basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, and adenocarcinoma). may be included. In some embodiments, TGFβ1 is primarily a disease-associated isoform, whereas TGFβ3 supports homeostatic functions in tissues such as epithelia.

Aberrant activity of the TGFβ signaling pathway has been reported to affect gene expression involved in both fibrosis and cancer processes. For example, dysregulation of the TGFβ1 signaling pathway has been found to alter genes such as SNAI1, MMP2, MMP9, and TIMP1, all of which are important for cellular processes such as adhesion and extracellular matrix remodeling. It is involved in fibrosis and epithelial-to-mesenchymal transition (EMT) processes in cancer. Thus, in some embodiments, the methods of treatment described herein, e.g., for fibrosis-related cancer indications, include the administration of a TGFβ inhibitor that does not inhibit TGFβ3, e.g., the use of a TGFβ1 selective antibody, e.g., Ab6. including. Certain tumors, such as various carcinomas, are characterized by low mutational burden tumors (MBTs). Such tumors are often poorly immunogenic and are unable to elicit sufficient T cell responses. Cancer therapies, including chemotherapy, radiotherapy (such as radiotherapeutics), cancer vaccines, and/or oncolytic viruses, may be useful in inducing T cell immunity in such tumors. Accordingly, the TGFβ1 inhibition therapies detailed herein can be used in conjunction with one or more of these cancer therapies to enhance anti-tumor efficacy. Essentially, such combination therapy targets "hypoimmunogenic" tumors (e.g., tumors with low immunogenicity) by promoting neoantigens to make it easier for effector cells to attack the tumor. The aim is to transform the tumor into a "highly immunogenic" tumor. Examples of such tumors include breast cancer, ovarian cancer, and pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC). Accordingly, any one or more of the antibodies or fragments thereof described herein may be used to sensitize low-immunogenic tumors (e.g., "immune exclusion") with cancer therapy aimed at promoting T-cell immunity. tumors) can be treated.

In the case of immunocompromised tumors, where effector T cells are kept away from the tumor site (and thus "eliminated"), the immunosuppressive tumor environment can be mediated in a TGFβ1-dependent manner. These are tumors that are typically immunogenic; however, T cells are unable to adequately infiltrate, proliferate, and elicit cytotoxic effects due to the immunosuppressed environment. Typically, such tumors are poorly responsive to cancer treatments such as CBT. As the data provided herein and in PCT/U.S. Patent Application Publication No. 2019/041373 suggest, adjuvant therapy including TGFβ1 inhibitors can overcome the immunosuppressive phenotype and It enables T cell infiltration, proliferation, and anti-tumor function, thereby making such tumors more responsive to cancer therapies such as CBT.

A second study is therefore directed to the identification or selection of patients with immunosuppressive tumors who may benefit from TGFβ inhibitor therapy, eg, TGFβ1 inhibitors such as Ab6. The degree of presence or frequency of effector T cells in a tumor is indicative of anti-tumor immunity. Therefore, detecting anti-tumor cells such as CD8+ cells within a tumor provides useful information in assessing whether a patient may benefit from CBT and/or TGFβ1 inhibitor therapy.

Detection can be performed by known methods such as immunohistochemical analysis of tumor biopsy samples, including digital pathology methods. More recently, non-invasive imaging methods have been developed that allow the detection of cells of interest (eg, cytotoxic T cells) in vivo. For example, http://www. imagineab. com/technology/; Tavare et al. , (2014) PNAS, 111(3):1108-1113; Tavare et al. , (2015) J Nucl Med 56(8):1258-1264; Rashidian et al. , (2017) J Exp Med 214(8):2243-2255; Beckford Vera et al. , (2018) PLoS ONE 13(3):e0193832; and Tavare et al. , (2015) Cancer Res 76(1):73-82, each of which is incorporated herein by reference. Typically, antibodies or antibody-like molecules engineered with detection moieties (e.g., radioactive labels) can be injected into the patient, and these are subsequently distributed and localized to the site of specific markers (e.g., CD8+). become present. In this way, it can be determined whether a tumor has an immune exclusion phenotype. If a tumor is determined to have an immune exclusion phenotype, cancer therapy (such as CBT) alone may not be effective because the tumor lacks sufficient cytotoxic cells within the tumor environment. Add-on therapy with TGFβ inhibitors as described herein can reduce immunosuppression, thereby making cancer therapy-resistant tumors more responsive to cancer therapy.

Non-invasive in vivo imaging techniques can be used to diagnose patients; select or identify patients who may benefit from TGFβ inhibitor therapy, e.g., TGFβ inhibitor therapy; and/or monitor patients for therapeutic response during treatment. It can be applied to various suitable methods for this purpose. Any cell with a known cell surface marker can be detected/localized by using antibodies or similar molecules that specifically bind to the cell marker. Typically, the cells to be detected using such techniques include immune cells, such as cytotoxic T lymphocytes, regulatory T cells, MDSCs, tumor-associated macrophages, NK cells, dendritic cells, and It is a medium ball. Antibodies or engineered antibody-like molecules that recognize such markers can be coupled to detection moieties.

Non-limiting examples of suitable immune cell markers include monocyte markers, macrophage markers (e.g. M1 and/or M2 macrophage markers), CTL markers, suppressive immune cell markers, MDSC markers (e.g. G- and/or or M-MDSC markers) including, but not limited to, CD8, CD3, CD4, CD11b, CD33, CD163, CD206, CD68, CD14, CD15, CD66b, CD34, CD25 and CD47. In some embodiments, in vivo imaging involves tracking T cells, such as cytotoxic CD8-positive T cells. Accordingly, any one of the TGFβ inhibitors of the present disclosure can be used to treat cancer in a subject having a solid tumor, the treatment comprising: i) performing an in vivo imaging analysis to detect T cells in the subject; (optionally, the T cells are CD8+ T cells) and if the solid tumor is determined to be an immunocompromised solid tumor based on the in vivo imaging analysis of (i), then administering a therapeutically effective amount of and administering to the subject a TGFβ inhibitor, such as Ab6. In some embodiments, the subject is undergoing CBT, in which case the solid tumor is optionally resistant to CBT. In some embodiments, the subject is administered CBT along with a TGFβ1 inhibitor as a combination therapy. This combination may involve administration of a single formulation containing both a checkpoint inhibitor and a TGFβ inhibitor. TGFβ inhibitors include TGFβ1 selective inhibitors such as Ab6, isoform non-selective inhibitors such as low molecular weight ALK5 antagonists, neutralizing antibodies that bind to two or more of TGFβ1/2/3 such as GC1008 and mutants, Antibodies that bind to TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors and/or integrin inhibitors (e.g., bind to αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 or α8β1 integrins and inhibit the downstream activities of TGFβ It may be a TGFβ1 inhibitor, such as an antibody that inhibits TGFβ1 and/or TGFβ3 selective inhibition. Alternatively, combination therapy may involve administration of a first formulation comprising a checkpoint inhibitor and a second formulation comprising a TGFβ inhibitor, where the TGFβ inhibitor is a TGFβ1 selective inhibitor, such as Ab6, Form non-selective inhibitors, such as low molecular weight ALK5 antagonists, neutralizing antibodies that bind to more than one of TGFβ1/2/3, such as GC1008 and variants, antibodies that bind to TGFβ1/3, ligand traps, such as 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 for TGFβ1 and/or TGFβ3) Inhibition).

In some embodiments, in vivo imaging includes tracking MDSCs, such as G-MDSCs and M-MDSCs. For example, MDSCs may be enriched at disease sites (such as fibrotic tissues and solid tumors) at baseline. Upon treatment (eg, TGFβ1 inhibitor therapy), a decrease in MDSCs can be observed, as measured by a decrease in the intensity of labels (such as radioisotopes and fluorescence), which is indicative of therapeutic efficacy.

In some embodiments, in vivo imaging comprises tracking or localizing LRRC33 positive cells. LRRC33-positive cells include, for example, MDSCs and activated M2-like macrophages (eg, TAMs and activated macrophages associated with fibrotic tissue). For example, LRRC33-positive cells may be enriched in diseased sites (such as fibrotic tissue and solid tumors) at baseline. Upon treatment (e.g., TGFβ1 inhibitor therapy), a decrease in cells expressing cell surface LRRC33 can be observed, as measured by a decrease in the intensity of the label (such as radioisotopes and fluorescence), which may interfere with the therapeutic effect. It shows.

In some embodiments, in vivo imaging includes PET-SPECT, Including the use of MRI and/or fluorescence/bioluminescence.

In some embodiments, labeling the antibody or antibody-like molecule with a detection moiety can include a direct label or an indirect label.

In some embodiments, the detection moiety can be a tracer. In some embodiments, the tracer can be a radioisotope, in which case the radioisotope can optionally be a positron emitting isotope. In some embodiments, the radioisotope is selected from the group consisting of ¹⁸ F, ¹¹ C, ¹³ N, ¹⁵ O, ⁶⁸ Ga, ¹⁷⁷ Lu, ¹⁸ F, and ⁸⁹ Zr.

Thus, using such methods, in vivo imaging using labeled antibodies in immuno-PET can be performed.

In some embodiments, such in vivo imaging is performed to monitor therapeutic response to TGFβ1 inhibition therapy in the subject. For example, a therapeutic response may include the transformation of an immunocompromised tumor to an inflammatory tumor, which correlates with increased immune cell infiltration into the tumor. This can be visualized by an increase in the frequency of intratumoral immune cells or the detectability of signals (such as radiolabels and fluorescence).

Accordingly, the present disclosure includes a method of treating cancer, the method comprising: i) selecting a patient diagnosed with a cancer comprising a solid tumor, the solid tumor being an immunocompromised tumor; ii) administering to the patient an amount of an antibody or fragment thereof, as encompassed herein, effective to treat the cancer. In some embodiments, the patient has undergone cancer therapy, such as immune checkpoint inhibition therapy (e.g., PD-(L)1 antibodies), chemotherapy, radiation therapy, engineered immune cell therapy, and cancer vaccine therapy. or are candidates for such therapy. In some embodiments, the selection step (i) comprises detection of the immune cell or one or more markers thereof, in which case the detection optionally includes tumor biopsy analysis, serum marker analysis, and/or Including in vivo imaging.

In some embodiments, the patient is diagnosed with a cancer for which CBT has been approved, in which case, optionally, statistically similar patient populations with the particular cancer are In contrast, the response rate is relatively low, eg, less than 25%. For example, the response rate for CBT can be about 10-25%, such as about 10-15%. Examples of such cancers include ovarian cancer, gastric cancer, and triple negative breast cancer. The TGFβ inhibitors of the present disclosure can be used to treat such cancers, in which case the subject has not yet received CBT. A TGFβ1 inhibitor may be administered to a subject in combination with CBT. In some embodiments, the subject is receiving or has received additional cancer therapy, such as chemotherapy and radiation therapy (including radiotherapeutic agents).

The in vivo imaging techniques described above can be used to detect, localize, and/or track specific MDSCs in patients diagnosed with TGFβ-related diseases, such as cancer. Healthy individuals have no or infrequent MDSCs in their circulation. With the onset or progression of such diseases, increased levels of circulating and/or disease-localized MDSCs can be detected. For example, CCR2-positive M-MDSCs have been reported to accumulate in tissues associated with inflammation and may cause the progression of tissue fibrosis (such as pulmonary fibrosis), which is correlated with TGFβ1 expression. has been revealed. Similarly, MDSCs are enriched in several solid tumors, including triple-negative breast cancer, and contribute in part to the immunosuppressive phenotype of the TME. Accordingly, in accordance with the present disclosure, therapeutic responses to TGFβ inhibition, such as TGFβ1 inhibition, can be monitored by localizing or tracking circulating MDSCs. A reduced level or lower frequency of circulating MDSCs is typically indicative of therapeutic benefit or better prognosis. Accordingly, the present disclosure predicts and monitors the therapeutic efficacy of TGFβ inhibitor therapy, such as combination therapy with a TGFβ1 inhibitor and checkpoint inhibitor, by measuring circulating MDSCs in the blood or blood components of a subject. provide a method. The present disclosure also provides methods for selecting a patient, eg, a patient with an immunosuppressive cancer, and determining a treatment regimen based on measured levels of circulating MDSC. TGFβ inhibitors include TGFβ1 selective inhibitors, such as Ab6, isoform non-selective inhibitors, such as low molecular weight ALK5 antagonists, neutralizing antibodies that bind to two or more of TGFβ1/2/3, such as GC1008 or variants, Antibodies that bind to TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors and/or integrin inhibitors (e.g., αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 or α8β1 integrin, downstream of TGFβ It may be a TGFβ1 inhibitor, such as an antibody that inhibits activation, such as selective inhibition of TGFβ1 and/or TGFβ3.

The TGFβ inhibitors of the present disclosure can be used to treat cancer in a subject, where the cancer is characterized by immunosuppression, where the cancer optionally comprises a solid tumor that is TGFβ1 positive and TGFβ3 positive. Such a subject may be diagnosed as having carcinoma. In some embodiments, the carcinoma is a breast cancer, in which case the breast cancer is optionally a triple negative breast cancer (TNBC). Such treatments further include cancer therapies including, but not limited to, chemotherapy, radiotherapy, cancer vaccines, engineered immune cell therapies (such as CAR-T), and immune checkpoint inhibition therapies such as anti-PD(L)-1 antibodies. be able to. TGFβ inhibitors include TGFβ1 selective inhibitors, such as Ab6, isoform non-selective inhibitors, such as low molecular weight ALK5 antagonists, neutralizing antibodies that bind to two or more of TGFβ1/2/3, such as GC1008 or variants, Antibodies that bind to TGFβ1/3, ligand traps, such as TGFβ1/3 inhibitors and/or integrin inhibitors (e.g., αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 or α8β1 integrin, downstream of TGFβ It may be a TGFβ1 inhibitor, such as an antibody that inhibits activation, such as selective inhibition of TGFβ1 and/or TGFβ3.

In some embodiments, a hypoimmunogenic tumor is known to be a type of cancer characterized by few effector cells or low immunogenicity both inside and outside the tumor. (e.g., tumors characterized as immune deserts) are identified. Subjects/patients with such tumors may be treated with chemotherapy, radiotherapy (such as radiotherapeutics), oncolytic virotherapy and cancer therapy to induce stronger T cell responses against tumor antigens (e.g. neoantigens). Treated with immunosensitizing cancer therapies such as vaccines. This step can convert low immunogenic tumors into "immune eliminated" tumors. Subjects optionally further receive CBT, such as anti-PD-(L)1. The subject is further treated with a TGFβ1 inhibitor, such as an antibody disclosed herein. This allows the conversion of hypoimmunogenic or immunocompromised tumors into "inflammatory" or "highly immunogenic" tumors, thus rendering them responsive to immunotherapy. Non-limiting examples of less immunogenic cancers include breast cancer (such as TNBC), prostate cancer (such as castration-resistant prostate cancer (CRPC)), and pancreatic cancer (such as pancreatic adenocarcinoma (PDAC)).

As shown to Applicants, the high affinity isoform selective inhibitors of TGFβ1 of the present disclosure, such as Ab6, are capable of inhibiting plasmin-induced activation of TGFβ1. The plasmin-plasminogen system is involved in specific tumorigenesis, invasion and/or metastasis of various cancer types, especially carcinomas such as breast cancer. Therefore, it is possible that TGFβ inhibitors such as those described herein may exert inhibitory effects via this mechanism in tumors or tumor models such as epithelial-involved EMT6. Indeed, plasmin-dependent destruction or remodeling of the epithelium is thought to contribute to the pathogenesis of diseases involving epithelial damage and carcinoma invasion/dissemination. The latter can be triggered by epithelial-to-mesenchymal transition (“EMT”). Plasminogen activation and plasminogen-dependent invasion are more pronounced in epithelial-like cells and have been reported to be directed, in part, by S100A10 and PAI-1 expression (Bydoun et al., (2018) Scientific Reports, : 14091).

The TGFβ inhibitors (eg, TGFβ1 inhibitors, eg, Ab6) of the present disclosure can be used to treat anemia in a subject in need thereof. In some embodiments, the subject is diagnosed with cancer. In some embodiments, the subject is diagnosed with a myeloproliferative disorder (eg, myelofibrosis). In some embodiments, a TGFβ inhibitor (eg, Ab6) is used alone to treat anemia. In some embodiments, TGFβ inhibitors are used in combination with additional agents, such as BMP antagonists (eg, BMP6 inhibitors, eg, RGMc inhibitors). In some embodiments, a combination comprising a TGFβ1 inhibitor (e.g., Ab6) and a BMP antagonist (e.g., a BMP6 inhibitor, e.g., an RGMc inhibitor) results from insufficient erythropoiesis, iron deficiency, and/or chemotherapy. Used to improve anemia. In some embodiments, treating anemia further comprises administering one or more JAK inhibitors (eg, a Jak1/2 inhibitor, a Jak1 inhibitor, and/or a Jak2 inhibitor).

The present disclosure includes methods for selecting patient populations or subjects that are likely to respond to treatments including TGFβ inhibitors such as those described herein. A subject selected according to such methods may be a subject receiving treatment according to various aspects of the present disclosure. Such methods include providing a biological sample (e.g., a clinical sample) taken from a subject; determining (e.g., measuring or assaying) the relative levels of TGFβ1, TGFβ2, and TGFβ3 in the sample; and determining whether TGFβ1 is , is the dominant isoform for TGFβ2 and TGFβ3; and/or if TGFβ1 is significantly overexpressed or upregulated compared to a control, a TGFβ inhibitor such as the TGFβ1 inhibitors described herein. administering to the subject a composition comprising: In some embodiments, such a method comprises: obtaining information regarding predetermined relative expression levels of TGFβ1, TGFβ2, and TGFβ3; and identifying a subject with a TGFβ1-positive (preferably TGFβ1-dominant) disease. and administering to a subject a composition comprising a TGFβ inhibitor disclosed herein. In some embodiments, such a subject has a disease (such as cancer) that is resistant to treatment (such as cancer therapy). In some embodiments, such a subject exhibits intolerance to the treatment and must or may discontinue treatment. Addition of a TGFβ inhibitor to the treatment regimen may allow the initial treatment dose to be reduced and still achieve the clinical benefits of the combination. In some embodiments, TGFβ inhibitors can delay or reduce the need for surgery. In some embodiments, the TGFβ inhibitor is a TGFβ1 inhibitor described herein, such as Ab6.

Relative levels of isoforms can be determined by RNA-based assays and/or protein-based assays that are well known in the art. In some embodiments, the step of administering may also include another therapy, such as an immune checkpoint inhibitor or other agents provided elsewhere herein. Such methods may optionally include assessing treatment response by monitoring changes in relative levels of TGFβ1, TGFβ2, and TGFβ3 at two or more time points. In some embodiments, clinical samples (such as biopsies) are collected both before and after administration. In some embodiments, clinical samples (such as biopsies) are collected multiple times after treatment to assess in vivo effects over time.

In addition to the above studies, a third study examines the range of TGFβ functions, including TGFβ1 functions, that are involved in specific diseases. In particular, this may be expressed by the number of TGFβ1 contexts, ie the number of TGFβ1 contexts in which the presenting molecule mediates disease-associated TGFβ1 functions. Broad inhibitors, such as TGFβ1-specific, context-independent inhibitors, would be advantageous in treating diseases involving both ECM and immune components of TGFβ1 function. Such diseases are thought to be associated with dysregulation in the ECM and perturbations in immune cell function or immune responses. Accordingly, the TGFβ1 inhibitors described herein can target ECM-associated TGFβ1 (e.g., presented by LTBP1 or LTBP3) as well as immune cell-associated TGFβ1 (e.g., presented by GARP or LRRC33). . Such inhibitors may target therapeutic targets (e.g., "context-independent" inhibitors): GARP-associated pro/latent TGFβ1; LRRC33-associated pro/latent TGFβ1; LTBP1-associated pro/latent TGFβ1; and LTBP3-associated pro/latent TGFβ1; All four types of TGFβ1 are inhibited to broadly inhibit TGFβ1 function in these contexts.

Whether a particular condition in a patient involves or is driven by multiple aspects of TGFβ1 function can be assessed by evaluating the expression profile of the presenting molecule in clinical samples collected from the patient. Can be done. A variety of assays are known in the art, including RNA-based assays and protein-based assays, and can be performed to obtain expression profiles. The relative expression levels of LTBP1, LTBP3, GARP, and LRRC33 (and/or changes/modifications thereof) in a sample may indicate the source and/or context of TGFβ1 activity associated with the condition. For example, a biopsy sample taken from a solid tumor can show high expression of all four presenting molecules. For example, LTBP1 and LTBP3 can be highly expressed in CAFs within the tumor stroma, whereas GARP and LRRC33 can be highly expressed by tumor-associated immune cells such as Tregs and leukocyte infiltrates, respectively.

Accordingly, the present disclosure includes methods for determining (eg, testing or confirming) the involvement of TGFβ1 in a disease relative to TGFβ2 and TGFβ3. In some embodiments, the method further comprises identifying the source (or context) of the disease-associated TGFβ1. In some embodiments, the source/context is assessed by determining the expression of TGFβ presenting molecules, such as LTBP1, LTBP3, GARP and LRRC33, in a clinical sample taken from a patient. In some embodiments, such methods are performed a posteriori.

Regarding LRRC33-positive cells, the applicant of the present disclosure has recognized that there can be a significant discrepancy between LRRC33 RNA and protein expression. In particular, selected cell types appear to express LRRC33 at the RNA level, but only a subset of such cells express LRRC33 protein on the cell surface. It is believed that LRRC33 expression may be highly regulated via protein trafficking/localization, for example with respect to plasma membrane insertion and rapid internalization. Thus, in certain embodiments, LRRC33 protein expression can be used, for example, as a marker associated with activated/M2-like macrophages and diseased tissues (such as tumor tissues) enriched with MDSCs.

In a related aspect, the disclosure provides therapeutic uses and related treatment methods involving immune checkpoint inhibitors, such as PD-(L)1 antibodies. Non-limiting examples of useful checkpoint inhibitors include 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®). can be mentioned.

According to the present disclosure, a method of treating cancer can include a checkpoint inhibitor for use in treating cancer in a subject, wherein the treatment comprises administering a checkpoint inhibitor to a subject undergoing treatment with a TGFβ inhibitor. administration or vice versa (i.e., administration of a TGFβ inhibitor to a subject undergoing treatment with a checkpoint inhibitor); MDSC level decreases. The sample may be a blood sample or a sample of a blood component. The checkpoint inhibitor can be a PD-1 antibody. The checkpoint inhibitor can be a PD-L1 antibody. The checkpoint inhibitor can be a CTLA4 antibody. In some embodiments, the checkpoint inhibitor is 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®). selected from.

According to the present disclosure, a method for treating cancer includes treating cancer in a subject that is poorly responsive to a checkpoint inhibitor, or where the subject has a cancer that has primary resistance to said checkpoint inhibitor. The treatment may include administering a TGFβ inhibitor to a subject, measuring circulating MDSC levels before and after administration of the TGFβ inhibitor, If circulating MDSCs are reduced after administration of the inhibitor, further administering to the subject an amount of checkpoint inhibitor sufficient to treat the cancer. The checkpoint inhibitor can be a PD-1 antibody. The checkpoint inhibitor can be a PD-L1 antibody. The checkpoint inhibitor may be a CTLA4 antibody. In some embodiments, the checkpoint inhibitor is 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®). selected from. Optionally, the TGFβ inhibitor is an isoform-selective inhibitor of TGFβ1, in which case the inhibitor is optionally an activation inhibitor of TGFβ1 or a neutralizing antibody that selectively binds to TGFβ1; or It is an isoform non-selective inhibitor (eg, an inhibitor of TGFβ1/2/3, TGFβ1/3, TGFβ1/2).

COMBINATION THERAPY Described herein is a pharmaceutical composition of a TGFβ inhibitor, e.g., an antibody or antigen-binding portion thereof described herein, for treating a subject who would benefit from TGFβ inhibition in vivo. Related methods for use as or in connection with combination therapy are disclosed. In any of these embodiments, such subject receives a first composition comprising at least one TGFβ inhibitor, e.g., a first composition comprising Ab6, for treatment of the same or overlapping disease or clinical condition. One may receive a combination therapy comprising together with at least one second composition comprising at least one additional treatment of interest. In some embodiments, such a subject may receive an additional third composition comprising at least one additional treatment aimed at treating the same or overlapping disease or clinical condition. TGFβ inhibitors include TGFβ1 selective inhibitors (e.g., those that do not inhibit TGFβ2 and/or TGFβ3 signaling at therapeutically effective doses), such as Ab6, or isoform non-selective inhibitors, such as low molecular weight ALK5 antagonists, TGFβ1 Neutralizing antibodies that bind to two or more of /2/3, e.g. GC1008 or variants, TGFβ1/3, ligand traps, e.g. TGFβ1/3 inhibitors and/or integrin inhibitors (e.g. αVβ1, αVβ3, αVβ5, αVβ6 , αVβ8, α5β1, αIIbβ3 or α8β1 integrin and inhibits downstream activation of TGFβ1, such as selective inhibition of TGFβ1 and/or TGFβ3). The first, second and third compositions may all act on the same cellular target or on separate cellular targets. In some embodiments, the first, second, and third compositions may treat or alleviate the same or overlapping set of symptoms or aspects of a disease or clinical condition. In some embodiments, the first, second, and third compositions may treat or alleviate a separate set of symptoms or aspects of a disease or clinical condition. In some embodiments, the combination therapy includes four or more that can act on the same target or distinct cellular targets and can treat or alleviate the same or overlapping set of symptoms or aspects of a disease or clinical condition. The composition may include a composition of For example, a first composition can treat a disease or condition associated with TGFβ signaling, whereas a second composition can treat a disease or condition associated with the same disease, such as inflammation or fibrosis. can do. As another example, the first composition can treat a disease or condition associated with TGFβ signaling, whereas the second and third compositions have anti-tumor activity and/or or promote reversal of immunosuppression. In certain embodiments, the first composition can be a TGFβ inhibitor (e.g., a TGFβ1 inhibitor described herein), the second composition can be a checkpoint inhibitor, and the third composition can be a checkpoint inhibitor. The composition can be a different checkpoint inhibitor than the second composition. In certain embodiments, a first composition comprising a TGFβ inhibitor (eg, a TGFβ1 inhibitor described herein) is combined with a checkpoint inhibitor and a chemotherapeutic agent. In certain embodiments, a first composition comprising a TGFβ inhibitor (eg, a TGFβ1 inhibitor described herein) is combined with two different checkpoint inhibitors and a chemotherapeutic agent. Such combination therapies may be administered in conjunction with each other. As noted above, the phrase "in combination with" in the context of combination therapy means that the effect of the first treatment in the subject receiving the combination therapy is temporally and/or spatially related to the therapeutic effect of the second treatment. It means overlapping. The first, second and/or additional compositions may be administered contemporaneously (eg, simultaneously), individually or sequentially. Accordingly, combination therapies may be formulated as a single formulation for synchronous or simultaneous administration, or as separate formulations for synchronous (eg, simultaneous), separate or sequential administration. As used herein, combination therapy is administered in a single bolus or administration, or in a single patient visit (e.g., to or in the presence of a health care professional) but in two or more separate doses. It may include two or more therapies (eg, compositions) provided in one bolus or administration or in separate patient outpatient visits (and, for example, in two or more separate boluses or administrations). For example, the therapeutic agent can be administered at intervals of less than about 5 minutes, or at intervals of 1 minute. The therapeutic agent may be administered less than about 30 minutes or hourly apart (eg, in a single patient visit). In some embodiments, the therapeutic agent lasts about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours. , at intervals of about 6 hours, about 8 hours, about 10 hours, about 1 day, about 2 days, about 3 days, about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month or more. can be administered. In some embodiments, therapeutic agents may be administered at intervals of about one day or more (eg, in separate outpatient visits). The therapeutic agents may be administered within 3 months (eg, within 1 month) of each other. In some embodiments, the therapeutic agent may be administered according to the dosing schedule of one or more approved therapeutic agents to treat the condition (e.g., for approved checkpoint inhibitors or other chemotherapeutic agents). (administered at the same frequency).

In certain embodiments, a TGFβ inhibitor (eg, a TGFβ1 inhibitor described herein) can be administered in an amount of about 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is administered in an amount of about 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less, e.g., once every two weeks, once every three weeks. , or once every two or three weeks (e.g., once every four weeks, once every six weeks), wherein the TGFβ inhibitor (e.g., Ab6) alone or checkpoint inhibitor therapy (e.g., 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), or T administered in combination with approved checkpoint inhibitor therapy, including but not limited to antibodies or other agents directed against the V-domain immunoglobulin (Ig)-containing inhibitor of cell activation (VISTA).

In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 3000 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 2400 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2400 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 2000 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 1600 mg once every 6 weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 800 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 240 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 240 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 80 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 80 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone in an amount of less than 80 mg once every 6 weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered in an amount less than 80 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) Administered in combination with 1 therapy. In some embodiments, the TGFβ inhibitor (e.g., Ab6) is administered in doses of 50-3000 mg, such as 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-3000 mg, every 6 weeks. It may be administered in doses of 2000mg, 250-2500mg, 1000-3000mg, 1500-2500mg, 2000-3000mg.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 3000 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 2400 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2400 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 2000 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 1600 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 800 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 240 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 240 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 80 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 80 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone in an amount of less than 80 mg once every four weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered in an amount less than 80 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) Administered in combination with 1 therapy. In some embodiments, the TGFβ inhibitor (e.g., Ab6) is administered at a dose of 50-3000 mg, such as 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-3000 mg, every 4 weeks. It may be administered in doses of 2000mg, 250-2500mg, 1000-3000mg, 1500-2500mg, 2000-3000mg.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 3000 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg as often as any multiple of 3 weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 2400 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2400 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 2400 mg at any multiple of three weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 2000 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg as often as any multiple of 3 weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 1600 mg once every three weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg as often as any multiple of 3 weeks, wherein the TGFβ inhibitor (e.g., Ab6) is ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 800 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg as often as any multiple of 3 weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 240 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 240 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) may be administered at 240 mg as often as any multiple of three weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 80 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 80 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 80 mg at any multiple of three weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone in an amount of less than 80 mg once every three weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered in an amount less than 80 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) Administered in combination with 1 therapy. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) may be administered at less than 80 mg as often as any multiple of three weeks, wherein the TGFβ inhibitor (e.g., Ab6) L) is administered in combination with 1 therapy. In some embodiments, the TGFβ inhibitor (e.g., Ab6) is administered in doses of 50-3000 mg, such as 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-300 mg, every 3 weeks. It may be administered in doses of 2000mg, 250-2500mg, 1000-3000mg, 1500-2500mg, 2000-3000mg.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 3000 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 2400 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2400 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) may be administered at 2400 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is ) is administered in combination with one therapy. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 2000 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 1600 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 800 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 240 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 240 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 240 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 80 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 80 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) may be administered at 80 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone in an amount of less than 80 mg once every two weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered in an amount less than 80 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) Administered in combination with 1 therapy. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at less than 80 mg at any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) L) is administered in combination with 1 therapy. In some embodiments, the TGFβ inhibitor (e.g., Ab6) is administered in doses of 50-3000 mg, such as 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-300 mg, every two weeks. It may be administered in doses of 2000mg, 250-2500mg, 1000-3000mg, 1500-2500mg, 2000-3000mg.

In certain embodiments, a TGFβ inhibitor (eg, a TGFβ1 inhibitor described herein, eg, Ab6) may be administered in combination with or in conjunction with genotoxic therapy. While not wishing to be bound by theory, in some embodiments, a subject receiving genotoxic therapy may have elevated levels of TGFβ1, e.g. may have lower TGFβ1 levels. Accordingly, in some embodiments, when a TGFβ inhibitor (e.g., a TGFβ1 inhibitor described herein, e.g., Ab6) is administered in combination with genotoxic therapy, post-treatment with genotoxic therapy This increased binding of TGFβ1 may provide additional therapeutic benefit. In some embodiments, genotoxic therapy can be chemotherapy and/or radiation therapy.

In certain embodiments, a TGFβ inhibitor (e.g., a TGFβ1 inhibitor described herein, e.g., Ab6) is used in combination with or in conjunction with checkpoint inhibitor therapy, e.g., anti-PD-(L)1 therapy. may be administered to a subject who is a non-responder to checkpoint inhibitor therapy, eg, anti-PD-(L)1 therapy. In certain embodiments, the subject has not previously received checkpoint inhibitor therapy. Exemplary checkpoint inhibitors include, but are not limited to, nivolumab (e.g., Opdivo®, an anti-PD-1 antibody), pembrolizumab (Keytruda®, an anti-PD-1 antibody), cemiplimab ( Libtayo®, anti-PD-1 antibody), Budigalimab (ABBV-181, anti-PD-1 antibody); BMS-936559 (anti-PD-L1 antibody), atezolizumab (e.g., Tecentriq®, anti-PD-1 antibody); L1 antibody), avelumab (e.g. Bavencio®, anti-PD-L1 antibody), durvalumab (e.g. Imfinzi®, anti-PD-L1 antibody), ipilimumab (e.g. Yervoy®, anti-CTLA4 tremelimumab (anti-CTLA4 antibody), IMP-321 (eftiragimod α or “ImmuFact®”, anti-LAG3 large molecule), BMS-986016 (leratorimab, anti-LAG3 antibody) and rililumab (anti-KIR2DL-1, - 2, -3 antibodies).

In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered alone to a subject with an advanced solid tumor. In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with checkpoint inhibitor therapy to a subject with an advanced solid tumor. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) is administered in combination with checkpoint inhibitor therapy to a subject with an advanced solid tumor, wherein the subject is non-responsive to previous checkpoint inhibitor therapy. Be the responder. In some embodiments, the subject has urothelial cancer (UC), including non-small cell lung cancer (NSCLC), melanoma (MEL), or metastatic urothelial carcinoma (mUC). In some embodiments, the subject has ovarian cancer, colorectal cancer (CRC), bladder cancer, renal cell carcinoma (RCC) (clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified (including RCC), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC), or oropharyngeal cancer). In some embodiments, the subject has esophageal cancer, gastric cancer, hepatocellular carcinoma (HCC), triple negative breast cancer (TNBC), cervical cancer, endometrial cancer, basal cell carcinoma (BCC), cutaneous squamous cell carcinoma ( CSCC), Merkel cell carcinoma (MCC), small cell lung cancer (SCLC), primary mediastinal large B-cell lymphoma (PMBCL), Hodgkin lymphoma, microsatellite instability-high carcinoma (MSI-H) (e.g., MSI- H CRC), mismatch repair-deficient cancer (dMMR) (eg, dMMR CRC), high tumor mutational burden (TMB-H) cancer, or malignant pleural mesothelioma (MPM). In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is administered to a subject with urothelial carcinoma (UC), including metastatic urothelial carcinoma (mUC), melanoma (MEL), or non-small cell lung cancer NSCLC. is given in combination with checkpoint inhibitor therapy. In certain embodiments, the subject is a non-responder to checkpoint inhibitor therapy. In certain embodiments, the checkpoint inhibitor therapy is pembrolizumab (eg, Keytruda®). In certain embodiments, the checkpoint inhibitor therapy is nivolumab (eg, Opdivo®). In certain embodiments, the checkpoint inhibitor therapy is cemiplimab (eg, Libtayo®). In certain embodiments, the checkpoint inhibitor therapy is atezolizumab (eg, Tecentriq®). In certain embodiments, the checkpoint inhibitor therapy is avelumab (eg, Bavencio®). In certain embodiments, the checkpoint inhibitor therapy is durvalumab (eg, Imfinzi®). In certain embodiments, the checkpoint inhibitor therapy is budigalimab (eg, ABBV-181).

In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less in combination with pembrolizumab once every two weeks, once every three weeks. , or once every two weeks or any multiple of three weeks (eg, once every four weeks, once every six weeks). In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is effective against NSCLC, UC, MEL, esophageal cancer, gastric cancer, HNSCC, HCC, cervical cancer, SCLC, PMBCL, Hodgkin's lymphoma, MSI-H or dMMR cancer, or Administered in combination with pembrolizumab to subjects with TMB-H cancer. In certain embodiments, the subject is a non-responder to pembrolizumab. In certain embodiments, the subject has not previously received pembrolizumab. In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with pembrolizumab to a subject with NSCLC who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject with NSCLC has not previously received pembrolizumab. In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with pembrolizumab to a subject with MEL who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject with MEL has not previously received pembrolizumab. In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with pembrolizumab to a subject with UC or mUC who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject with UC or mUC has not previously received pembrolizumab.

In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less in combination with nivolumab once every two weeks, once every three weeks. , or once every two weeks or any multiple of three weeks (eg, once every four weeks, once every six weeks). In certain embodiments, a TGFβ inhibitor (e.g., Ab6) is combined with nivolumab in subjects with NSCLC, UC, MEL, esophageal cancer, HNSCC, HCC, RCC, Hodgkin lymphoma, MSI-H or dMMR CRC, or MPM. administered. In certain embodiments, the subject is a non-responder to nivolumab. In certain embodiments, the subject has not previously received nivolumab.

In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less in combination with cemiplimab once every two weeks, once every three weeks. , or once every two weeks or any multiple of three weeks (eg, once every four weeks, once every six weeks). In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with cemiplimab to a subject with BCC or CSCC. In certain embodiments, the subject is a non-responder to cemiplimab. In certain embodiments, the subject has not previously received cemiplimab.

In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less in combination with atezolizumab once every two weeks, once every three weeks. or once every two or three weeks (eg, once every four weeks, once every six weeks). In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with atezolizumab to a subject with NSCLC, MEL, HCC, TNBC, or SCLC. In certain embodiments, the subject is a non-responder to atezolizumab. In certain embodiments, the subject has not previously received atezolizumab.

In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less in combination with avelumab once every two weeks, once every three weeks. , or once every two weeks or any multiple of three weeks (eg, once every four weeks, once every six weeks). In embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with avelumab to a subject with UC or MCC. In certain embodiments, the subject is a non-responder to avelumab. In certain embodiments, the subject has not previously received avelumab.

In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is administered at 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less in combination with durvalumab once every two weeks, once every three weeks. , or once every two weeks or any multiple of three weeks (eg, once every four weeks, once every six weeks). In embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with durvalumab to a subject with NSCLC or SCLC. In certain embodiments, the subject is a non-responder to durvalumab. In certain embodiments, the subject has not previously received durvalumab.

In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less, and the TGFβ inhibitor (e.g., ABBV-181) (e.g., 250 mg, 375 mg, or 500 mg dose), once every two weeks, once every three weeks, or once every multiple of two weeks or three weeks (e.g., once every four weeks, once every six weeks). administered in In certain embodiments, Budigalimab (eg, ABBV-181) is administered at 250 mg once every two weeks. In certain embodiments, budigalimab (eg, ABBV-181) is administered at 375 mg once every three weeks. In certain embodiments, budigalimab (eg, ABBV-181) is administered at 500 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with budigalimab to a subject with a locally advanced or metastatic solid tumor. See, e.g., NCT 03821935 (Safety, tolerability, and drug efficacy of ABBV-151 as a single agent and in combination with ABBV-181 in participants with locally advanced or metastatic solid tumors. Study to determine kinetics and recommended phase 2 dose (RP2D). https://clinicaltrials.gov/ct2/show/NCT03821935). In certain embodiments, budigalimab is administered once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) is administered in combination with budigalimab to a subject with triple negative breast cancer (TNBC), pancreatic adenocarcinoma, urothelial carcinoma, or hepatocellular carcinoma (HCC). . In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with budigalimab to a subject with non-small cell lung cancer or head and neck squamous cell carcinoma. Italiano et al. Cancer Immunol Immunother. 2022 Feb; 71(2):417-431. . In certain embodiments, the subject is a non-responder to budigalimab. In certain embodiments, the subject has not previously received budigalimab.

In certain embodiments, combination therapy provides a synergistic effect in treating the disease. The term "synergistic" refers to an effect that is greater than the additive effect (eg, greater efficacy) of each single agent in the ensemble.

In some embodiments, a combination therapy comprising a pharmaceutical composition described herein produces an efficacy that is overall equivalent to that produced by another therapy (such as monotherapy of a second agent). but with fewer concomitant undesirable adverse effects or less severe toxicity associated with the second agent compared to second agent monotherapy. In some embodiments, such combination therapy allows for a reduction in the dosage of the second agent while maintaining overall effectiveness. Such combination therapy may be particularly suitable for patient populations where long-term treatment is required and/or includes pediatric patients.

The present disclosure provides medicaments for use in and as a combination therapy for the reduction of TGFβ1 protein activation and the treatment or prevention of diseases or conditions associated with TGFβ1 signaling, as described herein. Compositions and methods are provided. Accordingly, the method or pharmaceutical composition may further include a second therapy. In some embodiments, the methods or pharmaceutical compositions disclosed herein can further include a third therapy. In some embodiments, the second therapy and/or the third therapy may be useful for treating or preventing a disease or condition associated with TGFβ1 signaling. The second treatment and/or the third treatment may alleviate or treat at least one symptom associated with the target disease. The first, second, and third therapies may exert their biological effects by similar or unrelated mechanisms of action; or either or both of the first and second therapies may exert multiple effects. may exert their biological effects by mechanisms. In some embodiments, the second therapy and the TGFβ inhibitor disclosed herein (e.g., the TGFβ1 selective inhibitor disclosed herein) are in a single formulation or in a single package. or in a separate formulation contained within a kit. In some embodiments, the second therapy, the third therapy, and the TGFβ inhibitor disclosed herein (e.g., the TGFβ1 selective inhibitor disclosed herein) are in a single formulation. or in separate formulations contained within a single package or kit. In some embodiments, the second therapy and a TGFβ inhibitor disclosed herein (e.g., a TGFβ1 selective inhibitor disclosed herein) are present in a single molecule, e.g. Included in a bispecific antibody or other multispecific construct, or in this case, the checkpoint inhibitor is a small molecule in an antibody-drug conjugate. In some embodiments, the second therapy, the third therapy, and the TGFβ inhibitors disclosed herein (e.g., the TGFβ1 selective inhibitors disclosed herein) are in a single molecule, e.g. Included in a bispecific antibody or other multispecific construct, or in this case, the checkpoint inhibitor is a small molecule in an antibody-drug conjugate. Examples of engineered constructs with TGFβ inhibitory activity include M7824 (Bintrafusp alfa) and AVID200. M7824 is a bifunctional fusion protein consisting of the two extracellular domains of TGF-βRII (TGF-β “trap”) fused to a human IgG1 monoclonal antibody against PD-L1. AVID200 is an engineered TGF-β ligand trap consisting of the TGF-β receptor ectodomain fused to a human Fc domain.

The pharmaceutical compositions described herein contain the first and second therapeutic agents in the same pharmaceutically acceptable carrier or in different pharmaceutically acceptable carriers for each of the described embodiments. You should understand that it is possible. Additionally, it is to be understood that the first and second therapeutic agents can be administered contemporaneously (eg, simultaneously), individually, or sequentially in the described embodiments.

One or more anti-TGFβ antibodies or antigen-binding portions thereof of the present disclosure can be used in conjunction with one or more additional therapeutic agents. Examples of additional therapeutic agents that can be used with the anti-TGFβ antibodies of the present disclosure include, but are not limited to, cancer vaccines, engineered immune cell therapies, chemotherapeutic agents, radiotherapy (e.g., radiotherapeutic agents), myostatin inhibitors. (e.g., myostatin inhibitors disclosed in WO 2016/073853 and WO 2017/049011, the contents of which are incorporated herein in their entirety) and members of the TGFβ superfamily, such as GDF11 inhibitors. Modulators; VEGF agonists; VEGF inhibitors (such as bevacizumab); IGF1 agonists; FXR agonists; CCR2 inhibitors; CCR5 inhibitors; dual CCR2/CCR5 inhibitors; CCR4 inhibitors, lysyl oxidase-like-2 inhibitors; ASK1 inhibitors ; acetyl-CoA carboxylase (ACC) inhibitors; p38 kinase inhibitors; pirfenidone; nintedanib; M-CSF inhibitors (e.g., M-CSF receptor antagonists and M-CSF neutralizers); MAPK inhibitors (e.g., Erk immune checkpoint agonists or antagonists; IL-11 antagonists; and IL-6 antagonists. Other examples of additional therapeutic agents that can be used with TGFβ inhibitors include, but are not limited to, indoleamine 2,3-dioxygenase (IDO) inhibitors, arginase inhibitors, tyrosine kinase inhibitors, Ser/ Thr kinase inhibitors, dual-specific kinase inhibitors. In some embodiments, such agents can be PI3K inhibitors, PKC inhibitors or JAK inhibitors.

Checkpoint inhibitor (CPI) therapy has transformed the treatment of solid tumors, but fewer than half of cancer patients are eligible to receive treatment with approved CPIs; <13% (Haslam 2019). Given these data, there remains significant unmet need across solid tumor indications with approved and unapproved therapies.

Recent data suggest that the effectiveness of immunomodulatory strategies requires the presence of a baseline immune response. Tumors lacking a pre-existing immune response or with low numbers of T cells in the tumor core and enriched in T cells at the infiltrating margin or stroma (e.g. in immunocompromised tumors) have a poor response to CPI. (Galon and Bruni 2019. Nat Rev Drug Discov. 18(3): 197-218). The TGFβ pathway is involved in mediating primary resistance to CPI therapy, and therefore combination therapy with anti-latent TGFβ monoclonal antibodies may increase efficacy in patients with inadequate response to CPI monotherapy. .

The present disclosure discloses that, for example, Ab6, alone or Including use in combination with other therapies. In some embodiments, a combination therapy comprising a TGFβ inhibitor, e.g., Ab6, and at least one additional agent is used to treat advanced solid tumors, e.g., cutaneous melanoma, urothelial carcinoma (UC), non-small cell lung cancer ( NSCLC) and head and neck cancer. In some embodiments, a combination therapy comprising a TGFβ inhibitor, e.g., Ab6, and at least one additional agent is administered to immunocompromised tumors, e.g., non-small cell lung cancer, melanoma, renal cell carcinoma, triple negative breast cancer, gastric cancer. may be effective in patients with microsatellite-stable colon cancer, pancreatic cancer, small cell lung cancer, HER2-positive breast cancer or prostate cancer.

In some embodiments, at least one additional agent (eg, a cancer therapy agent) used in a method or composition disclosed herein is a checkpoint inhibitor. In some embodiments, the at least one additional agent is a PD-1 antagonist, a PD-L1 antagonist, a PD-L1 or PD-L2 fusion protein, a CTLA4 antagonist, a GITR agonist, an anti-ICOS antibody, an anti-ICOSL antibody, an anti-B7H3 Antibodies, anti-B7H4 antibodies, anti-TIM3 antibodies, anti-LAG3 antibodies, anti-OX40 antibodies (OX40 agonists), anti-CD27 antibodies, anti-CD70 antibodies, anti-CD47 antibodies, anti-41BB antibodies, anti-PD-1 antibodies, oncolytic viruses, and selected from the group consisting of PARP inhibitors. Exemplary checkpoint inhibitors include, but are not limited to, nivolumab (e.g., Opdivo®, an anti-PD-1 antibody), pembrolizumab (Keytruda®, an anti-PD-1 antibody), cemiplimab ( Libtayo®, anti-PD-1 antibody), Budigalimab (ABBV-181, anti-PD-1 antibody); BMS-936559 (anti-PD-L1 antibody), atezolizumab (e.g., Tecentriq®, anti-PD-1 antibody); L1 antibody), avelumab (e.g. Bavencio®, anti-PD-L1 antibody), durvalumab (e.g. Imfinzi®, anti-PD-L1 antibody), ipilimumab (e.g. Yervoy®, anti-CTLA4 tremelimumab (anti-CTLA4 antibody), IMP-321 (eftiragimod α or “ImmuFact®”, anti-LAG3 large molecule), BMS-986016 (leratorimab, anti-LAG3 antibody) and rililumab (anti-KIR2DL-1, - 2, -3 antibodies). In some embodiments, the TGFβ inhibitors disclosed herein are used to treat cancer in subjects who are poor responders or non-responders to checkpoint inhibition therapies such as those listed herein. be done. In some embodiments, checkpoint inhibitors and TGFβ inhibitors (e.g., the TGFβ1 selective inhibitors disclosed herein) are administered in a single molecule, e.g., by a bispecific antibody or other multispecific antibody. in the construct or in an antibody-drug conjugate if the checkpoint inhibitor is a small molecule.

In some embodiments, the present disclosure provides at least one checkpoint inhibitor therapy for the treatment of solid tumors and/or hematological malignancies in which dysregulated TGFβ signaling is implicated as a mediator of the disease process. including the use of TGFβ inhibitors, such as Ab6, in combination with Ab6. In certain embodiments, combination therapy can be administered to patients who do not respond to checkpoint inhibitor therapy (eg, anti-PD-1 or anti-PD-L1 therapy). Such patients may include, but are not limited to, patients diagnosed with non-small cell lung cancer, urothelial bladder cancer, melanoma, triple negative breast cancer or other advanced solid cancers. In certain embodiments, a combination therapy may include a TGFβ inhibitor, eg, Ab6, and a checkpoint inhibitor therapy (eg, pembrolizumab). In certain embodiments, combination therapy is administered to immunotherapy-naïve patients diagnosed with a cancer that has received FDA approval for treatment with checkpoint inhibitor therapy (e.g., has not previously received checkpoint inhibitor therapy). patients). These cancers include gastric cancer (e.g. metastatic gastric cancer), urothelial bladder cancer, lung cancer, triple negative breast cancer, renal cell carcinoma (including clear cell RCC or papillary RCC), cervical cancer or head and neck squamous cell carcinoma. It can be. In certain embodiments, a combination therapy may include a TGFβ inhibitor, eg, Ab6, and a checkpoint inhibitor therapy (eg, pembrolizumab). In certain embodiments, the combination therapy may further include additional agents, such as additional checkpoint inhibitors and/or another chemotherapeutic agent. In certain embodiments, combination therapy may be used in immunotherapy-naïve patients diagnosed with cancers that have not received FDA approval for treatment with checkpoint inhibitor therapy (e.g., have not previously received checkpoint inhibitor therapy). patients). Such cancer may be microsatellite stable colon cancer or pancreatic cancer. In certain embodiments, the combination therapy comprises a TGFβ inhibitor, e.g., Ab6, a checkpoint inhibitor therapy (e.g., pembrolizumab), and at least one chemotherapeutic agent (e.g., axitinib, paclitaxel, cisplatin, and/or 5-fluorouracil). may be included. In certain embodiments, checkpoint inhibitor therapy can be pembrolizumab, nivolumab and/or atezolizumab. In certain embodiments, combination therapy is administered to patients with cancers characterized by exhibiting an immune exclusion phenotype. In certain embodiments, additional analyzes of a patient's cancer may be performed to provide further information about treatment, including microsatellite instability levels, PD-1 and/or PD-L1 expression levels, and/or Alternatively, known cancer-specific markers may be used, including the presence of mutations in known cancer driver genes such as EGFR, ALK, ROS1, BRAF. In certain embodiments, tumor PD-L1 expression can be used as a biomarker of treatment response.

In certain embodiments, a TGFβ inhibitor, such as Ab6, may be administered in an amount of about 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is administered in an amount of about 3000 mg, 2400 mg, 2000 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less, e.g., once every 6 weeks, once every 4 weeks. , once every three weeks, once every two weeks, where the TGFβ inhibitor (e.g., Ab6) is administered alone or in combination with checkpoint inhibitor therapy, e.g., anti-PD-(L) Administered in combination with 1 therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 3000 mg once every three weeks.

In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 3000 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 2400 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2400 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 2000 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 1600 mg once every 6 weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 800 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 240 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 240 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 80 mg once every 6 weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 80 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone in an amount of less than 80 mg once every 6 weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered in an amount less than 80 mg once every six weeks, wherein the TGFβ inhibitor (e.g., Ab6) Administered in combination with 1 therapy. In some embodiments, the TGFβ inhibitor (e.g., Ab6) is administered in doses of 50-3000 mg, such as 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-3000 mg, every 6 weeks. It may be administered in doses of 2000mg, 250-2500mg, 1000-3000mg, 1500-2500mg, 2000-3000mg.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 3000 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 2400 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2400 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 2000 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 1600 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 800 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 240 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 240 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 80 mg once every four weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 80 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone in an amount of less than 80 mg once every four weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered in an amount less than 80 mg once every four weeks, wherein the TGFβ inhibitor (e.g., Ab6) Administered in combination with 1 therapy. In some embodiments, the TGFβ inhibitor (e.g., Ab6) is administered at a dose of 50-3000 mg, such as 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-3000 mg, every 4 weeks. It may be administered in doses of 2000mg, 250-2500mg, 1000-3000mg, 1500-2500mg, 2000-3000mg.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 3000 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg as often as any multiple of 3 weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 2400 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2400 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 2400 mg at any multiple of three weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 2000 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg as often as any multiple of 3 weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 1600 mg once every three weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg as often as any multiple of 3 weeks, wherein the TGFβ inhibitor (e.g., Ab6) is ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 800 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg as often as any multiple of 3 weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 240 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 240 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) may be administered at 240 mg as often as any multiple of three weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 80 mg once every three weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 80 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 80 mg at any multiple of three weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone in an amount of less than 80 mg once every three weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered in an amount less than 80 mg once every three weeks, wherein the TGFβ inhibitor (e.g., Ab6) Administered in combination with 1 therapy. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) may be administered at less than 80 mg as often as any multiple of three weeks, wherein the TGFβ inhibitor (e.g., Ab6) L) is administered in combination with 1 therapy. In some embodiments, the TGFβ inhibitor (e.g., Ab6) is administered in doses of 50-3000 mg, such as 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-300 mg, every 3 weeks. It may be administered in doses of 2000mg, 250-2500mg, 1000-3000mg, 1500-2500mg, 2000-3000mg.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 3000 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 3000 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 2400 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2400 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) may be administered at 2400 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is ) is administered in combination with one therapy. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 2000 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 2000 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 1600 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 1600 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 800 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 800 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, a TGFβ inhibitor (eg, Ab6) may be administered alone at 240 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 240 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 240 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone at 80 mg once every two weeks. In certain embodiments, a TGFβ inhibitor (e.g., Ab6) can be administered at 80 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) is combined with anti-PD-(L)1 therapy. Administered in combination. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered at 80 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) ) is administered in combination with one therapy. In certain embodiments, the TGFβ inhibitor (eg, Ab6) may be administered alone in an amount of less than 80 mg once every two weeks. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) can be administered in an amount less than 80 mg once every two weeks, wherein the TGFβ inhibitor (e.g., Ab6) Administered in combination with 1 therapy. In certain embodiments, the TGFβ inhibitor (e.g., Ab6) may be administered at less than 80 mg as often as any multiple of two weeks, wherein the TGFβ inhibitor (e.g., Ab6) L) is administered in combination with 1 therapy. In some embodiments, the TGFβ inhibitor (e.g., Ab6) is administered at 50-3000 mg, such as 200-3000 mg, 200-1000 mg, 250-750 mg, 500-2000 mg, 750-2000 mg, 1000-300 mg, every two weeks. It may be administered in doses of 2000mg, 250-2500mg, 1000-3000mg, 1500-2500mg, 2000-3000mg.

In certain embodiments, the TGFβ inhibitor (e.g., Ab6) is effective against NSCLC, UC, MEL, esophageal cancer, gastric cancer, HNSCC, HCC, cervical cancer, SCLC, PMBCL, Hodgkin's lymphoma, MSI-H or dMMR cancer, or Administered in combination with pembrolizumab to subjects with TMB-H cancer. In certain embodiments, the subject is a non-responder to pembrolizumab. In certain embodiments, the subject has not previously received pembrolizumab. In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with pembrolizumab to a subject with NSCLC who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject with NSCLC has not previously received pembrolizumab. In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with pembrolizumab to a subject with MEL who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject with MEL has not previously received pembrolizumab. In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with pembrolizumab to a subject with UC or mUC who is a non-responder to pembrolizumab treatment. In certain embodiments, the subject with UC or mUC has not previously received pembrolizumab.

In certain embodiments, a TGFβ inhibitor (e.g., Ab6) is combined with nivolumab in subjects with NSCLC, UC, MEL, esophageal cancer, HNSCC, HCC, RCC, Hodgkin lymphoma, MSI-H or dMMR CRC, or MPM. administered. In certain embodiments, the subject is a non-responder to nivolumab. In certain embodiments, the subject has not previously received nivolumab.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with cemiplimab to a subject with BCC or CSCC. In certain embodiments, the subject is a non-responder to cemiplimab. In certain embodiments, the subject has not previously received cemiplimab.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with atezolizumab to a subject with NSCLC, MEL, HCC, TNBC, or SCLC. In certain embodiments, the subject is a non-responder to atezolizumab. In certain embodiments, the subject has not previously received atezolizumab.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with avelumab to a subject with UC or MCC. In certain embodiments, the subject is a non-responder to avelumab. In certain embodiments, the subject has not previously received avelumab.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with durvalumab to a subject with NSCLC or SCLC. In certain embodiments, the subject is a non-responder to durvalumab. In certain embodiments, the subject has not previously received durvalumab.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with checkpoint inhibitor therapy to a subject with a solid tumor for which checkpoint inhibitor therapy has been approved. In certain embodiments, the subject has a tumor type that has been approved for treatment with a combination of checkpoint inhibitor therapy and chemotherapy. In certain embodiments, the subject has a tumor type that typically exhibits immune clearance in greater than 50% of the tumor area (eg, tumor foci). In certain embodiments, the immune-excluded tumor type comprises triple negative breast cancer or renal cell carcinoma.

In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with checkpoint inhibitor therapy to a subject with a solid tumor for which checkpoint inhibitor monotherapy has been approved. In certain embodiments, the subject has tumors that typically exhibit immune clearance in greater than 50% of the tumor area (e.g., tumor foci), such as non-small cell lung cancer, urothelial cancer, gastric cancer, and renal cell carcinoma. Has a type. In certain embodiments, the subject has a tumor type, such as small cell lung cancer or melanoma, that typically exhibits immune clearance in less than 50% of the tumor area (eg, tumor foci).

In certain embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with checkpoint inhibitor therapy to a subject with a solid tumor for which checkpoint inhibitor therapy is not approved. In certain embodiments, the subject has a tumor type that typically exhibits immune clearance in greater than 50% of the tumor area (e.g., tumor foci), such as microsatellite stable colorectal cancer, pancreatic cancer, and prostate cancer. . Without wishing to be bound by theory, tumor types without approved checkpoint inhibitor therapy may have a poor response to checkpoint inhibitor therapy, with or without conventional chemotherapy. It is therefore conceivable that such tumor types, particularly those exhibiting immune clearance, may have a better response to checkpoint inhibitor therapy when combined with TGFβ inhibitors (eg, Ab6).

Thus, a TGFβ inhibitor (e.g., Ab6) can be used in conjunction (e.g., in combination) with checkpoint inhibitor therapy for the treatment of cancer in a subject, where the cancer has an immunosuppressive tumor. including, immunosuppressive tumors are resistant to checkpoint inhibitor therapy. Non-limiting examples of immunosuppressive tumors include acute myeloid leukemia, adrenocortical carcinoma, low-grade brain glioma, cholangiocarcinoma, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma, renal chromophobe cells, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pheochromocytoma and paraganglioma, prostatic adenocarcinoma, rectal adenocarcinoma, sarcoma, testicular germ cells Tumors include thymoma, thyroid cancer, uterine carcinosarcoma, endometrioid carcinoma, or uveal melanoma.

In some embodiments, at least one additional agent binds to a T cell costimulatory molecule, such as an inhibitory costimulatory molecule and an activating costimulatory molecule. In some embodiments, the at least one additional agent is selected from the group consisting of anti-CD40 antibodies, anti-CD38 antibodies, anti-KIR antibodies, anti-CD33 antibodies, anti-CD137 antibodies, and anti-CD74 antibodies.

In some embodiments, the at least one additional therapy is radiation. In some embodiments, the at least one additional agent is a radiotherapeutic agent. In some embodiments, at least one additional agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is taxol. In some embodiments, the at least one additional agent is an anti-inflammatory drug. In some embodiments, the at least one additional agent inhibits the process of monocyte/macrophage recruitment and/or tissue infiltration. In some embodiments, the at least one additional agent is an inhibitor of hepatic stellate cell activation. In some embodiments, the at least one additional agent is a chemokine receptor antagonist, such as a CCR2 antagonist and a CCR5 antagonist. In some embodiments, such chemokine receptor antagonists are bispecific antagonists, such as CCR2/CCR5 antagonists. In some embodiments, at least one additional agent administered as a combination therapy is or includes a member of the TGFβ superfamily of growth factors or modulators thereof. In some embodiments, such agents are selected from modulators (eg, inhibitors and activators) of GDF8/myostatin and GDF11. In some embodiments, such an agent is an inhibitor of GDF8/myostatin signaling. In some embodiments, such an agent is a monoclonal antibody that specifically binds to the pro/latent myostatin complex and blocks activation of myostatin. In some embodiments, a monoclonal antibody that specifically binds the pro/latent myostatin complex and blocks myostatin activation does not bind free mature myostatin; e.g., WO 2017/049011 Please refer to the brochure.

In some embodiments, the additional therapy includes cell therapy, such as CAR-T therapy and CAR-NK therapy.

In some embodiments, the additional therapy comprises administering a VEGF inhibitor, eg, an anti-VEGF therapy such as bevacizumab. In some embodiments, inhibitors of TGFβ contemplated herein can be used in conjunction with VEGF inhibitors (e.g., bevacizumab) for the treatment of solid cancers (e.g., ovarian cancer), e.g. , combination therapy, add-on therapy, etc.). In some embodiments, inhibitors of TGFβ contemplated herein may be used in conjunction with VEGF inhibitors (e.g., bevacizumab, etc.) for the treatment of hematopoietic cancers (e.g., combination therapy, add-on therapy, etc.).

In some embodiments, the additional therapy is a cancer vaccine. Many clinical trials have tested peptide cancer vaccines in hematologic malignancies (blood cancers), melanoma (skin cancer), breast cancer, head and neck cancer, gastroesophageal cancer, lung cancer, pancreatic cancer, prostate cancer, ovarian cancer, and colorectal cancer. Targeting cancer. Antigens included peptides from HER2, telomerase (TERT), survivin (BIRC5) and Wilms tumor 1 (WT1). In some studies, "individualized" mixtures of 12-15 different peptides were also used. That is, they contain a mixture of peptides from the patient's tumor to which the patient exhibits an immune response. Several trials have targeted solid tumors, gliomas, glioblastomas, melanomas, and breast, cervical, ovarian, colorectal, and non-small lung cell carcinomas, including MUC1, IDO1 (indoleamine 2,3-dioxygenase), CTAG1B, and two VEGF receptors, FLT1 and KDR. In particular, the IDO1 vaccine will be tested in melanoma patients in combination with the immune checkpoint inhibitor ipilimumab and the BRAF (gene) inhibitor vemurafenib.

Non-limiting examples of tumor antigens useful as cancer vaccines include NY-ESO-1, HER2, HPV16 E7 (Papillomaviridae #E7), CEA (carcinoembryonic antigen), WT1, MART-1 , gp100, tyrosinase, URLC10, VEGFR1, VEGFR2, surviving, MUC1 and MUC2.

However, activated immune cells primed by such cancer vaccines can be cleared from the TME partially through TGFβ1-dependent mechanisms. For the purpose of solving immunosuppression, the use of the TGFβ1 inhibitors of the present disclosure can be considered to unlock the potential of vaccines.

Combination therapies contemplated herein may advantageously utilize lower doses of therapeutic agents administered, thereby avoiding potential toxicities or complications associated with various monotherapies. . In some embodiments, the use of isoform-specific inhibitors of TGFβ1 described herein makes those who are hypo-responsive or non-responsive to therapy (e.g., standard therapy) more responsive. It can be sexual. In some embodiments, the use of isoform-specific inhibitors of TGFβ1 described herein allows for a reduction in the dosage of a therapeutic agent (e.g., standard of care), yet still achieves comparable clinical outcomes in patients. effect and associated drug-related toxicity or adverse events are reduced or their severity is reduced.

In some embodiments, inhibitors of TGFβ contemplated herein may be used in conjunction with selective inhibitors of myostatin (GDF8) (eg, combination therapy, add-on therapy, etc.). In some embodiments, the selective inhibitor of myostatin is an inhibitor of pro/latent myostatin activation. For example, see antibodies disclosed in WO 2017/049011 pamphlet, such as apitegromab.

Advantages of TGFβ1 Inhibitors as Therapeutics It is recognized that various diseases involve heterogeneous cell populations as a source of TGFβ1 that collectively contribute to disease pathogenesis and/or progression. Multiple types of TGFβ1-containing complexes (“contexts”) can coexist within the same disease microenvironment. In particular, such diseases may involve both the ECM (or "matrix") component of TGFβ1 signaling (eg, ECM dysregulation) and the immune component of TGFβ1 signaling. In such situations, selectively targeting only a single TGFβ1 context (eg, TGFβ1 associated with one particular type of presentation molecule) may provide limited relief. Therefore, broadly inhibitory TGFβ1 antagonists are desirable for therapeutic use. Previously described inhibitory antibodies that broadly targeted multiple latent complexes of TGFβ1 showed skewed binding profiles between target complexes (e.g., WO 2018/129329 and WO 2019/075090). Please refer to the brochure). Therefore, the present inventors set out to more uniformly identify inhibitory antibodies that selectively inhibit TGFβ1 activation, independent of the specific presentation molecule linked thereto. It was reasoned that potent inhibition of both matrix-associated and immune cell-associated TGFβ1 would be advantageous, especially for immuno-oncology applications.

In various embodiments, context-independent inhibitors of TGFβ1 are used in the treatments and methods disclosed herein to target the pro/latent form of TGFβ1. More specifically, in some manners, the inhibitor targets ECM-associated TGFβ1 (LTBP1/3-TGFβ1 complex). In another manner, the inhibitor targets immune cell-associated TGFβ1. This includes GARP-presenting TGFβ1, such as the GARP-TGFβ1 complex expressed on Treg cells and the LRRC33-TGFβ1 complex expressed on macrophages and other myeloid/lymphoid cells, as well as certain cancer cells. included.

Such antibodies inhibit pro/latent TGFβ1 complexes in a context-independent manner such that the antibodies can inhibit activation (or release) of TGFβ1 associated with multiple types of presentation molecules. Can include isoform-specific inhibitors of TGFβ1 that bind to and prevent activation (or release) of mature TGFβ1 growth factor. In particular, the present disclosure provides antibodies that can block ECM-associated TGFβ1 (LTBP-presenting complex and LTBP3-presenting complex) and cell-associated TGFβ1 (GARP-presenting complex and LRRC33-presenting complex).

Various disease states have been suggested to involve dysregulation of TGFβ signaling as a contributing factor. Indeed, the pathogenesis and/or progression of certain human conditions appears to be primarily driven by or dependent on TGFβ1 activity. Notably, many of these diseases and disorders involve both ECM and immune components of TGFβ1 function, which involve TGFβ1 activation in multiple contexts (e.g., mediated by multiple types of presentation molecules). This suggests that Furthermore, there appears to be cross-talk between TGFβ1-responsive cells. In some cases, interactions between the pleiotropic activities of the TGFβ1 system can trigger a cascade of events that lead to disease progression, exacerbation, and/or suppression of the host's ability to fight the disease. Certain disease microenvironments, such as the tumor microenvironment (TME) and the fibrotic microenvironment (FME), contain multiple different presenting molecules, such as LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, LRRC33-proTGFβ1, and It may be associated with TGFβ1 presented by any combination of these. TGFβ1 activity in one context may in turn modulate or influence TGFβ1 activity in another context, which, if dysregulated, increases the likelihood that this will cause worsening of the disease state. Therefore, it is desirable to inhibit broadly across multiple modes (ie, multiple contexts) of TGFβ1 function while selectively limiting the inhibitory effect to TGFβ1 isoforms. The aim is not to perturb homeostatic TGFβ signaling mediated by other isoforms such as TGFβ3, which play an important role in wound healing.

The immune component of TGFβ1 activity is primarily mediated by cell-associated TGFβ1 (eg, GARP-proTGFβ1 and LRRC33-proTGFβ1). Both the GARP- and LRRC33 arms of TGFβ1 function are associated with immunosuppressive features that contribute to the progression of many diseases. Accordingly, TGFβ1 inhibitors, such as the TGFβ1 inhibitors described herein, can be used to inhibit TGFβ1 associated with immunosuppressive cells. Immunosuppressive cells include regulatory T cells (Tregs), M2 macrophages/tumor-associated macrophages, and MDSCs. TGFβ inhibitors of the present disclosure can inhibit, reduce, or reverse immunosuppressive phenotypes at disease sites, such as the tumor microenvironment.

In some embodiments, the TGFβ1 inhibitor inhibits TGFβ1 associated with cells expressing the GARP-TGFβ1 complex or the LRRC33-TGFβ1 complex, where optionally the cells are T cells, fibrotic It can be a blast, myofibroblast, macrophage, monocyte, dendritic cell, antigen presenting cell, neutrophil, myeloid-derived suppressor cell (MDSC), lymphocyte, mast cell or microglial cell. The T cell can be a regulatory T cell (eg, an immunosuppressive T cell). A neutrophil can be an activated neutrophil. Macrophages can be activated (eg, polarized) macrophages, including pro-fibrotic and/or tumor-associated macrophages (TAM), such as M2c subtype and M2d subtype macrophages. In some embodiments, macrophages are exposed to tumor-derived factors (eg, cytokines, growth factors, etc.) that can further induce a pre-cancerous phenotype in macrophages. In some embodiments, such tumor-derived factor is CSF-1/M-CSF.

In some embodiments, the cells expressing the GARP-TGFβ1 complex or the LRRC33-TGFβ1 complex are cancer cells, such as circulating cancer cells and tumor cells.

TGFβ Inhibitors Useful in Practicing the Compositions and Methods of the Disclosure TGFβ inhibitors suitable for the therapeutic uses and related methods disclosed herein include small molecule (i.e., low molecular weight) antagonists and biologics. include. Such inhibitors include isoform selective inhibitors and isoform non-selective inhibitors. Biologic inhibitors include antibodies, antigen-binding fragments thereof, antibody-based molecules or immunoglobulin-like molecules as well as other engineered constructs, typically fusion proteins, such as ligand traps. Ligand traps typically include a ligand binding moiety derived from one or more ligand binding moieties of a TGFβ receptor. Such biologics can be multifunctional constructs such as bifunctional fusion proteins and bispecific antibodies.

In some embodiments, the methods disclosed herein include a low molecular weight antagonist of the TGFβ receptor, e.g., an ALK5 antagonist, e.g., galunisertib (LY2157299 monohydrate); a monoclonal antibody that inhibits all three isoforms ( neutralizing antibodies) (“pan-inhibitor” antibodies) (see e.g. WO 2018/134681); monoclonal antibodies that preferentially inhibit two of the three isoforms (e.g. TGFβ1/ 2 (e.g. WO 2016/161410 pamphlet) and TGFβ1/3 (e.g. WO 2006/116002 pamphlet); and manipulative molecules such as ligand traps (e.g. fusion proteins) (e.g. No. 2018/029367; No. 2018/129331 and No. 2018/158727). In some embodiments, the methods disclosed herein , Battle and Massague (Immunity, 2019. Apr 16;50(4):924-940), the contents of which are incorporated herein in their entirety. be able to.

In some embodiments, the TGFβ inhibitor is a TGFβ1 selective inhibitor, such as a TGFβ1 selective inhibitor disclosed herein, eg, Ab6. In some embodiments, the TGFβ1 selective inhibitor is one disclosed in PCT/US2019/041390, PCT/US2019/041373, or PCT/US2021/012930, each of which is incorporated herein by reference in its entirety. is incorporated herein by reference.

In some embodiments, the low molecular weight antagonist of the TGFβ receptor can include bactosertib (TEW-7197, EW-7197), LY3200882, PF-06952229, AZ 12601011, and/or AZ 12799734.

In some embodiments, the neutralizing pan-TGFβ antibody is GC1008, also known as fresolimumab, or a derivative thereof. In some embodiments, such an antibody comprises a sequence according to the disclosure of WO 2018/134681. In some embodiments, the pan-TGFβ antibody is SAR439459 or a derivative thereof.

In some embodiments, the TGFβ1/2 antibody comprises XPA-42-089 or a derivative thereof.

In some embodiments, the antibody is a neutralizing antibody that specifically binds both TGFβ1 and TGFβ3. In some embodiments, such antibodies preferentially bind TGFβ1 over TGFβ3. For example, the antibody comprises a sequence according to the disclosure of WO 2006/116002. In some embodiments, the antibody is 21D1.

In some embodiments, the antibody is a neutralizing antibody that specifically binds both TGFβ1 and TGFβ2. In some embodiments, the antibody comprises a sequence according to the disclosure of WO 2016/161410. In some embodiments, the antibody is XOMA-089, or NIS-793.

In some embodiments, the antibody is an activation-inhibiting antibody that is selective for TGFβ1. In some embodiments, the antibody comprises a sequence according to the disclosure of WO 2015/015003, WO 2019/075090, or WO 2016/115345.

In some embodiments, the antibody is a neutralizing antibody that is selective for TGFβ1. In some embodiments, the antibody comprises a sequence according to the disclosure of WO 2013/134365 or WO 2018/043734.

In some embodiments, the TGFβ inhibitor is a ligand trap. In some embodiments, the ligand trap comprises a structure according to the disclosure of WO 2018/158727. In some embodiments, the ligand trap comprises a structure according to the disclosures of WO 2018/029367; WO 2018/129331. In some embodiments, the ligand trap is a construct known as CTLA4-TGFbRII. In some embodiments, the ligand trap is a bifunctional fusion protein that includes checkpoint inhibitor function and TGFβ inhibitor function. In some embodiments, the bifunctional fusion protein is a construct known as M7824 or PDL1-TGFbRII. In some embodiments, the TGFβ inhibitor is a receptor-based TGFβ trap, such as AVID200.

In some embodiments, the TGFβ inhibitor is an integrin inhibitor. In some embodiments, the TGFβ inhibitor is an inhibitor of an integrin, such as αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3, and/or α8β1. Integrin inhibitors include small molecule inhibitors and antibodies that bind to integrins and/or inhibit the binding of integrins to the RGD motif of proTGFβ1 and/or proTGFβ3.

In some embodiments, the TGFβ inhibitor is an inhibitor of latent TGFβ (eg, latent TGFβ1 or latent TGFβ3). In some embodiments, the TGFβ inhibitor is an inhibitor that binds to the RGD motif of proTGFβ1 and/or proTGFβ3.

In some embodiments, the TGFβ inhibitor is an LTBP selective inhibitor, such as the LTBP selective inhibitor disclosed in PCT/US2020/015915. In some embodiments, the TGFβ inhibitor is an LTBP1 selective inhibitor. In some embodiments, the TGFβ inhibitor is an LTBP3 selective inhibitor. In some embodiments, the TGFβ inhibitor is an LTBP-1 and LTBP3 selective inhibitor.

Isoform-Selective Antibodies of proTGFβ1 Preferably, therapeutic uses and related methods according to the present disclosure are directed to isoform-selective inhibitors of TGFβ1, such as Ab6 (the sequence of which is described in PCT/U.S. Patent Application Publication No. 2019/041373). , the contents of which are incorporated herein by reference in their entirety.

Applicants have previously disclosed improved antibodies that embody all or most of the following features: 1) maintain selectivity for TGFβ1 to minimize unnecessary toxicities associated with pan-inhibition; “isoform selectivity”) (see e.g. PCT/US Patent Application Publication No. 2017/021972); 2) broad binding across various biological contexts or both matrix-related and cell-related categories 3) exhibiting activity (“context-independent”) (see e.g. WO 2018/129329); 3) achieving more uniform or unbiased affinities across multiple antigen complexes; “uniformity”); 4) exhibiting strong binding activity for each of the antigen complexes (“high affinity”) and having reliable inhibitory activity (“potency”) for each context (e.g., PCT and 5) a preferred mechanism of action is to inhibit the activation step so that the inhibitor targets the latent TGFβ1 complex tethered to the tissue. rather than directly targeting soluble/free growth factors, thereby pre-emptively preventing downstream activation events to achieve a sustained effect (“sustained”). As disclosed in PCT/US Patent Application Publication No. 2019/041373, these TGFβ1 inhibitors are highly potent and highly selective inhibitors of latent TGFβ1 activation. The data presented therein demonstrate, inter alia, that this mechanism of isoform-selective inhibition closely reproduces some of the features of primary resistance to CBT observed in human cancers. proved to be sufficient to solve the problem. Additionally, 6) such inhibitors have an improved safety profile compared to pan-inhibitors or other isoform non-selective inhibitors of TGFβ. Taken together, these efficacy and safety data support the use of selective TGFβ1 to expand and enhance clinical responses to checkpoint inhibition in cancer immunotherapy, as well as to treat a number of additional TGFβ1-related indications. Provides a rationale for exploring the therapeutic use of inhibition.

General Characteristics of Certain TGFβ1 Inhibitors Exemplary antibodies that may be used to practice the present disclosure are disclosed in WO2020014460, the contents of which are incorporated herein by reference in their entirety.

Preferred antibodies and corresponding nucleic acid sequences encoding such antibodies useful in carrying out the present disclosure include one or more of the CDR amino acid sequences shown in Tables 1 and 2. Each set of H-CDRs (H-CDR1, H-CDR2 and H-CDR3) listed in Table 1 is compatible with the L-CDRs (L-CDR1, L-CDR2 and L-CDR3) provided in Table 2. Can be combined.

Accordingly, the present disclosure provides antibodies or antigen-binding antibodies thereof comprising the six CDRs listed in Table 1 (e.g., H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3). providing a fragment, such as an isolated antibody or antigen-binding fragment thereof, and L-CDR1 comprises QASQDITNYLN (SEQ ID NO: 78), L-CDR2 comprises DASNLET (SEQ ID NO: 79), and L-CDR1 comprises QASQDITNYLN (SEQ ID NO: 78); CDR3 may include QQADNHPPWT (SEQ ID NO: 6); optionally, H-CDR1 may include FTFSSFSMD (SEQ ID NO: 80); H-CDR-2 may include YISPSADTIYYADSVKG (SEQ ID NO: 76); and/ Or H-CDR3 may include ARGVLDYGDMLMP (SEQ ID NO: 3). In some embodiments, the antibody or fragment has an H-CDR1 having the amino acid sequence FTFSSFSMD (SEQ ID NO: 80), an H-CDR2 having the amino acid sequence YISPSADTIYYADSVKG (SEQ ID NO: 76), and an amino acid sequence ARGVLDYGDMLMP (SEQ ID NO: 3). H-CDR-3; includes L-CDR1 having the amino acid sequence QASQDITNYLN (SEQ ID NO: 78), L-CDR2 having the amino acid sequence DASNLET (SEQ ID NO: 79), and L-CDR3 having the amino acid sequence QQADNHPPWT (SEQ ID NO: 6). Further examples of antibodies or antigen-binding fragments thereof encompassed by this disclosure are provided in PCT/US2019/041390, PCT/US2019/041373, and PCT/US2021/012930, the contents of each of which are incorporated herein by reference in their entirety. Incorporated herein by reference.

Determination of CDR sequences within an antibody depends on the particular numbering scheme utilized. Commonly used systems include Kabat numbering, IMTG numbering, Chothia numbering, and Lu. (Lu X et al., MAbs. 2019 Jan; 11(1):45-57). To illustrate, six CDR sequences of Ab6 as defined by four different numbering schemes are illustrated below. Any art-recognized CDR numbering system can be used to define the CDR sequences of the antibodies of the present disclosure.

The amino acid sequences of the heavy and light chain variable domains of exemplary antibodies of the present disclosure are provided in Table 4. Thus, in some embodiments, the isoform-selective TGFβ1 inhibitors of the present disclosure can be antibodies or antigen-binding fragments thereof that include a heavy chain variable domain ( _VH ) and a light chain variable domain ( _VL ). , V _H and V _L sequences are selected from any one of the sets of V _H and V _L sequences listed in Table 4 below.

In some embodiments, an antibody or antigen-binding fragment thereof is disclosed that includes a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain is Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% and 100%); and the light chain variable domains have sequence identities of Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25 , Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33 and Ab34; and/or the light chain variable domains may have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% and 100%) sequence identity. may have the same identity. In some embodiments, the heavy chain variable domain of the antibody or fragment has at least 90% sequence identity with SEQ ID NO: 7, and optionally the light chain variable domain of the antibody or fragment has at least 90% sequence identity with SEQ ID NO: 8. has at least 90% sequence identity. In some embodiments, the heavy chain variable domain of the antibody or fragment has at least 95% sequence identity with SEQ ID NO: 7, and optionally the light chain variable domain of the antibody or fragment has at least 95% sequence identity with SEQ ID NO: 8. has at least 95% sequence identity. In some embodiments, the heavy chain variable domain of the antibody or fragment has at least 98% sequence identity with SEQ ID NO: 7, and optionally the light chain variable domain of the antibody or fragment has at least 98% sequence identity with SEQ ID NO: 8. has at least 98% sequence identity. In some embodiments, the heavy chain variable domain of the antibody or fragment has 100% sequence identity with SEQ ID NO: 7, and optionally the light chain variable domain of the antibody or fragment has 100% sequence identity with SEQ ID NO: 8. Has 100% sequence identity.

In various embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise six CDRs from the heavy and light chain variable domains of SEQ ID NOs: 7 and 8, respectively, or the complete sequence thereof. . In some embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable domain sequence and a light chain variable domain sequence that have at least 90% sequence identity (e.g., at least 95% identity) with SEQ ID NOs: 7 and 8, respectively. Contains domain sequences. For example, an antibody or antigen-binding fragment thereof can contain six corresponding H-CDRs and L-CDRs selected from those listed in Tables 1 and 2 above. In some specific embodiments, the antibody or antigen-binding fragment thereof comprises a set of six corresponding H-CDRs and L-CDRs listed in Table 3 (e.g., using the method of Lu et al.). include.

Alternatively or additionally, antibodies or antigen-binding fragments thereof used in connection with the present disclosure are antibodies having at least 90% sequence identity (e.g., at least 95% identity) with SEQ ID NOs: 7 and 8, respectively. a first binding region that may include a chain variable domain and a light chain variable domain and that includes (i) at least a portion of Latent Lasso (SEQ ID NO: 126); and ii) at least one of Finger-1 (SEQ ID NO: 124). The antibody or fragment specifically binds to the proTGFβ1 complex with a second binding region that includes a second binding region; It is characterized in that it protects the binding area from solvent exposure. The first binding region may include PGPLPEAV (SEQ ID NO: 134) or a portion thereof, and the second binding region may include RKDLGWKW (SEQ ID NO: 143) or a portion thereof. As used herein, protection of a binding region refers to the extent to which a protein (e.g., a region of a protein containing an epitope) is exposed to solvent as assessed by HDX-MS-based assays of protein-protein interactions. refers to protein-protein interactions such as antibody-antigen binding. Protection of binding can be judged by the level of proton exchange that occurs at the binding site, which is inversely related to the extent of binding/interaction. Thus, when the antibodies described herein bind to a region of an antigen, protein-protein interactions prevent the binding region from becoming accessible by the surrounding solvent; be “protected” from. Therefore, the protected region represents the site of interaction. The antibody or fragment may further bind to the proTGFβ1 complex with one or more of the following binding regions or portions thereof: LVKRKRIEA (SEQ ID NO: 132); LASPPSQGEVPPGPL (SEQ ID NO: 126); LALYNSTR (SEQ ID NO: 135); REAVPEPVL ( YQKYSNNSWR (SEQ ID NO: 137); RKDLGWKWIHE (SEQ ID NO: 144); HEPKGYHANF (SEQ ID NO: 145); LGPCPYIWS (SEQ ID NO: 139); ALEPLPIV (SEQ ID NO: 140); and VGRKPKVEQL (SEQ ID NO: 141).

In some embodiments, the antibody or antigen-binding fragment is a TGFβ1 (e.g., pro- and/or latent-TGFβ1) antibody having a heavy chain variable domain of SEQ ID NO: 7 and a light chain variable domain of SEQ ID NO: 8. can be further characterized in that it cross-blocks (cross-competes) binding to. In some embodiments, the cross-blocking or cross-competing antibodies have heavy and light chain variable domains that are at least about 90% (e.g., 95% or 99%) identical to those of SEQ ID NOs: 7 and 8, respectively. including.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex is SEQ ID NO: 7 A nucleic acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with a nucleic acid sequence set forth in chain variable domain amino acid sequence, and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence set forth in SEQ ID NO:8. The light chain variable domain amino acid sequence encoded by the nucleic acid sequence having the following properties. In some embodiments, the antibody or antigen-binding portion thereof has a heavy chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 7, and a light chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 8. Contains chain variable domain amino acid sequences.

In some examples, any of the antibodies of the present disclosure that specifically binds to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex binds to CDRH1, CDRH2 , including antigen-binding portions thereof, having one or more CDR (eg, CDRH or CDRL) sequences substantially similar to CDRH3, CDRL1, CDRL2, and/or CDRL3. For example, antibodies include SEQ ID NOs: 3, 6, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 , 97, 98 and 99. It may contain CDR sequences. In some embodiments, one or more of the six CDR sequences contains up to three amino acid changes compared to the sequence provided in Table 1. Such antibody variants containing up to three amino acid changes per CDR are encompassed by this disclosure. In some embodiments, such variant antibodies are generated by an optimization process such as affinity maturation. Complete amino acid sequences for the heavy and light chain variable regions of the antibodies listed in Table 4 (e.g., Ab6) and the nucleic acid sequences encoding the heavy and light chain variable regions of particular antibodies are provided below. Ru.
Ab6-heavy chain variable region amino acid sequence
Ab6-light chain variable region amino acid sequence
Ab6-heavy chain amino acid sequence
Ab6-heavy chain nucleic acid sequence
Ab6-light chain amino acid sequence
Ab6-light chain nucleic acid sequence (human kappa)

In some embodiments, the "percent identity" of two amino acid sequences is as described in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990. Such an algorithm is described by Altschul, et al. , J. Mol. Biol. 215:403-10, 1990, into the NBLAST and XBLAST programs (version 2.0). BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecule of interest. If a gap exists between two sequences, gapped BLAST is performed as described by Altschul et al. , Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing the BLAST and gapped BLAST programs, each program's default parameters (eg, XBLAST and NBLAST) may be used.

In any of the antibodies or antigen-binding fragments described herein, one or more conservative mutations are introduced in the CDR or framework sequences at positions where the residue is not likely to participate in antibody-antigen interactions. can be done. In some embodiments, such conservative variations include the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex as the residues are determined based on the crystal structure. It can be introduced into CDR or framework sequences at positions that are not likely to participate in interaction with the complex. In some embodiments, potential interactions (eg, residues involved in antigen-antibody interactions) can be deduced from known structural information for another antigen that shares structural similarity.

As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be found in references summarizing such methods, eg, Molecular Cloning: A Laboratory Manual, J. Mol. Sambrook, et al. , eds. , Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biolog y, F. M. Ausubel, et al. , eds. , John Wiley & Sons, Inc. , New York, according to methods for modifying polypeptide sequences known to those skilled in the art. Conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the antibodies provided herein include mutations that confer desirable properties to the antibody. For example, to avoid potential complications due to Fab-arm exchange known to occur with natural IgG4 mAbs, antibodies provided herein may contain stabilizing "Adair" mutations. (Angal et al., “A single amino acid submission abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody,” Mol Immun ol 30, 105-108; 1993), serine 228 (EU numbering; residue 241 Kabat numbering ) is converted to proline resulting in an IgG1-like (CPPCP (SEQ ID NO: 43)) hinge sequence. Thus, any of the antibodies may contain a stabilizing "Adair" mutation or amino acid sequence (CPPCP (SEQ ID NO: 43)).

The isoform-specific context-independent inhibitors of TGFβ1 of the present disclosure may optionally include antibody constant regions or portions thereof. For example, a VL domain can be linked at its C-terminus to a light chain constant domain-like Cκ or Cλ. Similarly, a VH domain or portion thereof may be attached to all or part of a heavy chain such as IgA, IgD, IgE, IgG and IgM and any isotype subclass. The antibody may include a suitable constant region (see, eg, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Public cations, Bethesda, Md. (1991)). Thus, antibodies within the scope of this disclosure may include VH and VL domains, or antigen-binding portions thereof, in combination with any suitable constant region.

Additionally or alternatively, such antibodies include the antibodies of SEQ ID NOs: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, and 8. may or may not include framework regions. In some embodiments, the antibody that specifically binds to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex is a murine antibody and has murine framework region sequences. including.

In some embodiments, such antibodies have a relatively high affinity, e.g., 10 ⁻ Binds with a KD of less than ⁹ M, 10 ⁻¹⁰ M, 10 ⁻¹¹ M or less. For example, such antibodies can be applied to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex at a concentration of 5 pM to 1 nM, such as 10 pM to 1 nM, such as 10 pM to 500 pM. may bind with affinity. The present disclosure competes with any of the antibodies described herein for binding to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex and/or LRRC33-TGFβ1 complex, Also included are antibodies or antigen-binding fragments having a KD value of, for example, 1 nM or less, 500 pM or less, 100 pM or less. The affinity and binding kinetics of antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex can be determined using biosensor-based techniques (e.g., OCTET ® or Biacore®) and solution equilibrium titration-based techniques (eg, MSD-SET). In some embodiments, affinity and binding kinetics are measured by SPR, such as the Biacore system. In preferred embodiments, such antibodies dissociate from each of the aforementioned large latent complexes with an OFF rate of 10e-4 or less.

In some embodiments, inhibitors of cell-bound TGFβ1 (e.g., GARP-presented TGFβ1 and LRRC33-presented TGFβ1) according to the present disclosure inhibit such complexes (e.g., GARP-pro/latent TGFβ1 and LRRC33-pro/latent). type TGFβ1) and induces internalization of the complex. This mechanism of action causes the removal or depletion of inactive TGFβ1 complexes (e.g., GARP-proTGFβ1 and LRRC33-proTGFβ1) from the cell surface (e.g., Tregs, macrophages, etc.), thus making them available for activation. Decrease TGFβ1. In some embodiments, such an antibody or fragment thereof is such that binding occurs at neutral or physiological pH, but the antibody dissociates from its antigen at acidic pH; or the rate of dissociation is greater than at neutral pH. It binds to target complexes in a pH-dependent manner such that it is higher at acidic pH. Such antibodies or fragments thereof can function as recycling antibodies.

Antibodies that Compete with Preferred Antibodies for TGFβ1 Aspects of the present disclosure pertain to antibodies that compete or cross-compete with any of the antibodies provided herein. As used herein, the term "competing" with respect to antibodies means that a first antibody binds to an epitope (e.g., GARP-proTGFβ1 complex) in a manner that is sufficiently similar or overlapping with the binding of a second antibody. LTBP1-proTGFβ1 complex, LTBP3-proTGFβ1 complex, and LRRC33-proTGFβ1 complex), so that the result of the binding of the first antibody to that epitope is that the second antibody binding of the first antibody in the absence of is detectably reduced in the presence of the second antibody. Alternatively, binding of the second antibody to its epitope may also be detectably reduced in the presence of the first antibody, but this need not be the case. That is, the first antibody can inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its epitope or ligand, whether to the same extent, to a greater extent, or to a lesser extent, then the antibody They are said to "cross-compete" with each other. Both competing and cross-competing antibodies are within the scope of this disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change or binding to a common epitope or portion thereof), one skilled in the art will recognize that such competing antibodies and/or cross-competition It will be appreciated that antibodies can be included and useful in the methods and/or compositions provided herein. The terms "cross-blocking" and "cross-competition" may be used interchangeably.

Two different monoclonal antibodies (or antigen-binding fragments) that bind the same antigen can bind to the antigen simultaneously if the binding sites are far enough apart in three-dimensional space that each bond does not interfere with the other. There is. In contrast, two different monoclonal antibodies may have the same or overlapping antigen binding regions, in which case binding of the first antibody does not allow the second antibody to bind the antigen. It may disappear or vice versa. In the latter case, the two antibodies are said to "cross-block" each other with respect to the same antigen.

Antibody "binning" experiments are useful for sorting multiple antibodies raised against the same antigen into various "bins" based on their relative cross-blocking activities. Each "bin" therefore represents a separate binding region of antigen. Antibodies in the same bin cross-block each other by definition. Binning can be performed using standard test conditions, e.g., according to the manufacturer's instructions (e.g., binding assayed at room temperature, about 20-25°C), using a standard assay such as Biacore or Octet®. Can be tested by in vitro binding assay.

Aspects of the present disclosure relate to antibodies that compete or cross-compete with any of the particular antibodies or antigen-binding portions thereof as provided herein. In some embodiments, the antibody or antigen-binding portion thereof binds at or near the same epitope as any of the antibodies provided herein. In some embodiments, the antibody or antigen-binding portion thereof binds in close proximity to the epitope if it binds within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibodies as provided herein, or antigen-binding portions thereof, are 1, 2, 3, or 3 of the epitopes bound by any of the antibodies provided herein. Bonds within 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.

In another embodiment, an antigen provided herein (e.g., a GARP-TGFβ1 complex) has an equilibrium dissociation constant, K _D , between the antibody and the protein of less than 10 ⁻⁸ M. , LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex). In other embodiments, the antibodies compete or cross-compete for binding to any of the antigens provided herein with a KD in the range of 10 ⁻¹² M to 10 ⁻⁹ M. In some embodiments, provided herein are anti-TGFβ1 antibodies or antigen-binding portions thereof that compete for binding with the antibodies or antigen-binding portions thereof described herein. In some embodiments, provided herein is an anti-TGFβ1 antibody or antigen-binding portion thereof that binds to the same epitope as an antibody or antigen-binding portion thereof described herein.

Any of the antibodies provided herein can be characterized using any suitable method. For example, one method is identifying the epitope to which an antigen binds or "epitope mapping." See, for example, Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.C. Y. mapping the location of epitopes on proteins, including solving crystal structures of antibody-antigen complexes, competition assays, gene fragment expression assays, and synthetic peptide-based assays as described in Chapter 11 of , 1999; There are many suitable methods for characterization. In additional examples, epitope mapping can be used to determine the sequences to which an antibody binds. The epitope may be a linear epitope, i.e., it may be contained in a stretch of amino acids, or it may be a conformation formed by three-dimensional interactions of amino acids that may not necessarily be contained in a stretch (primary structural linear sequence). It can be an epitope. In some embodiments, the epitope is present in an antibody described herein or its It is a TGFβ1 epitope available only upon binding by the antigen binding moiety. Peptides of various lengths (eg, at least 4-6 amino acids in length) can be isolated or synthesized (eg, recombinantly) and used for binding assays with antibodies. In another example, the epitope to which an antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to gene fragment expression assays, the open reading frame encoding the target antigen is fragmented randomly or for specific genetic constructs, and the reactivity of the expressed fragment of the antigen with the antibody to be tested is determined. is determined. Gene fragments can be generated, for example, by PCR and subsequently transcribed in vitro in the presence of radioactive amino acids and translated into proteins. Binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In additional examples, mutagenesis of antigen binding domains, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. For example, domain swapping experiments show that various fragments of the GARP-proTGFβ1 complex, LTBP1-proTGFβ1 complex, LTBP3-proTGFβ1 complex, and/or proLRRC33-TGFβ1 complex are linked to different members of the TGFβ family (e.g., GDF11). This can be carried out using variants of the target antigen that are replaced (exchanged) with sequences from closely related but antigenically distinct proteins, such as.

Alternatively, competition assays can be performed using other antibodies known to bind the same antigen to determine whether the antibody binds to the same epitope as the other antibody. Competition assays are well known to those skilled in the art.

In some embodiments, the pharmaceutical composition comprises an antibody having a heavy chain variable domain of SEQ ID NO: 7 and a light chain variable domain of SEQ ID NO: 8 for binding to TGFβ1 (e.g., pro-TGFβ1 and/or latent TGFβ1). can be produced by a process that includes selecting antibodies or antigen-binding fragments thereof that cross-compete with the antigen-binding fragment thereof.

In some embodiments, the pharmaceutical composition is selected from the group consisting of Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34. and selecting an antibody or antigen-binding fragment thereof that cross-competes with the antibody to be used; and formulating it into a pharmaceutical composition.

Preferably, the antibodies selected by the process are such that the antibodies or antigen-binding fragments are of human LLC (e.g., hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1 and hLRRC33-proTGFβ1), as determined by solution equilibrium titration. They are high affinity binders characterized in that they are capable of binding to each with a K _D of ≦1 nM. Such cross-competing antibodies can be used in the treatment of TGFβ1-related symptoms in a subject according to the present disclosure.

and Variations Non-limiting variations, modifications, and features of any of the antibodies or antigen-binding fragments thereof encompassed by this disclosure are briefly discussed below. Related analytical method embodiments are also provided.

Various Modifications of Antibodies The naturally occurring structural units of antibodies usually constitute tetramers. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair consisting of one full-length "light" chain (in certain embodiments, about 25 kDa) and one full-length "light" chain (in certain embodiments, about 25 kDa) and one full-length has a "heavy" chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain usually contains a variable region of about 100-110 or more amino acids that is typically involved in antigen recognition. The carboxy-terminal portion of each chain usually defines a constant region that can be involved in effector functions. Human antibody light chains are commonly classified as kappa and lambda light chains. Heavy chains are usually classified as mu, delta, gamma, alpha, or epsilon and define the antibody's isotype. Antibodies may be of any type (e.g., IgM, IgD, IgG, IgA, IgY, and IgE) and class (e.g., IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgM ₁ , IgM ₂ , IgA ₁ , and IgA). ₂ ). In full-length light and heavy chains, the variable and constant regions are typically joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. (See, eg, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (incorporated by reference in its entirety). The variable region of each light/heavy chain pair typically forms an antigen binding site.

The variable regions usually exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are usually aligned by framework regions, allowing binding to specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chain variable regions typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is usually determined by the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Ch. othia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. , (1989) Nature 342:878-883. Light chain CDRs may also be referred to as CDR-L1, CDR-L2 and CDR-L3, and heavy chain CDRs may also be referred to as CDR-H1, CDR-H2 and CDR-H3. In some embodiments, the antibody may contain a small number of amino acid deletions from the carboxy terminus of the heavy chain. In some embodiments, the antibody comprises a heavy chain with a 1-5 amino acid deletion at the carboxy terminus of the heavy chain. In certain embodiments, the final designation of the CDRs and identification of the residues comprising the binding site of the antibody is accomplished by solving the structure of the antibody and/or solving the structure of the antibody-ligand complex. In certain embodiments, it may be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, various methods of analysis may be utilized to identify or approximate CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the definition described by Lu et al. (see above), and the contact definition.

An "affinity matured" antibody is an antibody that has one or more modifications in one or more of its CDRs that result in an improvement in the antibody's affinity for its antigen compared to a parent antibody that does not have those modifications. . Exemplary affinity matured antibodies will have nanomolar or picomolar affinities (eg, K _D in the range of about 10 ⁻⁹ M to 10 ⁻¹² M) for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. , (1992) Bio/Technology 10:779-783 describe affinity maturation by domain shuffling of VH and VL. Random mutagenesis of CDR and/or framework residues is described by Barbas, et al. , (1994) Proc Nat. Acad. Sci. USA 91:3809-3813; Schier et al. , (1995) Gene 169:147-155; Yelton et al. , (1995) J. Immunol. 155:1994-2004; Jackson et al. , (1995) J. Immunol. 154(7):3310-9; and Hawkins et al. , (1992) J. Mol. Biol. 226:889-896; selective mutagenesis with activity-enhancing amino acid residues; selective mutations at contact or hypermutation positions are described in U.S. Patent No. 6,914,128; be written. Typically, the parent antibody and its affinity-matured progeny (e.g., derivatives) retain the same binding region within the antigen, but certain interactions at the molecular level are due to the amino acid residues introduced by affinity maturation. Can be changed due to modification.

The term "CDR-grafted antibody" is derived from certain species, such as antibodies having murine heavy and light chain variable regions in which one or more murine CDRs (e.g., CDR3) are replaced with human CDR sequences. Refers to an antibody that contains heavy and light chain variable region sequences, but in which one or more sequences of the VH and/or VL CDR regions have been replaced with CDR sequences of another species.

The term "chimeric antibody" refers to heavy and light chain variable region sequences derived from one species and constant regions derived from another species, such as antibodies that have murine heavy and light chain variable regions linked to human constant regions. Refers to an antibody that contains the sequence.

As used herein, the term "framework" or "framework sequences" refers to the sequences that remain from the variable regions minus the CDRs. Since the precise definition of a CDR sequence may be determined in different ways, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2 and -L3 of the light chain and CDR-H1, -H2 and -H3 of the heavy chain) also link the framework regions on the light and heavy chains to the four lower chains of each chain. It is divided into regions (FR1, FR2, FR3 and FR4), in which CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. located between. Unless the particular subregion is identified as FR1, FR2, FR3 or FR4, the framework regions include the combined FRs within the variable region of a single naturally occurring immunoglobulin chain, as referenced elsewhere. represent. As used herein, FR represents one of four subregions and FRs represents two or more of the four subregions that make up the framework region.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 1 (H-FR1) having the following amino acid sequence, optionally with 1, 2 or 3 amino acid changes.
For example, the Gly residue at position 16 may be replaced with Arg (R); and/or the Ala residue at position 23 may be replaced with Thr (T).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 2 (H-FR2) having the following amino acid sequence, optionally with 1, 2 or 3 amino acid changes: WVRQAPGKGLEWVS (SEQ ID NO: 148).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 3 (H-FR3) having the following amino acid sequence, optionally with 1, 2 or 3 amino acid changes: RFTISRDNAKNSLYLQMNSLRAEDTAVYYC (SEQ ID NO: 149). For example, the Ser residue at position 12 can be replaced with Thr (T).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 4 (H-FR4) having the following amino acid sequence, optionally with 1, 2 or 3 amino acid changes: WGQGTLVTVSS (SEQ ID NO: 150).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 1 (L-FR1) having the following amino acid sequence, optionally with 1, 2 or 3 amino acid changes: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 151).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 2 (L-FR2) having the following amino acid sequence, optionally with one, two or three amino acid changes: WYQQKPGKAPKLLIY (SEQ ID NO: 152).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 3 (L-FR3) having the following amino acid sequence, optionally with one, two or three amino acid changes: GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 153).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (L-FR4) having the following amino acid sequence, optionally with one, two or three amino acid changes: FGGGTKVEIK (SEQ ID NO: 154).

In some embodiments, the antibody or antigen-binding portion thereof is a heavy chain immunoglobulin constant domain of a human IgM constant domain, a human IgG constant domain, a human IgG1 constant domain, a human IgG2 constant domain, a human IgG2A constant domain, a human IgG2B constant domain. a human IgG2 constant domain, a human IgG3 constant domain, a human IgG3 constant domain, a human IgG4 constant domain, a human IgA constant domain, a human IgA1 constant domain, a human IgA2 constant domain, a human IgD constant domain, or a human IgE constant domain. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain immunoglobulin constant domain of a human IgG1 constant domain or a human IgG4 constant domain. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain immunoglobulin constant domain of a human IgG4 constant domain. In some embodiments, the antibody or antigen-binding portion thereof is a heavy chain immunoglobulin of a human IgG4 constant domain with a Ser to Pro backbone substitution that creates an IgG1-like hinge and allows for the formation of an intrachain disulfide bond. Contains constant domains.

In some embodiments, the antibody or antigen-binding portion thereof further comprises a light chain immunoglobulin constant domain, including a human Ig lambda constant domain or a human Ig kappa constant domain.

In some embodiments, the antibody is an IgG with four polypeptide chains, two heavy chains and two light chains.

In some embodiments, the antibody is a humanized antibody, a diabody, or a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody comprises a framework with human germline amino acid sequences.

In some embodiments, the antigen binding portion is a Fab fragment, F(ab')2 fragment, scFab fragment or scFv fragment.

In some embodiments, the antibody contains one or more amino acid modifications in the Fc region. In some embodiments, modifications in the Fc region can provide changes in properties, such as changes in circulating half-life, for example, by changing affinity for Fc receptors such as FcRn (Front Immunol. 2019 Jun 7;10:1296). In some embodiments, modifications to the Fc region of an antibody provide for increased FcγR binding. In some embodiments, modifications in the Fc region provide improved antibody effector function. In some embodiments, modifications in the Fc region increase the in vivo half-life of the antibody. In some embodiments, the half-life of an antibody can be extended by increasing the affinity of binding to FcRn at low pH (eg, pH < 6.5). In some embodiments, such Fc modifications may include Met252Tyr, Ser254Thr, and Thr256Glu substitutions (see, eg, US Pat. No. 7,083,784).

As used herein, the term "germline antibody gene" or "gene fragment" refers to non-lymphoid cells that have not undergone the maturation process that results in genetic rearrangements and mutations for the expression of specific immunoglobulins. (e.g., Shapiro et al., (2002) Crit. Rev. Immunol. 22(3):183-200; Marshallonis et al., (2001) Adv. Exp. Med. .484:13-30). One of the advantages provided by various embodiments of the present disclosure is that germline antibody genes are more likely than mature antibody genes to conserve the essential amino acid sequence structure characteristic of individuals within a species, and thus stems from the recognition that when used therapeutically in the United States, it is less likely to be recognized as originating from an exogenous source.

As used herein, the term "neutralizing" refers to counteracting the biological activity of an antigen (eg, a target protein) when the binding protein specifically binds to the antigen. In certain embodiments, the neutralizing binding protein binds an antigen/target, such as a cytokine, kinase, growth factor, cell surface protein, soluble protein, phosphatase or receptor ligand, and reduces its biological activity by at least about 20%, 40%. , 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the neutralizing antibody to the growth factor binds to its receptor that specifically binds to the soluble mature growth factor released from the latent complex, thereby inducing downstream signaling. hinder ability. In some embodiments, the mature growth factor is TGFβ1 or TGFβ3. The term "binding protein," as used herein, refers to any polypeptide that specifically binds to an antigen (e.g., TGFβ1), including but not limited to antibodies or antigen-binding portions thereof, as well as antigen-binding regions. (eg, a region capable of binding TGFβ1) and one or more additional antigens or additional epitopes on a single antigen. Examples include DVD-IgTM, TVD-Ig, RAb-Ig, bispecific antibodies and bispecific antibodies. A binding protein can also include an antibody-drug conjugate, e.g., a second agent (e.g., a small molecule checkpoint inhibitor) capable of binding TGFβ1 (e.g., capable of binding pro- and/or latent-TGFβ1). ) linked to an antibody or antigen-binding fragment thereof.

The term "monoclonal antibody" or "mAb" when used in connection with a composition comprising the same may refer to an antibody preparation obtained from a substantially homogeneous population of antibodies, i.e., comprising the population. Individual antibodies are identical except for a few naturally occurring variations that may be present. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.

The term "recombinant human antibody" as used herein refers to an antibody (further described in Section IIC below) that is expressed using a recombinant expression vector transfected into a host cell; Antibodies isolated from recombinant combinatorial human antibody libraries (Hoogenboom, H.R. (1997) TIB Tech. 15:62-70; incorporated herein by reference; Azzazy, H. and Highsmith, W.E. (2002) Clin.Biochem.35:425-445; Gavilondo, J.V. and Larick, J.W. (2002) BioTechniques 29:128-145; Hoogenboom, H. and Chames, P. (2000) ) Immunol. Today 21:371-378), antibodies isolated from animals (e.g. mice) transgenic for human immunoglobulin genes (Taylor, L.D. et al., (1992) Nucl. Acids Res. 20:6287 -6295; Kellermann, S-A. and Green, L.L. (2002) Cur. Opin. in Biotechnol. 13: 593-597; Little, M. et al., (2000) Immunol. Today 21: 364- 370) or by any other means, including splicing human immunoglobulin gene sequences into other DNA sequences. It is intended to include all human antibodies that are isolated. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if animals transgenic with human Ig sequences are used) and are therefore The amino acid sequences of the VH and VL regions of antibodies are sequences that are derived from and related to human germline VH and VL sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

As used herein, a "dual variable domain immunoglobulin" or "DVD-IgTM" or the like includes a binding protein that includes a paired heavy chain DVD polypeptide and a light chain DVD polypeptide, and each of the paired The heavy and light chains provide two antigen binding sites. Each binding site contains a total of six CDRs involved in antigen binding per antigen binding site. DVD-IgTM typically has two arms that are linked together at least in part by dimerization of the CH3 domain, and each arm of DVD is bispecific and has four binding sites. Provides immunoglobulin. DVD-IgTM is provided in US Patent Application Publication Nos. 2010/0260668 and 2009/0304693, each of which is incorporated herein by reference, including the sequence listing.

As used herein, a "triple variable domain immunoglobulin" or "TVD-Ig" or the like is a binding protein that includes a paired heavy chain TVD binding protein polypeptide and a light chain TVD binding protein polypeptide, respectively. The paired heavy and light chains of provide three antigen binding sites. Each binding site contains a total of six CDRs involved in antigen binding per antigen binding site. The TVD-binding protein has two arms linked together at least in part by dimerization of the CH3 domain, and each arm of the TVD-binding protein is trispecific and has six binding sites. May provide protein.

As used herein, a "receptor-antibody immunoglobulin" or "RAb-Ig" or the like is a binding protein that includes a heavy chain RAb polypeptide and a light chain RAb polypeptide, for a total of three Forms antigen binding site. One antigen-binding site is formed by the pairing of heavy and light antibody variable domains present in each of the heavy and light chain RAb polypeptides to form a single binding site, with a total of six CDRs. A first antigen binding site is provided. Each heavy and light chain RAb polypeptide contains a receptor sequence that independently binds the ligand, providing second and third "antigen" binding sites. RAb-Ig typically has two arms linked together at least in part by dimerization of the CH3 domain, and each arm of RAb-Ig is trispecific and contains six binding sites. Provided is an immunoglobulin comprising: RAb-Ig is described in US Patent Application Publication No. 2002/0127231, the entire contents of which are incorporated herein by reference, including the sequence listing.

In various embodiments, the present disclosure can be used alone, linked to one or more additional agents (e.g., as an ADC), or as a larger macromolecule (e.g., a bispecific antibody, bispecific antibody, or e.g. as part of a construct in combination with a TGFB ligand trap such as M7824 (Merck) and AVID200 (Forbius)) or a bifunctional or multifunctional engineered construct (e.g. The present invention provides partially novel antibodies and antigen-binding fragments that can be used as part of pharmaceutical compositions, fusion proteins, and ligand traps) and administered as part of pharmaceutical compositions or combination therapies.

The term "bispecific antibody" as used herein and to be distinguished from "bispecific half-Ig binding protein" or "bispecific (half-Ig) binding protein" refers to the quadroma technique (See Milstein, C. and Cuello, A.C. (1983) Nature 305(5934): p. 537-540), chemical conjugation of two different monoclonal antibodies (Staerz, U.D. et al. , (1985) Nature 314(6012):628-631) or by introducing mutations in the Fc region that do not inhibit CH3-CH3 dimerization, resulting in bispecific antibodies in which only one is functional. (see Holliger, P. et al., (1993) Proc. Natl. Acad. Sci USA 90(14):6444-6448). Refers to full-length antibodies produced by By molecular function, a bispecific antibody binds to one antigen (or epitope) on one of its two binding arms (one pair of HC/LC) and on its second arm (one pair of HC/LC). bind to different antigens (or epitopes) on different pairs of antigens (or epitopes). By this definition, a bispecific antibody has two separate antigen-binding arms (in both specificity and CDR sequences) and is monovalent for each antigen it binds. For example, a bispecific antibody comprising two binding arms directed against TGFβ1 and PD-1 combines a TGFβ1 inhibitor (Ab6 or an Ab6-derived binding moiety) and a checkpoint inhibitor (e.g., an anti-PD1 antibody or moiety). can be used. Such bispecific antibodies may be used as an exemplary form of treatment for patients selected to receive combination therapy of TGFβ1 inhibitor and checkpoint inhibitor.

The term "bispecific antibody," as used herein and to be distinguished from bispecific half-Ig binding protein or bispecific binding protein, refers to its two binding arms (a pair of HC/LC ) refers to a full-length antibody capable of binding two different antigens (or epitopes) in each case (see WO 02/02773). Thus, a bispecific binding protein has two identical antigen-binding arms with the same specificity and the same CDR sequences, and is bivalent for each antigen it binds.

The term "multispecific antibody" refers to an antibody or antigen-binding fragment that exhibits binding specificity for more than one epitope, with each binding site being distinct and recognizing a different epitope (on the same or different antigens). Bispecific antibodies are an exemplary type of multispecific antibody. Higher order multispecific antibodies (ie, antibodies exhibiting three or more specificities) include, but are not limited to, trispecific antibodies, triple bodies, and tribodies in TriMAb. For exemplary types of multispecific antibodies and/or methods of making the same, see, eg, Castoldi et al., herein incorporated by reference in its entirety with respect to such types and methods. , Protein Eng Des Sel 2012; 25:551-9; Schubert et al. , MAbs 2011; 3:21-30; Kuegler et al. , Br J Haematol 2010;150:574-86; Schoonjans et al. , J Immunol 2000; 165:7050-7; and Egan et al. , MAbs 2017;9(1):68-84.

The term "Kon" as used herein refers to the rate constant for the binding of a binding protein (e.g., an antibody) to an antigen, e.g., to form an antibody/antigen complex, as is known in the art. is intended to refer to. "Kon" is also known by the term "association rate constant" or "ka", which are used interchangeably herein. This value, which indicates the rate of binding of an antibody to its target antigen or the rate of complex formation between an antibody and an antigen, is also given by the formula: Antibody (“Ab”) + Antigen (“Ag”) → Ab-Ag. It will be done.

The term "Koff" as used herein is intended to refer to the off rate constant for the dissociation of a binding protein (e.g., antibody) from, e.g., an antibody/antigen complex, as is known in the art. Ru. "Koff" is also known by the term "dissociation rate constant" or "kd", which are used interchangeably herein. This value indicates the rate of dissociation of an antibody from its target antigen or the separation of the Ab-Ag complex into free antibody and antigen over time as shown by the formula Ab+Ag←Ab-Ag.

The term "equilibrium dissociation constant" or "K _D ", used interchangeably herein, refers to the value obtained in a titration measurement at equilibrium or the dissociation rate constant (koff) divided by the association rate constant (kon). refers to the value obtained by Association rate constant, dissociation rate constant and equilibrium dissociation constant are used to describe the binding affinity of a binding protein, such as an antibody, for an antigen. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and ability to test samples in physiological buffers at equilibrium. Other experimental techniques and equipment may be used, such as the Biacore® (biomolecular interaction analysis) assay (e.g., equipment available from Biacore International AB, GE Healthcare company, Uppsala, Sweden). Additionally, the KinExA® (Binding Equilibrium Exclusion Method) assay available from Sapidyne Instruments (Boise, Id.) may also be used.

The terms "crystal" and "crystallized" as used herein refer to a binding protein (eg, an antibody) or antigen-binding portion thereof that exists in crystalline form. A crystal is a form of solid state of matter, which is different from other forms such as an amorphous solid state or a liquid crystalline state. Crystals are composed of regularly repeating three-dimensional arrays of atoms, ions, molecules (eg, proteins such as antibodies), or molecular assemblies (eg, antigen/antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well understood in the art. A basic unit, or building block, that is repeated in a crystal is called an asymmetric unit. The repetition of asymmetric units in an arrangement according to a given well-defined crystallographic symmetry results in a "unit cell" of the crystal. Repetition of the unit cell by regular translation in all three dimensions results in a crystal. Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ed. , pp. 201-16, Oxford University Press, New York, New York, (1999). The term "linker" is used to refer to a polypeptide that includes two or more amino acid residues joined by peptide bonds and is used to join one or more antigen-binding moieties. Such linker polypeptides are well known in the art (eg, Holliger, P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J. et al., (1994) Structure 2:1121-1123). Exemplary linkers include ASTKGPSVFPLAP (SEQ ID NO: 44), ASTKGP (SEQ ID NO: 45); TVAAPSVFIFPP (SEQ ID NO: 46); TVAAP (SEQ ID NO: 47); AKTTPKLEEGEFSEAR (SEQ ID NO: 48); AKTTPKLEEGEFSEARV (SEQ ID NO: 49); AKTTP KLGG (SEQ ID NO: 50); SAKTTPKLGG (SEQ ID NO: 51); SAKTTP (SEQ ID NO: 52); RADAAP (SEQ ID NO: 53); RADAAPTVS (SEQ ID NO: 54); RADAAAAGGPGS (SEQ ID NO: 55); RADAAA(G4S)4 (SEQ ID NO: 56) ); SAKTTPKLEEGEFSEARV (57); ADAAP (SEQ ID NO: 58); ADAAPTVSIFPP (SEQ ID NO: 59); QPKAAP (SEQ ID NO: 60); QPKAAPSVTLFPP (SEQ ID NO 61); SEquilization number 62); AKTTPPSVTPLAP (SEQ ID NO: 63) ; AKTTAP (SEQ ID NO: 64); AKTTAPSVYPLAP (SEQ ID NO: 6576); GGGGSGGGGSGGGGS (SEQ ID NO: 66); GENKVEYAPALMALS (SEQ ID NO: 67); GPAKELTPLKEAKVS (SEQ ID NO: 68); GHEAAAVMQVQYPAS (SEQ ID NO: 69) ;TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO: 70); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 71).

"Label" and "detectable label" or "detectable moiety" means detecting a reaction between a member of a particular binding pair, e.g. an antibody and an analyte, and a particular binding partner, e.g. an antibody or an analyte. Labeled refers to a moiety that is bound to a specific binding partner, such as an antibody or an analyte, and is therefore referred to as "detectably labeled." Accordingly, the term "labeled binding protein" as used herein refers to a protein that has an incorporated label that allows identification of the binding protein. In certain embodiments, the label is a detectable marker capable of producing a signal detectable by visual or instrumental means, such as incorporation of a radiolabeled amino acid or marked avidin (e.g., optically or a fluorescent marker that can be detected by colorimetric methods or streptavidin containing enzymatic activity). Examples of labels for polypeptides include radioisotopes or radionuclides (e.g., ¹⁸ F, ¹¹ C, ¹³ N, ¹⁵ O, ⁶⁸ Ga, ¹⁸ F, ⁸⁹ Zr, ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵³ Sm); chromogens; fluorescent labels (e.g., FITC, rhodamine, and lanthanide fluorophores); enzymatic labels (e.g. horseradish peroxidase, luciferase, and alkaline phosphatase); chemiluminescent markers; biotinyl groups; defined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains); , and epitope tags); and magnetic agents such as gadolinium chelate compounds. Representative examples of labels commonly utilized for immunoassays include light-generating moieties, such as acridinium compounds, and fluorescence-generating moieties, such as fluorescein. Other labels are described herein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with yet another moiety. The use of "detectably labeled" is intended to encompass the latter detectable label.

In some embodiments, the binding affinity of an antibody or antigen-binding portion thereof to an antigen (e.g., protein complex), such as a presenting molecule-proTGFβ1 complex, is determined using BLI (e.g., an Octet® assay). Determined by A BLI (eg, Octet®) assay is an assay that determines one or more kinetic parameters indicative of binding between an antibody and an antigen. In some embodiments, the Octet® system (ForteBio®, Menlo Park, CA) is used to determine the binding affinity of an antibody or antigen-binding portion thereof to the presenting molecule-proTGFβ1 complex. . For example, the binding affinity of antibodies can be determined using a ForteBio Octet® QKe dip and readout label-free assay system that utilizes biolayer interferometry. In some embodiments, the antigen is immobilized on a biosensor (e.g., a streptavidin-coated biosensor), and the antibody and complex (e.g., a biotinylated presentation molecule-proTGFβ1 complex) are bound Presented in solution at high concentration (50 μg/mL) to measure interactions. In some embodiments, the binding affinity of the antibody or antigen binding portion thereof to the presentation molecule-proTGFβ1 complex is determined using the protocols outlined herein.

Characterizing Binding Profiles of Exemplary Antibodies to proTGFβ1 Exemplary antibodies according to the present disclosure include those with enhanced avidity (eg, subnanomolar KD). Includes a class of high affinity context-independent antibodies that can selectively inhibit TGFβ1 activation. The term "context independent" is used herein in a stricter sense compared to its more conventional usage. According to the present disclosure, the term confers a level of uniformity (ie, unbiased) in the relative affinities that antibodies can exert for different antigen complexes. Thus, the context-independent antibodies of the present disclosure can target multiple types of TGFβ1 precursor complexes (e.g., presentation molecule-proTGFβ1 complexes) and, as measured by, for example, MSD-SET, be able to bind to each such complex with equal _affinity (i.e., a <3-fold difference in relative affinity). As shown below, many antibodies encompassed by this disclosure have K _D values in the sub-nanomolar range.

Therefore, the antibody is a human presentation molecule-proTGFβ1 complex (sometimes referred to as the "large latent complex", which is a ternary complex composed of proTGFβ1 dimers bound to a single presentation molecule). That is, it can specifically bind to each of LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. Typically, recombinantly produced, purified protein complexes are used as antigens (e.g., antigen complexes) to assess or confirm the ability of antibodies to bind to antigen complexes in a suitable in vitro binding assay. be done. Such assays are well known in the art and include biolayer interferometry (BLI)-based assays (such as Octet®) and solution equilibrium titration-based assays (such as MSD-SET). , but not limited to.

BLI-based binding assays are widely known in the art for measuring the affinity and kinetics of antibodies to antigens. It is a label-free technique in which biomolecular interactions are analyzed based on optical interference. A protein, eg one of the antibodies being tested, can be immobilized on a biosensor chip. When other proteins in solution, such as antigens, bind to the immobilized antibody, it causes a change in the interference pattern that can be measured in real time. This allows monitoring of binding specificity, rates of association and dissociation, and concentration dependence. BLI is therefore a kinetic measure that reveals the dynamics of a system. Due to their ease of use and rapid results, BLI-based assays, such as the Octet® system (available from ForteBio®/Molecular Devices®, Fremont California), are a valuable tool in the screening process. It is particularly useful when used as an initial screening method to identify and separate a pool of "binders" from a pool of "non-binders" or "weak binders."

A BLI-based binding assay revealed that the novel antibody is characterized as a "context-biased/context-independent" antibody when binding affinity is measured by Octet®. As can be seen from Table 5, which summarizes the BLI-based binding profiles of non-limiting examples of antibodies, these antibodies exhibit relatively uniform sub-nanomolar KD values among the four target complexes, with relative with a low matrix-to-cell difference (<5-fold bias) (see column (H)). This can be contrasted against the previously identified antibody Ab3, which served as a reference antibody, showing significantly higher relative affinity (more than 27-fold bias) for matrix-bound complexes than for cell-bound complexes. .

Table 5 below provides non-limiting examples of context-independent proTGFβ1 antibodies encompassed by this disclosure. The table provides representative results from in vitro binding assays as measured by Octet®. Similar results are obtained with SPR-based techniques (Biacore® series).

Column (A) of the table lists monoclonal antibodies with distinct amino acid sequences. Ab3 (shown in bold) is a previously identified reference antibody that has been shown to be potent in cell-based assays; is effective in a variety of animal models; and has a safe toxicology profile. (Disclosed in International Publication No. 2018/129329 pamphlet). Columns (B), (D), (E) and (F) provide the affinity of each of the listed antibodies measured in _KD . Column (B) shows the affinity of each of the antibodies for the recombinant human LTBP1-proTGFβ1 complex; column (C) shows the affinity of each of the antibodies for the recombinant human LTBP3-proTGFβ1 complex; (E ) shows the affinity of each of the antibodies for the recombinant human GARP-proTGFβ1 complex; and (F) shows the affinity of each of the antibodies for the recombinant human LRRC33-proTGFβ1 complex. The average K _D values of (B) and (C) are shown in the corresponding column (D) and collectively represent the affinity of the antibody for ECM or matrix-bound proTGFβ1 complexes. Similarly, the average K _D values of (E) and (F) are shown in the corresponding column (G) and collectively represent the affinity of the antibody for cell surface or cell-bound proTGFβ1 complexes. Finally, the relative ratio between the average K _D values from columns (D) and (G) is expressed as "˜fold bias" in column (H). Therefore, when comparing the binding preferences of antibodies for matrix-bound complexes and cell surface complexes, the larger the number in column (H), the greater the bias exists for a particular antibody. This is one way to quantitatively represent and compare the inherent bias of antibodies toward their target complexes. Such analysis can be useful in guiding the selection process for candidate antibodies for particular therapeutic uses.

The present disclosure provides a group of high-affinity, context-independent antibodies, each of which supports four known display molecule-proTGFβ1 complexes: LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33. - can bind to each of proTGFβ1 with equal affinity. In some embodiments, the antibody binds each of the display molecule-proTGFβ1 complexes with equal or higher affinity compared to the previously described reference antibody, Ab3. According to the present disclosure, such antibodies have an affinity (determined by K _D ) of ≦5 nM as measured by a suitable in vitro binding assay such as biolayer interferometry and surface plasmon resonance. Binds specifically to each of the aforementioned complexes. In some embodiments, the antibody or fragment binds to the human LTBP1-proTGFβ1 complex with an affinity of ≦5nM, ≦4nM, ≦3nM, ≦2nM, ≦1nM, ≦5nM or ≦0.5nM. . In some embodiments, the antibody or fragment binds to the human LTBP3-proTGFβ1 complex with an affinity of ≦5nM, ≦4nM, ≦3nM, ≦2nM, ≦1nM, ≦5nM or ≦0.5nM. . In some embodiments, the antibody or fragment binds to the human GARP-proTGFβ1 complex with an affinity of ≦5nM, ≦4nM, ≦3nM, ≦2nM, ≦1nM, ≦5nM or ≦0.5nM. . In some embodiments, the antibody or fragment binds to the human LRRC33-proTGFβ1 complex with an affinity of ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, or ≦0.5 nM.

In certain embodiments, such antibodies are human- and murine-cross-reactive. Thus, in some embodiments, the antibody or fragment has an affinity for the murine LTBP1-proTGFβ1 complex with an affinity of ≦5nM, ≦4nM, ≦3nM, ≦2nM, ≦1nM, ≦5nM or ≦0.5nM. Join. In some embodiments, the antibody or fragment binds the murine LTBP3-proTGFβ1 complex with an affinity of ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, or ≦0.5 nM. In some embodiments, the antibody or fragment binds the murine GARP-proTGFβ1 complex with an affinity of ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, or ≦0.5 nM. In some embodiments, the antibody or fragment binds the murine LRRC33-proTGFβ1 complex with an affinity of ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, or ≦0.5 nM.

As shown, the proTGFβ1 antibodies of the present disclosure have particularly high affinity for matrix-bound proTGFβ1 complexes. In some embodiments, the average K _D value of the matrix-bound complexes (ie, LTBP1-proTGFβ1 and LTBP3-proTGFβ1) is ≦1 nM or ≦0.5 nM.

As shown, the proTGFβ1 antibodies of the present disclosure have high affinity for cell-bound proTGFβ1 complexes. In some embodiments, the average K _D value of the cell-associated complexes (ie, GARP-proTGFβ1 and LRRC33-proTGFβ1) is ≦2 nM or ≦1 nM.

The high affinity proTGFβ1 antibodies of the present disclosure are characterized by their uniform (unbiased) affinity for all four antigen complexes (eg, compared to Ab3). None of the single antigen complexes among the four known presentation molecule-proTGFβ1 complexes described herein deviate significantly in _KD . In other words, more uniform avidity compared to previously described proTGFβ1 antibodies (including Ab3) in that each such antibody exhibits comparable affinities across the four antigen complexes; This has been achieved by the present disclosure. In some embodiments, the antibody or fragment has a difference ( _{or range) in affinity of the antibody or fragment across the four proTGFβ1 antigen complexes between the lowest and highest K D value} of 5. It exhibits an unbiased or uniform binding profile, characterized by less than a fold. In some embodiments, the relative difference (or range) in affinity is 3-fold or less.

The concept of "uniformity" or lack of bias is further illustrated in Table 5. The average K _D values between the two matrix-bound and cell-bound complexes are calculated, respectively (see columns (D) and (G)). These average _K values are then used to ask whether a bias in avidity exists between complexes bound to the matrix and complexes bound to the cell surface (e.g., immune cells). can be done. The bias can be expressed as a "fold difference" in the mean _KD , as shown in Table 5. Compared to the previously described antibody, Ab3, the high-affinity context-independent proTGFβ1 antibodies encompassed by the present disclosure have many lower average K _D between matrix-bound and cell-bound complexes. It is not significantly biased in that it exhibits less than a 3-fold difference in value (compared to the 25-fold or more bias for Ab3).

Thus, a group of context-independent monoclonal antibodies or fragments are provided, each of which has an affinity of ≦1 nM as determined by biolayer interferometry or surface plasmon resonance for the following molecule-proTGFβ1 complexes: can bind with equal affinity to each: LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. Such antibodies specifically bind to each of the aforementioned complexes with an affinity of ≦5 nM as determined by biolayer interferometry or surface plasmon resonance, and monoclonal antibodies or fragments bind to other complexes. shows less than a 3-fold bias in affinity for any one of the above complexes in comparison, and the monoclonal antibody or fragment inhibits the release of mature TGFβ1 growth factor from each of the proTGFβ1 complexes. , from the proTGFβ2 complex or the proTGFβ3 complex.

While the kinetics of binding profiles (e.g., "on" and "off" rates) obtained from BLI-based assays provide useful information, applicants of the present disclosure Based on the mechanism of action of the activation inhibitors, i.e., antibodies, disclosed herein, which function by binding to an inactive (e.g., latent) target and thereby preventing its activation, It was contemplated that binding properties measured at equilibrium may more accurately reflect their in vivo behavior and potency. With this in mind, by way of example, an antibody with a fast "on" rate ("K _on ") that would be reflected in the binding measurements obtained by BLI would be useful as a neutralizing antibody (e.g., as an effective inhibitor). Antibodies that must directly target and rapidly capture active soluble growth factors themselves in order to function may provide suitable parameters for evaluating them. However, it may not necessarily apply to antibodies that function as activation inhibitors such as those disclosed herein. As described, the mechanism of action of the novel TGFβ1 inhibitors of the present disclosure is through inhibition of the activation step, as opposed to sequestration of soluble post-activation growth factors. This is accomplished by targeting tethered latent complexes. This is because activation inhibitors of TGFβ1 target inactive precursors localized in the respective tissues (e.g., within the ECM, on the surface of immune cells, etc.), thereby releasing the mature growth factor from the complex. This is to prevent this from happening. This mechanism of action allows the inhibitor to achieve target saturation in vivo (e.g. , equilibrium).

In view of this difference in mechanism of action, further evaluation of the binding properties was performed by the use of an alternative format of in vitro binding assay that allows determination of equilibrium affinities.

In view of this, it is believed that assays that measure the binding affinity of such antibodies at equilibrium may more accurately represent the mode of target binding in vivo. Accordingly, a MSD-SET-based binding assay (or other suitable assay) can be performed as illustrated in Table 6 below.

Solution equilibrium titration (“SET”) is an assay in which the binding between two molecules (such as an antigen and an antibody that binds to the antigen) can be measured at equilibrium in solution. For example, Meso-Scale Discovery (“MSD”)-based SET, or MSD-SET, is a useful modality to determine dissociation constants for particularly high-affinity protein-protein interactions at equilibrium (e.g., Ducata et al. (2015) J Biomolecular Screening 20(10):1256-1267). SET-based assays are particularly useful for determining KD values for antibodies with subnanomolar (eg, picomolar) affinities.

Table 6 also includes three previously described TGFβ1 selective antibodies (C1, C2 and Ab3) as reference antibodies. C1 and C2 were first disclosed in PCT/US Patent Application Publication No. 2017/021972, published as WO 2017/156500 pamphlet (where they correspond to "Ab1" and "Ab2"), Ab3 was described in PCT/US Patent Application Publication No. 2018/012601, published as WO 2018/129329 (corresponding there to "Ab3").

As can be seen from the affinity data provided in Table 6, the avidity of the novel antibodies according to the present disclosure is significantly higher than the previously identified reference antibodies. Furthermore, the novel TGFβ1 antibodies are “in context” in that they bind to each of the human LLC complexes with comparable affinity (e.g., with a K _D of approximately subnanomolar range, e.g., <1 nM). Not dependent.” The high-affinity, context-independent binding profile may make these antibodies advantageous for use in the treatment of TGFβ1-related indications that involve dysregulation of both ECM-related and immune components, such as cancer. It suggests that there is.

For binding assays based on solution equilibrium titrations, a protein complex comprising one of the presentation molecules such as those shown above can be utilized as the antigen (presentation molecule-TGFβ1 complex or LLC). The test antibody forms an antigen-antibody complex in solution. The antigen-antibody reaction mixture is incubated to reach equilibrium; the amount of antigen-antibody complex present in the assay reaction can be determined by any suitable means well known in the art. Compared to BLI-based assays, SET-based assays are less affected by the on/off kinetics of antigen-antibody complexes, thus allowing delicate detection of very high affinity interactions. As shown in Table 6, in the present disclosure, certain high affinity inhibitors of TGFβ1 are present at sub-nanomolar (e.g., picomolar) concentrations across all large latent complexes tested, as determined by SET-based assays. concentration) range of affinities.

Accordingly, a group of context-independent monoclonal antibodies or fragments are provided, each of which has a K _D of ≦1 nM as determined by solution equilibrium titration assay for the following human-presenting molecule-proTGFβ1 complexes, e.g., MSD -SET: can bind with equal affinity to each of hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. Such antibodies specifically bind to each of the aforementioned complexes with a K _D of ≦1 as determined by MSD-SET, and the monoclonal antibodies or fragments bind mature TGFβ1 proliferation derived from each of the proTGFβ1 complexes. It inhibits the release of factors, but not those derived from proTGFβ2 or proTGFβ3 complexes. In certain embodiments, such antibodies or fragments bind to each of the aforementioned complexes with a K _D of 500 pM or less (i.e., ≦500 pM), 250 pM or less (i.e., ≦250 pM), or 200 pM or less (i.e., ≦200 pM). . Even more preferably, such antibodies or fragments bind to each of the aforementioned complexes with a K _D of 100 pM or less (ie ≦100 pM). In some embodiments, the antibody or fragment does not bind free TGFβ1 growth factor that is not bound to the prodomain complex. In some embodiments, the antibody or fragment does not bind to LTBP1/TGFβ2 or LTBP3/TGFβ3 LLC. This can be tested or confirmed by suitable in vitro binding assays known in the art, such as biolayer interferometry.

In further embodiments, such antibodies or fragments are also cross-reactive with murine (eg, rat and/or mouse) and/or non-human primate (eg, cynomolgus monkey) counterparts. By way of example, Ab6 can bind with high affinity to each of the large latent complexes of multiple species, including human, murine, rat, and cynomolgus monkey, as illustrated in Table 7 below.

Surface plasmon resonance (SPR) provides useful binding kinetic information with good resolution and sensitivity that allows detection of unlabeled biomolecular interactions (such as antibody-antigen interactions) in real time. SPR-based biosensors (such as the Biacore system) can be used in determining active concentrations and characterizing molecular interactions with respect to both affinity and chemical kinetics. In addition to having a high overall affinity (usually expressed as the equilibrium dissociation constant or _KD ), it targets the latent prodomain complex and inhibits the activation step of growth factors derived from the latent complex. For antibodies that inhibit K OFF, it may be particularly advantageous to have a slow off rate, or K _OFF . As exemplified in Example 1 of PCT/U.S. Patent Application Publication No. 2019/041373, Ab6, an activation inhibitor of TGFβ1, has a K _D of less than 0.5 nM and 10 Binds to each LLC with a K _OFF of less than .0E-4 (1/s). Thus, the off-rate of an antibody may be an important binding kinetic criterion for selection considerations for therapeutic antibodies to be produced and for use in human therapy as described herein.

Accordingly, the present invention includes TGFβ inhibitors that are antibodies or antigen-binding fragments thereof for use in the treatment of cancer in a subject (according to the present disclosure), with a K _OFF of less than 10.0E-4 (1/s). An antibody or fragment is selected that has a K _D of less than 0.5 nM, as measured by SPR.

The present invention is a method for producing a pharmaceutical composition comprising a TGFβ inhibitor that is an antibody or antigen-binding fragment thereof for use in the treatment of cancer in a subject (according to the present disclosure), comprising: 10.0E-4 The method further comprises selecting an antibody or antigen-binding fragment that has a K _OFF of less than (1/s) and, optionally, a K _D of less than 0.5 nM, as measured by SPR.

Efficacy The antibodies disclosed herein can be broadly characterized as "functional antibodies" with respect to their ability to inhibit TGFβ1 signaling. As used herein, a "functional antibody" confers one or more biological activities to a target protein (eg, an antigen) through its ability to bind in a manner that modulates its function. Thus, functional antibodies broadly include those capable of modulating the activity/function of a target molecule (ie, antigen). Such regulatory antibodies include inhibitory antibodies (or inhibitory antibodies) and activating antibodies. The present disclosure relates to antibodies capable of inhibiting biological processes mediated by TGFβ signaling associated with multiple contexts of TGFβ1. Inhibitory agents used to practice the present disclosure, such as the antibodies described herein, are administered at therapeutically effective doses (doses at which sufficient efficacy is achieved within acceptable toxicity levels). It is intended to be selective for TGFβ1 and not target or interfere with TGFβ2 and TGFβ3. The novel antibodies of the present disclosure have enhanced inhibitory activity (potency) compared to previously identified activation inhibitors of TGFβ1.

In some embodiments, the efficacy of inhibitory antibodies can be measured in a suitable cell-based assay, such as the CAGA reporter cell assay described herein. Generally, cultured cells, such as xenogeneic cells and primary cells, can be used to perform cell-based efficacy assays. Cells expressing endogenous TGFβ1 and/or presentation molecules such as LTBP1, LTBP3, GARP and LRRC33 of interest may be used. Alternatively, endogenous nucleic acids encoding proteins of interest, such as TGFβ1 and/or presentation molecules of interest such as LTBP1, LTBP3, GARP and LRRC33, can be used, for example, by transfection (e.g., stable transfection or transient transfection). ) or by viral vector-based infection. In some embodiments, LN229 cells are utilized for such assays. Cells expressing TGFβ1 and presentation molecules of interest (e.g., LTBP1, LTBP3, GARP, and LRRC33) are grown in culture and harbor large latent complexes (when bound to GARP or LRRC33) on the cell surface. present' or accumulate in the ECM (when bound to LTBP). Activation of TGFβ1 can be induced by integrins expressed on the surface of other cells. The integrin-expressing cell can be the same cell or another cell type that co-expresses the large latent complex. Reporter cells are added to the assay system incorporating TGFβ response elements. Thus, the extent of TGFβ activation can be measured by detecting a signal from a reporter cell (eg, a TGFβ-responsive reporter gene, such as luciferase, coupled to a TGFβ-responsive promoter element) upon TGFβ activation. Using such cell-based assay systems, the inhibitory activity of an antibody is determined by determining the inhibitory activity of a reporter signal (e.g., luciferase activity as measured by fluorescence readout) either in the presence or absence of the test antibody. It can be determined by measuring the change (reduction) or difference. Such an assay is illustrated in Example 2 of PCT/US Patent Application Publication No. 2019/041373.

Accordingly, in some embodiments, the novel The inhibitory potency (IC ₅₀ ) of the antibody can be 5 nM or less, measured against each of the hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1 and hLRRC33-proTGFβ1 complexes. In some embodiments, the antibody has an IC ₅₀ measured against each of LLC of 2 nM or less (ie, ≦2 nM). In certain embodiments, the IC ₅₀ of the antibody measured against each of the LLC complexes is 1 nM or less. In some embodiments, the antibody has an IC ₅₀ for each of hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1 and hLRRC33-proTGFβ1 complexes of less than 1 nM.

Activation of TGFβ1 can be induced by integrin-dependent or protease-dependent mechanisms. Inhibitory activity (eg, potency) of antibodies according to the present disclosure can be assessed for their ability to block TGFβ1 activation induced by one or both modes of activation. The reporter cell assay described above is designed to measure the ability of antibodies to block or inhibit integrin-dependent activation of TGFβ1 activation. Inhibitory efficacy can also be assessed by measuring the ability of an antibody to block protease-induced activation of TGFβ1. Non-limiting embodiments of such assays have been previously disclosed. See, for example, Example 3 of PCT/US2019/041373. Thus, in some embodiments of the present disclosure, isoform-selective inhibitors according to the present disclosure can inhibit integrin-dependent activation of TGFβ1 and protease-dependent activation of TGFβ1. Such inhibitors can be used to treat TGFβ1-related conditions characterized by EDM dysregulation, including protease activity. For example, such TGFβ1-related symptoms may be associated with increased myofibroblasts, increased stiffness of the ECM, excessive or abnormal collagen deposition, or any combination thereof. Such conditions include, for example, fibrotic disorders and solid tumors or cancers including myelofibrosis (such as metastatic carcinoma).

In some embodiments, efficacy can be assessed in a suitable in vivo model as a measure of efficacy and/or pharmacokinetic effects. For example, if a first antibody is effective in an in vivo model at a particular concentration, and a second antibody is equally effective at a lower concentration than the first antibody in the same in vivo model, then the second antibody is , can be said to be more potent than the first antibody. Any suitable disease model known in the art can be used to assess the relative efficacy of TGFβ1 inhibitors depending on the particular indication of interest, eg, cancer models and fibrosis models. Preferably, multiple doses or concentrations of each test antibody are included in such a test.

Similarly, pharmacokinetic (PD) effects can be measured to determine the relative potency of inhibitory antibodies. Commonly used PD measures for the TGFβ signaling pathway include phosphorylation of SMAD2/3 and expression of downstream effector genes, TGFβ activity, such as those with TGFβ-responsive promoter elements (e.g., Smad-binding elements). Examples include, but are not limited to, transcription that is sensitive to oxidation. In some embodiments, antibodies of the present disclosure are capable of completely blocking disease-induced SMAD2/3 phosphorylation in preclinical fibrosis models when animals are administered at a dose of 3 mg/kg or less. can. In some embodiments, antibodies of the present disclosure can reduce and/or completely block disease-induced SMAD2/3 phosphorylation. In some embodiments, antibodies of the present disclosure, e.g., in the nucleus, can reduce and/or completely block disease-induced SMAD2 phosphorylation (e.g., independent of any changes in SMAD3). ). In some embodiments, the reduction is measured as the ratio of phosphorylated SMAD2/3 to total SMAD2/3. In some embodiments, the reduction is measured as the ratio of phosphorylated SMAD2 to total SMAD2. In some embodiments, antibodies of the present disclosure are capable of reducing nuclear localization of phosphorylated SMAD2, as measured, for example, by IHC.

In some embodiments, measurement of nuclear localization of phosphorylated Smad2 (P-Smad2) is performed using one or more digital image analysis parameters (e.g., individual Digital image analysis parameters that allow visualization and measurement of nuclear IHC signal intensity, such as nuclear masks) can be used. P-Smad2 positive nuclei can be classified into categories (eg, 1+, 2+, 3+) based on color intensity relative to normalized scan parameters. This analysis allows data to be considered in multiple contexts (e.g., % nuclear positivity, H-score, density) and further analysis parameters (e.g., compartment area, total number of cells quantified, number of P-Smad2 positive cells). and/or the number or percentage of 0 P-Smad2, 1+P-Smad2, 2+P-Smad2, or 3+P-Smad2 cells). A pseudocolor image mask can be applied to each stain category for visualization and QC purposes. Exemplary tools for analyzing nuclear masking include, but are not limited to, software developed by Visiopharm, Indica Labs (e.g., HALO® image analysis platform), and Flagship Biosciences. Without being bound by theory, in some embodiments, measuring SMAD2 phosphorylation, e.g., nuclear SMAD2 phosphorylation (without measurement of SMAD3), may be used to detect treatment-related effects (e.g., therapeutic effects and/or accurate detection of target binding). Denis et al. , Development 143:3481-90 (2016); Liu et al. , J. Biol. Chem. 278:11721-8 (2003); David et al. , Oncoimmunology 6: e1349589 (2017).

In some embodiments, P-Smad2 nuclear translocation can be used in a method of determining therapeutic efficacy in a subject receiving one or more doses of a TGFβ inhibitor (eg, Ab6). In some embodiments, the level of P-Smad2 nuclear translocation is determined in a tumor sample obtained from a subject before and after administration of one or more doses of a TGFβ inhibitor (e.g., Ab6), wherein: A decrease in P-Smad2 nuclear translocation after administration compared to before administration indicates a therapeutic effect. In some embodiments, P-Smad2 nuclear translocation can be determined using immunohistochemistry. In some embodiments, nuclear translocation of P-Smad2 can be determined using nuclear masking. In some embodiments, nuclear translocation of P-Smad2 can be determined using digital image analysis tools, such as those developed by Flagship Biosciences, Visiopharm, or Indica Labs. In some embodiments, the level of P-Smad2 nuclear translocation after administration compared to before administration is at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1. 6x, 1.7x, 1.8x, 1.9x, 2x, 2.2x, 2.4x, 2.6x, 2.8x, 3x, 3.5x, 4 A fold, 4.5-fold, 5-fold, or greater reduction indicates a therapeutic effect. In some embodiments, if a decrease is detected, one or more additional doses of a TGFβ inhibitor (eg, Ab6) are administered.

In some embodiments, P-Smad2 nuclear translocation can be used in a method to determine target binding in a subject who has been administered one or more doses of a TGFβ inhibitor (eg, Ab6). In some embodiments, the level of P-Smad2 nuclear translocation is determined in a tumor sample obtained from a subject before and after administration of one or more doses of a TGFβ inhibitor (e.g., Ab6), wherein: A decrease in P-Smad2 nuclear translocation after administration compared to before administration indicates target binding. In some embodiments, P-Smad2 nuclear translocation can be determined using immunohistochemistry. In some embodiments, nuclear translocation of P-Smad2 can be determined using nuclear masking. In some embodiments, nuclear translocation of P-Smad2 can be determined using digital image analysis tools, such as those developed by Flagship Biosciences, Visiopharm, or Indica Labs. In some embodiments, the level of P-Smad2 nuclear translocation after administration compared to before administration is at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1. 6x, 1.7x, 1.8x, 1.9x, 2x, 2.2x, 2.4x, 2.6x, 2.8x, 3x, 3.5x, 4 A fold, 4.5-fold, 5-fold, or greater decrease indicates target binding. In some embodiments, if a decrease is detected, one or more additional doses of a TGFβ inhibitor (eg, Ab6) are administered.

In some embodiments, P-Smad2 nuclear translocation is detected in tumor CD8+ cells to determine therapeutic efficacy and/or target binding in subjects administered one or more doses of a TGFβ inhibitor (e.g., Ab6). (e.g., percentage of CD8+ cells in various tumor compartments, e.g., tumor nest, stroma, margin). In some embodiments, the therapeutic effect and/or target binding is, inter alia, a decrease in P-Smad2 nuclear translocation in a tumor sample obtained from a subject after administration of a TGFβ inhibitor (e.g., Ab6) compared to before administration. It can be shown by In some embodiments, therapeutic efficacy and/or target binding may be indicated by a decrease in P-Smad2 nuclear translocation and an increase in CD8+ cells in a tumor sample obtained from the subject after administration compared to before administration. In some embodiments, the therapeutic effect and/or target binding is a decrease in P-Smad2 nuclear translocation and total tumor area including immunoinflammatory tumor foci in tumor samples obtained from the subject after administration compared to before administration. may be indicated by an increase in . In some embodiments, two or more additional markers are used to assess therapeutic efficacy and/or target binding associated with P-Smad2 nuclear translocation. In some embodiments, a TGFβ inhibitor (eg, Ab6) is administered in combination with checkpoint inhibitor therapy. In some embodiments, additional doses of a TGFβ inhibitor (eg, Ab6) may be administered to patients whose tumors exhibit therapeutic response and/or target binding.

In some embodiments, the antibodies of the present disclosure are administered to a series of antibodies including Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, Loxl2 when the animal is administered at a dose of 10 mg/kg or less in a UUO model of renal fibrosis. The expression of marker genes induced by fibrosis can be significantly suppressed.

Accordingly, in some embodiments, the process of selecting antibodies or antigen-binding fragments thereof for therapeutic use includes identifying antibodies or fragments that exhibit sufficient inhibitory potency. For example, the selection process may include performing a cell-based TGFβ1 activation assay to determine the potency (e.g., IC ₅₀ ) of one or more test antibodies or fragments thereof; and candidate antibodies or fragments thereof exhibiting the desired potency. and selecting. In some embodiments, the IC ₅₀ for each human LLC is 5 nM or less. The selected antibodies or fragments can then be used in the treatment of TGFβ1-related conditions described herein.

Binding Region In the context of this disclosure, the “binding region” of an antigen provides the structural basis for antibody-antigen interaction. As used herein, a "binding region" refers to a proTGFβ1 complex (an "antigen") in physiological solution, as determined by a suitable technique such as hydrogen deuterium exchange mass spectrometry (HDX-MS). When bound, the antibody or fragment refers to the region of the interface between the antibody and antigen that protects the binding region from solvent exposure. Identification of binding regions is useful in gaining insight into antigen-antibody interactions and mechanisms of action for particular antibodies. Identification of additional antibodies with similar or overlapping binding regions can be facilitated by cross-blocking experiments that allow epitope binning. Optionally, X-ray crystallography can be utilized to identify the precise amino acid residues of the epitope that mediate antigen-antibody interactions.

The technique is well known for HDX-MS, a widely used technique for probing protein conformations or protein-protein interactions in solution. This method relies on the exchange of hydrogen in the amide of the protein backbone by deuterium present in solution. By measuring the hydrogen/deuterium exchange rate, information about protein dynamics and tertiary structure can be obtained (Wei et al., (2014) “Hydrogen/deuterium exchange mass spectrometry for probing higher order”, incorporated by reference). Structure of protein therapeutics: methodology and applications.”Drug Discov Today. 19(1):95-102). The application of this technique is based on the premise that when antibody-antigen complexes occur, the interface between the binding partners can reduce or impede the exchange rate through steric exclusion of the solvent by excluding it.

The present disclosure includes antibodies or antigen-binding fragments thereof that bind to human LLC with a region that includes latent Lasso or a portion thereof (a "binding region"). Latent Lasso is a protein module within the prodomain. It is believed that many potent activation inhibitors can bind to this region of the proTGFβ1 complex in such a way that antibody binding “locks in” the growth factor and thereby prevents its release. Interestingly, this is the section of the complex where the butterfly-like elongated regions of the growth factor (eg, corresponding to finger-1 and finger-2) interact closely with the cage-like structure of the prodomain. Based on the data disclosed in PCT/U.S. Patent Application Publication No. 2019/041373, antibodies that wrap tightly around the identified binding region cause the proTGFβ1 complex to dissociate (i.e., release the growth factor). It is anticipated that activation can be blocked by effectively preventing

Using HDX-MS technology, the binding region of proTGFβ1 can be determined. In some embodiments, the portion on proTGFβ1 identified as important in binding the antibody or fragment includes at least a portion of the prodomain and at least a portion of the growth factor domain. Antibodies or fragments that bind to the first binding region (“region 1” in FIG. 41) containing at least a portion of latent Lasso are preferred. More preferably, such an antibody or fragment further binds to a second binding region (“Region 2” in FIG. 41) comprising at least a portion of the growth factor domain at finger-1 of the growth factor domain. Such antibodies or fragments may further bind to a third binding region ("region 3" in FIG. 41) that includes at least a portion of finger-2 of the growth factor domain.

Additional regions within proTGFβ1 may also directly or indirectly contribute to the high affinity interactions of these antibodies disclosed herein. Regions considered important for mediating high affinity binding of antibodies to the proTGFβ1 complex are LVKRKRIEA (SEQ ID NO: 132); LASPPSQGEVP (SEQ ID NO: 133); PGPLPEAV (SEQ ID NO: 134); LALYNSTR (SEQ ID NO: 134); REAVPEPVL (SEQ ID NO: 136); YQKYSNNSWR (SEQ ID NO: 137); RKDLGWKWIHEPKGYHANF (SEQ ID NO: 138); LGPCPYIWS (SEQ ID NO: 139); ALEPLPIV (SEQ ID NO: 140); oTGFβ1 (based on native sequences).

Among the regions that may contribute to antibody-antigen interaction, in some embodiments, high affinity antibodies of the present disclosure contain at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL (“Region 1”) (SEQ ID NO: 142). can bind to epitopes containing epitopes.

In some embodiments, high affinity antibodies of the present disclosure may bind to an epitope comprising at least one residue of the amino acid sequence RKDLGWKWIHEPKGYHANF (“Region 2”) (SEQ ID NO: 138).

In some embodiments, high affinity antibodies of the present disclosure can bind to an epitope comprising at least one residue of the amino acid sequence VGRKPKVEQL (“Region 3”) (SEQ ID NO: 141).

In some embodiments, high affinity antibodies of the present disclosure have at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL ("Region 1") (SEQ ID NO: 142) and the amino acid sequence RKDLGWKWIHEPKGYHANF ("Region 2") (SEQ ID NO: 138). ) may bind to an epitope comprising at least one residue of

In some embodiments, high affinity antibodies of the present disclosure have at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL ("Region 1") (SEQ ID NO: 142) and the amino acid sequence VGRKPKVEQL ("Region 3") (SEQ ID NO: 141). ) may bind to an epitope comprising at least one residue of

In some embodiments, the high affinity antibodies of the present disclosure have at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL ("Region 1") (SEQ ID NO: 142), the amino acid sequence RKDLGWKWIHEPKGYHANF ("Region 2") (SEQ ID NO: 138). ) and at least one residue of the amino acid sequence VGRKPKVEQL (“Region 3”) (SEQ ID NO: 141).

In addition to contributions from regions 1, 2 and/or 3, such epitopes include LVKRKRIEA (SEQ ID NO: 132); LASPPSQGEVP (SEQ ID NO: 133); PGPLPEAV (SEQ ID NO: 134); LALYNSTR (SEQ ID NO: 135); REAVPEPVL (SEQ ID NO: 136); YQKYSNNSWR (SEQ ID NO: 137); RKDLGWKWIHEPKGYHANF (SEQ ID NO: 138); LGPCPYIWS (SEQ ID NO: 139); ALEPLPIV (SEQ ID NO: 140); and VGRKPKVEQL (SEQ ID NO: 141); may further include at least one amino acid residue derived from

Remarkably, many of the binding regions identified in structural studies using four representative isoform-selective TGFβ1 antibodies were found to be overlapping, indicating the potential of the proTGFβ1 complex. Refers to specific regions within the proTGFβ1 complex that may be particularly important for maintenance. Thus, advantageously, antibodies or fragments thereof may be selected based at least in part on their binding regions that contain overlap identified across multiple inhibitors described herein. These overlapping portions of binding regions include, for example, SPPSQGEVPPGPLPEAVL (SEQ ID NO: 165), WKWIHEPKGYHANF (SEQ ID NO: 166), and PGPLPEAVL (SEQ ID NO: 167). Accordingly, high affinity isoform-selective TGFβ1 inhibitors according to the present disclosure contain one or more amino acid residues of SPPSQGEVPPGPLPEAVL (SEQ ID NO: 165), WKWIHEPKGYHANF (SEQ ID NO: 166) and/or PGPLPEAVL (SEQ ID NO: 167). may bind to the proTGFβ1 complex (eg, human LLC) at an epitope containing the proTGFβ1 complex.

Accordingly, any of the antibodies or antigen-binding fragments encompassed by this disclosure, such as Categories 1-5 antibodies or fragments disclosed herein, bind to one or more of the binding regions identified herein. It is possible. Such antibodies can be used in treating TGFβ1 symptoms in a subject as described herein. Accordingly, a selection of antibodies or antigen-binding fragments thereof suitable for therapeutic use according to the present disclosure include those that bind SPPSQGEVPPGPLPEAVL (SEQ ID NO: 165), WKWIHEPKGYHANF (SEQ ID NO: 166), PGPLPEAVL (SEQ ID NO: 167), or any portion thereof. identifying or selecting an antibody or fragment thereof that

Non-limiting examples of protein domains or motifs of human proTGFβ1 as previously described (WO 2014/182676) are provided in Table 9.

Safety/Toxicity The development of TGFβ inhibitors remains a challenge due to the need to identify therapies with the desired pharmacological effects and sufficient therapeutic window that also eliminate on-target toxicity. The majority of TGFβ inhibitors, including monoclonal antibodies and small molecule kinase inhibitors (SMIs), are nonselective for either multiple TGFβ isoforms or TGFβ receptors that mediate signaling from all three TGFβ isoforms. be targeted. Unfortunately, these inhibitors have been shown to have limited efficacy, mainly due to lack of efficacy (Akhurst 2017; Cohn 2014; Voelker 2017), unfavorable safety profile, or both (Tolcher 2017; Volker 2017; Cohn 2014). did not demonstrate promising clinical data in cancer patients. Toxicity associated with these molecules includes cardiovascular abnormalities, epithelial thickening, gastrointestinal tract abnormalities, and skin lesions. Each of these toxicities has been characterized in multiple animal species (e.g., rodents, dogs, and cynomolgus monkeys) in studies ranging in duration from 1-2 weeks to 6 months (Lonning 2011; Stauber 2014; Mitra 2020). Among these toxicities, irreversible cardiovascular inflammatory lesions, bleeding and thickening in heart valves, and arterial lesions, including the aorta and coronary arteries, are of major concern.

Conventional pan-inhibitors of TGFβ, which can attenuate multiple isoforms, have been shown to reduce cardiovascular toxicity, including cardiovascular toxicity (heart lesions, most notably valvular disease), which have been reported across multiple species, including dogs and rats. known to cause toxicity. These include thickening in the aortic valve, right AV valve, and left AV valve; inflammation in the aortic valve, left AV valve, and ascending aorta; bleeding in the ascending aorta, aortic valve, and left AV valve; connective tissue degeneration in the ascending aorta ( For example, Strauber et al., (2014) “Nonclinical safety evaluation of a Transforming Growth Factor β receptor I kinase inhibitor in Fisch” er 344 rats and beagle dogs” J. Clin. Pract 4(3):1000196).

In addition, neutralizing antibodies that bind all three TGFβ isoforms are associated with specific epithelial toxicities observed across multiple species, some of which are summarized below.

Previous recognition by the applicant of the present disclosure that the lack of isoform specificity of conventional TGFβ antagonists may underlie the cause of toxicity associated with TGFβ inhibition (PCT/U.S. Patent Application Publication No. 2017/021972) (see specification), the present inventors aimed to treat various diseases exhibiting pleiotropic TGFβ1 dysregulation while maintaining the safety/tolerability profile of isoform-selective inhibitors. We further sought to achieve therapeutic broad-spectrum TGFβ1 inhibition.

In clinical practice, therapeutic efficacy is achieved only when the minimum effective concentration (MEC) of a drug (eg, a monoclonal antibody) is less than the minimum toxic concentration (MTC) of the drug. This was not achieved by most, if not all, of the conventional pan-inhibitors of TGFβ, and in fact appeared to cause dose-limiting toxicity. Applicant's previous studies have demonstrated that isoform-selective inhibitors of TGFβ1 showed a significantly improved safety profile compared to traditional pan-inhibitors such as small molecule receptor antagonists and neutralizing antibodies. Described. WO 2017/156500 discloses isoform-selective inhibitors of TGFβ1 activation that exhibit test article-related toxicity when administered at doses up to 100 mg/kg per week for 4 weeks in rats. was not observed, establishing the NOAEL for the antibody as the highest dose tested, ie 100 mg/kg. Applicants' subsequent studies have also shown that antibodies with enhanced function exhibit a comparable safety profile. Herein, one of the objectives was to identify antibodies with even higher affinity and potency, but with at least the same or comparable level of safety.

Results of a 4-week rat toxicity study are described in PCT/US Patent Application Publication No. 2019/041373, see for example the data provided in FIG. 24B. Two isoform-selective TGFβ1 inhibitors (Ab3 and Ab6) were tested in separate studies, along with a small molecule ALK5 inhibitor and a monoclonal neutralizing antibody as controls. No toxicity associated with the test article was observed with any of the isoform-selective antibodies, while, as expected, the non-selective inhibitors caused a variety of adverse events, consistent with published studies. caused it. In contrast to treatments that broadly block TGFβ signaling, Ab6 did not exhibit cardiotoxicity in a 4-week non-GLP pilot toxicity study in rats, which may be due to selective activation of the TGFβ1 isoform. suggesting that inhibition may have an improved safety profile compared to pan-TGFβ inhibitors. Furthermore, Ab6 was shown to be safe (eg, no adverse events were observed) at dose levels as high as 300 mg/kg in cynomolgus monkeys when administered weekly for 4 weeks. Ab6 has been shown to be effective in several in vivo models at dose levels as low as 3 mg/kg, thus providing up to a 100-fold therapeutic window. Importantly, this demonstrates that high efficacy does not necessarily mean greater risk of toxicity. Without being bound to any particular theory, it is believed that the highly selective nature of the antibodies disclosed herein is likely the reason for the observed lack of toxicity.

Thus, in some embodiments, novel antibodies according to the present disclosure have a maximum tolerated dose (MTD) of >100 mg/kg when administered weekly for at least 4 weeks. In some embodiments, novel antibodies according to the present disclosure have a no observed adverse effect level (NOAEL) of up to 100 mg/kg when administered weekly for at least 4 weeks. Suitable animal models to be used to perform safety/toxicity testing for TGFβ inhibitors and TGFβ1 inhibitors include, but are not limited to, rats, dogs, cynomolgus monkeys, and mice. . In certain embodiments, the minimum effective amount of the antibody based on suitable preclinical efficacy studies is below the NOAEL. More preferably, the minimum effective amount of antibody is about one-third or less of the NOAEL. In certain embodiments, the minimum effective amount of antibody is about one-sixth of the NOAEL or less. In some embodiments, the minimum effective amount of antibody is about 10 times less than the NOAEL.

In some embodiments, the present disclosure encompasses isoform-selective antibodies that are capable of inhibiting TGFβ1 signaling and, when administered to a subject, are administered to the heart at a dose effective to treat TGFβ1-related symptoms. Does not cause vascular or known epithelial toxicity. In some embodiments, the antibody has a minimum effective amount of about 3-10 mg/kg administered weekly, biweekly, or monthly. Preferably, the antibody causes no to minimal toxicity at a dose that is at least 6 times the minimally effective dose (eg, 6 times the therapeutic range). More preferably, the antibody causes no to minimal toxicity at a dose that is at least 10 times the minimally effective dose (eg, a 10 times therapeutic range). Even more preferably, the antibody causes no to minimal toxicity at a dose that is at least 15 times the minimum effective dose (eg, a 15 times therapeutic range).

Therapeutic agents that bind to immune cells pose a potential risk of activating immune cells when administered to a patient. Therefore, when selecting a TGFβ inhibitor for therapeutic use, determine that the candidate inhibitor does not induce a proinflammatory cytokine response (e.g., cytokine release) in human peripheral blood mononuclear cells (PBMCs). It is important to confirm whether Inflammatory cytokines include, for example, IFNγ, IL-2, IL-1β, TNFα, CCL2 and IL-6. In some embodiments, an acceptable level of cytokine release induced by a test agent (candidate inhibitor) is a response within 2.5-fold compared to a solvent control (eg, IgG).

Accordingly, the present disclosure provides: i) the selection of TGFβ inhibitors that have not been shown to induce dangerous levels of pro-inflammatory cytokine release in human PBMC; and ii) the administration of therapeutically effective amounts to patients to treat the condition. TGFβ inhibitors are provided for use in the treatment of TGFβ-related conditions (eg, cancer, myelofibrosis, fibrosis, etc.) in human patients, including administration of compositions comprising the TGFβ inhibitor. In some embodiments, the TGFβ inhibitor does not induce dangerous levels of cytokine release from human PBMC at an amount that is at least three times the therapeutically effective amount. Preferably, at least 5 times the therapeutically effective amount of TGFβ inhibitor does not cause dangerous levels of cytokine release in human PBMC.

It has been reported that human platelets express latent TGFβ1. Pharmacological interventions that target platelets can cause undesired effects on platelet function, such as platelet aggregation and activation, and can lead to coagulation dysregulation. Therefore, it is important to determine or ensure that a candidate inhibitor does not cause unwanted platelet activation or interfere with the normal function of platelets.

Accordingly, the present disclosure provides: i) selection of TGFβ inhibitors that have been shown not to cause platelet aggregation or activation; and ii) compositions comprising therapeutically effective amounts of TGFβ inhibitors to patients for treating conditions. TGFβ inhibitors are provided for use in the treatment of TGFβ-related conditions (eg, cancer, myelofibrosis, fibrosis, etc.) in human patients, including administration of TGFβ inhibitors. In some embodiments, the TGFβ inhibitor does not cause spontaneous or ADP-induced platelet activation in a dose-dependent manner at an amount that is at least three times the therapeutically effective amount. Preferably, at least five times the therapeutically effective amount of TGFβ inhibitor does not cause platelet activation. In certain embodiments, the TGFβ inhibitor does not inhibit ADP-induced platelet activation in a dose-dependent manner at an amount that is at least three times the therapeutically effective amount. Preferably, at least five times the therapeutically effective amount of TGFβ inhibitor does not inhibit platelet activation.

The present disclosure includes TGFβ inhibitors for use in the treatment of cancer in human patients, the treatment comprising: i) selecting a TGFβ inhibitor that has been shown to be effective and safe in preclinical models; and ii) administering to the human patient an effective dose of a TGFβ inhibitor, optionally the TGFβ inhibitor being effective in reducing tumor burden when used in combination with a checkpoint inhibitor. , further optionally, the TGFβ inhibitor does not induce platelet activation in human blood samples and does not cause inflammatory cytokine release in PBMCs at doses greater than the minimum effective dose; and further optionally, the TGFβ inhibitor , does not cause unacceptable adverse events, as assessed in standard toxicity studies in one or more preclinical models, where the NOAEL is at least 10 times the lowest effective dose.

Accordingly, selection of antibodies or antigen-binding fragments thereof for therapeutic use involves selecting antibodies or antigen-binding fragments that meet one or more of the criteria in categories 1-5 described herein; or performing in vivo efficacy studies in a suitable preclinical model to determine an effective amount of the fragment; in vivo safety/toxicity testing in a suitable model to determine the amount of antibody that is safe or toxic; performing tests (e.g., MTD, NOAEL, cytokine release, effect on platelets, or any art-recognized parameter to assess safety/toxicity); and at least a 3-fold therapeutic range (preferably The method may include selecting antibodies or fragments that provide a 6-fold, more preferably 10-fold therapeutic window, and even more preferably a 15-fold therapeutic window. In a preferred embodiment, in vivo efficacy testing is performed in two or more suitable preclinical models that reproduce the human condition. In some embodiments, such preclinical models include TGFβ1 positive cancers and may optionally include immunosuppressive tumors. Immunosuppressive tumors can be resistant to cancer therapies such as CBT, chemotherapy and radiotherapy (such as radiotherapeutic agents). In some embodiments, the preclinical model is selected from MBT-2, Cloudman S91 and EMT6 tumor models.

The selected antibodies or fragments can be used in the manufacture of pharmaceutical compositions containing the antibodies or fragments. Such pharmaceutical compositions can be used in treating TGFβ1 symptoms in a subject as described herein. For example, TGFβ1 symptoms can be proliferative and/or fibrotic disorders.

Mechanism of Action The antibodies of the present disclosure useful as therapeutic agents are inhibitory antibodies of TGFβ1. Furthermore, the antibody is an activation inhibitor, ie, the antibody blocks the activation step of TGFβ1, rather than following directly after already activating the growth factor.

In a broad sense, the term "inhibitory antibody" refers to an antibody that attenuates or neutralizes a target function, such as growth factor activity. Advantageously, certain inhibitory antibodies of the present disclosure are capable of inhibiting mature growth factor release from latent complexes, thereby reducing growth factor signaling. Inhibitory antibodies include antibodies that target any epitope that reduces growth factor release or activity when bound by such antibodies. Such epitopes can be on the prodomain of TGFβ proteins (eg, TGFβ1), growth factors, or other epitopes that result in reduced growth factor activity when bound by antibodies. Inhibiting antibodies of the present disclosure include, but are not limited to, TGFβ1-inhibiting antibodies. In some embodiments, the inhibitory antibodies of the present disclosure specifically bind to a combinatorial epitope, ie, an epitope formed by two or more components/portions of an antigen or antigen complex. For example, a combinatorial epitope can be formed by contributions from amino acid residues from multiple parts of a single protein, ie, two or more non-contiguous compartments of the same protein. Alternatively, a combinatorial epitope can be formed by contributions from multiple protein components of an antigen complex. In some embodiments, the inhibitory antibodies of the present disclosure are specific for conformational epitopes (or conformation-specific epitopes), e.g., epitopes that are sensitive to the three-dimensional structure (i.e., conformation) of an antigen or antigen complex. to combine.

Traditional approaches to attenuating TGFβ signaling include: i) reducing the amount of free ligand available for receptor binding (e.g., released from its latent precursor complex) after it has already become active; directly neutralizing the factor; ii) utilizing soluble receptor fragments capable of capturing free ligand (e.g. so-called ligand traps); or iii) targeting its cell surface receptors to release ligand- The goal was to block the receptor interaction. Each of these conventional approaches requires an antagonist to compete against its endogenous counterpart. Furthermore, the first two approaches (i and ii) above target active ligands that are transient species. Therefore, such antagonists need to be able to kinetically eliminate endogenous receptors within a short time frame. A third approach is that many growth factors (e.g., up to about 20) signal through the same receptor, which can lead to relatively long-lasting effects, but can also result in unintentionally unwanted inhibitory effects (and thus , potential toxicity).

To provide a solution to these shortcomings and further enable greater selectivity and localization of action, the underlying mechanism of action of inhibitory antibodies such as those described herein is Acts upstream of TGFβ1 activation and ligand-receptor interaction. Accordingly, high-affinity isoform-specific context-independent inhibitors of TGFβ1 suitable for carrying out the present disclosure preferably inhibit the activation step at its source (such as in the disease microenvironment, e.g., in the TME). ), it is believed that inactive (e.g., latent) precursor TGFβ1 complexes (e.g., complexes containing pro/latent TGFβ1) should be targeted prior to their activation. According to certain embodiments of the present disclosure, such inhibitors are not free ligands that are transiently available for receptor binding, but rather ECM-bound and cell surface-tethered pro/latent TGFβ1 complexes. Targets both of the body with equal affinity.

Recent studies have shown that the benefits of locally targeting complexes tethered to tissues/cells at the source, as opposed to soluble active species (i.e., mature growth factors after release from the source), are Further support. Ishihara et al. , (Sci. Transl. Med. 11, eaau3259 (2019) “Targeted antibody and cytokine cancer immunotherapies through collagen affinity”) was administered systemically. Drugs are targeted to tumor sites by binding to collagen binding moieties reported that when administered, they were able to enhance anti-tumor immunity and reduce treatment-related toxicity compared to non-targeted counterparts.

The mechanism of action achieved by the antibodies of the present disclosure may further contribute to enhanced durability of effect and greater overall efficacy and safety.

Interestingly, these antibodies may exert additional inhibitory activity against cell-bound TGFβ1 (LRRC33-proTGFβ1 and GARP-proTGFβ1). Applicants have found that LRRC33-binding antibodies tend to internalize upon binding to cell surface LRRC33. It is unclear whether internalization is actively induced by antibody binding or whether this phenomenon is instead due to the natural (eg, passive) endocytic activity of macrophages. However, the high affinity isoform-selective TGFβ1 inhibitor Ab6 can be rapidly internalized into cells transfected with LRRC33 and proTGFβ1, and the rate of internalization achieved with Ab6 is significantly higher than that with the reference antibody that recognizes cell surface LRRC33. Similar results are obtained with primary human macrophages. These observations raise the possibility that Ab6 can induce internalization upon binding to its target LRRC33-proTGFβ1, thereby removing the LRRC33-containing complex from the cell surface. At disease sites, this may reduce the availability of latent LRRC33-proTGFβ1 levels that can be activated. Therefore, isoform-selective TGFβ1 inhibitors have two parallel mechanisms of action: i) release of mature growth factors from latent complexes; and ii) release of LRRC33-proTGFβ1 from the cell surface via internalization. The LRRC33 arm of TGFβ1 can be inhibited through removal of the complex. It is possible that a similar inhibitory mechanism of action may apply to GARP-proTGFβ1.

In some embodiments, the antibody is a pH-sensitive antibody that binds its antigen with higher affinity at neutral pH (such as a pH of approximately 7) than at acidic pH (such as a pH of approximately 5). Such antibodies may have a higher dissociation rate under acidic conditions than under neutral or physiological conditions. For example, the ratio between the dissociation rate measured at acidic pH and the dissociation rate measured at neutral pH (e.g., K _off at pH 5 relative to K _off at pH 7) can be at least 1.2. . Optionally, the ratio is at least 1.5. In some embodiments, the ratio is at least 2. Such pH-sensitive antibodies may be useful as recycling antibodies. Upon target binding on the cell surface, antibodies can induce antibody-dependent internalization (and thus removal) of membrane-bound proTGFβ1 complexes (associated with LRRC33 or GARP). The antibody-antigen complex then dissociates in acidic intracellular compartments such as lysosomes, and free antibody can be transported to the extracellular domain.

Thus, in some embodiments, the selection of antibodies or antigen-binding fragments for therapeutic use may be based in part on the ability to induce antibody-dependent internalization and/or on the pH dependence of the antibody. .

Antigen Complexes and Components Thereof The novel antibodies of the present disclosure are specific for each of the four known human large latent complexes (e.g., hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1 and hLRRC33-proTGFβ1). specifically inhibits TGFβ1 activation.

Screening (eg, identification and selection) for such antibodies typically involves the use of suitable antigen complexes produced recombinantly. Useful protein components are provided that can include such antigen complexes, including TGFβ isoforms and related polypeptides, fragments and variants, presentation molecules (e.g., LTBP, GARP, LRRC33) and related polypeptides, fragments and variants. Ru. These components can be expressed and purified to form protein complexes (such as large latent complexes) that can be used in antibody screening processes. Screening can include a positive selection, in which desirable binders are selected from a pool or library of binders and non-binders, and a negative selection, in which undesirable binders are removed from the pool. Typically, at least one matrix binding complex (e.g. LTBP1-proTGFβ1 and/or LTBP1-proTGFβ1) and at least one cell binding complex (e.g. GARP-proTGFβ1 and/or LRRC33-proTGFβ1) are present for positive screening. included to ensure that the binder selected has affinity for both such biological contexts.

In some embodiments, TGFβ1 comprises a naturally occurring mammalian amino acid sequence. In some embodiments, TGFβ1 comprises naturally occurring human amino acid sequences. In some embodiments, TGFβ1 comprises a human, monkey, rat or mouse amino acid sequence. In some embodiments, the antibodies or antigen-binding portions thereof described herein do not specifically bind TGFβ2. In some embodiments, the antibodies or antigen-binding portions thereof described herein do not specifically bind TGFβ3. In some embodiments, the antibodies or antigen-binding portions thereof described herein do not specifically bind TGFβ2 or TGFβ3. In some embodiments, the antibodies or antigen-binding portions thereof described herein specifically bind TGFβ1 comprising the amino acid sequence set forth in SEQ ID NO:23. The amino acid sequence of TGFβ2 and TGFβ3 are set forth in SEQ ID NOs: 27 and 21, respectively. In some embodiments, the antibodies described herein or antigen-binding portions thereof include TGFβ1 (otherwise referred to herein as non-naturally occurring TGFβ1) that comprises a non-naturally occurring amino acid sequence. specifically binds to. For example, a non-naturally occurring TGFβ1 may include one or more recombinantly generated mutations to the naturally occurring TGFβ1 amino acid sequence. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequence comprises an amino acid sequence as set forth in SEQ ID NOs: 13-24 as shown in Table 11. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequence comprises an amino acid sequence as set forth in SEQ ID NOs: 25-32 as shown in Table 12.

TGF-β1 (prodomain + growth factor domain)

TGF-β2 (prodomain + growth factor domain)

TGF-β3 (prodomain + growth factor domain)

In some embodiments, the antigenic protein complex (e.g., LTBP-TGFβ1 complex) is one of the LTBP proteins (e.g., LTBP1, LTBP2, LTBP3 and LTBP4), GARP protein, LRRC33 protein, or a fragment thereof. or more display molecules. Typically, the minimum required fragment suitable for practicing embodiments disclosed herein will contain at least 50 cysteine residues that are capable of forming a disulfide bond with the proTGFβ1 complex. of amino acids, preferably at least 100 amino acids. Specifically, these Cys residues form a covalent bond with a cysteine residue present near the N-terminus of each monomer of the proTGFβ1 complex. In the three-dimensional structure of the proTGFβ1 dimeric complex, the so-called “alpha-1 helix” at the N-terminus of each monomer is brought into close proximity to each other and to the corresponding pair of cysteines present in the display molecule. Adjust the distance between the two cysteine residues (from each helix) required to form a productive covalent bond (e.g., Cuende et al., (2015) Sci. Trans. Med. 7:284ra56 Please refer to ). Therefore, when fragments of display molecules are used to form LLCs in screening processes (e.g., immunization, library screening, identification, and selection), such fragments are separated by cysteine residues by appropriate distances. group should be included to allow proper disulfide bond formation with the proTGFβ1 complex in order to preserve the correct conformation of the resulting LLC. For example, LTBPs (eg, LTBP1, LTBP3, and LTBP4) may contain a "cysteine-rich domain" that mediates covalent interaction with proTGFβ1.

Antibodies or antigen binding portions thereof as described herein are capable of binding to the LTBP1-TGFβ1 complex. In some embodiments, the LTBP1 protein is a naturally occurring protein or a fragment thereof. In some embodiments, the LTBP1 protein is a non-naturally occurring protein or a fragment thereof. In some embodiments, the LTBP1 protein is a recombinant protein. Such recombinant LTBP1 protein may comprise LTBP1, alternatively spliced variants thereof and/or fragments thereof. Recombinant LTBP1 protein can also be modified to include one or more detectable labels. In some embodiments, the LTBP1 protein includes a leader sequence (eg, a natural or non-natural leader sequence). In some embodiments, the LTBP1 protein does not include a leader sequence (ie, the leader sequence has been processed or cleaved). Such detectable labels may include, but are not limited to, biotin labels, polyhistidine tags, myc tags, HA tags, and/or fluorescent tags. In some embodiments, the LTBP1 protein is a mammalian LTBP1 protein. In some embodiments, the LTBP1 protein is a human, monkey, mouse, or rat LTBP1 protein. In some embodiments, the LTBP1 protein comprises the amino acid sequence as set forth in SEQ ID NO: 35 and 36 in Table 12. In some embodiments, the LTBP1 protein comprises the amino acid sequence as set forth in SEQ ID NO: 39 in Table 14.

Antibodies or antigen binding portions thereof as described herein are capable of binding to the LTBP3-TGFβ1 complex. In some embodiments, the LTBP3 protein is a naturally occurring protein or a fragment thereof. In some embodiments, the LTBP3 protein is a non-naturally occurring protein or a fragment thereof. In some embodiments, the LTBP3 protein is a recombinant protein. Such recombinant LTBP3 protein may comprise LTBP3, alternatively spliced variants thereof and/or fragments thereof. In some embodiments, the LTBP3 protein includes a leader sequence (eg, a natural or non-natural leader sequence). In some embodiments, the LTBP3 protein does not include a leader sequence (ie, the leader sequence has been processed or cleaved). Recombinant LTBP3 protein can also be modified to include one or more detectable labels. Such detectable labels may include, but are not limited to, biotin labels, polyhistidine tags, myc tags, HA tags, and/or fluorescent tags. In some embodiments, the LTBP3 protein is a mammalian LTBP3 protein. In some embodiments, the LTBP3 protein is human, monkey, mouse, or rat LTBP3 protein. In some embodiments, the LTBP3 protein comprises the amino acid sequence as set forth in SEQ ID NO: 33 and 34 in Table 12. In some embodiments, the LTBP1 protein comprises the amino acid sequence as set forth in SEQ ID NO: 40 in Table 14.

Antibodies or antigen binding portions thereof as described herein are capable of binding to the GARP-TGFβ1 complex. In some embodiments, the GARP protein is a naturally occurring protein or a fragment thereof. In some embodiments, the GARP protein is a non-naturally occurring protein or a fragment thereof. In some embodiments, the GARP protein is a recombinant protein. Such GARP may be recombinant, referred to herein as recombinant GARP. Some recombinant GARPs may contain one or more modifications, truncations and/or mutations compared to wild-type GARP. Recombinant GARP can be modified to become soluble. In some embodiments, the GARP protein includes a leader sequence (eg, a natural or non-natural leader sequence). In some embodiments, the GARP protein does not include a leader sequence (ie, the leader sequence has been processed or cleaved). In other embodiments, the recombinant GARP protein is modified to include one or more detectable labels. In further embodiments, such detectable labels may include, but are not limited to, biotin labels, polyhistidine tags, flag tags, myc tags, HA tags, and/or fluorescent tags. In some embodiments, the GARP protein is a mammalian GARP protein. In some embodiments, the GARP protein is human, monkey, mouse, or rat GARP protein. In some embodiments, the GARP protein comprises an amino acid sequence as set forth in SEQ ID NOs: 37-38 in Table 12. In some embodiments, the GARP protein comprises the amino acid sequence as set forth in SEQ ID NOs: 41 and 42 in Table 14. In some embodiments, the antibodies or antigen-binding portions thereof described herein do not bind TGFβ1 in a context-dependent manner, e.g., binding to TGFβ1 is caused by the TGFβ1 molecule being linked to a specific protein, such as GARP. This will occur only when a complex is formed with the presentation molecule. Instead, antibodies, and antigen-binding portions thereof, bind TGFβ1 in a context-independent manner. In other words, an antibody or antigen binding portion thereof binds TGFβ1 when bound to any presenting molecule: GARP, LTBP1, LTBP3 and/or LRRC33.

Antibodies or antigen binding portions thereof as described herein are capable of binding to the LRRC33-TGFβ1 complex. In some embodiments, the LRRC33 protein is a naturally occurring protein or a fragment thereof. In some embodiments, the LRRC33 protein is a non-naturally occurring protein or a fragment thereof. In some embodiments, the LRRC33 protein is a recombinant protein. Such LRRC33 may be recombinant, referred to herein as recombinant LRRC33. Some recombinant LRRC33 proteins may contain one or more modifications, truncations and/or mutations compared to wild-type LRRC33. Recombinant LRRC33 protein can be modified to become soluble. For example, in some embodiments, the ectodomain of LRRC33 can be expressed with a C-terminal His tag to express soluble LRRC33 protein (sLRRC33; see, eg, SEQ ID NO: 73). In some embodiments, the LRRC33 protein includes a leader sequence (eg, a natural or non-natural leader sequence). In some embodiments, the LRRC33 protein does not include a leader sequence (ie, the leader sequence has been processed or truncated). In other embodiments, the recombinant LRRC33 protein is modified to include one or more detectable labels. In further embodiments, such detectable labels may include, but are not limited to, biotin labels, polyhistidine tags, flag tags, myc tags, HA tags, and/or fluorescent tags. In some embodiments, the LRRC33 protein is a mammalian LRRC33 protein. In some embodiments, the LRRC33 protein is a human, monkey, mouse or rat LRRC33 protein. In some embodiments, the LRRC33 protein comprises an amino acid sequence as set forth in SEQ ID NOs: 72, 73, and 74 in Table 14.

Pharmaceutical Compositions and Formulations The present disclosure further provides pharmaceutical compositions for use as medicaments suitable for administration in human and non-human subjects. One or more high affinity context-independent antibodies encompassed by the present disclosure can be formulated or mixed with pharmaceutically acceptable carriers (excipients), including, for example, buffers, into pharmaceutical compositions. can be formed. Such formulations can be used for the treatment of diseases or disorders involving TGFβ signaling. In certain embodiments, such formulations may be used for immuno-oncology applications.

Pharmaceutical compositions of the present disclosure can be administered to a patient to alleviate TGFβ-related conditions (eg, fibrosis, immunodeficiency, and/or cancer). "Acceptable" means that the carrier is compatible with (and preferably capable of stabilizing) the active ingredient of the composition and is not deleterious to the subject to be treated. . Examples of pharmaceutically acceptable excipients (carriers), including buffers, will be apparent to those skilled in the art and have been previously described. For example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. See Hoover. In one example, a pharmaceutical composition described herein comprises two or more antibodies that specifically bind to GARP-proTGFβ1 complex, LTBP1-proTGFβ1 complex, LTBP3-proTGFβ1 complex, and LRRC33-proTGFβ1 complex. The antibodies recognize different epitopes/residues of the complex.

Pharmaceutical compositions to be used in the present method may include pharmaceutically acceptable carriers, excipients or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include phosphates, citrates, and other organic acids. buffering agents; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben); ; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulin; polymers; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sucrose, mannitol, trehalose, or sugars such as sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS® or polyethylene glycol (PEG). may be included. Pharmaceutically acceptable excipients are further described herein.

The present disclosure also includes a pharmaceutical composition comprising an antibody or fragment thereof according to the present disclosure and a pharmaceutically acceptable excipient.

Accordingly, antibodies or molecules containing antigen-binding fragments of such antibodies can be formulated into pharmaceutical compositions suitable for human administration.

Pharmaceutical formulations may include one or more excipients. In some embodiments, the excipient is available at https://www. accessdata. fda. gov/scripts/cder/iig/index. Cfm? event=browseByLetter. It can be selected from the list provided in page&Letter=A.

Pharmaceutical compositions are typically formulated to a final concentration of active biological agent (eg, monoclonal antibodies, engineered binding molecules, including antigen-binding fragments, etc.) that will be from about 20 mg/mL to about 200 mg/mL. For example, the final concentration (wt/vol) of the formulation may be about 20-200, 20-180, 20-160, 20-150, 20-120, 20-100, 20-80, 20-70, 20-60, 20-50, 20-40, 30-200, 30-180, 30-160, 30-150, 30-120, 30-100, 30-80, 30-70, 30-60, 30-50, 30- 40, 40-200, 40-180, 40-160, 40-150, 40-120, 40-100, 40-80, 40-70, 40-60, 40-50, 50-200, 50-180, 50-160, 50-150, 50-120, 50-100, 50-80, 50-70, 50-60, 60-200, 60-180, 60-160, 60-150, 60-120, 60- 100, 60-80, 60-70, 70-200, 70-180, 70-160, 70-150, 70-120, 70-100, 70-80 mg/mL. In some embodiments, the final concentration of biologic in the formulation is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/mL.

Pharmaceutical compositions of the present disclosure are preferably formulated in a suitable buffer. Suitable buffers include, but are not limited to, phosphate buffer, citrate buffer, and histidine buffer.

The final pH of the formulation is usually between pH 5.0 and 8.0. For example, the pH of the pharmaceutical composition is about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7. 2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8.

Pharmaceutical compositions of the present disclosure may include surfactants such as nonionic detergents approved for use in pharmaceutical formulations. Such surfactants include, for example, polysorbates such as polysorbate 20 (Tween™-20), polysorbate 80 (Tween-80), and NP-40.

Pharmaceutical compositions of the present disclosure may include stabilizers. For liquid-protein preparations, stability may be enhanced by the choice of pH buffering salts, and often amino acids may also be used. Often it is interactions at the liquid/air or liquid/solid interfaces (with packing) that lead to aggregation after protein adsorption and unfolding. Suitable stabilizers include, but are not limited to, sucrose, maltose, sorbitol and certain amino acids such as histidine, glycine, methionine and arginine.

Pharmaceutical compositions of the present disclosure may contain one or any combination of the following excipients: sodium phosphate, arginine, sucrose, sodium chloride, tromethamine, mannitol, benzyl alcohol, histidine, sucrose, polysorbate 80, Sodium citrate, glycine, polysorbate 20, trehalose, poloxamer 188, methionine, trehalose, hyaluronidase, sodium succinate, potassium phosphate, disodium edetate, sodium chloride, potassium chloride, maltose, histidine acetate, sorbitol, pentetate, human Serum albumin, pentetate.

In some embodiments, pharmaceutical compositions of the present disclosure may contain a preservative.

Pharmaceutical compositions of the present disclosure are typically provided in liquid or lyophilized form. Typically, the product may be provided in a vial (eg, a glass vial). Products available in syringes, pens, or autoinjectors may be provided as prefilled liquids in these containers/closed systems.

In some examples, the pharmaceutical compositions described herein contain antibodies that specifically bind to GARP-proTGFβ1 complex, LTBP1-proTGFβ1 complex, LTBP3-proTGFβ1 complex, and LRRC33-proTGFβ1 complex. containing liposomes as described by Epstein et al. , Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al. , Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation times are disclosed in US Pat. No. 5,013,556. Particularly useful liposomes can be made by reverse phase evaporation using a lipid composition that includes phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). To obtain liposomes with the desired diameter, the liposomes are extruded through a filter of defined pore size.

In some embodiments, liposomes with targeting properties are selected to preferentially deliver or localize the pharmaceutical composition to particular tissues or cell types. For example, certain nanoparticle-based carriers with bone marrow targeting properties, such as lipid-based nanoparticles or liposomes, can be utilized. See, eg, Sou (2012) “Advanced drug carriers targeting bone marrow”, ResearchGate publication 232725109.

In some embodiments, the pharmaceutical compositions of the present disclosure may include or be used in combination with an adjuvant. Certain adjuvants can, for example, boost a subject's immune response to tumor antigens and promote T effector function, DC differentiation from monocytes, enhanced antigen uptake and presentation by APCs, etc. Conceivable. Suitable adjuvants include retinoic acid adjuvants and their derivatives, oil-in-water emulsion adjuvants such as MF59 and other squalene-containing adjuvants, toll-like receptor (TRL) ligands (e.g. CpG), alpha-tocopherol (vitamin E). and derivatives thereof, but are not limited to these.

Antibodies described herein may also be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization. can be obtained, incorporated into colloidal drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules, or incorporated into macroemulsions. Exemplary techniques have been previously described, eg, in Remington, The Science and Practice of Pharmacy 20th Ed. See Mack Publishing (2000).

In other examples, the pharmaceutical compositions described herein can be formulated in a sustained release format. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g. poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), L- Degradable lactic acids such as copolymers of glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate copolymers, LUPRON DEPOT™ (injectable microspheres composed of lactic-glycolic acid copolymers and leuprolide acetate) -Glycolic acid copolymers, sucrose acetate isobutyrate and poly-D-(-)-3-hydroxybutyric acid.

Pharmaceutical compositions to be used for in vivo administration must be sterile. This is easily accomplished by, for example, filtration through a sterile filter membrane. The therapeutic antibody composition is generally packaged in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle.

The pharmaceutical compositions described herein may be in the form of tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral or rectal administration, or for administration by inhalation or insufflation. may be present in unit dosage form, such as.

Suitable surfactants include, in particular, polyoxyethylene sorbitans (e.g. Tween 20, 40, 60, 80 or 85) and other sorbitans (e.g. Span 20, 40, 60, 80 or Examples include nonionic drugs such as 85). Compositions with surfactants will conveniently contain 0.05-5% surfactant and may be present at 0.1-2.5%. It will be appreciated that other ingredients may be added if desired, such as, for example, mannitol or other pharmaceutically acceptable vehicles.

Suitable emulsions may be prepared using commercially available fat emulsions such as Intralipid(TM), Liposyn(TM), Infonutrol(TM), Lipofundin(TM) and Lipiphysan(TM). The active ingredient can be dissolved in a premixed emulsion composition, or alternatively it can be dissolved in an oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and a phospholipid (e.g. , egg phospholipids, soy phospholipids or soy lecithin) and water. It will be appreciated that other ingredients may be added to adjust the osmotic pressure of the emulsion, such as, for example, glycerol or glucose. Suitable emulsions will normally contain up to 20%, such as 5-20%.

Emulsion compositions can be prepared by mixing Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water) and antibodies of the present disclosure.

Kits for Use in Detecting, Monitoring or Mitigating TGFβ-Related Symptoms The present disclosure also provides kits for use in treating, diagnosing, or predicting response in a subject having a disease/disorder associated with a TGFβ-related indication. I will provide a. Such kits can be used to recognize TGFβ (e.g., TGFβ1 or latent TGFβ1), phosphorylated SMAD (e.g., phosphorylated SMAD2), or immune cells such as MDSCs (e.g., mMDSCs and/or gMDSCs) or CD8+ T cells. may contain one or more containers containing an antibody or antigen-binding portion thereof that specifically binds to any one of the surface markers described herein.

In some embodiments, the kit may include instructions for use with any of the methods described herein. The included instructions describe how TGFβ (e.g., TGFβ1 or latent TGFβ1), phosphorylated SMAD (e.g., phosphorylated SMAD2) may be used for treatment, diagnosis, or prediction of response in a subject having a target disease described herein. ), or an antibody or antigen-binding antibody thereof that specifically binds to any one of the surface markers described herein for recognizing immune cells such as MDSCs (e.g., mMDSCs and/or gMDSCs) or CD8+ T cells. May include instructions for using the portion. In some embodiments, the kits determine whether an individual has a target disease or treatment with a particular regimen, e.g., a TGFβ1 inhibitor, e.g., Ab6, alone or in combination with checkpoint inhibitor therapy or chemotherapy. Instructions may be included for selecting suitable individuals for treatment based on identification of the likelihood of clinical benefit following the included treatment. In yet other embodiments, the instructions include instructions for determining target binding or therapeutic efficacy in an individual treated with a TGFβ1 inhibitor, such as Ab6.

In some embodiments, the instructions include a biomarker described herein, e.g., TGFβ (e.g., TGFβ1 or latent TGFβ1), phosphorylated SMAD (e.g., phosphorylated SMAD2), as part of a treatment regimen. ), or the use of antibodies or antigen-binding fragments that bind to any one of the surface markers described herein to recognize immune cells such as MDSCs (e.g., mMDSCs and/or gMDSCs) or CD8+ T cells. information. The instructions provided in the kits of the present disclosure are typically instructions provided on a label or package insert (e.g., paper included with the kit), but are machine-readable instructions (e.g., magnetic or Instructions retained on optical storage disks) are also acceptable.

The label or package insert indicates that the composition is used to treat, diagnose, or predict response to a disease or disorder associated with TGFβ-related symptoms. Instructions may be provided for practicing any of the methods described herein.

The kits of the present disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like. Packaging for use in conjunction with specific devices such as inhalers, nasal administration devices (eg atomizers) or infusion devices such as mini-pumps is also contemplated. The kit may have a sterile access port (eg, the container may be an intravenous fluid bag or vial with a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (eg, the container may be an intravenous fluid bag or vial with a stopper pierceable by a hypodermic needle).

Kits may optionally provide additional components such as buffers and explanatory information. Typically, a kit will include a container and a label or insert on or associated with the container. In some embodiments, the present disclosure provides an article of manufacture that includes the contents of the kit described above.

Processes for Screening, Identification and Production of Preferred Isoform-Specific Inhibitors of TGFβ1 The present disclosure provides screening/selection methods, production methods and GARP-proTGFβ1 complexes, LTBP1-proTGFβ1 complexes, LTBP3- The present invention encompasses processes for producing antibodies or fragments thereof capable of binding to each of the proTGFβ1 complex and the LRRC33-proTGFβ1 complex, as well as pharmaceutical compositions and associated kits containing the same. In some embodiments, at least one of LTBP1-proTGFβ1 and LTBP3-proTGFβ1 complexes and at least one of GARP-proTGFβ1 and LRRC33-proTGFβ1 complexes are included for screening. The antibodies or fragments thereof identified in the screening process are preferably further tested to confirm their ability to bind each of the LLCs of interest with high affinity.

A number of methods can be used to obtain antibodies or antigen-binding fragments thereof of the present disclosure. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies can also be produced by hybridoma production according to known methods (eg, Kohler and Milstein (1975) Nature, 256:495-499). Hybridomas formed in this manner are then screened using standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (e.g. OCTET® or BIACORE) analysis and One or more hybridomas that produce antibodies that specifically bind to a designated antigen are identified. Any form of the specified antigen may be an immunogen, such as a recombinant antigen, a naturally occurring form, any variant or fragment thereof, as well as an antigenic peptide thereof (e.g., an epitope described herein as a linear epitope). or any of the epitopes described herein within the framework as a conformational epitope). One exemplary method of producing antibodies involves screening protein expression libraries, such as phage or ribosome display libraries, that express antibodies or fragments thereof (eg, scFv). Phage display is described, for example, by Ladner et al. , US Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. , (1991) Nature, 352:624-628; Marks et al. , (1991) J. Mol. Biol. , 222:581-597; International Publication No. 92/18619 pamphlet; International Publication No. 91/17271 pamphlet; International Publication No. 92/20791 pamphlet; International Publication No. 92/15679 pamphlet; International Publication No. 93/01288 It is described in pamphlets; WO 92/01047 pamphlet; WO 92/09690 pamphlet; and WO 90/02809 pamphlet.

In addition to the use of display libraries, designated antigens (e.g., presentation molecule-TGFβ1 complexes) can be used in non-human hosts such as rabbits, guinea pigs, rats, mice, hamsters, sheep, goats, chickens, camelids and It can be used to immunize non-mammalian hosts such as sharks. In one embodiment, the non-human animal is a mouse.

Immunization of non-human hosts can be carried out by the use of purified recombinant protein complexes, with or without a display molecule (or fragment thereof) attached thereto as an immunogen, such as proTGFβ1. These include, but are not limited to, LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1 and LRRC33-proTGFβ1. The display molecule that is attached need not be a full-length counterpart, but preferably contains two cysteine residues that form a covalent bond with the proTGFβ1 dimeric complex.

Alternatively, immunization of non-human hosts can be carried out through the use of cell-based antigens. The term cell-based antigen refers to cells (eg, xenogeneic cells) that express the proTGFβ1 protein complex. This can be achieved by overexpression of proTGFβ1, optionally with co-expression of presentation molecules. In some embodiments, endogenous counterparts may be utilized as cell-based antigens. Cell surface expression of proteins forming proTGFβ1-containing protein complexes can be confirmed by known methods such as FACS. Upon immunization of a host with such cells (cell-based antigens), an immune response against the antigen is elicited in the host, allowing antibody production and subsequent screening. In some embodiments, suitable knockout animals are used to promote a stronger immune response to the antigen. Alternatively, structural differences between different species may be sufficient to induce antibody production in the host.

In another embodiment, the monoclonal antibody is obtained from a non-human animal and subsequently modified, eg, is a chimera using suitable recombinant DNA techniques. Various techniques for making chimeric antibodies have been described. For example, Morrison et al. , Proc. Natl. Acad. Sci. U. S. A. 81:6851, 1985; Takeda et al. , Nature 314:452, 1985, Cabilly et al. , US Pat. No. 4,816,567; Boss et al. , U.S. Pat. No. 4,816,397; Tanaguchi et al. , EP 171,496; EP 0,173,494; GB 2,177,096B.

For additional antibody generation techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al. , Cold Spring Harbor Laboratory, 1988. This disclosure is not necessarily limited to any particular source, method of production, or other particular characteristics of the antibodies.

Some aspects of the present disclosure relate to host cells transformed with polynucleotides or vectors. Host cells can be prokaryotic or eukaryotic. A polynucleotide or vector present in a host cell can be integrated into the host cell's genome or it can be maintained extrachromosomally. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. In some embodiments, the eukaryotic cell is, for example, of the genus Saccharomyces, particularly S. It is of the species S. cerevisiae. The term "prokaryotic" includes all bacteria that can be transformed or transfected with DNA or RNA molecules for the expression of antibodies or the corresponding immunoglobulin chains. Prokaryotic hosts include, for example, E. coli (E. coli), S. coli. It can include Gram-negative and Gram-positive bacteria such as S. typhimurium, Serratia marcescens, and Bacillus subtilis. The term "eukaryotic" includes yeast, higher plant, insect and vertebrate cells, such as mammalian cells such as NSO and CHO cells. Depending on the host utilized in the recombinant production procedure, the antibody or immunoglobulin chain encoded by the polynucleotide may or may not be glycosylated. The antibody or corresponding immunoglobulin chain may also include an initial methionine amino acid residue.

In some embodiments, once the vector is introduced into a suitable host, the host can be maintained under conditions suitable for high level expression of the nucleotide sequence and optionally contain immunoglobulin light chains, heavy chains, light chains, etc. Recovery and purification of /heavy chain dimers or intact antibodies, antigen-binding fragments or other immunoglobulin forms may follow; Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.; Y. , (1979). Accordingly, the polynucleotide or vector is subsequently introduced into cells that produce the antibody or antigen-binding fragment. Large scale production of antibodies or antibody fragments (e.g., about 250 L or more, e.g., 1000 L, 2000 L, 3000 L, 4000 L or more) is suitable for commercial scale production of pharmaceutical compositions comprising antibodies, and typically includes: It is carried out in a culture system such as a suspension cell culture. Such a culture may be a eukaryotic cell culture, and optionally the eukaryotic cell culture is a mammalian cell culture, a plant cell culture or an insect cell culture. In some embodiments, the mammalian cell culture comprises CHO cells, MDCK cells, NS0 cells, Sp2/0 cells, BHK cells, mouse C127 cells, Vero cells, HEK293 cells, HT-1080 cells, or PER. Contains C6 cells.

Transformed host cells can be grown in fermentors and cultured using any suitable technique to achieve optimal cell growth. Once expressed, complete antibodies, their dimers, individual light and heavy chains, other immunoglobulin forms or antigen-binding fragments can be produced by various techniques including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc. It may be purified according to standard procedures in the art; Scopes, Protein Purification, Springer Verlag, N.M. Y. (1982). Antibodies or antigen-binding fragments can then be isolated from growth media, cell lysates, or cell membrane fractions. For example, the isolation and purification of antibodies or antigen-binding fragments expressed in microorganisms may include, for example, preparative chromatographic separation and the use of monoclonal or polyclonal antibodies directed against the constant region of the antibody. It may be by any conventional means such as chemical separation.

Aspects of the present disclosure relate to hybridomas, which provide an indefinitely extended source of monoclonal antibodies. Instead of obtaining immunoglobulins directly from hybridoma cultures, immortalized hybridoma cells can be used as a source of reconstituted heavy and light chain sites for subsequent expression and/or genetic manipulation. Rearranged antibody genes can be reverse transcribed from the appropriate mRNA to generate cDNA. In some embodiments, the heavy chain constant region can be replaced with one of a different isotype or removed entirely. The variable regions can be linked to encode a single chain Fv region. Multiple Fv regions can be linked to confer binding ability to more than one target, or chimeric heavy and light chain combinations can be utilized. Any suitable method can be used for cloning antibody variable regions and producing recombinant antibodies.

In some embodiments, suitable nucleic acids encoding heavy and/or light chain variable regions are obtained and inserted into expression vectors that can be transfected into standard recombinant host cells. A variety of such host cells can be used. In some embodiments, mammalian host cells may be advantageous for efficient processing and production. Typical mammalian cell lines useful for this purpose include CHO cells, 293 cells or NSO cells. Production of antibodies or antigen-binding fragments can be carried out by culturing modified recombinant hosts under culture conditions suitable for growth of the host cells and expression of the coding sequences. The antibody or antigen-binding fragment can be recovered by isolating it from culture. Expression systems can be designed to include a signal peptide so that the resulting antibody is secreted into the culture medium; however, intracellular production is also possible.

The present disclosure also includes polynucleotides encoding at least the variable regions of the immunoglobulin chains of the antibodies described herein. In some embodiments, the variable region encoded by the _{polynucleotide} comprises at least one _{complementarity} determining region ( CDR).

Polynucleotides encoding antibodies or antigen-binding fragments can be recombinantly produced, including, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or any of these polynucleotides alone or in combination. It can be a chimeric nucleic acid molecule. In some embodiments, the polynucleotide is part of a vector. Such vectors may further contain genes such as marker genes that allow selection of the vector under suitable conditions in suitable host cells.

In some embodiments, the polynucleotide is operably linked to expression control sequences that allow expression in prokaryotic or eukaryotic cells. Expression of a polynucleotide involves transcription of the polynucleotide into translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may contain regulatory sequences that promote initiation of transcription and optionally poly-A signals that promote termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional and translational enhancers and/or naturally associated or heterologous promoter regions. Suitable regulatory elements allowing expression in prokaryotic host cells include, for example, E. Examples of regulatory elements allowing expression in eukaryotic host cells include the PL, Lac, Trp or Tac promoters in E. coli, the AOX1 or GAL1 promoters in yeast or the AOX1 or GAL1 promoters in mammalian and other animal cells. CMV-promoter, SV40-promoter, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or globin intron.

In addition to elements involved in the initiation of transcription, such regulatory elements may also include transcription termination signals, such as SV40-poly-A sites or tk-poly-A sites downstream of the polynucleotide. Additionally, depending on the expression system utilized, a leader sequence capable of directing the polypeptide to an intracellular compartment or causing it to be secreted into the culture medium may be added to the coding sequence of the polynucleotide and previously Are listed. The leader sequence is assembled at the appropriate time with translation, initiation and termination sequences and preferably a leader sequence capable of directing secretion of the translated protein or portion thereof, eg into the extracellular medium. Optionally, a heterologous polynucleotide sequence can be used that encodes a fusion protein containing a desired characteristic, such as a C-terminal or N-terminal identification peptide that provides for stabilization or simplified purification of the expressed recombinant product.

In some embodiments, a polynucleotide encoding at least a variable domain of a light chain and/or a heavy chain may encode the variable domain of both or only one immunoglobulin chain. Similarly, polynucleotides can be under the control of the same promoter or can be separately controlled for expression. Additionally, some embodiments include polynucleotides encoding variable domains of immunoglobulin chains of antibodies or antigen-binding fragments; optionally genetically engineered in combination with polynucleotides encoding variable domains of other immunoglobulin chains of antibodies. vectors, in particular plasmids, cosmids, viruses and bacteriophages, customarily used in the field of medicine.

In some embodiments, expression control sequences are provided as eukaryotic promoter systems in vectors that can transform or transfect eukaryotic host cells, although control sequences for prokaryotic hosts can also be used. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes virus, or bovine papilloma virus can be used for delivery of polynucleotides or vectors to targeted cell populations (e.g., antibodies or to engineer cells that express antigen-binding fragments). Recombinant viral vectors can be constructed using a variety of suitable methods. In some embodiments, polynucleotides and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing polynucleotides (e.g., sequences encoding heavy and/or light variable domains of immunoglobulin chains and expression control sequences) can be introduced into host cells by suitable methods that vary depending on the type of cell host. .

Screening methods can include the step of evaluating or confirming the desired activity of the antibody or fragment thereof. In some embodiments, the step includes selecting for the ability to inhibit a target function, eg, inhibition of release of mature/soluble growth factor (eg, TGFβ1) from a latent complex. In certain embodiments, such steps include cell-based potency assays in which the inhibitory activity of one or more test antibodies is determined by the activity when the proTGFβ complex is expressed on the cell surface. The growth factor is assayed by measuring the level of growth factor released into the medium (e.g., assay solution) upon oxidation. The level of growth factors released into the medium/solution can be assayed, for example, by measuring TGFβ activity. Non-limiting examples of useful cell-based potency assays are described, eg, in Example 2 of PCT/US Patent Application Publication No. 2019/041373.

In some embodiments, screening for desirable antibodies or fragments includes selecting for antibodies or fragments thereof that promote internalization and subsequent removal of antibody-antigen complexes from the cell surface. In some embodiments, the step includes selecting for antibodies or fragments thereof that induce ADCC. In some embodiments, the step includes selecting for antibodies or fragments thereof that accumulate at the desired site (eg, cell type, tissue, or organ) in vivo. In some embodiments, the step includes selecting for antibodies or fragments thereof that have the ability to cross the blood-brain barrier. The methods optionally include stability, binding affinity, functionality (e.g., inhibitory activity, Fc function, etc.), immunogenicity, pH sensitivity, and developability (e.g., high solubility, low self-association, etc.). It can include optimizing one or more antibodies or fragments thereof to yield a variant counterpart with a desired profile, as determined by criteria.

The process for making compositions comprising antibodies or fragments according to the present disclosure can include optimization of one or more antibodies identified as having desirable binding and functional (eg, inhibitory) properties. Optimization may include affinity maturation of the antibody or fragment thereof. Further optimization steps may be performed to provide advantageous physicochemical properties to the therapeutic composition. Such steps may include, but are not limited to, mutagenesis or manipulation that results in improved solubility, lack of self-aggregation, stability, pH sensitivity, Fc function, and the like. The resulting optimized antibodies are preferably fully human or humanized antibodies suitable for human administration.

Manufacturing processes for pharmaceutical compositions containing such antibodies or fragments thereof may include steps such as purification, formulation, sterile filtration, packaging, and the like. Certain steps, such as sterile filtration, are performed according to guidelines set forth by relevant regulatory authorities, such as, for example, the FDA. Such compositions can be made available in the form of single-use containers, such as prefilled syringes, or multi-dose containers, such as vials.

Modifications The antibodies or antigen-binding portions thereof of the present disclosure may be enzymes, prosthetic molecules for the detection and isolation of GARP-proTGFβ1 complexes, LTBP1-proTGFβ1 complexes, LTBP3-proTGFβ1 complexes, and/or LRRC33-proTGFβ1 complexes. They may be modified with detectable labels or detectable moieties, including, but not limited to, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, non-radioactive paramagnetic metal ions, and affinity labels. A detectable substance or moiety can be attached to a polypeptide of the present disclosure either directly or indirectly via an intermediate moiety (e.g., a linker, e.g., a cleavable linker, etc.) using any suitable technique. can be obtained or conjugated. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase; non-limiting examples of suitable prosthetic group complexes include Non-limiting examples of suitable fluorescent materials include: avidin/biotin and avidin/biotin; non-limiting examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein, isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include radioactive metals. Ions such as alpha-emitters or such as iodine (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In) and technetium (99Tc). , 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, Lu (177Lu), 159Gd, 149Pm, 140La, 175Yb , 166Ho, 90Y, 47Sc, 86R, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, zirconium ( ^89Zr ) and tin (113Sn, 117S) n) etc. Other radioactive isotopes include. In some embodiments, the radiolabel may be selected from the group consisting of ¹¹ C, ¹³ N, ¹⁵ O, ⁶⁸ Ga, ¹⁷⁷ Lu, ¹⁸ F, and ⁸⁹ Zr. In some embodiments, useful labels are positron emitting isotopes that can be detected by positron emission tomography. The detectable substance can be directly attached to an antibody of the present disclosure that specifically binds to the GARP-proTGFβ1 complex, LTBP1-proTGFβ1 complex, LTBP3-proTGFβ1 complex, and/or LRRC33-proTGFβ1 complex, or a suitable may be indirectly linked or conjugated through an intermediate moiety (eg, a linker, etc.) using techniques. Any of the antibodies provided herein that are conjugated to a detectable substance can be used for any suitable diagnostic assay, such as those described herein.

Additionally, the antibodies of the present disclosure, or antigen-binding portions thereof, can also be modified with drugs. A drug can be attached to a polypeptide of the present disclosure directly or indirectly through an intermediate moiety (e.g., a linker, e.g., a cleavable linker, etc.) or conjugated using any suitable technique. Can be gated.

Targeting Agents In some embodiments, the methods of the present disclosure provide methods for modulating mature TGFβ release from GARP-proTGFβ1 complexes, LTBP1-proTGFβ1 complexes, LTBP3-proTGFβ1 complexes, and/or LRRC33-proTGFβ1 complexes. involves the use of one or more targeting agents to target an antibody, or antigen-binding portion thereof, as disclosed herein, to a specific site in a subject. For example, LTBP1-proTGFβ1 and LTBP3-proTGFβ1 complexes are normally localized to the extracellular matrix. Thus, in some embodiments, the antibodies disclosed herein can be conjugated to extracellular matrix targeting agents to localize the antibody to sites where LTBP-bound TGFβ1 complexes are present. In such embodiments, selective targeting of antibodies results in selective modulation of LTBP1-proTGFβ1 and LTBP3-proTGFβ1 complexes. In some embodiments, extracellular matrix targeting agents include heparin binding agents, matrix metalloproteinase binding agents, lysyl oxidase binding domains, fibrillin binding agents, hyaluronic acid binding agents, and the like.

Similarly, GARP-proTGFβ1 and LRRC33-proTGFβ1 complexes are normally localized and tethered to the surface of cells. The former is expressed on activated FOXP3+ regulatory T cells (Tregs), while the latter is expressed on certain bone marrow cells and some cancer cells such as AML. Thus, in some embodiments, the antibodies disclosed herein are used to target immune cells (e.g., Treg cells, activated (such as macrophages) may be conjugated to a binding agent. In such embodiments, selective targeting of the antibody results in selective inhibition of cell-bound proTGFβ1 complexes (e.g., selective inhibition of release of mature TGFβ1 for, e.g., immunomodulation in the treatment of cancer). . In such embodiments, the immune cell targeting agent may include, for example, CCL22 and CXCL12 proteins or fragments thereof.

In some embodiments, the bispecific antibody has a first portion that selectively binds to the proTGFβ1 complex and a component of the target site, such as a component of the ECM (e.g., fibrillin) or a Treg cell (e.g. , CTLA-4).

As further detailed herein, the present disclosure provides that isoform-selective TGFβ1 inhibitors such as those described herein can be used to promote or restore hematopoiesis in the bone marrow. intend to. Accordingly, in some embodiments, compositions comprising such inhibitors (eg, high affinity isoform selective inhibitors of TGFβ1) can be targeted to the bone marrow. One way to achieve bone marrow targeting is the use of specific carriers that preferentially target bone marrow localization or accumulation. For example, certain nanoparticle-based carriers with bone marrow targeting properties, such as lipid-based nanoparticles or liposomes, can be utilized. See, eg, Sou (2012) “Advanced drug carriers targeting bone marrow”, ResearchGate publication 232725109.

In some embodiments, the targeting agent includes an immune-enhancing agent, such as an adjuvant that includes squalene and/or alpha-tocopherol and an adjuvant that includes a TLR ligand/agonist (such as a TLR3 ligand/agonist). For example, squalene-containing adjuvants can preferentially target specific immune cells such as monocytes, macrophages and antigen presenting cells to enhance priming, antigen processing and/or immune cell differentiation to boost host immunity. In some embodiments, such adjuvants can stimulate a host immune response against the neo-epitope for T cell activation.

Therapeutic Targets and In Vivo Mechanisms of Action Accordingly, the TGFβ inhibitors disclosed herein (e.g., high affinity isoform-selective TGFβ1 inhibitors) can be used in any system, including in vitro, ex vivo and/or in vivo systems. can be used to inhibit TGFβ1 in any suitable biological system. Related methods can include contacting the biological system with a TGFβ1 inhibitor. A biological system can be an assay system, a biological sample, a cell culture, etc. In some cases, these methods involve altering the levels of free growth factors in the biological system.

Such pharmaceutical compositions and formulations can therefore be used to target TGFβ-containing latent complexes accessible by inhibitors in vivo. Accordingly, the antibodies of the present disclosure aim to target the following complexes at the disease site (e.g., TME), which preventively prevent the release of growth factors by binding to latent complexes. : i) proTGFβ1 presented by GARP; ii) proTGFβ1 presented by LRRC33; iii) proTGFβ1 presented by LTBP1; and iv) proTGFβ1 presented by LTBP3. Typically, complexes (i) and (ii) above are because both GARP and LRRC33 are transmembrane proteins that tether or can tether latent proTGFβ1 on the extracellular surface of cells expressing LRRC33. While present on the cell surface, complexes (iii) and (iv) are components of the extracellular matrix. Thus, the inhibitors embodied herein do not need to complete binding to endogenous high affinity receptors to exert an inhibitory effect. Additionally, targeting upstream of the ligand/receptor interaction provides a longer period of target reach than traditional inhibitors that transiently target soluble growth factors only after they have been released from the latent complex. It is longer and more localized to relevant tissues, which may allow for more sustained effects. Therefore, targeting latent complexes tethered to specific microenvironments is important because of their short half-life as soluble (free) growth factors, e.g., approximately 2 minutes after release from latent complexes. may promote improved target binding in vivo compared to conventional neutralizing antibodies that are supposed to compete for binding to sexual receptors.

Several studies have elucidated the mechanism of TGFβ1 activation. Three integrins, αVβ1, αVβ6, αVβ8, and αVβ1, have been demonstrated to be important activators of latent TGFβ1 (Reed, N.I., et al., Sci Transl Med, 2015. 7(288): p.288ra79; Travis, M.A. and D. Sheppard, Annu Rev Immunol, 2014.32: p.51-82; Munger, J.S., et al., Cell, 1999.96 (3): p.319-28; Sheppard. Cancer Metastasis Rev, 2005.24(3):395-402). αV integrin binds with high affinity to the RGD sequence present in TGFβ1 and TGFβ1 LAP (Dong, X., et al., Nat Struct Mol Biol, 2014.21(12): p. 1091-6). Transgenic mice with mutations in the TGFβ1 RGD site that prevent integrin binding but not secretion mimic the phenotype of TGFβ1 −/− mice (Yang, Z., et al., J Cell Biol, 2007.176 ( 6): p.787-93). Mice deficient in both β6 and β8 integrins recapitulate all the essential phenotypes of TGFβ1 and TGFβ3 knockout mice, including multisystem inflammation and cleft palate, and these two types of mutations in TGFβ1 activation during development and homeostasis The important role of integrins was confirmed (Aluwihare, P., et al., J Cell Sci, 2009.122 (Pt 2): p. 227-32). The key to integrin-dependent activation of latent TGFβ1 is covalent restraint of the presenting molecule; disruption of the disulfide bond between GARP and TGFβ1 LAP by mutagenesis does not impair complex formation; TGFβ1 activation by αVβ6 is completely abolished (Wang, R., et al., Mol Biol Cell, 2012.23(6): p.1129-39). Recent structural studies of latent TGFβ1 reveal how integrins enable release of active TGFβ1 from the latent complex: covalent binding of latent TGFβ1 to its presentation molecules Tethering latent TGFβ1 to the ECM via LTBP or to the cytoskeleton via GARP or LRRC33. Integrins that bind to RGD sequences cause force-dependent changes in the structure of LAP, allowing release of active TGFβ1 and binding to nearby receptors (Shi, M., et al., Nature, 2011.474(7351):p.343-9). The importance of integrin-dependent TGFβ1 activation in disease has also been well documented. Small molecule inhibitors of αVβ1 protect against bleomycin-induced pulmonary fibrosis and carbon tetrachloride-induced liver fibrosis (Reed, N.I., et al., Sci Transl Med, 2015.7(288): p .288ra79), αVβ6 blockade or loss of integrin β6 expression by antibodies suppresses bleomycin-induced pulmonary fibrosis and radiation-induced fibrosis (Munger, J.S., et al., Cell, 1999.96 (3 ): p. 319-28); Horan, G. S. , et al. , Am J Respir Crit Care Med, 2008.177(1): p. 56-65).

In addition to integrins, thrombospondin-1 and plasmin, matrix metalloproteases (MMPs, e.g. MMP2, MMP9 and MMP12), cathepsin D, kallikrein, thrombin and the ADAM family of zinc proteases (e.g. ADAM10, ADAM12 and ADAM17), etc. Other mechanisms of TGFβ1 activation have been implicated, including activation by proteases. Knockout of thrombospondin-1 recapitulates some aspects of the TGFβ1-/- phenotype in several tissues but is not protective in bleomycin-induced pulmonary fibrosis, which is known to be TGFβ-dependent. (Ezzie, M.E., et al., Am J Respir Cell Mol Biol, 2011.44(4): p.556-61). Furthermore, knockout of candidate proteases did not result in the TGFβ1 phenotype (Worthington, J.J., J.E. Klementowicz, and M.A. Travis, Trends Biochem Sci, 2011.36(1): p. 47 -54). This could be explained by redundancy or the importance of these mechanisms in certain diseases rather than in development and homeostasis.

Accordingly, the TGFβ inhibitors described herein (eg, high affinity isoform-specific inhibitors of TGFβ1) include inhibitors that act by interfering with the step of TGFβ1 activation. In some embodiments, such inhibitors can inhibit integrin-dependent (eg, mechanical or force-driven) activation of TGFβ1. In some embodiments, such inhibitors can inhibit protease-dependent or protease-induced activation of TGFβ1. The latter include inhibitors that inhibit the TGFβ1 activation step in an integrin-independent manner. In some embodiments, such inhibitors are capable of inhibiting TGFβ1 activation regardless of the mode of activation, e.g., both integrin-dependent and protease-dependent activation of TGFβ1. can be inhibited. Non-limiting examples of proteases that can activate TGFβ1 include kallikrein, chemotrypsin, trypsin, elastase, plasmin, thrombin and zinc metalloproteases (MMP family) such as MMP-2, MMP-9 and MMP-13. ) and other serine proteases. Kallikreins include plasma-kallikreins and tissue kallikreins such as KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14 and KLK15. The data presented in PCT/U.S. Patent Application Publication No. 2019/041373 demonstrate examples of isoform-specific TGFβ1 inhibitors that can inhibit kallikrein-dependent activation of TGFβ1 in vitro. ing. In some embodiments, the inhibitors of the present disclosure prevent the release or dissociation of active (mature) TGFβ1 growth factor from the latent complex. In some embodiments, such inhibitors may act by stabilizing the inactive (eg, latent) conformation of the complex. Applicants' previous data further demonstrated that high affinity context-independent TGFβ1 inhibitors (eg, Ab6) can also inhibit plasmin-dependent TGFβ1 activation. However, surprisingly, the context-biased TGFβ1 inhibitor (Ab3) was less effective at inhibiting this process. Both Ab3 and Ab6 have similar affinities for matrix-bound proTGFβ1 complexes. However, Ab3 has significantly weaker binding affinity for cell-bound proTGFβ1 complexes. The relative difference between the two categories is more than 20 times ("bias"). In comparison, Ab6 shows equally high affinity for both categories of antigen complexes. One possible explanation is that the observed functional differences may arise from the biased characteristics of Ab3. Another possible explanation is that it is mediated by differences in epitopes or binding regions.

The present disclosure is particularly useful for therapeutic use for certain diseases associated with multiple biological roles of TGFβ signaling that are not limited to a single context of TGFβ function. In such situations, inhibiting TGFβ1 effects across multiple contexts may be beneficial. Accordingly, the present disclosure provides methods for targeting and inhibiting TGFβ1 in an isoform-specific rather than a context-specific manner. Such agents may also be referred to as "isoform-specific context-independent" TGFβ1 modulators.

Mounting evidence indicates that many diseases demonstrate complex perturbations of TGFβ signaling, which may involve the involvement of disparate cell types conferring differential effects on TGFβ function mediated by interactions with so-called presenting molecules. I support this idea. At least four such presentation molecules have been identified and are capable of "presenting" TGFβ in a variety of extracellular microenvironments allowing its activation in response to local stimuli. In one category, TGFβ accumulates in the ECM in association with ECM-bound presenting molecules such as LTBP1 and LTBP3 that mediate ECM-bound TGFβ activity. In another category, TGFβ is tethered to the surface of immune cells through presentation molecules such as GARP and LRR33 that mediate specific immune functions. These presenting molecules show differential expression, localization and/or function in different tissues and cell types, and the triggering events and consequences of TGFβ activation will vary depending on the microenvironment. It shows. Based on the idea that many TGFβ effects can interact and contribute to disease progression, therapeutic agents that can attenuate multiple aspects of TGFβ function may offer greater efficacy.

LRRC33-proTGFβ1 as a target
LRRC33 is expressed on selective cell types, particularly those of the myeloid lineage, including monocytes and macrophages. Monocytes are derived from progenitor cells in the bone marrow, circulate in the bloodstream, and reach peripheral tissues. Circulating monocytes then migrate to tissues where they are exposed to a local environment (e.g., tissue-specific, disease-related, etc.) that contains a range of different factors, such as cytokines and chemokines, and monocytes It can induce differentiation of cells into macrophages, dendritic cells, etc. These include, for example, alveolar macrophages in the lungs, osteoclasts in the bone marrow, microglia in the CNS, histiocytes in connective tissue, Kupffer cells in the liver, and brown adipose tissue macrophages in brown adipose tissue. In solid tumors, infiltrating macrophages can be tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), and myeloid-derived suppressor cells (MDSCs). Such macrophages can be activated and/or associated with activated fibroblasts, such as carcinoma-associated (or cancer-associated) fibroblasts (CAFs) and/or corneal stroma. Thus, inhibitors of TGFβ1 activation described herein that inhibit the release of mature TGFβ1 from LRRC33-containing complexes target any of these cells that express LRR33-proTGFβ1 on the cell surface. be able to. In the fibrotic microenvironment, LRRC33-expressing cells may include M2 macrophages, tissue-resident macrophages, and/or MDSCs.

In some embodiments, the LRRC33-TGFβ1 complex is present on the outer surface of profibrotic (M2-like) macrophages. In some embodiments, profibrotic (M2-like) macrophages are present in a fibrotic microenvironment. In some embodiments, targeting the LRRC33-TGFβ1 complex at the external surface of profibrotic (M2-like) macrophages is compared to simply targeting the LTBP1-TGFβ1 and/or LTBP1-TGFβ1 complex. This will give you better results. In some embodiments, M2-like macrophages are further polarized into multiple subtypes with different phenotypes, such as M2c and M2d TAM-like macrophages. In some embodiments, the macrophage comprises TGFβ1, CCL2 (MCP-1), CCL22, SDF-1/CXCL12, M-CSF (CSF-1), IL-6, IL-8, IL-10, IL- 11, including but not limited to CXCR4, VEGF, PDGF, arachidonic acid and prostaglandin modulating agents such as cyclooxygenase-2 (COX-2), parathyroid hormone-related protein (PTHrP), RUNX2, HIF1α, and metalloproteases , can be activated by various factors present in the tumor microenvironment, such as growth factors, chemokines, cytokines, and ECM remodeling molecules. Exposure to one or more such factors may further drive monocytes/macrophages toward a tumor-promoting phenotype. As an example, co-expression of CCL2 and VEGF in tumors has been shown to correlate with increased TAM and underdiagnosis. Additionally, activated tumor-associated cells may also promote the recruitment and/or differentiation of other cells into tumor-promoting cells, such as CAFs, TANs, MDSCs, etc. Stromal cells also respond to macrophage activation and can influence ECM remodeling and ultimately angiogenesis, invasion and metastasis. For example, CCL2 not only functions as a monocyte attractant but also promotes cell adhesion by upregulating MAC-1, the receptor for ICAM-1 expressed in activated endothelium. This may lead to CCL2-dependent arteriogenesis and cancer progression. Accordingly, the TGFβ1 inhibitors described herein can be used in methods to inhibit arteriogenesis by interfering with the CCL2 signaling system.

Subsets of myeloid cells, including M2-polarized macrophages and myeloid-derived suppressor cells (MDSCs), both of which have an immunosuppressive phenotype and are enriched in disease settings (e.g., TME and FME), possess cell surface LRRC33. Express. Bone marrow-derived circulating monocytes do not appear to express cell surface LRRC33. The limited expression of LRRC33 makes it a particularly attractive therapeutic target. Most of the studies available in the literature focus on the biology of effector T cells (e.g., CD8+ cytotoxic cells) in cancer, but point to the important role of suppressive myeloid cell populations in disease. Evidence (such as data presented in PCT/US Patent Application Publication No. 2019/041373) is also increasing. Importantly, the highly selective TGFβ1 inhibitory antibodies disclosed herein are capable of targeting this arm of TGFβ1 function in vivo. More specifically, tumor-associated M2 macrophages and MDSCs express cell surface LRRC33 with strong correlation to disease progression. The high-affinity TGFβ1-selective antibodies disclosed herein can overcome initial resistance to tumor checkpoint blocker therapy (CBT) in multiple pharmacological models. Indeed, antitumor efficacy coincided with a significant reduction in tumor-associated macrophage and MDSC levels, suggesting that targeting this aspect of TGFβ1 function may contribute to therapeutically beneficial effects. There is. This may also be applicable to other diseases where these immunosuppressive cells are enriched. Some fibrotic conditions are also associated with increased local frequencies of these cell populations. Therefore, high affinity TGFβ1 selective antibodies would be expected to exert similar in vivo effects in such indications. Of note, the new findings provided herein identify LRRC33 as a novel cell surface marker of circulating MDSCs, which suggests that i) the extent of tumor burden correlates with tumor-associated MDSCs; and ii) combined with Applicants' previous findings that tumor-associated MDSCs correlate with circulating MDSCs, suggests a new use of LRRC33 as a blood-based biomarker, e.g. indicative of immunosuppression. As described herein, immunosuppressed tumors are treated with a TGFβ inhibitor (e.g., a TGFβ1 selective inhibitor, e.g., Ab6), optionally in combination with checkpoint inhibitor therapy. Especially responsive.

General Characteristics of TGFβ1-Related Symptoms TGFβ1 inhibitors, such as the isoform-selective inhibitors described herein, can be used to treat a wide variety of symptoms associated with TGFβ1 dysregulation (i.e., “TGFβ1-associated symptoms”) in human subjects. It may be used to treat diseases, disorders and/or conditions. As used herein, "a disease (disorder or condition) associated with TGFβ1 dysregulation" or "TGFβ1-related symptom" refers to any disease, disorder and/or condition associated with the expression, activity and/or metabolism of TGFβ1. means any disease, disorder and/or condition that may benefit from inhibition of the condition or activity and/or level of TGFβ1. There is a large amount of evidence in the literature directed towards dysregulation of the TGFβ signaling pathway in pathological conditions such as cancer and fibrosis.

Based on the inventors' recognition that TGFβ1 appears to be the predominant disease-associated isoform, the present disclosure relies on an isoform-selective context in methods for treating TGFβ1-related symptoms in human subjects. including the use of TGFβ1 inhibitors that do not. Such inhibitors are usually formulated into pharmaceutical compositions that further include pharmaceutically acceptable excipients. Advantageously, the inhibitor targets both ECM-bound TGFβ1 and immune cell-bound TGFβ1, but not TGFβ2 or TGFβ3 in vivo. In some embodiments, the inhibitor inhibits the activation step of TGFβ1. The disease is associated with the following attributes: a) regulatory T cells (Tregs); b) effector T cell (Teff) proliferation or function; c) myeloid cell proliferation or differentiation; d) monocyte recruitment or differentiation; e) macrophage function; f) epithelial-mesenchymal transition (EMT) and/or endothelial-mesenchymal transition (EndMT); g) a marker selected from the group consisting of PAI-1, ACTA2, CCL2, Col1a1, Col3a1, FN-1, CTGF, and TGFB1. It may be characterized by dysregulation or impairment in at least two of: gene expression in one or more of the following: h) ECM components or function; i) fibroblast differentiation. A therapeutically effective amount of such an inhibitor is administered to a subject suffering from or diagnosed with a disease.

In some embodiments, such therapeutic uses incorporate diagnosing and/or monitoring therapeutic response as detailed herein. For example, circulating MDSCs and/or circulating latent TGFβ1 can be used as biomarkers according to the present disclosure. Such therapeutic uses may further include selecting a suitable TGFβ inhibitor for therapy and/or selecting a patient or patient population that may benefit from such therapy.

In some embodiments, the diseases treated herein can include dysregulation or disorders of ECM components or functions that exhibit increased collagen I deposition. In some embodiments, the ECM dysregulation includes increased stiffness of the matrix. In some embodiments, the ECM dysregulation involves fibronectin and/or fibrillin.

In some embodiments, the dysregulation or disorder of fibroblast differentiation comprises an increase in myofibroblasts or myofibroblast-like cells. In some embodiments, the myofibroblast or myofibroblast-like cell is a cancer-associated fibroblast (CAF). In some embodiments, CAFs may be associated with tumor stroma to produce CCL2/MCP-1 and/or CXCL12/SDF-1. In some embodiments, the myofibroblasts or myofibroblast-like cells are localized to fibrotic tissue.

In some embodiments, the regulatory T cell dysregulation or disorder comprises increased Treg activity.

In some embodiments, the dysregulation or impairment of effector T cell (Teff) proliferation or function comprises suppression of CD4+/CD8+ cell proliferation.

In some embodiments, the dysregulation or disorder of bone marrow cell proliferation or differentiation comprises increased proliferation of bone marrow progenitor cells. Increased proliferation of bone marrow cells can occur in the bone marrow.

In some embodiments, dysregulation or impairment of monocyte differentiation results in increased differentiation of bone marrow-derived and/or tissue-resident monocytes into macrophages at disease sites such as fibrotic tissues and/or solid tumors. include.

In some embodiments, dysregulation or impairment of monocyte recruitment includes increased recruitment of bone marrow-derived monocytes to disease sites, such as the TME, with increased macrophage differentiation and M2 polarization followed by increased TAM. bring about.

In some embodiments, the dysregulation or impairment of macrophage function includes increased polarization of macrophages to the M2 phenotype.

In some embodiments, the dysregulation or disorder of bone marrow cell proliferation or differentiation comprises an increase in the number of Tregs, MDSCs and/or TANs.

TGFβ-related manifestations are immune-eliminated diseases such as tumors or cancerous tissues that partially suppress the body's normal defense mechanisms/immunity by eliminating effector immune cells (e.g., CD4+ and/or CD8+ T cells). may include conditions involving the microenvironment. In some embodiments, such immunocompromising conditions are associated with inadequate responsiveness to treatment (eg, cancer therapy). Non-limiting examples of cancer therapies for patients who respond poorly include, but are not limited to, checkpoint inhibitor therapy, cancer vaccines, chemotherapy, and radiation therapy (such as radiotherapeutic agents). Without intending to be bound by any particular theory, TGFβ inhibitors, such as those described herein, promote immune cell access, e.g., T cell proliferation and/or tumor infiltration. countering the tumor's ability to evade or eliminate anti-cancer immunity by re-establishing the access of T cells (e.g., CD8+ cells) and macrophages (e.g., F4/80+ cells, M1-polarized macrophages) by It is thought that there is a possibility of promoting

Therefore, inhibition of TGFβ may lead to therapeutic resistance (e.g., immune checkpoint resistance, cancer vaccine resistance, CAR-T resistance, chemotherapy resistance, radiotherapy resistance (such as resistance to radiotherapeutic agents), etc.). Such intensification of inhibition of TGFβ may further result in long-lasting immune memory mediated by, for example, CD8+ T cells.

In some embodiments, the tumor is poorly immunogenic (eg, a "desert" or "non-inflammatory" tumor). Patients may benefit from cancer therapies that induce neo-antigens or promote immune responses. Such therapies include chemotherapy, radiotherapy (including radiotherapeutic agents), oncolytic virotherapy, oncolytic peptides, tyrosine kinase inhibitors, neo-epitope vaccines, anti-CTLA4, instability-inducing agents, and DDR agents. , NK cell activators, and TLR ligands/agonists. TGFβ1 inhibitors such as those described herein can be used in combination to boost the effectiveness of cancer therapy. One mechanism of action of TGFβ1 inhibitors may be to promote T cell immunity by normalizing or reconstituting MHC expression.

Non-limiting examples of TGFβ-related symptoms include organ fibrosis (e.g., renal fibrosis, liver fibrosis, cardiac/cardiovascular fibrosis, myofibrosis, skin fibrosis, uterine fibrosis/endometriosis, and fibrosis, including pulmonary fibrosis, scleroderma, Alport syndrome, cancer (blood cancers such as leukemia, myelofibrosis, multiple myeloma, colon cancer, kidney cancer, breast cancer, malignant melanoma, and glioblastoma). fibrosis associated with solid tumors (e.g., cancerous fibrosis such as fibrous melanoma, pancreatic cancer-associated fibrosis, and breast cancer fibrosis), interstitial fibrosis (e.g., fibrosis between the breasts); radiation-induced fibrosis (e.g. radiation fibrosis syndrome), promotion of rapid hematopoietic development after chemotherapy, bone healing, wound healing, dementia, myelofibrosis, bone marrow dysplasia (e.g. myelodysplastic syndrome or MDS), renal disease (e.g. end-stage renal disease or ESRD), unilateral ureteral ligation (UUO), tooth loss and/or degeneration, endothelial growth syndrome, asthma and allergies, gastrointestinal disorders, aging. Anemia, aortic aneurysm, orphan signs (such as Marfan syndrome and Kamrachi-Engelman disease), obesity, diabetes, arthritis, multiple sclerosis, muscular dystrophy, bone disorders, amyotrophic lateral sclerosis (ALS), Parkinson's disease , osteoporosis, osteoarthritis, osteopenia, metabolic syndrome, nutritional disorders, organ atrophy, chronic obstructive pulmonary disease (COPD), and anorexia.

Evidence suggests that the ectonucleotidases CD39 and CD73 may contribute, at least in part, to elevated levels of adenosine in disease states. Of note, the CD39/CD73-TGFβ axis may play a role in regulating immune cells involved in TGFβ signaling, including Tregs and MDSCs. Both regulatory T cells (Tregs) and myeloid-derived suppressive cells (MDSCs) generally exhibit an immunosuppressive phenotype. In many pathological conditions (eg, cancer, fibrosis), these cells become concentrated at the disease site and can contribute to creating and/or maintaining an immunosuppressive environment. This is mediated, at least in part, by the ectonucleotidases CD39 and CD73, which are involved in the breakdown of ATP to the nucleoside adenosine, and increases the local concentration of adenosine in disease environments, such as the tumor microenvironment and fibrotic environment. could lead to an increase. Adenosine can bind to its receptors expressed on target cells such as T cells and NK cells, thereby suppressing the antitumor functions of these target cells.

Diseases with Abnormal Gene Expression; Biomarkers Aberrant activation of the TGFβ signaling pathway in various disease states has been observed to be associated with changes in gene expression of a number of markers. These gene expression markers (e.g., measured by mRNA) include Serpin 1 (encodes PAI-1), MCP-1 (also known as CCL2), Col1a1, Col3a1, FN1, TGFB1, CTGF, ACTA2 ( encodes α-SMA), SNAI1 (drives EMT in fibrosis and metastasis by downregulating E-cadherin (Cdh1)), MMP2 (matrix metalloprotease associated with EMT), MMP9 (associated with EMT) TIMP1 (matrix metalloprotease associated with EMT), FOXP3 (marker of Treg induction), CDH1 (E-cadherin (marker of epithelial cells) downregulated by TGFβ) and CDH2 (upregulated by TGFβ) including, but not limited to, N-cadherin (a marker for mesenchymal cells). Interestingly, many of these genes have been suggested to play a role in a diverse array of disease states, including various types of organ fibrosis, as well as many cancers, including myelofibrosis. Indeed, a pathophysiological link between fibrotic pathology and abnormal cell proliferation, tumorigenesis and metastasis has been suggested. For example, Cox and Erler (2014) Clinical Cancer Research 20(14):3637-43 “Molecular pathways: connecting fibrosis and solid tumor metastases” is”, Shiga et al. , (2015) Cancers 7:2443-2458 “Cancer-associated fibroblasts: their characteristics and their roles in tumor growth”; Wynn and Ba rron (2010) Semin. Liver Dis. 30(3):245-257 "Macrophages: master regulators of inflammation and fibrosis". The contents of these documents are incorporated herein by reference. While not wishing to be bound by any particular theory, the inventors of the present disclosure believe that the TGFβ1 signaling pathway may indeed be an important link between these broad pathologies.

The ability of chemotactic cytokines (or chemokines) to mediate leukocyte recruitment (eg, monocytes/macrophages) to damaged or diseased tissues has crucial consequences in disease progression. Monocyte chemoattractant protein 1 (MCP-1) also known as CCL2, macrophage inflammatory protein 1-alpha (MIP-1α) also known as CCL3 and MIP-1β also known as CCL4 and also known as CXCL2 Members of the CC chemokine family, such as MIP-2α, are involved in this process.

For example, MCP-1/CCL2 is thought to play a role in both fibrosis and cancer. MCP-1/CCL2 is characterized as a profibrotic chemokine and is a monocyte chemoattractant, and evidence suggests that it may be involved in both cancer initiation and progression. In fibrosis, MCP-1/CCL2 has been shown to play an important role in the inflammatory phase of fibrosis. For example, neutralization of MCP-1 dramatically reduced glomerular crescent formation and type I collagen deposition. Similarly, passive immunotherapy with anti-MCP-1 antibodies or anti-MIP-1 alpha antibodies was shown to significantly reduce the accumulation of mononuclear phagocytes in bleomycin-challenged mice, which was associated with MIP-1 alpha and MCP-1 contribute to leukocyte recruitment during pulmonary inflammatory responses (Smith, Biol Signals. 1996 Jul-Aug; 5(4): 223-31, “Chemotactic cytokines mediate leukocyte recruitment in fibrotic disease”). MIP-1 alpha levels have been reported to be elevated in patients with cystic fibrosis and multiple myeloma (e.g., Mrugacz et al, J Interferon Cytokine Res. 2007 Jun;27(6):491- 5), supporting the idea that MIP-1α is associated with localized or systemic inflammatory responses.

A body of evidence indicates the involvement of CC chemokines in tumor progression/metastasis. For example, tumor-derived MCP-1/CCL2 can promote a "cancer-inducing" phenotype in macrophages. For example, in lung cancer, MCP-1/CCL2 is produced by stromal cells and has been shown to promote metastasis. In human pancreatic cancer, tumors secrete CCL2 and immunosuppressive CCR2-positive macrophages infiltrate these tumors. Patients with tumors exhibiting high expression of CCL2/low infiltration of CD8 T cells have significantly decreased survival. While not wishing to be bound by any particular theory, monocytes recruited to an injured or diseased tissue environment subsequently undergo polarization in response to local cues (e.g., in response to tumor-derived cytokines). It is thought that this may lead to further progression of the disease. These M2-like macrophages likely contribute to immune evasion by suppressing effector cells such as CD4+ and CD8+ T cells. In some embodiments, this process is partially mediated by LRRC33-TGFβ1 expressed by activated macrophages. In some embodiments, this process is partially mediated by GARP-TGFβ1 expressed by Tregs.

Similarly, in certain carcinomas such as breast cancer (e.g., triple-negative breast cancer), CXCL2/CCL22-mediated recruitment of MDSCs has been shown to promote angiogenesis and metastasis (e.g., Kumar et al., (2018) J Clin Invest 128(11):5095-5109). Therefore, this process is thought to be mediated, at least in part, by TGFβ1, such as LRRC33-TGFβ1. Furthermore, since proteases such as MMP9 are involved in matrix remodeling processes that contribute to tumor invasion and metastasis, the same or overlapping signaling pathways may also play a role in fibrosis.

PAI-1/serpin1 has been implicated in various fibrotic pathologies, cancer, angiogenesis, inflammation and neurodegenerative diseases (eg Alzheimer's disease). Elevated expression of PAI-1 in tumors and/or serum has been linked to poor prognosis (e.g., shortened survival, metastasis) in various cancers, such as breast cancer and bladder cancer (e.g., transitional cell carcinoma), as well as myelofibrosis. increase). In the context of fibrotic pathologies, PAI-1 has been recognized as an important downregulated effector of TGFβ1-induced fibrosis, and increased PAI-1 expression is associated with pulmonary fibrosis (such as IPF), renal fibrosis, and hepatic fibrosis. It has been observed in various forms of tissue fibrosis, including scleroderma and scleroderma. In some embodiments, this process is partially mediated by ECM-associated TGFβ1, such as LTBP1-proTGFβ1 and/or LTBP3-proTGFβ1.

In some embodiments, the in vivo effects of TGFβ1 inhibitor therapy can be assessed by measuring changes in the expression levels of suitable genetic markers. Suitable markers include TGFβ (eg, TGFB1, TGFB2 and TGFB3). Suitable markers may also include one or more presenting molecules for TGFβ (eg, TGFβ1, TGFβ2 and TGFβ3), such as LTBP1, LTBP3, GARP (or LRRC32) and LRRC33. In some embodiments, suitable markers include mesenchymal transition genes (e.g., AXL, ROR2, WNT5A, LOXL2, TWIST2, TAGLN, and/or FAP), immunosuppressive genes (e.g., IL10, VEGFA, VEGFC), Monocyte and macrophage chemotaxis genes (eg, CCL2, CCL3, CCL4, CCL7, CCL8, CCL13 and CCL22) and/or various fibrotic markers discussed herein are included. An exemplary marker is a plasma/serum marker.

As described in PCT/U.S. Patent Application Publication No. 2019/041373, isoform-specific, context-independent inhibitors of TGFβ1 are capable of inhibiting mechanisms such as UUO, which have been shown to be TGFβ1-dependent. It can be used to reduce the expression levels of many of these markers in suitable preclinical models, including clinical animal models. Such inhibitors may therefore be used to treat diseases or disorders characterized by aberrant expression (e.g., overexpression/upregulation or underexpression/downregulation) of one or more gene expression markers of the disease. can be done.

Accordingly, in some embodiments, isoform-specific, context-independent inhibitors of TGFβ1 include: PAI-1 (encoded by Serpin 1), MCP-1 (also known as CCL2), Used in the treatment of diseases associated with overexpression of one or more of Col1a1, Col3a1, FN1, TGFB1, CTGF, α-SMA, ITGA11 and ACTA2, which therapy uses this inhibitor to treat the disease. comprising administering an effective amount to a subject suffering from a disease. In some embodiments, inhibitors are used to treat diseases associated with overexpression of PAI-1, MCP-1/CCL2, CTGF, and/or α-SMA. In some embodiments, the disease is myelofibrosis. In some embodiments, the disease is cancer, eg, cancer, including solid tumors.

The involvement of the TGFβ1 pathway in the regulation of important aspects of both ECM and immune components may explain the observation that a significant number of dysregulated genes are shared across a wide range of disease states, such as proliferative and fibrotic disorders. This supports the idea that abnormal expression patterns in genes involved in TGFβ1 signaling are likely to be a generalizable phenomenon. These marker genes fall into several categories: genes involved in mesenchymal transition (e.g., EndMT and EMT), genes involved in angiogenesis, genes involved in hypoxia, and wound healing. genes involved in; and genes involved in tissue injury-induced inflammatory responses.

Hugo et al. , (Cell, 165(1):35-44) revealed differential gene expression patterns of these classes of markers and responsiveness to checkpoint blockade therapy (CBT) in metastatic melanoma. The correlation between the two was clearly demonstrated. The authors show that co-enrichment analysis of a set of genes, coined the "IPRES signature," defines transcriptome subsets within all major common human malignancies analyzed, not just melanoma. I found it. In fact, this study demonstrated that CBT could influence tumor cell phenotypic plasticity (i.e., mesenchymal transition) and the resulting effects on the microenvironment (e.g., ECM remodeling, cell adhesion, and angiogenic function of immunosuppressive wound healing). Linked to resistance. In addition to IPRES, TIDE (Jing et al., Nat Med. 2018 Oct; 24 (10): 1550-1558), TIS (Danaher et al., J Immunother Cancer. 2018 Jun 22; 6 (1): 63) , F-TBRS (Mariathasan et al., Nature. 2018 Feb 22; 554 (7693): 544-548), IMPRES (Auslander et al., Nat Med. 2018 Oct; 24 (10): 1545-1549) and xCell Other gene signatures such as (Aran et al. Genome Biol. 2017 Nov 15;18(1):220) can also be used to assess the tumor immune microenvironment.

Applicants have recognized that each of these IPRES gene categories are involved in diseases involving TGFβ dysregulation and have previously shown that TGFβ1 isoforms may specifically mediate these processes in disease states. I thought about it (for example, please refer to International Publication No. 2017/156500 pamphlet). Previously disclosed studies further support this idea (e.g., Example 11 of PCT/U.S. Patent Application Publication No. 2019/041373, FIG. 37A) and show that therapeutics that selectively target TGFβ1 ( (as opposed to non-selective alternatives), further confirming that it may offer advantages in terms of both efficacy and safety.

Accordingly, the present disclosure provides a method/process for selecting or identifying a candidate patient or population of candidates likely to respond to TGFβ1 inhibition therapy and administering to the patient an effective amount of a high affinity isoform selective inhibitor of TGFβ1. including. Observation of a patient's lack of responsiveness (eg, resistance) to CBT may indicate that the patient is a candidate for TGFβ1 inhibition therapy as described herein. Thus, isoform-selective inhibitors of TGFβ1, such as Ab6, may be used to treat cancer in subjects who have a poor response to CBT. The subject may have advanced cancer, such as a locally advanced solid tumor or metastatic cancer. After a period of time that would be expected to be sufficient to demonstrate a significant therapeutic effect of a particular therapy, no or little significant therapeutic effect is obtained (e.g., according to standard guidelines such as RECIST and iRECIST). A patient is said to be a "poor responder" if the patient does not meet the criteria for a partial or complete response (based on the criteria for partial response or complete response). Typically such a period for CBT is at least about 3 months of treatment, with or without the addition of both such as chemotherapy. Such patients are sometimes referred to as "refractory" or "non-responders." If such a patient has a poor response to initial CBT, the patient may be referred to as a "primary non-responder." Cancers (or patients with such cancers) belonging to this category can be characterized as having "primary resistance" to CBT. In some embodiments, the subject is a primary non-responder after receiving at least about 3 months of CBT treatment (optionally after receiving at least about 4 months of CBT treatment). In some embodiments, the subject is also receiving additional therapy in combination with CBT, such as chemotherapy.

Once a subject has been identified as a non-responder to CBT, a high affinity isoform selective inhibitor of TGFβ1 can be administered to the subject in conjunction with CBT. In this case, the CBT may or may not contain the same checkpoint inhibitor as the initial CBT to which the subject did not respond. Any suitable immune checkpoint inhibitor can be used, including approved checkpoint inhibitors. In some embodiments, a high affinity isoform selective inhibitor of TGFβ1 is administered to a subject in conjunction with a CBT comprising an anti-PD-1 antibody or an anti-PD-L1 antibody. High-affinity isoform-selective inhibitors of TGFβ1 aim to overcome resistance by increasing the sensitivity of cancers to CBT.

The process of selecting or identifying candidate patients or candidate patient populations who are likely to respond to or otherwise benefit from TGFβ1 inhibitory therapy involves the use of biopsy samples collected from patients (or patient populations). may include testing a biological sample, such as, for expression of one or more of the markers discussed herein. Similarly, such genetic markers can be used for purposes of monitoring patient responsiveness to therapy. Monitoring involves testing two or more biological samples collected from a patient, e.g., before and after treatment is administered, and over time during the course of a treatment regimen to indicate treatment response or efficacy. may include assessing changes in gene expression levels of markers of. In some embodiments, liquid biopsy may be used.

In some embodiments, the method of selecting candidate patients or candidate patient populations that are likely to respond to TGFβ1 inhibitory therapy is based on previously tested for genetic markers such as those described herein and their abnormal The method may include identifying a patient or patient population that exhibits the expression. These same methods can later be applied to confirm or correlate a patient's response to treatment.

In some embodiments, the aberrant expression of the marker comprises elevated levels of at least one of the following: TGFβ1, LRRC33, GARP, LTBP1, LTBP3, CCL2, CCL3, PAI-1/Serpen1. In some embodiments, the patient or patient population (eg, a biological sample taken therefrom) exhibits elevated TGFβ1 activation, phospho-Smad2, phospho-Smad2/3, or a combination thereof. In some embodiments, the patient or patient population (eg, a biological sample taken therefrom) exhibits elevated MDSC. In some embodiments, such a patient or patient population has a cancer that may include a solid tumor that is TGFβ1 positive. Solid tumors are TGFβ1-dominant tumors, in which TGFβ1 is the predominant isoform expressed compared to other isoforms. In some embodiments, the solid tumor can be a TGFβ1-codominant tumor where TGFβ1 is expressed in the tumor in a codominant isoform, eg, TGFβ1+/TGFβ3+. In some embodiments, such patients or patient populations receive chemotherapy, radiation therapy (such as radiotherapeutic agents), and/or immune checkpoint therapy, such as anti-PD-1 (e.g., pembrolizumab, nivolumab, and spartalizumab). ), anti-PD-L1 (eg, atezolizumab), anti-CTLA4 (eg, ipilimumab), genetically engineered immune cell therapy (eg, CAR-T), and cancer vaccines. According to the present disclosure, the TGFβ1 inhibitors provided herein, such as Ab6, induce resistance by releasing immunosuppression and allowing effector cells to invade cancer cells, thereby obtaining antitumor effects. Overcome. Therefore, TGFβ1 inhibitor therapy may promote effector cell infiltration and/or expansion in tumors. Furthermore, TGFβ1 inhibitor therapy can reduce the frequency of immunosuppressive immune cells such as Tregs and MDSCs in tumors.

In some embodiments, the aberrant expression of markers includes one or more panels of genes such as: mesenchymal transition markers (e.g., AXL, ROR2, WNT5A, LOXL2, TWIST2, TAGLN, FAP); Immunosuppressive genes (e.g. IL10, VEGFA, VEGFC), monocyte and macrophage chemotaxis genes (e.g. CCL2, CCL7, CCL8, CCL13), genes involved in angiogenesis and wound healing (e.g. suppressive T cells) , cell adhesion markers, ECM remodeling; skeletal system and bone development markers and genes involved in tissue damage-induced inflammatory responses.

In some embodiments, lack or downregulation of MHC expression (such as MHC class 1) may serve as a biomarker for TGFβ1-related pathologies for which antibodies or antigen-binding fragments encompassed by this disclosure may be used therapeutically. Decreased MHC levels may be a signal of immune escape, which may correlate with poor patient response to immunotherapy such as CBT. Therefore, selective inhibition of TGFβ1 may at least partially restore effector cell function.

The present disclosure provides a TGFβ inhibitor (e.g., a TGFβ1 selective inhibitor such as Ab6) for use in treating a TGFβ-related disorder involving aberrant expression of a gene (e.g., as described herein) in a patient. and the treatment includes administering a composition comprising a TGFβ inhibitor (eg, a TGFβ1 inhibitor) selected based at least in part on its immunosafety profile. A favorable immunosafety profile of a TGFβ inhibitor is that it: i) does not induce unacceptable levels of cytokine release (e.g., within 2.5 times the control); ii) does not promote unacceptable levels of platelet aggregation; or Both are characterized in accepted cell-based assays and/or in vivo assays (such as those described herein).

Diseases Associated with Mesenchymal Transition Mesenchymal transition is a process in which the phenotype of cells such as epithelial cells and endothelial cells shifts to a mesenchymal phenotype (such as myofibroblasts). Examples of genetic markers indicative of mesenchymal transition include AXL, ROR2, WNT5, LOXL2, TWIST2, TAGLN and FAP. For example, in cancer, mesenchymal transition (eg, increased EndMT and EMT signatures) indicates phenotypic plasticity of tumor cells. Therefore, inhibitors of TGFβ, eg TGFβ1 inhibitors such as Ab6, can be used to treat diseases initiated or driven by mesenchymal transition such as EMT and EndMT.

EMT (epithelial-mesenchymal transition) is a process in which epithelial cells in tight junctions change to mesenchymal characteristics (phenotype), such as loosening and cell-cell contacts. This process is observed in many normal biological processes and pathological conditions, including embryonic development, wound healing, cancer metastasis, and fibrosis (e.g., Shiga et al., (2015) “Cancer-Associated Fibroblasts : Their Characteristics and Their Roles in Tumor Growth.”Cancers, 7:2443-2458). It is generally believed that EMT signals are mainly induced by TGFβ. For example, many types of cancer appear to involve transdifferentiation of cells toward a mesenchymal phenotype (such as myofibroblasts and CAFs) that correlates with a poorer prognosis. Therefore, isoform-specific, context-independent inhibitors of TGFβ1, such as those described herein, can be used to treat diseases initiated or driven by EMT. Indeed, data illustrated in PCT/U.S. Patent Application Publication No. 2019/041373 (e.g., FIGS. 4-6) show that such inhibitors It has been shown that it has the ability to suppress the in vivo expression of myofibroblast/CAF markers such as FN (fibronectin). Therefore, TGFβ inhibitors, for example TGFβ1 inhibitors such as Ab6, can be used in the treatment of diseases characterized by EMT. A therapeutically effective amount of an inhibitor can be an amount sufficient to reduce the expression of markers such as α-SMA/ACTA2, LOXL2, Col1 (type I collagen) and FN (fibronectin). In some embodiments, the disease is a proliferative disease such as cancer.

Similarly, TGFβ is also an important regulator of endothelial-to-mesenchymal transition (EndMT) observed in normal development such as heart formation. However, the same or similar phenomenon is seen in many disease-related tissues, such as cancer stroma and fibrotic sites. In some disease processes, endothelial markers such as CD31 are downregulated upon TGFβ1 exposure, and instead the expression of mesenchymal markers such as FSP-1, α-SMA/ACTA2 and fibronectin is induced. In fact, stromal CAFs may be derived from vascular endothelial cells. Therefore, TGFβ inhibitors, eg TGFβ1 inhibitors such as Ab6, can be used in the treatment of diseases characterized by EndMT. A therapeutically effective amount of an inhibitor can be an amount sufficient to reduce the expression of markers such as FSP-1, α-SMA/ACTA2 and fibronectin. In some embodiments, the disease is a proliferative disease such as cancer.

The present disclosure provides TGFβ inhibitors (e.g., TGFβ1 selective inhibitors such as Ab6) for use in the treatment of TGFβ-related disorders involving mesenchymal transition (e.g., as described herein) in patients. The providing and treatment includes administering a composition comprising a TGFβ inhibitor (eg, a TGFβ1 inhibitor) selected based at least in part on its immunosafety profile. A favorable immunosafety profile of a TGFβ inhibitor is that it: i) does not induce unacceptable levels of cytokine release (e.g., within 2.5 times the control); ii) does not promote unacceptable levels of platelet aggregation; or Both are characterized in accepted cell-based assays and/or in vivo assays (such as those described herein).

Diseases involving matrix stiffening and remodeling The progression of various TGFβ1-related indications such as fibrotic pathologies and cancer (e.g., tumor growth and metastasis) is associated with increased levels of matrix components deposited in the ECM and/or maintenance of the ECM. /Includes remodeling. Increased deposition of ECM components such as collagen can alter the mechanophysical properties of the ECM (e.g., matrix/substrate stiffness), and this phenomenon has been reported to be associated with TGFβ1 signaling. There is. Applicants have previously reported that TGFβ demonstrated the role of matrix stiffness on integrin-dependent activation of As disclosed in WO 2018/129329, matrices with higher stiffness enhance TGFβ1 activation, which can be suppressed by isoform-specific inhibitors of TGFβ1. These observations suggest that TGFβ1 influences ECM properties (such as stiffness), which in turn may further induce TGFβ1 activation that reflects disease progression.

Therefore, TGFβ1 inhibitors such as Ab6 can be used to block this process and counter disease progression that involves ECM changes such as fibrosis, tumor growth, invasion, metastasis and desmoplasia. The LTBP arm of such inhibitors can directly target the ECM-associated pro/latent TGFβ1 complex presented by LTBP1 and/or LTBP3, thereby reducing the activity of growth factors from the complex in disease niches. Prevents oxidation/release. In some embodiments, TGFβ1 inhibitors may normalize ECM stiffness to treat diseases involving integrin-dependent signaling. In some embodiments, the integrin comprises an α11 chain, a β1 chain, or both. The structure of the ECM, eg, ECM components and organization, can also be altered by matrix-associated proteases. Thus, in some embodiments, TGFβ1 inhibitors can normalize ECM stiffness to treat diseases associated with disease-associated ECM, such as protease-dependent signaling in tumors and fibrotic tissues. .

Lampi and Reinhart-King (Science Translational Medicine, 10(422): eaao0475, “Targeting extracellular matrix stiffness to attenu ate disease: From molecular mechanisms to clinical trials”), the increase in tissue ECM stiffness , occurring during the pathological progression of cancer, fibrosis and cardiovascular diseases. Mechanical properties associated with this process involve phenotypically transformed myofibroblasts, TGFβ and matrix crosslinks. A major cause of increased ECM stiffness in cancer and fibrotic diseases is dysregulated matrix synthesis and remodeling by activated fibroblasts that dedifferentiate into myofibroblasts (eg, CAF and FAF). Tumor stromal remodeling and organ fibrosis show striking similarities to the wound healing response, except that the response persists in pathological conditions. Myofibroblasts are a heterogeneous cell population with pathology-specific progenitors derived from multiple cell sources, including bone marrow-derived cells and tissue-resident cells. Commonly used myofibroblast markers include alpha smooth muscle actin (α-SMA). As shown in PCT/U.S. Patent Application Publication No. 2019/041373, high affinity isoform-specific TGFβ1 inhibitors inhibit ACTA2 expression (encoding α-SMA), collagen, and FN (fibronectin) can be reduced. Fibronectin is important in anchoring the LTBP-associated proTGFβ1 complex to matrix structures.

The importance of the TGFβ pathway in ECM regulation is well established. Integrin-TGFβ1 interaction has become a therapeutic target because TGFβ1 (and TGFβ3) can be activated mechanically by certain integrins (eg, αv integrin). For example, monoclonal antibodies against αvβ6 are being investigated for idiopathic pulmonary fibrosis. However, such an approach would also be expected to interfere with TGFβ3 signaling, which shares the same integrin-binding motif RGD, and furthermore, such antibodies would inhibit TGFβ1 that is activated by other methods, such as protease-induced activation. would not be effective in blocking. In contrast, high affinity isoform-specific TGFβ1 inhibitors such as Ab6 can block both protease-dependent activation of TGFβ1 (FIG) and integrin-dependent activation of TGFβ1. Such TGFβ1 inhibitors may therefore offer superior properties. Based on Applicant's previous work, high affinity isoform selective inhibitors of TGFβ1 may be effective in treating diseases associated with ECM stiffness.

Accordingly, the present disclosure includes the therapeutic use of isoform-selective inhibitors of TGFβ1 in the treatment of diseases associated with matrix stiffness or in methods of reducing matrix stiffness in a subject. Such uses include administering a therapeutically effective amount of an isoform selective inhibitor of TGFβ1, such as Ab6.

The present disclosure provides that TGFβ inhibitors (e.g., TGFβ1 selective inhibitors such as Ab6) for use in treating TGFβ-related disorders involving matrix stiffening and remodeling (e.g., as described herein) in a patient. ), and the treatment includes administering a composition comprising a TGFβ inhibitor (eg, a TGFβ1 inhibitor) selected based at least in part on its immunosafety profile. A favorable immunosafety profile of a TGFβ inhibitor is that it: i) does not induce unacceptable levels of cytokine release (e.g., within 2.5 times the control); ii) does not promote unacceptable levels of platelet aggregation; or Both are characterized in accepted cell-based assays and/or in vivo assays (such as those described herein).

Diseases involving proteases Activation of TGFβ from its latent complex can be induced force-dependently by integrins and/or mechanically by proteases. Certain classes of proteases, such as, but not limited to, Ser/Thr proteases, such as kallikrein, chemotrypsin, elastase, plasmin, thrombin, and zinc metalloproteases of the MMP family, such as MMP-2, MMP-9 and MMP-13. Evidence suggests that Adam family proteases such as Adam10 and Adam17 may be involved in this process. MMP-2 degrades collagen IV, the most abundant component of the basement membrane, raising the possibility that it plays a role in ECM-associated TGFβ1 regulation. MMP-9 has been suggested to play a central role in tumor progression, angiogenesis, stromal remodeling, and metastasis in cancers such as breast cancer. Therefore, protease-dependent activation of TGFβ1 in the ECM may be important for the treatment of ECM-related diseases such as fibrosis and cancer.

Kallikrein (KLK) is a trypsin-like or chymotrypsin-like serine protease, including plasma kallikrein and tissue kallikrein. The ECM plays a role in tissue homeostasis, acting as a structural and signaling scaffold and barrier that suppresses malignant growth. KLK may play a role in the degradation of ECM proteins and other components that can promote tumor expansion and invasion. For example, KLK1 is greatly upregulated in certain breast cancers and can activate proMMP-2 and proMMP-9. KLK2 activates latent TGFβ1, bringing prostate cancer adjacent to fibroblasts and allowing cancer growth. KLK3 has been widely studied as a diagnostic marker (PSA) for prostate cancer. KLK3 can directly activate TGFβ1 by processing plasminogen to plasmin, which proteolytically cleaves LAP, thereby releasing the TGFβ1 growth factor from the latent complex. KLK6 may be a potential marker for Alzheimer's disease.

Furthermore, data previously provided by the applicant indicates that such a protease may be kallikrein. Accordingly, the present disclosure encompasses the use of isoform-specific, context-independent inhibitors of TGFβ1 in methods of treating diseases associated with kallikrein or kallikrein-like proteases. In some embodiments, the TGFβ1 inhibitor is Ab6 or a derivative thereof.

Known activators of TGFβ1, such as plasmin, TSP-1 and αVβ6 integrin, all interact directly with LAP. It is hypothesized that proteolytic cleavage of LAP may destabilize the LAP-TGFβ interaction, thereby releasing active TGFβ1 (growth factor domain) from the latent complex. It has been suggested that the region containing the amino acid stretch 54-LSKLRL-59 is important for maintaining TGFβ1 latency. Therefore, agents (eg, antibodies) that stabilize the interaction or block proteolytic cleavage of LAP may prevent activation of TGFβ1.

Many of these proteases associated with pathological conditions (eg, cancer) function by different mechanisms of action. Thus, targeted inhibition of particular proteases, or combinations of proteases, may provide therapeutic benefit in the treatment of pathologies involving the protease-TGFβ axis. Therefore, it is believed that inhibitors that selectively inhibit protease-induced activation of TGFβ1 (eg, TGFβ1 antibodies) may be advantageous in the treatment of such diseases (eg, cancer). Similarly, selective inhibition of TGFβ1 activation by one protease over another may also provide therapeutic benefit depending on the disease state being treated.

Plasmin is a serine protease produced as a precursor form called plasminogen. Once released, plasmin enters the blood circulation and is therefore detected in the serum. Elevated serum plasmin concentrations appear to correlate with cancer progression, possibly through mechanisms involving disruption of the extracellular matrix (e.g., basement membrane and stromal barrier), which promotes tumor cell motility, invasion, and metastasis. be. Plasmin can also affect adhesion, proliferation, apoptosis, cancer nutrition, oxygen supply, blood vessel formation and activation of VEGF (Didiasova et al., Int. J. Mol. Sci, 2014, 15, 21229- 21252). Furthermore, plasmin can promote the migration of macrophages into the tumor microenvironment (Philips et al., Cancer Res. 2011 Nov 1;71(21):6676-83 and Choong et al., Clin. Orthop. Relat. Res. 2003, 415S, S46-S58). Indeed, tumor-associated macrophages (TAMs) are well-characterized drivers of tumorigenesis due to their ability to promote tumor growth, invasion, metastasis, and angiogenesis.

Plasmin activity is primarily associated with ECM destruction. However, there is increasing evidence that plasmin also regulates downstream activation of MMPs and TGFβ. Specifically, plasmin causes activation of TGFβ by proteolytic cleavage of the latent-associated peptide (LAP) derived from the N-terminal region of the TGFβ gene product (Horiguchi et al., J Biochem. 2012 Oct; 152 ( 4):321-9), which has been suggested to result in the release of active growth factors. Since TGFβ1 can promote cancer progression, this raises the possibility that plasmin-induced activation of TGFβ mediates this process, at least in part.

TGFβ1 has also been shown to regulate the expression of uPA, which plays an important role in the conversion of plasminogen to plasmin (Santibanez, Juan F., ISRN Dermatology, 2013:597927). uPA can independently promote cancer progression (e.g., adhesion, proliferation, migration) by binding to its cell surface receptor (uPAR) and promoting the conversion of plasminogen to plasmin. It is shown. Furthermore, the expression of uPA and/or plasminogen activation inhibitor-1 (PAI-1) is associated with colorectal cancer (D.Q. Seetoo, et al., Journal of Surgical Oncology, vol. 82, no. 3, pp. 184-193, 2003), breast cancer (N. Harbeck et al., Clinical Breast Cancer, vol. 5, no. 5, pp. 348-352, 2004) and skin cancer (Santibanez, Juan F., ISRN Dermat) ology , 2013:597927) is a predictor of poor prognosis. Therefore, without wishing to be bound by any particular theory, the interaction between plasmin, TGFβ1 and uPA may form a positive feedback loop towards promoting cancer progression. Therefore, inhibitors that selectively inhibit plasmin-dependent TGFβ1 activation may be particularly suitable for the treatment of cancers that rely on the plasmin/TGFβ1 signaling axis.

In one aspect of the disclosure, TGFβ inhibitors, such as the isoform-specific inhibitors of TGFβ1 described herein, can inhibit protease-dependent activation of TGFβ1. In some embodiments, the inhibitor can inhibit protease-dependent TGFβ1 activation in an integrin-independent manner. In some embodiments, such inhibitors can inhibit activation of TGFβ1 regardless of the mode of activation, e.g., both integrin-dependent and protease-dependent activation of TGFβ1. can be inhibited. In some embodiments, the protease is selected from the group consisting of serine proteases such as kallikrein, chemotrypsin, trypsin, elastase, plasmin and zinc metalloproteases (MMP family) such as MMP-2, MMP-9 and MMP-13. be done.

In some embodiments, a TGFβ inhibitor (eg, a TGFβ1 antibody) can inhibit plasmin-induced activation of TGFβ1. In some embodiments, the inhibitor can inhibit plasmin- and integrin-induced TGFβ1 activation. In some embodiments, the antibody is a monoclonal antibody that specifically binds proTGFβ1. In some embodiments, the antibody binds latent proTGFβ1, thereby inhibiting release of mature growth factor from the latent complex. In some embodiments, a high affinity, context-dependent inhibitor of TGFβ1 activation suitable for use in the method of inhibiting plasmin-dependent activation of TGFβ1 is Ab6 or a derivative or variant thereof.

In some embodiments, the TGFβ inhibitor (eg, TGFβ1 antibody) inhibits cancer cell migration. In some embodiments, the inhibitor inhibits macrophage migration. In some embodiments, the inhibitor inhibits TAM accumulation.

In another aspect, a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a TGFβ inhibitor (e.g., a TGFβ1 antibody), wherein the inhibitor Provided herein are methods of inhibiting protease-induced activation (eg, plasmin) and thereby treating cancer in a subject.

In another aspect, a method of reducing cancer growth in a subject in need thereof, comprising administering to the subject an effective amount of a TGFβ inhibitor (e.g., a TGFβ1 antibody), the method comprising: Provided herein are methods in which an agent inhibits protease-induced activation of TGFβ1 (eg, plasmin), thereby reducing the growth of a cancer in a subject.

The present disclosure provides TGFβ inhibitors (e.g., TGFβ1 selective inhibitors such as Ab6) for use in treating TGFβ-related disorders associated with proteases (e.g., as described herein) in a patient. and the treatment includes administering a composition comprising a TGFβ inhibitor (eg, a TGFβ1 inhibitor) selected based at least in part on its immunosafety profile. A favorable immunosafety profile of a TGFβ inhibitor is that it: i) does not induce unacceptable levels of cytokine release (e.g., within 2.5 times the control); ii) does not promote unacceptable levels of platelet aggregation; or Both are characterized in accepted cell-based assays and/or in vivo assays (such as those described herein).

Myeloproliferative Disorders/Myelofibrosis The present disclosure provides therapeutic use of TGFβ1 inhibitors, such as Ab6, in the treatment of myeloproliferative disorders. These include, for example, myelodysplastic syndromes (MDS) and myelofibrosis (eg, primary myelofibrosis and secondary myelofibrosis).

Myelofibrosis, also known as bone marrow fibrosis, is a relatively rare bone marrow proliferative disorder (cancer) that belongs to a group of diseases called myeloproliferative disorders. Myelofibrosis is classified as a Philadelphia chromosome negative (-) branch of myeloproliferative neoplasms. Myelofibrosis is characterized by clonal bone marrow proliferation, abnormal cytokine production, extramedullary hematopoiesis, and fibrosis of the bone marrow. Abnormal clonal proliferation of hematopoietic stem cells in the bone marrow and other sites results in fibrosis, or replacement of the bone marrow by scar tissue. The term myelofibrosis refers to primary myelofibrosis (PMF) unless otherwise specified. This is sometimes called chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary are used in these cases to indicate that the cause of the disease is unknown or that the disease is spontaneous). means). This is in contrast to myelofibrosis, which develops secondary to polycythemia vera or essential thrombocythemia. Myelofibrosis is a form of myeloid metaplasia, which refers to changes in cell types in the hematopoietic tissue of the bone marrow, and the two terms are often used interchangeably. The term myelofibrosis with unexplained myeloid metaplasia and myeloid metaplasia (MMM) is also used to refer to primary myelofibrosis. In some embodiments, blood proliferative disorders that may be treated according to the present disclosure include myeloproliferative diseases such as myelofibrosis. The so-called "classic" BCR-ABL (Ph)-negative chronic myeloproliferative disease group includes essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). .

Myelofibrosis inhibits the body's normal production of blood cells. The result is extensive scarring of the bone marrow, severe anemia, weakness, fatigue, and often splenomegaly. Production of cytokines such as fibroblast growth factors by abnormal hematopoietic cell clones (particularly by megakaryocytes) leads to replacement of hematopoietic tissue in the bone marrow by connective tissue through collagen fibrosis. Depletion of hematopoietic tissue impairs the patient's ability to produce new blood cells, resulting in progressive pancytopenia (lack of all blood cell types). However, fibroblast proliferation and collagen deposition are considered secondary phenomena, and the fibroblasts themselves may not be part of the abnormal cell clone.

Myelofibrosis can be caused by abnormalities in blood stem cells in the bone marrow. Abnormal stem cells create mature, poorly differentiated cells that proliferate rapidly and take over the bone marrow, causing both fibrosis (formation of scar tissue) and chronic inflammation.

Primary myelofibrosis is characterized by constitutive activation of the JAK-STAT pathway, progressive scarring, or fibrosis of the bone marrow, which can lead to Janus kinase 2 (JAK2), thrombopoietin receptor (MPL), and carreti It is associated with mutations in CALR. Patients may develop extramedullary hematopoiesis, ie, the formation of blood cells at sites other than the bone marrow, as hematopoietic cells are forced to migrate to other areas, particularly the liver and spleen. This causes these organs to enlarge. In the liver, abnormal size is called hepatomegaly. Enlargement of the spleen is called splenomegaly, which also contributes to pancytopenia, particularly thrombocytopenia, and anemia. Another complication of extramedullary hematopoiesis is osteocytosis, or the presence of dysmorphic red blood cells.

The main site of extramedullary hematopoiesis in myelofibrosis is the spleen, which is usually markedly enlarged in patients suffering from myelofibrosis. Multiple subcapsular infarcts of the spleen often occur as a result of the extremely enlarged spleen. This means that partial or complete tissue death occurs as the oxygen supply to the spleen is cut off. At the cellular level, the spleen contains erythroid progenitors, granulocyte progenitors, and megakaryocytes, with megakaryocytes being prominent in their number and their abnormal shape. Megakaryocytes may be involved in causing the secondary fibrosis seen in this condition.

It has been suggested that TGFβ may be involved in the fibrotic aspect of the pathogenesis of myelofibrosis (e.g., Agarwal et al., “Bone marrow fibrosis in primary myelofibrosis: pathogenic mechanisms and the le of TGFβ” (2016 ) Stem Cell Investig 3:5). Bone marrow lesions in primary myelofibrosis are characterized by fibrosis, angiogenesis, and osteosclerosis, and fibrosis is associated with increased production of collagen deposited in the ECM.

A large number of biomarkers have been described, changes in which are indicative of or correlated with disease. In some embodiments, the biomarker is a cellular marker. Such disease-related biomarkers are useful in diagnosing and/or monitoring disease progression and treatment efficacy (eg, patient responsiveness to treatment). These biomarkers include numerous fibrotic and cellular markers. For example, in lung cancer, it has been reported that TGFβ1 concentration in bronchoalveolar lavage (BAL) fluid is significantly higher (approximately 2+ fold increase) in lung cancer patients compared to patients with benign disease; It may also serve as a biomarker for diagnosing and/or monitoring progression or treatment efficacy.

Because myelofibrosis is associated with the abnormal development of megakaryocytes, specific cellular markers of megakaryocytes and their progenitors of the stem cell lineage are useful markers for diagnosing and/or monitoring disease progression and treatment efficacy. It may be useful as In some embodiments, useful markers include differentiated megakaryocyte cell markers (e.g., CD41, CD42, and Tpo R), megakaryocyte-erythroid progenitor cell markers (e.g., CD34, CD38, and CD45RA-), general cell markers of bone marrow progenitor cells (e.g., IL-3α/CD127, CD34, SCF R/c-kit and Flt-3/Flk-2) and cell markers of hematopoietic stem cells (e.g., CD34, CD38-, Flt- 3/Flk-2), but are not limited to these. In some embodiments, useful biomarkers include fibrotic markers. Fibrous markers include, but are not limited to, TGFβ1/TGFB1, PAI-1 (also known as Serpin 1), MCP-1 (also known as CCL2), Col1a1, Col3a1, FN1, CTGF, α-SMA , ACTA2, Timp1, Mmp8 and Mmp9. In some embodiments, useful biomarkers are serum markers (eg, proteins or fragments found and detected in serum samples).

Based on the knowledge that TGFβ is a component of the leukemic bone marrow niche, targeting the bone marrow microenvironment with TGFβ inhibitors could effectively target leukemic cells expressing molecules that modulate local TGFβ availability in tissues. This may be a promising approach to reduce

Indeed, due to the pleiotropic nature of the pathology, which expresses TGFβ-dependent dysregulation in both myeloproliferative and fibrotic aspects (as the term "myelofibrosis" itself suggests), the Such isoform-specific TGFβ inhibitors may provide particularly advantageous therapeutic effects for patients suffering from myelofibrosis. It is believed that the LTBP arm of such inhibitors can target ECM-associated TGFβ1 complexes in the bone marrow, while the LRRC33 arm of the inhibitors can block myeloid cell-associated TGFβ1. Moreover, both GARP-mediated and LTBP-mediated TGFβ1 activity may be involved in the biological abnormalities of megakaryocytes associated with myelofibrosis. Therefore, TGFβ inhibitors, such as the isoform-specific, context-independent inhibitors of TGFβ1 disclosed herein, target such complexes and thereby inhibit the release of active TGFβ1 in the niche. can be done.

TGFβ inhibitors, such as the TGFβ1 selective inhibitors described herein, have not responded well to or are not tolerated by other (or standard of care) treatments such as hydroxyurea and JAK inhibitors. It is useful in the treatment of patients with primary and secondary myelofibrosis. Such inhibitors are also useful in the treatment of patients with intermediate or high risk myelofibrosis (MF), such as primary MF, post-polycythemia vera MF, and post-essential thrombocythemia MF.
In some embodiments, such TGFβ inhibitors may be used in combination with checkpoint inhibitor therapy.

Accordingly, one aspect of the present disclosure relates to a method of treating primary myelofibrosis. The method includes administering to a patient suffering from primary myelofibrosis a therapeutically effective amount of a composition comprising a TGFβ inhibitor that causes a decrease in TGFβ availability. In some embodiments, an isoform-specific, context-dependent monoclonal antibody inhibitor of TGFβ1 activation is administered to a patient with myelofibrosis. Such antibodies may be administered at doses ranging from 0.1 to 100 mg/kg, such as from 1 to 30 mg, such as 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, It may be administered at a dose such as 30 mg/kg. For example, a suitable dosing regimen includes administering 1-30 mg/kg weekly. In some embodiments, the TGFβ1 inhibitor is administered at about 10 mg/kg/week. Optionally, the frequency of dosing can be adjusted after the initial phase, for example from about once a week (during the initial phase) to once a month (during the maintenance phase). In some embodiments, TGFβ inhibitors (eg, TGFβ1 inhibitors) may be administered in combination with checkpoint inhibitor therapy.

Exemplary routes of administration of pharmaceutical compositions comprising antibodies are intravenous or subcutaneous administration. When the composition is administered intravenously, the patient may, for example, administer the drug over a suitable period of time, e.g., for about 30 to 120 minutes (e.g., 30 minutes, 60 minutes, 75 minutes, 90 minutes, and 120 minutes) for each treatment. The therapeutic agent is then administered every few weeks, e.g., every 3 weeks, every 4 weeks, every 6 weeks, etc., for a total number of cycles, e.g., 4 cycles, 6 cycles, 8 cycles, 10 cycles, 12 cycles, etc. , may be repeated. In some embodiments, patients receive dose levels of 1 to 10 mg/kg (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg) of the inhibitory antibody. In some embodiments, such treatment is administered as a chronic (long-term) treatment (e.g., continued indefinitely as long as it is deemed beneficial), instead of discontinuing after a set number of dosing cycles. be done.

Although myelofibrosis is considered a type of leukemia, it is also characterized by the development of fibrosis. It is desirable to inhibit ECM-associated TGFβ activity, as TGFβ is known to regulate aspects of ECM homeostasis, the dysregulation of which can lead to tissue fibrosis. Accordingly, antibodies or fragments thereof that bind to and inhibit proTGFβ presented by LTBPs (such as LTBP1 and LTBP3) are encompassed by the present disclosure. In some embodiments, antibodies or fragments thereof suitable for treating myelofibrosis bind to multiple contexts of the proTGFβ complex, such as those associated with LRRC33, GARP, LTBP1, LTBP3, or any combination thereof. It is ``context-independent'' in that it can be In some embodiments, such an antibody is a context-independent inhibitor of TGFβ activation, which is characterized in that the antibody is capable of binding to and inhibiting any of the following latent complexes: LTBP1-proTGFβ, LTBP3-proTGFβ, GARP-proTGFβ and LRRC33-proTGFβ. In some embodiments, such antibodies are isoform-specific antibodies that bind to and inhibit such latent complexes comprising one but not other isoforms of TGFβ. Such antibodies include, for example, LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1 and LRRC33-proTGFβ1. In some embodiments, such antibodies are isoform selective antibodies that preferentially bind with high affinity and inhibit TGFβ1 signaling.

Early in vivo data show that TGFβ inhibitors, such as the isoform-selective context-independent inhibitor of TGFβ1 described herein, treat myelofibrosis in convertible mouse models of primary myelofibrosis. It shows that it can be used to Unlike current standard of care JAK2 inhibitors, which only provide symptomatic relief and no clinical or survival benefit, TGFβ inhibitors (e.g., TGFβ1 isoform-selective as described herein) context-independent inhibitors) produced significant anti-fibrotic effects in the bone marrow of affected mice and also prolonged survival, suggesting that TGFβ1 inhibitors may be effective in treating myeloproliferative disorders in human patients. support.

Suitable patient populations with myeloproliferative neoplasms that may be treated with the compositions and methods described herein include, but are not limited to, a) patient populations that are Philadelphia (+); c) a patient population classified as “classic” (PV, ET and PMF); d) a patient population carrying the mutation JAK2V617f(+); e) a patient population carrying JAK2V617f(-). f) a patient population with JAK2 exon 12(+), g) a patient population with MPL(+), and h) a patient population with CALR(+).

In some embodiments, the patient population includes patients with intermediate-2 or high risk myelofibrosis. In some embodiments, the patient population includes subjects with myelofibrosis that are refractory to, or are not candidates for, available treatments. In some embodiments, the subject has a platelet count of 100-200×10 ⁹ /L. In some embodiments, the subject has a platelet count >200×10 ⁹ /L before receiving treatment.

In some embodiments, the subject receiving (and benefiting from) isoform-specific, context-independent TGFβ1 inhibitor therapy has primary myelofibrosis (PMF) moderate to -1 or higher; The patient was diagnosed with post-polycythemia vera/post-essential thrombocythemia myelofibrosis (post-PV/ET MF). In some embodiments, the subject has confirmed myelofibrosis prior to treatment. In some embodiments, the subject is MF-2 or higher as assessed by the European Consensus Grading Score and Grade 3 or higher on the Modified Bauermeister Scale prior to treatment. In some embodiments, the subject has an ECOG performance status of 1 prior to treatment. In some embodiments, the subject has a white blood cell count ranging from 5 to 120 (10 ⁹ /L) before treatment. In some embodiments, the subject has a JAK2V617F allelic burden ranging from 10 to 100%.

In some embodiments, subjects receiving (and who may benefit from) isoform-specific, context-independent TGFβ1 inhibitor therapy receive 8.5 g Transfusion dependent (pre-treatment) characterized by the subject having a history of at least 2 units of red blood cell transfusion in the past month for a hemoglobin level below /dL.

In some embodiments, the subject receiving (and who may benefit from) isoform-specific, context-independent TGFβ1 inhibitor therapy has previously been treated for myelofibrosis. In some embodiments, subjects include, but are not limited to, AZD1480, panobinostat, EPO, IFNα, hydroxyurea, pegylated interferon, thalidomide, prednisone, and JAK2 inhibitors (e.g., lestaurtinib, CEP-701) , treated with one or more therapies.

In some embodiments, the patient has extramedullary hematopoiesis. In some embodiments, the extramedullary hematopoiesis is in the liver, lung, spleen, and/or lymph nodes. In some embodiments, pharmaceutical compositions of the present disclosure are administered locally to one or more local sites of disease manifestation.

In some embodiments, a TGFβ inhibitor, such as an isoform-specific, context-independent TGFβ1 inhibitor described herein, alone or in combination with checkpoint inhibitor therapy, is used to treat myelofibrosis. administered to the patient in an effective amount. A therapeutically effective amount may be effective in treating one or more symptoms and/or complications of myelofibrosis in a patient, including, but not limited to, excessive accumulation of ECM in the bone marrow stroma (fibrosis of the bone marrow), angiogenesis, bone sclerosis, splenomegaly, hepatomegaly, anemia, bleeding, bone pain and other bone-related conditions, extramedullary hematopoiesis, thrombocytosis, leukopenia, cachexia, infections, thrombosis, and mortality. It's a sufficient amount. Accordingly, TGFβ inhibition therapy comprising antibodies or antigen-binding fragments of the present disclosure can obtain clinical benefits, including, among others, anti-fibrotic effects and/or blood cell count normalization. Such therapy may prolong survival and/or reduce the need for bone marrow transplantation.

In some embodiments, the amount of TGFβ inhibitor is effective to reduce the expression and/or secretion of TGFβ1 (eg, of megakaryocytic cells) in the patient. Such inhibitors may therefore reduce TGFβ1 mRNA levels in treated patients. In some embodiments, such inhibitors reduce TGFβ1 mRNA levels in bone marrow, such as mononuclear cells. PMF patients typically have a concentration of more than about 2,500 pg/mL, e.g. , 9,000, and 10,000 pg/mL (as opposed to the normal range of approximately 600 to 2,000 pg/mL as measured by ELISA) (e.g., Mascaremhas et al., (See Leukemia & Lymphoma, 2014, 55(2): 450-452). Zingariello (Blood, 2013, 121(17):3345-3363) quantified the bioactivity and total TGFβ1 content in plasma of PMF patients and controls. According to this reference, the median bioactive TGFβ1 in PMF patients was 43 ng/mL (range 4-218 ng/mL) and total TGFβ1 was 153 ng/mL (32-1000 ng/mL); In matched controls, those values were 18 (0.05-144) and 52 (8-860), respectively. Accordingly, based on these reports, plasma TGFβ1 content in PMF patients is elevated several times, such as 2-fold, 3-fold, 4-fold, 5-fold, etc., compared to control or healthy plasma samples. For example, every 4 weeks, a monoclonal antibody described herein is administered at a dose of 0.1 to 100 mg/kg, such as 1 to 30 mg/kg, for 4 to 12 cycles (eg, 2, 4, 6, 8 , 10, 12 cycles) or after chronic or long-term treatment, treatment with an inhibitor may increase plasma TGFβ1 levels by at least 10% compared to the corresponding baseline (pre-treatment), e.g. It can be reduced by 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%.

Some of the therapeutic effects may be observed relatively quickly, eg, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after initiation of treatment. For example, an inhibitor can effectively increase the number of stem cells and/or progenitor cells within the bone marrow of patients treated with the inhibitor within 1-8 weeks. Such cells include hematopoietic stem cells and blood progenitor cells. A bone marrow biopsy may be performed to assess changes in the frequency/number of bone marrow cells. In response, patients may show improvement in symptoms such as bone pain and fatigue.

Subjects suffering from a myeloproliferative disease (eg, myelofibrosis) may exhibit elevated levels of white blood cell counts (eg, leukemia). In some embodiments, a therapeutically effective amount of a TGFβ inhibitor (eg, a TGFβ1 inhibitor) is an amount effective to normalize blood cell counts. In some embodiments, the amount is effective to reduce the subject's total white blood cell count compared to before treatment. In some embodiments, the amount is effective to reduce the subject's total platelet count compared to before treatment. In some embodiments, the amount is effective to increase (eg, normalize or restore) the subject's hemoglobin level compared to before treatment. In some embodiments, the amount is effective to increase (eg, normalize or restore) the subject's hematocrit compared to before treatment.

One of the morphological features of myelofibrosis is fibrosis of the bone marrow (eg, bone marrow stroma), characterized in part by abnormal ECM. In some embodiments, the amount of TGFβ inhibitor (eg, TGFβ1 inhibitor) is effective to reduce fibrosis characterized by excessive collagen deposition, eg, by mesenchymal stromal cells. In some embodiments, the TGFβ inhibitor is effective in reducing the number of CD41-positive cells, such as megakaryocytes, in a treated subject compared to a control subject that does not receive treatment. In some embodiments, the baseline frequency of megakaryocytes in PMF bone marrow ranges from 200 to 700 cells per square millimeter ( ^mm ) when measured in randomly selected sections, and in PMF spleen. may range from 40 to 300 megakaryocytes per square millimeter (mm ² ). In contrast, the frequency of megakaryocytes in the bone marrow and spleen of normal donors is less than 140 and less than 10, respectively. Treatment with a TGFβ inhibitor (eg, a TGFβ1 inhibitor) can reduce the number (eg, frequency) of megakaryocytes in the bone marrow and/or spleen. In some embodiments, treatment with an inhibitor may reduce or inhibit autocrine TGFβ1 signaling in megakaryocytes. In some embodiments, treatment with an inhibitor may cause a decrease in the level of downstream effector signaling, such as phosphorylation of SMAD2/3, e.g., phosphorylation of SMAD2. In some embodiments, TGFβ inhibitors (eg, TGFβ1 inhibitors) are effective in reducing the expression levels of fibrotic markers such as those described herein. Patients with myelofibrosis may have splenomegaly. Therefore, the clinical efficacy of treatment can be assessed by monitoring changes in spleen size. Spleen size may be examined by known techniques such as assessing spleen length by palpation and/or assessing spleen volume by ultrasound. In some embodiments, the subject being treated with an isoform-specific, context-independent inhibitor of TGFβ1 has a baseline spleen length of 5 cm or more, such as in the range of 5 to 30 cm, as assessed by palpation (treatment previous). In some embodiments, the subject treated with an isoform-specific, context-independent inhibitor of TGFβ1 has a baseline spleen volume of 300 mL or more, such as in the range of 300-1500 mL, as assessed by ultrasound (treatment previous). For example, after administration of a monoclonal antibody described herein at a dose of 0.1 to 30 mg/kg for 4 to 12 cycles (e.g., 2, 4, 6, 8, 10, 12 cycles) every 4 weeks. Treatment with an inhibitor of can reduce a subject's spleen size. In some embodiments, the effective amount of the inhibitor increases the spleen size in the patient population receiving inhibitor treatment by at least 10%, 20%, 30%, 35%, 40% compared to the corresponding baseline value. %, 50%, and 60%. For example, the treatment is effective in reducing spleen volume by ≧35% from baseline, as measured by MRI or CT scan, in 12-24 weeks compared to a placebo control. In some embodiments, the treatment reduces spleen volume by ≧35% from baseline as measured by MRI or CT scan at 24-48 weeks compared to the best available treatment control. It is valid. The best available treatments include hydroxyurea, glucocorticoids and no medication, anagrelide, epoetin alfa, thalidomide, lenalidomide, mercaptopurine, thioguanine, danazol, peginterferon alfa-2a, interferon-alpha, melphalan, and acetylsalicylic acid. , cytarabine and colchicine.

In some embodiments, patient populations treated with TGFβ inhibitors, such as the isoform-specific, context-independent TGFβ1 inhibitors described herein, are used for the study and treatment of myelofibrosis, for example. International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) criteria, modified Bauermeister scale and European consensus grading after treatment (e.g., 4, 6, 8, or 12 cycles) The degree of change in myelofibrosis grade as measured by score, as assessed by symptom response using the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF), indicates statistically improved treatment response.

In some embodiments, treatment with an isoform-specific, context-independent TGFβ1 inhibitor, such as those described herein, is evaluated by, for example, the Modified Myelofibrosis Symptom Assessment Form (MFSAF). , achieving statistically improved treatment responses. In this case, symptoms were measured using the MFSAF tool (v2.0), a 0-10 scale (0 = no symptoms, 10 = worst possible symptoms), and debilitating symptoms of myelofibrosis (abdominal discomfort, Measured by a daukt diary capturing early satiety, left subcostal pain, itching, night sweats, and bone/muscle pain. In some embodiments, the treatment is effective to reduce the total MFSAF score by ≧50% from baseline, eg, in 12-24 weeks. In some embodiments, a significant proportion of patients receiving treatment have a ≧50% improvement in total symptom score compared to patients receiving placebo. For example, the percentage of the patient pool achieving ≧50% improvement can exceed 40%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

In some embodiments, a therapeutically effective amount of an inhibitor is an amount sufficient to achieve clinical improvement as measured by anemic response. For example, improvement in anemic response may include longer periods of transfusion independence, such as 8 weeks or more, after 4 to 12 cycles, such as 6 cycles, of treatment.

In some embodiments, a therapeutically effective amount of an inhibitor is an amount sufficient to maintain stable disease for a period of time, such as 6 weeks, 8 weeks, 12 weeks, 6 months, etc. In some embodiments, progression of the disease can be assessed by changes in overall bone marrow cellularity, degree of reticulin or collagen fibrosis, and/or changes in JAK2V617F allele burden.

In some embodiments, a patient population treated with an isoform-specific, context-independent TGFβ1 inhibitor, such as those described herein, has a statistically significant demonstrated improved (prolonged) survival. For example, in control groups, the median survival time for PMF patients is about 6 years (about 16 months for high-risk patients), and less than 20% of patients are expected to survive more than 10 years after diagnosis. Treatment with isoform-specific, context-independent TGFβ1 inhibitors, such as those described herein, can improve survival by at least 6 months, 12 months, 18 months, 24 months, 30 months, 36 months, or 48 months. Can be extended. In some embodiments, the treatment achieves an improvement in overall survival at 26 weeks, 52 weeks, 78 weeks, 104 weeks, 130 weeks, 144 weeks, or 156 weeks compared to patients receiving a placebo. It is valid.

Clinical benefits of treatments such as those exemplified above may be seen in patients with or without new onset of anemia.

One advantageous feature of isoform-specific, context-independent TGFβ1 inhibitors is that they offer improved safety, enabled by isoform selectivity, compared to conventional TGFβ antagonists that lack selectivity. Maintain your profile. Therefore, treatment with isoform-specific, context-independent inhibitors such as those described herein may be comparable to those treated with conventional TGFβ antagonists in terms of the frequency and/or severity of such events. It is expected that adverse events may be reduced in the patient population compared to the patient population. Thus, isoform-specific, context-independent TGFβ1 inhibitors may offer a larger therapeutic window in terms of dosage and/or duration of treatment.

Adverse events can be graded by any suitable art-recognized method, such as Common Terminology Criteria for Adverse Events (CTCAE) version 4. Previously reported adverse events in human patients receiving TGFβ antagonists, such as GC1008, include increased white blood cell counts (grade 3), fatigue (grade 3), hypoxia (grade 3), and cardiac arrest (grade 5). ), leukopenia (grade 1), recurrent, transient, tender, erythematous, nodular skin lesions, purulent dermatitis, and herpes zoster.

In patients with myelofibrosis, TGFβ1 inhibitor therapy is effective against, for example, anemia, thrombocytopenia, neutropenia, hypercholesterolemia, alanine transaminase (ALT) elevation, aspartate transaminase (AST) elevation, bruising, dizziness, and headache. , compared to JAK inhibitor therapy, may cause less frequent and/or less severe adverse events (side effects) and may provide a safer treatment option.

It is contemplated that inhibitors of TGFβ signaling may be used as a combination (eg, "add-on") therapy in conjunction with one or more therapeutic agents to treat myelofibrosis. In some embodiments, the TGFβ inhibitor is an inhibitor of TGFβ activation, e.g., TGFβ1 activation, e.g., Ab6, and a patient suffering from myelofibrosis is treated with one of the compounds disclosed herein. It is administered in combination with the above checkpoint inhibitors. In some embodiments, a TGFβ inhibitor, such as Ab6, is administered to a patient suffering from myelofibrosis who is receiving or is a candidate for administration of a JAK1 inhibitor, JAK2 inhibitor, or JAK1/JAK2 inhibitor. administered. In some embodiments, such patients respond to JAK1 inhibitors, JAK2 inhibitors or JAK1/JAK2 inhibitor therapy, while in other embodiments, such patients respond to JAK1 inhibitors, JAK2 inhibitors. or JAK1/JAK2 inhibitor therapy. In some embodiments, the use of a TGFβ inhibitor, such as the isoform-specific inhibitors of TGFβ1 described herein, is responsive to JAK1 inhibitor, JAK2 inhibitor, or JAK1/JAK2 inhibitor therapy. may increase responsiveness in patients with low or no response. In some embodiments, use of a TGFβ inhibitor, such as the isoform-specific inhibitors of TGFβ1 described herein, reduces the dose of a JAK1 inhibitor, a JAK2 inhibitor, or a JAK1/JAK2 inhibitor. can still provide the same or meaningful clinical efficacy or benefit to the patient, but with less or a lower degree of drug-related toxicity or adverse events (such as those described above). In some embodiments, treatment with a TGFβ activation inhibitor described herein in combination with a JAK1 inhibitor, JAK2 inhibitor, or JAK1/JAK2 inhibitor therapy provides a synergistic or additive treatment to the patient. can have an effect. In some embodiments, treatment with a TGFβ activation inhibitor described herein is a JAK1 inhibitor, JAK2 inhibitor, or JAK1/JAK2 inhibitor or other therapy given to treat myelofibrosis. can increase profits. In some embodiments, the patient may further receive treatment to address anemia associated with myelofibrosis.

In some embodiments, the TGFβ inhibitors described herein, such as the TGFβ1 selective inhibitors described herein (e.g., Ab6), are checkpoint inhibitors for the treatment of myelofibrosis. Can be used in conjunction with therapy to provide therapeutic benefit. Primary cells isolated from patients with JAK2 mutations exhibit higher PD-L1 expression compared to primary cells from healthy donors. This suggests that constitutive activation of the JAK2/STAT pathway in megakaryocytes and platelets contributes to PD-L1-mediated T cell activation, metabolic activity, and immune escape through reduced T cell cell cycle progression. It shows the possibility (Prestipino et al., Sci Transl Med 2018; 10 (429)). Additionally, activation of the TGFβ signaling pathway has been shown to increase PD-1 expression on cytotoxic T cells and reduce their susceptibility to PD-1/PD-L1-mediated checkpoint inhibition. (Chen et al., Int J Cancer 2018; 143:2561). Without being bound by theory, these findings, along with the low response rates to checkpoint inhibitor therapy (e.g., anti-PD-1 therapy) observed in patients with myelofibrosis, suggest that clinical This supports the potential importance of TGFβ signaling in mediating clinical tolerance.

In some embodiments, a TGFβ inhibitor, such as a TGFβ1 inhibitor (e.g., Ab6), is combined with a BMP antagonist (e.g., a BMP6 inhibitor) to treat anemia in patients with a myeloproliferative disease, such as myelofibrosis. , for example, an RGMc inhibitor). Without wishing to be bound by theory, TGFβ1 inhibitors (e.g., Ab6) may be useful in promoting hematopoiesis, while BMP antagonists (e.g., BMP6 inhibitors, e.g., RGMc inhibitors) may be useful in promoting hematopoiesis. It is believed that iron deficiency (eg, deficiency resulting from cancer and/or chemotherapy) may be reduced. In some embodiments, a treatment comprising a TGFβ1 inhibitor (e.g., Ab6) and a BMP antagonist (e.g., a BMP6 inhibitor, e.g., an RGMc inhibitor) reduces one or more anemia-related symptoms and/or complications. may be administered in a therapeutically effective amount sufficient to In some embodiments, a therapy comprising a TGFβ1 inhibitor (e.g., Ab6) and a BMP antagonist (e.g., a BMP6 inhibitor, e.g., an RGMc inhibitor) is administered in a patient with a myeloproliferative disease (e.g., myelofibrosis). can be administered in a therapeutically effective amount to increase red blood cell production and/or decrease iron limitation. In some embodiments, treating anemia further comprises administering one or more JAK inhibitors (eg, a Jak1/2 inhibitor, a Jak1 inhibitor, and/or a Jak2 inhibitor). In some embodiments, improved anemic response may include a longer duration of transfusion independence, eg, lasting for 8 weeks or more after 4 to 12 cycles, eg, 6 cycles of treatment. In some embodiments, the treatment further includes one or more checkpoint inhibitors, such as anti-PD1 antibodies, anti-PD-L1 antibodies, and/or anti-CTLA-4 antibodies.

Accordingly, the present disclosure provides TGFβ inhibitors (e.g., TGFβ1 selective inhibitors such as Ab6) for use in the treatment of myeloproliferative diseases, such as primary myelofibrosis, in patients, wherein the treatment administering a composition comprising a TGFβ inhibitor (eg, a TGFβ1 inhibitor) selected based at least in part on its safety profile. A favorable immunosafety profile of a TGFβ inhibitor is that it: i) does not induce unacceptable levels of cytokine release (e.g., within 2.5 times the control); ii) does not promote unacceptable levels of platelet aggregation; or Both are characterized in accepted cell-based assays and/or in vivo assays (such as those described herein).

Pathological conditions involving MHC downregulation or mutations TGFβ-related indications also include pathological conditions in which major histocompatibility complex (MHC) class I is deleted or deficient (e.g., downregulated). obtain. Such conditions include genetic disorders in which one or more components of MHC-mediated signaling are impaired, as well as conditions in which MHC expression is altered by other factors such as cancer, infection, fibrosis, and medications. included.

For example, downregulation of MHC I in tumors is associated with tumor evasion from immune surveillance. Indeed, immune evasion strategies aimed at evading T cell recognition, including loss of tumor MHC class I expression, are commonly found in malignant cells. Tumor immune evasion has been observed to have a negative effect on clinical outcomes of cancer immunotherapies, including treatment with antibodies that block immune checkpoint molecules (e.g., Garrido et al., (2017) Curr Opin Immunol 39:44-51. “The urgent need to recover MHC class I in cancers for effective immunotherapy”, which is incorporated herein by reference). Accordingly, isoform-selective, context-independent TGFβ1 inhibitors encompassed by the present disclosure may be used as monotherapy to release or enhance anti-cancer immunity and/or increase responsiveness or efficacy to other therapies. or in combination with another therapy, such as checkpoint inhibitors, chemotherapy, radiation therapy (such as radiotherapeutics).

In some embodiments, MHC downregulation is associated with acquired resistance to cancer treatments such as CBT. It is believed that isoform-selective inhibitors of TGFβ1 could be used to treat patients who are first responders to cancer treatments such as CBT to reduce the probability of developing acquired resistance. Therefore, in patients treated with TGFβ1 inhibitors who are primary responders to cancer therapy, the development of secondary or acquired resistance to cancer therapy may be reduced over time.

Downregulation of MHC class I proteins has also been associated with certain infections, including viral infections such as HIV. For example, Cohen et al. , (1999) Immunity 10(6):661-671 “The selective downregulation of class I major histocompatibility complex proteins by HIV-1 "protects HIV-infected cells from NK Cells". This document is incorporated herein by reference. Accordingly, isoform-selective context-independent TGFβ1 inhibitors encompassed by the present disclosure can be used as monotherapy to release or enhance host immunity and/or increase responsiveness or effectiveness to other therapies. , or in combination with another therapy (antiviral therapy, protease inhibitor therapy, etc.).

The present disclosure provides that TGFβ inhibitors (e.g., TGFβ1 selective inhibitors such as Ab6) for use in the treatment of TGFβ-related disorders involving MHC downregulation or mutations (e.g., as described herein) in a patient. and the treatment includes administering a composition comprising a TGFβ inhibitor (eg, a TGFβ1 inhibitor) selected based at least in part on its immunosafety profile. A favorable immunosafety profile of a TGFβ inhibitor is that it: i) does not induce unacceptable levels of cytokine release (e.g., within 2.5 times the control); ii) does not promote unacceptable levels of platelet aggregation; or Both are characterized in accepted cell-based assays and/or in vivo assays (such as those described herein).

Pathological conditions involving stem cell self-renewal, tissue regeneration, and stem cell repopulation suggest that the TGFβ pathway plays a role in regulating the homeostasis of various stem cell populations and their differentiation/repopulation within tissues. There is evidence. During homeostasis, tissue-specific stem cells remain largely quiescent, but are induced to enter the cell cycle when subjected to certain stresses. TGFβ1 is thought to function as a "blocker" in processes that tightly regulate stem cell differentiation and reconstitution, and stress that induces entry into the cell cycle can cause TGFβ1 to remove the "blocker". Consistent with inhibition. Therefore, isoform-selective inhibitors of TGFβ1, such as those described herein, can be used to skew or correct the cell cycle and G0 entry decisions of stem/progenitor cells within specific tissues. It is thought that it can be done.

Accordingly, the inventors of the present disclosure contemplate the use of isoform-selective TGFβ1 inhibitors in the following pathological conditions: i) stem cell/progenitor cell differentiation/reconstitution is arrested or perturbed due to the disease; ii) the patient is undergoing a treatment or intervention that kills or depletes healthy cells; iii) the patient undergoes increased differentiation/reconstitution of stem/progenitor cells; iv) the disease is associated with abnormal stem cell differentiation/reconstitution;

In self-renewing tissues such as bone marrow (blood cell production) and epithelium, the balance between proliferation and differentiation processes is tightly regulated to ensure the maintenance of stem cell populations throughout life. D’Archangel et al. , (2017) Int. J Mol Sci. 18(7):1591. TGFβ1 acts as a potent negative regulator of cell cycle and tumor suppressors, in part through induction of the cyclin-dependent kinase inhibitors p15/INK4b, p21 and p57. There is evidence to suggest that TGFβ1 contributes to the induction of p16/INK4a and p19/ARF to mediate growth arrest and senescence of certain cell types. Accordingly, in some embodiments, isoform-selective inhibitors of TGFβ1 activation, such as those described herein, are used to modulate p16/INK4a-dependent cellular senescence and stem cell dynamics in various stem cell populations. do.

For example, mesenchymal stromal/stem cells (MSCs) are a small population of stromal cells present in most adult connective tissues such as bone marrow, adipose tissue, and umbilical cord blood. Although MSCs are maintained in a relative quiescent state in vivo, they proliferate in response to a variety of physiological and pathological stimuli and then become osteoblasts, chondrocytes, adipocytes or smooth muscle cells (SMCs) and myocardial cells. cells that can differentiate into other mesodermal lineages. Multiple signaling networks orchestrate the differentiation of MSCs into functional mesenchymal lineages, among which TGF-β1 has emerged as an important player (e.g., Zhao & Hantash (2011. Vitamin Horm 87:127 -41)).

Similarly, hematopoietic stem cells are required for lifelong blood cell production, and to prevent loss, the majority of hematopoietic stem cells remain quiescent during steady-state hematopoiesis. However, during hematological stress, these cells are rapidly recruited into the cell cycle and undergo extensive self-renewal and differentiation to meet increased hematopoietic demands. TGFβ1 may act as a “switch” to control the quiescence-repopulation transition/balance.

Therefore, isoform-selective inhibitors of TGFβ1 can be used to treat pathological conditions involving hematopoietic stem cell deficiency and bone marrow failure. In some embodiments, depletion or impairment of hematopoietic stem cell reservoirs results in hematopoietic failure or hematologic malignancy. In some embodiments, such a condition is a DNA repair disorder characterized by progressive bone marrow failure. In some embodiments, such pathologies are caused by stem and progenitor cell attrition. In some embodiments, such conditions are associated with anemia. In some embodiments, such condition is Fanconi anemia (FA). In some embodiments, such pathologies are characterized by an overactive TGFβ pathway that suppresses survival of certain cell types upon DNA damage. Therefore, isoform-selective inhibitors of TGFβ1 could be used to rescue FA hematopoietic stem cell proliferation defects and/or bone marrow failure in subjects with FA. For example, Zhang et al. , (2016), Cell Stem Cell, 18:668-681, “TGF-β inhibition rescues hematopoietic stem cell defects and bone marrow failure in Fanc. oni Anemia"

The present disclosure provides that TGFβ inhibitors (such as those described herein) for use in the treatment of TGFβ-related disorders involving stem cell self-renewal, tissue regeneration, and/or stem cell repopulation (e.g., as described herein) in a patient. e.g., a TGFβ1 selective inhibitor such as Ab6), the treatment comprises administering a composition comprising a TGFβ1 inhibitor (e.g., a TGFβ1 inhibitor) selected based at least in part on its immunosafety profile. Including. A favorable immunosafety profile of a TGFβ inhibitor is that it: i) does not induce unacceptable levels of cytokine release (e.g., within 2.5 times the control); ii) does not promote unacceptable levels of platelet aggregation; or Both are characterized in accepted cell-based assays and/or in vivo assays (such as those described herein).

Conditions involving therapy-induced hematopoietic dysregulation. Certain drugs designed to treat various disease states are often found to induce or worsen anemia in the patients being treated. (e.g. treatment- or drug-induced anemia such as chemotherapy-induced anemia and radiotherapy-induced anemia). In some embodiments, the patient is treated with myelosuppressive drugs that can cause side effects including anemia. Such patients may benefit from pharmacological TGFβ1 inhibition to enhance hematopoiesis. In some embodiments, TGFβ1 inhibitors can promote hematopoiesis in a patient by preventing entry into a quiescent state. In some embodiments, the patient can receive G-CSF therapy (eg, filgrastim).

Accordingly, the present disclosure describes the use of isoform-selective inhibitors of TGFβ1, such as those disclosed herein, administered to patients undergoing myelosuppressive therapy (e.g., therapy with side effects including myelosuppressive effects). including. Examples of myelosuppressive therapies include peginterferon alfa-2a, interferon alfa-n3, peginterferon alfa-2b, aldesleukin, gemtuzumab ozogamicin, interferon alfa-1, rituximab, ibritumomab tiuxetan, tositumomab. , alemtuzumab, bevacizumab, L-phenylalanine, bortezomib, cladribine, carmustine, amsacrine, chlorambucil, raltitrexed, mitomycin, bexarotene, vindesine, floxuridine, thioguanine, vinorelbine, dexrazoxane, sorafenib, streptozocin, gemcitabine, teniposide, epirubicin, chloram Phenicol, lenalidomide, altretamine, zidovudine, cisplatin, oxaliplatin, cyclophosphamide, fluorouracil, propylthiouracil, pentostatin, methotrexate, carbamazepine, vinblastine, linezolid, imatinib, clofarabine, pemetrexed, daunorubicin, irinotecan, methimazole, etoposide, Dacarbazine, temozolomide, tacrolimus, sirolimus, mechlorethamine, azacytidine, carboplatin, dactinomycin, cytarabine, doxorubicin, hydroxyurea, busulfan, topotecan, mercaptopurine, thalidomide, melphalan, fludarabine, flucytosine, capecitabine, procarbazine, arsenic trioxide, idarubicin , ifosfamide, mitoxantrone, lomustine, paclitaxel, docetaxel, dasatinib, decitabine, nelarabine, everolimus, vorinostat, thiotepa, ixabepilone, nilotinib, belinostat, trabectedin, trastuzumab emtansine, temsirolimus, bosutinib, bendamustine, cabazitaxel, eribulin, ruxoli tinib, carfilzomib , tofacitinib, ponatinib, pomalidomide, obinutuzumab, tedizolid phosphate, blinatumomab, ibrutinib, palbociclib, olaparib, dinutuximab and colchicine.

Additional TGFβ-related indications include any of those disclosed in U.S. Patent Application Publication No. 2013/0122007, U.S. Patent No. 8,415,459, or WO 2011/151432. may be included. The contents of each of these documents are incorporated herein by reference in their entirety.

In certain embodiments, the antibodies, antigen-binding portions thereof, and compositions of the present disclosure can be used to treat a wide variety of diseases, disorders, and/or conditions associated with TGFβ1 signaling. In some embodiments, the target tissue/cell preferentially expresses the TGFβ1 isoform over other isoforms. Accordingly, the present disclosure describes the use of pharmaceutical compositions comprising antibodies or antigen-binding portions thereof described herein to treat such pathological conditions associated with TGFβ1 expression (e.g., dysregulation of TGFβ1 signaling and/or TGFβ1 upregulation of expression).

In some embodiments, the disease comprises TGFβ1 associated with (eg, displayed on or produced from) multiple cellular sources. In some embodiments, such diseases involve both immune and ECM components of TGFβ1 function. In some embodiments, such diseases include i) ECM dysregulation (e.g., overproduction/deposition of ECM components such as collagen and proteases; changes in ECM matrix stiffness; myofibroblasts, fibrocytes; and abnormal or pathological activation or differentiation of fibroblasts such as CAFs); ii) immunosuppression by increasing Tregs and/or suppressing effector T cells (Teffs), e.g. by increasing the Treg/Teff ratio; CD4 and / or increased leukocyte infiltration (e.g., macrophages and MDSCs) causing suppression of CD8 T cells; and/or iii) macrophages (e.g., bone marrow-derived monocytes/macrophages and tissue-resident macrophages), monocytes, myeloid-derived suppressor cells. (MDSC), including aberrant or pathological activation, differentiation and/or recruitment of myeloid cells such as neutrophils, dendritic cells and NK cells.

In some embodiments, the condition comprises TGFβ1 presented by two or more types of presentation molecules (eg, two or more of GARP, LRRC33, LTBP1 and/or LTBP3). In some embodiments, a diseased tissue/organ/cell comprising TGFβ1 from multiple cell sources. As an example, solid tumors (which can also include proliferative diseases involving the bone marrow, such as myelofibrosis and multiple myeloma) contain cancer cells, stromal cells, and surrounding healthy cells that contain different types of presenting molecules. It may include TGFβ1 from multiple sources, such as cells, and/or infiltrating immune cells (eg, CD45+ leukocytes). Associated immune cells include myeloid and lymphoid cells, such as neutrophils, eosinophils, basophils, lymphocytes (e.g., B cells, T cells, and NK cells) and monocytes. However, it is not limited to these. Context-independent inhibitors of TGFβ1 may be useful in treating such conditions.

In some embodiments, hematopoietic dysregulation associated with cancer can cause anemia. TGFβ therapy may further include a BMP6 inhibitor, such as an RGMc inhibitor. In some embodiments, cancer-related anemia may be caused by the disease itself, while in some embodiments, anemia may be caused by cancer treatment (such as chemotherapy).

The present disclosure describes TGFβ inhibitors (e.g., TGFβ1, such as Ab6) for use in the treatment of TGFβ-related disorders involving hematopoietic dysregulation induced by therapy (e.g., as described herein) in a patient. selective inhibitors), and the treatment includes administering a composition comprising a TGFβ inhibitor (eg, a TGFβ1 inhibitor) selected based at least in part on its immunosafety profile. A favorable immunosafety profile of a TGFβ inhibitor is that it: i) does not induce unacceptable levels of cytokine release (e.g., within 2.5 times the control); ii) does not promote unacceptable levels of platelet aggregation; or Both are characterized in accepted cell-based assays and/or in vivo assays (such as those described herein).

Non-limiting examples of disease conditions or disorders that can be treated with isoform-specific, context-independent inhibitors of TGFβ1, such as the antibodies or fragments thereof described herein, are provided below.

Treatment Regimens, Administration To carry out the methods disclosed herein, an effective amount of the pharmaceutical composition described above is administered to a subject (e.g., a human) in need of treatment by a suitable route, e.g., intravenously. can be administered by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation or topical routes, for example as a bolus or by continuous infusion over a period of time. . Commercially available nebulizers for liquid formulations are useful for administration, including jet nebulizers and ultrasonic nebulizers. Liquid formulations can be directly atomized, and lyophilized powders can be atomized after reconstitution. Alternatively, antibodies or antigen-binding portions thereof that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex can be used in combination with fluorocarbon formulations and metered dose inhalers. It can be used to aerosolize or inhaled as a lyophilized ground powder.

The subject treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats. A human subject in need of treatment may be a human patient having, at risk of, or suspected of having a TGFβ-related indication as described above. Subjects with TGFβ-related indications can be identified by routine medical tests, such as laboratory tests, organ function tests, CT scans, or ultrasound. A subject suspected of having any of such indications will exhibit one or more symptoms of that indication. A subject at risk for an indication may be a subject who has one or more risk factors for that indication.

As used herein, the terms "effective amount" and "effective dose" refer to the intended purpose, i.e., the desired biological or drug response in a tissue or subject at an acceptable benefit/risk ratio. refers to the amount or dose of a compound or composition sufficient to satisfy For example, in certain embodiments of the invention, the intended goal may be to inhibit activation of TGFβ-1 in vivo and achieve clinically significant outcomes associated with TGFβ-1 inhibition. As will be recognized by those skilled in the art, an effective amount will depend on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex and weight, the duration of treatment, the combination It will vary depending on the nature of the therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of the health care professional. These factors are well known to those skilled in the art. It can be dealt with only by routine experimentation. It is generally preferable to use the maximum dosage of the individual components or combinations thereof, ie, the highest safe dose according to sound medical judgment. However, one skilled in the art will appreciate that a patient may require a lower or tolerated dose for medical reasons, psychological reasons or virtually any other reason.

Empirical considerations such as half-life will generally assist in determining dosage. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, can be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration can be determined and adjusted depending on the course of treatment, and generally (but not necessarily) is used to treat and/or suppress and/or ameliorate and/or treat TGFβ-related indications. Based on delay. Alternatively, sustained release formulations of antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex may be appropriate. Various formulations and devices for achieving sustained release will be apparent to those skilled in the art and are within the scope of this disclosure.

In one example, the dosage of an antibody described herein can be determined empirically in an individual who has received one or more administrations of the antibody. Individuals are given increasing doses of the antagonist. To assess efficacy, indicators of TGFβ-related indications can be followed. For example, methods for measuring muscle fiber damage, muscle fiber repair, levels of inflammation in muscle, and/or levels of fibrosis in muscle are well known to those skilled in the art.

The present disclosure encompasses the recognition that agents capable of modulating the activation process of TGFβ in an isoform-specific manner may provide a good safety profile when used as a pharmaceutical. Accordingly, the present disclosure includes methods that specifically bind to and inhibit activation of TGFβ1, but not TGFβ2 and TGFβ3, thereby specifically inhibiting TGFβ1 signaling in vivo, but not TGFβ2 and/or or antibodies and antigen-binding fragments thereof that minimize undesirable side effects by affecting TGFβ3 signaling.

In some embodiments, the antibodies or antigen-binding portions thereof described herein are non-toxic when administered to a subject. In some embodiments, the antibodies or antigen-binding portions thereof described herein have reduced toxicity when administered to a subject compared to antibodies that specifically bind to both TGFβ1 and TGFβ2. In some embodiments, the antibodies or antigen-binding portions thereof described herein have reduced toxicity when administered to a subject compared to antibodies that specifically bind to both TGFβ1 and TGFβ3. In some embodiments, the antibodies or antigen-binding portions thereof described herein have reduced toxicity when administered to a subject compared to antibodies that specifically bind TGFβ1, TGFβ2, and TGFβ3.

In general, for administration of any of the antibodies described herein, the initial candidate dosage can be about 1-20 mg/kg per administration, and the frequency of administration can be, for example, once a week (QW ), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once a month, etc. For example, the patient may receive an infusion of about 1-10 mg/kg per week, per 2 weeks, per 3 weeks, or per 4 weeks, in an amount effective to treat the disease (e.g., cancer). and the amount is well tolerated (within acceptable toxicity or adverse events).

For purposes of this disclosure, typical dosages (per administration, such as injections and infusions) may range from about 0.1 mg/kg to 30 mg/kg, depending on the factors mentioned above. When administered repeatedly over several days or more depending on the condition, treatment continues until the desired suppression of symptoms occurs or until a therapeutic level sufficient to alleviate the TGFβ-related indication or symptoms thereof is achieved. sustained. For example, a suitable effective dosage of Ab6 is 1 mg/kg to 30 mg/kg (e.g., 1-10 mg/kg, 1-15 mg/kg, 3-20 mg/kg, 5-30 mg/kg, etc.) twice weekly. Administration may be once a week, every two weeks, every four weeks, or once a month. Suitable effective doses of Ab6 include administration of about 1 mg/kg, about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, eg, weekly.

An exemplary dosing regimen includes administering an initial dose followed by one or more maintenance doses. For example, the initial dose can be about 1-30 mg/kg, eg, once a week or twice a week. This may be followed by a maintenance dose, eg, about 0.1-20 mg/kg, eg, weekly, biweekly, monthly, etc. However, other dosing regimens may be useful depending on the pattern of pharmacokinetic decay that the physician desires to achieve. Pharmacokinetic experimental results indicate that serum concentrations of antibodies disclosed herein (e.g., Ab3) remain stable for at least 7 days after administration to a preclinical animal model (e.g., a mouse model). Ta. While not wishing to be bound by any particular theory, this post-administration stability suggests that the antibody may be less stable while maintaining clinically effective serum concentrations in the subject to which it is administered (e.g., a human subject). It may be advantageous because the antibodies may be administered at frequent intervals. In some embodiments, the dosing frequency is once every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks. or once every month, every two months, or every three months, or once every month or more. The progress of this treatment is easily monitored by conventional techniques and assays. Dosage regimens (including antibodies used) may vary over time.

In some embodiments, in normal weight adult patients, doses ranging from about 1 mg/kg to 30 mg/kg, or 80 mg to 3000 mg, such as 30, 50, 100, 500, 1000, 2000 or 3000 mg, are administered. obtain. The particular dosing regimen, eg, dose, timing and repetition, will depend on the particular individual and that individual's medical history as well as the characteristics of the individual drug, such as the drug's half-life and other relevant considerations.

Serum concentrations of TGFβ inhibitor antibodies that are therapeutically effective for treating TGFβ1-related indications according to the present disclosure can be at least about 10 μg/mL, such as about 10 μg/mL to 1.0 mg/mL. In some embodiments, the effective amount of antibody, as measured by serum concentration, is about 20-400 μg/mL. In some embodiments, the effective amount of antibody, as measured by serum concentration, is about 100-800 μg/mL. In some embodiments, the effective amount of antibody as measured by serum concentration is at least about 20 μg/mL, such as at least about 50 μg/mL, 100 μg/mL, 150 μg/mL, or 200 μg/mL. As previously shown, a single dose of Ab6 administered intravenously at 1, 3, 10, 30 or 37.5 mg/kg resulted in Cmax values of about 25 μg/mL to about 900 μg/mL. Furthermore, in non-human primates, after maintaining serum concentration levels of approximately 2,000-3,000 μg/mL for at least 8 weeks, Ab6-related toxicities (e.g., cardiotoxicity, hyperplasia and inflammation, (data provided in Example 12 of this PCT/US Patent Application Publication No. 2019/041373). Thus, a therapeutic window of about 10-100 times can be achieved.

For purposes of this disclosure, appropriate dosages of antibodies that specifically bind to GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex are used. the specific antibody (or composition thereof) to be administered, the type and severity of the indication, whether the antibody is administered prophylactically or therapeutically, previous treatments, the patient's clinical history and response to antagonists; It will depend on the discretion of the attending physician. In some embodiments, the clinician uses antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex to achieve the desired result. Administer until the resulting dose is reached. Antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and/or the LRRC33-TGFβ1 complex can be used depending on, for example, the physiological condition of the recipient, the purpose of administration being therapeutic, etc. It may be administered continuously or intermittently, depending on whether it is prophylactic or prophylactic, and other factors known to the skilled practitioner. The antibody that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex is administered substantially continuously over a preselected period of time. and may be administered in a series of spaced doses, eg, before, during or after the onset of a TGFβ-related indication.

To minimize the potential risks and adverse events associated with TGFβ inhibition, a TGFβ inhibitor, such as any one of the antibodies disclosed herein, can be administered intermittently. For example, a TGFβ inhibitor can be administered “as needed” in a therapeutically effective amount sufficient to obtain and maintain clinical benefit (eg, reduction in tumor volume and/or reversal or reduction of immunosuppression). In some embodiments, administration of a TGFβ inhibitor, e.g., any one of the antibodies disclosed herein (e.g., a TGFβ1 inhibitor, e.g., Ab6), is used to determine or monitor therapeutic efficacy (e.g., can be used in combination with methods (measuring circulating MDSCs). In some embodiments, the TGFβ inhibitor is administered to the patient only when a clinical benefit from further doses of the TGFβ inhibitor is determined, eg, only when an increase in circulating MDSCs is detected.

As used herein, the term "treating" refers to a TGFβ-related indication, a symptom of an indication, or a predisposition to a subject suffering from an indication, a symptom of an indication, or applying or administering a composition containing one or more active agents for the purpose of curing, repairing, alleviating, alleviating, altering, eliminating, ameliorating, ameliorating, or influencing a predisposition to an indication. refers to doing.

Alleviating TGFβ-related indications with antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and/or LRRC33-TGFβ1 complex can be done according to the indication. This includes delaying the onset or progression, or reducing the severity of an indication. Alleviation of indications does not necessarily require a curative outcome. As used herein, "delaying" the onset of an indication related to a TGFβ-related indication refers to slowing, preventing, retarding, delaying, stabilizing, and/or earlier the progression of an indication. It means to extend to. This delay can be of varying lengths of time depending on the history of the indication and/or the individual being treated. A method of "slowing" or mitigating the progression of an indication, or delaying the onset of an indication, improves one or more of the indications within a given time frame compared to not using the method. A method that reduces the probability that a condition will progress and/or reduces the severity of the condition within a given time frame. Such comparisons are usually based on clinical trials with a sufficient number of subjects to obtain statistically significant results.

Diagnosis, Patient Selection, Monitoring Treatment methods involving TGFβ inhibition therapy may include diagnosis of TGFβ indications and/or selection of patients likely to respond to such treatment. Additionally, patients receiving TGFβ inhibitors may be monitored for treatment efficacy, generally by measuring one or more suitable parameters that are indicative of disease state and may be measured (e.g., assayed) before or after treatment. and evaluating changes in treatment-related parameters. For example, such parameters may include the level of a biomarker present in a biological sample taken from a patient. Biomarkers can be RNA, protein, cell, and/or tissue based. For example, genes that are overexpressed in a particular disease state can serve as biomarkers for diagnosing and/or monitoring response to disease or therapy. Cell surface proteins of disease-associated cell populations can function as biomarkers. Such methods may involve directly measuring disease parameters indicative of the extent of a particular disease, such as tumor size/volume. Suitable sampling methods can be used, such as serum/blood samples, biopsies, and imaging. Biopsies can include tissue biopsies (eg, tumor biopsies, eg, core needle biopsies) and liquid biopsies.

Although biopsies have traditionally been the standard for diagnosing and monitoring various diseases such as proliferative disorders (eg, cancer), less invasive alternatives may be preferred. For example, many non-invasive in vivo imaging techniques can be used to diagnose, monitor, and select patients for treatment. Accordingly, the present disclosure includes the use of in vivo imaging techniques to diagnose and/or monitor disease in a patient or subject. In some embodiments, the patient or subject is receiving an isoform-specific TGFβ1 inhibitor described herein. In other embodiments, in vivo imaging techniques can be used to select patients for treatment with isoform-specific TGFβ1 inhibitors. In some embodiments, such techniques can be used to determine whether or how a patient will respond to a treatment, eg, a TGFβ1 inhibition treatment.

Exemplary in vivo imaging techniques used in the method include x-ray radiography, magnetic resonance imaging (MRI), ultrasonography, endoscopy, elastography, Examples include, but are not limited to, photography, tactile imaging, thermography, and medical photography. Other imaging techniques include, for example, functional nuclear medicine imaging such as positron emission tomography (PET) and single photon emission tomography (SPECT). Methods of implementing these techniques and analyzing the results are known in the art.

Non-invasive imaging techniques commonly used for cancer diagnosis and monitoring include magnetic resonance imaging (MRI), computed tomography (CT), ultrasound, positron emission tomography (PET), and single photon emission. Examples include, but are not limited to, tomography (SPECT), fluorescence reflectance imaging (FRI), and fluorescence tomography (FMT). Hybrid imaging platforms can also be used for cancer diagnosis and monitoring. For example, hybrid techniques include, but are not limited to, PET-CT, FMT-CT, FMT-MRI, and PET-MRI. Dynamic contrast-enhanced MRI (DCE-MRI) is another imaging technique commonly used to detect breast cancer. Methods of implementing these techniques and analyzing the results are known in the art.

More recently, non-invasive imaging methods have been developed that allow detection of cells of interest (eg, cytotoxic T cells, macrophages, and cancer cells) in vivo. For example, www. imagineab. com/technology/; Tavare et al. , (2014) PNAS, 111(3):1108-1113; Tavare et al. , (2015) J Nucl Med 56(8):1258-1264; Rashidian et al. , (2017) J Exp Med 214(8):2243-2255; Beckford Vera et al. , (2018) PLoS ONE13(3):e0193832; and Tavare et al. , (2015) Cancer Res 76(1):73-82. Each of these documents is incorporated herein by reference. So-called "T cell tracking" aims to detect and localize anti-tumor effector T cells in vivo. This may provide useful insights for understanding the immunosuppressive phenotype of solid tumors. Tumors that are well infiltrated by cytotoxic T cells ("inflammatory" or "hot" tumors) are more likely to respond to cancer treatments such as checkpoint blockade therapy (CBT). On the other hand, tumors exhibiting an immunosuppressive phenotype tend to have poor T cell infiltration even when there is an anti-tumor immune response. These so-called "immune-excluded" tumors are unlikely to respond to cancer treatments such as CBT. T cell tracking techniques can reveal these different phenotypes and provide information to guide therapeutic approaches likely to benefit patients. For example, patients with "immune-excluded" tumors are likely to benefit from TGFβ inhibitor therapy, such as TGFβ1 inhibitor therapy, which facilitates reversing the immunosuppressive phenotype. It is believed that similar techniques can be used to diagnose and monitor other diseases, such as fibrosis. Typically, an antibody or antibody-like molecule engineered with a detection moiety (e.g., radioactive label, fluorescent, etc.) can be injected into a patient, which then detects specific markers (e.g., CD8+ and M2 macrophages). It is distributed and localized in the following areas.

Non-invasive in vivo imaging techniques can be used to diagnose patients; to select or identify patients who are likely to benefit from TGFβ inhibitor therapy, such as TGFβ1 inhibitor therapy; and/or to assess patient therapeutic response during treatment. It can be applied to various suitable methods for monitoring purposes. Cells with known cell surface markers can be detected/localized by using antibodies or similar molecules that specifically bind to the cell marker. Typically, cells detected through the use of such techniques include cytotoxic T lymphocytes, regulatory T cells, MDSCs, disease-associated macrophages (M2 macrophages such as TAM and FAM), NK cells, dendritic cells, etc. cells, and immune cells such as neutrophils.

Non-limiting examples of suitable immune cell markers include monocyte markers, macrophage markers (e.g. M1 and/or M2 macrophage markers), CTL markers, suppressive immune cell markers, MDSC markers (e.g. G-MDSC and and/or markers for M-MDSC), including, but not limited to, CD8, CD3, CD4, CD11b, CD33, CD163, CD206, CD68, CD14, CD15, CD66b, CD34, CD25 and CD47. In some embodiments, levels of circulating MDSCs (e.g., circulating mMDSCs and/or gMDSCs) are used to determine therapeutic agents, including doses of TGFβ inhibitors (e.g., TGFβ1 selective inhibitors, e.g., Ab6). Physical effects can be predicted, determined, and monitored. In some embodiments, circulating mMDSCs are identified by surface markers CD11b+, HLADR-/low, CD14+, CD15-, CD33+/high, and CD66b-. In some embodiments, gMDSCs are identified by surface markers CD11b+, HLADR-, CD14-, CD15+, CD33+/low, and CD66b+. In some embodiments, LRRC33 is used as a marker for MDSC. In some embodiments, LRRC33 is used as a marker of circulating MDSCs. In some embodiments, a decrease in circulating MDSC levels after treatment with a TGFβ inhibitor (eg, a TGFβ1 selective inhibitor, eg, Ab6) can be indicative of therapeutic efficacy. In some embodiments, levels of circulating MDSCs are used to predict, determine, and monitor the pharmacological effects of a treatment that includes a dose of a TGFβ inhibitor (e.g., a TGFβ1 selective inhibitor, e.g., Ab6). It is possible. Therefore, LRRC33 levels in blood samples may serve as a predictive biomarker for assessing treatment response in conditions associated with immunosuppression.

The in vivo imaging techniques described above can be used to detect, localize and/or track specific MDSCs in patients diagnosed with TGFβ1-related diseases such as cancer and fibrosis. Healthy individuals have no or low frequency of MDSCs in circulation. With the onset or progression of such diseases, increased levels of circulating and/or disease-associated MDSCs can be detected. For example, CCR2-positive M-MDSCs have been reported to accumulate in tissues with inflammation and can cause the progression of fibrosis (such as pulmonary fibrosis) in tissues, which is correlated with TGFβ1 expression. It is shown. Similarly, MDSCs are abundant in many solid tumors (including triple negative breast cancer) and contribute in part to the immunosuppressive phenotype of the TME. Accordingly, therapeutic response to TGFβ inhibition therapy, such as TGFβ1 inhibitor therapy according to the present disclosure, can be monitored by localizing or tracking MDSCs. A decrease or low frequency of detectable MDSCs generally indicates therapeutic benefit or good prognosis.

Accordingly, the present disclosure also includes a method for treating a TGFβ1-related disease or condition, which may include the steps of: i) selecting a patient diagnosed with a TGFβ1-related disease or condition; and ii ) Administering to the patient an antibody or fragment thereof, as encompassed herein, in an amount effective to treat the disease or condition. In some embodiments, selecting step (i) comprises detecting a disease marker (e.g., a marker of fibrosis or cancer as described herein), and optionally, detecting is a biopsy analysis, a serum marker analysis. , and/or in vivo imaging. In some embodiments, selection step (i) includes in vivo imaging techniques described herein.

The present disclosure also includes methods for treating cancer, which may include the steps of: i) selecting a patient diagnosed with a cancer that includes a solid tumor, the solid tumor being immunocompromised; suspected of having a tumor or an immunocompromised tumor; and ii) administering to the patient an antibody or fragment thereof, as encompassed herein, in an amount effective to treat the cancer. Preferably, the patient has received or is undergoing cancer therapy, such as immune checkpoint inhibition therapy (e.g. PD-(L)1 antibody), chemotherapy, radiotherapy, genetically engineered immune cell therapy and cancer vaccine therapy; Or is a candidate to receive it. In some embodiments, the selection step (i) comprises detection of the immune cell or one or more markers thereof, optionally the detection comprising tumor biopsy analysis, serum marker analysis, and/or in vivo imaging. including. In some embodiments, selection step (i) includes in vivo imaging techniques described herein. In some embodiments, the method further comprises monitoring response to a treatment described herein. In certain embodiments, circulating MDSCs, such as G-MDSCs and M-MDSCs, receive a therapeutically effective amount of a TGFβ inhibitor, such as a TGFβ1 inhibitor described herein, as an indicator of therapeutic efficacy and/or a predictor of response. (eg, 1 to 7 days or 1 to 10 weeks before or after administration).

In some embodiments, in vivo imaging is performed to monitor therapeutic response to TGFβ1 inhibition therapy in a subject. In vivo imaging can include any one of the imaging techniques described herein and can measure any one of the markers and/or parameters described herein. In the case of cancer, the therapeutic response may include conversion of an immunocompromised tumor to an inflammatory tumor (which correlates with increased immune cell infiltration into the tumor), reduction in tumor size, and/or reduction in disease progression. Increased immune cell infiltration can be visualized by an increase in intratumoral immune cell frequency or the extent of detection signals such as radiolabel and fluorescence.

In some embodiments, in vivo imaging used to diagnose, select, treat, or monitor patients involves MDSC tracking, such as tracking of G-MDSCs (also known as PMN-MDSCs) and M-MDSCs. include. For example, MDSCs (eg, mMDSCs and/or gMDSCs) may be enriched at baseline disease sites, such as fibrotic tissue and solid tumors. Upon administration of therapy (eg, TGFβ1 inhibitor therapy), MDSC levels may decrease, as measured by a decrease in the intensity of the label (such as radioisotopes and fluorescence), indicating a therapeutic effect. In some embodiments, circulating MDSCs, including circulating G-MDSCs and M-MDSCs, can be detected in the blood or blood components of a subject receiving a TGFβ inhibitor, such as a TGFβ1 inhibitor, such as Ab6. In some embodiments, MDSCs are detected, measured, or quantified through the use of antibodies that bind LRRC33. In some embodiments, mMDSCs and/or gMDSCs are detected, measured, or quantified by detecting a set of cell surface markers such as those provided herein.

In certain embodiments, assays useful for determining efficacy and/or therapeutic response in subjects treated with TGFβ inhibitors (e.g., Ab6) include circulating MDSCs (e.g., G-MDSCs and M-MDSCs). immunohistochemical or immunofluorescent analysis of certain immune cell markers known in the art (e.g., flow cytometry or immunohistochemical analysis) to measure the levels of Not done. In some embodiments, human G-MDSCs can be identified by expression of the surface markers CD11b+, CD14-, CD33+/low, CD15+, HLADR-, and CD66b+. In some embodiments, human G-MDSCs may also express HLA-DR, LOX-1 and/or arginase. In some embodiments, M-MDSCs can be identified by expression of surface markers: CD11b+, CD33+/high, CD15-, HLADR-/low, CD66-, and CD14+. In some embodiments, TGFβ inhibitors, such as those encompassed herein, can be used to detect a decrease in circulating MDSCs, but not other circulating MDSCs, after administration to a patient in need thereof. Not used to detect monocyte levels. In some embodiments, circulating MDSCs are measured by the expression of cell surface expressed LRRC33.

In some embodiments, in vivo imaging includes tracking or localization of LRRC33 positive cells. LRRC33-positive cells include, for example, MDSCs and activated M2-like macrophages (eg, activated macrophages associated with TAMs and fibrotic tissue). For example, LRRC33-positive cells may be enriched at baseline disease sites (such as solid tumors). Upon treatment (eg, TGFβ1 inhibitor therapy), a decrease in cells expressing cell surface LRRC33 can be observed, as measured by a decrease in the intensity of the label (such as radioisotope and fluorescence), indicating a therapeutic effect.

In some embodiments, the in vivo imaging techniques described herein may include the use of PET-SPECT, MRI, and/or optical fluorescence/bioluminescence to detect cells of interest.

In some embodiments, labeling of an antibody or antibody-like molecule with a detection moiety can include direct labeling or indirect labeling.

In some embodiments, the detection moiety can be a tracer. In some embodiments, the tracer can be a radioisotope, and optionally the radioisotope can be a positron emitting isotope. In some embodiments, the radioisotope is selected from the group consisting of 18F, 11C, 13N, 15O, 68Ga, 177Lu, 18F, and 89Zr.

Such methods can therefore be used to perform in vivo imaging through the use of labeled antibodies in immuno-PET.

Accordingly, the present disclosure also includes methods for treating TGFβ1 indications in a subject, which methods include using imaging techniques to diagnose, select patients, and/or monitor treatment efficacy. In some embodiments, a TGFβ inhibitor, such as an isoform-selective TGFβ1 inhibitor according to the present disclosure, is used to treat a TGFβ1 indication, and the treatment comprises an amount of a TGFβ inhibitor ( For example, Ab6) and further comprising monitoring the therapeutic effect in the subject by in vivo imaging. Optionally, the subject may be selected as a candidate for receiving TGFβ inhibitor therapy (eg, TGFβ1 inhibitor therapy) by a diagnostic or selection process that includes in vivo imaging. TGFβ1 indications can be proliferative diseases, such as cancer with solid tumors and myelofibrosis.

In some embodiments, the subject has cancer and the method includes: i) selecting a patient diagnosed with a cancer comprising a solid tumor, the solid tumor being an immunocompromised tumor. or suspected of having an immunocompromised tumor; and ii) administering to the patient an antibody or fragment encompassed herein in an amount effective to treat the cancer. Preferably, the patient is undergoing or is a candidate for cancer treatment, such as immune checkpoint inhibition therapy (e.g., PD-(L)1 antibodies), chemotherapy, radiotherapy, genetically engineered immune cell therapy, and cancer vaccine therapy. It is. In some embodiments, the selection step (i) comprises detection of the immune cell or one or more markers thereof, optionally the detection comprising tumor biopsy analysis, serum marker analysis, and/or in vivo imaging. including. In some embodiments, selection step (i) includes in vivo imaging techniques described herein. In some embodiments, the method further comprises monitoring response to a treatment described herein.

Cell-Based Assays for Measuring TGFβ Activation Activation of TGFβ (and its inhibition by TGFβ test inhibitors, such as antibodies) can be measured by any suitable method known in the art. For example, integrin-mediated activation of TGFβ can be utilized in cell-based potency assays, such as the “CAGA12” reporter (eg, luciferase) assay described in more detail herein. As indicated, such assay systems may include the following components: i) a source of TGFβ (recombinant, endogenous or transfected); ii) a source of activator such as an integrin (recombinant, endogenous or transfected); and iii) cells expressing TGFβ receptors that are responsive to TGFβ and capable of translating the signal into readable output (e.g., luciferase activity in CAGA12 cells or other reporter cell lines); Receptor systems that respond to activation. In some embodiments, the reporter cell line comprises a reporter gene (eg, a luciferase gene) under the control of a TGFβ-responsive promoter (eg, a PAI-1 promoter). In some embodiments, specific promoter elements that confer sensitivity can be incorporated into the reporter system. In some embodiments, such promoter element is a CAGA12 element. Reporter cell lines that can be used in the assay are described, for example, in Abe et al. , (1994) Anal Biochem. 216(2):276-84, which is incorporated herein by reference. In some embodiments, each of the aforementioned assay components is provided from the same source (eg, the same cell). In some embodiments, two of the aforementioned assay components are provided from the same source and the third assay component is provided from a different source. In some embodiments, all three assay components are provided from different sources. For example, in some embodiments, the integrin and latent TGFβ complex (proTGFβ and presentation molecule) are provided for assay from the same source (eg, the same transfected cell line). In some embodiments, integrin and TGF are provided for assay from separate sources (eg, a combination of two different cell lines, purified integrin and transfected cells). When cells are used as a source of one or more assay components, such assay components can be endogenous to the cells, stably expressed in the cells, or transiently transfected. or any combination thereof.

Those skilled in the art will be able to readily adapt such assays to a variety of suitable configurations. For example, various sources of TGFβ may be considered. In some embodiments, the source of TGFβ is a cell that expresses and deposits TGFβ, such as a primary cell, a proliferating cell, an immortalized cell or a cell line. In some embodiments, the source of TGFβ is purified and/or recombinant TGFβ immobilized in the assay system using any suitable means. In some embodiments, the TGFβ immobilized in the assay system is presented within an extracellular matrix (ECM) composition on the assay plate that mimics fibroblast-derived TGFβ, with or without decellularization. Ru. In some embodiments, TGFβ is displayed on the cell surface of the cells used in the assay. Additionally, selected display molecules can be included in the assay system to provide suitable latent TGFβ complexes. One of ordinary skill in the art can readily determine which display molecules may be present or expressed in a particular cell or cell type. Using such assay systems, relative changes in TGFβ activation in the presence or absence of a test agent (such as an antibody) can be readily measured to determine the effect of the test agent on TGFβ activation in vitro. can be evaluated. Data from exemplary cell-based assays are provided in the Examples section below.

Such cell-based assays can be modified or adjusted in many ways, depending on the TGFβ isoform being studied, the type of latent complex (eg, presentation molecule), etc. In some embodiments, cells known to express integrins capable of activating TGFβ can be used as a source of integrins in the assay. Such cells include SW480/β6 cells (eg, clone 1E7). In some embodiments, the integrin-expressing cell contains a plasmid encoding a presentation molecule of interest (e.g., GARP, LRRC33, LTBP (e.g., LTBP1 or LTBP3), etc.) and a pro form of the TGFβ isoform of interest (e.g., can be co-transfected with a plasmid encoding proTGFβ1). After transfection, the cells are incubated for a period sufficient to express the transfected gene (eg, about 24 hours), washed, and incubated with serial dilutions of the test agent (eg, antibody). A reporter cell line (eg, CAGA12 cells) is then added to the assay system followed by an appropriate incubation period to allow TGFβ signaling. After an incubation period (e.g., approximately 18-20 hours) following addition of the test agent, a signal/readout (e.g., luciferase activity) is detected using a suitable means (e.g., in a luciferase-expressing reporter cell line, Bright- Glo reagent (Promega) can be used). In some embodiments, luciferase fluorescence can be detected using a Bio Tek (Synergy H1) plate reader with autogain set.

Data showing that exemplary antibodies of the present disclosure can selectively inhibit activation of TGFβ1 in a context-independent manner can be found, for example, in PCT/US2019/041390, PCT/US2019/041373, and PCT/US2019/041373; /US2021/012930.

Nucleic Acids In some embodiments, the antibodies, antigen-binding portions thereof, and/or compositions of the present disclosure may be encoded by nucleic acid molecules. Such nucleic acid molecules include, but are not limited to, DNA molecules, RNA molecules, polynucleotides, oligonucleotides, mRNA molecules, vectors, plasmids, and the like. In some embodiments, the present disclosure may include cells programmed or generated to express nucleic acid molecules encoding the compounds and/or compositions of the present disclosure. In some examples, the nucleic acids of the present disclosure include codon-optimized nucleic acids. Methods for producing codon-optimized nucleic acids are known in the art and include U.S. Pat. (incorporated herein by reference in its entirety).

List of specific embodiments
Further non-limiting embodiments of the present disclosure are presented below.
1. A method of treating cancer in a subject, the treatment comprising administering to the subject an amount of a TGFβ inhibitor sufficient to reduce circulating MDSC levels.
2. The method of embodiment 1, wherein the reduced circulating MDSCs are G-MDSCs.
3. The method of embodiment 1 or 2, wherein the G-MDSC expresses one or more of LOX-1+, CD11b+, HLADR-, CD14-, CD15+, CD33+/low and CD66+.
4. 4. The method of any one of embodiments 1-3, wherein the treatment further comprises administering cancer therapy.
5. 5. The method of any one of embodiments 1-4, wherein the TGFβ inhibitor and cancer therapy are administered in combination (eg, simultaneously), separately, or sequentially.
6. The method of embodiment 4 or 5, comprising determining whether the subject has a reduction in circulating MDSC levels after administration of a TGFβ inhibitor, and administering cancer therapy if MDSC levels are reduced. .
7. Any of embodiments 1-6, wherein the cancer therapy comprises checkpoint inhibitor therapy, optionally an agent targeting PD-1 or PD-L1, optionally an anti-PD-1 or anti-PD-L1 antibody. The method described in one.
8. 1. A method of predicting treatment efficacy in a subject with cancer, the method comprising:
(i) determining circulating MDSC levels in a subject prior to administering a TGFβ inhibitor (alone or in combination with cancer therapy);
(ii) administering to the subject a therapeutically effective amount of a TGFβ inhibitor (alone or in combination with cancer therapy);
iii) determining circulating MDSC levels in the subject after administration; and
A method comprising: a reduction in circulating MDSC levels after administration compared to pre-administration circulating MDSC levels predicts pharmacological effect.
9. A method of treating cancer in a subject, the method comprising:
(i) determining circulating MDSC levels in the subject prior to administering the TGFβ inhibitor;
(ii) administering to the subject a first therapeutically effective dose of a TGFβ inhibitor;
(iii) determining circulating MDSC levels in the subject after administering the TGFβ inhibitor;
(iv) the circulating MDSC level measured after administering the first therapeutically effective dose of the TGFβ inhibitor compared to the circulating MDSC level measured before administering the first therapeutically effective dose of the TGFβ1 inhibitor; if lowered, administering to the subject a second therapeutically effective dose of the TGFβ inhibitor.
method including.
10. 10. The method of embodiment 8 or embodiment 9, comprising administering checkpoint inhibitor therapy in combination (e.g., simultaneously), separately or sequentially with a TGFβ inhibitor.
11. A method of treating cancer in a subject, the method comprising:
(i) determining circulating MDSC levels in the subject prior to administering a combination therapy comprising a therapeutically effective amount of a TGFβ inhibitor and a therapeutically effective amount of checkpoint inhibitor therapy;
(ii) administering the combination therapy to the subject;
(iii) determining circulating MDSC levels in the subject after administering the combination therapy;
(iv) the circulating MDSC level measured after administering the first therapeutically effective dose of the combination therapy is reduced compared to the circulating MDSC level measured before administering the first therapeutically effective dose; Process of continuing combination therapy
method including.
12. 12. The method of embodiment 11, wherein the combination therapy comprises administering the TGFβ inhibitor in combination (e.g., simultaneously), separately or sequentially with checkpoint inhibitor therapy.
13. A method of treating advanced cancer in a human subject, the method comprising:
i) selecting a subject with an advanced cancer, including a locally advanced tumor and/or a metastatic cancer, with primary resistance to checkpoint inhibitor therapy;
ii) administering a TGFβ inhibitor;
ii) administering checkpoint inhibitor therapy to the subject;
method including.
14. The method of embodiment 13, wherein the checkpoint inhibitor therapy is administered in combination (e.g., simultaneously), separately or sequentially with the TGFβ inhibitor.
15. A method for treating advanced cancer in a human subject, the method comprising:
i) selecting a subject with an advanced cancer, including a locally advanced tumor and/or a metastatic cancer, with primary resistance to CPI therapy, the subject receiving a TGFβ1 selective inhibitor or a TGFβ inhibitor that does not inhibit TGFβ3; being administered a TGFβ inhibitor that is;
ii) optionally administering CPI therapy to the subject in conjunction with a TGFβ inhibitor;
method including.
16. The method of any one of embodiments 13-15, further comprising measuring the level of circulating MDSC levels before and after administering the treatment, wherein a decrease in circulating MDSC levels is indicative of a therapeutic response.
17. 17. The method of embodiment 16, further comprising continuing treatment if a decrease in circulating MDSC levels is determined.
18. 18. The method of any one of embodiments 8-17, wherein the reduced circulating MDSCs are G-MDSCs.
19. 19. The method of embodiment 18, wherein the G-MDSC expresses one or more of LOX-1+, CD11b+, HLADR-, CD14-, CD15+, CD33+/low and CD66+.
20. 20. The method of any one of embodiments 1-19, wherein circulating MDSC levels are determined from whole blood or blood components collected from the subject.
21. The method of any one of embodiments 1-20, wherein the treatment reduces circulating MDSC levels by at least 10%, optionally by at least 15%, 20%, 25% or more.
22. 22. The method according to any one of embodiments 1-21, wherein the TGFβ inhibitor is a TGFβ1 inhibitor, optionally a TGFβ1-specific inhibitor.
23. The method of any one of embodiments 1-21, wherein the subject has, prior to treatment, a circulating MDSC level that is at least 2 times higher than circulating MDSC levels in a healthy subject.
24. A method of selecting a subject for treatment, wherein the subject has, prior to treatment, a circulating MDSC level that is at least twice as high as the circulating MDSC level in a healthy subject.
25. The method according to 24, wherein the subject has or is suspected of having cancer.
26. A method of treating cancer in a subject having, prior to treatment, a circulating MDSC level that is at least twice as high as a circulating MDSC level in a healthy subject, the subject comprising administering an amount of a TGFβ inhibitor sufficient to reduce the circulating MDSC level. A method comprising administering to
27. The level of circulating MDSC cells according to any one of embodiments 1 to 26 is determined within 3 to 6 weeks, such as within about 3 weeks or at about 3 weeks, after administration of the TGFβ inhibitor. Method.
28. A method according to any one of embodiments 1 to 27, wherein the level of circulating MDSC cells is determined within two weeks, such as within 10 days, after administration of the TGFβ inhibitor.
29.
(i) determining the level of tumor-associated immune cells in the subject prior to administering the treatment;
(ii) administering the treatment to the subject;
(iii) determining the level of tumor-associated immune cells in the subject after administering the treatment;
any one of embodiments 1-28, further comprising: 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 before administration is indicative of a therapeutic effect. The method described in.
30. 30. The method of embodiment 29, wherein the change in the level of tumor-associated immune cells in step (iii) indicates a reduction or amelioration of immunosuppression in cancer.
31. 31. The method of embodiment 29 or 30, wherein the tumor-associated immune cells include CD8+ T cells and/or tumor-associated macrophages (TAMs).
32. The method of embodiments 29-31, wherein the change in the level of tumor-associated immune cells comprises an increase in the level of CD8+ T cells by at least 10%, optionally at least 15%, 20%, 25% or more.
33. 33. The method of embodiments 29-32, wherein the change in the level of tumor-associated immune cells comprises an increase in the level of TAM by at least 10%, optionally at least 15%, 20%, 25% or more.
34. 34. The method of any one of embodiments 29-33, wherein the level of tumor-associated immune cells is determined in the sample taken from the subject by immunohistochemical analysis.
35. 35. The method of any one of embodiments 29-34, wherein the level of tumor-associated immune cells is determined by in vivo imaging.
36. 35. The method according to any one of embodiments 29-34, wherein the sample is a tumor biopsy sample.
37. The method of any one of embodiments 29-36, further comprising continuing to administer the treatment if a change in the level of one or more tumor-associated immune cell populations is detected.
38.
(i) determining the level of circulating latent TGFβ in the subject prior to administering the treatment;
(ii) administering the treatment to the subject;
(iii) determining the level of circulating latent TGFβ in the subject after administering the treatment;
38. The method of any one of embodiments 1-37, further comprising: wherein an increase in circulating latent TGFβ after administration of the inhibitor compared to circulating latent TGFβ before administration is indicative of a therapeutic effect.
39. 39. The method of embodiment 38, further comprising continuing to administer the treatment if a change in the level of circulating latent TGFβ is detected.
40. The method of 38 or 39, wherein the level of circulating latent TGFβ is determined in the sample obtained from the subject.
41. The method according to 40, wherein the sample is a whole blood sample or a blood component.
42. The method of any one of embodiments 39-41, wherein the circulating latent TGFβ is circulating latent TGFβ1.
43. A method of treating cancer, the method comprising: interferon gamma (IFN gamma), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1 beta) and administering to the subject a therapeutically effective amount of a TGFβ inhibitor that does not cause significant release of one or more cytokines selected from chemokine CC motif ligand 2 (CCL2)/monocyte chemoattractant protein 1 (MCP-1); A method that includes doing.
44. A method for determining whether a TGFβ inhibitor is tolerable in a patient, the method comprising: contacting a cell culture or fluid sample with a TGFβ inhibitor; -2), interleukin 6 (IL-6), tumor necrosis factor α (TNFα), interleukin 1β (IL-1β) and chemokine CC motif ligand 2 (CCL2)/monocyte chemoattractant protein 1 (MCP - determining whether it causes significant release of one or more cytokines selected from 1), wherein significant release indicates that the TGFβ inhibitor is not well tolerated. .
45. Cytokine release is assessed in an in vitro cytokine release assay, optionally in peripheral blood mononuclear cells (PBMCs) or whole blood, optionally in which the PBMCs or whole blood are administered to the subject prior to administering TGFβ inhibitor therapy. 45. The method according to embodiment 43 or 44, obtained from.
46. 46. The method of any one of embodiments 43-45, wherein cytokine release is assessed in an in vitro cytokine release assay of peripheral blood mononuclear cells (PBMC) or whole blood obtained from healthy subjects.
47. 47. The method of embodiment 45 or 46, wherein the cytokine release assay comprises soluble phase and/or solid phase assay formats.
48. 48. The method of any one of embodiments 45-47, wherein the cytokine release assay comprises i) a solid phase assay, ii) a high density PBMC pre-culture assay, and/or iii) a PBL-HUVEC co-culture assay.
49. 49. The method of any one of embodiments 45-48, wherein the cytokine release assay comprises a multiplexed array, such as a Luminex® array system.
50. The cytokine release assay comprises comparing cytokine release from a TGFβ inhibitor to release from one or more control antibodies selected from an anti-CD3 antibody and an anti-CD28 antibody; optionally, the CD28 antibody is optionally 50. The method of any one of embodiments 45-49, optionally comprising TGN1412.
51. TGFβ inhibitors increase IL-6 levels by more than 10-fold compared to levels in the absence of inhibitor or in the presence of a control antibody, optionally 2-fold, 4-fold, 6-fold increase in IL-6 levels compared to levels in the absence of inhibitor or in the presence of a control antibody. 51. The method of any one of embodiments 43-50, which does not induce a fold or less than an 8-fold increase.
52. The TGFβ inhibitor increases IFNγ levels by more than 10-fold compared to levels in the absence of the inhibitor or in the presence of a control antibody, optionally 2-fold, 4-fold, 6-fold or 8-fold in IFNγ levels. 52. The method of any one of embodiments 43-51, wherein the method does not induce an increase of less than
53. The TGFβ inhibitor increases TNFα levels by more than 10 times compared to levels in the absence of the inhibitor or in the presence of a control antibody, optionally 2 times, 4 times, 6 times or 8 times the TNFα levels. 53. The method of embodiments 43-52, which does not induce an increase of less than
54. 54. The method of any one of embodiments 43-53, wherein the TGFβ inhibitor is administered in a therapeutically effective amount that does not cause significant release of one or more cytokines in animal models, including non-human primates.
55. The method of embodiments 43-54, wherein the therapeutically effective amount of TGFβ inhibitor is an amount sufficient to reduce circulating MDSC levels.
56. 56. The method of embodiment 55, wherein the degraded MDSCs are G-MDSCs.
57. 57. The method of embodiment 56, wherein the G-MDSCs express one or more of LOX-1+, CD11b+, HLADR-, CD14-, CD15+, CD33+/low and CD66+.
58. 58. The method of any one of embodiments 55-57, wherein circulating MDSC levels are determined from whole blood or blood components collected from the subject.
59. 59. The method according to any one of embodiments 43-58, wherein the TGFβ inhibitor is a TGFβ1 inhibitor, optionally a TGFβ1 specific inhibitor, such as Ab6.
60. A TGFβ inhibitor for use in treating cancer in a subject, the treatment comprising administering a dose of said TGFβ inhibitor to a subject having cancer, said TGFβ inhibitor comprising 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 chemotaxis. A TGFβ inhibitor that does not cause significant release of one or more cytokines selected from catabolic protein 1 (MCP-1).
61. A combination therapy comprising a dose of a TGFβ inhibitor and a cancer therapeutic agent for use in the treatment of cancer, wherein the treatment comprises administering the doses of the TGFβ inhibitor and the cancer therapeutic agent to a subject simultaneously, separately or sequentially. The TGFβ inhibitor includes interferon γ (IFNγ), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor α (TNFα), interleukin 1β (IL-1β). and a combination therapy that does not cause significant release of one or more cytokines selected from the chemokine CC motif ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1).
62. A TGFβ inhibitor for use according to embodiment 60 or an embodiment, wherein the TGFβ inhibitor is administered in a therapeutically effective amount that does not cause significant release of one or more cytokines in animal models, including non-human primates. Combination therapy for use according to 61.
63. A TGFβ inhibitor for use according to embodiment 60 or 62 or a combination for use according to embodiment 61, wherein the TGFβ inhibitor is administered in a therapeutically effective amount sufficient to reduce circulating MDSC levels. Therapy.
64. A TGFβ inhibitor for use or a combination therapy for use according to embodiment 63, wherein the reduced MDSCs are G-MDSCs.
65 G-MDSCs express one or more of LOX-1+, CD11b+, HLADR-, CD14-, CD15+, CD33+/low and CD66+, or for use as described in embodiment 64. combination therapy.
66. A TGFβ inhibitor for use or a combination therapy for use according to any one of embodiments 63-65, wherein circulating MDSC levels are determined from whole blood or blood components collected from the subject.
67. In the method of embodiment 44 or any embodiment subordinate thereto, in embodiment 60 or any embodiment subordinate thereto, the TGFβ inhibitor is a TGFβ1 inhibitor, optionally a TGFβ1 specific inhibitor. A TGFβ inhibitor for the use described or a combination therapy for the use as described in embodiment 61 or any embodiment dependent thereon.
68. The TGFβ inhibitor does not cause significant release of one or more cytokines as determined by the method described in embodiment 44 or any embodiment dependent thereon. A TGFβ inhibitor for use as described in embodiment 61 or a combination therapy for use as described in embodiment 61 or any embodiment subordinate thereto.
69. A method of treating cancer comprising administering to a subject a therapeutically effective amount of a TGFβ inhibitor that does not induce a significant increase in platelet binding, activation and/or aggregation.
70. 70. The method of embodiment 69, wherein platelet binding, activation and/or aggregation is measured in a sample of plasma or whole blood.
71. A method for determining whether a TGFβ inhibitor causes a significant increase in platelet binding, activation and/or aggregation after exposure of a sample to said TGFβ inhibitor, the method comprising: and/or measuring aggregation.
72. The method of any one of embodiments 70 or 71, wherein the sample is obtained from the subject prior to administering TGFβ inhibitor therapy.
73. 73. The method of any one of embodiments 69-72, wherein the sample is obtained from a healthy subject.
74. Embodiment 69, wherein administering a TGFβ inhibitor does not increase platelet binding by more than 10% compared to binding in the absence of a TGFβ inhibitor and/or in the presence of a buffer or isotype control. -73. The method according to any one of 73.
75. according to any one of embodiments 69-74, wherein administering the TGFβ inhibitor does not increase platelet activation by more than 10% compared to activation in the absence of the inhibitor. Method.
76. Embodiments 69-75, wherein administering the TGFβ inhibitor does not increase platelet aggregation in vitro by more than 10% of the activation induced by known platelet aggregation agonists, such as adenosine diphosphate (ADP). The method described in any one of .
77. 77. The method of any one of embodiments 69-76, wherein administering the TGFβ inhibitor does not increase platelet aggregation by more than 10% compared to aggregation caused by the negative control.
78. 78. The method of any one of embodiments 69-77, wherein the therapeutically effective amount of TGFβ inhibitor is an amount sufficient to reduce the level of circulating MDSCs.
79. 79. The method of embodiment 78, wherein the degraded MDSCs are G-MDSCs.
80. 80. The method of embodiment 79, wherein the G-MDSC expresses one or more of LOX-1+, CD11b+, HLADR-, CD14-, CD15+, CD33+/low and CD66+.
81. 81. The method of any one of embodiments 78-80, wherein circulating MDSC levels are determined from whole blood or blood components collected from the subject.
82. 82. The method according to any one of embodiments 69-81, wherein the TGFβ inhibitor is a TGFβ1 inhibitor, optionally a TGFβ1-specific inhibitor.
83. A TGFβ inhibitor for use in the treatment of cancer by administering to a subject a dose of a TGFβ inhibitor that does not cause a significant increase in platelet binding, activation and/or aggregation.
84. A combination therapy comprising a dose of a TGFβ inhibitor and a cancer therapeutic agent for the treatment of cancer, the treatment comprising simultaneous, separate or sequential administration to a subject of a dose of a TGFβ inhibitor and a cancer therapeutic agent. A combination therapy comprising: said TGFβ inhibitor does not cause a significant increase in platelet binding, activation and/or aggregation.
85. A TGFβ inhibitor for use according to embodiment 83 or a combination therapy for use according to embodiment 84, wherein the TGFβ inhibitor is administered in a therapeutically effective amount sufficient to reduce circulating MDSC levels.
86. A TGFβ inhibitor for use or a combination therapy for use according to embodiment 85, wherein the reduced MDSCs are G-MDSCs.
87. a TGFβ inhibitor or for use according to embodiment 85, wherein the M-MDSC expresses one or more of LOX-1+, CD11b+, HLADR-, CD14-, CD15+, CD33+/low, and CD66+ combination therapy.
88. TGFβ inhibitor for use according to embodiment 85, wherein the reduced MDSCs are M-MDSCs expressing one or more of CD11b+, HLADR-/low, CD14+, CD15-, CD33+/high, and CD66b- or combination therapy for use.
89. A method according to embodiment 71 or any embodiment dependent thereon, wherein the TGFβ inhibitor is a TGFβ1 inhibitor, optionally a TGFβ1 specific inhibitor; A TGFβ inhibitor for the use described or a combination therapy for the use as described in embodiment 84 or any embodiment subordinate thereto.
90. Embodiment 83 or wherein the TGFβ inhibitor has been determined to not cause a significant increase in platelet binding, activation and/or aggregation by the method described in Embodiment 71 or any embodiment subordinate thereto. A TGFβ inhibitor for use as described in any embodiment dependent thereon or a combination therapy for use as described in embodiment 84 or any embodiment dependent thereon.
91. A TGFβ inhibitor for use according to embodiment 83 or any embodiment subordinate thereto, wherein the TGFβ inhibitor is a TGFβ inhibitor according to any one of embodiments 60 to 68 or embodiment 84. or a combination therapy for use as described in any embodiment subordinate thereto.
92. A method of producing a TGFβ inhibitor for treating cancer in a subject, comprising 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 least at doses greater than the minimum effective dose; and
c) the TGFβ inhibitor does not induce significant cytokine release from human PBMC or whole blood in an in vitro cytokine release assay at the lowest effective dose determined in one or more preclinical models of (a);
A method comprising the step of selecting a TGFβ inhibitor that satisfies one or more, such as all, of the following.
93. A method of producing a TGFβ inhibitor for treating cancer in a subject, comprising 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 least at doses greater than the minimum effective dose;
c) the TGFβ inhibitor does not induce significant cytokine release from human PBMC or whole blood in an in vitro cytokine release assay at the lowest effective dose determined in one or more preclinical models of (a);
d) the TGFβ inhibitor does not induce a significant increase in platelet binding, activation and/or aggregation as determined in one or more preclinical models of (a); and
e) the TGFβ inhibitor reduces circulating MDSCs at the lowest effective dose determined in one or more preclinical models of (a);
further comprising the step of selecting a TGFβ inhibitor that satisfies one or more, such as all, of
A method further comprising manufacturing a pharmaceutical composition comprising a TGFβ inhibitor and a pharmaceutically acceptable excipient.
94. 94. A method according to embodiment 92 or 93, wherein the TGFβ inhibitor is a TGFβ1 inhibitor, optionally a TGFβ1-specific inhibitor.
95. 95. A method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of a TGFβ inhibitor prepared according to the method of any one of embodiments 92-94.
96. A TGFβ inhibitor for use in an intermittent dosing regimen for cancer immunotherapy in a patient, the intermittent dosing regimen comprising:
(i) measuring circulating MDSCs in a first sample, e.g., a blood sample, taken from the patient prior to TGFβ inhibitor treatment;
(ii) administering a TGFβ inhibitor to a patient being treated with cancer therapy, the cancer therapy optionally being checkpoint inhibitor therapy;
(iii) measuring circulating MDSCs in a second sample taken from the patient after TGFβ inhibitor treatment;
(iv) if the second sample exhibits reduced levels of circulating MDSCs compared to the first sample, continuing the cancer therapy;
(v) repeating the process as necessary after further blood samples from the patient show elevated levels of circulating MDSC levels;
A TGFβ inhibitor.
97. Measuring circulating MDSC in a third sample and administering to the patient an additional dose of a TGFβ inhibitor if the third sample shows an elevated level of circulating MDSC compared to the second sample. 97. The method of embodiment 96, further comprising:
98. 98. The method of embodiment 96 or 97, wherein the TGFβ inhibitor inhibits TGFβ1 signaling.
99. 98. The method of embodiment 96 or 97, wherein the TGFβ inhibitor inhibits TGFβ1 signaling, but does not inhibit TGFβ2 signaling and/or TGFβ3 signaling at a therapeutically effective dose.
100. 98. The method of embodiment 96 or 97, wherein the TGFβ inhibitor is TGFβ1 selective.
101. 98. The method of embodiment 96 or 97, wherein the TGFβ inhibitor is an integrin inhibitor.
102. 102. The method according to 101, wherein the integrin inhibitor inhibits integrin αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α5β1, αIIbβ3 and/or α8β1.
103. 103. The method according to 101 or 102, wherein the integrin inhibitor inhibits downstream TGFβ1/3 activation.
104. A TGFβ1 selective inhibitor for use in the treatment of cancer in a subject, wherein the subject is being treated with a TGFβ inhibitor that inhibits TGFβ3 in conjunction with a checkpoint inhibitor.
105. 105. The method of 104, wherein the cancer is metastatic cancer, desmoplastic tumor, or myelofibrosis.
106. 106. The method of 104 or 105, wherein the subject has or is at risk of developing a disorder involving dysregulated extracellular matrix (ECM).
107. 107. The method of 106, wherein the disorder involving dysregulated ECM is NASH.
108. A TGFβ1 selective inhibitor for use according to any one of embodiments 104-107, wherein the former TGFβ inhibitor inhibits TGFβ1/2/3 or TGFβ1/3.
109. A non-isoform selective TGFβ inhibitor for use in the treatment of cancer in a subject, comprising:
(i) selecting a subject who has not been diagnosed with a fibrotic disease or who is not at high risk of developing a fibrotic disease;
(ii) administering to the subject an amount of a non-isoform selective TGFβ inhibitor effective to treat cancer;
A non-isoform selective TGFβ inhibitor comprising:
110. an isoform-nonselective TGFβ inhibitor for use in treating cancer in a subject, the treatment comprising: selecting a subject whose cancer is not a highly metastatic cancer; an isoform non-selective TGFβ inhibitor.
111. 111. The method according to 109 or 110, wherein the isoform non-selective TGFβ inhibitor is TGFβ1/2/3 or an antibody that inhibits TGFβ1/3.
112. The method according to any one of embodiments 109-111, wherein the isoform non-selective TGFβ inhibitor is an integrin inhibitor that binds to integrin αVβ1, αVβ6, αVβ8 and/or αVβ3.
113. The method according to 112, wherein the integrin inhibitor is an inhibitor of TGFβ1/3 activation.
114. 111. The method of 110, wherein the isoform non-selective TGFβ inhibitor is a recombinant construct comprising a TGFβ receptor ligand binding moiety.
115. Highly metastatic cancers include colorectal cancer, lung cancer (e.g., NSCLC), bladder cancer, renal cancer (e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC) (clear cell RCC, papillary RCC, chromophobe RCC). 111. An isoform-non-selective TGFβ inhibitor for use according to embodiment 110, wherein the isoform-non-selective TGFβ inhibitor is a uterine cancer, a prostate cancer, a gastric cancer, or a thyroid cancer.
116. A TGFβ1 selective inhibitor for use in treating cancer in a subject, the treatment comprising:
(i) selecting a subject whose cancer is highly metastatic;
(ii) administering to the subject an isoform-selective TGFβ1 inhibitor;
Highly metastatic cancers include colorectal cancer, lung cancer, bladder cancer, renal cancer (e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC) (clear cell RCC, papillary RCC, chromophobe RCC, collecting duct TGFβ1 selective inhibitors, including RCC, or unclassified RCC), uterine cancer, prostate cancer, gastric cancer or thyroid cancer.
117. A TGFβ1 selective inhibitor for use in treating cancer in a subject, the treatment comprising:
(i) selecting a subject having myelofibrosis, a fibrotic disease, or at risk of developing a fibrotic disease;
(ii) administering to the subject an isoform-selective TGFβ1 inhibitor in an amount effective to treat cancer;
A TGFβ1 selective inhibitor comprising.
118. A TGFβ1 selective inhibitor for use according to embodiment 117, wherein the subject is further treated with cancer therapy, and optionally, the cancer therapy comprises a checkpoint inhibitor.
119. A method, a medical use, a TGFβ inhibitor for use, or a combination therapy for use according to any one of the preceding embodiments, wherein the subject is a patient who has not received cancer therapy.
120. A method, a medical use, a TGFβ inhibitor for use or a combination therapy for use according to any one of the preceding embodiments, wherein the subject is undergoing cancer therapy.
121. A method, a medical use, a TGFβ inhibitor for use, or a combination therapy for use according to any one of the preceding embodiments, wherein the subject has previously undergone cancer therapy.
122. A method, a medical use, a TGFβ inhibitor for use, or a combination therapy for use according to any one of the preceding embodiments, wherein the subject is undergoing or will undergo cancer therapy.
123. A method, a medical use, a TGFβ inhibitor for use or a combination for use according to any one of the preceding embodiments, wherein the subject has a cancer that is resistant to a cancer therapy that does not include a TGFβ inhibitor. Therapy.
124. A method, a medical use, a TGFβ inhibitor for use, or a combination therapy for use according to any one of the preceding embodiments, wherein the subject has a reduced response to a cancer therapy that does not include a TGFβ inhibitor.
125. A method, medical use, TGFβ inhibitor for use or use according to any one of the preceding embodiments, wherein the subject is currently receiving or has previously received a cancer therapy that does not include a TGFβ inhibitor. Combination therapy for.
126. The method, medical use, use of a TGFβ inhibitor or combination therapy of any one of embodiments 121-125, wherein the cancer therapy does not include a TGFβ inhibitor.
127. The cancer therapy of embodiments 121-126 includes chemotherapy, radiotherapy (e.g., radiotherapeutic agents), recombinant immune cell therapy (e.g., CAR-T therapy), oncolytic virus therapy, and/or cancer vaccine therapy. Any one of the methods, medical uses, use of a TGFβ inhibitor or use of a combination therapy.
128. 128. The method, medical use, use of a TGFβ inhibitor or combination therapy of any one of embodiments 121-127, wherein the cancer therapy comprises immunotherapy, including checkpoint inhibitor therapy.
129. A method of treating a subject having a solid tumor, the method comprising: determining the level of cytotoxic T cells (e.g., CD8+ T cells) in a sample obtained from the subject prior to administering a TGFβ inhibitor. The level of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor is lower than the level of cytotoxic T cells (e.g., CD8+ T cells) outside the tumor before treatment. , administering to a subject a therapeutically effective amount of a TGFβ inhibitor, the therapeutically effective amount increasing the level of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor as compared to the level outside the tumor. and administering an amount sufficient to increase.
130. A method of treating a subject with a solid tumor comprising: determining levels of cytotoxic T cells (e.g., CD8+ T cells) within and outside the tumor in a sample obtained from the subject; 1. A method comprising selecting a subject having a ratio of the level of cytotoxic T cells (e.g., CD8+ T cells) within a tumor to less than 1, and administering to the subject a therapeutically effective amount of a TGFβ inhibitor.
131. 131. The method of embodiment 129 or 130, wherein the level of cytotoxic T cells (eg, CD8+ T cells) outside the tumor is determined from the tumor margin and/or stroma.
132. 132. The method of any one of embodiments 129-131, wherein the level of cytotoxic T cells (eg, CD8+ T cells) outside the tumor is determined from the margin.
133. 133. The method of embodiment 131 or embodiment 132, wherein the edge is about 10-100 μm wide (eg, 50 μm wide).
134. The level of cytotoxic T cells (e.g., CD8+ T cells) in the margin and/or stroma is at least 2, 3, 4, 5, 7, or 10 times higher than the level inside the tumor. , the method of any one of embodiments 129-133.
135. A method of treating a subject having a solid cancer, the method comprising: measuring the level of CD8+ cells in one or more tumor foci from at least one tumor tissue sample obtained from the subject; and the area of the sample measured. more than 50% of the tumor nests have a lower level of CD8+ cells inside the tumor nests relative to the level of CD8+ cells outside the tumor nests (e.g., less than 5% of the inside the tumor nests). administering to the subject a therapeutically effective amount of a TGFβ inhibitor if the tumor nest comprises CD8+ cells and greater than 5% CD8+ cells outside the tumor nest.
136. 1. A method of treating a subject having a solid cancer, the method comprising:
(i) determine the levels of cytotoxic T cells (e.g., CD8+ T cells) inside and outside the tumor in a first sample, and determine the density of cytotoxic T cells (e.g., CD8+ T cells) inside the tumor relative to the density outside the tumor; , CD8+ T cell) density ratio is less than 1;
(ii) administering to the subject a first dose of a TGFβ inhibitor;
(iii) determining the level of cytotoxic T cells (e.g., CD8+ T cells) within the tumor in the second sample;
(iv) the level of cytotoxic T cells (e.g., CD8+ T cells) within the tumor determined in step (iii) is such that the level of cytotoxic T cells (e.g., CD8+ T cells) within the tumor determined in step (i) is , CD8+ T cells), administering to the subject a second dose of the TGFβ inhibitor;
method including.
137. A method for determining the therapeutic efficacy of cancer treatment in a subject, the method comprising:
(i) determining the level of cytotoxic T cells (e.g., CD8+ T cells) within the tumor in the first sample;
(ii) administering to the subject a dose of a TGFβ inhibitor;
(iii) determining the level of cytotoxic T cells (e.g., CD8+ T cells) within the tumor in the second sample;
(iv) determining whether the level of cytotoxic T cells (e.g., CD8+ T cells) determined in step (iii) is increased compared to step (i); The increase indicates and determines the therapeutic effect of cancer treatment.
method including.
138. 138. The method of embodiment 136 or embodiment 137, wherein step (ii) further comprises administering to the subject an additional cancer therapy simultaneously, separately or sequentially with the TGFβ inhibitor.
139. 139. The method of embodiment 138, wherein the cancer therapy comprises checkpoint inhibitor therapy (eg, an agent targeting PD-1 or PD-L1 or an anti-PD-1 or anti-PD-L1 antibody).
140. In any one of embodiments 136-139, the level of cytotoxic T cells (e.g., CD8+ T cells) in the tumor is increased by at least 10%, 15%, 20%, 25% or more. Method described.
141. 141. The method according to any one of embodiments 129-140, wherein the TGFβ inhibitor is a TGFβ1 selective inhibitor, such as Ab6.
142. 142. The method of any one of embodiments 129-141, wherein the sample is a tumor biopsy sample.
143. 143. The method of embodiment 142, wherein the tumor biopsy sample is a core needle biopsy sample of a tumor.
144. 144. The method of any of embodiments 129-143, wherein the level of cytotoxic T cells (eg, CD8+ T cells) is determined by immunohistochemical analysis.
145. further comprising determining the level of circulating MDSC before and after administration of the first dose of the TGFβ inhibitor, wherein the decrease in the MDSC level is determined after administration of the first dose of the TGFβ inhibitor, step (iii) The level of cytotoxic T cells (e.g., CD8+ T cells) within the tumor determined in step (i) is the level of cytotoxic T cells (e.g., CD8+ T cells) within the tumor determined in step (i). 137. The method of embodiment 136, wherein the second dose of the TGFβ inhibitor is administered when the second dose is increased compared to the TGFβ inhibitor.
146. further comprising determining the level of circulating MDSC before and after administration of the TGFβ inhibitor, including a decrease in the level of MDSC and/or an increase in the level of cytotoxic T cells (e.g., CD8+ T cells) within the tumor after administration. 138. The method of embodiment 137, wherein the method exhibits a therapeutic effect.
147. The method of embodiment 145 or 146, wherein the circulating MDSC is a G-MDSC.
148. 148. The method of embodiment 147, wherein the G-MDSC expresses one or more of LOX-1+, CD11b+, HLADR-, CD14-, CD15+, CD33+/low and CD66+.
149. 149. The method of any one of embodiments 145-148, wherein circulating MDSC levels are determined from whole blood or blood components collected from the subject.
150. 150. The method of any one of embodiments 145-149, wherein the level of circulating MDSC levels is reduced by at least 10%, optionally by at least 15%, 20%, 25% or more.
151. The level of cytotoxic T cells (e.g., CD8+ T cells) within the tumor is increased by at least 10%, 15%, 20%, 25% or more, the level of circulating MDSC is increased by at least 15%, The method of any one of embodiments 145-150, wherein the method is reduced by 20%, 25% or more.
152. Any of embodiments 129-151, wherein the level of cytotoxic T cells (e.g., CD8+ T cells) is a percentage of CD8+ T cells or a CD8+ cell density (e.g., number of CD8+ T cells per square millimeter). The method described in one.
153. 153. The method of any one of embodiments 129-152, wherein the therapeutically effective amount of TGFβ inhibitor is from 0.1 mg/kg to 30 mg/kg per administration.
154. 154. The method of any one of embodiments 129-153, wherein the therapeutically effective amount of TGFβ inhibitor is from 1 mg/kg to 10 mg/kg per administration.
155. 155. The method of any one of embodiments 129-154, wherein the therapeutically effective amount of TGFβ inhibitor is from 2 mg/kg to 7 mg/kg per administration.
156. TGFβ inhibitors are administered once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 6 weeks, once every 8 weeks, and once every 2 months. , once every 10 weeks, once every 12 weeks, once every 3 months, once every 4 months, once every 6 months, once every 8 months, once every 10 months, or once a year , the method of any one of embodiments 129-155.
157. 157. The method of any one of embodiments 129-156, wherein the TGFβ inhibitor is administered about once every three weeks.
158. 158. The method of any one of embodiments 129-157, wherein the TGFβ inhibitor is administered intravenously or subcutaneously.
159. Embodiments 129-158, wherein the cancer is non-small cell lung cancer, melanoma, renal cell cancer, triple negative breast cancer, gastric cancer, microsatellite stable colorectal cancer, pancreatic cancer, small cell lung cancer, HER2 positive breast cancer or prostate cancer. The method described in any one of .
160. A method of determining the therapeutic effect of a cancer treatment in a subject, the treatment comprising administering to the subject a combination therapy for simultaneous, separate or sequential administration comprising a dose of a TGFβ inhibitor and a cancer therapy; The method is
(i) determining the level of circulating myeloid-derived suppressor cells (MDSC) in a sample obtained from the subject prior to administering the TGFβ inhibitor, optionally the MDSC level being determined by an antibody that binds to LRRC33; a process determined by the use of;
(ii) determining circulating MDSC levels in a sample obtained from the subject after administration of the TGFβ inhibitor, optionally the MDSC levels being determined by use of an antibody that binds to LRRC33; ;as well as
(iii) determining whether the level determined in step (ii) is reduced compared to the level determined in step (i), such reduction being indicative of a therapeutic effect of the cancer treatment;
Including, methods.
161. Embodiments wherein the level of circulating MDSC cells is determined within 3 to 6 weeks after administration of the dose of TGFβ inhibitor, optionally within or about 3 weeks after administration of the dose of TGFβ inhibitor. 160.
162. 161. The method of embodiment 160, wherein the level of circulating MDSC cells is determined within two weeks after administration of the TGFβ inhibitor dose, optionally at about 10 days after administration of the TGFβ inhibitor dose.
163. The subject in step (i) or (ii) has not received any previous cancer therapy, and optionally the subject in steps (i) and (ii) has not received any previous cancer therapy. 163. The method according to any one of embodiments 160-162.
164. 164. The method of any one of embodiments 160-163, wherein the subject will receive cancer therapy if circulating MDSC levels are determined to be reduced.
165. The subject in steps (i) or (ii) has received or is undergoing a previous cancer therapy, and optionally the subject in steps (i) and (ii) has received or is undergoing a previous cancer therapy. or undergoing cancer therapy, the method of any one of embodiments 160-164.
166. 166. The method of embodiment 165, wherein if it is determined that circulating MDSC levels have been reduced, the subject will receive additional cancer therapy.
167. 167. The method of any one of embodiments 160-166, wherein the subject receives two or more administrations of a TGFβ inhibitor before step (ii).
168. 168. The method of any one of embodiments 160-167, wherein the sample is a whole blood sample or a blood component.
169. A cancer therapeutic agent for use in treating cancer in a subject, the subject receiving a dose of a TGFβ inhibitor, wherein the level of circulating MDSC in the subject measured after administration of the TGFβ inhibitor is has been determined to be reduced compared to a circulating MDSC level measured in the subject, and optionally, the circulating MDSC level is measured using an antibody that binds to LRRC33. cancer therapy.
170. A combination therapy comprising a dose of a TGFβ inhibitor and a cancer therapeutic agent for use in the treatment of cancer, wherein the treatment comprises the simultaneous, separate or sequential administration to a subject of a dose of the TGFβ inhibitor and the cancer therapeutic agent. comprising that the circulating MDSC level in the subject measured after administration of the TGFβ inhibitor was reduced compared to the circulating MDSC level measured in the subject before administering the dose of the TGFβ inhibitor. and, optionally, circulating MDSC levels are measured using an antibody that binds LRRC33.
171. A combination for use according to embodiment 170, wherein the subject has not received previous cancer therapy and it has been determined that circulating MDSC levels in the subject have been reduced prior to administration of the cancer therapeutic agent. Therapy.
172. 172. A combination therapy for use according to embodiment 170 or embodiment 171, wherein the subject has not received previous cancer therapy and the subject is administered the TGFβ inhibitor before the cancer therapeutic agent.
173. Combination therapy for use according to embodiment 170, wherein the subject is administered the cancer therapeutic agent before the TGFβ inhibitor.
174. A TGFβ inhibitor for use in treating cancer in a subject, wherein the subject has been administered at least a first dose of a TGFβ inhibitor, and the treatment comprises administering an additional dose of a TGFβ inhibitor. , provided that the level of circulating MDSC in the subject measured after administration of at least the first dose of the TGFβ inhibitor is reduced compared to the level of circulating MDSC measured in the subject prior to administration of the dose of the TGFβ inhibitor. , optionally, circulating MDSC levels are measured using an antibody that binds to LRRC33, a TGFβ inhibitor.
175. A TGFβ inhibitor for use in treating cancer in a subject, wherein the subject is administered a dose of a TGFβ inhibitor, the TGFβ inhibitor reduces or reverses immunosuppression in the cancer, and the TGFβ inhibitor reduces or reverses immunosuppression in the cancer, Reversed immunosuppression is determined by a decrease in circulating MDSC levels in a subject measured after administration of a TGFβ inhibitor compared to circulating MDSC levels measured in the subject before administering a dose of TGFβ inhibitor. and, optionally, circulating MDSC levels are measured using an antibody that binds to LRRC33, a TGFβ inhibitor.
176. A cancer therapeutic agent for use according to embodiment 169, embodiment 170 or any subordinate thereto, wherein the subject has received two or more administrations of a TGFβ inhibitor prior to the determination that circulating MDSC levels have been reduced. A combination therapy for use as described in any embodiment or a TGFβ inhibitor for use as described in embodiment 174 or embodiment 175.
177. The level of circulating MDSC cells is within 3-6 weeks after administration of the dose of TGFβ inhibitor, optionally within or about 3 weeks after administration of the dose of TGFβ inhibitor, optionally 2 weeks after administration of the dose of TGFβ inhibitor. Embodiment 169 or any practice dependent thereon, wherein the dose of TGFβ inhibitor is determined within or about 10 days, and optionally, the dose of TGFβ inhibitor is the first dose of TGFβ inhibitor administered to the subject. A cancer therapeutic agent for use as described in Embodiment 170 or any embodiment dependent thereon, or a combination therapy for use as described in Embodiment 174 or Embodiment 175 or any embodiment dependent thereon. A TGFβ inhibitor for use as described in .
178. The subject has not received previous cancer therapy, a cancer therapeutic agent for use as described in embodiment 169 or any embodiment subordinate thereto, embodiment 170 or any embodiment subordinate thereto. A combination for a use as described or a TGFβ inhibitor for a use as described in embodiment 174 or embodiment 175 or any embodiment subordinate thereto.
179. The subject is undergoing cancer therapy, a cancer therapeutic agent for the use as described in embodiment 169 or any embodiment subordinate thereto, of the use as described in embodiment 170 or any embodiment subordinate thereto. A TGFβ inhibitor for combination or use as described in embodiment 174 or embodiment 175 or any embodiment subordinate thereto.
180. A cancer therapeutic agent for the use according to embodiment 169 or any embodiment subordinate thereto, wherein the subject is scheduled to undergo cancer therapy, a use according to embodiment 170 or any embodiment subordinate thereto. or a TGFβ inhibitor for use as described in embodiment 174 or embodiment 175 or any embodiment subordinate thereto.
181. Cancer therapy may include immunotherapy, chemotherapy, radiotherapy, recombinant immune cell therapy (e.g., CAR-T therapy), cancer vaccine therapy, and/or oncolytic virus therapy, according to embodiment 169 or any dependent thereon. A cancer therapeutic agent for the use described in embodiment 170, a combination for the use described in embodiment 170 or any embodiment subordinate thereto, or for the use described in any one of embodiments 178-180. TGFβ inhibitor.
182. The cancer therapy is an immunotherapy including checkpoint inhibitor therapy, optionally the checkpoint inhibitor targeting programmed cell death protein 1 (PD-1) or programmed cell death protein 1 ligand (PD-L1). a cancer therapeutic agent for use as described in embodiment 169 or any embodiment subordinate thereto, wherein the checkpoint inhibitor comprises an anti-PD-(L)1 antibody; A combination for use as described in embodiment 170 or any embodiment subordinate thereto or a TGFβ inhibitor for use as described in any one of embodiments 178-180.
183. For the method of determining therapeutic efficacy as described in embodiment 160 or any embodiment subordinate thereto, the use as described in embodiment 169 or any embodiment subordinate thereto, wherein the circulating MDSCs are G-MDSCs. a cancer therapeutic agent, a combination for use as described in embodiment 170 or any embodiment subordinate thereto, or TGFβ for use as described in embodiment 174 or embodiment 175 or any embodiment subordinate thereto. inhibitor.
184. 184, wherein the G-MDSCs express one or more of LOX-1+, CD11b+, HLADR-, CD14-, CD15+, CD33+/low and CD66+. A therapeutic agent, combination therapy for use or a TGFβ inhibitor for use.
185. Circulating MDSC levels are reduced by at least 10%, optionally by at least 15%, 20%, 25% or more, determining the therapeutic effect of embodiment 160 or any embodiment subordinate thereto. A method for treating cancer, a cancer therapeutic agent for use as described in embodiment 169 or any embodiment subordinate thereto, a combination for use as described in embodiment 170 or any embodiment subordinate thereto, or embodiment 174 or a TGFβ inhibitor for use as described in embodiment 175 or any embodiment subordinate thereto.
186. A cancer therapeutic agent for use according to embodiment 169 or any embodiment dependent thereon, wherein the circulating MDSC level is determined from a whole blood sample or blood component obtained from the subject, embodiment 170 or A combination for use as described in any dependent embodiment or a TGFβ inhibitor for use as described in embodiment 174 or embodiment 175 or any embodiment dependent thereon.
187. A method of determining a therapeutic effect as described in embodiment 160 or any embodiment subordinate thereto, wherein the TGFβ inhibitor is a TGFβ1 inhibitor, and optionally the TGFβ inhibitor is a TGFβ1-specific inhibitor; A cancer therapeutic agent for use as described in Embodiment 169 or any embodiment subordinate thereto, a combination for use as described in Embodiment 170 or any embodiment subordinate thereto, or Embodiment 174 or any embodiment 175 or any embodiment subordinate thereto.
188. Embodiment 169 A method of determining the efficacy of a treatment as described in embodiment 160 or any embodiment subordinate thereto, wherein the subject has, prior to treatment, a circulating MDSC level that is at least two times higher than a circulating MDSC level in a healthy subject. or a cancer therapeutic agent for use as described in any embodiment subordinate thereto, a combination for use as described in embodiment 170 or any embodiment subordinate thereto, or embodiment 174 or embodiment 175 or thereof. A TGFβ inhibitor for use according to any dependent embodiment.
189. The subject has or is suspected of having cancer. a cancer therapeutic agent for use, a combination for use as described in embodiment 170 or any embodiment subordinate thereto, or a combination for use as described in embodiment 174 or embodiment 175 or any embodiment subordinate thereto; TGFβ inhibitor for.
190. The level of tumor-associated immune cells in the subject measured after administration of the first dose of the TGFβ inhibitor is compared to the level of tumor-associated immune cells in the subject measured before administration of the first dose of the TGFβ inhibitor. A method for determining therapeutic efficacy as described in embodiment 160 or any embodiment subordinate thereto, or a cancer therapeutic agent for use as described in embodiment 169 or any embodiment subordinate thereto, wherein , a combination for use as described in embodiment 170 or any embodiment subordinate thereto, or a TGFβ inhibitor for use as described in embodiment 174 or embodiment 175 or any embodiment subordinate thereto.
191. 160 or any embodiment subordinate thereto, comprising:
(iv) determining the level of one or more tumor-associated immune cells in a sample obtained from the subject prior to administering the TGFβ inhibitor;
(v) determining the level of one or more tumor-associated immune cells in a sample obtained from the subject after administration of the TGFβ inhibitor;
(vi) determining whether the level determined in step (v) is changed compared to the level determined in step (iv), wherein such change To show and determine the effect
How to further include.
192. The method of embodiment 191, wherein the change in the level of one or more tumor-associated immune cells indicates a reduction or amelioration of immunosuppression in cancer.
193. 193. The method of embodiment 191 or embodiment 192, wherein the tumor-associated immune cells include CD8+ T cells and/or tumor-associated macrophages (TAMs).
194. Embodiments 191-193, wherein the change in the level of one or more tumor-associated immune cells comprises an increase in the level of CD8+ T cells by at least 10%, optionally at least 15%, 20%, 25% or more. The method described in any one of .
195. The change in the level of one or more tumor-associated immune cells comprises an increase in the level of TAM by at least 10%, optionally at least 15%, 20%, 25% or more. Any one of the methods.
196. The method of any one of embodiments 191-195, wherein the level of one or more tumor-associated immune cells is determined in the sample obtained from the subject by immunohistochemical analysis.
197. The method of any one of embodiments 191-196, wherein the level of one or more tumor-associated immune cells is determined by in vivo imaging.
198. 198. The method of any one of embodiments 191-197, wherein the sample is a tumor biopsy sample, and the tumor biopsy sample is optionally a core needle biopsy sample of a tumor.
199. The level of circulating latent TGFβ (e.g., circulating latent TGFβ1) in a subject measured after administration of a first dose of a TGFβ inhibitor is equal to the level of circulating latent TGFβ (e.g., circulating latent TGFβ1) in a subject measured before administration of a first dose of a TGFβ inhibitor A cancer therapeutic agent for use according to embodiment 169 or any embodiment dependent thereon, wherein the cancer therapeutic agent for use according to embodiment 169 or any embodiment dependent thereon is altered relative to the level of circulating latent TGFβ; A combination for a use as described or a TGFβ inhibitor for a use as described in embodiment 174 or embodiment 175 or any embodiment subordinate thereto.
200. A method of determining therapeutic efficacy as described in embodiment 160 or embodiment 191 or any embodiment subordinate thereto, comprising:
(vii) determining the level of circulating latent TGFβ in a sample obtained from the subject prior to administering the TGFβ inhibitor;
(viii) determining the level of circulating latent TGFβ in a sample obtained from the subject after administration of the TGFβ inhibitor;
(ix) determining whether the level determined in step (viii) is increased compared to the level determined in step (vii), wherein such increase To show and determine the effect
How to further include.
201. 201. The method of embodiment 200, wherein the level of circulating latent TGFβ is determined in a sample obtained from the subject, the sample being a whole blood sample or a blood component.
202. 201. The method of embodiment 200, wherein the circulating latent TGFβ is circulating latent TGFβ1.
203. A method for determining therapeutic efficacy as described in embodiment 160 or any embodiment dependent thereon, wherein the subject has a cancer that is resistant to a cancer therapy that does not include a TGFβ inhibitor. a cancer therapeutic agent for use as described in embodiment 170 or any embodiment dependent thereon; or a combination for use as described in embodiment 174 or embodiment 175 or any embodiment dependent thereon; A TGFβ inhibitor for use as described in the form.
204. The subject has a low response to cancer therapy that does not include a TGFβ inhibitor, the method of determining therapeutic efficacy as described in embodiment 160 or any embodiment subordinate thereto, or the practice of embodiment 169 or any embodiment subordinate thereto. a cancer therapeutic agent for the use described in Embodiment 170 or any embodiment dependent thereon; or a combination for the use described in Embodiment 174 or 175 or any embodiment subordinate thereto; TGFβ inhibitors for the described uses.
205. A method for determining therapeutic efficacy as described in embodiment 160 or any embodiment subordinate thereto, embodiment 169 or a cancer therapeutic agent for use as described in any dependent embodiment, a combination for use as described in embodiment 170 or any embodiment dependent thereon, or embodiment 174 or embodiment 175 or dependent thereon; A TGFβ inhibitor for use according to any embodiment.
206. A method for determining therapeutic efficacy as described in embodiment 160 or any embodiment dependent thereon, wherein the subject has a cancer that is resistant to a cancer therapy that does not include a TGFβ inhibitor. a cancer therapeutic agent for use as described in embodiment 170 or any embodiment dependent thereon; or a combination for use as described in embodiment 174 or embodiment 175 or any embodiment dependent thereon; A TGFβ inhibitor for use as described in the form.
207. The TGFβ inhibitor is administered intravenously to a subject in the method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use or for use of any one of the preceding embodiments, wherein the TGFβ inhibitor is administered intravenously to a subject. combination therapy.
208. The TGFβ inhibitor is about 37.5 mg/kg, 30 mg/kg, 20 mg/kg, 10 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg A method, a medical use, a cancer therapeutic agent for use, a TGFβ inhibitor for use or a combination for use according to any one of the preceding embodiments, administered at a concentration of 1 kg or less. Therapy.
209. The method, medical use, use in cancer according to any one of the preceding embodiments, wherein the TGFβ inhibitor is administered in an amount of about 3000 mg, 2400 mg, 1600 mg, 800 mg, 240 mg, 80 mg or less. A therapeutic agent, a TGFβ inhibitor for use or a combination therapy for use.
210. The method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use or for use of embodiment 204 or embodiment 205, wherein the TGFβ inhibitor is administered about once every three weeks. combination therapy.
211. A method, a medical use, a cancer therapeutic for use, a TGFβ inhibitor for use, or a combination therapy for use as in any one of the preceding embodiments, wherein the cancer comprises an immunoablated tumor.
212. The method, medical use, cancer treatment for use according to any one of the preceding embodiments, wherein the cancer is a myeloproliferative disease, and optionally the myeloproliferative disease is myelofibrosis. agent, a TGFβ inhibitor for use or a combination therapy for use.
213. A method, a medical use, a cancer therapeutic agent for use, a TGFβ inhibitor for use, or a combination therapy for use according to any one of the preceding embodiments, wherein the cancer is a highly metastatic cancer. .
214. Cancers include colorectal cancer, lung cancer, bladder cancer, renal cancer (e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC) (clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC), uterine cancer, prostate cancer, gastric cancer or thyroid cancer), uterine cancer, prostate cancer, gastric cancer or thyroid cancer. TGFβ inhibitors or combination therapy for use.
215. The subject is at risk of developing aortic stenosis, the method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use or use of any one of the preceding embodiments. Combination therapy for.
216. A method, a medical use, a cancer therapeutic for use, a TGFβ inhibitor for use, or a combination therapy for use as in any one of the preceding embodiments, wherein the cancer is TGFβ1 positive.
217. The method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use or combination for use of any one of the preceding embodiments, wherein the cancer co-expresses TGFβ1 and TGFβ3. Therapy.
218. A method, a medical use, a cancer therapeutic for use, a TGFβ inhibitor for use, or a combination therapy for use as in any one of the preceding embodiments, wherein the tumor is a TGFβ1-dominant tumor.
219. 1. A method for manufacturing a pharmaceutical composition comprising a TGFβ inhibitor, the method comprising:
i) The following criteria:
a) the TGFβ inhibitor is a monoclonal antibody, antigen-binding fragment or multispecific construct thereof capable of binding TGFβ;
b) When the TGFβ inhibitor is measured by an SPR-based assay (e.g. Biacore), a K of <1.0 nM _D , preferably K _D binding to TGFβ and inhibiting TGFβ1 at <500 pM;
c) the TGFβ inhibitor is effective in vivo in preclinical models at doses that do not cause the toxicity associated with pan-inhibition of TGFβ when administered at least 10 times the minimum effective dose for at least 4 weeks in the animal model;
providing a TGFβ inhibitor that satisfies;
ii) Below:
a) Cytokine release assay (in vitro and/or in vivo); and/or
b) Platelet assay
a step of performing immunosafety including;
iii) producing the TGFβ inhibitor on a scale of 250 L or more;
iv) formulating the TGFβ inhibitor with one or more excipients into a pharmaceutical composition;
method including.
220. 220. The method of embodiment 219, wherein the TGFβ is TGFβ1.
221. 220. The method of embodiment 219, wherein the TGFβ is a proTGFβ complex, a mature TGFβ growth factor, or a ligand binding domain of a TGFβ receptor.
222. TGFβ inhibitors cause tumor growth regression, prolonged survival and/or normalized gene expression of PAI-1, CCL2, FN-1, ACTA2, Col1a1, Col3a1, FN-1, CTGF and/or TGFβ1 220. The method of embodiment 219, wherein the method is effective for.
223. 223. The method of embodiment 222, wherein the tumor is a TGFβ1 predominant tumor and optionally the tumor further expresses TGFβ3.
224. 220. The method of embodiment 219, wherein the toxicity associated with pan-inhibition of TGFβ includes one or more of cardiovascular toxicity (e.g., valvular heart disease), epithelial hyperplasia, hemorrhage, and skin lesions.
225. 220. The method of embodiment 219, wherein the immunosafety assessment comprises an in vitro cytokine release assay.
226. 220. The method of embodiment 219, wherein the scale of production is at least 500L, at least 1000L, at least 2000L.
227. Any one of embodiments 219-226, wherein the production comprises a eukaryotic cell culture, and optionally the eukaryotic cell culture is a mammalian cell culture, a plant cell culture, or an insect cell culture. The method described in.
228. Mammalian cell cultures include CHO cells, MDCK cells, NS0 cells, Sp2/0 cells, BHK cells, mouse C127 cells, Vero cells, HEK293 cells, HT-1080 cells or PER. 228. The method of embodiment 227, comprising C6 cells.
229. A method of treating a TGFβ-related disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a TGFβ inhibitor to treat the disease, the therapeutically effective amount reducing the level of circulating latent TGFβ after administration. A method that is sufficient to increase the amount.
230. A method of treating a TGFβ-related disease in a subject, the method comprising administering a TGFβ inhibitor and monitoring the level of circulating latent TGFβ after administration.
231. The method of embodiment 229 or 230, wherein the TGFβ-related disease is a TGFβ1-related disease.
232. 232. The method of embodiment 231, wherein the TGFβ1-related disease is cancer.
233. 232. The method of embodiment 231, wherein the TGFβ1-related disease is an immune disorder.
234. The level of circulating latent TGFβ after administration of the TGFβ inhibitor is, for example, at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold compared to the level before administration. 234. The method of any one of embodiments 229-233, wherein an additional dose of the TGFβ inhibitor is administered.
235. 235. The method of any one of embodiments 229-234, wherein the level of circulating latent TGFβ after administration of the TGFβ inhibitor is increased to a maximum of at least 1000 pg/ml.
236. 236. The method of any one of embodiments 229-235, wherein the level of circulating latent TGFβ after administration of the TGFβ inhibitor is increased to a maximum of about 1000 pg/ml to about 8000 pg/ml.
237. 237. The method of any one of embodiments 229-236, wherein the level of circulating latent TGFβ after administration of the TGFβ inhibitor is increased to a maximum of about 2000 pg/ml to about 6500 pg/ml.
238. 238. The method of any one of 229-237, wherein the level of circulating latent TGFβ after administration of the TGFβ inhibitor is increased by a minimum of about 1.5-fold.
239. 239. The method of any one of embodiments 229-238, wherein the level of circulating latent TGFβ is measured from about 8 to about 672 hours after administration of the TGFβ inhibitor.
240. 240. The method of any one of embodiments 229-239, wherein the level of circulating latent TGFβ is measured from about 24 hours to about 336 hours after administration of the TGFβ inhibitor.
241. 241. The method of any one of embodiments 229-240, wherein the level of circulating latent TGFβ is measured from about 72 hours to about 240 hours after administration of the TGFβ inhibitor.
242. 242. The method of any one of embodiments 229-241, wherein the TGFβ inhibitor is administered at a dose of about 1 mg/kg to about 30 mg/kg.
243. 243. The method of any one of embodiments 229-242, wherein the TGFβ inhibitor is administered at a dose of about 5 mg/kg to about 20 mg/kg.
244. 244. The method of any one of embodiments 229-243, wherein the TGFβ inhibitor is administered at a dose of about 2 mg/kg to about 7 mg/kg.
245. 246. The method of any one of embodiments 229-245, wherein the TGFβ inhibitor is administered about once every three weeks.
246. A method of determining the effectiveness of cancer treatment in a subject, the method comprising: determining the level of circulating latent TGFβ1 in a first sample from the subject; and administering to the subject a dose of a TGFβ1 inhibitor; determining the level of circulating latent TGFβ in a second sample from the subject after administration, at least two times, three times the level of circulating latent TGFβ between the first sample and the second sample. , a 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more increase is indicative of therapeutic efficacy.
247. A method of treating a subject with a solid cancer, the method comprising: determining the level of circulating latent TGFβ1 in a first sample from the subject; administering to the subject a dose of a TGFβ1 inhibitor; later determining the level of circulating latent TGFβ in a second sample from the subject.
248. 248. The method of embodiment 247, wherein the additional dose of the TGFβ inhibitor is administered if the subject has a ratio of circulating latent TGFβ post-administration to pre-administration of at least 1.2.
249. 249. The method of any one of embodiments 246-248, wherein the second sample is collected from the subject 24 hours to 56 days after administration.
250. The method of any one of embodiments 229-249, wherein the TGFβ inhibitor is a TGFβ activation inhibitor, such as a TGFβ1 selective inhibitor.
251. The method of embodiment 2250, wherein the TGFβ inhibitor is Ab6.
252. 252. The method of any one of embodiments 229-251, wherein the therapeutically effective amount of TGFβ inhibitor is from 0.1 mg/kg to 30 mg/kg per administration.
253. 253. The method of any one of embodiments 229-252, wherein the therapeutically effective amount of TGFβ inhibitor is from 1 mg/kg to 10 mg/kg per administration.
254. 254. The method of any one of embodiments 229-253, wherein the therapeutically effective amount of TGFβ inhibitor is from 2 mg/kg to 7 mg/kg per administration.
255. TGFβ inhibitors are administered once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 6 weeks, once every 8 weeks, and once every 2 months. , once every 10 weeks, once every 12 weeks, once every 3 months, once every 4 months, once every 6 months, once every 8 months, once every 10 months, or once a year , the method of any one of embodiments 229-254.
256. 256. The method of any one of embodiments 229-255, wherein the TGFβ inhibitor is administered about once every three weeks.
257. 257. The method of any one of embodiments 229-256, wherein the TGFβ inhibitor is administered intravenously or subcutaneously.
258. 258. The method of any one of embodiments 229-257, wherein the latent TGFβ is latent TGFβ1.
259. 259. The method of any one of embodiments 229-258, wherein the level of circulating latent TGFβ is measured in a blood sample.
260. 260. The method of any one of embodiments 229-259, wherein the blood sample is a serum sample or a plasma sample.
261. 261. The method of any one of embodiments 229-260, wherein circulating latent TGFβ levels are measured by ELISA.
262. The method of embodiments 229-261 further comprising determining the level of circulating MDSC in the subject before and after administration of the TGFβ inhibitor, optionally the circulating MDSC level being determined by use of an antibody that binds LRRC33. Any one of the methods.
263. 263. The method of embodiment 262, wherein a reduction in the level of circulating MDSCs after administration compared to pre-administration indicates a therapeutic effect, and optionally one or more additional treatments comprising a TGFβ inhibitor are administered.
264. 263. The method of embodiment 262, wherein the circulating MDSC is a G-MDSC.
265. G-MDSC includes CD11b, CD33, CD15, LOX-1, CD66b and HLA-DR ^lo/- 265. The method of embodiment 264, wherein the method expresses one or more of the following.
266. 247. The method of any one of embodiments 243-246, wherein circulating MDSC levels are determined from whole blood or blood components collected from the subject.
267. The method of any one of embodiments 262-266, wherein administration of the TGFβ inhibitor reduces circulating MDSC levels by at least 10%, optionally by at least 15%, 20%, 25% or more. .
268. according to any one of embodiments 262-267, wherein circulating latent TGFβ levels are increased by at least 50% and circulating MDSC levels are reduced by at least 15%, 20%, 25% or more. Method.
269. The TGFβ inhibitor is a method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use or for use in any one of the preceding embodiments, which inhibits TGFβ1 signaling. Combination therapy.
270. The method, medical use, cancer therapeutic agent for use according to any one of the preceding embodiments, wherein the TGFβ inhibitor inhibits TGFβ1 signaling, but not TGFβ3 signaling, at a therapeutically effective dose. A TGFβ inhibitor for use or a combination therapy for use.
271. The method, medical use, cancer therapeutic agent for use, according to any one of the preceding embodiments, wherein the TGFβ inhibitor does not inhibit TGFβ2 signaling and TGFβ3 signaling at a therapeutically effective dose. TGFβ inhibitors or combination therapy for use.
272. The TGFβ inhibitor does not bind free TGFβ growth hormone, the method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use or for use according to any one of the preceding embodiments. combination therapy.
273. The method, medical use, cancer therapeutic agent for use, TGFβ inhibition for use according to any one of the preceding embodiments, wherein the TGFβ inhibitor binds pro-TGFβ1 and/or latent TGFβ1. agent or combination therapy for use.
274. The method, medical use, cancer therapeutic agent for use, TGFβ inhibitor of any one of the preceding embodiments, wherein the TGFβ inhibitor binds at least a portion of latent lasso in TGFβ1. Inhibitors or combination therapy for use.
275. The TGFβ inhibitor binds to at least a portion of the finger-1 domain in TGFβ1. TGFβ inhibitors or combination therapy for use.
276. The method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use or use of any one of the preceding embodiments, wherein the TGFβ inhibitor is a neutralizing antibody or a ligand trap. Combination therapy for.
277. The method, medical use, according to any one of the preceding embodiments, wherein the TGFβ inhibitor selectively binds TGFβ1 and optionally selectively binds pro-TGFβ1 and/or latent TGFβ1; A cancer therapeutic agent for use, a TGFβ inhibitor for use or a combination therapy for use.
278. The TGFβ inhibitor has (i) a first binding region comprising at least a portion of Latent Lasso (SEQ ID NO: 126); and ii) a second binding region comprising at least a portion of finger-1 (SEQ ID NO: 124). The method, medical use, cancer therapeutic agent for use according to any one of the preceding embodiments, wherein the isolated antibody or antigen-binding fragment thereof is capable of specifically binding to the proTGFβ1 complex in , a TGFβ inhibitor for use or a combination therapy for use.
279. The method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use or use of embodiment 278, wherein the first binding region further comprises the amino acid sequence of SEQ ID NO: 134 or a portion thereof. Combination therapy for.
280. The method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use or use of embodiment 278, wherein the second binding region further comprises the amino acid sequence of SEQ ID NO: 143 or a portion thereof. Combination therapy for.
281. TGFβ inhibitors have three heavy chains comprising the amino acid sequences of SEQ ID NO: 1 (H-CDR1), SEQ ID NO: 2 (H-CDR2) and SEQ ID NO: 3 (H-CDR3) as defined by the IMTG numbering system. or A method, a medical use, a cancer therapeutic for use, a TGFβ inhibitor for use, or a combination therapy for use as in any one of the preceding embodiments, comprising an antigen-binding fragment thereof.
282. The TGFβ inhibitor comprises three heavy chain complementarity determining regions (H-CDR1, H-CDR2 and H-CDR3) from the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8. as described in any one of the preceding embodiments, comprising an isolated antibody or antigen-binding fragment thereof comprising three light chain complementarity determining regions (L-CDR1, L-CDR2 and L-CDR3) from a chain variable region A method for a medical use, a cancer therapeutic agent for use, a TGFβ inhibitor for use, or a combination therapy for use.
283. The TGFβ inhibitor comprises three heavy chain complementarity determining regions (H-CDR1, H-CDR2 and H-CDR3) from the heavy chain variable region that are at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8. an isolated antibody or antigen-binding fragment thereof comprising three light chain complementarity determining regions (L-CDR1, L-CDR2 and L-CDR3) from a light chain variable region that is at least 90% identical in amino acid sequence; A method, a medical use, a cancer therapeutic for use, a TGFβ inhibitor for use, or a combination therapy for use according to any of the embodiments.
284. The TGFβ inhibitor is an isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8. A method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use, or combination therapy for use according to any of the preceding embodiments, comprising:
285. Any of the preceding embodiments, wherein the TGFβ inhibitor comprises an isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. The method, medical use, use of a cancer therapeutic agent, use of a TGFβ inhibitor or use of a combination therapy as described in .
286. The method according to any of the preceding embodiments, wherein the TGFβ inhibitor comprises an isolated antibody or antigen-binding fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 11. , a medical use, a cancer therapeutic agent for use, a TGFβ inhibitor for use or a combination therapy for use.
287. The TGFβ inhibitor cross-blocks and/or competes for binding to TGFβ1 with an antibody or antigen-binding fragment comprising the heavy chain variable domain of SEQ ID NO: 7, the light chain variable domain of SEQ ID NO: 8, according to any of the preceding embodiments. The method, medical use, use of a cancer therapeutic agent, use of a TGFβ inhibitor, or use of a combination therapy according to any of the preceding claims.
288. The method, medical use, cancer therapeutic agent for use according to any one of the preceding embodiments, wherein the TGFβ inhibitor is a monoclonal antibody, optionally a fully human or humanized antibody or an antigen-binding fragment thereof. , a TGFβ inhibitor for use or a combination therapy for use.
289. The TGFβ inhibitor is present in a multispecific or bispecific construct, a method, a medical use, a cancer therapeutic agent for use, a TGFβ inhibitor for use according to any one of the preceding embodiments. Inhibitors or combination therapy for use.
290. The method of embodiment 289, wherein the multispecific or bispecific construct is also capable of binding an immune cell surface antigen, medical use, cancer therapeutic agent for use, TGFβ inhibition for use agents or combination therapy for use.
291. The method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use or use of embodiment 290, wherein the immune cell surface antigen is PD-1, PD-L1, CTLA4 or LAG3. Combination therapy for.
292. The method, medical use of embodiment 290 or 291, wherein the immune cell surface antigen is optionally PD-1 or PD-L1, including an anti-PD-1 or anti-PD-L1 antibody or antigen-binding fragment thereof. , a cancer therapeutic agent for use, a TGFβ inhibitor for use or a combination therapy for use.
293. TGFβ inhibitors are human IgG ₄ or IgG ₁ A method, medical use, cancer therapeutic for use, TGFβ inhibitor for use, or combination therapy for use as in any of the preceding embodiments, comprising a constant region.
294. The subjects are human patients with solid tumors previously demonstrated to be metastatic or locally advanced for which standard therapy does not exist, has failed in the patient, or is not well tolerated by the patient. The method, medical use, use of any of the preceding embodiments, wherein the patient is not a suitable candidate for or otherwise unsuitable for standard therapy. A cancer therapeutic agent for use, a TGFβ inhibitor for use or a combination therapy for use.
295. The subjects are human patients who have progressed after at least three cycles of treatment with an anti-PD-(L)1 antibody therapy approved for their tumor type (optionally alone or in combination with chemotherapy). with a history of primary anti-PD-(L)1 antibody nonresponse, manifesting either as a sexually transmitted disease or stable condition (e.g., clinically or radiographically not improving, but also not worsening). A method, a medical use, a cancer therapeutic for use, a TGFβ inhibitor for use, or a combination therapy for use according to any of the embodiments.
296. The method according to any of the preceding embodiments, wherein the subject is a human patient, and the patient has received recent administration of anti-PD-(L)1 antibody therapy within 6 months of administration of the TGFβ inhibitor. , a medical use, a cancer therapeutic agent for use, a TGFβ inhibitor for use or a combination therapy for use.
297. The subject is a human patient who has NSCLC and has a genomic tumor aberration for which targeted therapy (optionally, the targeted therapy targets anaplastic lymphoma kinase and/or EGFR) is available. , and further optionally, the patient has progressed on or is intolerant of approved therapies for these abnormalities, or is incapable of treatment with approved therapies for these abnormalities. A method, medical use, cancer therapeutic agent for use, TGFβ inhibitor for use or for use according to any of the preceding embodiments that is not considered a candidate or otherwise ineligible. combination therapy.
298. The method, medical use, of any of the preceding embodiments, wherein the subject is a human patient, and the patient has measurable disease as determined by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. A cancer therapeutic agent for use, a TGFβ inhibitor for use or a combination therapy for use.
299. The subject is a human patient, and the patient has a US East Coast Cancer Clinical Trial Group Performance Status (PS) of 0-1. A therapeutic agent, a TGFβ inhibitor for use or a combination therapy for use.
300. The subject is a human patient, the patient has a predicted life expectancy of ≧3 months, the method, medical use, cancer therapeutic agent for use, TGFβ of any of the preceding embodiments. Inhibitors or combination therapy for use.
301. The method of embodiment 1, wherein the reduced circulating MDSCs are M-MDSCs.
302. The method of embodiment 1 or embodiment 2, wherein the M-MDSC expresses one or more of CD11b+, HLADR-/low, CD14+, CD15-, CD33+/high and CD66b-.
303. TGFβ inhibitors may be administered at up to 100, 200 or 300 μg/kg once a week for 4, 8 or up to 12 weeks, as assessed by standard toxicity assays or in accordance with the present disclosure. A composition, composition for use, or method according to any one of the preceding embodiments, which is shown not to cause significant adverse events (e.g., dose-limiting toxicity) in clinical animal models.
304. The selection of compositions or TGFβ inhibitors includes in vivo efficacy and safety criteria, which include i) lack of platelet aggregation, activation and/or binding when evaluated under conditions according to the present disclosure; and ii) lack of significant (e.g., within 2.5-fold of control) cytokine release when evaluated under conditions according to the present disclosure. Compositions, TGFβ inhibitors for use or methods.
305. Any of the preceding embodiments, wherein the pharmacodynamics of the TGFβ inhibitor are evaluated by measuring circulating latent TGFβ1 levels in a blood (serum) sample taken from the subject before and after administration of the TGFβ1 inhibitor. A composition, TGFβ inhibitor or method for use as described in one.

List of Certain Other Embodiments 1. A TGFβ inhibitor for use in treating cancer in a subject, the treatment comprising determining the level of circulating TGFβ in a blood sample taken from the subject, and administering the TGFβ inhibitor to the subject; Circulating TGFβ levels are or have been determined by processing a blood sample below room temperature in a sample tube containing an anticoagulant, a TGFβ inhibitor.
2. A method of treating cancer in a subject, the method comprising:
(i) determining the level of circulating TGFβ in the subject prior to administering the TGFβ inhibitor;
(ii) administering to the subject a therapeutically effective dose of a TGFβ inhibitor;
(iii) determining the level of circulating TGFβ in the subject after administration;
A method in which circulating TGFβ levels are or have been determined by processing a blood sample from a subject at below room temperature in a sample tube containing an anticoagulant.
3. A method for determining treatment efficacy in a subject undergoing treatment for cancer, the method comprising:
(i) determining the level of circulating TGFβ in the subject prior to administering the TGFβ inhibitor;
(ii) administering to the subject a therapeutically effective dose of a TGFβ inhibitor; and (iii) determining the level of circulating TGFβ in the subject after administration;
An increase in circulating TGFβ levels after administration of an inhibitor compared to before administration indicates a therapeutic effect, with circulating TGFβ levels determined by processing a blood sample from a subject at below room temperature in a sample tube containing an anticoagulant. the method by which it is done or determined.
4. An increase of at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more in circulating TGFβ levels after administration of the inhibitor compared to pre-administration is considered therapeutic. The method of embodiment 3 exhibiting an effect.
5. 1. A method of determining target binding in a subject with cancer, the method comprising:
(i) determining the level of circulating TGFβ in the subject prior to administering the TGFβ inhibitor;
(ii) administering to the subject a therapeutically effective dose of a TGFβ inhibitor;
(iii) determining the level of circulating TGFβ in the subject after administration;
An increase in circulating TGFβ levels after inhibitor administration compared to pre-administration indicates target binding of the TGFβ inhibitor, and circulating TGFβ levels are increased by processing blood samples from subjects at below room temperature in sample tubes containing anticoagulant. method of determining or being determined by
6. An increase of at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more in circulating TGFβ levels after administration of an inhibitor compared to pre-administration indicates that TGFβ 5. The method of embodiment 4 demonstrating targeted binding of an inhibitor.
7. The level of circulating TGFβ after administration is at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, or more than the level of circulating TGFβ before administration. 7. The method according to any one of embodiments 2-6, wherein administration of the TGFβ inhibitor is continued if the increase exceeds .
8. A method of treating cancer in a subject, the method comprising administering a second dose of a TGFβ inhibitor to a subject having an elevated level of circulating TGFβ after receiving a first dose of a TGFβ inhibitor; A method, wherein the level of TGFβ is measured by processing a blood sample from a subject at below room temperature in a sample tube containing an anticoagulant.
9. A method of treating cancer in a subject, the method comprising:
(i) determining the level of circulating TGFβ in the subject prior to administering the TGFβ inhibitor;
(ii) administering to the subject a first dose of a TGFβ inhibitor;
(iii) determining the level of circulating TGFβ in the subject after the administration; and (iv) if the circulating TGFβ level is elevated, administering to the subject a second dose of the TGFβ inhibitor;
The method wherein determining the level of TGFβ comprises processing a blood sample from the subject at less than room temperature in a sample tube containing an anticoagulant.
10. the level of circulating TGFβ after the first dose of the TGFβ inhibitor is at least 1.5 times, at least 2 times, at least 2.5 times as compared to the level of circulating TGFβ before the first dose of the TGFβ inhibitor; , at least 3-fold, at least 4-fold, at least 5-fold, or more.
11. The cancer includes a solid tumor, and optionally the solid tumor includes melanoma (e.g., metastatic melanoma), renal cell carcinoma, triple-negative breast cancer, HER2-positive breast cancer, colorectal cancer (e.g., microsatellite-stable colon cancer). rectal cancer), lung cancer (e.g., metastatic non-small cell lung cancer, small cell lung cancer), esophageal cancer, pancreatic cancer, bladder cancer, kidney cancer (e.g., transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC) including cellular RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC), uterine cancer, prostate cancer, gastric cancer (e.g., gastric cancer), head and neck squamous cell carcinoma, urothelial carcinoma, hepatocellular carcinoma The TGFβ inhibitor or method according to any one of embodiments 1-10, selected from cancer or thyroid cancer.
12. The TGFβ inhibitor or method according to any one of embodiments 1-11, comprising administering checkpoint inhibitor therapy contemporaneously (e.g., simultaneously), separately or sequentially with the TGFβ inhibitor. , the checkpoint inhibitor is optionally an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG3 antibody, or an antigen-binding fragment thereof.
13. The cancer is characterized as containing less than 5% intratumoral CD8+ cells and more than 5% marginal CD8+ cells, as assessed by, for example, immunohistochemical analysis that can detect individual tumor foci within the tumor. 13. The TGFβ inhibitor or method of embodiment 12, wherein the TGFβ inhibitor or method has an immune exclusion phenotype.
14. The cancer may be associated with lower levels of CD8+ cells inside the tumor nest relative to levels of CD8+ cells outside the tumor nest, e.g. less than 5% of CD8+ cells inside the tumor nest and outside the tumor nest, e.g. at the edges. The TGFβ inhibitor or method according to embodiment 13, characterized in that the TGFβ inhibitor or method has a tumor area of more than 50%, comprising tumor foci, containing more than 5% of CD8+ cells.
15. Embodiments 1-14, wherein processing the blood sample comprises one or more centrifugation steps at a speed greater than 100 x g, and/or one or more centrifugation steps at a speed less than 15000 x g. A TGFβ inhibitor or method according to any one of .
16. Processing blood samples is
i) a first step of 5-25 minutes at 100-500×g and a second step of 10-40 minutes at 1000-5000×g; or ii) a second step of 5-25 minutes at 1000-5000×g. or ii) a first step of 5-25 minutes at 500-3000 x g and 2-10 minutes at 7500-15000 x g. A TGFβ inhibitor or method according to any one of embodiments 1-15, comprising a centrifugation protocol comprising a second step for 30 minutes.
17. Processing blood samples is
i) a first step of 10 minutes at 150 x g and a second step of 2500 x g for 20 minutes; or ii) a first step of 10 minutes at 2500 x g and a second step of 20 minutes at 2500 x g. or iii) a centrifugation protocol comprising a first step at 1500 x g for 10 minutes and a second step at 12000 x g for 5 minutes. The TGFβ inhibitor or method described in .
18. Any one of embodiments 1-17, wherein processing the blood sample comprises a centrifugation protocol comprising a first step at 2500 x g for 10 minutes and a second step at 2500 x g for 20 minutes. The TGFβ inhibitor or method described in .
19. A TGFβ inhibitor or method according to any one of embodiments 1-18, wherein the anticoagulant comprises 0.1-0.5M buffered trisodium citrate.
20. A TGFβ inhibitor or method according to any one of embodiments 1-19, wherein the anticoagulant comprises 10-20M theophylline.
21. A TGFβ inhibitor or method according to any one of embodiments 1-20, wherein the anticoagulant comprises 2-5M adenosine.
22. A TGFβ inhibitor or method according to any one of embodiments 1-21, wherein the anticoagulant comprises 0.1-0.25M dipyridamole.
23. The anticoagulant includes a citric acid-theophylline-adenosine-dipyridamole (CTAD) solution, optionally a buffered trisodium citrate solution of about 0.11M, about 15M theophylline, about 3.7M adenosine, and about A TGFβ inhibitor or method according to any one of embodiments 1-22, comprising 0.198M dipyridamole.
24. A TGFβ inhibitor or method according to any one of embodiments 1 to 23, wherein the collection tube is coated with a solution having a pH of 4.0 to 6.0, such as about 5.0.
25. A TGFβ inhibitor or method according to any one of embodiments 1-24, wherein the collection tube is glass or plastic.
26. A TGFβ inhibitor or method according to any one of embodiments 1-25, wherein the collection tube comprises a silicone coating.
27. A TGFβ inhibitor or method according to any one of embodiments 1-26, wherein the collection tube has a Hemogard™ closure.
28. A TGFβ inhibitor or method according to any one of embodiments 1-27, wherein the collection tube has a volume capacity of 2-3 mL, such as 2.7 mL.
29. A TGFβ inhibitor or method according to any one of embodiments 1-28, wherein the collection tube is sterile.
30. A TGFβ inhibitor or method according to any one of embodiments 1-29, wherein the collection tube is a BD Vacutainer™ CTAD blood collection tube.
31. A TGFβ inhibitor or method according to any one of embodiments 1-30, wherein the blood sample is processed at 2-8°C.
32. A TGFβ inhibitor or method according to any one of embodiments 1-31, wherein the blood sample is processed at 4°C.
33. Processing the blood sample includes determining a level of platelet factor 4 (PF4) in the same sample in which circulating TGFβ is determined, the sample optionally measuring circulating TGFβ levels in the sample. A TGFβ inhibitor or method according to any one of embodiments 1 to 32, wherein the TGFβ inhibitor or method according to any one of embodiments 1 to 32 is used for PF4 and is normalized based on the PF4 level determined from the same sample if the PF4 level is 500 ng/ml or less. .
34. A TGFβ inhibitor or method according to any one of embodiments 1-33, wherein the circulating TGFβ is circulating TGFβ1.
35. A TGFβ inhibitor or method according to any one of embodiments 1-34, wherein the circulating TGFβ is circulating latent TGFβ1.
36. Embodiments 1-35, wherein the TGFβ inhibitor is administered at a dose of 2000 mg or 3000 mg and at a frequency of once every two weeks, once every three weeks, or once every multiple of two weeks or three weeks. TGFβ inhibitor or method according to any one.
37. A method of treating cancer in humans, comprising administering a TGFβ inhibitor at a dose of 2000 mg or 3000 mg once every two weeks, once every three weeks, or once every multiple of two weeks or three weeks, e.g. once every 4 weeks, once every 6 weeks), wherein the subject is not receiving checkpoint inhibitor therapy.
38. A TGFβ inhibitor for use in the treatment of cancer in humans, the treatment comprising administering a TGFβ inhibitor at a dose of 2000 mg or 3000 mg once every two weeks, once every three weeks, or for two weeks or three weeks. a TGFβ inhibitor at any multiple of frequency (e.g., once every 4 weeks, once every 6 weeks), and the subject is not receiving checkpoint inhibitor therapy.
39. A method of treating cancer in a subject, the method comprising administering to the subject a TGFβ inhibitor and checkpoint inhibitor therapy, the TGFβ inhibitor at a dose of 2000 mg or 3000 mg and once every two weeks for three weeks. A method in which the method is administered once or once every two or three weeks.
40. A TGFβ inhibitor for use in treating cancer in a subject, the treatment comprising administering to the subject a TGFβ inhibitor and checkpoint inhibitor therapy, the TGFβ inhibitor at a dose of 2000 mg or 3000 mg, and A TGFβ inhibitor administered once every two weeks, once every three weeks, or once every multiple of two weeks or three weeks.
41. A TGFβ inhibitor for the method or use according to any one of embodiments 37-40, wherein the subject is a non-responder to checkpoint inhibitor therapy.
42. A TGFβ inhibitor for the method or use according to any one of embodiments 37-41, wherein the subject has not previously been administered checkpoint inhibitor therapy.
43. A TGFβ inhibitor for the method or use according to any one of embodiments 37-42, wherein the checkpoint inhibitor therapy is pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, budigalimab, or durvalumab.
44. 44. A TGFβ inhibitor for the method or use according to any one of embodiments 37-43, wherein the TGFβ inhibitor is administered once every two weeks.
45. 44. A TGFβ inhibitor for the method or use according to any one of embodiments 37-43, wherein the TGFβ inhibitor is administered once every three weeks.
46. 44. A TGFβ inhibitor for the method or use according to any one of embodiments 37-43, wherein the TGFβ inhibitor is administered at a frequency of once every four weeks.
47. 44. A TGFβ inhibitor for the method or use according to any one of embodiments 37-43, wherein the TGFβ inhibitor is administered once every six weeks.
48. A TGFβ inhibitor for the method or use according to any one of embodiments 37-47, wherein the TGFβ inhibitor is administered at a dose of 2000 mg.
49. 48. A TGFβ inhibitor for the method or use according to any one of embodiments 37-47, wherein the TGFβ inhibitor is administered at a dose of 3000 mg.
50. The TGFβ inhibitor is administered sequentially or in combination with a checkpoint inhibitor, and the checkpoint inhibitor is optionally an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti- A TGFβ inhibitor for the method or use according to any one of embodiments 37-49, which is a LAG3 antibody, or antigen-binding fragment thereof.
51. 51. A TGFβ inhibitor for the method or use of embodiment 50, wherein the TGFβ inhibitor is administered at the same frequency as checkpoint inhibitor therapy.
52. A method for determining treatment efficacy in a subject undergoing treatment for cancer, the method comprising:
(i) determining the level of P-Smad2 nuclear translocation in a tumor sample obtained from the subject prior to administering the TGFβ inhibitor;
(ii) administering to the subject one or more doses of a TGFβ inhibitor; and (iii) determining the level of P-Smad2 nuclear translocation in a tumor sample obtained from the subject after administration;
A method, wherein a decrease in P-Smad2 nuclear translocation after administration compared to before administration indicates a therapeutic effect.
53. A decrease of at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more in the level of P-Smad2 nuclear translocation after administration compared to before administration, 53. The method of embodiment 52, which exhibits therapeutic efficacy.
54. 1. A method of determining target binding in a subject with cancer, the method comprising:
(i) determining the level of P-Smad2 nuclear translocation in a tumor sample obtained from the subject prior to administering the TGFβ inhibitor;
(ii) administering to the subject one or more doses of a TGFβ inhibitor;
(iii) determining the level of P-Smad2 nuclear translocation in a tumor sample obtained from the subject after administration;
A method, wherein a decrease in P-Smad2 nuclear translocation after administration compared to pre-administration indicates target binding of the TGFβ inhibitor.
55. A decrease of at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more in the level of P-Smad2 nuclear translocation after administration compared to before administration, 55. The method of embodiment 54 showing target binding.
56. A method of treating cancer in a subject, the method comprising:
(i) determining the level of P-Smad2 nuclear translocation in a tumor sample obtained from the subject prior to administering the TGFβ inhibitor;
(ii) administering to the subject a first dose of a TGFβ inhibitor;
(iii) determining the level of P-Smad2 nuclear translocation in a tumor sample obtained from the subject after administration; and (iv) determining the level of p-Smad2 nuclear translocation following administration of the first dose. A method comprising administering to the subject one or more additional doses of a TGFβ inhibitor if p-Smad2 nuclear translocation is reduced compared to pre-administration.
57. The method of embodiment 56, wherein P-Smad2 nuclear translocation is reduced by at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more. .
58. 58. A TGFβ inhibitor for the method or use of any one of embodiments 54-57, wherein the determination of nuclear translocation of p-Smad2 comprises immunohistochemistry.
59. 59. A TGFβ inhibitor for the method or use of any one of embodiments 54-58, wherein determining nuclear translocation of p-Smad2 comprises nuclear masking.
60. A TGFβ inhibitor or method according to any one of embodiments 1-59, wherein the TGFβ inhibitor inhibits TGFβ1 signaling.
61. 61. A TGFβ inhibitor or method according to any one of embodiments 1-60, wherein the TGFβ inhibitor selectively binds pro/latent TGFβ.
62. 61. The TGFβ inhibitor or method of embodiment 60, wherein the TGFβ inhibitor selectively binds pro/latent TGFβ1.
63. 63. A TGFβ inhibitor or method according to embodiment 61 or embodiment 62, wherein the TGFβ inhibitor does not bind mature TGFβ1.
64. as in any one of embodiments 1-63, wherein the TGFβ1 selective inhibitor is a neutralizing antibody that binds mature TGFβ1 or an activating inhibitor that binds proTGFβ1. TGFβ inhibitor or method as described.
65. TGFβ1 selective inhibitors are
a) a monoclonal antibody designated herein as Ab6, a TGFβ1 selective variant thereof or an antigen-binding fragment thereof, or an antibody comprising CDRs and/or variable domains from Ab6; or b) competes for binding with Ab6, and 65. The TGFβ inhibitor or method according to embodiment 64, wherein the TGFβ inhibitor or method is an antibody or antigen-binding fragment thereof that binds to the same epitope as Ab6.
66. TGFβ inhibitors have three heavy chains comprising the amino acid sequences of SEQ ID NO: 1 (H-CDR1), SEQ ID NO: 2 (H-CDR2) and SEQ ID NO: 3 (H-CDR3) as defined by the IMTG numbering system. or optionally, the TGFβ inhibitor comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence 90% identical thereto. 66. A TGFβ inhibitor or method according to any one of embodiments 1-65, comprising an isolated antibody or antigen-binding fragment thereof, comprising:
67. 67. The TGFβ inhibitor or method according to any one of embodiments 1-66, wherein the TGFβ inhibitor is a TGFβ1/2 inhibitor, and optionally the inhibitor is NIS793/XOMA-089 or GC1008.
68. The TGFβ inhibitor according to any one of embodiments 1 to 66, wherein the TGFβ inhibitor is a TGFβ1/3 inhibitor, and optionally, the inhibitor is M7824 (bintrafsp alpha) or AVID200; or Method.
69. 69. The TGFβ inhibitor or method according to any one of embodiments 1-68, wherein the TGFβ inhibitor is a low molecular weight antagonist of TGFβ receptor kinase, and optionally the antagonist is an ALK5 inhibitor.
70. A method of treating cancer in a human subject having a solid tumor, the method comprising administering to the subject an amount of a checkpoint inhibitor effective to reduce tumor growth, wherein the solid tumor is isolated from individual tumors within the tumor. A method of having an inflammatory phenotype characterized by containing more than 5% intratumoral CD8+ cells, as assessed by immunohistochemical analysis capable of detecting foci.
71. 71. The method of embodiment 70, wherein the tumor has a tumor area of greater than 50%, including tumor foci containing greater than 5% CD8+ cells within the tumor foci.
72. A TGFβ inhibitor for use in treating cancer in a subject, the treatment comprising administration of a TGFβ inhibitor, cancer therapy, or a combination thereof, optionally the cancer therapy comprising checkpoint inhibitor therapy or genotoxic therapy, the treatment comprising determining the level of MDSC in a blood sample obtained from the subject, optionally the MDSC level being measured by the use of an antibody that binds to LRRC33, and further optionally, Antibodies that bind to LRRC33 are TGFβ inhibitors used in FACS-based assays or ELISA-based assays.
73. Embodiments wherein the treatment further comprises determination of CD8+ cells in the tumor biopsy sample obtained from the subject, optionally using an image analysis method that allows resolution of individual tumor foci in accordance with the disclosure herein. 72. A TGFβ inhibitor for use according to 72.
74. The treatment further comprises determining circulating TGFβ levels in a blood sample obtained from the subject, the blood sample optionally collected and/or processed at below room temperature in accordance with the disclosure herein, and optionally blood 74. A TGFβ inhibitor for use according to embodiment 72 or 73, wherein the sample is processed to prepare a platelet poor plasma (PPP) sample.
75. The TGFβ inhibitor is an isoform-selective inhibitor of TGFβ1, and optionally the isoform-selective inhibitor is a neutralizing antibody that selectively binds to TGFβ1 or an antibody that binds to latent TGFβ1 complexes. or the TGFβ inhibitor is an inhibitor of TGFβ1 and TGFβ2, and optionally the inhibitor is NIS793/XOMA-089 or GC1008. TGFβ inhibitor for.
76. For use according to any one of embodiments 48-50, the TGFβ inhibitor is a TGFβ1 and TGFβ3 inhibitor, and optionally the inhibitor is M7824 (vintrafusp alpha) or AVID200. TGFβ inhibitor.
77. A TGFβ inhibitor is a pan-inhibitor that inhibits TGFβ1, TGFβ2, and TGFβ3, and optionally the pan-inhibitor is GC1008 or a derivative thereof, SAR439459, LY3022859, or an agent that blocks the ligand binding domain of the TGFβ receptor. A TGFβ inhibitor or method for use according to any one of embodiments 1-66 and 69-74.
78. A TGFβ inhibitor is an activation inhibitor of the TGFβ pathway that interferes with the integrin-TGFβ interaction, optionally the inhibitor is an agent that binds to an integrin or an RGD motif present in latent TGFβ1 and/or TGFβ3. and further optionally, the agent is a low molecular weight (small molecule) compound, or an antibody or antigen-binding fragment thereof. TGFβ inhibitor or method for use.
79. A TGFβ inhibitor or method for use according to any one of embodiments 1-66 and 69-74, wherein the TGFβ inhibitor is a low molecular weight (small molecule) antagonist of ALK5.
80. A TGFβ inhibitor or method for use according to any one of embodiments 1-66 and 69-74, wherein the TGFβ inhibitor is an RNA-based inhibitor of TGFβ1 expression.
81. A TGFβ inhibitor is a soluble ligand trap comprising a ligand-binding fragment of a TGFβ receptor that is capable of sequestering a mature growth factor, the mature growth factor being TGFβ1, TGFβ2, TGFβ3, or a combination thereof, optionally A TGFβ inhibitor or method for use according to any one of embodiments 1-66 and 69-74, wherein the ligand trap is M7824 (bintrafsp alpha) or AVID200.
82. according to any one of embodiments 70-81, wherein the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG3 antibody, or an antigen-binding fragment thereof. TGFβ inhibitor or method for use.
83. 73. A TGFβ inhibitor for use according to embodiment 72, wherein the cancer therapy is genotoxic therapy.
84. TGFβ inhibition for the method or use according to any one of embodiments 1-35, 52-69, wherein the TGFβ inhibitor is administered in conjunction (e.g., simultaneously or sequentially) with genotoxic therapy. agent.
85. A method of treating solid cancer, the method comprising:
i) selecting a subject having a cancer with elevated TGFβ levels and/or TGFβ activity; and ii) administering to the subject a therapeutically effective dose of a TGFβ inhibitor;
A method, wherein the TGFβ inhibitor is administered in combination (eg, simultaneously or sequentially) with genotoxic therapy.
86. A TGFβ inhibitor for use in the treatment of solid tumors, the agent comprising:
i) selecting a subject having a cancer with elevated TGFβ levels and/or signaling; and ii) administering to the subject a therapeutically effective dose of a TGFβ inhibitor;
The TGFβ inhibitor is administered in conjunction (eg, simultaneously or sequentially) with genotoxic therapy.
87. 87. A TGFβ inhibitor for the method or use of embodiment 85 or embodiment 86, wherein the subject has intrinsic or acquired resistance to genotoxic therapy.
88. 87. A TGFβ inhibitor for the method or use of embodiment 85 or embodiment 86, wherein the TGFβ inhibitor provides a synergistic effect with genotoxic therapy.
89. 89. A TGFβ inhibitor for the method or use according to any one of embodiments 83-88, wherein the genotoxic therapy is chemotherapy or radiotherapy.
90. Subjects include ovarian cancer, breast cancer, bladder cancer, pancreatic cancer, such as pancreatic adenocarcinoma, prostate cancer, such as prostate adenocarcinoma, melanoma, such as cutaneous melanoma, lung cancer, such as lung squamous cell carcinoma and lung adenocarcinoma. , liver cancer, e.g., hepatocellular carcinoma, uterine cancer, e.g., uterine endometrial cancer, renal cancer, e.g., kidney clear cell carcinoma, head and neck cancer, e.g., head and neck squamous cell carcinoma, colon cancer, e.g. A TGFβ inhibitor for the method or use according to any one of embodiments 83-89, with adenocarcinoma, esophageal cancer, or tenosynovial giant cell tumor (TGCT).
91. TGFβ inhibitors have three heavy chains comprising the amino acid sequences of SEQ ID NO: 1 (H-CDR1), SEQ ID NO: 2 (H-CDR2) and SEQ ID NO: 3 (H-CDR3) as defined by the IMTG numbering system. or optionally, the TGFβ inhibitor comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence 90% identical thereto. 91. A TGFβ inhibitor for the method or use of any one of embodiments 83-90, comprising an isolated antibody or antigen-binding fragment thereof.
92. A method of identifying an MDSC population from a biological sample obtained from a patient, wherein the gMDSC population is identified by cell surface markers of CD11b+, HLA-DR-, CD14-, CD15+, CD33+/low, and CD66b+; A method wherein the mMDSC population is identified by cell surface markers of CD11b+, HLA-DR-/low, CD14+, CD15-, CD33+/high, and CD66b-.
93. 93. The method of embodiment 92, wherein the identification includes binary intensity selection.
94. 94. The method of embodiment 93, wherein the binary intensity selection comprises binning the signal intensity according to one or more of the cutoff points shown in Table 38B.
95. 95. The method as in any one of embodiments 92-94, wherein the identification comprises sequential application of signal filtering.
96. Identification included signal binning according to HLD-DR signal intensity, with signal intensities <0.105 binned as negative signals, signal intensities between 0.105 and 0.125 binned as low signals, and 0.125 96. The method of any one of embodiments 92-95, wherein signal intensities of ˜0.155 are binned as medium signals and signal intensities greater than 0.155 are binned as high signals.
97. Identification included signal binning according to CD33 signal intensity, with signal intensities <0.17 binned as negative signals, signal intensities 0.17-0.18 binned as low signals, and 0.18-0. 97. The method according to any one of embodiments 92-96, wherein a signal strength of .19 is binned as a medium signal and a signal strength of greater than .19 is binned as a high signal.

Other suitable modifications and variations of the compositions and methods described herein may be made using suitable equivalents without departing from the scope of this disclosure or the embodiments disclosed herein. It will be readily apparent to those skilled in the art what can be obtained. The present disclosure is further illustrated by the following examples, which should not be construed as limiting.

We previously observed that tumor-associated MDSCs express cell surface LRRC33 in tumor-bearing mice. Here, we investigated whether circulating MDSCs could also express cell surface LRRC33. Previously, we and other researchers discovered that the presence of LRRC33 mRNA transcripts does not necessarily correlate with protein expression of LRRC33, particularly cell surface LRRC33. Therefore, FACS experiments were performed to assess whether circulating MDSCs of tumor-bearing mice express cell surface LRRC33. A monoclonal antibody that binds to cell surface LRRC33 and an IgG control were used to isolate/enrich MDSCs, including G-MDSC and M-MDSC pools, from blood samples taken from MBT-2 mice. The data show substantially uniform and robust expression of LRRC33 in circulating MDSCs. In fact, nearly all MDSCs, including G-MDSCs and M-MDSCs in circulation, appear to express high levels of cell surface LRRC33. Taken together, these data demonstrate that LRRC33 is expressed not only within tumors but also on the cell surface by circulating MDSCs (i.e., tumor-associated MDSCs), that circulating monocytes do not express LRRC33, and that LRRC33 is expressed on the cell surface by circulating MDSCs (i.e., tumor-associated MDSCs); has been identified as a novel blood-based antigen. This raises the possibility that LRRC33 functions as an alternative blood-based biomarker for immunosuppression, particularly in cancer.

Example 1: Detection of Circulating MDSCs The effect of Ab6 treatment on circulating immune cell subsets in vivo was determined using the MBT-2 mouse model. Tumor-bearing mice were administered 10 mg/kg of Ab6 alone or in combination with anti-PD-1 antibody administered on days 1, 4, and 8 at 10 mg/kg.

Whole blood was collected on day 10 and processed for flow cytometry analysis. Levels ^of circulating G-MDSCs and M-MDSCs were determined based on the expression of surface protein markers for G-MDSCs (CD45+CD11b+Ly6G+Ly6Clow) and M-MDSCs (CD45+CD11b+Ly6G- ^Ly6Chigh ). Values were expressed as a percentage of total CD45+ cells detected in the blood. Circulating G-MDSC levels were reduced in the groups treated with both Ab6 alone and combination therapy (i.e. Ab6 in combination with anti-PD-1), whereas circulating M-MDSC levels in the Ab6-treated group were lower than IgG control or were not different compared to the group treated with anti-PD-1 therapy alone. Tumor and circulating MDSC levels assessed 10 days after initiation of treatment are shown in Figures 1 and 2.

Flow cytometric analysis of T cells was also performed on whole blood at 10 days after the start of treatment. Circulating T cell levels were determined based on the expression of T cell surface protein markers. CD8+ and CD3+ T cell levels and values were normalized to total circulating CD45+ cells detected in whole blood. The group treated with Ab6 alone showed a slight increase in both CD8+ and CD3+ circulating T-cell levels compared to IgG controls. Ab6 and anti-PD-1 combination treatment did not result in significant changes in either CD8+ or CD3+ circulating T cell levels.

A second in vivo study was performed to further evaluate the effect of Ab6 treatment on circulating and intratumoral MDSC populations. MBT-2 mice were treated with IgG control, Ab6 alone (10 mg/kg), anti-PD-1 antibody (10 mg/kg), or Ab6 (1 mg/kg, 3 mg/kg or 10 mg/kg) plus anti-PD-1 antibody (10 mg/kg). /kg). Treatments were administered on days 1 and 8. Whole blood was collected 17 days before administering the first dose of treatment and on days 3, 6, and 10. Tumor volume was monitored throughout the study and intratumoral MDSC analysis was performed on day 10.

Measurements of tumor volume on days 1, 4, 7, and 10 were statistically significant in all animals treated with anti-PD-1 antibody alone and with the combination of anti-PD-1 antibody and Ab6. but not in animals treated with Ab6 alone before day 10 (Figure 3). The lack of therapeutic response observed in animals treated with Ab6 alone before day 10 could be due to inappropriate dosing, as pharmacokinetic results confirmed that all animals received the appropriate Ab6 dose. This was unlikely to be the result. These results demonstrate that for MBT-2 tumors, simultaneous inhibition of the PD-1 and TGFβ1 pathways may reduce tumor growth (e.g., delay or regression) to a greater extent than inhibition of the PD-1 or TGFβ1 pathways alone. show.

Levels of circulating immune cell populations were determined from whole blood samples by FACS analysis. Total CD11b+ bone marrow cells were identified from whole blood, the M-MDSC population derived therefrom was then identified by expression of the cell surface marker Ly6C (Ly6C ^high ), and the G-MDSC population was identified by the expression of the cell surface marker Ly6G (Ly6G+ ) was identified by the expression of Baseline circulating MDSC levels were determined from whole blood samples taken from non-tumor-bearing mice and consisting of 17.8% bone marrow cells, 30% G-MDSCs and 29.1% M-MDSCs. (percentage of total bone marrow population) (Figure 4). Levels of circulating immune cells were assessed on day 10 from tumor-bearing mice. Compared to baseline levels of non-tumor-bearing mice, tumor-bearing mice showed significantly increased levels of total bone marrow population and circulating G-MDSC, but not M-MDSC cells (Figure 6). Blood samples from tumor-bearing mice were found to consist of 64.9% bone marrow cells, which contained 70% G-MDSCs and 6.95 M-MDSCs. Furthermore, G-MDSCs also constitute 45.4% of the total CD45+ immune cell population in the blood of tumor-bearing mice compared to 5.45% of the total CD45+ cells in the blood of non-tumor-bearing mice. Got it (Figure 5).

Circulating MDSC populations were assessed in tumor-bearing animals throughout treatment. As shown in Figure 6, the level of M-MDSC remained low throughout the treatment, whereas the level of G-MDSC showed a decreasing trend, with a statistically significant decrease in G-MDSC level. , detected in all groups by day 10. No reduction in circulating G-MDSC levels in animals treated with Ab6 alone was observed by day 10 (FIGS. 7A and 7B). This suggests that Ab6 treatment alone may be sufficient to reduce circulating MDSC levels, albeit at a slower rate compared to the combination of Ab6 and anti-PD-1 treatment.

The association of circulating G-MDSC levels with tumor volume was also evaluated on day 10. Figure 8 shows a linear correlation between circulating G-MDSC levels and tumor volume in all groups.

Circulating and intratumoral MDSC levels were compared to tumor volume measurements on day 10. Intratumoral M-MDSC levels in treated animals were similar across all treatment groups and were not reduced compared to control animals. In contrast, intratumoral G-MDSC levels in animals treated with anti-PD-1 antibody alone or in combination with anti-PD-1 antibody and Ab6 were reduced compared to control animals (FIG. 9A). While Ab6 treatment alone resulted in a decrease in circulating G-MDSC levels, intratumoral MDSC levels were not affected by Ab6 treatment alone (Figure 9B). The correlation of relative MDSC levels in tumor and circulation is shown in FIG. 10. Furthermore, reduced intratumoral G-MDSC levels at day 10 were found to correlate with increased tumor CD8+ cells across all treatment groups (Figure 11), suggesting reduced overall tumor immunosuppression. There is.

Example 2: In Vivo Assessment of LRRC33 Expression on Circulating MDSCs in MBT2 Syngeneic Bladder Cancer Mouse Model LRRC33 expression levels on circulating MDSCs were determined using FACS analysis using whole blood samples collected from MBT2 tumor-bearing mice. Decided. M-MDSC and G-MDSC populations were identified based on Ly6C and Ly6G expression, respectively, and then further characterized using a monoclonal antibody that binds to cell surface LRRC33 and an IgG control. Almost all circulating MDSCs, including both G-MDSCs and M-MDSCs, appeared to express LRRC33. As shown in Figure 12A, LRRC33 expression was detected in 89.1% of G-MDSCs and 100% of M-MDSCs from whole blood of tumor-bearing mice. Consistent with previous observations, LRRC33 was also detected on intratumoral MDSCs, including G-MDSCs and M-MDSCs (FIG. 12B), although at different levels. In contrast, no LRRC33 expression was detected on monocytes.

Example 3: Ab6 treatment modulates circulating TGFβ1 levels Circulating TGFβ1 levels were assessed before and after treatment with Ab6 and/or anti-PD-1 antibodies in the MBT-2 mouse model. Tumor-bearing mice received 1 mg/kg, 3 mg/kg, or 10 mg/kg of Ab6 alone, 10 mg/kg of PD-1 antibody alone, or 1 mg/kg of Ab6 in combination with 10 mg/kg of anti-PD1 antibody. It was administered on the 8th day. Control animals received an IgG control.

Blood samples were taken 14 days before tumor implantation (day −14), 1 day before treatment, and 3, 6, and 10 days after treatment. Samples were processed, including an acid treatment step, and circulating TGFβ1 levels (pg/mL) were determined using an enzyme-linked immunosorbent assay (ELISA, eg, R&D Systems Quantikine® assay). The acid treatment step liberates TGFβ1 from its latent complex and, without being bound by theory, it is believed that the majority of circulating TGFβ1 resides in the latent complex. Blood samples were also analyzed for platelet activation as a quality control measure. Samples with significant platelet activation were not further analyzed for circulating TGFβ levels as they were likely to contain TGFβ released from activated platelets and therefore likely to result in inaccurate TGFβ readings. Ta. Platelet factor 4 (PF4) concentration was used as a surrogate marker to identify samples containing significant platelet activation. Samples with high PF4 levels (>500 ng/ml) were identified as having significant platelet activation and were therefore excluded from further analysis of TGFβ concentrations. Pharmacodynamic readouts such as assessment of tumor size, target binding, and immune infiltration are performed using tumor samples.

Results showed a trend towards increased circulating TGFβ1 levels in animals treated with Ab6 (alone or in combination with anti-PD-1 antibody) compared to circulating TGFβ1 levels in pre-transplant and IgG control treated animals . In contrast, animals treated with anti-PD1 antibody alone did not show increased circulating TGFβ1 levels compared to controls (Figure 13A). A statistically significant correlation was found between circulating TGFβ1 levels and plasma concentrations of Ab6 for each treatment group (R ² =0.714) (FIGS. 13B and 13C).

To avoid measuring additional circulating TGFβ1 released as sample collection and processing artifacts, plasma platelet factor 4 (PF4) levels were determined in each sample by ELISA and the resulting PF4 levels were used to determine circulating TGFβ1. Emissions were normalized. PF4 levels can be used as an indicator of platelet activation induced during sample collection, which may contribute to TGFβ1 release. PF4 levels (ng/mL) were found to be low across drug-treated and IgG control samples. In comparison, pre-transplant samples showed higher PF4 levels (Figure 14, left panel). Sample outliers were identified using interquartile range and had an upper PF4 level of 60.45 ng/mL and a lower limit of 42.41 ng/mL (Figure 14, right panel). Results corrected for PF4 outliers showed a statistically significant, dose-dependent increase in circulating TGFβ1 levels after Ab6 treatment (alone or in combination with anti-PD-1 antibodies). Additionally, outlier-corrected results also showed elevated circulating TGFβ1 levels in tumor-bearing animals compared to non-tumor-bearing controls (pre-transplant) (Figure 15). As shown in Figure 15, outlier-corrected total circulating TGFβ1 levels were approximately 2000 pg/mL before transplantation and approximately 3000 pg/mL in mice treated with IgG control and treated with anti-PD-1 alone. approximately 2500 pg/mL in mice treated with 10 mg/kg Ab6 alone, and approximately 9000 pg/mL in mice treated with 10 mg/kg Ab6 alone, and with combination therapy containing 1 mg/kg, 3 mg/kg, and 10 mg/kg Ab6, respectively. 6000 pg/mL, 7200 pg/mL and >9000 pg/mL in treated mice.

Without wishing to be bound by theory, PF4 levels may be useful in identifying and removing samples contaminated by platelet activation during sample collection and processing. In some embodiments, a sample with a PF4 concentration greater than 500 ng/mg may indicate that the sample contains platelet activation. In some embodiments, samples with PF4 concentrations greater than 500 ng/mg may be treated as outlier samples.

A similar trend of dose-dependent increases in circulating TGFβ1 levels was also observed in non-human primates and rats treated with a single dose of Ab6. In non-human primates treated with 1 mg/kg, 3 mg/kg, 10 mg/kg, or 30 mg/kg Ab6, the magnitude and duration of the increase in circulating TGFβ1 levels was dose-dependent. Circulating TGFβ1 levels were measured approximately 72-240 days after Ab6 administration, with peak circulating TGFβ1 levels ranging from approximately 2000 pg/ml to approximately 4000 pg/ml (Figure 16). Similarly, rats treated with 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, or 30 mg/kg Ab6 also showed a dose-dependent increase in circulating TGFβ1 levels. Circulating TGFβ1 levels were detected approximately 24-360 hours after Ab6 administration, with peak circulating TGFβ1 levels detected ranging from about 5000 pg/ml to about 7500 pg/ml. The duration of circulating TGFβ1 elevation for each treatment group appeared to be dose-dependent (Figure 17).

Example 4: Sample collection and processing methods for assessing circulating latent TGFβ1 in human blood samples Pilot study identifies improved collection and processing conditions for assessing circulating latent TGFβ1 in human plasma samples It was carried out to Whole blood samples were collected from six healthy donors and processed under various conditions, including variations in temperature, collection tube type, pre-processing time, and centrifugation conditions, to generate platelet-poor plasma (PPP). (Figure 18, Table 15). PPP treatment was evaluated using tubes coated with the anticoagulants citrate-theophylline-adenosine-dipyridamole (CTAD) or sodium citrate (TSC). Samples were processed after 2 or 4 hours of incubation after sample collection. Three centrifugation protocols: first step at 150 x g for 10 min, followed by a second step (P1) at 2500 x g for 20 min, first step at 2500 x g for 10 min, then at 2500 x g. Samples were centrifuged according to either a second step (P2) for 20 minutes at 1500×g, and a first step for 10 minutes at 1500×g followed by a second step (P3) for 5 minutes at 12000×g. The acceleration and deceleration of the centrifugation process were set at 20% of the centrifuge maximum. Following the first centrifugation step, the supernatant portion of each tube is transferred to another tube, approximately 70% of the volume is removed without disturbing the buffy coat and red blood cell layer, and the supernatant is transferred to a second centrifugation step. further processing. The resulting supernatant was used to measure circulating TGFβ1 levels. Sample incubation and processing was performed at 4°C or room temperature (RT).

Circulating TGFβ1 was assessed by ELISA after acidification of PPP using the R&D System Quantikine assay. The average concentration of TGFβ1 ranged from 500 pg/ml to 1500 pg/ml (Figure 19A). Samples treated at 4°C were shown to have significantly lower TGFβ1 levels compared to samples treated at room temperature. As a control for platelet-induced TGFβ1 activation, platelet factor 4 (PF4) levels were assessed. Significantly higher levels of TGFβ1 and PF4 were detected in samples treated at room temperature compared to samples treated at 4°C (Figure 19B). The lowest levels of PF4 were observed in samples collected in CTAD tubes and processed under centrifugation conditions P2 (Figure 19B). A correlation between PF4 and TGFβ1 was observed in samples treated at room temperature (Figure 20). An exemplary analysis of outlier removal based on high PF4 levels is shown in FIGS. 21A-21C. The regression analysis used in FIG. 21B was performed by Motulsky and Brown. BMC Bioinformatics. 2006 Mar 9;7:123.

Example 5: In Vivo Evaluation of Circulating Latent TGFβ1 in Human Plasma Samples Circulating latent TGFβ1 levels were evaluated in human platelet-poor plasma samples both before and 1 hour after Ab6 administration. As a quality control of platelet activation in each sample, PF4 levels were analyzed either at baseline or 1 hour post-dose (Figure 22). A general method for determining circulating latent TGFβ levels is shown in Example 3 above. Circulating TGFβ levels were measured at additional time points after Ab6 administration, and the results are shown in Figures 23A-C. Tables 16A-C below show exemplary baseline TGFβ and PF4 concentrations from plasma samples collected from patients treated with Ab6.

Example 6: Measurement of latent TGFβ activation The inhibitory activity of Ab6 was determined as described by Martin et al. , 2020. Briefly, LN229 cells (ATCC) were transfected with plasmids encoding either human, rat, or cynomolgus proTGFβ1. Approximately 24 hours after cell transfection, Ab6 was added to the transformants along with CAGA12 reporter cells (Promega, Madison, Wis.). Approximately 16-20 hours after incubation of the co-culture, the assay was developed and the luminescence was read in a plate reader. Dose-response activities were non-linear fits to a 3-parameter log inhibitor-pair-response model using Prism 8 and best-fit IC50 values were calculated.

Modulation of TGFβ signaling in Ab6-treated tumors was detected using a P-Smad2 IHC assay, where decreased expression in P-Smad2 indicates TGFβ inhibition. As shown in Figure 24, a P-Smad2 IHC assay was developed using commercially available melanoma samples and digital nuclear masking analysis. Nuclear mask is a digital image analysis parameter that allows visualization and measurement of IHC signal intensity of individually marked cells or nuclei. This assay was established using commercially available melanoma samples and allows discrimination of P-Smad2 nuclear staining intensities ranging from high (red), medium (orange), low (yellow) to negative (blue). did. Cells were sorted into categories (eg, 0, 1+, 2+, 3+) based on color intensity versus scan parameters. The variables analyzed during assay development are shown in Table 17 below. Analysis was performed using software developed by Flagship Biosciences.

pSmad2 phosphorylation was measured in MBT2 mice, further supporting the rationale for measuring tumor phosphorylated Smad2 (p-Smad2) as a biomarker of SRK-181-mediated TGFβ1 inhibition. As shown in Figure 25, p- Smad2 levels were analyzed. Ab6-mIgG1 treatment reduced tumor p-Smad2 phosphorylation, thereby supporting p-Smad2 as a pharmacodynamic biomarker.

Briefly, samples from MBT-2 mice were fixed, processed, embedded, and sectioned at 4 um thickness. Slides were deparaffinized and stained using a Leica Bond Rx autostainer. Sections were blocked using 1× diluted Animal Free Blocking solution from Cell Signaling Technologies (#15019). Staining was performed using phospho-Smad2 rabbit monoclonal antibody from Cell Signaling Technologies (#3108, 138D4) at a final concentration of 0.174 ug/mL. Heat-induced epitope repair was performed at 95°C for 20 min using a citric acid-based solution. Polymer-Rabbit HRP purchased from CellSignaling Technologies (#8114S) was incubated as post-primary for 25 minutes. Secondary fluorophores, spectral DAPI, and tyramide signal amplification reagents were purchased from Akoya Biosciences (NEL741001KT, FP1490). Sections were dried, coverslipped with antifade mounting medium (ThermoFisher, P36961), and imaged on a Zeiss Axio Scan z1 with an additional Cy5 band to further separate autofluorescent signals.

Image quantification was analyzed using the Visiopharm analysis platform. Nuclei were first segmented based on DAPI staining intensity and the resulting subjects were reclassified as pSmad2 strong, moderate, or weak positive. These classifications are thresholded by FITC intensity with a background threshold of 15000 ABU and require at least 30% nuclear coverage. Segmented objects with an area less than 23 um2 or more than 230 um2 were discarded before pSmad2 classification was applied. Strong, moderate, and weak classifications were used for QC purposes, and the total % pSmad2 positivity is reported as the sum of these groups divided by the total number of cells multiplied by 100.

Example 7: Analysis of CD8-positive cells in Ab6-treated tumors Investigation of CD8 T-cell status in biopsy tissue typically predicts each as one of three main phenotypes: immunodepleted, immunoexcluded or inflammatory. Represents an organization. The immunodepleted phenotype does not express appreciable levels of CD8 across tissues. Immune-excluded tissue shows CD8 expression, but expression is primarily localized to the stroma or the stromal margin surrounding tumor foci. Inflamed tissue exhibits significant levels of CD8 expression or CD8-positive cells within tumor foci of tissue. While this phenotypic classification is informative, these percentages of expression are often calculated as an average of expression across tissues and do not take into account the heterogeneous nature of tumor biology. This may cause a tumor containing one highly inflammatory tumor foci to be averaged together with multiple depleted tumor foci to classify the tissue as being removed or depleted even though inflammation is present.

To better represent the inflammatory heterogeneity that exists within tumor tissue, an algorithm based on image analysis was developed that not only separates tumor, stroma, and tumor/stromal margin, but also Each tumor focus was identified as its own separate object. This analysis allowed enumeration of the number and size of all tumor foci within the tissue and further quantified the percentage of CD8 expression within and outside of each tumor foci. Each tumor foci was given its own phenotypic classification of inflammatory, cleared, or depleted, and the percentage of tumor foci exhibiting each phenotype was calculated to represent the heterogeneous inflammation within the tumor.

Tumor samples from 36 subjects diagnosed with bladder cancer or melanoma were obtained using core needle biopsy. Three to four core biopsy samples were taken from each tumor using a 16-18 gauge needle. Samples were fixed in formalin containers at room temperature for 24-48 hours. Once fixation was complete, the biopsy was transferred to a Histocassette between sponges pre-soaked with PBS. The Histocassettes were then submerged in cold PBS and stored for up to 3-4 days before analysis.

The percentage of CD8+ cells was determined by immunohistochemical analysis in 23 whole tissue tumor resection samples of bladder cancer and 13 samples of melanoma. Data from whole tissue and tumor, stroma, and margin (25 μm in each direction from the tumor/stromal interface) sections of the whole tissue were evaluated. The margin and marginal zone between the tumor nest and the surrounding stroma were identified by pathologist evaluation and digital analysis of the stained samples using automated machine learning software developed by Flaship Biosciences (Westminster, Colorado). It was done. The percentage of CD8+ cells was also assessed for individual tumor foci across samples containing tumor foci that may have mixed immunophenotypes. For samples with ill-defined or poorly defined analyzable tumors, only whole tissue was analyzed. Results from compartment analysis demonstrated variation in the percentage of CD8+ cells between different compartments in bladder cancer (Figure 26A) and melanoma (Figure 26B) samples. Cell number, compartment area, CD8+ cell density (average number of CD8+ cells/mm ² ) and CD8+ cell clustering were determined. Results from tumor nest analysis are shown in Figures 27-29E. Cell counts based on Figure 29C are provided below.

To determine the immunophenotype, the percentage of CD8+ cells in the tumor compartment was compared with that in the stromal and marginal compartments. The ratio of CD8+ cells in the tumor compartment to those in the stromal or marginal compartment varied across immunophenotypes. As an example, Figure 30A shows that bladder cancer samples #26, #30, and #9 showed different percentages of CD8+ cells across compartments, indicating that these tumors have different immunophenotypes. It was shown that there is a high possibility that Bladder sample #26 (Figure 30A, left) has low CD8+ staining across all three compartments (0.8% CD8+ staining in the tumor, 1.9% in the stroma, and 1.3% in the margin). exhibited an immunodepleted phenotype as demonstrated by CD8+ staining. Bladder sample #9 (FIG. 30A, right), which showed an immunoinflammatory phenotype, showed CD8+ staining in all three compartments (11% CD8+ staining in the tumor, 8.7% in the stroma, and 12% in the margin). %) showed similarly high percentages of CD8+ staining. In contrast, bladder sample #30 (Fig. 30A, middle), which exhibited an immunoexcluded phenotype, showed a higher percentage of CD8+ cells in the stroma and margins compared to the tumor (39 in the stroma). % and CD8+ staining in the tumor compared with 24% in the margin). In some cases, further analysis of CD8+ expressed in the stroma and limbus by subdividing the limbal compartment may provide further information for immunophenotyping. For example, as shown in Figure 30B, 18.3% and 4.8% of the CD8+ cells outside the tumor are located in the stromal and limbal compartments, respectively, and subdivision of the limbal component shows that CD8+ cells in the limbal It is further shown that almost all of the cells are on the stroma-facing side of the limbus, with very few CD8+ cells seen on the tumor-facing side. Similarly, Figure 30C shows strong CD8 staining at the tumor margin of bladder sample #30, and when the margin component is subdivided, almost all of the CD8 positivity is localized to the stromal side of the margin compartment, and to the tumor side. It is further demonstrated that very few CD8 positives are localized. These observations indicate that the tumor is likely to be immunocompromised.

Compartmental proportions of CD8+ expression for tumor, stroma, and margin were compared to absolute percent CD8 positivity in all tissues. As demonstrated in Figure 31, tumors exhibiting similar percentages of CD8+ cells present distinct CD8+ expression profiles in each tumor compartment, indicating that the compartmental proportion of CD8+ expression is greater than the absolute percent CD8+ of all tumors only. Comparison with the data suggests that it may provide further context for immunophenotyping. Similarly, the CD8+ cell density in each tumor compartment, determined by the number of CD8+ cells per square millimeter, was compared to the percent CD8+ expression of the total tissue. The IHC staining data in Figure 32 shows that the two different tumor stromal sections have approximately 10-fold differences in CD8+ cell density, despite showing similar overall percentages of CD8+ expression (Figure 32). These findings demonstrate that cell density may be used as an additional or alternative measure to absolute CD8 positivity and that, in some cases, cell density data may better represent the tumor immune population for immunophenotyping. It suggests that you get something.

Tumor depth was determined for two bladder samples by determining the distance available for CD8+ T cell penetration within the tumor nest of a given sample. Bladder sample #22 had a tumor depth greater than 8 (measurements continued towards the opposite side of the tumor nest), while bladder sample #30 had a tumor depth less than 2. (Figure 33). Given that percent CD8 expression in these samples is known to be similar, these results demonstrate that tumor depth can be used in combination with other parameters such as percent CD8 expression to determine tumor immunophenotype. Suggests that it may be a useful parameter for determining/confirming the test.

Localized CD8 expression was further analyzed for melanoma sample #30, which showed a low overall CD8 percentage. The focal area of CD8 expression showed a concentration of CD8 cells near the necrotic area, which could indicate a potential therapeutic effect (Figure 34).

CD8 positivity (eg, percentage of CD8+ cells) was determined for individual tumor foci and compared to CD8 positivity measured in tumor compartments. As shown in Figure 27, further decomposition of the tumor compartment into tumor foci reveals varying degrees of CD8 positivity in different parts of the tumor, providing further insight into the immunophenotype of the tumor. For example, first measuring the CD8 positivity of 74 tumor foci and then assigning a relative phenotype (e.g., immunoinflammatory, immunoeliminated, or immunodepleted) to each individual tumor foci based on CD8 positivity; The immunophenotypes of the bladder tumor samples were determined by finally calculating the combined tumor area exhibiting each immunophenotype (Figure 28). In some cases, the immunophenotype determined based on tumor nest analysis was different from the immunophenotype determined based on analysis of tumor compartments alone (FIGS. 29A-E).

Example 7: Prevalence of tumor MDSCs in various solid tumors A signal intensity filter was established to detect cell surface markers of human MDSCs. Multiple markers have been selected and developed to distinguish MDSC subtypes from other monocytes. Cell surface markers for differentiating human MDSCs include CD11b, CD33, CD66b, CD14, CD15, and HLA-DR. Chromogenic assays were performed to define the immunofluorescence dynamic range of each antibody used for detection of cell surface markers. Two types of signal intensity filters were used to identify MDSCs: 1) binary intensity selection, i.e., based on positive versus negative signals, e.g. to distinguish CD14+mMDSCs from CD15+gMDSCs; and 2) e.g. HLA-DR ^low-neg. Categorical binning of signal intensities to define ranges of signal intensities to differentiate mMDSCs from HLA-DR ^neg gMDSCs. Signal intensity filters were applied sequentially and the classification was reviewed and confirmed by a pathologist. Exemplary thresholds for detecting cell surface markers after application of signal intensity filters are shown in Tables 19A-C below.

An exemplary analysis of signal intensity filtering of MDSC markers in bladder cancer is shown in Figure 35A. An example of HLA-DR detection using a threshold cutoff is shown in Figure 35B. All cells were plotted to show threshold cutoffs. Here, the x-axis shows staining intensity (binned columns) and the y-axis shows cell number. Signal intensity visualization was normalized per channel to reduce background and bleed-through (spectral separation). Therefore, the threshold cutoff was different for each channel. An example of signal normalization is shown in the panel of Figure 35C.

gMDSC populations in tumor samples were first identified by filtering for CD15 and CD66b positivity (Figure 36A). This filtering distinguished gMDSCs from CD15- and CD66b-negative mMDSCs. A sequential application of signal filters was then applied to identify gMDSCs according to the markers and thresholds described above (Figure 36B). Original tumor image from a bladder cancer sample without signal filtering (right panel), image after applying CD15+/CD66b ^high filtering (top left panel), and CD15 ⁺ /CD66b ^high /CD14 ⁻ /CD33 ^low /HLADR ⁻ /CD11b ⁺ A comparison of the images after filtering (bottom left panel) is shown in Figure 36C. The majority of the gMDSC population appeared to be at the tumor edge near the stromal compartment.

The same signal filtering as described above was used to analyze gMDSC and mMDSC populations in samples from bladder cancer, melanoma, NSCLC, and ovarian cancer (Figures 37A-C). The samples had a much larger gMDSC population compared to the mMDSC population. Ovarian cancer showed the lowest gMDSC levels and the highest mMDSC levels among the various indications tested. mMDSC levels were lowest in melanoma samples.

Example 8: Phase 1 Clinical Trial Safety, tolerability, and efficacy of Ab6 administered alone and in combination with anti-PD-(L)1 therapy to adult patients with locally advanced or metastatic solid tumors. A multicenter, open-label, phase 1, first-in-human, dose escalation and expansion study (DRAGON; NCT04291079) is underway to evaluate pharmacokinetics, pharmacodynamics, and efficacy. The study is divided into three treatment parts: Part A1, Part A2, and Part B, as well as a long-term extension phase (LTEP). Part A1 (dose escalation) determines the maximum tolerated dose (MTD) or maximum dose (MAD) of Ab6 as a single agent and the recommended phase 2 dose (RP2D) of Ab6 as a single agent. Part A2 (dose escalation) includes the MTD or MAD of Ab6 in combination with anti-PD-(L)1 antibody therapy and the RP2D of Ab6 in combination with anti-PD-(L)1 antibody therapy for use in Part B. Determine. Part B (dose expansion) includes non-small cell lung cancer (NSCLC), urothelial carcinoma (UC), cutaneous melanoma (MEL), renal cell carcinoma, or another advanced or metastatic solid that is not NSCLC, UC, or MEL. Parallel cohorts of patients with tumor types were enrolled to confirm the tolerability of Ab6's RP2D (determined in part A2) and assess the antitumor activity of Ab6 in combination with anti-PD-(L)1 antibody therapy. be done. Patients in Part A1 may continue treatment with Ab6 as a single agent in RP2D of LTEP after three cycles of treatment with Ab6 as a single agent in Part A1. Patients in Part A2 received 3 cycles of treatment with Ab6 in RP2D in combination with anti-PD-(L)1 antibody therapy in Part A2, followed by Ab6 in combination with anti-PD-(L)1 antibody therapy in LTEP. Treatment may be continued. Patients in Part B will receive 9 cycles of treatment with Ab6 in combination with anti-PD-(L)1 antibody therapy in Part B, followed by continued treatment with Ab6 in combination with anti-PD-(L)1 antibody therapy in LTEP. You may.

In Part A1, patients with advanced solid tumors receive 80 mg, 240 mg, 800 mg, 1600 mg, 2400 mg, or 3000 mg of Ab6 by intravenous infusion once every three weeks. In Part A2, patients who are non-responders to previous anti-PD-(L)1 therapy are administered Ab6 in combination with an approved anti-PD-(L)1 therapy based on tumor type, where: Ab6 is administered intravenously at 240 mg, 800 mg, 1600 mg, or 2400 mg once every three weeks. In Part B, patients who are non-responders to previous anti-PD-(L)1 therapy will receive a combination of anti-PD-(L)1 therapy and Ab6 by intravenous infusion. Cohort A of Part B consists of patients with non-small cell lung cancer (NSCLC) receiving Ab6 in combination with pembrolizumab. Cohort B of Part B consists of patients with urothelial carcinoma (UC) receiving Ab6 in combination with pembrolizumab. Cohort C of Part B consists of patients with melanoma (MEL) receiving Ab6 in combination with pembrolizumab. Cohort D of Part B will consist of cancer patients who are not NSCLC, UC, or MEL and will receive Ab6 in combination with an approved anti-PD-(L)1 therapy based on their tumor type. In Part B, blood chemistry assays were performed as a safety assessment, including the cytokines interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNBα), interleukin 6 (IL-6), Includes measuring blood levels of leukin 8 (IL-8), interferon gamma (IFNγ), and interleukin 10 (IL-10).

The primary outcome measurements of the study included 1) evaluation of the safety and tolerability of Ab6 (Part A1) or in combination with anti-PD-(L)1 therapy (Part A2); and 2) MTD or MAD; Includes determination of RP2D, and dose-limiting toxicity (DLT). DLT responses will be assessed during the first 21 days of the study. MTD, MAD, RP2D, and DLT were assessed using RECIST v1.1 and did not include toxicity clearly related to disease progression and concomitant diseases. Safety endpoints include adverse events, clinical observations (eg, vital signs, physical examination), laboratory tests, electrocardiograms, echocardiograms.

Secondary outcome measures of the study include evaluation of pharmacokinetics of Ab6 alone or in combination with anti-PD-(L)1 therapy. Specific pharmacokinetic parameters include maximum drug concentration (C _max ), time to C _max (T _max ), last verified plasma concentration (C _last ), time to C _last (T _last ), and Half-life (T _1/2 ) is included. The time frame for evaluating pharmacokinetic parameters is cycles 1 and 3, where each cycle is 21 days. Antitumor activity of Ab6 alone or in combination with anti-PD-(L)1 therapy will also be assessed as a secondary outcome. Specific clinical response parameters include objective response defined as complete response (CR) or partial response (PR) as determined by RECIST v1.1 or iRECIST v1.1. Other secondary outcome measures include assessment of anti-drug antibodies and biomarkers. Biomarker evaluation includes assessment of immune status (eg, identification of immunoinflammatory, immune clearance, and immune desert tumors) and assessment of TGFβ pathway status within the tumor. Orthogonal biomarker strategies are also applied to the evaluation of multiple biologically relevant pathways, including tumor-based multiplexed immunohistochemistry (IHC), next-generation sequencing, and blood-based biomarkers. This includes evaluations, etc.

The main inclusion criteria for this study include patients aged 18 years or older with an expected life expectancy of 3 months or more, and patients with measurable disease according to RECIST v1.1 at the time of screening. Additionally, patients must have an Eastern Cooperative Oncology Group performance status (PS) of 0-1. To qualify for Part A1, a patient must be eligible for a standard therapy for which no standard therapy exists, has failed or is not tolerated by the patient, or for which the patient is a suitable candidate for a standard therapy. Must have a histologically demonstrated solid tumor that is metastatic or locally advanced, as assessed by the investigator as non-eligible or otherwise ineligible. To meet Part A2 and Part B eligibility, patients must have received at least 3 cycles of anti-PD-(L)1 antibody therapy (alone or in combination with chemotherapy) approved for their tumor type. , a history of primary anti-PD-(L)1 antibody nonresponse, manifesting as either progressive disease or stable disease (e.g., clinically or radiographically not improving, but also not worsening) (based on the assessment of the responsible physician). Additionally, to meet Part B eligibility, patients must have received their most recent anti-PD-(L)1 antibody therapy within 6 months of enrollment (or within 9 months for the UC cohort). must be maintained.

Key exclusion criteria include ECOG performance status ≥2, concomitant anticancer therapy, history of active metastatic CNS disease, current or past history of autoimmune disease, hypersensitivity to anti-PD-(L)1 antibody therapy or Patients with the presence of anti-drug antibodies and/or concomitant secondary malignancy are included. Additional exclusion criteria include appropriately treated non-invasive cancers, such as cervical cancer, non-melanoma skin cancer, breast cancer with discordant bilateral simultaneous evaluation, or other sites of cancer other than low-grade prostate cancer under observation. Patients with secondary malignancies will be included. Past history of other malignancies indicates that the patient has not had a recurrence for more than 2 years or that the patient has received curative-intent treatment within the past 2 years and that, in the opinion of the investigator, there is no possibility of recurrence. Permissible only if the possibility is low. Women who are pregnant or breastfeeding are also excluded. Exclusion criteria for Part A2 and Part B include receipt of concurrent anticancer therapy, other than approved or investigational Part A2 or Part B anti-PD-(L)1 antibody therapy, within 28 days prior to Ab6 administration. patients who received biologic therapy (other than Part A2 or Part B anti-PD-(L)1 antibody therapy) less than 28 days before Ab6 administration, or systemic cytotoxicity less than 28 days before Ab6 administration. patients who have received sexual chemotherapy (except in combination with anti-PD-(L)1 antibody therapy), patients who have received targeted small molecule therapy within 5 half-lives of the compound prior to Ab6 administration, or severe irAEs or previous Patients with a history of intolerance or treatment discontinuation due to other adverse reactions to anti-PD-(L)1 antibody therapy are included.

Twenty patients were dosed (14 patients in part A1 and 6 patients in part A2). The dose escalation phase of the study is underway. Initial results from Part A1 showed that increasing the dose of Ab6 from 80 mg to 2400 mg did not result in DLTs. Similarly, increasing the dose of Ab6 from 240 mg to 800 mg did not result in DLTs in part A2 when administered in combination with anti-PD-(L)1 therapy. A dose of 3000 mg Ab6 is currently being evaluated in Part A2.

Example 9: Effect of TGFβ1 inhibition on MK differentiation in cells isolated from myelofibrosis patients TGFβ can promote proliferation and collagen deposition of bone marrow stromal cells and endothelial cell proliferation, thereby promoting MF Promotes microenvironmental changes similar to those observed in bone marrow. Furthermore, increased levels of TGFβ in myelofibrosis patients have been implicated in both the development of anemia and thrombocytopenia as well as disease development in patients with myelofibrosis. It is therefore hypothesized that treatment with TGFβ inhibitors may be able to reverse the undesirable effects due to increased TGFβ levels in myelofibrosis patients and myelofibrosis cancers. Because megakaryocytes (MKs) and platelets are the main sources of TGFβ1 (Blood 2007;110:986-993), the therapeutic potential of the TGFβ1 inhibitor Ab6 is limited to bone marrow production under conditions that generate megakaryocyte-enriched populations. It is explored by culturing mononuclear cells or CD34+ cells from fibrosis patients.

Culture conditions to generate MK-enriched populations were described by Mosoyan et al. (Leukemia.2017 November; 31(11):2458-2467), the entire contents of which are incorporated herein by reference. Briefly, cells are cultured using a two-step liquid culture system of either mononuclear cells or CD34+ cells derived from healthy donors or myelofibrosis patients (MF-MNC). Cells were treated with 1% penicillin/streptomycin, 1% L-glutamine (Invitrogen, Grand Island, NY), 20 mM β-mercaptoethanol, and 1% bovine serum albumin (BSA) fraction V (Sigma, St. Louis). , MO), 30% serum replacement BIT 9500, 100 ng/ml recombinant human stem cell factor (hSCF), 50 nM/ml recombinant human thrombopoietin (hTPO) (R&D Systems, Minneapolis, MN, USA) (the entire content is available at Iancu-Rubin et al., Exp Hematol. 2012 Jul; 40(7):564-74), incorporated herein by Iancu-Rubin et al., herein incorporated by reference. MK colony forming unit (CFU-MK) assays are performed using the MegaCult System and Detection Kit according to the manufacturer's instructions (Stem Cell Technologies, Vancouver, BC, Canada). Isolation of CD61+ MKs is performed using the Immunomagnetic Selection Kit according to the manufacturer's recommendations (Miltenyi Biotech Inc., Auburn, CA, USA).

MF-MNC or CD34+ cells from healthy individuals or myelofibrosis patients are cultured with SCF and TPO for 7 days, after which the cells are cultured with TPO alone for an additional 2-8 days. Ab6, negative control antibody (eg, isotype control) or the TGFβR1 kinase inhibitor galunisertib are added for 24-72 hours during cell culture conditioning as described. Conditioned media is collected from each of the cultures and assayed for levels of TGFβ1, TGFβ2 and TGFβ3 by ELISA. The effects of conditioned media treated with negative control antibody, Ab6, or galnisertib on normal fibroblast and endothelial cell proliferation and collagen deposition are evaluated. The effect of conditioned media on normal and myelofibrotic CD34+ colony formation in the presence of cytokine combinations is also evaluated.

To determine the effect of negative control, Ab6, or conditioned medium treated with galnisertib on malignant hematopoiesis, hematopoietic colonies cloned from myelofibrosis CD34+ cells were genotyped for myelofibrosis-driving mutations. be. To determine whether TGFβ1 inhibition abolishes the downstream effects of excessive TGFβ signaling, target cells such as fibroblasts, endothelial cells, and hematopoietic cells are analyzed for SMAD activation.

MK cultures are analyzed using flow cytometry and MK cells are identified based on expression of CD41 and CD42 protein markers. Treatment of MK cultures with up to 100 nM Ab6 inhibits autocrine TGFβ1 signaling in MKs from MF-MNCs but does not inhibit pSMAD2 activation by recombinant TGFβ1. TGFβ activation occurs cell autonomously in patient cell cultures where Smad phosphorylation is demonstrated. Inhibition of phosphorylation by Ab6 confirms that phosphorylation is induced by TGFb1. Addition of exogenous TGFβ1 growth factor to cell cultures is used to demonstrate Smad signaling capacity in the cultures.

Example 10: Exacerbation of ECM Dysregulation in Mice Treated with TGFβ3 Inhibitors From a safety perspective, it is widely recognized that pan-inhibition of TGFβ can cause toxicity; This confirms the fact that development has not been successful up to this point. To avoid potentially dangerous adverse effects, many groups have recently begun to identify inhibitors that target subsets, but not all, of the isoforms and remain effective.

A profibrotic phenotype (eg, increased collagen deposition in the ECM) has been associated not only with fibrosis but also with aspects of cancer progression such as invasion and metastasis. For example, Chakravarthy et al. (Nature Communications, (2018) 9:4692. “TGF-β-associated extracellular matrix genes link cancer-associated fibroblasts to immuno e evasion and immunotherapy failure”). Diseased tissues with dysregulated ECM, including the stroma of various cancer types, can express both TGFβ1 and TGFβ3. Indeed, recently in 2019, multiple groups are working on developing TGFβ inhibitors that target both of these isoforms, such as ligand traps and integrin inhibitors.

Previously, we showed that inhibition of TGFβ1 alone is sufficient to overcome primary resistance to checkpoint blockade therapy in tumor models. To further investigate the in vivo role of TGFβ3 in regulating ECM, TGFβ1 and TGFβ3 selective inhibitors were tested in a diet-induced mouse liver fibrosis model.

In control animals fed a normal diet, the baseline fibrosis score, as measured by percentage of PSR-positive area by histology, was less than 2%. After 12 weeks of fibrosis-inducing diet (maintaining diet but antibody treatment for the last 8 weeks), control animals treated with IgG alone showed approximately 6.5% PSR-positive area by histology. . Animals treated with TGFβ1 selective inhibitor reduced it to approximately 4% PSR positive area (p<0.001 vs. IgG control group). While animals treated with TGFβ3-selective inhibitors were found to develop significantly worse fibrosis with approximately 12.5% PSR-positive area (p<0.001 vs. IgG control group) , animals treated with a combination of TGFβ3-selective inhibitor and TGFβ1-selective inhibitor showed milder fibrosis with approximately 8% PSR-positive area (p<0.001 vs. IgG control group). Ta.

These results suggest that inhibition of TGFβ3 exacerbated ECM dysregulation as indicated by increased collagen accumulation. The data also show that simultaneous inhibition of 1/3 isoforms actually weakens the efficacy of TGFβ1 inhibition in vivo, raising the possibility that TGFβ3 inhibition may be detrimental to ECM regulation.

Equivalents The various features and embodiments of the disclosure mentioned in individual sections above apply mutatis mutandis to other sections as appropriate. As a result, features described in one section may be combined with features described in other sections, as appropriate.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be covered by the following claims.

RELATED APPLICATIONS This application is filed in U.S. Provisional Patent Application No. 63/166,824, filed March 26, 2021, entitled “TGF-BETA INHIBITORS AND USE THEREOF,” June 3, 2021, respectively. Specification No. 63/202,260 filed on January 25, 2022; Specification No. 63/302,999 filed on January 25, 2022; and No. 63 filed on February 24, 2022 No. 313,386, the contents of which are hereby expressly incorporated by reference in their entirety.
Sequence list
This application contains a Sequence Listing filed electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy created on July 1, 2022 is 15094.0013-00304_SL. txt and has a size of 251,422 bytes.

Shows tumor MDSC levels measured in MBT-2 tumors. Tumor volume and circulating G-MDSC and M-MDSC levels in MBT-2 mice are shown. Tumor volumes in MBT-2 mice across treatment groups are shown. Baseline levels of circulating MDSC in non-tumor-bearing mice are shown. Levels of circulating MDSC in tumor-bearing mice are shown. Comparison of circulating MDSC levels in non-tumor-bearing and tumor-bearing mice is shown. Figure 7A shows a comparison of circulating M-MDSC and G-MDSC levels from days 3 to 10. Figure 7B shows the time course of circulating M-MDSC and G-MDSC levels from days 3 to 10. Figure 2 is a plot of circulating MDSC levels and tumor volume at day 10 across treatment groups. Figure 9A shows tumor MDSC levels in different treatment groups. Figure 9B shows a comparison of circulating G-MDSC and tumor MDSC levels at day 10 across treatment groups. Correlation between circulating and tumor MDSC levels is shown. Tumor G-MDSC and tumor CD8+ cells across all treatment groups are shown. Figure 12A shows circulating gMDSC and mMDSC levels in whole blood of mice bearing MBT2 tumors. Figure 12B shows intratumoral gMDSC and mMDSC levels in mice bearing MBT2 tumors. Figure 13A shows circulating TGFβ1 levels (pg/mL) in MBT-2 mice. Figure 13B shows plasma levels of Ab6 (μg/mL, left) and TGFβ1 (pg/mL, right). Figure 13C shows the correlation between plasma levels of Ab6 (μg/mL) and TGFβ1 (pg/mL) in MBT-2 mice treated with AB6 alone or in combination with anti-PD1 antibody. Plasma platelet factor 4 levels (ng/mL) in MBT-2 mice (right) and sample outliers determined by interquartile range (left) are shown. Identified sample outliers (left) and outlier correction levels (pg/mL) (right) of circulating TGFβ1 are shown. Circulating TGFβ levels in NHPs after a single administration of Ab6. Circulating TGFβ levels in rats after a single administration of Ab6. 1 illustrates an exemplary sample collection and processing method for assessing circulating TGFβ1 levels in blood. Circulating TGFβ1 levels in blood samples evaluated under various sample processing conditions. Figure 2 shows platelet factor 4 (PF4) levels in blood samples evaluated under various sample processing conditions. Figure 2 shows the correlation between circulating TGFβ1 and PF4 levels in blood samples evaluated under various sample processing conditions. FIG. 21A shows PF4 levels in blood samples evaluated under various sample processing conditions. 21B and 21C show an exemplary outlier analysis based on measurements of PF4 levels. PF4 versus TGFβ1 levels are shown before and 1 hour after administration. Figure 23A shows the fold change in TGFβ levels over time in subjects treated with 80-240 mg of Ab6. FIG. 23B shows the fold change in TGFβ levels over time in subjects treated with 800 mg of Ab6. Figure 23C shows the fold change in TGFβ levels over time in subjects treated with 1600 mg of Ab6. P-Smad2 IHC analysis of melanoma samples is shown. pSmad-2 signaling in MBT2 tumors after treatment with Ab6-mIgG1. FIG. 26A shows tissue compartmentalization data for bladder cancer samples. Figure 26B shows tissue compartmental data for melanoma samples. Figure 2 shows the density of CD8+ cells in bladder cancer samples analyzed based on tumor foci. Immunophenotypic analysis of a single bladder cancer sample based on the density of CD8+ cells measured within tumor nests. Figure 29A shows the average percentage of CD8+ cells and immunophenotypes in bladder cancer and melanoma samples analyzed by tumor compartment (left) and tumor focus (right). Underlined phenotypes indicate differences between assays. Figure 29B shows the average percentage of CD8+ cells and immunophenotypes in bladder cancer and melanoma samples analyzed by tumor compartment (left) and tumor focus (right). Underlined phenotypes indicate differences between assays. Figure 29C shows tumor nest data and immunophenotype of individual tumor nests identified from bladder cancer samples. Figure 29D shows tumor focal CD8+ data and immunophenotypes of bladder cancer and melanoma samples. Figure 29E shows the percentage of CD8+ cells in the tumor, tumor margin, and stromal compartment of commercially available bladder cancer samples. Figure 30A shows representative CD8+ staining in bladder cancer samples. FIG. 30B shows subdivision of CD8+ staining in tumor margin compartments. Figure 30C shows subdivision of CD8+ staining in the tumor margin compartment of bladder samples. Comparison of compartment CD8+ ratio and absolute percent CD8 positivity is shown. Comparison of CD8+ cell density and absolute percentage of CD8 positivity rate is shown. The tumor invasion depth of the bladder sample is shown. CD8 density in melanoma samples is shown. Figures 35A-C show exemplary analysis of MDSCs by signal filtering. Figures 36A-C show the identification of tumor MDSC populations in various solid tumor samples. Figures 37A-C show analysis of gMDSC and mMDSC populations in various solid tumor samples. FIG. 3 depicts a schematic of an exemplary pathological analysis of a tumor tissue sample. FIG. 3 depicts a schematic of an exemplary pathological analysis of a tumor tissue sample. 1 shows a schematic diagram of an exemplary TGFβ inhibitor treatment regimen. Identification of three binding regions (region 1, region 2, and region 3) after statistical analysis is shown. Region 1 overlaps with a region called "latent lasso" within the prodomain of proTGFβ1, whereas regions 2 and 3 are within the growth factor domain. FIG. 41 discloses SEQ ID NO: 172. Various domains and motifs of proTGFβ1 are shown with respect to the three binding regions involved in Ab6 binding. A sequence alignment between the three isoforms is also provided. FIG. 42 discloses SEQ ID NOs: 142, 173-174, 138, 175-176, 141, and 177-178, respectively, in order of publication.

The terms "crystal" and "crystallized" as used herein refer to a binding protein (eg, an antibody) or antigen-binding portion thereof that exists in crystalline form. A crystal is a form of solid state of matter, which is different from other forms such as an amorphous solid state or a liquid crystalline state. Crystals are composed of regularly repeating three-dimensional arrays of atoms, ions, molecules (eg, proteins such as antibodies), or molecular assemblies (eg, antigen/antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well understood in the art. A basic unit, or building block, that is repeated in a crystal is called an asymmetric unit. The repetition of asymmetric units in an arrangement according to a given well-defined crystallographic symmetry results in a "unit cell" of the crystal. Repetition of the unit cell by regular translation in all three dimensions results in a crystal. Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ed. , pp. 201-16, Oxford University Press, New York, New York, (1999). The term "linker" is used to refer to a polypeptide that includes two or more amino acid residues joined by peptide bonds and is used to join one or more antigen-binding moieties. Such linker polypeptides are well known in the art (eg, Holliger, P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J. et al., (1994) Structure 2:1121-1123). Exemplary linkers include ASTKGPSVFPLAP (SEQ ID NO: 44), ASTKGP (SEQ ID NO: 45); TVAAPSVFIFPP (SEQ ID NO: 46); TVAAP (SEQ ID NO: 47); AKTTPKLEEGEFSEAR (SEQ ID NO: 48); AKTTPKLEEGEFSEARV (SEQ ID NO: 49); AKTTP KLGG (SEQ ID NO: 50); SAKTTPKLGG (SEQ ID NO: 51); SAKTTP (SEQ ID NO: 52); RADAAP (SEQ ID NO: 53); RADAAPTVS (SEQ ID NO: 54); RADAAAAGGPGS (SEQ ID NO: 55); RADAAA(G4S)4 (SEQ ID NO: 56) ); SAKTTPKLEEGEFSEARV (57); ADAAP (SEQ ID NO: 58); ADAAPTVSIFPP (SEQ ID NO: 59); QPKAAP (SEQ ID NO: 60); QPKAAPSVTLFPP (SEQ ID NO 61); SEquilization number 62); AKTTPPSVTPLAP (SEQ ID NO: 63) ; AKTTAP (SEQ ID NO: 64); AKTTAPSVYPLAP (SEQ ID NO: 6 5) ; GGGGSGGGGSGGGGS (SEQ ID NO: 66); GENKVEYAPALMALS (SEQ ID NO: 67); GPAKELTPLKEAKVS (SEQ ID NO: 68); GHEAAAVMQVQYPAS (SEQ ID NO: 69) ;TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO: 70) ; and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 71).

Known activators of TGFβ1, such as plasmin, TSP-1 and αVβ6 integrin, all interact directly with LAP. It is hypothesized that proteolytic cleavage of LAP may destabilize the LAP-TGFβ interaction, thereby releasing active TGFβ1 (growth factor domain) from the latent complex. It has been suggested that the region containing the amino acid stretch 54-LSKLRL-59 (SEQ ID NO: 171) is important for maintaining TGFβ1 latency. Therefore, agents (eg, antibodies) that stabilize the interaction or block proteolytic cleavage of LAP may prevent activation of TGFβ1.

Claims (21)

A TGFβ inhibitor for use in treating cancer in a subject, said treatment comprising:
(i) determining the level of circulating TGFβ in a blood sample from the subject prior to administering the TGFβ inhibitor;
(ii) administering to said subject a first dose comprising a therapeutically effective dose of said TGFβ inhibitor; and (iii) determining the level of circulating TGFβ in a blood sample from said subject after said administration. including,
A second dose of said TGFβ inhibitor is administered to the subject after step (iii) if said level of circulating TGFβ after the first administration is increased compared to said level of circulating TGFβ before said administration. is,
Optionally, a TGFβ inhibitor, wherein said increase is at least 1.5-fold. A method for determining treatment efficacy in a subject undergoing treatment for cancer, the method comprising:
(i) determining the level of circulating TGFβ in a blood sample taken from said subject prior to administering a TGFβ inhibitor; and (ii) determining the level of circulating TGFβ in a blood sample taken from said subject after said administration. including determining the level;
an increase in the circulating TGFβ level after administration compared to pre-administration is indicative of a therapeutic effect;
Optionally, the increase is at least 1.5 times. 1. A method of determining target binding in a subject with cancer, the method comprising:
(i) determining the level of circulating TGFβ in a blood sample taken from said subject prior to administering a TGFβ inhibitor; and (ii) determining the level of circulating TGFβ in a blood sample taken from said subject after said administration. including determining the level;
an increase in the circulating TGFβ level after the administration compared to before the administration is indicative of target binding of the TGFβ inhibitor;
Optionally, the increase is at least 1.5 times. The circulating TGFβ level is
(a) treating said blood sample in a sample tube containing an anticoagulant at a temperature of 2 to 8°C;
(b) one or more centrifugation steps at a speed greater than 100 x g, and/or one or more centrifugation steps at a speed less than 15000 x g; and/or (c) a centrifugation protocol comprising: :
i) a first step of 10 minutes at 150 x g and a second step of 20 minutes at 2500 x g; or ii) a first step of 10 minutes at 2500 x g and a second step of 20 minutes at 2500 x g. or iii) a first step of 1500 x g for 10 minutes and a second step of 12000 x g for 5 minutes and optionally , the method comprises citric acid-theophylline-adenosine-dipyridamole (CTAD) solution, 0.11 M buffered trisodium citrate solution, 15 M theophylline, 3.7 M adenosine, and 0.198 M dipyridamole at pH 5.0. 4. The method or use according to any one of claims 1 to 3, further comprising using a collection tube coated with an anticoagulant containing TGFβ. determining the level of circulating TGFβ comprises determining the level of platelet factor 4 (PF4) in the same sample in which the circulating TGFβ is determined; A TGFβ inhibitor for the method or use according to any one of claims 1 to 4, which is used to determine circulating TGFβ levels only if said PF4 level is less than 500 ng/ml. A TGFβ inhibitor for the method or use according to any one of claims 1 to 5, wherein said circulating TGFβ1 is circulating TGFβ1, and optionally said circulating TGFβ1 is latent circulating TGFβ1. A TGFβ inhibitor for use in treating cancer in a subject, said treatment comprising:
(i) determining the level of circulating MDSC in a blood sample from the subject prior to administering the TGFβ inhibitor;
(ii) administering to said subject a first dose comprising a therapeutically effective dose of said TGFβ inhibitor; and (iii) determining the level of circulating TGFβ in a blood sample from said subject after said administration. including,
a second dose of said TGFβ inhibitor after step (iii) if said level of circulating MDSCs after administration of said first dose is reduced by at least 10% compared to said level of circulating TGFβ before said administration. is administered to the subject,
Optionally, said MDSC level is determined by measuring LRRC33 surface expression, optionally said LRRC33 surface expression is determined by a FACS-based assay or an ELISA-based assay. determining the level of circulating MDSCs comprises determining the level of circulating mMDSCs and/or the level of circulating gMDSCs;
The circulating mMDSC level is determined by measuring the level of cells expressing CD11b+, HLADR-/low, CD14+, CD15-, CD33+/high, and CD66b-, and the circulating mMDSC after administration of the first dose. said second dose of said TGFβ inhibitor is administered to the subject after step (iii) if said level of circulating TGFβ is reduced by at least 10% compared to said level of circulating TGFβ before said administration, and/or or said circulating gMDSC level is determined by measuring the level of cells expressing CD11b+, HLADR-, CD14-, CD15+, CD33+/low, and CD66+, said circulating gMDSC level after administration of said first dose. 7. The second dose of the TGFβ inhibitor is administered to the subject after step (iii) if the level is reduced by at least 10% compared to the level of circulating TGFβ before the administration. A TGFβ inhibitor for use as described in . A method for determining treatment efficacy in a subject undergoing treatment for cancer, the method comprising:
(i) determining the level of circulating MDSC in a blood sample taken from said subject prior to administering a TGFβ inhibitor; and (ii) determining the level of circulating MDSC in a blood sample taken from said subject after said administration. including determining the level;
a decrease in the circulating MDSC level after the administration compared to before the administration is indicative of a therapeutic effect;
Optionally, the reduction is at least 10%. determining the level of circulating MDSCs comprises determining the level of circulating mMDSCs and/or the level of circulating gMDSCs;
The circulating mMDSC level is determined by measuring the level of cells expressing CD11b+, HLADR-/low, CD14+, CD15-, CD33+/high, and CD66b, and the circulating mMDSC after the administration compared to before the administration. a decrease of at least 10% in said level of is indicative of a therapeutic effect; and/or said circulating gMDSC level measures the level of cells expressing CD11b+, HLADR-, CD14-, CD15+, CD33+/low, and CD66+. 10. The method of claim 9, wherein a decrease in the level of circulating gMDSCs by at least 10% after the administration compared to before the administration is indicative of a therapeutic effect. 1. A method of determining target binding in a subject with cancer, the method comprising:
(i) determining the level of circulating MDSC in a blood sample taken from said subject prior to administering a TGFβ inhibitor; and (ii) determining the level of circulating MDSC in a blood sample taken from said subject after said administration. including determining the level;
a decrease in the circulating MDSC level after the administration compared to before the administration is indicative of target binding of the TGFβ inhibitor;
Optionally, the reduction is at least 10%. determining the level of circulating MDSCs comprises determining the level of circulating mMDSCs and/or the level of circulating gMDSCs;
The mMDSC level is determined by measuring the levels of cells expressing CD11b+, HLADR-/low, CD14+, CD15-, CD33+/high, and CD66b, and the level of circulating mMDSC after the administration compared to before the administration. a decrease of at least 10% in said level is indicative of target binding; and/or said gMDSC level is determined by measuring the level of cells expressing CD11b+, HLADR-, CD14-, CD15+, CD33+/low, and CD66+. 12. The method of claim 11, wherein a decrease in the level of circulating gMDSCs of at least 10% after the administration compared to before the administration is indicative of target binding. A TGFβ inhibitor for use in treating cancer in a subject, the agent comprising:
(i) determining the level of P-Smad2 nuclear translocation in a tumor sample obtained from said subject prior to administering a TGFβ inhibitor;
(ii) administering to said subject a first dose comprising a therapeutically effective dose of said TGFβ inhibitor;
(iii) determining the level of P-Smad2 nuclear translocation in a tumor sample obtained from the subject after said administration; and (iv) determining the nuclear translocation level of P-Smad2 after said administration of said first dose. administering to the subject one or more additional doses of the TGFβ inhibitor if the P-Smad2 nuclear translocation is reduced compared to before the administration of the first dose;
Optionally, a TGFβ inhibitor, wherein said reduction is at least 1.3-fold. A method for determining treatment efficacy in a subject undergoing treatment for cancer, the method comprising:
(i) determining the level of phosphorylated Smad2 (P-Smad2) nuclear translocation in a tumor sample obtained from said subject prior to administering the TGFβ inhibitor; and (ii) determining the level of phosphorylated Smad2 (P-Smad2) nuclear translocation in a tumor sample obtained from said subject after said administration; determining the level of P-Smad2 nuclear translocation in a tumor sample obtained by
A decrease in P-Smad2 nuclear translocation after said administration compared to before said administration is indicative of a therapeutic effect;
Optionally, the reduction is at least 1.3 times. A method for determining target binding in a subject undergoing treatment for cancer, the method comprising:
(i) determining the level of P-Smad2 nuclear translocation in a tumor sample obtained from said subject prior to administering a TGFβ inhibitor;
(ii) administering to said subject one or more doses comprising a therapeutically effective dose of said TGFβ inhibitor;
(iii) determining the level of P-Smad2 nuclear translocation in a tumor sample obtained from the subject after said administration;
a decrease in P-Smad2 nuclear translocation after said administration compared to before said administration indicates target binding of said TGFβ inhibitor;
Optionally, the reduction is at least 1.3 times. said cancer has an immunoexclusion phenotype characterized by less than 5% intratumoral CD8+ cells and more than 5% marginal CD8+ cells as assessed by immunohistochemical analysis of CD8+ cells, said CD8+ cells is evaluated in individual tumor foci within said tumor, optionally said cancer being characterized in that said tumor area has more than 50% of said tumor foci comprising less than 5% of tumor foci with CD8+ cells. A TGFβ inhibitor for the method or use according to any one of claims 1 to 15. further comprising administering a checkpoint inhibitor and/or genotoxic therapy in combination with said TGFβ inhibitor, optionally said checkpoint inhibitor being an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti- a CTLA-4 antibody, an anti-LAG3 antibody, or an antigen-binding fragment thereof; and/or said genotoxic therapy is chemotherapy or radiotherapy, and optionally said chemotherapy is PARP inhibitor therapy. A TGFβ inhibitor for the method or use according to any one of claims 1 to 16. The subject has an advanced cancer and/or solid cancer, optionally said advanced cancer and/or solid cancer is melanoma (e.g. metastatic melanoma), renal cell carcinoma, breast cancer, e.g. triple negative breast cancer, HER2 positive breast cancer, colorectal cancer, e.g. microsatellite stable colorectal cancer and colon adenocarcinoma, lung cancer (e.g. metastatic non-small cell lung cancer, small cell lung cancer), esophageal cancer, pancreatic cancer, bladder cancer, kidney cancer, For example, transitional cell carcinoma, renal sarcoma, and renal cell carcinoma (RCC) (including clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, or unclassified RCC), uterine cancer, e.g. Selected from endometrial cancer, prostate cancer, gastric cancer (e.g. gastric cancer), head and neck cancer, e.g. head and neck squamous cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, thyroid cancer, or tenosynovial giant cell tumor (TGCT). A TGFβ inhibitor for the method or use according to any one of claims 1 to 17. The TGFβ inhibitor is an inhibitor of TGFβ1 and TGFβ2; an inhibitor of TGFβ1 and TGFβ3; a pan-inhibitor that inhibits TGFβ1, TGFβ2, and TGFβ3; a drug that binds to the RGD motif present in latent TGFβ1 and/or TGFβ3; an RNA-based inhibitor; or a soluble ligand trap;
Optionally, said inhibitor of TGFβ1 and TGFβ2 is NIS793/XOMA-089 or GC1008; said inhibitor of TGFβ1 and TGFβ3 is M7824 (vintrafsp alpha) or AVID200; said pan-inhibitor is: GC1008 or its derivatives, SAR439459, LY3022859, or an agent that blocks the ligand-binding domain of the TGFβ receptor; the agent that binds to the RGD motif present in latent TGFβ1 and/or TGFβ3 is a low molecular weight ) compound or antibody or antigen-binding fragment; said RNA-based inhibitor is an inhibitor of TGFβ1 expression; and said soluble ligand trap is M7824 (bintrafusp alpha) or AVID200. A TGFβ inhibitor for the method or use according to any one of . When the TGFβ inhibitor is defined by the IMTG numbering system, it has three overlapping amino acid sequences comprising SEQ ID NO: 1 (H-CDR1), SEQ ID NO: 2 (H-CDR2) and SEQ ID NO: 3 (H-CDR3). An antibody comprising a chain complementarity determining region and three light chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO: 4 (L-CDR1), SEQ ID NO: 5 (L-CDR2) and SEQ ID NO: 6 (L-CDR3), or an antibody thereof. optionally, the TGFβ inhibitor comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence with 90% identity thereto, and an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having 90% identity thereto. A TGFβ inhibitor for the method or use according to any one of claims 1 to 18, comprising a light chain variable region comprising an amino acid sequence with 90% identity. A method for identifying an mMDSC population and a gMDSC population from a biological sample obtained from a patient, wherein the mMDSC population is CD11b+, HLA-DR-/low, CD14+, CD15-, CD33+/high, and CD66b-. identified by cell surface markers; the gMDSC population is identified by cell surface markers of CD11b+, HLA-DR-, CD14-, CD15+, CD33+/low, and CD66b+.

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