High affinity, isoform-selective tgfβ1 inhibitors and uses thereof

Abstract

Disclosed herein are monoclonal antibodies and antigen-binding fragments thereof capable of selectively inhibiting TGFβ1 with high potency. Also disclosed are related compositions, methods, and therapeutic uses.

Description

[0001] Related applications

[0002] This application claims the benefits and priorities of the following applications: U.S. Provisional Patent Application No. 62 / 696,752, filed July 11, 2018; U.S. Provisional Patent Application No. 62 / 718,196, filed August 13, 2018; U.S. Provisional Patent Application No. 62 / 737,534, filed September 27, 2018; U.S. Provisional Patent Application No. 62 / 758,180, filed November 9, 2018; U.S. Provisional Patent Application No. 62 / 810,263, filed February 25, 2019; and U.S. Provisional Patent Application No. 62 / 827,552, filed April 1, 2019, each entitled “High Affinity, Background-Independent TGFβ1 Inhibitors and Use Thereof,” the entire contents of which are expressly incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list, which has been electronically submitted in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy created on July 11, 2019, is named 127036-03520_SL.txt and is 261,252 bytes in size. Background Technology

[0005] Transforming growth factor β1 (TGFβ1), along with two other structurally related isoforms (i.e., TGFβ2 and TGFβ3), is a member of the TGFβ superfamily of growth factors, each encoded by an independent gene. These TGFβ isoforms act as pleiotropic cytokines, regulating cell proliferation, differentiation, immune regulation (e.g., adaptive immune responses), and a variety of other biological processes in both homeostatic and disease contexts. These three TGFβ isoforms signal through the same cell surface receptors and trigger similar, typical downstream signaling events, including the SMAD2 / 3 pathway. However, gene knockout studies in mice have revealed distinct phenotypes, suggesting that each isoform plays a discrete role in vivo. This can be partly attributed to the differential expression patterns of the three isoforms.

[0006] In the immune system, T cells are considered the primary direct target of TGFβ. TGFβ signaling is important in effector cell proliferation, as well as in regulating effectors and T cell differentiation. For example, TGFβ is a potent inhibitor of both Th1 and Th2 effector T cells. TGFβ has also been shown to inhibit the effector function of cytotoxic T cells through multiple mechanisms. Furthermore, there is evidence that other cell types in the immune system, such as dendritic cells (Langhans cells) and natural killer (NK) cells, are also regulated by the TGFβ signaling pathway. TGFβ dysregulation is associated with many disease conditions, such as cancer, fibrosis, and immune disorders.

[0007] Many biological processes involving the extracellular matrix are related to TGFβ signaling. To name just a few, TGFβ is involved in wound healing, tumor invasion and metastasis, and the progression of fibrosis.

[0008] For these and other reasons, TGFβ has become a promising therapeutic target for treating immune disorders, various proliferative disorders, and fibrotic conditions. However, observations from preclinical studies (including in rats and dogs) have shown serious toxicities associated with systemic inhibition of TGFβ in vivo. Furthermore, although several TGFβ inhibitors have been developed to date, most clinical protocols targeting TGFβ have been discontinued due to the risk of serious side effects (summarized in, for example, WO2017 / 156500). Therefore, despite direct and indirect evidence that TGFβ signaling is involved in the progression of diseases such as cancer and fibrosis, no TGFβ therapy has yet been deemed safe and effective on the market.

[0009] Previously, the applicant described a class of monoclonal antibodies with a novel mechanism of action for regulating growth factor signaling (see, for example, WO 2014 / 182676). These antibodies were designed to take advantage of the fact that TGFβ1 is expressed as a potential pro-protein complex containing a pre-domain and growth factor, requiring an activation step to release growth factor from the potential complex. Instead of the conventional approach of directly targeting the mature growth factor itself after activation (as with neutralizing antibodies), this new class of inhibitory antibodies specifically targets the inactive pro-protein complex itself, thereby preemptively blocking the activation step upstream of the ligand-receptor interaction. It is reasonable to believe that this unique mechanism of action should provide an advantage in achieving temporal and spatial benefits, as it works at the source—by targeting the potential proTGFβ1 complex in the disease microenvironment prior to activation.

[0010] Using this method, monoclonal antibodies were produced that specifically bind to and inhibit the TGFβ1 activation step (i.e., the release of mature growth factors from the potential complex) in a subtype-selective manner (see WO 2017 / 156500). The data provided therein support the view that subtype-specific inhibition of TGFβ (as opposed to pan-inhibition) can improve the safety properties of antagonizing TGFβ in vivo. With this in mind, the applicant subsequently sought to develop TGFβ1 inhibitors that i) are subtype-specific; and ii) can target multiple TGFβ1 signaling complexes associated with different presenting molecules, as therapeutic agents for conditions driven by multi-level TGFβ1 effects and their dysregulation.

[0011] Such antibodies were subsequently described in PCT / US2018 / 012601 (submitted January 5, 2018). In fact, the subtype-specific inhibitors described therein target both ECM-associated TGFβ1 and immune cell-associated TGFβ1, thereby blocking multiple sources of TGFβ1 in various biological contexts while maintaining subtype specificity. Data from numerous in vivo models have been published demonstrating the efficacy and safety of subtype-selective TGFβ1 activation inhibitors, suggesting that such inhibitors may be useful for treating diseases involving in vivo dysregulation of both ECM-associated and immune cell-associated TGFβ1.

[0012] Although the earlier work cited above demonstrated the utility of antibodies capable of targeting every known proTGFβ1 complex and inhibitory activity, improved subtype-selective TGFβ1 inhibitors with higher in vivo potency are expected. Summary of the Invention

[0013] This disclosure provides a novel class of high-affinity, subtype-selective antibodies capable of efficiently inhibiting TGFβ1 activation. These include antibodies (including immunoglobulins and their antigen-binding fragments or portions, and engineered molecules incorporating such fragments) capable of targeting multiple presenting molecules—proTGFβ1 complexes (referred to as “large potential complexes” or “LLCs”) with high affinity. These antibodies retain equivalent selectivity and safety properties and demonstrate improved in vivo efficacy in a variety of preclinical models that can be translated into human conditions. These properties of TGFβ1 inhibitors provide opportunities for the development of safe and effective TGFβ1 therapies to treat diseases involving TGFβ1 dysregulation.

[0014] The following selection criteria were considered in generating the proTGFβ1 antibodies of this disclosure: 1) subtype selectivity; 2) high affinity for human LLCs (e.g., LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1); 3) potent inhibitory potency; 4) favorable in vivo safety / toxicological properties; and 5) in vivo efficacy in preclinical models that generalize human disease. Furthermore, when evaluating the efficacy of TGFβ1 inhibitors used as combination therapies (e.g., adjunctive therapies), the ability to achieve synergistic effects (rather than simple additive effects) should be weighed. Based on these criteria, the inventors of this disclosure have identified a class of high-affinity monoclonal antibodies and fragments thereof that specifically target the proTGFβ1 complex and effectively block TGFβ1 activation. In some embodiments, the novel antibodies disclosed herein exhibit high affinity (e.g., nanomolar to sub-nanomolar KD) on all target LLCs. In a preferred embodiment, such antibodies are unbiased across different proTGFβ1 complexes, thus exhibiting equivalent affinity for all target complexes. This invention covers related compositions, therapeutic uses, preparations, formulations, processes, and methods.

[0015] Therefore, in some embodiments, the present invention includes a monoclonal antibody or an antigen-binding fragment thereof, which, as measured by solution equilibrium titration, is capable of binding each of the following human LLC complexes with a KD of ≤10 nM: LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. In some embodiments, the antibody binds each of the human LTBP1-proTGFβ1 and LTBP3-proTGFβ1 complexes with a KD of ≤1 nM. Preferably, the antibody has a KD of ≤1 nM for each of the four human LLCs.

[0016] In some embodiments, the antibody or fragment binds to the latent lasso of proTGFβ1 or a fragment thereof. In some embodiments, the antibody or fragment also binds to one or more portions of growth factor domains (such as finger-1 and finger-2). For example, the antibody or fragment may bind to an epitope comprising one or more amino acid residues of the latent lasso. Optionally, the epitope may further comprise one or more amino acid residues of the growth factor domain. Thus, such epitopes may be combinatorial epitopes. In a preferred embodiment, the antibody does not bind to free TGFβ1 growth factor not associated with the proTGFβ1 complex.

[0017] The TGFβ1 inhibitors of this invention are functional antibodies because they possess inhibitory activity against TGFβ1. The potency of such antibodies is subtype-specific, as measured by suitable in vitro potency assays, such as the cell-based reporter factor assays described herein. Therefore, the antibodies do not bind to or inhibit TGFβ2 or TGFβ3 counterparts.

[0018] The TGFβ1 inhibitor of the present invention can prevent the release of mature growth factors from potential LLC complexes. In some embodiments, the TGFβ1 inhibitor can inhibit integrin-dependent activation of TGFβ1 and / or protease-dependent activation of TGFβ1. In some embodiments, the protease is kallikrein, plasmin, or MMP protease. In some embodiments, the TGFβ1 inhibitor blocks integrin-dependent TGFβ1 activation without blocking the binding of integrin to LLC.

[0019] In some embodiments, the TGFβ1 inhibitors of the present invention can act through a dual inhibitory mode on cell-associated LLCs (e.g., GARP-proTGFβ1 and LRRC33-proTGFβ1). In one mechanism, such inhibitors block the activation step of TGFβ1 associated with membrane-anchored GARP and / or LRRC33. In a second mechanism, such inhibitors can induce antibody-dependent internalization (and thus removal) of LLCs from the cell surface after target engagement, thereby reducing TGFβ1 signaling at the niche. In some embodiments, the antibody is a pH-sensitive antibody, characterized in that the antibody binds the proTGFβ1 complex with higher affinity at neutral pH than at acidic pH.

[0020] In some embodiments, the TGFβ1 inhibitor of the present invention effectively reduces the expression of disease-related genes such as TGFB1, Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, Loxl2, CCL2, and Mmp2.

[0021] In some embodiments, the TGFβ1 inhibitor of the present invention effectively reduces the phosphorylation of downstream effectors SMAD2 / 3 in vivo.

[0022] In some embodiments, the TGFβ1 inhibitor of the present invention is effective in treating TGFβ1-related indications. Such indications include diseases involving abnormal gene expression, diseases involving ECM dysregulation, diseases characterized by an increase in immunosuppressive cells (e.g., Treg, MDSC, and / or M2 macrophages), diseases involving mesenchymal transition, diseases involving proteases, diseases associated with abnormal stem cell proliferation and / or differentiation, and so on, and these categories of diseases are not intended to be mutually exclusive. In some embodiments, TGFβ1-related indications are proliferative disorders, such as myeloproliferative disorders and solid tumor cancers. In some embodiments, TGFβ1-related indications are fibrotic disorders, such as organ fibrosis. Cancer can be advanced cancer, including locally advanced tumors / cancers and metastatic cancers.

[0023] In some embodiments, the TGFβ1 inhibitor of the present invention can reduce the number of immunosuppressive cell populations at disease sites (such as the tumor microenvironment and fibrotic microenvironment). In some embodiments, the immunosuppressive cell population may include M2-polarized macrophages and / or MDSCs.

[0024] In some embodiments, the TGFβ1 inhibitors of the present invention effectively achieve tumor control (e.g., partial and complete remission), wherein the tumor optionally has an immunosuppressive (e.g., immune exclusion) phenotype. In some embodiments, TGFβ1 inhibitors can achieve a synergistic antitumor effect when used in combination with cancer therapies (e.g., checkpoint blockade therapy, chemotherapy, and radiotherapy). Checkpoint blockade therapy may include, for example, one or more anti-PD-(L)1 antibodies. In such combination therapies, TGFβ1 inhibitors can overcome treatment resistance (e.g., primary resistance), thereby making the cancer more sensitive to cancer therapies. Therefore, TGFβ1 inhibitors can be used to treat cancers, including immunosuppressive tumors in subjects. Subjects may be i) those who are primary non-responders to cancer therapies (e.g., checkpoint inhibitors); or ii) those diagnosed with cancer for which at least one checkpoint inhibitor has been approved by a regulatory authority as a therapy. The response rate (the sum of partial and complete responses among those who have received treatment) for cancers for which at least one checkpoint inhibitor has been approved is less than 100%. Typically, the response rate is between about 10-60%. TGFβ1 inhibitors can increase response rates in the patient population. Furthermore, in the partial response group among primary responders, TGFβ1 inhibitors can provide improved clinical benefit. In some embodiments, TGFβ1 inhibitors can reduce acquired resistance to cancer therapies in the primary responder group. Immunosuppressive tumors can be locally advanced cancers / tumors or metastatic cancers. In some embodiments, cancer therapies may include, for example, checkpoint inhibitor therapy, chemotherapy, and / or radiation therapy.

[0025] In some embodiments, the TGFβ1 inhibitor of the present invention effectively provides a survival benefit in subjects with solid tumors, wherein the solid tumors are optionally locally advanced or metastatic cancers.

[0026] In some embodiments, the TGFβ1 inhibitor of the present invention effectively achieves a durable anti-tumor effect by inducing T cell memory function. Therefore, the TGFβ1 inhibitor can reduce or delay disease recurrence.

[0027] In some embodiments, the TGFβ1 inhibitor of the present invention can effectively achieve antitumor effects in tumors that primarily express TGFB1 and / or TGFβ3. In some embodiments, the tumors co-expressing TGFβ1 and TGFβ3 are cancers.

[0028] In some embodiments, the TGFβ1 inhibitor of the present invention can overcome primary tumor resistance to cancer therapy. In some embodiments, such tumors are infiltrated with immunosuppressive cell types, such as regulatory T cells, M2 macrophages, and / or myeloid-derived suppressor cells (MDSCs). Following treatment, the number of tumor-associated immunosuppressive cells decreases, and the number of anti-tumor effector T cells increases accordingly.

[0029] In some embodiments, the TGFβ1 inhibitor of the present invention promotes effector cell infiltration into the tumor. In some embodiments, effector cells can enter the tumor via the tumor's vascular system. In some embodiments, the TGFβ1 inhibitor of the present invention promotes effector cell expansion (e.g., proliferation). This can be mediated at least in part by the inhibition of GARP-positive regulatory T cells.

[0030] In some embodiments, the TGFβ1 inhibitor of the present invention is effective in treating myelofibrosis. In some embodiments, the TGFβ1 inhibitor achieves an anti-fibrotic effect on the bone marrow of a subject with myelofibrosis. In some embodiments, the TGFβ1 inhibitor effectively normalizes certain hematological parameters.

[0031] In some embodiments, the TGFβ1 inhibitor of the present invention effectively achieves anti-fibrotic effects in vivo. Anti-fibrotic effects may include reversing established fibrosis, which may be partial or complete.

[0032] In some embodiments, the TGFβ1 inhibitors of the present invention are well tolerable in preclinical safety / toxicology studies at doses up to 100, 200, or 300 mg / kg when administered weekly for at least 4 weeks. Such studies can be conducted in animal models known to be sensitive to TGFβ inhibition, such as rats and non-human primates. In some embodiments, the TGFβ1 inhibitors of the present invention do not cause visible toxicities associated with pan-inhibition of TGFβ, such as cardiovascular toxicity (e.g., valvular disease) and epithelial hyperplasia, as well as other toxicities known in the art.

[0033] In some embodiments, the TGFβ1 inhibitor of the present invention achieves a sufficient therapeutic window because in vivo efficacy studies show that the effective amount of the inhibitor is far below (e.g., at least 3 times, at least 6 times, or at least 10 times) the amount or concentration that would cause visible toxicity. In some embodiments, the therapeutically effective amount of the inhibitor is between about 1 mg / kg and about 30 mg / kg per week. Attached Figure Description

[0034] Figure 1 This is a graph showing the inhibition of LTBP1-proTGFβ activation in the LN229 assay.

[0035] Figure 2 This is a graph showing the inhibition of proTGFβ complex activation in the LN229 assay.

[0036] Figure 3 This is a graph showing the inhibition of GARP-proTGFβ1 activation in the SW480β6 assay.

[0037] Figure 4 This is a graph showing the inhibition of LRRC33-proTGFβ1 activation in the SW480β6 assay.

[0038] Figure 5A The inhibitory effects of Ab3 and Ab6 on kallikrein-induced TGFβ1 activation were demonstrated in vitro.

[0039] Figure 5B The inhibitory effects of Ab3 and Ab6 on plasmin-induced TGFβ1 activation were demonstrated in vitro.

[0040] Figure 6 A graph showing the rapid internalization of LRRC33-proTGFb1 after Ab6 binding in heterologous cells transfected with LRRC33 and proTGFβ1 is provided.

[0041] Figure 7Two graphs are provided showing the effects of Ab6 or Ab3 on the expression of collagen genes (Col1a1 and Col3a1) in UUO mice. Mice were treated with Ab3 at 3, 10, or 30 mg / kg / week, or Ab6 at 3 or 10 mg / kg / week. IgG alone was used as a control.

[0042] Figure 8 Two graphs are provided showing the effects of Ab3 or Ab6 on Fn1 and Loxl2 gene expression in UUO mice. Mice were treated with Ab3 at 3, 10, or 30 mg / kg / week, or Ab6 at 3 or 10 mg / kg / week. IgG alone was used as a control.

[0043] Figure 9 The statistical significance of gene expression changes (relative to UUO+IgG) after treatment in the UUO model was summarized.

[0044] Figure 10 This is a graph showing the percentage of survival over time (days) in the Cloudman S91 melanoma model after administration of Ab3 in combination with anti-PD-1 at doses of 30 mg / kg or 10 mg / kg. Anti-PD-1 alone, anti-SR-AB3 alone, and as a control are also shown.

[0045] Figure 11A Tumor growth (tumor volume in mm), expressed as median tumor progression in the Cloudman S91 melanoma model, is provided as a function of time (days) following administration of Ab3 or Ab6 (both in combination with anti-PD-1) at doses of 30 mg / kg or 10 mg / kg. 3 Five graphs showing the changes. Anti-PD-1 alone was used as a control. Dashed lines represent tumor ulceration reaching 2000 mm. 3 Animals that must be euthanized before reaching the final standard.

[0046] Figure 11B Two graphs are provided showing the median tumor volume of Cloudman S91 as a function of time after administration of Ab3 (left) or Ab6 (right) (in combination with anti-PD-1) at 30 mg / kg or 10 mg / kg. Anti-PD-1 alone, Ab3 alone, Ab6 alone, and IgG alone were used as controls.

[0047] Figure 11CSix graphs are provided showing changes in S91 tumor volume as a function of time in mice treated with (1) control IgG; (2) Ab6 only; (3) anti-PD1 only; (4) anti-PD1 / Ab6 (3 mg / kg); (5) anti-PD1 / Ab6 (10 mg / kg); and (6) anti-PD1 / Ab6 (30 mg / kg). The dashed line at the top indicates the endpoint tumor volume of 2,000 mm. 3 The dashed line below indicates a 25% threshold volume of 500 mm². 3 Respondents were defined as those whose tumor size was less than 25% of the endpoint volume.

[0048] Figure 11D Three graphs are provided showing changes in S91 tumor volume as a function of time in mice treated with a combination of anti-PD-1 and Ab6 at three dose levels (3, 10, and 30 mg / kg). Durable antitumor effects were observed after treatment.

[0049] Figure 11E A summary is provided from Figure 11C A graph of data expressed as median tumor volume.

[0050] Figure 11F Provides display from Figure 11C A graph showing the survival of animals over time in each treatment group.

[0051] Figure 12 This is a graph showing the ratio of phosphorylated to total SMAD2 / 3 (pSMAD / SMAD) in the MBT2 bladder cancer model. Animals were treated with the following: (1) anti-PD-1 antibody alone; (2) a combination of Ab5 (3 mg / kg) and anti-PD-1 antibody; (3) a combination of Ab5 (10 mg / kg) and anti-PD-1 antibody; (4) a combination of Ab3 (10 mg / kg) and anti-PD-1 antibody; and (5) a combination of Ab3 (30 mg / kg) and anti-PD-1 antibody.

[0052] Figure 13A and 13B Two sets of five graphs are provided, showing MBT2 tumor growth (tumor volume in mm) over time (days) after administration of Ab3 at 30 mg / kg or 10 mg / kg, or Ab6 at 3 mg / kg or 10 mg / kg (in combination with anti-PD-1). 3 Changes in ) were observed. Anti-PD-1 alone was used as a control. The results were measured on a logarithmic scale ( Figure 13A ) and linear scaling ( Figure 13B The figure represents the change in tumor volume as a function of time. The dashed line represents the change in tumor volume as a function of time, reaching 1200 mm. 3 Animals that must be euthanized before reaching the final standard.

[0053] Figure 13C A graph showing median tumor volume as a function of time after administration of Ab3 (top left) at 30 mg / kg or 10 mg / kg, or Ab6 (top right) at 10 mg / kg or 3 mg / kg (in combination with anti-PD-1) in an MBT2 syngeneic bladder cancer model is provided. Anti-PD-1 alone, Ab3 alone, Ab6 alone, and IgG alone were used as controls. The median tumor volume at day 15 is summarized in the figure below.

[0054] Figure 13D Five figures are provided showing the effect of Ab6 combined with anti-PD-1 in an MBT2 syngeneic bladder cancer model. Respondents were defined as those whose tumor size was less than 25% of the endpoint volume at the study endpoint.

[0055] Figure 14 This is a graph showing the percentage of survival over time (days) after administration of Ab3 at 10 mg / kg or Ab6 at 3 mg / kg or 10 mg / kg (in combination with anti-PD-1) in an MBT2 syngeneic bladder cancer model. Anti-PD-1 alone was used as a control.

[0056] Figure 15 A set of graphs is provided showing tumor growth (tumor volume in mm) measured over time (days) in a tumor re-attack study. 3 Changes in tumor size were observed. Animals that had previously cleared their tumors (achieving complete regression) were re-attacked with MBT2 tumor cells. Initially untreated animals were used as controls. Dashed lines indicate tumor ulceration reaching 1200 mm. 3 Animals that must be euthanized before reaching the final standard.

[0057] Figure 16 This is a heatmap showing the binding results of HDX-protected Ab5 Fab in the regions (regions 1 and 2) of proTGFβ1.

[0058] Figure 17 The region of the proTGFβ1 complex is shown, as measured by HDX after Ab5 binding (see [link]). Figure 16 It is protected from solvent exchange.

[0059] Figure 18A This is a heatmap showing the protective effect of Ab6 Fab binding to proTGFβ1(C4S). Regions affected by antibody-antigen interactions are indicated by red boxes (1, 2a, 2b, 2c, 3, 4, 5a, 5b, 6a, and 6b).

[0060] Figure 18B HDX data overlaid on the TGFβ1 crystal structure is provided. (Shown...) Figure 18A The area identified in the Chinese identification process.

[0061] Figure 19A The three binding regions (regions 1, 2, and 3) identified after statistical analysis are shown. Region 1 overlaps with the so-called "latent lasso" within the proTGFβ1 predomain, while regions 2 and 3 are within the growth factor domain.

[0062] Figure 19B The various domains and motifs of proTGFβ1 relative to the three binding regions involved in Ab6 binding are described. Sequence alignments between the three isotypes are also provided.

[0063] Figure 20A-20D The relative RNA expression of TGFβ isoforms in various tissues and cells is shown. Figure 20A The expression of TGFβ subtypes in various human cancer tissues compared with normal controls (by cancer type) is shown. Figure 20B The frequency of TGFβ subtype expression by human cancer type was shown based on an analysis of more than 10,000 samples from 33 tumor types. Figure 20C The expression of TGFβ subtypes in a single tumor sample is shown by cancer type. Figure 20D The expression of the TGFβ isoform was shown in a mouse syngeneic cancer cell model line.

[0064] Figure 20E Four gene expression plots are provided, showing all the presenting molecules (LTBP1, LTBP3, GARP, and LRRC33) that are highly expressed in most human cancer types.

[0065] Figure 20F Expression analysis of TGFβ and related signaling pathway genes from syngeneic mouse tumor models Cloudman S91, MBT-2, and EMT-6 was provided.

[0066] Figure 20G Three graphs are provided comparing the protein expression of three TGFβ isoforms in Cloudman S91, MBT-2, and EMT-6 tumor models using ELISA.

[0067] Figure 20H A graph comparing RNA expression levels using whole tumor lysates of presenting molecules in Cloudman S91, MBT-2, and EMT-6 tumor models is provided.

[0068] Figure 21AThe results of a microcardiac study based on a 1-week toxicology study using a pan-TGFβ antibody are described. Figure 21B The results of a microcardiac study derived from Ab3, based on a 4-week rat toxicology study, were described compared with ALK5 inhibitors or pan-TGFβ antibodies. Figure 21C The results of a microcardiac study from Ab6, based on a 4-week rat toxicology study, are described compared to ALK5 inhibitors or pan-TGFβ antibodies.

[0069] Figure 22 A graph showing the median tumor volume of S91 as a function of time is provided. The combination group (arm) represents four different subtypes of selective, background-independent TGFβ1 inhibitors at two dose levels, all in combination with anti-PD-1 therapy.

[0070] Figures 23A-23B Representative immunohistochemical sections of S91 tumors stained with CD8+ cell markers are provided. Figure 23A These are tumor slices from animals that received only anti-PD-1 treatment. Figure 23B These are tumor slices from animals treated with anti-PD-1 and representative, background-independent TGFβ1 inhibitors.

[0071] Figures 24A-24D Representative immunohistochemical sections of S91 tumors stained with macrophage markers are provided. Figure 24A These are tumor slices from animals that received only anti-PD-1 treatment. Figure 24B These are tumor slices from animals treated with anti-PD-1 and representative, background-independent TGFβ1 inhibitors. Figure 24C These are tumor slices from animals treated with anti-PD-1 and Ab3 (30 mg / kg) using anti-F4 / 80 as a macrophage marker. Figure 24D The slides, which used anti-CD163 as a marker for M2 macrophages, showed that most cells were CD163 negative.

[0072] Figure 25 This is a graph showing the log2 fold change of CD8+ T lymphocyte genes (CD8α, perforin, and granzyme B) after 1 week of treatment with anti-PD-1 / Ab3 in animals with MBT2 tumors compared to animals treated with anti-PD-1 alone.

[0073] Figure 26A FACS data showing CD3 / CD28-induced GARP upregulation in peripheral human regulatory T cells are provided.

[0074] Figure 26B This graph shows the effect of Ab3 or Ab6 on Treg-mediated Teff proliferation inhibition. IgG was used as a control.

[0075] Figure 27A A gating strategy for sorting T cell subsets in MBT2 tumors is shown.

[0076] Figure 27B A set of graphs is provided showing the T cell subsets at day 13, expressed as a percentage of CD45+ cells.

[0077] Figure 28A A gating strategy for sorting myeloid subsets in MBT2 tumors is provided.

[0078] Figure 28B A set of figures showing the myeloid cell subsets at day 13 is provided.

[0079] Figure 28C FACS data are provided for tumor-associated macrophages in MBT-2 cells expressing LRRC33 on the cell surface.

[0080] Figure 28D The results showed that MBT-2 tumor-infiltrating MDSCs expressed LRRC33 on the cell surface.

[0081] Figures 29A-29C provide additional FACS data analyses showing the role of Ab6 and anti-PD-1 therapy in MBT2 tumors.

[0082] Figures 30A-30D provide IHC images of representative MBT2 tumor sections showing CD8-positive T cells within the tumor.

[0083] Figure 30E Quantification of IHC data from Figures 30A-30D is provided, expressed as the fraction of CD8-positive cells in each treatment group. Necrotic areas in the sections were excluded from the analysis.

[0084] Figure 30F Immunohistochemical analysis of the effects of Ab6 and anti-PD-1 therapy in MBT2 tumors is provided. Tumor sections were visualized targeting phosphorylated SMAD3 (top panel) or CD8 and CD31 (bottom panel) in animals from the three treatment groups shown.

[0085] Figure 30G Data provided confirm that the combination of Ab6 and anti-PD-1 appears to trigger CD8+ T cell mobilization and infiltration from CD31+ vessels into MBT2 tumors.

[0086] Figures 31A-31D show the gene expression of immune response markers Ptprc (Figure 31A), CD8a (Figure 31B), CD4 (Figure 31C), and Foxp3 (Figure 31D) collected from MBT2 tumors in the four treatment groups, as illustrated.

[0087] Figures 32A-32C provide the gene expression of the functional markers Ifng (Figure 32A), Gzmb (Figure 32B), and Prf1 (Figure 32C) at day 10 and / or day 13, as shown.

[0088] Figure 32D A set of graphs is provided showing the expression of four gene markers (granzyme B, perforin, IFNγ, and Klrk1) in MBT2 tumor samples as measured by qPCR on day 10. Each graph shows the fold change in expression across the three treatment groups: anti-PD-1 alone (left); Ab6 alone (middle); and a combination of anti-PD-1 and Ab6 (right).

[0089] Figure 33A The in vitro binding of Ab6 to four large potential complexes, as measured by solution equilibrium titration-based assay (MSD-SET), is shown. The measured K values ​​are shown on the right. D Value (in picomoles).

[0090] Figure 33B A potency assay based on LN229 cells is shown, and a graph is provided showing the concentration-dependent potency of Ab6 against four large potential complexes as indicated. It is also shown that Ab6 does not inhibit proTGFβ3.

[0091] Figure 34A A set of nine figures is provided to show the effect of Ab6 with or without anti-PD1 and / or anti-TGFβ3 on tumor growth / regression over time in EMT6 (Study 1). The dashed line at the top of each figure indicates the endpoint tumor volume of 2,000 mmHg. 3 The dashed line at the bottom of each image represents 25% of the final volume (i.e., 500 mm). 3 ).

[0092] Figure 34B The study provided percentages of survival over time (days after treatment initiation) in EMT6 (Study 1). The treatment group containing both anti-PD-1 and Ab6 showed a significant survival benefit compared to anti-PD-1 alone.

[0093] Figure 34C The study provided percentages of survival in EMT6 over time (days after treatment initiation) (Study 2). The treatment group containing both anti-PD-1 and Ab6 showed a significant survival benefit compared to anti-PD-1 alone, and the antitumor effect was durable after treatment.

[0094] Figure 34DThe effect of the combination of anti-PD-1 and Ab6 on survival in the EMT6 breast cancer model was provided.

[0095] Figure 35 Two graphs are provided showing the relative expression of the three TGFβ isoforms in EMT6 tumors as measured by mRNA level (left) and protein level (right).

[0096] Figure 36A A set of histological images showing silver staining of reticulin, a marker of myelofibrosis phenotype in a mouse model of myeloproliferative disease, is provided.

[0097] Figure 36B MPL was provided, showing a high disease burden from two independent duplicate studies. W515L Two graphs showing histopathological analysis of myelofibrosis in mice and the inhibitory effect of TGFβ1.

[0098] Figure 36C Provided MPL values ​​showing the results of treatment with Ab6 or control IgG. W515L A set of graphs showing hematological parameters in mice.

[0099] Figure 36D Provided MPL values ​​showing the results of treatment with Ab6 or control IgG. W515L A set of graphs showing other hematological parameters in mice.

[0100] Figure 37A A genomic variation analysis (GSVA) ​​was provided to show the correlation between TGFβ isotype expression and the IPRES genome.

[0101] Figure 37B Genomic variation analysis (GSVA) ​​was provided to show the correlation between TGFβ isoform expression and the Plasari genome. TGFb1 isoform expression is associated with TGFβ pathway activation. The expression of TGFb1 RNA isoforms in the Plasari genome of TGFβ-responsive genes was significantly and closely associated with various TCGA-annotated tumor types. The correlation between TGFB1 mRNA and TGFβ signaling markers was also presented. Detailed Implementation

[0102] definition

[0103] To facilitate understanding of this invention, certain terms are first defined. These definitions should be read in accordance with the remainder of this disclosure and as understood by one of ordinary skill 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 set forth throughout the detailed description.

[0104] Advanced cancer, advanced malignancy: As used herein, the terms “advanced cancer” or “advanced malignancy” have the meaning understood in the relevant field, such as as an oncologist understands in the context of diagnosing or treating a subject / patient with cancer. Advanced malignancy of a solid tumor can be locally advanced or metastatic. The term “locally advanced cancer” is used to describe cancer (e.g., a tumor) that has begun to grow outside the organ but has not yet spread to distant parts of the body. Thus, the term includes cancer that has spread from its origin to nearby tissues or lymph nodes. Conversely, “metastatic cancer” is cancer that has spread from its originating part of the body (primary lesion) to other parts of the body (e.g., distant parts).

[0105] Affinity: Affinity is the strength of the binding between a molecule (such as an antibody) and its ligand (such as an antigen). It is usually determined by the equilibrium dissociation constant (K). D Measurement and reporting. In the context of antibody-antigen interactions, K... D It is the antibody dissociation rate ("dissociation rate (off rate)" or k). off The rate at which an antibody dissociates from its antigen is related to the antibody-antibody binding rate (“binding rate” or Kon). on The ratio of the rate at which an antibody binds to its antigen to the rate at which it binds. For example, the KA of an antibody with an affinity ≤5 nM, determined by a suitable in vitro binding assay. D The value is 5 nM or lower (i.e., 5 nM or higher affinity). Suitable in vitro assays can be used to measure the KD value of an antibody against its antigen, such as biolayer interferometry (BLI) and solution equilibrium titration (e.g., MSD-SET).

[0106] Antibody: The term “antibody” includes any naturally occurring, recombinant, modified, or engineered immunoglobulin or immunoglobulin-like structure or its antigen-binding fragment or portion, or derivatives thereof, as further described elsewhere herein. Thus, the term refers to an immunoglobulin molecule that specifically binds to a target antigen and includes, for example, chimeric, humanized, fully human, and bispecific antibodies. A complete antibody typically contains at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains, such as antibodies naturally occurring in camels that may contain only heavy chains. Antibodies may be derived from a single source or may be “chimeric,” meaning that different portions of the antibody may be derived from two different antibodies. Antibodies or their antigen-binding portions can be produced in hybridomas using recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. As used herein, the term antibody includes, respectively, monoclonal antibodies, bispecific antibodies, microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimics”), chimeric antibodies, humanized antibodies, human antibodies, and antibody fusions (sometimes referred to herein as “antibody conjugates”). In some implementations, the term also includes peptide bodies.

[0107] Antigen: The term "antigen" broadly includes any molecule containing an antigenic determinant in one or more binding regions that are specifically bound by an antibody or fragment. An antigen can be a single-unit molecule (such as a protein monomer or fragment) or a complex consisting of multiple components. An antigen provides an epitope, such as a molecule or a portion of a molecule, or a complex of molecules or portions of molecules, which can be bound by selective binders, such as antigen-binding portions (including, for example, antibodies). Thus, selective binders can specifically bind to antigens in the form of complexes formed from two or more components. In some embodiments, the antigen can be used in animals to generate antibodies capable of binding to the antigen. An antigen may have one or more epitopes capable of interacting with different antigen-binding proteins (e.g., antibodies). In the context of this disclosure, a suitable antigen is a complex containing a proTGF dimer that binds to a presenting molecule (e.g., a multimeric complex consisting of bound multiple 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 dimer complex (see Figure 19). This is then covalently linked to the presenting molecule via a disulfide bond involving a cysteine ​​residue near the N-terminus of each proTGF monomer. Such a multi-complex formed by the binding of the proTGF dimer to the presenting molecule is commonly referred to as a large latent complex. Antigen complexes suitable for screening antibody or antigen-binding fragments include, for example, presenting molecule components comprising large latent complexes. Such presenting molecule components can be full-length presenting molecules or one or more fragments thereof. The minimum required portion of the presenting molecule typically contains at least 50 amino acids, but more preferably at least 100 amino acids, of the presenting molecule polypeptide, including two cysteine ​​residues capable of forming a covalent bond with the proTGFβ1 dimer.

[0108] Antigen-binding moiety / fragment: As used herein, the term “antigen-binding moiety” or “antigen-binding fragment” of an antibody means one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TGFβ1). 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 an antigen to form a complex. In some embodiments, the antigen-binding moiety of an antibody may be derived from the whole antibody molecule, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and optionally constant domains of the antibody. Non-limiting examples of antigen-binding moieties include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of an antibody single arm; (v) single-chain Fv (scFv) molecules (see, for example, 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, for example, Ward et al., (1989) NATURE (341:544-546); and (vii) the smallest recognizing unit (e.g., a separated complementarity-determining region (CDR)) consisting of amino acid residues of a simulated hypervariable region of the antibody. Other forms of single-chain antibodies are also included, such as biantibodies. The term "antigen-binding portion of an antibody" includes a "single-chain Fab fragment," also known as "scFab," which comprises an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; and wherein the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids.

[0109] Bias: In the context of this disclosure, the term "bias" refers to a skewed or uneven affinity relative to or against a group of antigens to which the antibody can specifically bind. For example, an antibody is considered biased when its affinity for one antigen complex is unequal to its affinity for another antigen complex. The background-independent antibodies according to this disclosure have equal affinity for such antigen complexes (i.e., unbiased).

[0110] Binding region: As used herein, a "binding region" is a portion of an antigen that, when bound to an antibody or a fragment thereof, forms an interface for antibody-antigen interaction. After antibody binding, the binding region becomes unprotected and exposed, and can be detected by suitable techniques such as HDX-MS. Antibody-antigen interactions can be mediated by multiple (e.g., two or more) binding regions. Binding regions may contain antigenic determinants or epitopes.

[0111] Biolayer Interferometry (BLI): BLI is a label-free technique used for the optical measurement of interactions between biomolecules, such as between ligands immobilized on the surface of a biosensor tip and analytes in solution. BLI provides the ability to monitor binding specificity, binding and dissociation rates, or concentrations with precision and accuracy. BLI platform instruments are available, for example, from ForteBio and are commonly referred to as… system.

[0112] Cancer: As used herein, the term “cancer” refers to a physiological condition in multicellular eukaryotes characterized by unregulated cell proliferation and malignancy. Therefore, the term broadly encompasses both solid and fluid malignancies, including tumors, blood cancers (e.g., leukemia, lymphoma, and myeloma), and myelofibrosis.

[0113] Cell-associated proTGFβ1: This term refers to membrane-bound (e.g., ligated to the cell surface) TGFβ1 or its signaling complex (e.g., pre / potential TGFβ1). Typically, such cells are immune cells. TGFβ1 presented by GARP or LRRC33 is cell-associated TGFβ1. GARP and LRRC33 are transmembrane molecules expressed on the cell surface of certain cells. GARP-proTGFβ1 and LRRC33 can be collectively referred to as “cell-associated” (or “cell-surface”) proTGFβ1 complexes that mediate cell-associated (e.g., immune cell-associated) TGFβ1 activation / signaling. The term also includes recombinant, purified GARP-proTGFβ1 and LRRC33-proTGFβ1 complexes in solution that are not physically attached to the cell membrane (e.g., in in vitro assays). The average KD value of antibodies (or fragments thereof) against the GARP-proTGFβ1 complex and the LRRC33-proTGFβ1 complex can be calculated to collectively represent affinity for the cell-associated (e.g., immune cell-associated) proTGFβ1 complex. See, for example, Table 8, column (G). Human counterparts of presenting molecules or presenting molecule complexes can be indicated by an "h" preceding the protein or protein complex, such as "hGARP", "hGARP-proTGFβ1", "hLRRC33", and "hLRRC33-proTGFβ1". In addition to preventing the release of active TGFβ1 growth factor from the cell line chain complex, cell-associated proTGFβ1 may be a target for internalization (e.g., endocytosis) and / or cell killing, such as ADCC, ADCP, or ADC-mediated exhaustion of target cells expressing this cell surface complex.

[0114] Checkpoint inhibitors: In the context of this disclosure, checkpoint inhibitors mean immune checkpoint inhibitors and have the meaning as understood in the art. Typically, the targets are receptor molecules on T cells or NK cells, or corresponding cell surface ligands on antigen-presenting cells (APCs) or tumor cells. Immune checkpoints are activated in immune cells to prevent the development of inflammatory immunity against “self.” Therefore, altering the balance of the immune system through checkpoint inhibition should allow it to be fully activated to detect and eliminate cancer. The most well-known inhibitory receptors involved in the control of the immune response are cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-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 T-cell activation inhibitory factor (VISTA) containing the V domain of immunoglobulin (Ig). Non-limiting examples of checkpoint inhibitors include: nivolumab, pembrolizumab, BMS-936559, atezolizumab, avelumab, duvamarab, ipilimumab, tremelimumab, IMP-321, BMS-986016, and lirilumab. This is an example of a PD-1 inhibitor. Therapies using one or more immune checkpoint inhibitors can be called checkpoint blockade therapy (CBT).

[0115] Clinical benefit: As used herein, the term “clinical benefit” is intended to include both the efficacy and safety of a treatment. Therefore, a therapeutic treatment that achieves the desired clinical benefit is both effective (e.g., achieving a therapeutically beneficial effect) and safe (e.g., having a tolerable or acceptable level of toxicity or adverse events).

[0116] Combination therapy: "Combination therapy" refers to a treatment regimen for a clinical indication that comprises two or more therapeutic agents. Therefore, the term signifies a treatment regimen in which a first therapy comprising a first composition (e.g., an active ingredient) is administered to a patient in conjunction with a second therapy comprising a second composition (an active ingredient) to treat the same or overlapping disease or clinical condition. The first and second compositions may both act on the same cellular target or different cellular targets. In the context of combination therapy, the phrase "in conjunction with" means that the therapeutic effect of the first therapy overlaps with the therapeutic effect of the second therapy in the subject receiving the combination therapy in time and / or space. Therefore, combination therapies can be formulated as single preparations for simultaneous administration or as separate preparations for sequential administration. When a second therapy is administered to a subject who has already been treated with a first therapy for the same disease, the second therapy may be referred to as an adjunct or supplementary therapy.

[0117] Combinatory or combinatorial epitopes: Combinatory epitopes are epitopes recognized and bound by a combinatorial antibody at a site (i.e., a non-antigenic determinant) formed by discontinuous portions of one or more components of an antigen, which are tightly bound together in a three-dimensional structure to form an epitope. Therefore, the antibodies of the present invention can bind epitopes formed by two or more components (e.g., portions or segments) of a pre / potential TGFβ1 complex. Combinatory epitopes may contain amino acid residues from a first component of the complex, and amino acid residues from a second component of the complex, and so on. Each component may be a single protein of the antigen complex or two or more proteins. Combinatory epitopes are formed by structural contributions from the antigen or two or more components (e.g., portions or segments, such as amino acid residues) of the antigen or antigen complex.

[0118] Competition or cross-competition; cross-blocking: When used in the context of competing antigen-binding proteins for the same epitope, the term "competition" (e.g., antibody or its antigen-binding portion) means competition between antigen-binding proteins measured by an assay, wherein the tested antigen-binding protein prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein to a common antigen (e.g., TGFβ1 or a fragment thereof). Many types of competitive binding assays can be used to determine whether one antigen-binding protein binds to another competing antigen, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay, solid-phase direct biotin-avidin EIA, solid-phase direct labeling assay, and solid-phase direct labeling sandwich assay. Typically, when a competitive antigen-binding protein is present in excess, it inhibits (e.g., reduces) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen-binding protein to the common antigen. In some cases, at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more of the binding is inhibited.

[0119] In some embodiments, the first antibody or its antigen-binding portion and the second antibody or its antigen-binding portion "cross-block" each other for the same antigen, for example, by Biacor or The assay is performed using standard test conditions. For example, according to the manufacturer's instructions (e.g., measuring binding at room temperature, ~20-25°C). In some embodiments, the first antibody or a fragment thereof and the second antibody or a fragment thereof may have the same epitope. In other embodiments, the first antibody or a fragment thereof and the second antibody or a fragment thereof may have overlapping epitopes that are not exactly the same. In a further embodiment, the first antibody or a fragment thereof and the second antibody or a fragment thereof may have separate (different) epitopes that are very close in three-dimensional space, such that antibody binding is cross-blocked by steric hindrance. "Cross-blocking" means that the binding of the first antibody to the antigen prevents the binding of the second antibody to the same antigen, and similarly, the binding of the second antibody to the antigen prevents the binding of the first antibody to the same antigen.

[0120] Antibody binning (sometimes called epitope binning or epitope mapping) can be performed to characterize and sort collections (e.g., “libraries”) of monoclonal antibodies prepared against a target protein or protein complex (i.e., antigen). Such antibodies against the same target are tested in pairs against all other antibodies in the library to assess whether the antibodies block each other’s binding to the antigen. Closely related binning profiles indicate that the antibodies have the same or closely related (e.g., overlapping) epitopes and are “grouped” together. Binning provides useful structure-function properties for antibodies that share similar binding regions within the same antigen, because the biological activity (e.g., intervention; potency) achieved by binding to the antibody’s intended target may transfer to another antibody in the same bin. Therefore, among antibodies within the same antigen bin, those with higher affinity (lower KD) generally have higher potency.

[0121] Complementarity-Determining Regions: As used herein, the term “CDR” refers to the complementarity-determining region within the variable sequence of an antibody. Three CDRs exist in each variable region of the heavy and light chains, 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 present in a single variable region capable of binding the antigen. The exact boundaries of these CDRs are defined differently depending on the system. The system described by Kabat (Kabat et al., (1987; 1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.)) provides not only a definitive residue numbering system applicable to any variable region of an antibody but also precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; and Chothia et al. (1989) Nature 342: 877-883) found that certain subregions of the Kabat CDR adopted nearly identical peptide skeleton architectures, despite their great diversity at the amino acid sequence level. These subregions were named L1, L2, and L3 or H1, H2, and H3, where “L” and “H” represent the light chain and heavy chain regions, respectively. These regions can be referred to as the Chothia CDR, which has boundaries that overlap with the Kabat CDR. Other boundaries defining CDRs that overlap with the Kabat CDR have been described in Padlan (1995) FASEB J. 9: 133-139 and MacCallum (1996) J. Mol. Biol. 262(5): 732-45. Other CDR boundary definitions may not strictly follow any of those in the system described herein, but will still overlap with the Kabat CDR, although they may be shortened or lengthened based on predictions or experimental findings that specific residues or groups of residues or even the entire CDR do not significantly affect antigen binding (see, for example: Lu X et al., MAbs. 2019 Jan; 11(1):45-57). The methods used herein can utilize CDRs defined according to any of these systems, although some implementations use CDRs defined by Kabat or Chothia.

[0122] Conformational epitopes: Conformational epitopes are epitopes that are recognized and bound by conformational antibodies in a three-dimensional conformation, but not in an unfolded peptide with the same amino acid sequence. Conformational epitopes can be called conformation-specific epitopes, conformation-dependent epitopes, or conformation-sensitive epitopes. The corresponding antibodies or fragments that specifically bind to such epitopes can be called 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.

[0123] Constant domains: Immunoglobulin constant domains refer to the constant domains of the heavy or light chain. The amino acid sequences of the constant domains of the human IgG heavy and light chains are known in the art.

[0124] Background-biased: As used herein, a “background-biased antibody” refers to a conformational antibody type that binds an antigen with different affinities when the antigen binds to (i.e., binds to or attaches to) an interacting protein or fragment thereof. Therefore, background-permitted antibodies that specifically bind to epitopes within proTGFβ1 may bind to LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1 with different affinities. For example, an antibody is termed “matrix-biased” if it has a higher affinity for matrix-associated proTGFβ1 complexes (e.g., LTBP1-proTGFβ1 and LTBP3-proTGFβ1) than for cell-associated proTGFβ1 complexes (e.g., GARP-proTGFβ1 and LRRC33-proTGFβ1). [Matrix-associated complexes]: The relative affinity of [cell-associated complexes] can be obtained by taking the average KD value of the former and the average KD value of the latter, and calculating the ratio between the two, as illustrated in the examples below.

[0125] Background-independent: According to this disclosure, the “background-independent antibody” binding to proTGFβ1 exhibits equivalent affinity among four known presenting molecule-proTGFβ1 complexes (i.e., LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1). The background-independent antibodies disclosed in this application may also be described as unbiased. Typically, background-independent antibodies exhibit equivalent (i.e., bias not exceeding five-fold) affinity such that the relative ratio of the measured KD values ​​between the matrix-associated complex and the cell-associated complex, as measured by suitable in vitro binding assays (e.g., surface plasmon resonance, biolayer interferometry (BLI), and / or solution equilibrium titration (e.g., MSD-SET)), does not exceed 5.

[0126] ECM-associated TGFβ1 / proTGFβ1: This term refers to TGFβ1 or its signaling complex (e.g., pre / potential TGFβ1) that is a component of the extracellular matrix (e.g., deposited into it). TGFβ1 presented by LTBP1 or LTBP3 is ECM-associated TGFβ1 (referred to as LTBP1-proTGFβ1 and LTBP3-proTGFβ1, respectively). LTBPs are essential for the proper deposition and subsequent bioavailability of TGFβs in the ECM, with fibrinogen (Fbn) and fibronectin (FN) considered the main matrix proteins responsible for LTBP binding to the ECM. Such matrix-associated potential complexes are enriched in connective tissues as well as certain disease-associated tissues, such as tumor stroma and fibrotic tissue. Human counterparts of presenting molecules or presenting molecule complexes may be indicated by an "h" preceding the protein or protein complex, such as "hLTBP1", "hLTBP1-proTGFβ1", "hLTBP3", and "hLTBP3-proTGFβ1".

[0127] Effective dose: An effective dose (or therapeutically effective dose, or therapeutic dose) is the dose or administration regimen that achieves a statistically significant clinical benefit (e.g., efficacy) in a patient population. For example, in preclinical models, Ab6 has shown efficacy at doses as low as 3 mg / kg and as high as 30 mg / kg. Therefore, it can be said that the effective dose of Ab6 is between approximately 3 and 30 mg / kg.

[0128] Effective tumor control: The term "effective tumor control" can be used to refer to the degree of tumor regression achieved in response to treatment, where, for example, tumor regression is a defined fraction of the endpoint tumor volume (e.g., <25%). For example, in a particular model, if the endpoint tumor volume is set to 2,000 mm... 3 The tumor will shrink to less than 500mm. 3 (Assuming a threshold <25%), effective tumor control is achieved. Therefore, effective tumor control includes complete regression. Clinically, effective tumor control includes partial remission (PR) and complete remission (CR) based on recognized standards in the art (such as RECIST 1.1 and the corresponding iRECIST). In some implementations, effective tumor control in a clinical context also includes disease stabilization, where tumor growth that would normally be expected to occur at a certain rate is prevented through treatment, even if shrinkage cannot be achieved.

[0129] Effector T cells: As used herein, effector T cells are T lymphocytes that immediately and actively respond to stimuli (such as co-stimuli), and include, but are not limited to, CD4+ T cells (also known as helper T cells or Th cells) and CD8+ T cells (also known as cytotoxic T cells). Th cells assist other leukocytes in the immune process, 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 referred to as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when they are presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist active immune responses. These cells can differentiate into one of several subtypes, including Th1, Th2, Th3, Th17, Th9, or TFh, which secrete different cytokines to promote different types of immune responses. Signaling from APCs directs T cells to specific subtypes. Cytotoxicity (killing). On the other hand, cytotoxic T cells (TC cells, CTLs, killer T cells, cytotoxic T cells) destroy virus-infected cells and cancer cells and are also associated with transplant rejection. These cells are also called 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 cells (e.g., CD8+ cells) include, for example, perforin and granzyme B.

[0130] Epitope: The term "eptope," also known as an antigenic determinant, is a molecular determinant (e.g., a peptide determinant) that can be specifically bound by a binder, immunoglobulin, or T-cell receptor. Epitope determinants include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. An epitope recognized by an antibody or an antigen-binding fragment of an antibody is a structural element of an antigen that interacts with the CDR (e.g., complementary site) of the antibody or fragment. An epitope can be formed by contributions from several amino acid residues that interact with the antibody's CDR to produce specificity. An antigen fragment may contain more than one epitope. In some embodiments, an antibody specifically binds to its target antigen when it recognizes it in a complex mixture of proteins and / or macromolecules. For example, if antibodies cross-competitive (one antibody prevents the binding or modulation of another antibody), these antibodies are referred to as "binding to the same epitope."

[0131] Equivalent affinity: In the context of this disclosure, the term "equivalent affinity" is intended to mean: i) as determined by suitable in vitro binding assays (e.g., solution equilibrium titration (e.g., MSD-SET), biolayer interferometry (e.g., ... As measured by surface plasmon resonance (e.g., the Biacore system), the antibody binds to the matrix-associated proTGFβ1 complex and the cell-associated proTGFβ1 complex with an affinity bias of less than 5-fold; and / or ii) the relative affinity of the antibody to the four complexes is uniform, wherein: the lowest affinity (highest KD value) exhibited by the antibody among the four antigen complexes is not more than five-fold lower than the average calculated from the other three affinities; or, the highest affinity (lowest KD value) exhibited by the antibody among the four antigen complexes is not more than five-fold higher than the average calculated from the other three affinities. Antibodies with the same affinity can achieve a more uniform inhibitory effect, regardless of the specific presenting molecule bound to the proTGFβ1 complex (and therefore "independent of background"). In a particularly preferred embodiment, the average affinity bias observed between the matrix-associated complex and the cell-associated complex does not exceed three-fold.

[0132] Extended Latent Lasso: As used herein, the term "extended latent lasso" refers to a portion of the anterior domain comprising a latent lasso and an α-2 helix (e.g., LASPPSQGEVPPGPLPEAVLALYNSTR (SEQ ID NO:154)). In some embodiments, the extended latent lasso also comprises a portion of an α-1 helix (e.g., LVKRKRIEA (SEQ ID NO:159)) or a portion thereof.

[0133] Fibrosis: The term “fibrosis” or “fibrotic symptoms / condition” refers to the process or manifestation characterized by the pathological accumulation of extracellular matrix (ECM) components (such as collagen) within tissues or organs.

[0134] Fibrotic microenvironment: The term "fibrotic microenvironment" refers to a localized disease niche within a tissue, in which fibrosis occurs in vivo. The fibrotic microenvironment can include disease-associated molecular markers (a set of chemokines, cytokines, etc.), disease-associated cell populations (such as activated macrophages, MDSCs, etc.), and a disease-associated ECM environment (with altered ECM composition and / or structure). It is believed that the fibrotic microenvironment supports the TGFβ-dependent transformation of fibroblasts into α-smooth muscle actin-positive myofibroblasts. The fibrotic microenvironment can be further characterized by the infiltration of certain immune cells (such as macrophages and MDSCs).

[0135] (TGFβ1 growth factor) Finger-1: As used herein, “finger-1” is a domain within the TGFβ1 growth factor domain. In its unmutated form, the finger-1 of human proTGFβ1 contains the following amino acid sequence: CVRQLYIDFRKDLGWKWIHEPKGYHANFC (SEQ ID NO: 151). In the 3D structure, the finger-1 domain (partially shown as region “5a” in Figures 18 and 19) is very close to the latent lasso.

[0136] (TGFβ1 growth factor) Finger-2: 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: 152). Finger-2 includes “binding regions 6” (i.e., “6a” and “6b”) as described in Figures 18 and 19, which are spatially very close to the latent lasso.

[0137] GARP-proTGFβ1 complex: As used herein, the term "GARP-TGFβ1 complex" refers to a protein complex containing the proprotein form or potential form of transforming growth factor-β1 (TGFβ1) protein and a glycoprotein-A repeat-dominant protein (GARP) or a fragment or variant thereof. In some embodiments, the proprotein form or potential form of TGFβ1 protein may be referred to as "pre / potential TGFβ1 protein". In some embodiments, the GARP-TGFβ1 complex comprises GARP covalently linked to pre / potential TGFβ1 via one or more disulfide bonds. Essentially, such covalent bonds are formed by cysteine ​​residues present near the N-terminus (e.g., amino acid position 4) of the proTGFβ1 dimer complex. In other embodiments, the GARP-TGFβ1 complex comprises GARP non-covalently linked to pre / potential TGFβ1. In some embodiments, the GARP-TGFβ1 complex is a naturally occurring complex, such as the GARP-TGFβ1 complex in cells. The term "hGARP" refers to human GARP.

[0138] High affinity: As used herein, the term "high affinity" in "high affinity proTGFβ1 antibody" refers to K D The binding activity is ≤5 nM (more preferably ≤1 nM). Therefore, the high-affinity, background-independent proTGFβ1 antibody covered herein has a KB value of ≤5 nM (more preferably ≤1 nM) for each of the following antigen complexes. DValues: LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1 and LRRC33-proTGFβ1.

[0139] Human Antibody: As used herein, the term "human antibody" is intended to include antibodies derived from human germline immunoglobulin sequences having variable and constant regions. Human antibodies of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDRs, particularly CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which a germline CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0140] Humanized Antibody: The term "humanized antibody" means an antibody that contains variable heavy and light chain domain sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences have been altered to be more "human-like," i.e., more similar to human lineages. One type of humanized antibody is a CDR transplantation antibody, in which a human CDR sequence is introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Furthermore, a "humanized antibody" is an antibody, or a variant, derivative, analog, or fragment thereof, that binds immunospecifically to an antigen of interest and comprises an FR region having substantially the amino acid sequence of a human antibody and a CDR region having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR means a CDR having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical amino acid sequence to the CDR of a non-human antibody. Humanized antibodies substantially comprise all of at least one, typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all CDR regions correspond to those of non-human immunoglobulins (i.e., donor antibodies), and all or substantially all FR regions are those of human immunoglobulin common sequences. In one embodiment, the humanized antibody further comprises at least a portion of the immunoglobulin Fc region, typically a portion of the Fc region of a human immunoglobulin. In some embodiments, the humanized antibody contains a light chain and at least a variable domain of the heavy chain. The antibody may also include CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only the humanized light chain. In some embodiments, the humanized antibody contains only the humanized heavy chain. In a specific embodiment, the humanized antibody contains only the humanized light chain variable domain and / or the humanized heavy chain.

[0141] Hydrogen / deuterium exchange mass spectrometry (HDX-MS): HDX-MS is a well-known technique for querying protein identification and protein-protein interactions in solution by measuring solvent accessibility. See, for example, Wei et al., (2014) DrugDiscov Today 19(1):95-102. “Hydrogen / deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics:methodology and applications.” HDX-MS can be used to identify one or more regions of an antigen bound by an antibody (i.e., “one or more binding regions”). Thus, such one or more binding regions may contain or form epitopes.

[0142] Immune exclusion or immune-excluded tumors: As used herein, tumors characterized as “immune exclusion” are those that do not contain, or substantially do not contain, intratumoral anti-tumor lymphocytes. For example, a tumor with poor T-cell infiltration may have T cells surrounding the tumor, such as at the periphery of the tumor mass and / or near the tumor’s vascular system (“pervascular”), but these cells cannot effectively flood into the tumor to exert cytotoxic functions against cancer cells. In other cases, the tumor fails to elicit a strong immune response (so-called “cold” or “immune desert” tumors), resulting in the near-absence of T cells in the tumor environment. In contrast to immune-excluded tumors, tumors infiltrated with anti-tumor lymphocytes are sometimes referred to as “hot” or “inflammatory” tumors; these tumors tend to be more responsive to the immune system and are therefore targets for immune checkpoint blockade therapy (“CBT”). However, typically, only a small percentage of patients respond to CBT due to immune exclusion, thus making the tumor resistant to CBT.

[0143] Immunosuppression, immunosuppressive: This term refers to the ability to suppress immune cells (such as T cells, NK cells, and B cells). The gold standard for evaluating immunosuppressive function is the suppression of T cell activity, which can include antigen-specific and non-specific suppression. Regulatory T cells (Tregs) and MDSCs can be considered immunosuppressive cells. M2-polarized macrophages (e.g., disease-localized macrophages such as TAMs and FAMs) can also be characterized as immunosuppressive.

[0144] Immune memory: Immune memory refers to the immune system's ability to rapidly and specifically recognize previously encountered antigens and initiate an appropriate immune response. Normally, these are secondary, tertiary, and other subsequent immune responses to the same antigen. Immune memory is responsible for the adaptive portion of the immune system, namely specialized T cells and B cells—so-called memory T cells and B cells. Upon encountering homologous antigens in an environment containing MHC molecules on the surface of specialized antigen-presenting cells (e.g., dendritic cells), T cells that have not been exposed to the antigen proliferate and differentiate into memory and effector T cells. A single unifying theme across all memory T cell subtypes is their long lifespan and their ability to rapidly proliferate into large numbers of effector T cells upon re-exposure to their homologous antigens. Through this mechanism, the immune system is provided with a "memory" of previously encountered pathogens. Memory T cells can be CD4+ or CD8+ and typically express CD45RO. In a preclinical context, immune memory can be detected in the tumor re-attack paradigm.

[0145] Subtype Specificity: The term "subtype specificity" refers to the ability of a reagent to distinguish one subtype from other structurally related subtypes (i.e., selectivity). Subtype-specific TGFβ inhibitors exert their inhibitory activity against one subtype of TGFβ at a given concentration, but not against other subtypes of TGFβ. For example, subtype-specific TGFβ1 antibodies selectively bind to TGFβ1. TGFβ1-specific inhibitors (antibodies) target (bind and thus inhibit) the TGFβ1 subtype with significantly higher affinity than TGFβ2 or TGFβ3. For example, selectivity in this context can refer to the ability to determine selectivity by in vitro binding assays, for example... The difference is at least 500-1000 times from the corresponding affinity measured by Biacor. In some embodiments, the selectivity refers to the fact that when the inhibitor is used at a dose that effectively inhibits TGFβ1 in vivo, it does not inhibit TGFβ2 and TGFβ3. For such an inhibitor to be usable as a therapeutic agent, the dose that achieves the desired effect (e.g., a therapeutically effective dose) must fall within the window in which the inhibitor effectively inhibits the TGFβ1 subtype without inhibiting TGFβ2 or TGFβ3.

[0146] Isolated: As used herein, "isolated" antibody means an antibody that is substantially free of other antibodies that have different antigen specificities. In some embodiments, isolated antibodies are substantially free of other unintended cellular material and / or chemicals.

[0147] Large potential complexes: In the context of this disclosure, the term "large potential complex" ("LLC") refers to a complex consisting of a proTGFβ1 dimer bound to a so-called presenting molecule. Thus, large potential complexes are presenting 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 the complex. For screening purposes, the presenting molecule used to form such an LLC need not be a full-length polypeptide; however, it is generally required that a portion of the protein be able to form a disulfide bond with the proTGFβ1 dimer via a cysteine ​​residue near its N-terminal region.

[0148] Potentially related peptide (LAP): LAP is referred to as the “pre-domain” of proTGFβ1. As described in more detail herein, LAP consists of a “straight jacket” domain and an “arm” domain. The straight jacket itself is further divided into an α-1 helix and a latent lasso domain.

[0149] Latent lasso: As used herein, the "latent lasso," sometimes also referred to as the latent loop, is the domain flanking the α-1 helix and arm within the anterior domain of proTGFβ1. In its unmutated form, the latent lasso of human proTGFβ1 contains the amino acid sequence: LASPPSQGEVPPGPL (SEQ ID NO:153), and substantially corresponds to... Figure 18A The areas “2a” and “2b” shown are, and are Figure 19ARegion 1, as indicated in the diagram, spans [the region]. As used herein, the term "extended latent lasso region" refers to the latent lasso along with its nearest C-terminal motif, the α-2-helix (α2-helix) referred to as the pre-domain. Proline residues at the C-terminus of the latent lasso provide a vertical "turn," much like the "elbow" connecting the loop of the lasso to the α2-helix. The extended latent lasso comprises the regions shown as "2a," "2b," and "2c" in Figures 18 and 19. Certain high-affinity TGFβ1 activation inhibitors bind at least partially to the latent lasso or a portion thereof to confer inhibitory potency (e.g., the ability to block activation), wherein optionally, this portion of the latent lasso is ASPSQGEVPPGPL (SEQ ID NO: 266). In some embodiments, the antibody of this disclosure binds to the proTGFβ1 complex at ASPSQGEVPPGPL (SEQ ID NO: 266) or a portion thereof. Certain high-affinity TGFβ1 activation inhibitors bind at least partially to an extended latent laceration or a portion thereof to confer inhibitory potency (e.g., the ability to block activation), wherein optionally, the portion of the extended latent laceration is KLRLASPPSQGEVPPGPLPEAVL (SEQ ID NO:169).

[0150] Localized: In the context of this disclosure, the term "localized" (as in "local tumor," "local disease," etc.) refers to an anatomically isolated or separable abnormality, such as a solid tumor, rather than a systemic disease. For example, some leukemias may have both local components of the disease (e.g., bone marrow) and systemic components (e.g., circulating blood cells).

[0151] LRRC33-proTGFβ1 complex: As used herein, the term "LRRC33-TGFβ1 complex" refers to a complex formed between the pre-protein or potential form of transforming growth factor-β1 (TGFβ1) protein and a leucine-rich repeat sequence protein 33 (LRRC33; also known as a reactive oxygen species or a negative regulator of NRROS) or a fragment or variant thereof. In some embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 covalently linked to the pre- / potential TGFβ1 via one or more disulfide bonds. In practice, such covalent bonds are formed with cysteine ​​residues present near the N-terminus (e.g., amino acid position 4) of the proTGFβ1 dimer complex. In other embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 non-covalently linked to the pre- / potential TGFβ1. In some embodiments, the LRRC33-TGFβ1 complex is a naturally occurring complex, such as the LRRC33-TGFβ1 complex in cells. The term "hLRRC33" refers to human LRRC33. In vivo, LRRC33 on the cell surface and LRRC33-containing complexes can be internalized. LRRC33 is expressed on a subset of myeloid cells, including M2-polarized macrophages (such as TAMs) and MDSCs.

[0152] LTBP1-proTGFβ1 complex: As used herein, the term "LTBP1-TGFβ1 complex" refers to a protein complex comprising a proprotein or potential form of transforming growth factor-β1 (TGFβ1) protein with a potential TGF-β binding protein 1 (LTBP1) or a fragment or variant thereof. In some embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 covalently linked to the pro / potential TGFβ1 via one or more disulfide bonds. In practice, such covalent bonds are formed with cysteine ​​residues present near the N-terminus (e.g., amino acid position 4) of the proTGFβ1 dimer complex. In other embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 non-covalently linked to the pro / potential TGFβ1. In some embodiments, the LTBP1-TGFβ1 complex is a naturally occurring complex, such as the LTBP1-TGFβ1 complex in cells. The term "hLTBP1" refers to human LTBP1.

[0153] LTBP3-proTGFβ1 complex: As used herein, the term "LTBP3-TGFβ1 complex" refers to a protein complex comprising a proprotein or potential form of transforming growth factor-β1 (TGFβ1) protein and a potential TGF-β binding protein 3 (LTBP3) or a fragment or variant thereof. In some embodiments, the LTBP3-TGFβ1 complex comprises LTBP3 covalently linked to the pro / potential TGFβ1 via one or more disulfide bonds. Specifically, such covalent bonds are formed with cysteine ​​residues present near the N-terminus (e.g., amino acid position 4) of the proTGFβ1 dimer complex. In other embodiments, the LTBP3-TGFβ1 complex comprises LTBP1 non-covalently linked to the pro / potential TGFβ1. In some embodiments, the LTBP3-TGFβ1 complex is a naturally occurring complex, such as the LTBP3-TGFβ1 complex in cells. The term "hLTBP3" refers to human LTBP3.

[0154] M2 or M2-like macrophages: M2 macrophages represent a subset of activated or polarized macrophages and are present in both fibrotic and tumor microenvironments, serving as disease-associated macrophages. Cell surface markers of M2-polarized macrophages typically include CD206 and CD163 (i.e., CD206+ / CD163+). M2-polarized macrophages may also express cell surface LRRC33. Activation of M2 macrophages is primarily promoted by IL-4, IL-13, IL-10, and TGFβ; they secrete the same cytokines that activate them (IL-4, IL-13, IL-10, and TGFβ). These cells possess high phagocytic capacity and produce ECM components, angiogenesis, and chemokines. TGFβ release from macrophages can maintain myofibroblast activation, EMT, and EndMT induction in diseased tissues such as fibrotic tissue and tumor stroma. For example, M2 macrophages are essential for TGFβ-driven pulmonary fibrosis and are enriched in several tumor types.

[0155] Matrix-associated proTGFβ1: LTBP1 and LTBP3 presenting molecules are components of the extracellular matrix (ECM). LTBP1-proTGFβ1 and LTBP3-proTGFβ1 can be collectively referred to as “ECM-associated” (or “matrix-associated”) proTGFβ1 complexes, which mediate ECM-associated TGFβ1 activation / signaling. This term also includes recombinant, purified LTBP1-proTGFβ1 and LTBP3-proTGFβ1 complexes in solution (e.g., in in vitro assays) that are not physically attached to the matrix or substance.

[0156] Maximum Tolerated Dose (MTD): For safety / toxicology reasons, the term MTD generally refers to the highest amount of an analyte (such as a TGFβ1 inhibitor) evaluated with no visible adverse effect level (NOAEL). For example, in rats, the NOAEL for Ab6 is the highest evaluated dose (100 mg / kg), indicating that based on four weeks of toxicology studies, the MTD of Ab6 is >100 mg / kg. In non-human primates, the NOAEL for Ab6 is the highest evaluated dose (300 mg / kg), indicating that based on four weeks of toxicology studies, the MTD of Ab6 in non-human primates is >300 mg / kg.

[0157] Meso-Scale Discovery: "Meso-Scale Discovery," or "MSD," is a type of immunoassay that uses electrochemiluminescence (ECL) as the detection technology. Typically, a highly binding carbon electrode is used to capture proteins (e.g., antibodies). The antibody can be incubated with a specific antigen, and its binding can be detected using a secondary antibody conjugated with an electrochemiluminescence label. Once an electrical signal is generated, the light intensity can be measured to quantify the analyte in the sample.

[0158] Myelofibrosis: Myelofibrosis, also known as myeloproliferative disorders, is a relatively rare myeloproliferative disorder (e.g., cancer) that belongs to a group of diseases called myeloproliferative dysplasia. Myelofibrosis is classified as a Philadelphia chromosome-negative (-) branch of myeloproliferative neoplasms. Myelofibrosis is characterized by the proliferation of abnormal clones of hematopoietic stem cells in the bone marrow and other sites leading to fibrosis, or by scar tissue replacing bone marrow. The term myelofibrosis encompasses primary myelofibrosis (PMF), also known as chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary mean that in these cases the disease has an unknown or spontaneous origin), as well as secondary types of myelofibrosis, such as myelofibrosis secondary to polycythemia vera (PV) or essential thrombocythemia (ET). Myelofibrosis is a form of bone marrow metaplasia, which refers to changes in cell types in the blood-forming tissues of the bone marrow, and the two terms are often used synonymously. The terms homomyeloid metaplasia and myelofibrosis with myeloid metaplasia (MMM) can also be used to refer to primary myelofibrosis. Myelofibrosis is characterized by mutations that cause upregulation or overactivation of the downstream JAK pathway.

[0159] Myeloid cells: In hematopoiesis, myeloid cells are blood cells produced by progenitor cells of granulocytes, monocytes, erythrocytes, or platelets (commonly known as myeloid progenitor cells, i.e., CMP or CFU-GEMM), or more narrowly, especially those derived from the lineage of myeloid blasts (bone marrow cells, monocytes, and their progeny types). They are distinguished from lymphoid cells (i.e., lymphocytes) derived from common lymphoid progenitor cells that produce B cells and T cells. The following summarizes some myeloid cell types in mice and humans, their general morphology, typical cell surface markers, and immunosuppressive capabilities.

[0160]

[0161]

[0162] Bone marrow-derived suppressor cells (MDSCs): Bone marrow-derived suppressor cells (MDSCs) are a heterogeneous population of cells that arise under various pathological conditions and are considered to represent a pathological state of monocyte and relatively immature neutrophil activation. MDSCs comprise at least two cell types, referred to as i) “granulocytes” (G-MDSCs) or polymorphonuclear cells (PMN-MDSCs), which are phenotypically and morphologically similar to neutrophils; and ii) monocytes (M-MDSCs), which are phenotypically and morphologically similar to monocytes. MDSCs possess a unique set of genomic and biochemical characteristics and can be distinguished by specific surface molecules. For example, human G-MDSCs / PMN-MDSCs typically express the cell surface markers CD11b, CD33, CD15, and CD66. Furthermore, human G-MDSCs / PMN-MDSCs also express HLA-DR and / or arginase. In contrast, human M-MDSCs typically express the cell surface markers CD11b, CD33, and CD14. Additionally, human M-MDSCs may also express HLA-DR. In addition to these cell surface markers, MDSCs possess the ability to suppress immune cells such as T cells, NK cells, and B cells. The immunosuppressive function of MDSCs can include suppressing both antigen-nonspecific and antigen-specific functions. MDSCs can express cell surface LRRC33 and / or LRRC33-proTGFβ1.

[0163] Myofibroblasts: Myofibroblasts are cells that possess some phenotypes of fibroblasts and smooth muscle cells, typically expressing vimentin, α-smooth muscle actin (α-SMA; human gene ACTA2), and paladin. In various disease states involving extracellular matrix dysregulation (such as increased matrix stiffness), normal fibroblasts dedifferentiate into myofibroblasts in a TGFβ-dependent manner. Aberrant overexpression of TGFβ is common in myofibroblast-driven lesions. TGFβ is known to promote myofibroblast differentiation, cell proliferation, and matrix production. Myofibroblasts or myofibroblast-like cells in a fibrotic microenvironment can be termed fibrosis-associated fibroblasts (or "FAF"), while myofibroblasts or myofibroblast-like cells in a tumor microenvironment can be termed cancer-associated fibroblasts (or "CAF").

[0164] Pan-TGFβ Inhibitors / Pan-TGFβ Inhibition: The term "pan-TGFβ inhibitor" refers to any agent capable of inhibiting or antagonizing all three TGFβ isoforms. Such inhibitors can be small molecule inhibitors of TGFβ isoforms, as those known in the art. The term includes pan-TGFβ antibodies, which refer to any antibody capable of binding to each TGFβ isoform (i.e., TGFβ1, TGFβ2, and TGFβ3). In some embodiments, pan-TGFβ antibodies bind to and neutralize the activity of all three isoforms (i.e., 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 TGFβ isoforms. Examples of small molecule pan-TGFβ inhibitors include galunisertib (LY2157299 monohydrate), which is an antagonist of TGFβ receptor I kinase / ALK5 that mediates signaling of all three TGFβ isoforms.

[0165] Perivascular (infiltration): The prefix "peri-" means "around," "surrounding," or "near," so "perivascular" literally translates to "around the blood vessels." As used in this article in the context of tumor cell infiltration, the term "perivascular infiltration" refers to the pattern of entry of tumor-infiltrating immune cells (e.g., lymphocytes) through the blood vessels of a solid tumor.

[0166] Efficacy: As used herein, the term "efficacy" refers to the activity of a drug (such as an inhibitory antibody (or fragment) with inhibitory activity) relative to the concentration or amount of said drug that produces a defined effect. For example, an antibody capable of producing certain effects at a given dose is more potent than another antibody requiring twice the amount (dose) of said drug to produce an equivalent effect. Efficacy can be measured in cell-based assays (such as TGFβ activation / inhibition assays), thereby allowing the measurement of the degree of TGFβ activation (such as activation triggered by integrin binding) in a cell-based system with or without the analyte (e.g., inhibitory antibody). Generally, among those capable of binding to the same or overlapping binding regions of the antigen (e.g., cross-blocking antibodies), those with higher affinity (lower KB) have higher affinity. D Antibodies with a lower affinity (K0.05) tend to show higher affinity than those with a higher K0.05. D The antibody (value) has higher potency.

[0167] Presenting molecules: In the context of this disclosure, presenting molecules refer to proteins that form a covalent bond with a potential proprotein (e.g., proTGFβ1) and tether (“present”) an inactive complex to an extracellular niche (such as the ECM or the surface of immune cells), thereby maintaining its latency until an activation event occurs. Known presenting molecules for proTGFβ1 include LTBP1, LTBP3, GARP, and LRRC33, each of which can form presenting molecule proTGFβ1 complexes (i.e., LLCs), referred to as LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1, respectively. In fact, LTBP1 and LTBP3 are components of the extracellular matrix (ECM); therefore, LTBP1-proTGFβ1 and LTBP3-proTGFβ1 can be collectively referred to as “ECM-associated” (or “matrix-associated”) proTGFβ1 complexes that mediate ECM-associated TGFβ1 signaling / activity. On the other hand, GARP and LRRC33 are transmembrane proteins expressed on the cell surface of some cells; therefore, GARP-proTGFβ1 and LRRC33-proTGFβ1 can be collectively referred to as “cell-associated” (or “cell-surface”) proTGFβ1 complexes, which mediate cell-associated (e.g., immune cell-associated) TGFβ1 signaling / activity.

[0168] Protection (avoidance of solvent exposure): In the context of HDX-MS-based assessment of protein-protein interactions (such as antibody-antigen binding), protein exposure (e.g., regions of proteins containing epitopes) to solvents results in a negative correlation between the degree of proton exchange and the degree of binding / interaction. Therefore, when an antibody binds to a region of an antigen as described herein, the binding region is "protected" from solvent exposure because protein-protein interactions prevent the binding region from being approached by the surrounding solvent. Thus, the protected region indicates the site of interaction. Typically, a suitable solvent is a physiological buffer.

[0169] ProTGFβ1: As used herein, the term “proTGFβ1” is intended to encompass the inactive precursor form of the TGFβ1 complex, which contains the pre-domain sequence of TGFβ1 within the complex. Therefore, the term can include both the precursor and potential forms of TGFβ1. The expressions “pre / potential TGFβ1” are used interchangeably. The “pre-” form of TGFβ1 exists prior to proteolytic cleavage at the furin site. Once cleaved, the resulting form is referred to as the “potential” form of TGFβ1. The “potential” complex remains associated until further activation is triggered, such as integrin-driven activation events. The proTGFβ1 complex consists of a dimeric proTGFβ1 polypeptide linked by disulfide bonds. The potential dimeric complex is covalently linked to a single presenting molecule via a cysteine ​​residue at position 4 (Cys4) of each proTGFβ1 polypeptide. The adjective “potential” is commonly / widely used to describe the “inactive” state of TGFβ1 between integrin-mediated or other activation events. The proTGFβ1 polypeptide contains a pre-terminal domain (LAP) and a growth factor domain (SEQ ID NO:146).

[0170] Regression (tumor regression): The regression of tumor or tumor growth can be used as an indicator of efficacy in vivo. For example, in a preclinical context, the median tumor volume (MTV) and criteria for efficacy of regression response treatment can be determined by the tumor volume of the animals remaining on the last day of the study. Treatment efficacy can also be determined by the incidence and size of regression responses observed during the study period. In animals, treatment can result in partial regression (PR) or complete regression (CR). Complete regression achieved in response to a therapy (e.g., drug administration) can be referred to as “complete remission,” and subjects who achieve complete remission can be referred to as “complete responders.” Therefore, complete remission does not include spontaneous complete regression. In some implementations of preclinical tumor models, a PR response is defined as a tumor volume of 50% or less of its day 1 volume for three consecutive measurements during the study period, and equal to or greater than 13.5 mm for one or more of these three measurements. 3In some implementations, CR response is defined as three consecutive measurements during the study period showing a tumor volume of less than 13.5 mm. 3 In preclinical models, animals with a complete remission (CR) response at the end of the study can be further classified as tumor-free survivors (TFS). The term "effective tumor control" can be used to refer to the degree to which a response to treatment results in tumor regression, where, for example, the tumor volume shrinks to less than 25% of the endpoint tumor volume required to respond to treatment. For example, in a particular model, if the endpoint tumor volume is 2,000 mmHg... 3 If the tumor shrinks to less than 500mm 3 This means achieving effective tumor control. Therefore, effective tumor control encompasses both complete regression and partial regression that reaches a threshold reduction.

[0171] Regulatory T cells: "Regulatory T cells," or Tregs, are characterized by the expression of the biomarkers CD4, FOXP3, and CD25. Sometimes referred to as suppressor T cells, Tregs represent a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs are immunosuppressive and typically suppress or downregulate the induction and proliferation of effector T (Teff) cells. They can develop in the thymus (so-called CD4+Foxp3+ "natural" Tregs) or differentiate from immature CD4+ T cells in the periphery, for example, after exposure to TGFβ or retinoic acid. Tregs are capable of expressing cell surface GARP-proTGFβ1.

[0172] Resistance (to a specific therapy): Resistance to a particular therapy (such as CBT) can be attributed to an innate characteristic of the disease, such as cancer (“primary resistance”), or to an acquired phenotype that develops over time after treatment (“acquired resistance”). Patients who do not show a treatment response (e.g., are nonresponders or have a poor response to therapy) are said to have primary resistance to the therapy. Patients who initially show a treatment response but subsequently lose that response (e.g., experience progression or relapse despite continued treatment) are said to have acquired resistance to the therapy.

[0173] Response Evaluation Criteria in Solid Tumors (RECIST) and iRECIST: RECIST is a set of published rules defining when a tumor in a cancer patient improves (“response”), remains unchanged (“stable”), or worsens (“progress”) during treatment. The criteria were published in February 2000 by an international collaboration including the European Organisation for Research and Treatment of Cancer (EORTC), the National Cancer Institute of the United States, and the Clinical Trials Group of the National Cancer Institute of Canada. Subsequently, a revised version of the RECIST guideline (RECIST v 1.1) has been widely adopted (see: Eisenhauera et al., (2009), “New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1)” Eur J Cancer 45:228-247, incorporated herein by reference).

[0174] The response criteria are as follows: Complete remission (CR): All target lesions disappear; Partial remission (PR): The total LD ​​of the target lesions decreases by at least 30% relative to the baseline total LD; Stable disease (SD): From the start of treatment, the total LD ​​is neither sufficient to shrink to meet the PR criteria nor sufficient to increase to meet the PD criteria, using the minimum total LD ​​as a reference; Progressive disease (PD): From the start of treatment or the appearance of one or more new lesions, the total LD ​​of the target lesions increases by at least 20% relative to the recorded minimum total LD.

[0175] On the other hand, iRECIST provides a modified set of criteria that takes into account immune-related responses. See: www.ncbi.nlm.nih.gov / pmc / articles / PMC5648544 / (The content of which is incorporated herein by reference). The RECIST and iRECIST standards are standardized, subject to revision as more data becomes available, and are well understood in the field.

[0176] Solid tumor: The term "solid tumor" refers to a proliferative disease that results in a mass of tissue that grows abnormally or does not typically contain cystic or fluid-filled areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Solid tumors include advanced 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, and lymphocytes. Treatment of solid tumors with a subtype-selective inhibitor of TGFβ1, as described herein, typically results in TGFβ1-positive (TGFβ1+) tumors that may include multiple cell types that produce TGFβ1. In some embodiments, TGFβ1+ tumors may also express TGFβ3 (i.e., TGFβ3-positive). For example, some tumors are TGFβ1 / 3-codominant. In some embodiments, such tumors are caused by epithelial cell carcinomas (e.g., carcinomas).

[0177] Solution equilibrium titration (SET): SET is an assay by which the binding between two molecules (such as an antigen and an antibody that binds to the antigen) is measured at equilibrium in solution. For example, SET based on Meso-Scale Discovery (“MSD”) or MSD-SET is a suitable mode for determining the dissociation constant of particularly high-affinity protein-protein interactions (such as picomolar affinity antibodies bound to their antigens) at equilibrium (see, for example: Ducata et al., (2015) J Biomolecular Screening 20(10):1256-1267). SET-based assays are particularly suitable for determining the K of antibodies with sub-nanomolar (e.g., picomolar) affinity. D value.

[0178] Specific binding: As used herein, the term "specific binding" means that the interaction between an antibody or its antigen-binding moiety and an antigen depends on the presence of a specific structure (e.g., an antigenic determinant or epitope). For example, the antibody or its antigen-binding moiety binds to a specific protein rather than multiple proteins. In some embodiments, if the antibody binds to the K of the target... D For at least about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12If M or smaller, the antibody or its antigen-binding moiety specifically binds to the target, such as TGFβ1. In some embodiments, as used herein, the terms "specifically binds to an epitope of proTGFβ1," "specifically binds to an epitope of proTGFβ1," "specifically binds to TGFβ1," or "specifically binds to TGFβ1" or "means an antibody or its antigen-binding moiety that binds to proTGFβ1 and has a value of 1.0 x 10⁻⁶ as determined by a suitable in vitro binding assay (such as surface plasmon resonance and biolayer interference (BLI)). -8 M or a lower dissociation constant (K) D In one embodiment, the antibody or its antigen-binding portion may specifically bind to both human and non-human (e.g., mouse) orthologs of proTGFβ1.

[0179] Subject: In the context of therapeutic applications, the term "subject" refers to an individual receiving clinical care or intervention (such as treatment, diagnosis, etc.). Appropriate subjects include vertebrates, including but not limited to mammals (e.g., humans and non-human mammals). When the subject is a human subject, the term "patient" may be used interchangeably. In a clinical context, the terms "patient population" or "patient subgroup" are used to refer to a group of individuals belonging to a set of criteria, such as clinical criteria (e.g., disease presentation, disease stage, susceptibility to certain symptoms, response to treatment, etc.), medical history, health status, sex, age group, genetic criteria (e.g., carriers of certain mutations, polymorphisms, gene duplications, DNA sequence duplications, etc.) and lifestyle factors (e.g., smoking, alcohol consumption, exercise, etc.).

[0180] Surface plasmon resonance (SPR): Surface plasmon resonance is an optical phenomenon that allows for the real-time detection of unlabeled interactants. SPR-based biosensors, such as those commercially available from Biacore, can be used to measure interactions between biomolecules, including protein-protein interactions such as antigen-antibody binding. This technique is well-known in the art and can be used to determine parameters such as binding affinity, kinetic rate constants, and thermodynamics.

[0181] TGFβ1-related indications: “TGFβ1-related indications” means any disease or condition in which at least part of the pathogenesis and / or progression can be attributed to TGFβ1 signaling or its dysregulation. Some TGFβ1-related conditions are primarily driven by TGFβ1 subtypes. Subjects with TGFβ1-related indications may benefit from inhibition of TGFβ1 activity and / or levels. Some TGFβ1-related indications are primarily driven by TGFβ1 subtypes. TGFβ1-related indications include, but are not limited to: fibrotic conditions (such as organ fibrosis and tissue fibrosis involving chronic inflammation), proliferative conditions (such as cancers, such as solid tumors and myelofibrosis), diseases associated with ECM dysregulation (such as conditions involving matrix hardening and remodeling), diseases involving mesenchymal transition (e.g., EndMT and / or EMT), diseases involving proteases, and diseases with abnormal gene expression of certain markers described herein. These disease categories are not intended to be mutually exclusive.

[0182] TGFβ inhibitors: The term “TGFβ inhibitor” means any agent capable of antagonizing the biological activity, signaling, or function of TGFβ growth factors (e.g., TGFβ1, TGFβ2, and / or TGFβ3). This term is not intended to limit its mechanism of action and includes, for example, neutralizing inhibitors, receptor antagonists, soluble ligand traps, and TGFβ activation inhibitors. TGFβ inhibitors also include antibodies capable of reducing the availability of potential proTGFβ that can be activated in its niche, for example, by inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADPC), and antibodies that induce internalization of cell surface complexes containing potential proTGFβ, thereby removing the precursor from the plasma membrane without depleting the cell itself. Internalization can be a suitable mechanism of action against protein complexes containing LRRC33 (such as human LRRC33-proTGFβ1), which leads to a decrease in cellular levels expressing LRRC33-containing protein complexes on the cell surface.

[0183] The "TGFβ family" is a class within the TGFβ superfamily, comprising three members in humans: TGFβ1, TGFβ2, and TGFβ3, which are structurally similar. These three growth factors are known to signal through the same receptor.

[0184] TGFβ1-positive cancer / tumor: As used herein, this term refers to cancer / tumor exhibiting aberrant TGFβ1 expression (overexpression). Many human cancer / tumor types show predominant expression of a TGFβ1 subtype (note that "TGFB" is sometimes used to refer to the gene as opposed to the protein). In some cases, such cancers / tumors may show co-expression of other subtypes, such as TGFβ3. Many epithelial cancers (e.g., carcinomas) can co-express TGFβ1 and TGFβ3. In the tumor setting of TGFβ1-positive tumors, TGFβ1 can originate from a variety of sources, including, for example, cancer cells, tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), regulatory T cells (Tregs), bone marrow-derived suppressor cells (MDSCs), and the surrounding extracellular matrix (ECM). In the context of this disclosure, the preclinical cancer / tumor model that summarizes the human condition is a TGFβ1-positive cancer / tumor.

[0185] Therapeutic window: The term "therapeutic window" refers to the range of doses that produce a therapeutic response in a subject without causing significant / observable / unacceptable side effects (e.g., within the acceptable or tolerable range of side effects). The therapeutic window can be calculated as the ratio of the minimum effective concentration (MEC) to the minimum toxic concentration (MTC). For illustration, a TGFβ1 inhibitor that achieves efficacy in vivo at a dose of 10 mg / kg and shows tolerability or acceptable toxicity at a dose of 100 mg / kg provides a therapeutic window of at least 10 times (e.g., 10x). In contrast, pan-inhibitors of TGFβ are effective at 10 mg / kg but cause adverse reactions at doses below the effective dose, and are therefore considered to have "dose-limiting toxicity." Typically, the maximum tolerated dose (MTD) can be set as the upper limit of the therapeutic window.

[0186] For example, Ab6 has been shown to be effective in rats or non-human primates at doses of approximately 3–30 mg / kg / week, and has also shown no visible toxicity associated with pan-inhibition of TGFβ at doses of at least 100 or 300 mg / kg / week for 4 weeks. Based on this, Ab6 exhibits a therapeutic window of at least 3.3 times and at most 100 times.

[0187] Toxicity: As used herein, the term “toxicity” refers to a harmful in vivo effect in a subject (e.g., a patient) associated with a therapy administered to that subject (e.g., a patient), such as undesirable side effects and adverse events. “Tolerability” refers to the level of toxicity associated with a treatment or regimen that is reasonably tolerated by the patient without interruption of treatment due to toxicity. Typically, toxicity / toxicology studies are conducted in one or more preclinical models prior to clinical development to assess the safety properties of a drug candidate (e.g., a monoclonal antibody therapy). Toxicity / toxicology studies can help determine the “no visible adverse reaction level (NOAEL)” and “maximum tolerated dose (MTD)” of the test substance, from which the therapeutic window can be derived. Preferably, species sensitive to a particular intervention should be selected as preclinical animal models for safety / toxicity studies. In the case of TGFβ inhibition, suitable species include rats, dogs, and cynomolgus monkeys. Mice have been reported to be less sensitive to pharmacological inhibition of TGFβ and may not exhibit potentially dangerous toxicities in other species, including humans, although some studies have reported toxicities observed with pan-inhibition of TGFβ in mice. For the purposes of this disclosure, based on four-week toxicology studies, the NOAEL of Ab6 in rats was the highest dose evaluated (100 mg / kg), indicating an MTD > 100 mg / kg. Based on four-week toxicology studies, the MTD of Ab6 in non-human primates was > 300 mg / kg.

[0188] To determine NOAEL and MTD, it is preferable to select species sensitive to the specific intervention as preclinical animal models for safety / toxicology studies. Suitable species for TGFβ inhibition include, but are not limited to, rats, dogs, and cynomolgus monkeys. Mice have been reported to be less sensitive to pharmacological inhibition of TGFβ and may not exhibit potentially serious or dangerous toxicities in other species, including humans.

[0189] Transducibility: In the context of drug discovery and clinical development, the term "translatability" or "translatable" refers to certain qualities or characteristics of a preclinical model or data that can profile a human condition. As used herein, preclinical models profiled for a TGFβ1 indication typically show predominant expression of TGFB1 (or TGFβ1) relative to TGFB2 (or TGFβ2) and TGFB3 (or TGFβ3). In the combination therapy paradigm, for example, translatability may require the same emphasized mechanism of action as the combination of active ingredients intended to achieve its effect in the model. For example, many human tumors are immune-excluded, and TGFβ1-positive tumors exhibit primary resistance to checkpoint blockade therapy (CBT). A second therapy (such as a TGFβ1 inhibitor) can be used in combination to overcome resistance to CBT. In this case, a suitable translatable preclinical model includes TGFβ1-positive tumors that show primary resistance to checkpoint blockade therapy (CBT).

[0190] Treatment: The term "treatment" includes therapeutic treatment, preventative treatment, and any application that reduces the risk of a subject developing a disorder or other risk factors. Therefore, the term is intended to broadly refer to: eliciting a therapeutic benefit in a patient by, for example, enhancing or strengthening the body's immunity; reducing or reversing immunosuppression; reducing, removing, or eliminating harmful cells or substances in the body; reducing the burden of disease (e.g., tumor burden); preventing recurrence or relapse; prolonging the refractory period; and / or improving survival. The term includes therapeutic treatment, preventative treatment, and any application that reduces the risk of a subject developing a disorder or other risk factors. Treatment does not require a complete cure of the disorder and includes implementations that reduce symptoms or potential risk factors. In the context of combination therapy, the term may also refer to: i) the ability of a second therapy to reduce the effective dose of a first therapy to reduce side effects and increase tolerability; ii) the ability of a second therapy to make a patient more sensitive to a first therapy; and / or iii) the ability to achieve additive or synergistic clinical benefits.

[0191] Tumor-associated macrophages (TAMs): TAMs are polarized / activated macrophages with a pre-tumor phenotype (M2-like phenotype). TAMs can be bone marrow-derived monocytes / macrophages recruited to the tumor site or tissue-resident macrophages derived from erythrocyte-myeloid progenitors. The differentiation of monocytes / macrophages into TAMs is influenced by many factors, including local chemical signals such as cytokines, chemokines, growth factors, and other molecules acting as ligands, as well as cell-cell interactions within the niche (tumor microenvironment) between monocytes / macrophages. Typically, monocytes / macrophages can be polarized into so-called "M1" or "M2" subtypes, the latter being associated with a greater pre-tumor phenotype. In solid tumors, up to 50% of tumor mass may correspond to macrophages, which are preferably M2 polarized. In tumor-associated monocyte and myeloid cell populations, M1 macrophages typically express cell surface HLA-DR, CD68, and CD86, while M2 macrophages typically express cell surface HLA-DR, CD68, CD163, and CD206. Tumor-associated M2-like macrophages (such as the M2c and M2d subtypes) may express cell surface LRRC33 and / or LRRC33-proTGFβ1.

[0192] Tumor microenvironment: The term "tumor microenvironment (TME)" refers to a local disease niche in which a tumor (e.g., a solid tumor) resides within the body. The TME can include disease-associated molecular markers (a set of chemokines, cytokines, etc.), disease-associated cell populations (such as TAMs, CAFs, MDSCs, etc.), and disease-associated ECM environments (alterations in ECM components and / or structure).

[0193] Variable Region: The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chain of an antibody, typically comprising approximately 120 to 130 amino acids at the amino terminus in the heavy chain and approximately 100 to 110 amino acids at the amino terminus in the light chain. In some embodiments, even between antibodies of the same species, the variable regions of different antibodies can vary considerably in amino acid sequence. The variable region of an antibody typically determines the specificity of a particular antibody for its target.

[0194] Unless otherwise stated in the operational implementation or otherwise, all figures for the quantities of expressed components or reaction conditions used herein should be understood in all cases to be modified by the term "about". When used in conjunction with percentages, the term "about" may mean ±1%.

[0195] The indefinite article “a / an” used herein in the specification and claims, unless the opposite is explicitly stated, shall be understood to mean “at least one / an”.

[0196] As used herein in the specification and claims, the phrase “and / or” should be understood to mean “any one or both” of the elements so connected, i.e., elements that coexist in some cases and exist separately in others. In addition to the elements specifically identified by the “and / or” clause, other elements may optionally exist, whether related to or unrelated to those specifically identified, unless explicitly stated otherwise. Thus, as a non-limiting example, when used in conjunction with open-ended language such as “comprising,” in one embodiment a reference to “A and / or B” may mean A without B (optionally including elements other than B); in another embodiment, it may mean B without A (optionally including elements other than A); in yet another embodiment, it may mean both A and B (optionally including other elements); and so on.

[0197] As used herein in the specification and claims, the phrase “at least one” in relation to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list, but not necessarily including at least one of every element specifically listed in the list, and does not exclude any combination of elements in the list. This definition also allows for the optional presence of elements other than those specifically identified within the list of elements referred to by the phrase “at least one,” whether related to or unrelated to those specifically identified elements. Thus, as a non-limiting example, “at least one of A and B” (or equivalently, “at least one of A or B”, or equivalently, “at least one of A and / or B”) may mean, in one embodiment, at least one, optionally including more than one A, with no B (and optionally including elements other than B); in another embodiment, at least one, optionally including more than one B, with no A (and optionally including elements other than A); in yet another embodiment, at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0198] The use of ordinal terms such as "first," "second," and "third" to modify claim elements in claims does not imply any priority, order, or that one claim element is superior to another in terms of the chronological order of the actions of the method of execution. They are merely used as markers to distinguish one claim element with a specific name from another element with the same name (but for the use of ordinal terms), thus differentiating claim elements.

[0199] The ranges provided in this document should be understood as abbreviations of all values ​​within the range. For example, the range 1 to 50 should be understood as including any number, combination of numbers, or subrange derived from any group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, such as 10 to 20, 1 to 10, 30 to 40, etc.

[0200] Transforming growth factor-β (TGFβ)

[0201] The activity of transforming growth factor-β (TGFβ) was first described in the late 1970s and early 1980s, followed by partial purification of soluble growth factors, while the TGFβ field began 40 years ago. To date, 33 gene products have been identified, constituting a large TGFβ superfamily. Based on structural similarity, the TGFβ superfamily can be divided into at least three subclasses: TGFβ, growth differentiation factor (GDF), and bone morphogenetic protein (BMP). The TGFβ subclass consists of three highly conserved isoforms, namely TGFβ1, TGFβ2, and TGFβ3, which are encoded by three independent genes in humans.

[0202] TGFβ is believed to play a crucial role in multiple processes, such as inhibiting cell proliferation, extracellular matrix (ECM) remodeling, and immune homeostasis. The observation that TGFβ1- / - mice only survive for 3-4 weeks and suffer multiple organ failure due to massive immune activation demonstrates the importance of TGFβ1 for T cell homeostasis (Kulkarni, AB et al., Proc Natl Acad Sci USA, 1993.90(2):p.770-4; Shull, MM et al., Nature, 1992.359(6397):p.693-9). The roles of TGFβ2 and TGFβ3 remain unclear. Although these three TGFβ isoforms have different temporal and spatial expression patterns, they signal through the same receptors TGFβRI and TGFβRII, although in some cases, such as for TGFβ2 signaling, type III receptors (such as β-glycans) are also required (Feng, XH and R. Derynck, Annu Rev Cell Dev Biol, 2005.21:p.659-93; Massague, J., Annu Rev Biochem, 1998.67:p.753-91). Ligand-induced TGFβRI / II oligomerization triggers phosphorylation of SMAD transcription factors, leading to 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). The SMAD-independent TGFβ signaling pathway has also been described, for example, in cancer or in aortic lesions in Marfan mice (Derynck, R. and YE Zhang, Nature, 2003, 425(6958): p.577-84; Holm, TM et al., Science, 2011, 332(6027): p.358-61).

[0203] The biological importance of the TGFβ pathway in humans has been validated through genetic diseases. Camurati-Engelman's disease, caused by an autosomal dominant mutation in the TGFB1 gene, leads to osteodystrophy and consequently constitutive activation of TGFβ1 signaling (Janssens, K. et al., J Med Genet, 2006, 43(1): p. 1-11). Patients with Loeys / Dietz syndrome carry autosomal dominant mutations in components of the TGFβ signaling pathway, resulting in aortic aneurysms, hypertelorism, and bifid uvula (Van Laer, L., H. Dietz and B. Loeys, Adv Exp Med Biol, 2014, 802: p. 95-105). Because TGFβ pathway dysregulation is associated with a variety of diseases, several drugs targeting the TGFβ pathway have been developed and tested in patients, but with limited success rates.

[0204] Dysregulation of TGFβ signaling is associated with a variety of human diseases. In fact, in many disease conditions, this dysregulation may involve multiple aspects of TGFβ function. Diseased tissues (such as fibrotic and / or inflamed tissues and tumors) may generate TGFβ activation in the local environment, leading to disease exacerbation or progression. TGFβ is activated in an autocrine and / or paracrine manner, along with many other cytokines, chemokines, and growth factors that function in the specific disease context.

[0205] For example, in addition to cancer (e.g., malignant) cells, the tumor microenvironment (TME) also contains a variety of cell types that express TGFβ1, such as activated myofibroblast-like fibroblasts, stromal cells, infiltrating macrophages, MDSCs, and other immune cells. Therefore, the TME represents a heterogeneous population of cells that express and / or respond to TGFβ1 but are associated with more than one type of presenting cell (e.g., LTBP1, LTBP3, LRRC33, and GARP) within its niche.

[0206] Advances in immunotherapy have transformed the effective treatment of an increasing number of cancer patients. Most notably, checkpoint blockade therapy (CBT) has now become part of the standard of care for a growing number of cancers. While significant and durable responses to CBT have been observed in a growing number of cancer types, it is now clear that a significant proportion of tumors remain refractory to CBT even at the start of treatment. Therefore, addressing primary resistance remains a major challenge enabling the immune system to target and eliminate tumor cells in many patients. Efforts have been made to understand and address the underlying mechanisms conferring primary resistance to CBT in order to expand its therapeutic efficacy to more patients. However, this enthusiasm has been dampened by lackluster and failed clinical trials when CBT is combined with agents known to affect the same tumor type or modulate seemingly related components of the immune system. One possible reason is that the clear mechanistic principles underlying a given combination are often not derived from clinically sourced data, leading to unclear and confounding outcomes in trials aimed at enhancing approved monotherapy. Clearly, the design of combination immunotherapies should be based on scientific evidence relating to the underlying tumor and immune system biology.

[0207] Recently, a phenomenon known as "immune exclusion" has been coined to describe the tumor environment, where anti-tumor effector T cells (e.g., CD8+ T cells) are kept outside the tumor environment (and thus "excluded") by immunosuppressive local cues. Recent retrospective analyses of numerous clinically derived tumors have shown that activation of the TGFβ pathway mediates primary resistance to CBT. For example, transcriptional profiling and pre-treatment melanoma biopsy analyses have revealed enrichment of TGFβ-related pathways and biological processes in tumors unresponsive to anti-PD-1 CBT. In immune-excluded tumors, effector cells that would normally attack cancer cells by recognizing tumor antigens on their cell surface are prevented from entering the cancer cell site. In this way, cancer cells evade the host's immunity and immuno-oncology therapies that utilize and depend on this immunity, such as checkpoint inhibitors. Indeed, such tumors exhibit resistance to checkpoint inhibitors (such as anti-PD-1 and anti-PD-L1 antibodies) presumably because target T cells are prevented from entering the tumor and thus unable to exert their anti-cancer effects.

[0208] Retrospective analyses of tumors from numerous clinical sources have indicated that activation of the TGFβ pathway mediates primary resistance to CBT. For example, transcriptional profiling and pre-treatment melanoma biopsy analyses have shown enrichment of TGFβ-related pathways and biological processes in tumors unresponsive to anti-PD-1 CBT. Recently, similar analyses of tumors from patients with metastatic urothelial carcinoma have shown that the lack of response to PD-L1 blockade with atezolizumab is associated with transcriptional signatures of TGFβ signaling, particularly in tumors where CD8+ T cells appear to be excluded from the tumor. In the EMT-6 syngeneic mouse model of breast cancer, the crucial role of TGFβ signaling in mediating immune exclusion leading to anti-PD(L)1 resistance has been demonstrated. Although EMT-6 tumors respond poorly to treatment with anti-PD-L1 antibodies, combining this checkpoint inhibitor with 1D11 (a group of antibodies that block the activity of all TGFβ isoforms) significantly increases the frequency of complete remission compared to treatment with a single inhibitor. Synergistic antitumor activity is thought to result from altered cancer-associated fibroblast (CAF) phenotypes and disruption of the immune exclusion phenotype, leading to the infiltration of activated CD8+ T cells into tumors. Similar results were obtained in mouse models of colorectal cancer and metastasis using a combination of anti-PD-L1 antibody and galunisertib (a small molecule inhibitor of type I TGFβ receptor ALK5 kinase). Overall, 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 work has suggested a relationship between TGFβ pathway activation and primary CBT resistance, TGFβ signaling has long been associated with the characterization of cancer pathogenesis. As a potent immunosuppressive factor, TGFβ prevents the activation of antitumor T cells and promotes immunosuppressive macrophages. Malignant cells are typically resistant to TGFβ signaling as a mechanism to evade its growth and tumor-suppressive effects. TGFβ activates CAFs, inducing extracellular matrix production and promoting tumor progression. Finally, TGFβ induces EMT, thereby supporting tissue invasion and tumor metastasis.

[0209] Mammals possess distinct genes encoding and expressing three TGFβ growth factors: TGFβ1, TGFβ2, and TGFβ3. All of these growth factors signal through the same heteromeric TGFβ receptor complex. Despite the shared signaling pathway, each TGFβ isoform appears to possess a unique biological function, as demonstrated by the non-overlapping TGFβ knockout mouse phenotype. All three TGFβ isoforms are represented as inactive predomain growth factor complexes, where the TGFβ predomain is also known as the latent related peptide (LAP), which surrounds the growth factor and places it in a potential non-signaling state. Furthermore, latent TGFβ is co-expressed with latent TGFβ-binding proteins, forming a large latent complex (LLC) via disulfide bonds. Binding of latent TGFβ to latent TGFβ-binding protein-1 (LTBP1) or LTBP3 enables tethering to the extracellular matrix, while binding to the transmembrane proteins GARP or LRRC33 enables processing on the surface of Treg cells or macrophages, respectively. In vivo, latent TGFβ1 and latent TGFβ3 are activated by a group of αV integrins that bind to a shared RGD sequence on the LAP, triggering a conformational change to release growth factors. The mechanism of latent TGFβ2 activation is unclear because it lacks a shared RGD motif. TGFβ1 released via proteolytic cleavage of the LAP is also considered an activation mechanism, but its biological relevance remains unknown.

[0210] While the pathogenic role of TGFβ activation in several disease states is well-established, it is equally clear that therapeutic targeting of the TGFβ pathway is challenging due to its pleiotropic effects resulting from broad and persistent pathway inhibition. For example, multiple studies have shown that small-molecule-mediated inhibition of the TGFβ type I receptor kinase ALK5 (TGFBR1), or blocking all three highly associated TGFβ growth factors with high-affinity antibodies, leads to severe valvular heart disease in mice, rats, and dogs. These “pan-TGFβ” approaches, which block all TGFβ signaling, therefore have very narrow therapeutic windows, proving to hinder treatment of many disease-related processes with significant unmet medical needs. To date, no TGFβ-targeting therapies have been approved, and clinical trial results using this approach have largely been disappointing, likely due to the use of dosing regimens that have proven ineffective in addressing safety concerns.

[0211] Safety concerns regarding widespread TGFβ inhibition, and compelling evidence that this pathway plays a crucial role in various disease processes, suggest that a better understanding of the specific roles of one or more TGFβ family members in disease pathology could lead to feasible avenues for therapeutic intervention. Regarding TGFβ and responses to it, we observed widespread expression of TGFβ1 in many human tumors, indicating that this family member may be a major driver of this pathway's contribution to primary resistance.

[0212] As mentioned above, mounting evidence suggests that TGFβ may be a key player in establishing and / or maintaining immunosuppression in diseased tissues, including the immune-excluded tumor environment. Therefore, TGFβ inhibition can relieve immunosuppression and enable effector T cells (particularly cytotoxic CD8+ T cells) to enter and kill target cancer cells. In addition to tumor invasion, TGFβ inhibition can also promote CD8+ T cell proliferation. Such proliferation can occur in lymph nodes and / or within the tumor (intratumoral). Although the exact mechanisms elucidating this process are not yet clear, immunosuppression can be expected to be mediated at least in part by immune cell-associated TGFβ1 activation involving regulatory T cells and activated macrophages. TGFβ has been reported to directly promote the expression of Foxp3 in CD4+ T cells, thereby converting them to a regulatory (immunosuppressive) phenotype (i.e., Treg). Moreover, Tregs inhibit effector T cell proliferation (see, for example, Figure 26B This process reduces the immune response. It has been shown to be TGFβ1-dependent and may involve GARP-associated TGFβ1 signaling. Observations in both human and animal models have shown that increased Tregs in the tumor-associated macrophage (TME) are associated with poor prognosis in multiple cancer types. Furthermore, the applicant has previously found that M2-polarized macrophages exposed to tumor-derived factors (such as M-CSF) significantly upregulate the cell surface expression of LRRC33, a TGFβ1 presenting molecule (see, e.g., PCT / US2018 / 031759). These so-called tumor-associated macrophages (or TAMs) are thought to promote the TGFβ1-dependent immunosuppression observed in the TME and promote tumor growth.

[0213] Many solid tumors are characterized by an abundance of myofibroblasts or myofibroblast-like cells in their tumor stroma. These cells produce a collagenous matrix that surrounds or encapsulates the tumor (as in connective tissue formation), which can be at least partially caused by overactivation of TGFβ1 signaling. TGFβ1 activation is expected to be mediated in the tumor stroma by ECM-associated presenting molecules (e.g., LTBP1 and LTBP3).

[0214] The applicant has previously disclosed antibodies capable of inhibiting TGFβ1 activation in many of these biological contexts, which have shown promising activity both in vivo and in vitro (see, for example, PCT / US2018 / 012601). However, the following challenges exist: i) developing an improved antibody with less bias in affinity for a variety of antigen complexes to ensure consistent inhibitory activity in different biological contexts or in the niches where fundamentally related TGFβ1 resides, and / or, ii) developing an antibody that provides even greater potency than previously described counterparts.

[0215] For the work presented herein, it is conceivable that improved antibodies should embody all or most of the following characteristics: 1) maintain selectivity for TGFβ1 to minimize undesirable toxicity associated with pan-inhibition (“subtype selectivity”) (see, e.g., PCT / US2017 / 021972); 2) exhibit broad binding activity across a variety of biological backgrounds, or both matrix-related and cell-related types (“background-independent”); 3) achieve more uniform or unbiased affinity among multiple antigen complexes (“homogeneity”); 4) exhibit strong binding activity against each antigen complex (“high affinity”); and 5) possess potent inhibitory activity against each background (“potency”). Furthermore, a preferred mechanism of action is inhibition of the activation step, allowing the inhibitor to target the underlying TGFβ1 complex in the tissue lineage, thereby preemptively blocking downstream activation events for a durable effect, rather than directly targeting soluble / free growth factors (“persistence”). As further detailed herein, the novel, improved TGFβ1 inhibitors disclosed herein are potentially highly potent and selective inhibitors of TGFβ1 activation. The data presented herein, in particular, demonstrate that this mechanism of subtype-specific inhibition is sufficient to overcome primary resistance to anti-PD-1 in a syngeneic mouse model that adequately encapsulates some characteristics of primary resistance to CBT observed in human cancers. Compared to pan-TGFβ inhibitors, these antibodies exhibit improved preclinical safety properties, and these efficacy data provide a theoretical basis for exploring the use of selective TGFβ1 inhibition in cancer immunotherapy to broaden and enhance clinical responses to checkpoint blockade, as well as to manage a variety of other TGFβ1-related indications.

[0216] Novel, high-affinity, subtype-selective antibodies against proTGFβ1

[0217] General characteristics

[0218] This document discloses high-affinity, improved TGFβ1 inhibitors characterized by enhanced binding properties, increased inhibitory potency, and retention of desired safety properties and subtype selectivity compared to previously disclosed TGFβ1 selective inhibitors. These TGFβ1 selective inhibitors disclosed herein are monoclonal antibodies (e.g., immunoglobulins, engineered immunoglobulin-like molecules, or their antigen-binding fragments or portions) that specifically bind to at least a portion of the pre-domain (sometimes referred to as “LAP”) of a potential proTGFβ1 complex and exhibit subtype-selective inhibitory activity against TGFβ1 (see “Core Characteristics” in Table 1).

[0219] The enhanced binding properties of the antibodies according to this disclosure include increased affinity, as measured under equilibrium conditions. In some embodiments, as measured by MSD-SET, the antibody has a KD ≤1 nM against at least one human LLC complex (hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and / or hLRRC33-proTGFβ1). In some embodiments, as measured by MSD-SET, such antibodies have a KD ≤1 nM against two human LLC complexes selected from hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. In some embodiments, as measured by MSD-SET, such antibodies have a KD ≤1 nM against three human LLC complexes selected from the following: hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. In preferred embodiments, as measured by MSD-SET, such antibodies have a KD ≤1 nM against each of the following human LLC complexes: hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. According to this disclosure, at equilibrium, high-affinity antibodies may have a KD value of 1 nM or less (e.g., ≤1 nM, ≤0.5 nM, ≤400 pM, ≤300 pM, ≤200 pM, and ≤100 pM) against a specific antigen (e.g., an antigen complex).

[0220] The invention also includes antibodies or antigen-binding fragments thereof, which, as measured at equilibrium (e.g., MSD-SET), are capable of specifically binding to each human LLC complex (hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1) with a KD of ≤10 nM (e.g., ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.5 nM, and ≤0.1 nM). In some embodiments, as measured by solution equilibrium titration, the antibody binds to each of the aforementioned LLC complexes with a KD of ≤5 nM. In some embodiments, as measured by solution equilibrium titration, the antibody binds to each of the aforementioned LLC complexes with a KD of ≤1 nM.

[0221] For therapeutic use in treating TGFβ1-related indications involving dysregulation of both the extracellular matrix and immune components, antibodies with high affinity (e.g., KD ≤ 1 nM) for at least one of ECM-associated proTGFβ1 complexes (hLTBP1-proTGFβ1 and / or hLTBP3-proTGFβ1) and additionally at least one of cell-associated proTGFβ1 complexes (hGARP-proTGFβ1 and / or hLRRC33-proTGFβ1) are preferred to exert inhibitory effects on both conditions (e.g., at the ECM and attracted to immune cells). In some embodiments, the antibody has high affinity (e.g., KD ≤ 1 nM) for both hLTBP1-proTGFβ1 and hLTBP3-proTGFβ1 and additionally at least one cell-associated proTGFβ1 complex (hGARP-proTGFβ1 or hLRRC33-proTGFβ1). In other embodiments, the antibodies exhibit high affinity (e.g., KD ≤ 1 nM) for each of the aforementioned complexes (hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1). In a preferred embodiment, such antibodies have a KD ≤ 200 pM (e.g., ≤ 100 pM) for each human complex, as measured by a solution equilibrium titration method (e.g., MSD-SET).

[0222] Embodiments of this disclosure include high-affinity, background-independent antibodies. Such antibodies are capable of binding with equivalent affinity to four known presenting molecule-proTGFβ1 complexes, namely LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. Equivalent affinity may mean that the lowest affinity (highest KD value) exhibited by the antibody in the four antigen complexes is not less than five times the average calculated from the other three affinities; or that the highest affinity (lowest KD value) exhibited by the antibody in the four antigen complexes does not exceed five times the average calculated from the other three affinities. In some embodiments, such antibodies may be referred to as having equivalent affinity when the ratio of the average KD value of two ECM-associated complexes to the average KD value of two cell-associated complexes does not exceed three times.

[0223] Antibodies with equivalent affinity can achieve more uniform (e.g., unbiased) inhibition regardless of the specific presenting molecule associated with the proTGFβ1 complex (and thus "background-independent"). In a particularly preferred embodiment, the antibody is a high-affinity, background-independent antibody because the affinity for each of the four human LLCs, as measured by a solution equilibrium titration method, is 1 nM or less (e.g., 200 pM or less), and the antibody has equivalent affinity for all four human LLCs discussed above. For example, the bias observed in the average affinity between the matrix-associated complex and the cell-associated complex does not exceed three-fold.

[0224] In some implementations, such antibodies bind to each of the aforementioned complexes (hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1) with KD specificity ≤10 nM (e.g., ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.5 nM, and ≤0.1 nM), as measured by a solution equilibrium titration method (e.g., MSD-SET).

[0225] Any of the inhibitory antibodies covered herein can bind to each of the aforementioned large potential complexes (hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1) at one or more binding regions of at least a portion of a latent lasso within the proTGFβ1 complex's pre-domain. Such binding regions may also contain at least a portion of a growth factor domain. In a particularly preferred embodiment, such antibodies bind to each of the LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1 complexes at a KD value of ≤200 pM (e.g., ≤150 pM and ≤100 pM) within a binding region of an LLC complex containing at least a portion of the latent lasso and at least a portion of the growth factor domain.

[0226] In some embodiments, regardless of the activation mode, high-affinity, background-independent antibodies that selectively inhibit TGFβ1 can inhibit activated TGFβ1. For example, certain integrins are known to directly bind to the RGD motif within the predomain of LLC and mechanically “pull open” the cage-like predomain structure, thereby releasing TGFβ1 growth factor from the potential complex. Separately, certain proteases present in the extracellular environment have been shown to activate TGFβ1 in an integrin-independent manner. Antibodies that directly target the RGD motif to interfere with integrin binding may not inhibit protease-dependent activation of TGFβ1. Conversely, antibodies that directly target one or more of the protease recognition or cleavage sites may not inhibit integrin-dependent activation of TGFβ1. In contrast, in a preferred embodiment of the invention, high-affinity, background-independent antibodies can inhibit both integrin-dependent and protease-dependent activation of TGFβ1.

[0227] While high affinity binding to target proteins is an essential characteristic of antibody therapeutics, the ability to cross-react with counterparts of other species is also advantageous. In particular, given that most preclinical pharmacological models are in rodents, species cross-reactivity with mouse / rat proteins provides a suitable tool for preclinical studies as an alternative antibody. Therefore, in some embodiments, the high-affinity antibodies of this disclosure advantageously cross-react with other mammalian counterparts, such as mice, rats, and / or non-human primates.

[0228] Among the novel antibodies covered by this disclosure, particularly preferred antibody classes and their characteristics are classified and discussed below.

[0229] Preferred features

[0230] In some implementations, in addition to the core properties, the preferred antibodies disclosed herein also meet one or more of the antibody criteria in categories 1-5 as listed in Table 1 herein.

[0231] In some embodiments, other desired criteria for the antibodies of the present invention are defined by their binding properties (e.g., antibody affinity for antigens). In this context, "antigen" comprises at least four protein complexes, namely the human large potential complex (LLC) of TGFβ1, referred to as hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes. According to the invention disclosed herein, antibodies can be typically bound according to K... D A certain affinity is measured for each of these complexes. Category 1 and Category 2 antibodies are included within the scope of these implementations. For the purpose of defining criteria based on binding properties (e.g., Category 1 and 2), the affinity of the antibody at equilibrium is determined, rather than by kinetic assays (such as BLI).

[0232] Alternatively or concurrently, other desired criteria for the antibodies of the present invention are defined by their amino acid sequences. Category 3 and Category 4 antibodies are defined by their CDR sequences, while Category 5 antibodies are defined by their heavy and light chain variable domain sequences.

[0233] Table 1: Preferred features of the novel, high-affinity, selective TGFβ1 inhibitor of the present invention

[0234]

[0235]

[0236]

[0237]

[0238] The following provides non-limiting implementations for each category.

[0239] Category 1 Antibodies

[0240] The antibody disclosed in this article is a high-affinity, subtype-selective antibody that can specifically target the potential large complex of human TGFβ1.

[0241] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof specifically binding to each of the following human LLCs at a KD concentration of ≤200 pM: hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes, wherein affinity is measured in equilibrium using a suitable assay (e.g., an assay based on solution equilibrium titration).

[0242] As measured by solution equilibrium titration, such antibodies or fragments can bind each hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complex with a KD of ≤150 pM. More preferably, as measured by solution equilibrium titration, such antibodies or fragments can bind each hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complex with a KD of ≤100 pM. Any suitable in vitro affinity assay capable of measuring the KD value of the antibody under equilibrium conditions can be used, including, for example, MSD-SET, which is described in more detail elsewhere herein. Non-limiting examples of antibodies disclosed herein that meet the preferred antibody criteria of Category 1 include: Ab6, Ab22, Ab24, Ab26, Ab29, Ab30, Ab31, Ab32, and Ab33.

[0243] The antibody can also bind to the corresponding LLC in other species with high specificity and high affinity. In a preferred embodiment, the antibody exhibits species cross-reactivity with its mouse counterpart.

[0244] Category 2 antibodies

[0245] The antibody disclosed in this article is a high-affinity, subtype-selective antibody that can specifically target the potential large complex of human TGFβ1.

[0246] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to each of the following human LLCs at a KD concentration of ≤1 nM: hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes, wherein affinity is measured in equilibrium using a suitable assay (e.g., a solution equilibrium titration-based assay), and wherein the antibody or fragment binds to the human LLC in a binding region comprising at least a portion of a latent lasso. A latent lasso is a protein domain that forms part of a so-called “straight jacket” before forming a pre-domain structure. In its native form, the latent lasso of the human proTGFβ1 polypeptide has the amino acid sequence LASPPSQGEVPPGPL (SEQ ID NO: 153). Any suitable technique can be used to determine whether the antibody binds to the human TGFβ1 LLC in a region comprising at least a portion of the latent lasso. For example, a competitive assay using the corresponding polypeptide can be performed. In some implementations, the binding region can be determined by HD-X or X-ray crystallography.

[0247] In some embodiments, such antibodies or fragments, as measured by solution equilibrium titration, can bind each of the hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes at a KD of ≤500 pM (optionally ≤400 pM, ≤300 pM, ≤200 pM, or ≤100 pM), wherein said antibody or fragment binds to human LLC in a binding region comprising at least a portion of a latent lasso. Non-limiting examples of antibodies disclosed herein that meet the preferred antibody criteria of Category 2 include Ab5 and Ab6.

[0248] In some embodiments, such antibodies may further bind to LLC in one or more other binding regions comprising at least a portion of the growth factor domain within the proTGFβ1 complex. In some embodiments, the additional binding occurs only in the context of the potential complex, such that the antibody binds nonspecifically to free growth factor that does not bind to the pre-domain complex. One or more other binding regions within the LLC growth factor domain may comprise at least a portion of a protein domain referred to as “finger 1” and / or “finger 2”. Thus, such antibodies may bind a combinatorial epitope comprising at least one amino acid residue of a latent lasso and at least one amino acid residue of a growth factor domain.

[0249] The antibody can also bind to the corresponding LLC in other species with high specificity and high affinity. In a preferred embodiment, the antibody exhibits species cross-reactivity with its mouse counterpart.

[0250] Category 3 Antibodies

[0251] The antibody disclosed in this article is a high-affinity, subtype-selective antibody that can specifically target the potential large complex of human TGFβ1.

[0252] In a further aspect, the present invention provides an antibody or an antigen-binding fragment thereof comprising H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, wherein CDR-H1 has an amino acid sequence represented by FTF(X1)(X2)(X3)(X4)M(X5) (SEQ ID NO: 143). In some embodiments, X1 may be S, G, or A; X2 may be S or F; X3 may be F or Y; X4 may be S or A; and / or, X5 may be D, N, or Y, in any combination. In some embodiments in which H-CDR1 contains at least one amino acid substitution, position X1 may be substituted with S; position X2 may be substituted with S; position X3 may be substituted with F; position X4 may be substituted with S; and / or, position X5 may be substituted with D.

[0253] The antibody CDR-H2 has an amino acid sequence represented by YI(X1)(X2)(X3)A(X4)TIYYA(X5)SVKG (SEQ ID NO: 144). In some embodiments, X1 can be S or H; X2 can be P or S; X3 can be S or D; X4 can be D or S; and / or, X5 can be D or G, in any combination. In some embodiments where H-CDR2 contains at least one amino acid substitution, position X1 can be substituted with S; position X2 can be substituted with P; position X3 can be substituted with D; position X4 can be substituted with S; and / or, position X5 can be substituted with D.

[0254] The antibody's CDR-H3 has an amino acid sequence represented by (X1)R(X2)(X3)(X4)D(X5)GDML(X6)P (SEQ ID NO: 145). In some embodiments, X1 can be A or V; X2 can be G or A; X3 can be V or T; X4 can be L or W; X5 can be Y or M; and / or, X6 can be M or D, in any combination. In some embodiments where H-CDR3 contains at least one amino acid substitution, position X1 can be substituted with A; position X2 can be substituted with G; position X3 can be substituted with V; position X4 can be substituted with L; position X5 can be substituted with Y; and / or, position X6 can be substituted with D.

[0255] CDR-L1 has the amino acid sequence QASQDITNYLN (SEQ ID NO:105), which optionally has one or two amino acid changes.

[0256] CDR-L2 has the amino acid sequence DASNLET (SEQ ID NO:106), which optionally has one or two amino acid changes.

[0257] CDR-L3 has the amino acid sequence QQADNHPPWT (SEQ ID NO:12), which optionally has one or two amino acid changes.

[0258] Non-limiting examples of antibodies disclosed herein that meet the criteria for preferred antibodies of category 3 include: Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, and Ab34.

[0259] Table 2 below summarizes the common CDR sequences of Category 3 antibodies. In some embodiments, each CDR sequence may optionally contain one or more amino acid substitutions as shown below.

[0260] Table 2: Common CDR sequences and preferred amino acid substitutions in heavy and light chains

[0261]

[0262]

[0263] In some embodiments, the class 3 antibody or its antigen-binding fragment binds to each of the following human LLCs with a KD specificity of ≤1 nM: hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes, wherein the affinity is measured in equilibrium using a suitable assay (such as a solution equilibrium titration-based assay).

[0264] In some embodiments, the antibody or fragment binds to human LLC in a binding region containing at least a portion of a latent lasso. A latent lasso is a protein domain that forms part of a so-called "straight jacket" before the formation of the pre-domain structure. In its native form, the latent lasso of the human proTGFβ1 polypeptide has the amino acid sequence LASPPSQGEVPPGPL (SEQ ID NO: 153). Any suitable technique can be used to determine whether the antibody binds to human TGFβ1 LLC in a region containing at least a portion of the latent lasso. For example, a competitive assay using the corresponding polypeptide can be performed. In some embodiments, the binding region can be determined by HD-X or X-ray crystallography.

[0265] In some embodiments, such as those measured by solution equilibrium titration, such antibodies or fragments can bind each of the hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes at a KD of ≤500 pM (optionally ≤400 pM, ≤300 pM, ≤200 pM, or ≤100 pM), wherein said antibody or fragment binds to human LLC in a binding region comprising at least a portion of a latent lasso.

[0266] In some embodiments, such antibodies may further bind to LLC in one or more other binding regions comprising at least a portion of the growth factor domain within the proTGFβ1 complex. In some embodiments, the additional binding occurs only in the context of the potential complex, such that the antibody binds nonspecifically to free growth factor that does not bind to the pre-domain complex. One or more other binding regions within the LLC growth factor domain may comprise at least a portion of a protein domain referred to as “finger 1” and / or “finger 2”. Thus, such antibodies may bind a combinatorial epitope comprising at least one amino acid residue of a latent lasso and at least one amino acid residue of a growth factor domain.

[0267] The antibody can also bind to the corresponding LLC in other species with high specificity and high affinity. In a preferred embodiment, the antibody exhibits species cross-reactivity with its mouse counterpart.

[0268] This document includes cross-blocking antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or fragment thereof cross-blocks or cross-competes with one of Class 3 antibodies, wherein, as measured by MSD-SET, the antibody has a KD ≤1 nM against at least one human LLC complex (hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and / or hLRRC33-proTGFβ1). In some embodiments, as measured by MSD-SET, the antibody has a KD ≤1 nM against two human LLC complexes selected from hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. In some embodiments, as measured by MSD-SET, such antibodies have a KD ≤1 nM against three human LLC complexes selected from the group consisting of hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. In preferred embodiments, as measured by MSD-SET, such antibodies have a KD ≤1 nM against each of the following human LLC complexes selected from the group consisting of hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. According to this disclosure, at equilibrium, high-affinity antibodies may have a KD value of 1 nM or lower against a specific antigen (e.g., an antigen complex), such as ≤1 nM, ≤0.5 nM, ≤400 pM, ≤300 pM, ≤200 pM, and ≤100 pM.

[0269] Category 4 Antibodies

[0270] The antibody disclosed in this article is a high-affinity, subtype-selective antibody that can specifically target the potential large complex of human TGFβ1.

[0271] In a further aspect, the present invention provides an antibody or an antigen-binding fragment thereof comprising H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, wherein: H-CDR1 comprises FTFSSFSMD (SEQ ID NO: 107) or FTFSSSMMN (SEQ ID NO: 114), wherein optionally each may contain up to 4 amino acid changes (optionally up to 4, up to 3, up to 2, or 1 amino acid change); H-CDR2 comprises YISPDASTIYYADSVKG (SEQ ID NO: 111), wherein optionally H-CDR2 may contain up to 4 amino acid changes (optionally up to 4, up to 3, up to 2, or 1 amino acid change); and H-CDR3 comprises ARGVLDYGDMLDP (SEQ ID NO: 114). NO:110), wherein optionally H-CDR3 may contain up to 3 amino acid changes (optionally up to 3, up to 2 or 1 amino acid changes); L-CDR1QASQDITNYLN (SEQ ID NO:105), which has optionally 1 or 2 amino acid changes; L-CDR2 containing DASNLET (SEQ ID NO:106), which has optionally 1 or 2 amino acid changes; and L-CDR3 containing QQADNHPPWT (SEQ ID NO:12), which has optionally 1 or 2 amino acid changes.

[0272] Non-limiting examples of antibodies disclosed herein that meet the criteria for preferred antibodies of category 4 include: Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34.

[0273] Table 3 below summarizes the CDR sequences of category 4 antibodies. In some embodiments, each CDR sequence may optionally contain one or more amino acid substitutions as shown below.

[0274] Table 3: CDR sequences and variants

[0275]

[0276]

[0277] In some embodiments, the category 4 antibody or its antigen-binding fragment binds to each of the following human LLCs with a KD specificity of ≤1 nM: hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes, wherein the affinity is measured in equilibrium using a suitable assay (such as an assay based on solution equilibrium titration).

[0278] In some embodiments, the antibody or fragment binds to human LLC in a binding region containing at least a portion of a latent lasso. A latent lasso is a protein domain that forms part of a so-called "straight jacket" before the formation of the pre-domain structure. In its native form, the latent lasso of the human proTGFβ1 polypeptide has the amino acid sequence LASPPSQGEVPPGPL (SEQ ID NO: 153). Any suitable technique can be used to determine whether the antibody binds to human TGFβ1 LLC in a region containing at least a portion of the latent lasso. For example, a competitive assay using the corresponding polypeptide can be performed. In some embodiments, the binding region can be determined by HD-X or X-ray crystallography.

[0279] In some embodiments, such as those measured by solution equilibrium titration, such antibodies or fragments can bind each of the hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes at a KD of ≤500 pM (optionally ≤400 pM, ≤300 pM, ≤200 pM, or ≤100 pM), wherein said antibody or fragment binds to human LLC in a binding region comprising at least a portion of a latent lasso.

[0280] In some embodiments, such antibodies may further bind to LLC in one or more other binding regions comprising at least a portion of the growth factor domain within the proTGFβ1 complex. In some embodiments, the additional binding occurs only in the context of the potential complex, such that the antibody binds nonspecifically to free growth factor that does not bind to the pre-domain complex. One or more other binding regions within the LLC growth factor domain may comprise at least a portion of a protein domain referred to as “finger 1” and / or “finger 2”. Thus, such antibodies may bind a combinatorial epitope comprising at least one amino acid residue of a latent lasso and at least one amino acid residue of a growth factor domain.

[0281] The antibody can also bind to the corresponding LLC in other species with high specificity and high affinity. In a preferred embodiment, the antibody exhibits species cross-reactivity with its mouse counterpart.

[0282] This document includes cross-blocking antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or fragment thereof cross-blocks or cross-competes with one of Class 4 antibodies, wherein, as measured by MSD-SET, the antibody has a KD ≤1 nM against at least one human LLC complex (hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and / or hLRRC33-proTGFβ1). In some embodiments, as measured by MSD-SET, the antibody has a KD ≤1 nM against two human LLC complexes selected from hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. In some embodiments, as measured by MSD-SET, such antibodies have a KD ≤1 nM against three human LLC complexes selected from the group consisting of hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. In preferred embodiments, as measured by MSD-SET, such antibodies have a KD ≤1 nM against each of the following human LLC complexes selected from the group consisting of hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. According to this disclosure, at equilibrium, high-affinity antibodies may have a KD value of 1 nM or lower against a specific antigen (e.g., an antigen complex), such as ≤1 nM, ≤0.5 nM, ≤400 pM, ≤300 pM, ≤200 pM, and ≤100 pM.

[0283] Category 5 Antibodies

[0284] The antibody disclosed in this article is a high-affinity, subtype-selective antibody that can specifically target the potential large complex of human TGFβ1.

[0285] In a further aspect, the present invention provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain (V) having at least 90% sequence identity with the following. H ): EVQLVESGGGLVQPGGSLRLSCTASGFTFSSFSMDWVRQAPGKGLEWVSYISPSADTIYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARGVLDYGDMLMPWGQGTLVTVSS (SEQ ID NO:13); and, a light chain variable domain (V) having at least 90% sequence identity with the following. L): DIQMTQSPSSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQADNHPPWTFGGGTKVEIK (SEQ ID NO: 15).

[0286] In some embodiments, the heavy chain variable domain of the antibody is associated with the V shown in SEQ ID NO:13. H The sequences have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0287] In some embodiments, the heavy chain variable domain of the antibody is related to the V mentioned above. H The sequences have at least 95% identity.

[0288] In some embodiments, the heavy chain variable domain of the antibody is associated with the V shown in SEQ ID NO:15. L The sequences have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0289] In some embodiments, the variable domain of the antibody's light chain is related to the V described above. L The sequences have at least 95% identity.

[0290] Non-limiting examples of antibodies disclosed herein that meet the criteria for preferred antibodies in category 5 include: Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34.

[0291] In some embodiments, the class 5 antibody or its antigen-binding fragment binds to each of the following human LLCs with a KD specificity of ≤1 nM: hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes, wherein the affinity is measured in equilibrium using a suitable assay (such as an assay based on solution equilibrium titration).

[0292] In some embodiments, the antibody or fragment binds to human LLC in a binding region containing at least a portion of a latent lasso. A latent lasso is a protein domain that forms part of a so-called "straight jacket" before the formation of the pre-domain structure. In its native form, the latent lasso of the human proTGFβ1 polypeptide has the amino acid sequence LASPPSQGEVPPGPL (SEQ ID NO: 153). Any suitable technique can be used to determine whether the antibody binds to human TGFβ1 LLC in a region containing at least a portion of the latent lasso. For example, a competitive assay using the corresponding polypeptide can be performed. In some embodiments, the binding region can be determined by HD-X or X-ray crystallography.

[0293] In some embodiments, such as those measured by solution equilibrium titration, such antibodies or fragments can bind each of the hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes at a KD of ≤500 pM (optionally ≤400 pM, ≤300 pM, ≤200 pM, or ≤100 pM), wherein said antibody or fragment binds to human LLC in a binding region comprising at least a portion of a latent lasso.

[0294] In some embodiments, such antibodies may further bind to LLC in one or more other binding regions comprising at least a portion of the growth factor domain within the proTGFβ1 complex. In some embodiments, the additional binding occurs only in the context of the potential complex, such that the antibody binds nonspecifically to free growth factor that does not bind to the pre-domain complex. One or more other binding regions within the LLC growth factor domain may comprise at least a portion of a protein domain referred to as “finger 1” and / or “finger 2”. Thus, such antibodies may bind a combinatorial epitope comprising at least one amino acid residue of a latent lasso and at least one amino acid residue of a growth factor domain.

[0295] The antibody can also bind to the corresponding LLC in other species with high specificity and high affinity. In a preferred embodiment, the antibody exhibits species cross-reactivity with its mouse counterpart.

[0296] This document includes cross-blocking antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or fragment thereof cross-blocks or cross-competes with one of Class 5 antibodies, wherein, as measured by MSD-SET, the antibody has a KD ≤1 nM against at least one human LLC complex (hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and / or hLRRC33-proTGFβ1). In some embodiments, as measured by MSD-SET, the antibody has a KD ≤1 nM against two human LLC complexes selected from hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. In some embodiments, as measured by MSD-SET, such antibodies have a KD ≤1 nM against three human LLC complexes selected from the group consisting of hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. In preferred embodiments, as measured by MSD-SET, such antibodies have a KD ≤1 nM against each of the following human LLC complexes selected from the group consisting of hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. According to this disclosure, at equilibrium, high-affinity antibodies may have a KD value of 1 nM or lower against a specific antigen (e.g., an antigen complex), such as ≤1 nM, ≤0.5 nM, ≤400 pM, ≤300 pM, ≤200 pM, and ≤100 pM.

[0297] Exemplary antibodies of the present invention

[0298] Exemplary antibodies used to implement the present invention and corresponding nucleic acid sequences encoding such antibodies comprise one or more CDR amino acid sequences shown in Tables 4 and 5. Each set of H-CDRs (H-CDR1, H-CDR2, and H-CDR3) listed in Table 5 can be combined with L-CDRs (L-CDR1, L-CDR2, and L-CDR3) provided in Table 5.

[0299] Therefore, the present invention provides an isolated antibody or its antigen-binding fragment comprising six CDRs (e.g., H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3), wherein H-CDR1, H-CDR2, and H-CDR3 are selected from the set of H-CDRs of antibodies listed in Table 4, and wherein L-CDR1 comprises QASQDITNYLN (SEQ ID NO: 105), L-CDR2 comprises DASNLET (SEQ ID NO: 106), and L-CDR3 comprises QQADNHPPWT (SEQ ID NO: 12), wherein optionally, H-CDR1 may comprise FTFSSFSMD (SEQ ID NO: 107); H-CDR-2 may comprise YISPSADTIYYADSVKG (SEQ ID NO: 103); and / or, H-CDR3 may comprise ARGVLDYGDMLMP (SEQ ID NO: 6). In some embodiments, the antibody or fragment comprises H-CDR1 having the amino acid sequence FTFSSFSMD (SEQ ID NO:107), H-CDR2 having the amino acid sequence YISPSADTIYYADSVKG (SEQ ID NO:103), and H-CDR3 having the amino acid sequence ARGVLDYGDMLMP (SEQ ID NO:6); L-CDR1 having the amino acid sequence QASQDITNYLN (SEQ ID NO:105), L-CDR2 having the amino acid sequence DASNLET (SEQ ID NO:106), and L-CDR3 having the amino acid sequence QQADNHPPWT (SEQ ID NO:12).

[0300] Table 4: Complementarity-determining regions of the heavy chain of exemplary antibodies, as determined using the numbering scheme described in Lu et al.

[0301]

[0302]

[0303] Table 5: Complementarity-determining regions of the light chains of exemplary antibodies, as determined using the Kabat numbering scheme or the numbering system of Lu et al.

[0304]

[0305] The determination of the intra-antibody CDR sequence depends on the specific numbering scheme used. Commonly used systems include, but are not limited to, the Kabat numbering system, the IMTG numbering system, the Chothia numbering system, and others, such as the numbering scheme described by Lu et al. (Lu et al., MAbs. 2019 Jan; 11(1):45-57). For illustration, the following examples illustrate the six CDR sequences of Ab6 defined by four different numbering systems. Any CDR numbering system known in the art can be used to define the CDR sequences of the antibodies of this disclosure.

[0306] Table 6: Six CDRs for an exemplary antibody (Ab6) based on four numbering schemes

[0307]

[0308]

[0309] Table 7 provides the amino acid sequences of the heavy chain variable domain and light chain variable domain of exemplary antibodies of this disclosure. Therefore, in some embodiments, the high-affinity, subtype-selective TGFβ1 inhibitor of this disclosure may include a heavy chain variable domain (V... H ) and light chain variable structural domain (V L The antibody or its antigen-binding fragment, wherein V H and V L The sequence is selected from any of the V sets listed in Table 7 below. H and V L sequence.

[0310] Table 7: Heavy chain variable domains and light chain variable domains of exemplary antibodies

[0311]

[0312]

[0313] Therefore, the present invention provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) sequence identity with any sequence selected from the group consisting of: Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab3 4; and, wherein the light chain variable domain has at least 90% sequence identity with any sequence selected from the group consisting of: Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34, wherein, optionally, the heavy chain variable domain may optionally have at least 95% sequence identity, and / or, the light chain variable domain may have at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, and 100%) sequence identity. In some embodiments, the heavy chain variable domain of the antibody or fragment has at least 90% sequence identity with SEQ ID NO:13, and wherein, optionally, the light chain variable domain of the antibody or fragment has at least 90% sequence identity with SEQ ID NO:15. In some embodiments, the heavy chain variable domain of the antibody or fragment has at least 95% sequence identity with SEQ ID NO:13, and optionally, the light chain variable domain of the antibody or fragment has at least 95% sequence identity with SEQ ID NO:15. In some embodiments, the heavy chain variable domain of the antibody or fragment has at least 98% sequence identity with SEQ ID NO:13, and optionally, the light chain variable domain of the antibody or fragment has at least 98% sequence identity with SEQ ID NO:15. In some embodiments, the heavy chain variable domain of the antibody or fragment has 100% sequence identity with SEQ ID NO:13, and optionally, the light chain variable domain of the antibody or fragment has 100% sequence identity with SEQ ID NO:15.

[0314] In some embodiments, the antibody or its antigen-binding portion that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex comprises an amino acid sequence of a heavy chain variable domain encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleic acid sequence shown in SEQ ID NO: 14, and an amino acid sequence of a light chain variable domain encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleic acid sequence shown in SEQ ID NO: 16. In some embodiments, the antibody or its antigen-binding portion comprises an amino acid sequence of a heavy chain variable domain encoded by the nucleic acid sequence shown in SEQ ID NO: 14, and an amino acid sequence of a light chain variable domain encoded by the nucleic acid sequence shown in SEQ ID NO: 16.

[0315] In some instances, any antibody of this disclosure that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex includes any antibody (including its antigen-binding portion) having one or more CDR (e.g., CDRH or CDRL) sequences substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3. For example, the antibody may comprise one or more CDR sequences as shown in Table 4, which contain up to 5, 4, 3, 2, or 1 amino acid residue changes compared to the corresponding CDR region of any of the following: SEQ ID NO: 6, 12, 103, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, and 126. In some embodiments, one or more of the six CDR sequences contain up to three (3) amino acid changes compared to the sequences provided in Table 4. Such antibody variants, each containing up to three amino acid changes, are covered in this invention. In some embodiments, such variant antibodies are produced by an optimization method such as affinity maturation. The complete amino acid sequences of the heavy chain variable regions and light chain variable regions of the antibodies listed in Table 7 (e.g., Ab6), as well as the nucleic acid sequences encoding the heavy chain variable regions and light chain variable regions of certain antibodies, are provided below:

[0316] Ab3 – Heavy chain variable region amino acid sequence

[0317] EVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVSSISGSGGATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDFDYWGQGTLVTVSS(SEQ ID NO:95)

[0318] Ab6 – Heavy chain variable region amino acid sequence

[0319] EVQLVESGGGLVQPGGSLRLSCTAS GFTFSSFS MDWVRQAPGKGLEWVSY ISPSADTI YYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYC ARGVLDYGDMLMP WGQGTLVTVSS(SEQ ID NO:13)

[0320] Ab6 – Light chain variable region amino acid sequence

[0321] DIQMTQSPSSLSASVGDRVTITCQAS QDITNY LNWYQQKPGKAPKLLIY DAS NLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQADNHPPWT FGGGTKVEIK(SEQ ID NO:15)

[0322] Ab6 – Heavy chain amino acid sequence

[0323] EVQLVESGGGLVQPGGSLRLSCTASGFTFS SFSMD WVRQAPGKGLEWVS YISPSADTIYYADSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR GVLDYGDMLMPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:17)

[0324] Ab6 - heavy chain nucleic acid sequence

[0325]

[0326] Ab6 – Light chain amino acid sequence

[0327] DIQMTQSPSSLSASVGDRVTITC QASQDITNYLN WYQQKPGKAPKLLIY DASNLET GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQADNHPPWT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:19)

[0328] Ab6 – Light chain nucleic acid sequence (human κ)

[0329] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTACCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAGCAGGCCGACAATCACCCTCCTTGGACTTTTGGCGGAGGGACCAAGGTTGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT(SEQ ID NO:20)

[0330] In some implementations, the “percentage of identity” between two amino acid sequences is determined using an algorithm as described in Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. This algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al. J. Mol. Biol. 215:403-10, 1990. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the target protein molecule. When there are gaps between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used.

[0331] In any antibody or antigen-binding fragment described herein, one or more conserved mutations may be introduced into the CDR or frame sequence at locations where residues are unlikely to be involved in antibody-antigen interactions. In some embodiments, such conserved mutations may be introduced into the CDR or frame sequence at locations where residues, determined based on crystal structure, are unlikely to interact with the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex. In some embodiments, possible interfaces (e.g., residues involved in antigen-antibody interactions) may be deduced from known structural information of another antigen sharing structural similarity.

[0332] As used herein, “conservative amino acid substitution” means an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the substitution occurs. Variants can be prepared according to methods known to those skilled in the art for altering peptide sequences, for example, those summarized in references such as *Molecular Cloning: A Laboratory Manual*, J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or *Current Protocols in Molecular Biology*, F.A. Mosurubel et al., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions between amino acids in 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.

[0333] In some embodiments, the antibodies provided herein contain mutations that confer the desired properties to the antibody. For example, to avoid potential complications due to Fab arm exchanges known to occur naturally in IgG4 mAb, the antibodies provided herein may contain a stabilizing “Adair” mutation (Angal et al., “A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody,” Mol Immunol 30, 105-108; 1993), in which serine 228 (EU number; its Kabat number is residue 241) is converted to proline, resulting in an IgG1-like (CPPCP (SEQ ID NO: 54)) hinge sequence. Therefore, any said antibody may include a stabilizing 'Adair' mutation or the amino acid sequence CPPCP (SEQ ID NO: 54).

[0334] The subtype-specific, background-independent inhibitors of TGFβ1 disclosed herein may optionally include an antibody constant region or a portion thereof. For example, V LThe domain may be linked at its C-terminus to a light chain constant domain, such as Cκ or Cλ. Similarly, the VH domain or a portion thereof may be linked to all or part of the heavy chain, such as IgA, IgD, IgE, IgG, and IgM, as well as any subtype or subclass. Antibodies may contain suitable constant regions (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of this disclosure may contain a VH domain combined with any suitable constant region. H and V L The domain or its antigen-binding portion.

[0335] Alternatively or concurrently, such antibodies may or may not contain the frame regions of the antibodies in SEQ ID NOs: 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, and 15. In some embodiments, the antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex are mouse antibodies and contain mouse frame region sequences.

[0336] In some embodiments, such antibodies bind with relatively high affinity to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, for example, with a KD less than 10. -9 M, 10 -10 M, 10 -11M or lower. For example, such antibodies can bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex with an affinity between 5 pM and 1 nM (e.g., between 10 pM and 1 nM, or, for example, between 10 pM and 100 pM). This disclosure also includes antibodies or antigen-binding fragments that compete with any of the antibodies described herein for binding to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, and have a KD value of 1 nM or lower (e.g., 1 nM or lower, 500 pM or lower, 100 pM or lower). 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 tested using any suitable method, including but not limited to biosensor-based techniques (e.g., or BIACORE) and solution equilibrium titration-based techniques (e.g., MSD-SET).

[0337] In some embodiments, inhibitors of cell-associated TGFβ1 (e.g., GARP-presented TGFβ1 and LRRC33-presented TGFβ1) according to the invention comprise antibodies or fragments thereof that specifically bind to such complexes (e.g., GARP-pre / potential TGFβ1 and LRRC33-pre / potential TGFβ1) and trigger internalization of the complexes. This mode of action results in the removal or depletion of inactive TGFβ1 complexes from the cell surface (e.g., Tregs, macrophages, etc.), thereby reducing the amount of TGFβ1 available for activation. In some embodiments, such antibodies or fragments thereof bind to the target complex in a pH-dependent manner, such that binding occurs at neutral or physiological pH, but the antibody dissociates from its antigen at acidic pH; or, the dissociation rate is higher at acidic pH than at neutral pH. Such antibodies or fragments thereof can function as recirculating antibodies.

[0338] Antibodies that compete with high-affinity, subtype-specific inhibitory antibodies against TGFβ1

[0339] This disclosure relates to antibodies that compete with or cross-compete with any antibodies provided herein. As used herein, the term "competitive" in relation to an antibody means that a first antibody binds to an epitope (e.g., an epitope of the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex) in a manner sufficiently similar to the binding of a second antibody to that of the first antibody, such that the binding of the first antibody to its epitope in the presence of the second antibody is detectably reduced compared to the binding of the first antibody in the absence of the second antibody. Alternatively, the binding of the second antibody to its epitope may, but not necessarily, be detectably reduced in the presence of the first antibody. That is, the first antibody may 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, when each antibody detectably inhibits the binding of another antibody to its epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other to bind their respective epitopes. Both competing and cross-compete 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 a portion thereof), those skilled in the art will understand that such competitive and / or cross-competing antibodies are included herein and can be used in the methods and / or compositions provided herein. The term “cross-blocking” is used interchangeably.

[0340] If the binding sites are far enough apart in three-dimensional space that each binding does not interfere with other bindings, two different monoclonal antibodies (or antigen-binding fragments) binding to the same antigen may be able to bind to the antigen simultaneously. In contrast, two different monoclonal antibodies may have the same or overlapping antigen-binding regions. In this case, the binding of the first antibody may prevent the second antibody from binding to the antigen, or vice versa. In the latter case, the two antibodies are said to "cross-block" each other relative to the same antigen.

[0341] Antibody "binning" assays are used to classify multiple antibodies prepared against the same antigen into various "bins" based on relative cross-blocking activity. Each "bin" thus represents one or more discrete binding regions of the antigen. Antibodies in the same bin cross-block each other by definition. Standard in vitro binding assays (such as Biacor or...) can be used. Using standard testing conditions, for example, according to the manufacturer's instructions (e.g., performing binding determination at room temperature to 20-25°C), the groups were examined.

[0342] This disclosure relates to antibodies that compete with or cross-compete with any particular antibody or antigen-binding moiety thereof provided herein. In some embodiments, the antibody or antigen-binding moiety thereof binds to or near the same epitope as any antibody provided herein. In some embodiments, the antibody or antigen-binding moiety thereof binds to 15 or fewer amino acid residues within an epitope, meaning it binds near the epitope. In some embodiments, any antibody or antigen-binding moiety thereof provided herein binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues within an epitope to which any antibody provided herein binds.

[0343] In another embodiment, this document provides an antibody or its antigen-binding portion, which is based on the equilibrium dissociation constant K between the antibody and the protein. D Less than 10 -8 M competes or cross-competes with any antigen provided herein (e.g., GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex). In other embodiments, the antibody is in 10 -12 M to 10 -9 K in the range of M D Competitive or cross-competitive binding to any antigen provided herein. In some embodiments, an anti-TGFβ1 antibody or its antigen-binding portion is provided herein that competitively binds to the antibody or its antigen-binding portion described herein. In some embodiments, an anti-TGFβ1 antibody or its antigen-binding portion is provided herein that binds to the same epitope as the antibody or its antigen-binding portion described herein.

[0344] Any antibody described herein can be characterized using any suitable method. For example, one method is to identify the epitope to which the antigen binds, or “epitope localization.” Many suitable methods exist for localizing and characterizing the position of epitopes on proteins, including resolving the crystal structure of antibody-antigen complexes, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In another instance, epitope localization can be used to determine the sequence to which the antibody binds. The epitope can be a linear epitope, i.e., contained in a single amino acid chain, or a conformational epitope formed by the three-dimensional interactions of amino acids that do not necessarily need to be contained in a single amino acid chain (a linear sequence of primary structure). In some embodiments, the epitope is a TGFβ1 epitope, which, when TGFβ1 is in a GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, is only available for binding by the antibody or its antigen-binding portion as described herein. Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombined) and used for antibody binding assays. In another example, the epitope bound by the antibody can be identified in a systematic screening process by using overlapping peptides derived from the target antigen sequence and determining binding with the antibody. Based on gene fragment expression assays, open reading frames encoding the target antigen are randomly or through specific genetic constructs, and the reactivity of the expressed fragment of the antigen with the antibody to be tested is determined. For example, the gene fragment can be generated by PCR, then transcribed and translated into a protein in vitro in the presence of radioactive amino acids. The binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Epitopes can also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, the binding of a defined library of overlapping peptide fragments to a test antibody can be tested in a simple binding assay. In other instances, mutagenesis of antigen-binding domains, domain exchange assays, and alanine scan mutagenesis can be performed to identify the necessary, sufficient, and / or required residues for epitope binding. For example, domain exchange assays can be performed using mutants of the target antigen, where various fragments of the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex have been replaced (exchanged) with sequences from closely related but antigenically different proteins, such as another member of the TGFβ protein family (e.g., GDF11).

[0345] Alternatively, a competitive assay can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as other antibodies. Competitive assays are well known to those skilled in the art.

[0346] In some embodiments, the present invention includes antibodies (e.g., immunoglobulins, antigen-binding fragments, etc.) that cross-block (cross-competitively block) any of class 1, class 2, class 3, class 4, and / or class 5 antibodies. Therefore, in some embodiments, the pharmaceutical composition can be prepared by a method comprising the steps of: selecting an antibody or its antigen-binding fragment that cross-competes with a class 1 antibody; and formulating the antibody into a pharmaceutical composition.

[0347] In some embodiments, the pharmaceutical composition can be prepared by a method including the following steps: selecting an antibody or its antigen-binding fragment that cross-competes with a Class 2 antibody; and formulating the antibody into a pharmaceutical composition.

[0348] In some embodiments, the pharmaceutical composition can be prepared by a method including the following steps: selecting an antibody or its antigen-binding fragment that cross-competes with a class 3 antibody; and formulating the antibody into a pharmaceutical composition.

[0349] In some embodiments, the pharmaceutical composition can be prepared by a method including the following steps: selecting an antibody or its antigen-binding fragment that cross-competes with a class 4 antibody; and formulating the antibody into a pharmaceutical composition.

[0350] In some embodiments, the pharmaceutical composition can be prepared by a method including the following steps: selecting an antibody or its antigen-binding fragment that cross-competes with a Class 5 antibody; and formulating the antibody into a pharmaceutical composition.

[0351] In some embodiments, a pharmaceutical composition may be prepared by a method comprising the following steps: selecting an antibody or its antigen-binding fragment that cross-competes with an antibody selected from the group consisting of: Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34; and, formulating the pharmaceutical composition.

[0352] Preferably, the antibody selected by this method is a high-affinity conjugate, characterized in that the antibody or antigen-binding fragment can bind at a Kc concentration of ≤5 nM. DThe antibody binds to each LLC (e.g., hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1), as measured by solution equilibrium titration. In some embodiments, the antibody meets one or more criteria of category 1, category 2, category 3, category 4, and category 5. According to this disclosure, such cross-competitive antibodies can be used to treat TGFβ1-related indications in subjects.

[0353] Various modifications and mutations of antibodies

[0354] Non-limiting variations, modifications, and characteristics of any antibody or antigen-binding fragment thereof covered by this disclosure are briefly discussed below. Implementation methods for related analytical methods are also provided.

[0355] Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically consists of two pairs of identical polypeptide chains, each pair having a full-length "light" chain (approximately 25 kDa in some embodiments) and a full-length "heavy" chain (approximately 50-70 kDa in some embodiments). The amino-terminal portion of each chain typically contains a variable region of approximately 100 to 110 or more amino acids, which is typically responsible for antigen recognition. The carboxyl-terminal portion of each chain typically defines a constant region that can be responsible for effector function. Human antibody light chains are typically classified as κ and λ light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and define the antibody subtype. Antibodies can be of any type (e.g., IgM, IgD, IgG, IgA, IgY, and IgE) and class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, and IgA2). Typically, within both the full-length light and heavy chains, variable and constant regions are linked by “J” regions of about 12 or more amino acids, while the heavy chain also includes “D” regions of about 10 or more amino acids (see, for example, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (the entire contents of which are incorporated herein by reference). The variable region of each light / heavy chain pair typically forms an antigen-binding site.

[0356] The variable regions typically exhibit the same general structure as the relatively conserved framework regions (FRs) connected by three hypervariable regions, also known as complementarity-determining regions or CDRs. The CDRs from the two chains in each pair are usually aligned through the framework regions, allowing them to bind to specific epitopes. From the N-terminus to the C-terminus, the variable domains of both the light and heavy chains typically contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments of each domain generally conform to the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:878-883. The CDR of the light chain may also be referred to as CDR-L1, CDR-L2, and CDR-L3, and the CDR of the heavy chain 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 carboxyl terminus of the heavy chain. In some embodiments, the antibody comprises a heavy chain with 1-5 amino acid deletions from the carboxyl terminus of the heavy chain. In some embodiments, the precise definition of the CDR and the identification of the residues containing the antibody binding site are accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. In some embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, various analytical methods can be used to identify or roughly estimate the CDR region. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the definitions described by Lu et al. (see above), and the contact definition.

[0357] "Affinity-matured" antibodies are antibodies that have one or more modifications in one or more CDRs, which result in increased affinity for the antigen compared to their parent antibodies without these modifications. Exemplary affinity-matured antibodies have nanomolar or even picomolar affinity for the target antigen (e.g., ~10). -9 M-10 -12 K in the range of M D Affinity-matured antibodies are generated using methods known in the art. Marks et al., (1992) Bio / Technology 10:779-783, describe the generation of affinity-matured antibodies using V... H and V LAffinity maturation of domain mixing. 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 described random mutagenesis of CDR and / or framework residues; and US Patent No. 6,914,128 described selective mutagenesis at selective mutagenesis sites, contact sites, or hypermutation sites with activity-enhancing amino acid residues. Typically, parental antibodies and their affinity-matured progeny (e.g., derivatives) retain the same binding region within the antigen, although changes in amino acid residues introduced due to affinity maturation may alter some interactions at the molecular level.

[0358] The term "CDR grafted antibody" refers to an antibody that contains variable region sequences of both heavy and light chains derived from a single species, but in which V... H and / or V L An antibody in which one or more CDR regions are replaced by CDR sequences of another species, such as an antibody with variable regions of mouse heavy and light chains, wherein one or more mouse CDRs (e.g., CDR3) have been replaced by human CDR sequences.

[0359] The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies with mouse heavy and light chain variable regions linked to human constant regions.

[0360] As used herein, the term "frame" or "frame sequence" refers to the sequence remaining after subtracting the CDR from the variable region. Because the exact definition of the CDR sequence can be determined by different systems, the meaning of a frame sequence is influenced by correspondingly different interpretations. The six CDRs (CDRs -L1, -L2, and -L3 for the light chain and CDRs -H1, -H2, and -H3 for the heavy chain) also divide the frame region on each chain into four subregions (FR1, FR2, FR3, and FR4), where CDR1 lies between FR1 and FR2, CDR2 lies between FR2 and FR3, and CDR3 lies between FR3 and FR4. As mentioned by others, in the absence of a specific subregion designated as FR1, FR2, FR3, or FR4, a frame region represents a combination of FRs within the variable region of a single naturally occurring immunoglobulin chain. As used herein, the singular FR represents one of the four subregions, and the plural FR represents two or more of the four subregions that constitute the frame region.

[0361] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain framework region 1 (H-FR1) having the following amino acid sequence with optional 1, 2, or 3 amino acid alterations: EVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 174). For example, the Gly residue at position 16 may be replaced by Arg(R); and / or, the Ala residue at position 23 may be replaced by Thr(T).

[0362] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain framework region 2 (H-FR2) having the following amino acid sequence with optional 1, 2 or 3 amino acid alterations: WVRQAPGKGLEWVS (SEQ ID NO:175).

[0363] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain framework region 3 (H-FR3) having the following amino acid sequence with optional 1, 2, or 3 amino acid alterations: RFTISRDNAKNSLYLQMNSLRAEDTAVYYC (SEQ ID NO: 176). For example, the Ser residue at position 12 may be replaced by Thr(T).

[0364] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain framework region 4 (H-FR4) having the following amino acid sequence with optional 1, 2 or 3 amino acid alterations: WGQGTLVTVSS (SEQ ID NO:177).

[0365] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain framework region 1 (L-FR1) having the following amino acid sequence with optional 1, 2 or 3 amino acid alterations: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:178).

[0366] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain framework region 2 (L-FR2) having the following amino acid sequence with optional 1, 2 or 3 amino acid alterations: WYQQKPGKAPKLLIY (SEQ ID NO:179).

[0367] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain framework region 3 (L-FR3) having the following amino acid sequence with optional 1, 2 or 3 amino acid alterations: GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO:180).

[0368] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain framework region 4 (L-FR4) having the following amino acid sequence with optional 1, 2 or 3 amino acid changes: FGGGTKVEIK (SEQ ID NO:181).

[0369] In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain immunoglobulin constant domain comprising 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 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 its antigen-binding portion comprises a heavy chain immunoglobulin constant domain comprising a human IgG1 constant domain or a human IgG4 constant domain. In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain immunoglobulin constant domain comprising a human IgG4 constant domain, having a Ser-to-Pro backbone substitution that generates an IgG1-like hinge and allows the formation of interchain disulfide bonds.

[0370] In some embodiments, the antibody or its antigen-binding portion further includes a light chain immunoglobulin constant domain, which includes a human Igλ constant domain or a human Igκ constant domain.

[0371] In some embodiments, the antibody is IgG having four polypeptide chains, which are two heavy chains and two light chains.

[0372] In some embodiments, the antibody is a humanized antibody, a biantibody, 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 having a human germline amino acid sequence.

[0373] In some implementations, the antigen-binding portion is a Fab fragment, an F(ab')2 fragment, a scFab fragment, or a scFv fragment.

[0374] As used herein, the term “germ antibody gene” or “gene fragment” refers to an immunoglobulin sequence encoded by a non-lymphocyte that has not undergone maturation, resulting in genetic rearrangements and mutations to express a specific immunoglobulin (see, for example, Shapiro et al., (2002) Crit. Rev. Immunol. 22(3):183-200; Marhalonis et al., (2001) Adv. Exp. Med. Biol. 484:13-30). One of the advantages provided by the various embodiments of this disclosure stems from the recognition that germ antibody genes are more likely than mature antibody genes to preserve the essential amino acid sequence structure specific to an individual in the species, and are therefore less likely to be considered of external origin when used for treatment in that species.

[0375] As used herein, “neutralization” means the neutralization of the biological activity of an antigen (e.g., a target protein) when a binding protein specifically binds to it. In one embodiment, a neutralizing binding protein binds to 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, a neutralizing antibody against a growth factor specifically binds to a mature, soluble growth factor that has been released from a potential complex, thereby preventing the growth factor from binding to its receptor to stimulate downstream signaling. In some embodiments, the mature growth factor is TGFβ1 or TGFβ3.

[0376] As used herein, the term “binding protein” includes any polypeptide that specifically binds to an antigen (e.g., TGFβ1), including but not limited to antibodies or their antigen-binding portions, DVD-Ig™, TVD-Ig, RAb-Ig, bispecific antibodies, and bispecific antibodies.

[0377] The terms "monoclonal antibody" or "mAb," when used in the context of identical compositions, may refer to antibody preparations obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for possible natural variations that may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigen. Furthermore, unlike polyclonal antibody preparations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" should not be interpreted as requiring the antibody to be produced by any particular method.

[0378] As used herein, the term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant methods, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below in Part II C), antibodies isolated from recombinant combined human antibody libraries (Hoogenboom, HR (1997) TIB Tech. 15:62-70; Azzazy, H. and Highsmith, WE (2002) Clin. Biochem. 35:425-445; Gavilondo, JV and Larrick, JW (2002) BioTechniques 29:128-145; Hoogenboom, H. and Chames, P. (2000) Immunol. Today 21:371-378, which are incorporated herein by reference), antibodies isolated from animals that are transgenic human immunoglobulin genes (e.g., mice) (see Taylor, LD et al., (1992) Nucl. Acids Res. 20:6287-6295; Kellermann, SA. and Green, LL. (2002) Cur. Opin. in Biotechnol. 13:593-597; Little, M. et al., (2000) Immunol. Today 21:364-370) or antibodies prepared, expressed, produced, or isolated by any other method involving splicing a human immunoglobulin gene sequence into another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are mutagenized in vitro (or, when using transgenic animals expressing human Ig sequences, in vivo somatic cell mutagenization), thus the V of the recombinant antibody is reduced. H and V L The amino acid sequence of the region is as follows: although it originates from human lineage V H and V L The sequence is associated with it, but it may not naturally exist in the human antibody germline library in vivo.

[0379] As used herein, "dual variable domain immunoglobulin" or "DVD-IgTM" and similar terms are binding proteins comprising paired heavy-chain DVD polypeptides and light-chain DVD polypeptides, each pair of heavy and light chains providing two antigen-binding sites. Each binding site contains a total of six CDRs involved in antigen binding for each antigen-binding site. DVD-IgTM typically has two arms that bind to each other at least partially via dimerization of the CH3 domain, wherein each arm of the DVD is bispecific, providing an immunoglobulin with four binding sites. DVD-IgTM is provided in U.S. Patent Publications 2010 / 0260668 and 2009 / 0304693, the entire contents of which, including the sequence listing, are incorporated herein by reference.

[0380] As used herein, “triple variable domain immunoglobulin” or “TVD-Ig” and similar terms refer to a binding protein comprising paired heavy-chain TVD-binding polypeptides and light-chain TVD-binding polypeptides, each pair of heavy and light chains providing three antigen-binding sites. Each binding site includes a total of six CDRs involved in antigen binding. The TVD-binding protein may have two arms that bind to each other at least partially via dimerization of the CH3 domain, wherein each arm of the TVD-binding protein is trispecific, providing a binding protein with six binding sites.

[0381] As used herein, “receptor antibody immunoglobulin” or “RAb-Ig” and similar terms are binding proteins comprising heavy-chain RAB polypeptides and light-chain RAB polypeptides that together form a total of three antigen-binding sites. An antigen-binding site is formed by pairing the heavy-chain and light-chain antibody variable domains present in each heavy-chain and light-chain RAB polypeptide to form a single binding site with a total of six CDRs providing the first antigen-binding site. Each heavy-chain and light-chain RAB polypeptide includes independently binding ligands and receptor sequences providing the second and third “antigen” binding sites. RAb-Ig typically has two arms that bind to each other at least partially via dimerization of the CH3 domain, and each arm of RAb-Ig is trispecific, providing an immunoglobulin with six binding sites. RAb-Ig is described in U.S. Patent Application Publication No. 2002 / 0127231, the entire contents of which, including the sequence listing, is incorporated herein by reference.

[0382] As used herein, in distinction from “bispecific half-Ig binding protein” or “bispecific (half-Ig) binding protein”, the term “bispecific antibody” refers to a full-length antibody produced by the quadroma technique (see Milstein, C. and Cuello, AC (1983) Nature 305 (5934): p. 537-540), which produces a variety of different immunoglobulin species through the chemical conjugation of two different monoclonal antibodies (see Staerz, UD et al., (1985) Nature 314 (6012): 628-631), or through the introduction of a mutation in the Fc region that does not inhibit CH3-CH3 dimerization, or similar methods (see Holliger, P. et al., (1993) Proc. Natl. Acad. Sci USA 90 (14): 6444-6448), of which only one is a functional bispecific antibody. By molecular function, a bispecific antibody binds to one antigen (or epitope) on one of its two binding arms (a pair of HC / LCs) and to a different antigen (or epitope) on its second arm (a pair of distinct HC / LCs). By this definition, a bispecific antibody has two distinct antigen-binding arms (in terms of both specificity and CDR sequence) and is monovalent for each antigen it binds.

[0383] As used herein, and to distinguish it from bispecific half-Ig binding proteins or bispecific binding proteins, the term "bispecific antibody" refers to a full-length antibody that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC / LC) (see PCT Publication No. WO 02 / 02773). Thus, a bispecific binding protein has two identical antigen-binding arms with the same specificity and the same CDR sequence, and is bivalent for each antigen it binds.

[0384] As used herein, the term “Kon” is intended to refer to the rate constant of binding protein (e.g., antibody) to form, for example, an antibody / antigen complex as known in the art. The terms “binding rate constant” or “ka” are also known as “Kon” and are used interchangeably herein. This value represents the rate at which an antibody binds to its target antigen or the rate at which a complex forms between the antibody and the antigen, and is also expressed by the following equation: antibody (“Ab”) + antigen (“Ag”) → Ab - Ag.

[0385] As used herein, the term “Koff” is intended to refer to the dissociation rate constant of a bound protein (e.g., an antibody) from a known antibody / antigen complex, such as an antibody / antigen complex. The terms “dissociation rate constant” or “kd” are also known as “Koff” and are used interchangeably herein. This value represents the rate of dissociation of the antibody from its target antigen, or the time it takes for the Ab-Ag complex to separate into free antibody and antigen, expressed as: Ab+Ag ← Ab-Ag.

[0386] The term "equilibrium dissociation constant" or "K" D The terms “binding rate constant” and “dissociation rate constant” are used interchangeably in this document and refer to the value obtained in a titration measurement under equilibrium conditions, or by dividing the dissociation rate constant (koff) by the binding rate constant (kon). The binding rate constant, dissociation rate constant, and equilibrium dissociation constant are used to represent the binding affinity of a binding protein (e.g., an antibody) for an antigen. Methods for determining the binding and dissociation rate constants are well known in the art. Fluorescence-based techniques offer high sensitivity and the ability to examine samples in physiological buffers under equilibrium conditions. Other experimental methods and instruments, such as… (Biomolecular interaction analysis) assays (e.g., instruments are available from GE Healthcare's BIAcore International AB, Uppsala, Sweden). Alternatively, instruments purchased from Savyne Instruments (Boise, Idaho) can also be used. (Kinetic exclusion assay) determination.

[0387] As used herein, the term "crystal / crystallized" refers to a binding protein (e.g., an antibody) or its antigen-binding portion existing in crystalline form. A crystal is a form of solid matter that differs from other forms such as amorphous solids or liquid crystal states. Crystals consist of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g., proteins, such as antibodies), or molecular combinations (e.g., antigen / antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relations well known in the art. The basic units or building blocks that repeat in a crystal are called asymmetric units. The repetition of these asymmetric units in an arrangement that conforms to a given, well-defined crystallographic symmetry provides the "unit cell" of the crystal. The unit cell provides the crystal by repeating regular translations in all three dimensions. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 201-16, Oxford University Press, New York, New York (1999). The term "linker" is used to refer to a polypeptide comprising two or more amino acid residues linked by peptide bonds and used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger, P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al., (1994) Structure 2:1121-1123).Exemplary linkers include but are not limited to: ASTKGPSVFPLAP (SEQ ID NO:55); ASTKGP (SEQ ID NO:56); TVAAPSVFIFPP (SEQ ID NO:57); TVAAP (SEQ ID NO:58); AKTTPKLEEGEFSEAR (SEQ ID NO:59); AKTTPKLEEGEFSEARV (SEQ ID NO:60); AKTTPKLGG (SEQ ID NO:61); SAKTTPKLGG (SEQ ID NO:62); SAKTTP (SEQ ID NO:63); RADAAP (SEQ ID NO:64); RADAAPTVS (SEQ ID NO:65); RADAAAAGGPGS (SEQ ID NO:66); RADAAAA(G4S)4 (SEQ ID NO:67); SAKTTPKLEEGEFSEARV (SEQ ID NO:68); ADAAP (SEQ ID NO:69); ADAAPTVSIFPP (SEQ ID NO:70); QPKAAP (SEQ ID NO:71); QPKAAPSVTLFPP (SEQ ID NO:72); AKTTPP (SEQ ID NO:73); AKTTPPSVTPLAP (SEQ ID NO:74); AKTTAP (SEQ ID NO:75); AKTTAPSVYPLAP (SEQ ID NO:76); GGGGSGGGGSGGGGS (SEQ ID NO:77); GENKVEYAPALMALS (SEQ ID NO:78); GPAKELTPLKEAKVS (SEQ ID NO:79); GHEAAAVMQVQYPAS (SEQ ID NO:80); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO:81); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO:82).

[0388] The terms "label" and "detectable label" or "detectable moiety" refer to labels attached to a specific binding pair, such as an antibody or analyte, to make the reaction between members of a specific binding pair (e.g., antibodies and analytes) and a specific binding pair (e.g., antibodies or analytes) detectable; such labels are referred to as "detectable labels." Therefore, the term "labeled binding protein" as used herein refers to a protein having an incorporated label for identifying the binding protein. In one embodiment, the label is a detectable marker that can generate a signal detectable by visual or instrumental means, such as a polypeptide incorporated with a radiolabeled amino acid or attached to a biotinylated moiety, which can be detected by a labeled avidin protein (e.g., streptavidin containing a fluorescent marker or enzymatic activity, detectable by optical or colorimetric methods). Examples of polypeptide labels include, but are not limited to, 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); chromophores; fluorescent labels (e.g., FITC, rhodamine, and lanthanide phosphors); enzyme labels (e.g., horseradish peroxidase, luciferase, and alkaline phosphatase); chemiluminescent labels; biotin groups; predefined peptide epitopes recognized by a second reporter molecule (e.g., leucine zipper pairs, binding sites of second antibodies, metal-binding domains, and epitope tags); and magnetic reagents, such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-generating portions, such as acridine compounds, and fluorescence-generating portions, such as luciferin. Other labels are described herein. In this regard, the portion itself may not be detectably labeled, but may become detectable upon reaction with another portion. The use of “detectable label” is intended to include the latter type of detectable label.

[0389] In some embodiments, the Octet assay is used to determine the binding affinity of an antibody or its antigen-binding moiety to an antigen (e.g., a protein complex), such as the presenting molecule-proTGFβ1 complex. In some embodiments, the Octet assay is a method for determining one or more kinetic parameters that indicate the binding between the antibody and the antigen. In some embodiments, the Octet assay is used... The system (ForteBio, Menlo Park, CA) measures the binding affinity of an antibody or its antigen-binding moiety to the proTGFβ1 complex. For example, the binding affinity of the antibody can be determined using a fortéBio Octet QKe dip and a label-free readout assay system using biolayer interferometry. In some embodiments, the antigen is immobilized onto a biosensor (e.g., a streptavidin-coated biosensor), and the antibody and the complex (e.g., a biotinylated proTGFβ1 complex) are present in solution at a high concentration (50 μg / mL) to measure the binding interaction. In some embodiments, the binding affinity of the antibody or its antigen-binding moiety to the proTGFβ1 complex is determined using the protocols outlined herein.

[0390] Characterization of novel, high-affinity, background-independent proTGFβ1 antibodies

[0391] Combination properties

[0392] The antibodies disclosed herein exhibit enhanced binding activity. This includes a class of high-affinity, background-independent antibodies capable of selectively inhibiting TGFβ1 activation. Notably, the term "background-independent" is used herein with greater precision than its more general usage previously. According to this disclosure, this term confers on the antibody the ability to impose a uniform level of relative affinity (i.e., unbiased) on different antigen complexes. Therefore, the background-independent antibodies of this invention can target multiple types of precursor complexes (e.g., presenting molecule-proTGFβ1 complexes) and can bind each such complex with equivalent affinity (i.e., the relative affinity difference between complexes does not exceed three-fold), wherein, as measured by, for example, MSD-SET, K... D The value is below 10 nM, preferably below 5 nM, more preferably below 1 nM, and even more preferably below 100 pM. As shown below, many antibodies covered by this invention have K values ​​in the sub-nanomolar range. D value.

[0393] Therefore, antibodies can specifically bind to each human presenting molecule-proTGFβ1 complex (sometimes referred to as a "large potential complex," which is a ternary complex composed of a proTGFβ1 dimer coupled to a single presenting molecule, namely LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1). Typically, recombinantly produced, purified protein complexes are used as antigens (e.g., antigen complexes) to evaluate or confirm the antibody's ability to bind to antigen complexes in a suitable in vitro binding assay. Such assays are well known in the art and include, but are not limited to, biolayer interferometry (BLI)-based assays (such as...). ) and determinations based on solution equilibrium titration (such as MSD-SET).

[0394] BLI-based binding assays are widely used in the art to measure antibody affinity and kinetics to antigens. This is a label-free technique in which biomolecular interactions are analyzed based on optical interference. A protein (e.g., the antibody to be detected) can be immobilized on the tip of a biosensor. When other proteins (e.g., antigens) in solution begin to bind to the immobilized antibody, they cause changes in the interference pattern, which can be measured in real time. This allows for monitoring of binding specificity, binding and dissociation rates, and concentration dependence. Therefore, BLI is a kinetic indicator revealing system dynamics. Due to its ease of use and rapid results, BLI-based assays, such as… The system (available from ForteBio / MolecularDevices, Fremont, California) is particularly convenient as an initial screening method for identifying and separating mixtures of “binding agents” from mixtures of “non-binding agents” or “weakly binding agents” during the screening process.

[0395] BLI-based binding assays indicate that when via When measuring binding affinity, the novel antibodies were characterized as “background balanced / independent” antibodies. As can be seen in Table 8, which summarizes non-limiting examples of antibodies based on BLI, these antibodies exhibited relatively uniform K values ​​in the sub-nanomolar range across the four targeting complexes. D The values, and relatively low matrix-associated complex (matrix-associated complex) variability (not exceeding five-fold bias) (see column (H)). This contrasts with the previously identified antibody Ab3, which is provided in reference form and shows a significantly higher relative affinity for matrix-associated complexes (27+-fold bias) than for cell-associated complexes.

[0396] Table 8 below provides non-limiting examples of high-affinity, background-independent proTGFβ1 antibodies covered by this invention. This table provides examples such as those obtained by... The results of the in vitro binding assays were representative. Similar results were also obtained using the SPR-based technique (Biacore System).

[0397] Column (A) of the table lists monoclonal antibodies with discrete amino acid sequences. Ab3 (shown in bold) is a previously identified reference antibody that has shown potency in cell-based assays; efficacy in multiple animal models; and clear toxicological properties (disclosed in PCT / US2018 / 012601). Columns (B), (D), (E), and (F) provide information on monoclonal antibodies with K... D The affinity of each listed antibody was measured. Column (B) shows the affinity of each antibody for the recombinant human LTBP1-proTGFβ1 complex; column (C) shows the affinity for the recombinant human LTBP3-proTGFβ1 complex; column (E) shows the affinity for the recombinant human GARP-proTGFβ1 complex; and column (F) shows the affinity for the recombinant human LRRC33-proTGFβ1 complex. The average K in (B) and (C) D The values ​​are shown in the corresponding (D) column, which collectively represent the antibody affinity for the ECM or matrix-associated proTGFβ1 complex. Similarly, the average K values ​​in (E) and (F) are... D The values ​​are displayed in the corresponding (G) column, collectively representing the antibody's affinity for the cell surface or cell-associated proTGFβ1 complex. Finally, the relative ratio between the mean KD values ​​from columns (D) and (G) is expressed as "fold bias" in column (H). Therefore, when comparing antibody binding preferences against matrix-associated complexes and cell surface complexes, a larger value in column (H) indicates a greater bias against a particular antibody. This is one way to quantitatively present and compare the intrinsic bias of an antibody against its target complex. This analysis can be used to guide the selection process for candidate antibodies for specific therapeutic uses.

[0398] Table 8: Non-restricted examples of background-independent TGFβ1 antibodies and K as measured by BLI D value

[0399]

[0400] This invention provides a class of high-affinity, background-independent antibodies that bind with equivalent affinity to four known proTGFβ1-presenting molecule complexes: LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. In some embodiments, the antibodies bind to each proTGFβ1-presenting molecule complex with equivalent or higher affinity than the previously described reference antibody Ab3. According to the invention, such antibodies bind with an affinity ≤5 nM (as measured by suitable in vitro binding assays such as biolayer interferometry and surface plasmon resonance) with K... D (Specifically) binds to each of the aforementioned complexes. In some embodiments, the antibody or fragment binds to the human LTBP1-proTGFβ1 complex with an affinity of ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤5 nM, or ≤0.5 nM. In some embodiments, the antibody or fragment binds to the human LTBP3-proTGFβ1 complex with an affinity of ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤5 nM, or ≤0.5 nM. In some embodiments, the antibody or fragment binds to the human GARP-proTGFβ1 complex with an affinity of ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤5 nM, or ≤0.5 nM. In some embodiments, the antibody or fragment binds to the human LRRC33-proTGFβ1 complex with an affinity of ≤5nM, ≤4nM, ≤3nM, ≤2nM, ≤1nM, ≤5nM or ≤0.5nM.

[0401] In preferred embodiments, such antibodies are cross-reactive between humans and mice. Therefore, in some embodiments, the antibody or fragment binds to the mouse LTBP1-proTGFβ1 complex with an affinity of ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤5 nM, or ≤0.5 nM. In some embodiments, the antibody or fragment binds to the mouse LTBP3-proTGFβ1 complex with an affinity of ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤5 nM, or ≤0.5 nM. In some embodiments, the antibody or fragment binds to the mouse GARP-proTGFβ1 complex with an affinity of ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤5 nM, or ≤0.5 nM. In some embodiments, the antibody or fragment binds to the mouse LRRC33-proTGFβ1 complex with an affinity of ≤5nM, ≤4nM, ≤3nM, ≤2nM, ≤1nM, ≤5nM or ≤0.5nM.

[0402] As shown, the proTGFβ1 antibody of this disclosure exhibits particularly high affinity for matrix-associated proTGFβ1 complexes. In some embodiments, the average Ka of the matrix-associated complexes (i.e., LTBP1-proTGFβ1 and LTBP3-proTGFβ1) is... D The value is ≤1nM or ≤0.5nM.

[0403] As shown, the proTGFβ1 antibody of this disclosure has a high affinity for the cell-associated proTGFβ1 complex. In some embodiments, the average Kc of the cell-associated complex (i.e., GARP-proTGFβ1 and LRRC33-proTGFβ1) is... D The value is ≤2nM or ≤1nM.

[0404] The high-affinity proTGFβ1 antibody disclosed herein is characterized by its consistent (unbiased) affinity for all four antigen complexes (e.g., compared to Ab3). Of the four known presenting molecule-proTGFβ complexes described herein, none exhibits a Kg of the antigen complex. D Significant deviation. In other words, compared to previously described proTGFβ1 antibodies (including Ab3), this disclosure has achieved more uniform binding activity because each such antibody exhibits equivalent affinity in the four antigen complexes. In some embodiments, the antibody or fragment exhibits unbiased or uniform binding properties, characterized by the affinity difference (or range) of the antibody or fragment in the four proTGFβ1 antigen complexes between the lowest and highest K values. D The values ​​should not exceed five times. In some implementations, the relative difference (range) in affinity should not exceed three times.

[0405] Table 8 further illustrates the concept of "homogeneity" or lack of bias. The mean KD values ​​for the two matrix-related and cell-related complexes were calculated separately (see columns (D) and (G)). These mean KD values ​​can then be... D The value is used to inquire whether there is a bias in the binding activity between matrix-associated complexes and cell surface (e.g., immune cell)-associated complexes. As shown in Table 8, bias can be expressed as the average K. D The value shows a "fold difference". Compared to the previously described antibody Ab3, the high-affinity, background-independent proTGFβ1 antibody covered in this disclosure is significantly unbiased because many antibodies have an average K value between matrix-associated complexes and cell-associated complexes. D The difference in values ​​does not exceed three times (compared to a bias of 25 times or more for Ab3).

[0406] Therefore, a class of background-independent monoclonal antibodies or fragments is provided, all capable of binding with equivalent affinity to each of the following presenting molecules—proTGFβ1 complexes—with an affinity ≤1 nM, as measured by biolayer interferometry or surface plasmon resonance: LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1, and LRRC33-proTGFβ1. Such antibodies specifically bind to each of the aforementioned complexes with an affinity ≤5 nM, as measured by biolayer interferometry or surface plasmon resonance, wherein the monoclonal antibody or fragment shows an affinity bias of no more than three-fold relative to the other complexes for any of the aforementioned complexes, and wherein the monoclonal antibody or fragment inhibits the release of mature TGFβ1 growth factor from each of the proTGFβ1 complexes, but not from the proTGFβ2 or proTGFβ3 complexes.

[0407] While the kinetics (e.g., “binding” and “dissociation” rates) of binding curves obtained from BLI-based assays can provide useful information, the applicant of this invention anticipates that the binding properties measured in equilibrium for antibodies that act by binding to tethered (e.g., tissue-localized) inactive (e.g., potential) targets, thereby preventing their activation, based on the mechanisms of action of activation inhibitors disclosed herein, may more accurately reflect their in vivo behavior and potency. From this perspective, antibodies, for example, with rapid “binding” rates (“Kon”) that would be reflected in binding measurements obtained via BLI could provide relevant parameters for evaluating neutralizing antibodies (e.g., those that directly target and must rapidly isolate the active, soluble growth factor itself in order for it to function as an effective inhibitor). However, this may not necessarily apply to antibodies acting as activation inhibitors, such as those disclosed herein. As described, the mechanism of action of the novel TGFβ1 inhibitor of this invention is through the inhibition of the activation step, which, in contrast to the isolation of soluble, activated growth factors, is accomplished by targeting the potential complex of tissue / cell tethering. This is because TGFβ1 activation inhibitors target inactive precursors located in various tissues (e.g., within the ECM, on the surface of immune cells, etc.), thereby preemptively preventing the release of mature growth factors from the complex. This mechanism of action is thought to allow the inhibitor to reach target saturation (e.g., equilibrium) in vivo without having to compete with endogenous receptors for transient growth factor molecules as rapidly as conventional neutralizing inhibitors require.

[0408] Given this difference in mechanism of action, the binding properties were further evaluated using an alternative in vitro binding assay, which determined the affinity at equilibrium.

[0409] In view of this, it can be expected that assays measuring the binding affinity of such antibodies at equilibrium can more accurately represent the pattern of target binding in vivo. Therefore, binding assays based on MSD-SET (or other suitable assays) can be performed, as shown in Table 9 below.

[0410] Solution equilibrium titration (“SET”) is an assay that measures the binding between two molecules (such as an antigen and an antibody that binds to the antigen) in equilibrium in solution. For example, Meso-Scale Discovery (“MSD”) based SET or MSD-SET is a useful model for determining the dissociation constant of protein-protein interactions with particularly high affinity in equilibrium (see, e.g., Ducata et al., (2015) J Biomolecular Screening 20(10):1256-1267). SET-based assays are useful for determining the K-value of antibodies with sub-nanomolar (e.g., picomolar) affinity. D Values ​​are particularly useful.

[0411] Table 9: Non-restricted examples of high-affinity, background-independent TGFβ1 antibodies (hIgG4) and Kg as measured by MSD-SET D Value ("h" indicates human complex)

[0412]

[0413] Table 9 also includes three previously described TGFβ1 selective antibodies (C1, C2, and Ab3) as reference antibodies. C1 and C2 were first disclosed in PCT / US2017 / 021972, WO 2017 / 156500, and Ab3 was described in PCT / US2018 / 012601, WO 2018 / 129329.

[0414] As can be seen from the affinity data provided in Table 9, the binding activity of the novel antibody according to this disclosure is significantly higher than that of the previously identified reference antibody. Furthermore, the novel TGFβ1 antibody is "background-independent" because it exhibits equivalent affinity (e.g., ~ sub-nanomolar range, e.g., K0). D <1 nM) binds to each human LLC complex. The high affinity, background-independent binding properties suggest that these antibodies may be beneficial for treating TGFβ1-related indications, including ECM-related and immune component dysregulations, such as cancer.

[0415] For binding assays based on solution equilibrium titration, a protein complex containing one of the presenting molecules (as shown above) can be used as the antigen (presenting molecule-TGFβ1 complex, or LLC). The antibody to be tested is allowed to form 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 reactant can be measured by suitable methods well known in the art. Compared to BLI-based assays, SET-based assays are less affected by the binding / dissociation rate of the antigen-antibody complex, thus allowing for highly sensitive detection of very high affinity interactions. As shown in Table 9, in this disclosure, preferred high-affinity TGFβ1 inhibitors exhibit a sub-nanomolar (e.g., picomolar) affinity range across all detected large potential complexes, as determined by SET-based assays.

[0416] Therefore, a class of background-independent monoclonal antibodies or fragments is provided, each of which, as measured by solution equilibrium titration, can achieve K+ with equivalent affinity at ≤1 nM. D Binds to each of the following human-presenting molecules-proTGFβ1 complexes, as per MSD-SET: hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1. As measured by MSD-SET, these antibodies have a K+ concentration ≤1 nM. D Specifically binds to each of the aforementioned complexes, and wherein the monoclonal antibody or fragment inhibits the release of mature TGFβ1 growth factor from each proTGFβ1 complex but not from the proTGFβ2 or proTGFβ3 complexes. In a preferred embodiment, such antibodies or fragments are at a Kc concentration of 500 pM or lower (i.e., ≤500 pM), 250 pM or lower (i.e., ≤250 pM), or 200 pM or lower (i.e., ≤200 pM). D Combine each of the aforementioned complexes. Even more preferably, such antibodies or fragments are at 100 pM or lower (i.e., ≤100 pM) K. D Each of the aforementioned complexes is bound. In some embodiments, the antibody or fragment does not bind to free TGFβ1 growth factor that does not bind to the pre-domain complex. This can be detected or confirmed by suitable in vitro binding assays known in the art, such as biolayer interferometry.

[0417] In a further preferred embodiment, such antibodies or fragments also exhibit cross-reactivity with rodent (e.g., rats and / or mice) and / or non-human primate (e.g., cyno) counterparts. To cite just one example, Ab6 is capable of binding with high affinity to every large potential complex in multiple species, including humans, mice, rats, and cynomolgus monkeys, as shown in Table 10 and Example 9 below.

[0418] Table 10: Non-restricted examples of high-affinity TGFβ1 antibodies with cross-species reactivity independent of background, as measured by MSD-SET ("h" indicates human; "m" indicates mouse).

[0419]

[0420] efficacy

[0421] The antibodies disclosed herein can be broadly characterized as “functional antibodies” due to their ability to inhibit TGFβ1 signaling. As used herein, a “functional antibody” confers one or more biological activities due to its ability to bind to a target protein (e.g., an antigen) in a manner that modulates its function. Thus, functional antibodies broadly include those antibodies capable of modulating the activity / function of a target molecule (i.e., an antigen). Such modulating antibodies include inhibitory antibodies (or suppressive antibodies) and activating antibodies. This disclosure relates to antibodies capable of inhibiting biological processes mediated by TGFβ1 signaling associated with multiple backgrounds of TGFβ1. When administered at a therapeutically effective amount (a dose that achieves sufficient efficacy within acceptable levels of toxicity), the inhibitors used to carry out the invention (antibodies as described herein) are intended to have TGFβ1 selectivity, rather than targeting or interfering with TGFβ2 and TGFβ3. The novel antibodies of this disclosure exhibit enhanced inhibitory activity (potency) compared to previously identified TGFβ1 activating inhibitors.

[0422] In some embodiments, the potency of inhibitory antibodies can be measured in suitable cell-based assays, such as the CAGA reporter cell assay described herein. Typically, cultured cells (e.g., heterologous and primary cells) can be used for cell-based potency assays. Cells expressing endogenous TGFβ1 and / or target presenting molecules (e.g., LTBP1, LTBP3, GARP, and LRRC33) can be used. Alternatively, nucleic acids encoding one or more target proteins (e.g., TGFβ1) and / or target presenting molecules (e.g., LTBP1, LTBP3, GARP, and LRRC33) can be introduced into such cells for expression, for example, by transfection (e.g., stable or transient transfection) or by infection with a viral vector. In some embodiments, LN229 cells are used for such assays. Cells expressing TGFβ1 and target presenting molecules (e.g., LTBP1, LTBP3, GARP, or LRRC33) are grown in a culture medium, where they “present” a large potential complex on the cell surface or deposited in the ECM (when bound to LTBP). Activation of TGFβ1 can be triggered by integrin expressed on the surface of another cell. Cells expressing integrin can be the same cell type co-expressing a large potential complex or separate cell types. Reporter cells are incorporated into an assay system incorporating a TGFβ response element. In this way, the degree of TGFβ activation can be measured by detecting signals from the reporter cells (e.g., TGFβ response reporter genes coupled to TGFβ response promoter elements, such as luciferase) upon TGFβ activation. Using such cell-based assay systems, the inhibitory activity of an antibody can be determined by measuring changes (reductions) or differences in the reporter signal (e.g., luciferase activity measured by fluorescence readout) in the presence or absence of the test antibody. Such assays are illustrated in Example 2 of this document.

[0423] Therefore, in some embodiments, the inhibitory potency (IC50) of the novel antibody of this disclosure, measured against each of the hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes, calculated based on a cell-based reporter assay (such as the LN229 cell assay described elsewhere herein) for measuring TGFβ1 activation, is... 50 The IC50 value can be 5 nM or lower. In some embodiments, the antibody has an IC50 value of 2 nM or lower (i.e., ≤2 nM) measured for each LLC. 50 In a preferred embodiment, the IC50 of the antibody is measured for each LLC complex. 50It is 1 nM or lower. In some embodiments, the antibody has an IC50 of less than 1 nM against each of the hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes. 50 .

[0424] Table 11: Inhibitory efficacy of selected antibodies as measured by reporter cell assay (in IC50). 50 count)

[0425]

[0426] TGFβ1 activation can be triggered via an integrin-dependent mechanism or a protease-dependent mechanism. The inhibitory activity (e.g., potency) of antibodies according to this disclosure can be evaluated by their ability to block TGFβ1 activation induced by one or both activation modes. The reporter cell assays described above can be designed to measure the ability of antibodies to block or inhibit integrin-dependent activation of TGFβ1. Inhibitory potency can also be assessed by measuring the ability of antibodies to block protease-induced TGFβ1 activation. Example 3 of this disclosure provides a non-limiting implementation of such an assay. Results are summarized in... Figure 5A and Figure 5B Therefore, in some embodiments of the invention, the subtype-selective inhibitors according to this disclosure are capable of inhibiting both integrin-dependent activation and protease-dependent activation of TGFβ1. Such inhibitors can be used to treat TGFβ1-related indications characterized by EDM dysregulation associated with protease activity. For example, such TGFβ1-related indications may be associated with increased myofibroblasts, increased ECM rigidity, excessive or abnormal collagen deposition, or any combination thereof. Such conditions include, for example, fibrotic disorders and cancers, including solid tumors (such as metastatic cancer) or myelofibrosis.

[0427] In some implementations, efficacy can be evaluated in suitable in vivo models as an indicator of efficacy and / or pharmacodynamic effect. For example, if a first antibody is effective in an in vivo model at a certain concentration, and a second antibody is equally effective in the same in vivo model at a lower concentration, the second antibody can be considered more effective than the first antibody. Any suitable disease model known in the art can be used to evaluate the relative efficacy of TGFβ1 inhibitors, depending on the specific target indication, such as cancer models and fibrosis models. Preferably, such studies include multiple doses or concentrations of the antibody to be tested weekly.

[0428] Similarly, pharmacodynamic (PD) effects can be measured to determine the relative potency of inhibitory antibodies. Common PD markers targeting the TGFβ signaling pathway include, but are not limited to, phosphorylation of SMAD2 / 3 and expression of downstream effector genes whose transcription is sensitive to TGFβ activation, such as those using TGFβ-responsive promoter elements (e.g., Smad binding elements). In some embodiments, the antibodies of this disclosure, when administered to animals at doses of 3 mg / kg or lower, are able to completely block disease-induced SMAD2 / 3 phosphorylation in preclinical fibrosis models. In some embodiments, when administered to animals in a UUO model of renal fibrosis at doses of 10 mg / kg or lower, the antibodies of this disclosure are able to significantly inhibit fibrosis-induced expression of a group of marker genes, including Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, and Loxl2.

[0429] In some embodiments, the selection process for antibodies or antigen-binding fragments thereof for therapeutic use may therefore include identifying antibodies or fragments that exhibit sufficient inhibitory potency. For example, the selection process may include performing a cell-based TGFβ1 activation assay to measure the potency (e.g., IC50) of one or more test antibodies or fragments thereof. 50 The steps include selecting candidate antibodies or fragments thereof that exhibit the desired potency. In some embodiments, the IC50 of each human LLC... 50 It is 5 nM or lower. The selected antibody or fragment can then be used to treat the TGFβ1-related indications described herein.

[0430] Integration Zone

[0431] In the context of this disclosure, the “one or more binding regions” of an antigen provide the structural basis for antibody-antigen interactions. As used herein, a “binding region” refers to the interfacial region between the antibody and the antigen, such that when bound to the proTGFβ1 complex (“antigen”) in physiological solution, the antibody or fragment can protect the binding region from solvent exposure, as determined by suitable techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS). Identification of the binding region can be used to gain a deeper understanding of antigen-antibody interactions and the mechanisms of action of specific antibodies. Cross-blocking experiments capable of antigen grouping can help identify other antibodies with similar or overlapping binding regions. Optionally, X-ray crystallography can be used to identify the exact amino acid residues of the epitopes mediating antigen-antibody interactions.

[0432] HDX-MS is well-known in the field as a widely used technique for exploring protein conformation or protein-protein interactions in solution. This method relies on the exchange of hydrogen in the amide of the protein backbone with deuterium present in solution. By measuring the hydrogen-deuterium exchange rate, information about protein dynamics and conformation can be obtained (reviewed in: Wei et al., (2014) "Hydrogen / deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics:methodology and applications." Drug Disco Today. 19(1):95-102; incorporated by reference). The application of this technique is based on the premise that when antibody-antigen complexes form, the interface between binding partners may block the solvent, thereby reducing or preventing the exchange rate due to steric repulsion of the solvent.

[0433] This disclosure includes antibodies or antigen-binding fragments of human LLC binding to a region (“binding region”) containing a latent lasso or a portion thereof. The latent lasso is a protein module within the pre-domain. Many potent activation inhibitors are expected to bind to this region of the proTGFβ1 complex in such a manner that antibody binding “locks” the growth factor, thereby preventing its release. Interestingly, this is a portion of the complex in which butterfly-shaped elongated regions of the growth factor (e.g., corresponding to, for example, finger-1 and finger-2) interact closely with the cage-like structures of the pre-domain.

[0434] As in Figure 18B As shown, the latent lasso includes regions labeled 2a and 2b, which are part of the pre-domain. Notably, the region immediately adjacent to the latent lasso, labeled 5a, corresponds to the so-called finger-1 within the growth domain, while the region surrounding the opposite side, labeled 6b, is part of finger-2 within the growth factor domain. Based on this, it is easy to conceive that an antibody tightly encapsulating these regions could effectively prevent the proTGFβ1 complex from dislodging, thereby inhibiting activation.

[0435] HDX-MS technology can be used to determine the binding region of proTGFβ1. In some embodiments, a portion of proTGFβ1 identified as important for binding antibodies or fragments includes at least a portion of the prodomain and at least a portion of the growth factor domain. Binding is performed on a first binding region comprising at least a portion of the latent lasso (in... Figure 19A Antibodies or fragments of the “region 1” in the text are preferred. More preferably, such antibodies or fragments also bind to a second binding region ( Figure 19AThe second binding region (“Region 2”) contains at least a portion of the growth factor domain of the growth factor domain-1. Such antibodies or fragments can also bind to a third binding region (in… Figure 19A The third binding region ("region 3") contains at least a portion of the growth factor domain index-2.

[0436] Other regions within proTGFβ1 may also directly or indirectly contribute to the high-affinity interactions of these antibodies disclosed herein. It is believed that these regions mediate the antibody-proTGFβ1 complex (see...). Figure 18A High-affinity binding important regions may include, but are not limited to: LVKRKRIEA (SEQ ID NO:159); LASPPSQGEVP (SEQ ID NO:160); PGPLPEAV (SEQ ID NO:161); LALYNSTR (SEQ ID NO:162); REAVPEPVL (SEQ ID NO:163); YQKYSNNSWR (SEQ ID NO:164); RKDLGWKWIHEPKGYHANF (SEQ ID NO:165); LGPCPYIWS (SEQ ID NO:166); ALEPLPIV (SEQ ID NO:167); and VGRKPKVEQL (SEQ ID NO:168) (based on the natural sequence of human proTGFβ1).

[0437] In some embodiments, in regions that may facilitate antibody-antigen interactions, the high-affinity antibody of this disclosure may bind to an epitope containing at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL (“Region 1”) (SEQ ID NO:169).

[0438] In some embodiments, the high-affinity antibody of this disclosure can bind to an epitope containing at least one residue of the amino acid sequence RKDLGWKWIHEPKGYHANF (“Region 2”) (SEQ ID NO:165).

[0439] In some embodiments, the high-affinity antibody of this disclosure can bind to an epitope containing at least one residue of the amino acid sequence VGRKPKVEQL (“Region 3”) (SEQ ID NO:168).

[0440] In some embodiments, the high-affinity antibody of this disclosure can bind to an epitope comprising at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL (“Region 1”) (SEQ ID NO:169) and at least one residue of the amino acid sequence RKDLGWKWIHEPKGYHANF (“Region 2”) (SEQ ID NO:165).

[0441] In some embodiments, the high-affinity antibody of this disclosure can bind to an epitope comprising at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL (“Region 1”) (SEQ ID NO:169) and at least one residue of the amino acid sequence VGRKPKVEQL (“Region 3”) (SEQ ID NO:168).

[0442] In some embodiments, the high-affinity antibody of this disclosure can bind to an epitope comprising at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL (“Region 1”) (SEQ ID NO:169), at least one residue of the amino acid sequence RKDLGWKWIHEPKGYHANF (“Region 2”) (SEQ ID NO:165) and at least one residue of the amino acid sequence VGRKPKVEQL (“Region 3”) (SEQ ID NO:168).

[0443] In addition to contributions from regions 1, 2 and / or 3, such epitopes may also contain at least one amino acid residue selected from the following sequences: LVKRKRIEA (SEQ ID NO:159); LASPPSQGEVP (SEQ ID NO:160); PGPLPEAV (SEQ ID NO:161); LALYNSTR (SEQ ID NO:162); REAVPEPVL (SEQ ID NO:163); YQKYSNNSWR (SEQ ID NO:164); RKDLGWKWIHEPKGYHANF (SEQ ID NO:165); LGPCPYIWS (SEQ ID NO:166); ALEPLPIV (SEQ ID NO:167); and VGRKPKVEQL (SEQ ID NO:168).

[0444] Notably, it was found that many binding regions identified in structural studies using four representative isotype-selective TGFβ1 antibodies overlapped, suggesting that certain regions of the proTGFβ1 complex may be particularly important for maintaining the latency of the proTGFβ1 complex. Therefore, advantageously, antibodies or fragments thereof can be selected at least in part based on one or more of their binding regions, which include overlapping portions identified across the various inhibitors described herein. These overlapping portions of binding regions include, for example, SPPSQGEVPPGPLPEAVL (SEQ ID NO:201), WKWIHEPKGYHANF (SEQ ID NO:202), and PGPLPEAVL (SEQ ID NO:203). Therefore, the high-affinity, subtype-selective TGFβ1 inhibitors according to this disclosure can bind to a proTGFβ1 complex (e.g., human LLC) at an epitope containing one or more amino acid residues of SPPSQGEVPPGPLPEAVL (SEQ ID NO:201), WKWIHEPKGYHANF (SEQ ID NO:202), and / or PGPLPEAVL (SEQ ID NO:203).

[0445] Therefore, any antibody or antigen-binding fragment covered by this disclosure, such as those of categories 1 to 5 disclosed herein, may bind to one or more binding regions identified herein. As described herein, such antibodies may be used to treat TGFβ1 in subjects. Therefore, selecting an antibody or antigen-binding fragment thereof suitable for therapeutic use according to this disclosure may include identifying or selecting an antibody or fragment thereof that binds SPPSQGEVPPGPLPEAVL (SEQ ID NO: 201), WKWIHEPKGYHANF (SEQ ID NO: 202), PGPLPEAVL (SEQ ID NO: 203), or any portion thereof.

[0446] Table 12 provides non-limiting examples of protein domains or motifs of human proTGFβ1 as previously described (WO 2014 / 182676).

[0447] Table 12: Protein domains / motifs for the selection of human TGFβ1-related peptides

[0448]

[0449] Safety / Toxicity

[0450] Conventional pan-inhibitors of TGFβ, which antagonize multiple subtypes, are known to cause a variety of toxicities, including, for example, cardiovascular toxicity (cardiac injury, most notably valvular disease) reported in multiple species, including dogs and rats. These include hyperplasia in the aortic, right, and left aortic valves; inflammation in the aortic, left aortic, and ascending aorta; hemorrhage in the ascending aorta, aortic valve, and left aortic valve; and connective tissue degeneration in the ascending aorta (see, for example, Strauber et al., (2014) “Nonclinical safety evaluation of a Transforming GrowthFactorβ receptor I kinase inhibitor in Fischer 344 rats and beagle dogs” J. Clin. Pract 4(3):1000-196). See also Figure 21A .

[0451] In addition, neutralizing antibodies that bind to all three TGFβ isoforms have been associated with certain epithelial cell toxicities observed across multiple species, some of which are summarized below.

[0452] Table 13: Epithelial toxicity associated with pan-inhibitors of TGFβ

[0453]

[0454] Based on the applicant's earlier understanding (see PCT / US2017 / 021972), the lack of subtype specificity of conventional TGFβ antagonists can be a source of toxicity associated with TGFβ inhibition. The inventors seek to further achieve broad-spectrum TGFβ1 inhibition for the treatment of various diseases exhibiting multifaceted TGFβ1 dysregulation, while maintaining the safety / tolerability of subtype-selective inhibitors.

[0455] In a clinical context, therapeutic benefit is achieved only when the minimum effective concentration (MEC) of a drug (e.g., a monoclonal antibody) is below its minimum toxic concentration (MTC). Most (if not all) conventional TGFβ pan-inhibitors fail to achieve this, and in fact, pan-inhibitors appear to cause dose-limiting toxicities. Previous work by the applicant described subtype-selective inhibitors of TGFβ1 with significantly improved safety properties compared to conventional pan-inhibitors such as small molecule receptor antagonists and neutralizing antibodies. WO 2017 / 156500 discloses a subtype-selective inhibitor of TGFβ1 activation that, when administered to rats at doses up to 100 mg / kg per week for 4 weeks, showed no analyte-related toxicity, thus establishing the antibody's NOAEL as the highest detectable dose of 100 mg / kg. Subsequent work by the applicant has also shown that functionally enhanced antibodies possess the same safety properties. One objective here is to identify antibodies with higher affinity and potency, but at least the same or comparable level of safety.

[0456] exist Figure 21B and Figure 21C Results from a four-week rat toxicology study are provided. Two subtype-selective TGFβ1 inhibitors (Ab3 and Ab6), along with a small molecule ALK5 inhibitor and a monoclonal neutralizing antibody, were tested in separate studies. Neither subtype-selective antibody showed analyte-related toxicity, while the non-selective inhibitors, as expected, caused a variety of adverse events, consistent with published studies. Moreover, Ab6 was shown to be safe in cynomolgus monkeys at dose levels up to 300 mg / kg when administered weekly for four consecutive weeks (e.g., no adverse events were observed). Since Ab6 has shown efficacy in many in vivo models at doses as low as 3 mg / kg, it provides a therapeutic window of up to 100-fold. Importantly, this demonstrates that high potency does not necessarily imply a higher risk of toxicity. Without being bound by any particular theory, it can be expected that the highly selective nature of the antibodies disclosed herein may result in a lack of observed toxicity.

[0457] Therefore, in some embodiments, the novel antibody according to this disclosure has a maximum tolerated dose (MTD) of >100 mg / kg when administered weekly for at least 4 consecutive weeks. In some embodiments, the novel antibody according to this disclosure has a no-visible-adverse-effect level (NOAEL) of up to 100 mg / kg when administered weekly for at least 4 consecutive weeks. Suitable animal models for conducting safety / toxicology studies of TGFβ inhibitors and TGFβ1 inhibitors include, but are not limited to, rats, dogs, cynomolgus monkeys, and mice. In a preferred embodiment, the minimum effective amount of the antibody based on a suitable preclinical efficacy study is less than the NOAEL. More preferably, the minimum effective amount of the antibody is about one-third or less of the NOAEL. In a particularly preferred embodiment, the minimum effective amount of the antibody is about one-sixth or less of the NOAEL. In some embodiments, the minimum effective amount of the antibody is about one-tenth or less of the NOAEL.

[0458] In some embodiments, the present invention includes a subtype-selective antibody capable of inhibiting TGFβ1 signaling, which, when administered to a subject, does not cause cardiovascular or known epithelial toxicity at a dose effective for treating a TGFβ1-related indication. In some embodiments, the minimum effective dose of the antibody administered weekly, bi-weekly, or monthly is about 3-10 mg / kg. Preferably, the antibody does not cause minimum toxicity at a dose at least six times the minimum effective dose (e.g., six times the therapeutic window). More preferably, the antibody does not cause minimum toxicity at a dose at least ten times the minimum effective dose (e.g., ten times the therapeutic window). Even more preferably, the antibody does not cause minimum toxicity at a dose at least fifteen times the minimum effective dose (e.g., fifteen times the therapeutic window).

[0459] Therefore, the selection of antibodies or antigen-binding fragments thereof for therapeutic use may include: selecting antibodies or antigen-binding fragments that meet one or more criteria of categories 1-5 described herein; conducting in vivo efficacy studies in suitable preclinical models to determine the effective amount of the antibody or fragment; conducting in vivo safety / toxicology studies in suitable models to determine the amount of safe or toxic antibodies (e.g., MTD, NOAEL, or any parameter known in the art for evaluating safety / toxicity); and selecting antibodies or fragments that provide at least three times the therapeutic window (preferably six times, more preferably ten times, and even more preferably fifteen times). In a preferred embodiment, in vivo efficacy studies are conducted in two or more suitable preclinical models that generalize to a human condition. In some embodiments, such preclinical models include TGFβ1-positive cancers, which may optionally include immunosuppressive tumors. Immunosuppressive tumors may be resistant to cancer therapies such as CBT, chemotherapy, and radiotherapy. In some embodiments, the preclinical models are selected from MBT-2, Cloudman S91, and EMT6 tumor models.

[0460] The selected antibody or fragment can be used to prepare a pharmaceutical composition comprising the antibody or fragment. Such pharmaceutical compositions can be used to treat the TGFβ1 indication in subjects as described herein. For example, the indication for TGFβ1 can be proliferative disorders and / or fibrotic disorders.

[0461] Mechanism of action

[0462] The antibody of the present invention used for treatment is an inhibitory antibody against TGFβ1. Furthermore, the antibody is an activation inhibitor; that is, the antibody blocks the activation step of TGFβ1, rather than directly targeting the already activated growth factor.

[0463] In a broad sense, the term "inhibitory antibody" refers to an antibody that antagonizes or neutralizes the function of a target, such as growth factor activity. Advantageously, preferred inhibitory antibodies of this disclosure are capable of inhibiting the release of mature growth factors from a potential complex, thereby reducing growth factor signaling. Inhibitory antibodies include antibodies that target any epitope that, when bound to these antibodies, reduces growth factor release or activity. Such epitopes may be located on the predomain of a TGFβ protein (e.g., TGFβ1), growth factors, or other epitopes that, when bound by an antibody, result in reduced growth factor activity. Inhibitory antibodies of the present invention include, but are not limited to, antibodies that inhibit TGFβ1. In some embodiments, the inhibitory antibodies of this disclosure specifically bind to a combinatorial epitope, i.e., an epitope formed from two or more components / parts of an antigen or antigen complex. For example, a combinatorial epitope may be formed from contributions from multiple parts of a single protein, i.e., amino acid residues from more than one discontinuous segment of the same protein. Alternatively, a combinatorial epitope may be formed from contributions from multiple protein components of an antigen complex. In some embodiments, the inhibitory antibodies of this disclosure specifically bind to conformational epitopes (or conformation-specific epitopes), such as epitopes sensitive to the three-dimensional structure (i.e. conformation) of an antigen or antigen complex.

[0464] Conventional approaches to antagonizing TGFβ signaling have been used to: i) directly neutralize mature growth factors after they have become active, thereby depleting the free ligands available for receptor binding (e.g., releasing them from their potential precursor complexes); ii) use soluble receptor fragments capable of chelating free ligands (e.g., so-called ligand traps); or, iii) target their cell surface receptors to block ligand-receptor interactions. Each of these conventional approaches requires the antagonist to compete with the endogenous counterpart. Furthermore, the first two approaches (i and ii) target the active ligand, which is a transitional form. Therefore, such an antagonist must be kinetically able to overtake the endogenous receptor within a short window. In contrast, the third approach may provide a more durable effect, but inadvertently leads to undesirable inhibitory effects (and thus potential toxicity), since many growth factors (e.g., up to ~20) transduce signals via the same receptor.

[0465] To provide solutions to these drawbacks and further achieve greater selectivity and localization, the preferred mechanisms of action of inhibitory antibodies are, for example, those described herein that act upstream of TGFβ1 activation and ligand-receptor interactions. Therefore, it is contemplated that subtype-specific, background-permissive inhibitors of TGFβ1 suitable for carrying out the present invention should preferably target the inactive (e.g., potential) proTGFβ1 complex (e.g., a complex containing pre / potential TGFβ1) prior to its activation, to block the activation step at its source (e.g., in the disease microenvironment, e.g., the TME). According to a preferred embodiment of the invention, such inhibitors target the pre / potential TGFβ1 complex associated with the ECM and / or cell surface ligands, rather than free ligands that are transiently available for receptor binding.

[0466] Recent studies have further demonstrated the advantages of local targeting of tissue / cell tethering complexes at the source, in contrast to soluble active substances (i.e., mature growth factors released from the source). Ishihara et al. (Sci. Transl. Med. 11, eaau 3259 (2019) "Targeted antibody and cytokine cancer immune otherapies through collagen affinity") reported that when systemically administered drugs target tumor sites by binding to collagen-bound portions, they enhance antitumor immunity and reduce treatment-related toxicity compared to non-targeted counterparts.

[0467] The mechanism of action achieved by the antibodies disclosed herein may further contribute to enhancing the persistence of action, as well as greater overall efficacy and safety.

[0468] Interestingly, these antibodies may exhibit additional inhibitory activity against cell-associated TGFβ1 (LRRC33-proTGFβ1 and GARP-proTGFβ1). The applicant has found that antibodies binding to LRRC33 tend to be internalized upon binding to LRRC33 on the cell surface. It is unclear whether this internalization is actively induced by antibody binding or whether this phenomenon is caused by the macrophage's natural (e.g., passive) endocytic activity. However, the high-affinity, subtype-selective TGFβ1 inhibitor Ab6 was rapidly internalized in LRRC33 and proTGFβ1 transfected cells, and the internalization rate achieved by Ab6 was significantly higher than that of the reference antibody recognizing LRRC33 on the cell surface (…). Figure 6 Similar results were obtained from primary human macrophages. These observations increase the likelihood 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 potential LRRC33-proTGFβ1 levels. Therefore, subtype-selective TGFβ1 inhibitors can inhibit the LRRC33 arm of TGFβ1 through two parallel mechanisms of action: i) preventing the release of mature growth factors from the potential complex; and ii) removing the LRRC33-proTGFβ1 complex from the cell surface through internalization. Similar inhibitory mechanisms may apply to GARP-proTGFβ1.

[0469] In some embodiments, the antibody is a pH-sensitive antibody that binds its antigen with higher affinity at neutral pH (e.g., pH about 7) than at acidic pH (e.g., pH about 5). Such antibodies can exhibit a higher dissociation rate under acidic conditions than under neutral or physiological conditions. For example, the ratio of the dissociation rate measured at acidic pH to the dissociation rate measured at neutral pH (e.g., Kc at pH 5) is a significant difference. off Higher than K at pH 7 off The ratio 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 can be used as recovery antibodies. After the target binds to the cell surface, the antibody can trigger antibody-dependent internalization (and thus removal) of the membrane-bound proTGFβ1 complex (which binds to LRRC33 or GARP). Subsequently, in an acidic intracellular compartment (such as a lysosome), the antibody-antigen complex dissociates, and the free antibody can be transported back to the extracellular domain.

[0470] Therefore, in some implementations, the selection of antibody or antigen-binding fragments for therapeutic purposes may be based in part on the antibody’s ability to induce antibody-dependent internalization and / or pH-dependent internalization.

[0471] Antigen complexes and their components

[0472] The novel antibodies disclosed herein specifically bind to each of four known large human potential complexes (e.g., hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1), selectively inhibiting TGFβ1 activation. Preferred antibodies further satisfy one or more of the criteria for categories 1-5 listed in Table 1.

[0473] Screening (e.g., identification and selection) of such antibodies involves the use of suitable antigen complexes, which are typically recombinantly produced. Useful protein components that can contain such antigen complexes are provided, including TGFβ isoforms and associated peptides, fragments, and variants, presenting molecules (e.g., LTBP, GARP, LRRC33), and associated peptides, fragments, and variants. These components can be expressed, purified, and capable of forming protein complexes (such as large potential complexes) that can be used in antibody screening processes. Screening can include positive selection, where desired binders are selected from pools or libraries of binders and non-binders, and negative selection, where unwanted binders are removed from the pools. Typically, positive screening includes at least one matrix-associated complex (e.g., LTBP1-proTGFβ1 and / or LTBP1-proTGFβ1) and at least one cell-associated complex (e.g., GARP-proTGFβ1 and / or LRRC33-proTGFβ1) to ensure that the selected binders have affinity for both biological backgrounds.

[0474] In some embodiments, TGFβ1 comprises a naturally occurring mammalian amino acid sequence. In some embodiments, TGFβ1 comprises a naturally occurring amino acid sequence. In some embodiments, TGFβ1 comprises an amino acid sequence of a human, monkey, rat, or mouse. In some embodiments, the antibody or its antigen-binding portion described herein does not specifically bind to TGFβ2. In some embodiments, the antibody or its antigen-binding portion described herein does not specifically bind to TGFβ3. In some embodiments, the antibody or its antigen-binding portion described herein does not specifically bind to either TGFβ2 or TGFβ3. In some embodiments, the antibody or its antigen-binding portion described herein specifically binds to TGFβ1, which comprises the amino acid sequence shown in SEQ ID NO:34. The amino acid sequences of TGFβ2 and TGFβ3 are shown in SEQ ID NO:38 and 32, respectively. In some embodiments, the antibody or its antigen-binding portion described herein specifically binds to TGFβ1, which comprises a non-naturally occurring amino acid sequence (or, referred to herein as non-naturally occurring TGFβ1). For example, non-natural TGFβ1 may contain one or more recombination-induced mutations relative to the naturally occurring TGFβ1 amino acid sequence. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequences comprise amino acid sequences as shown in SEQ ID NO:24-35, as shown in Table 14. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequences comprise amino acid sequences as shown in SEQ ID NO:36-43, as shown in Table 15.

[0475] TGFβ1 (pre-structural domain+) Growth factor domain )

[0476] LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRR ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGP CPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO:24)

[0477] TGFβ2 (pre-structural domain+) Growth factor domain )

[0478] SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKR ALDAAYCFRNVQD NCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASPCCCVSQDLEPLTIL YYIGKTPKIEQLSNMIVKSCKCS (SEQ ID NO:28)

[0479] TGFβ3 (prodomain + Growth factor domain )

[0480] SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKR ALDTNYCFRNLEENCC VRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYV GRTPKVEQLSNMVVKSCKCS (SEQ ID NO:32)

[0481] Table 14: Exemplary TGFβ1, TGFβ2 and TGFβ3 Amino Acid Sequences

[0482]

[0483]

[0484] Table 15: Exemplary Non-Human Amino Acid Sequences

[0485]

[0486]

[0487]

[0488]

[0489] In some embodiments, the antigen protein complex (e.g., the LTBP-TGFβ1 complex) may comprise one or more presenting molecules, such as LTBP proteins (e.g., LTBP1, LTBP2, LTBP3, and LTBP4), GARP proteins, LRRC33 proteins, or fragments thereof. Typically, the minimum required fragment for implementing the embodiments disclosed herein comprises at least 50 amino acids, preferably at least 100 amino acids, of a presenting molecule protein containing at least two cysteine ​​residues capable of forming disulfide bonds with the proTGFβ1 complex. Specifically, these Cys residues form covalent bonds with cysteine ​​residues present near the N-terminus of each monomer of the proTGFβ1 complex. In the three-dimensional structure of the proTGFβ1 dimer complex, the so-called “α-1 helix” at the N-terminus of each monomer is very close to each other (see, e.g., Figure 18B The two helices (near the bottom of the gray structure) are positioned to set the distance between the two cysteine ​​residues (one per helix) required to form an effective covalent bond with the corresponding cysteine ​​pair present in the presenting molecule (see, for example, Cuende et al., (2015) Sci. Trans. Med. 7: 284ra56). Therefore, when fragments of the presenting molecule are used to form LLCs during screening processes (e.g., immunization, library screening, identification, and selection), such fragments should contain cysteine ​​residues spaced at the correct distance, which will allow for the formation of the correct disulfide bond with the proTGFβ1 complex to preserve the correct conformation of the resulting LLC. LTBPs (e.g., LTBP1, LTBP3, and LTBP4), for example, may contain "cysteine-rich domains" to mediate covalent interactions with proTGFβ1.

[0490] The antibody or its antigen-binding portion as described herein can bind 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 proteins may contain LTBP1, or its splice variants and / or fragments thereof. The recombinant LTBP1 protein may also be modified to contain one or more detectable tags. In some embodiments, the LTBP1 protein contains a leader sequence (e.g., a natural or non-natural leader sequence). In some embodiments, the LTBP1 protein does not contain a leader sequence (i.e., the leader sequence has been processed or cleaved). Such detectable tags may include, but are not limited to, biotinylate tags, 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 contains the amino acid sequences shown in SEQ ID NO:46 and 47 in Table 15. In some embodiments, the LTBP1 protein comprises the amino acid sequence shown in SEQ ID NO:50 in Table 17.

[0491] The antibody or its antigen-binding portion as described herein can bind 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 proteins may contain LTBP3, or its splice variants and / or fragments thereof. In some embodiments, the LTBP3 protein contains a leader sequence (e.g., a natural or non-natural leader sequence). In some embodiments, the LTBP3 protein does not contain a leader sequence (i.e., the leader sequence has been processed or cleaved). The recombinant LTBP3 protein may also be modified to contain one or more detectable tags. Such detectable tags may include, but are not limited to, biotinylate tags, polyhistidine tags, myc tags, HA tags, and / or fluorescent tags. In some embodiments, the LTBP3 protein is a mammalian LTBP3 protein. In some embodiments, the LTBP3 protein is a human, monkey, mouse, or rat LTBP3 protein. In some embodiments, the LTBP3 protein contains the amino acid sequences shown in SEQ ID NO:44 and 45 in Table 15. In some embodiments, the LTBP1 protein comprises the amino acid sequence shown in SEQ ID NO:51 in Table 17.

[0492] The antibody or its antigen-binding portion as described herein can bind 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 GARPs can be recombinant and are referred to herein as recombinant GARPs. Some recombinant GARPs may contain one or more modifications, truncations, and / or mutations compared to wild-type GARPs. Recombinant GARPs may be modified to be soluble. In some embodiments, the GARP protein contains a leader sequence (e.g., a natural or non-natural leader sequence). In some embodiments, the GARP protein does not contain a leader sequence (i.e., the leader sequence has been processed or cleaved). In other embodiments, the recombinant GARP may be modified to contain one or more detectable tags. In further embodiments, such detectable tags may include, but are not limited to, biotinylate tags, 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 a human, monkey, mouse, or rat GARP protein. In some embodiments, the GARP protein comprises the amino acid sequences shown in SEQ ID NO:48-49 of Table 15. In some embodiments, the GARP protein comprises the amino acid sequences shown in SEQ ID NO:52 and 53 of Table 18. In some embodiments, the antibody or its antigen-binding moiety described herein does not bind to TGFβ1 in a background-dependent manner, for example, binding to TGFβ1 only occurs when the TGFβ1 molecule complexes with a specific presenting molecule (such as GARP). Instead, the antibody or its antigen-binding moiety binds to TGFβ1 in a background-independent manner. In other words, the antibody or its antigen-binding moiety binds to TGFβ1 when bound to any of the following presenting molecules: GARP, LTBP1, LTBP3, and / or LRCC33.

[0493] The antibody or its antigen-binding moiety described herein can bind 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 can be recombinant and is 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 proteins can be modified to be soluble. For example, in some embodiments, the extracellular domain of LRRC33 may be expressed together with a C-terminal His tag to express soluble LRRC33 protein (sLRRC33; see, for example, SEQ ID NO:84). In some embodiments, the LRRC33 protein includes a leader sequence (e.g., a natural or non-natural leader sequence). In some embodiments, the LRRC33 protein does not include a leader sequence (i.e., the leader sequence has been processed or cleaved). In other embodiments, the recombinant LRRC33 protein may be modified to include one or more detectable tags. In further embodiments, such detectable tags may include, but are not limited to, biotinylate tags, 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 the amino acid sequences shown in SEQ ID NO: 83, 84, and 101 in Table 18.

[0494] Table 17: Exemplary LTBP amino acid sequences

[0495]

[0496]

[0497] Table 18: Exemplary GARP and LRRC33 amino acid sequences

[0498]

[0499]

[0500] Pharmaceutical compositions and formulations

[0501] The present invention further provides pharmaceutical compositions suitable for administration in human and non-human subjects. One or more high-affinity, background-independent antibodies covered by the present invention can be formulated or mixed with pharmaceutically acceptable carriers (excipients), including, for example, buffers, to form pharmaceutical compositions. Such formulations can be used to treat diseases or conditions involving TGFβ signaling. In a particularly preferred embodiment, such formulations can be used for immuno-oncology applications.

[0502] The pharmaceutical compositions of the present invention can be administered to patients to alleviate TGFβ-related indications (e.g., fibrosis, immune disorders, and / or cancer). "Acceptable" means that the carrier is compatible with (and preferably, capable of stabilizing) the active ingredient of the composition and is harmless 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 described above. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KEHoover. In one example, the pharmaceutical compositions described herein contain one or more antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex, wherein the antibodies recognize different epitopes / residues of the complex.

[0503] The pharmaceutical composition used in this method may contain pharmaceutically acceptable carriers, excipients, or stabilizers in lyophilized or aqueous form (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KEHoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and may contain buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butyl or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less) The following substances are considered as a whole: polypeptides (approximately 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; 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; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants, such as Tween. TM Prunnick TM Or polyethylene glycol (PEG). This article further describes pharmaceutically acceptable excipients.

[0504] The present invention also includes pharmaceutical compositions comprising an antibody or a fragment thereof according to the invention, and pharmaceutically acceptable excipients.

[0505] Therefore, antibodies or molecules containing antigen-binding fragments of such antibodies can be formulated into pharmaceutical compositions suitable for human administration.

[0506] Pharmaceutical formulations may contain one or more excipients. In some embodiments, one or more excipients may be selected from the list provided below: https: / / www.accessdata.fda.gov / scripts / cder / iig / index.Cfm?event=browseByLetter.page&Letter=A

[0507] Pharmaceutical compositions are typically formulated into active biological products (e.g., monoclonal antibodies, engineered conjugate molecules containing antigen-binding fragments, etc.) at final concentrations between about 2 mg / mL and about 200 mg / mL. For example, the final concentration (wt / vol) of the formulation can be approximately 2-200, 2-180, 2-160, 2-150, 2-120, 2-100, 2-80, 2-70, 2-60, 2-50, 2-40, 5-200, 5-180, 5-160, 5-150, 5-120, 5-100, 5-80, 5-70, 5-60, 5-50, 5-40, 10-200 mg / mL. 10-180, 10-160, 10-150, 10-120, 10-100, 10-80, 10-70, 10-60, 10-50, 10-40, 20-200, 20-180, 20-160, 20-150, 20-120, 20-100, 20-80, 20-70, 20-60, 20-50, 20-40, 30-200, 30-18 0, 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 The values ​​are within the range of -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, and 70-80 mg / mL. In some embodiments, the final concentration of the biological product 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.

[0508] The pharmaceutical compositions of the present invention are preferably formulated with suitable buffers. Suitable buffers include, but are not limited to, phosphate buffers, citrate buffers, and histidine buffers.

[0509] The final pH of the formulation is typically between pH 5.0 and 8.0. For example, the pH of a pharmaceutical composition may be about 5.0, 5.2, 5.5, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, 7.4, 7.5, 7.6, or 7.8.

[0510] The pharmaceutical compositions disclosed herein may contain 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.

[0511] The pharmaceutical compositions disclosed herein may contain stabilizers. For liquid-protein products, stability can be enhanced by selecting pH buffer salts, and amino acids are also commonly used. Interactions typically occur at the liquid / air interface or liquid / solid interface (with the packaging), leading to protein adsorption and aggregation upon unfolding. Suitable stabilizers include, but are not limited to, sucrose, maltose, sorbitol, and certain amino acids such as histidine, glycine, methionine, and arginine.

[0512] The pharmaceutical compositions disclosed herein 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, Rh hyaluronidase, sodium succinate, potassium phosphate, disodium EDTA, sodium chloride, potassium chloride, maltose, histidine acetate, sorbitol, penteacin, human serum albumin, and penteacin.

[0513] In some embodiments, the pharmaceutical compositions disclosed herein may contain preservatives.

[0514] The pharmaceutical compositions disclosed herein are typically available in liquid or lyophilized form. Typically, the product can be packaged in vials (e.g., glass vials). Products available in syringes, pens, or autoinjectors may be available as pre-filled liquids within these container / sealing systems.

[0515] In some instances, the pharmaceutical compositions described herein comprise liposomes containing antibodies that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex, which can be prepared by any suitable method, such as 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. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced cycle times are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with a defined pore size to produce liposomes with a desired diameter.

[0516] In some embodiments, liposomes with targeting properties are selected to preferentially deliver or target the drug composition to certain tissue or cell types. For example, certain nanoparticle-based carriers with bone marrow targeting properties, such as lipid-based nanoparticles or liposomes, can be used. See, for example, Sou (2012) “Advanced drug carriers targeting bone marrow”, ResearchGate publication 232725109.

[0517] In some embodiments, the pharmaceutical compositions of the present invention may contain or be used with adjuvants. Certain adjuvants are anticipated to enhance the immune response of a subject to, for example, tumor antigens, and to promote the function of effector T cells, monocyte-derived dendritic cell differentiation, antigen uptake, and enhanced APC presentation. Suitable adjuvants include, but are not limited to, retinoic acid-based adjuvants and their derivatives, oil-in-water emulsion-based adjuvants such as MF59 and other squalene-containing adjuvants, Toll-like receptor (TRL) ligands (e.g., CpG), α-tocopherol (vitamin E), and their derivatives.

[0518] The antibodies described herein can also be encapsulated in microcapsules, for example, prepared by coagulation techniques or by interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or crude emulsions. Exemplary techniques have been previously described; see, for example, Remington, The Science and Practice of Pharmacy, 20th Ed. Mack Publishing (2000).

[0519] In other instances, the pharmaceutical compositions described herein can be formulated into sustained-release forms. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being in the form of a molded article, such as a film or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (US Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl, non-degradable ethylene-vinyl acetate, and degradable lactic-glycolic acid copolymers, such as LUPRON DEPOT. TM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose isobutyrate acetate and poly-D-(-)-3-hydroxybutyric acid.

[0520] Pharmaceutical compositions intended for internal administration must be sterile. This can be readily achieved, for example, by filtration through a sterile filter membrane. Therapeutic antibody compositions are typically placed in containers with sterile access channels, such as intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle.

[0521] The pharmaceutical compositions described herein may be unit dosage forms, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or for inhalation or blowing in.

[0522] Suitable surfactants include, in particular, nonionic agents, such as polyoxyethylene sorbitan (e.g., Tween). TM 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span) TM 20, 40, 60, 80, or 85). Compositions containing surfactants will conveniently contain 0.05 to 5% surfactant, and may be 0.1 to 2.5%. It should be understood that other ingredients, such as mannitol or other pharmaceutically acceptable carriers, may be added if desired.

[0523] Suitable emulsions can be 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 it can be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and mixed with phospholipids (e.g., lecithin, soybean lecithin, or soybean lecithin) and water to form an emulsion. It should be understood that other ingredients, such as glycerol or glucose, can be added to adjust the emulsion tension. Suitable emulsions typically contain up to 20% oil, for example, between 5% and 20%.

[0524] The emulsion composition can be made by combining the antibody of the present invention with Intralipid. TM Those prepared by mixing their components (soybean oil, egg lecithin, glycerin, and water).

[0525] Kits for detecting, monitoring, or alleviating TGFβ-related indications

[0526] This disclosure also provides kits for alleviating diseases / symptoms associated with TGFβ-related indications. Such kits may comprise one or more containers containing an antibody or its antigen-binding moiety that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex, such as any of those described herein.

[0527] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include a description of administering an antibody or its antigen-binding moiety that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex to treat, delay the onset of, or alleviate the target disease described herein. The kit may also include a description of selecting individuals suitable for treatment based on identifying whether the individual has the target disease. In other embodiments, the instructions include a description of administering an antibody or its antigen-binding moiety to an individual at risk of the target disease.

[0528] Instructions for use regarding the use of antibodies or their antigen-binding moieties that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex generally include information on the intended therapeutic dose, dosing regimen, and route of administration. Containers may be unit dose, bulk packaging (e.g., multi-dose packaging), or subunit dose. Instructions provided with kits disclosed herein are typically written instructions on a label or packaging insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0529] The label or packaging insert indicates that the composition is used to treat, delay the onset of, and / or alleviate diseases or conditions associated with TGFβ-related indications. Instructions for use in practicing any of the methods described herein may be provided.

[0530] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth bottles, flexible packaging (e.g., sealed polyester film or plastic bags), etc. Packaging for use in combination with specific devices is also contemplated, such as inhalers, nasal application devices (e.g., nebulizers), or infusion devices, such as micropumps. The kit may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). The container may also have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an antibody or its antigen-binding moiety that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex (as described herein).

[0531] The kit may optionally include addi...

Claims

1. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment binding each of the following antigen complexes at a KD of ≤5 nM: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The KD mentioned therein was measured using the biological layer interferometry method. The antibody or its antigen-binding fragment thereof inhibits TGFβ1 activation; The antibody or its antigen-binding fragment mentioned therein is a fully human or humanized antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 107; H-CDR2 as shown in SEQ ID NO: 103; H-CDR3 as shown in SEQ ID NO: 6; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

2. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment binding each of the following antigen complexes at a KD of ≤5 nM: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The KD mentioned therein was measured using the biological layer interferometry method. The antibody or its antigen-binding fragment thereof inhibits TGFβ1 activation; The antibody or its antigen-binding fragment mentioned therein is a fully human or humanized antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 107; H-CDR2 as shown in SEQ ID NO: 111; H-CDR3 as shown in SEQ ID NO: 110; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

3. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment binding each of the following antigen complexes at a KD of ≤5 nM: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The KD mentioned therein was measured using the biological layer interferometry method. The antibody or its antigen-binding fragment thereof inhibits TGFβ1 activation; The antibody or its antigen-binding fragment mentioned therein is a fully human or humanized antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 114; H-CDR2 as shown in SEQ ID NO: 103; H-CDR3 as shown in SEQ ID NO: 110; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

4. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment binding each of the following antigen complexes at a KD of ≤5 nM: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The KD mentioned therein was measured using the biological layer interferometry method. The antibody or its antigen-binding fragment thereof inhibits TGFβ1 activation; The antibody or its antigen-binding fragment mentioned therein is a fully human or humanized antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 116; H-CDR2 as shown in SEQ ID NO: 111; H-CDR3 as shown in SEQ ID NO: 110; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

5. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment binding each of the following antigen complexes at a KD of ≤5 nM: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The KD mentioned therein was measured using the biological layer interferometry method. The antibody or its antigen-binding fragment thereof inhibits TGFβ1 activation; The antibody or its antigen-binding fragment mentioned therein is a fully human or humanized antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 119; H-CDR2 as shown in SEQ ID NO: 120; H-CDR3 as shown in SEQ ID NO: 121; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

6. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment binding each of the following antigen complexes at a KD of ≤5 nM: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The KD mentioned therein was measured using the biological layer interferometry method. The antibody or its antigen-binding fragment thereof inhibits TGFβ1 activation; The antibody or its antigen-binding fragment mentioned therein is a fully human or humanized antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 114; H-CDR2 as shown in SEQ ID NO: 103; H-CDR3 as shown in SEQ ID NO: 125; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

7. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to each of the following antigen complexes: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 107; H-CDR2 as shown in SEQ ID NO: 103; H-CDR3 as shown in SEQ ID NO: 6; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

8. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to each of the following antigen complexes: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 107; H-CDR2 as shown in SEQ ID NO: 111; H-CDR3 as shown in SEQ ID NO: 110; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

9. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to each of the following antigen complexes: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 114; H-CDR2 as shown in SEQ ID NO: 103; H-CDR3 as shown in SEQ ID NO: 110; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

10. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to each of the following antigen complexes: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 116; H-CDR2 as shown in SEQ ID NO: 111; H-CDR3 as shown in SEQ ID NO: 110; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

11. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to each of the following antigen complexes: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 119; H-CDR2 as shown in SEQ ID NO: 120; H-CDR3 as shown in SEQ ID NO: 121; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

12. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to each of the following antigen complexes: i) Human LTBP1-proTGFβ1; ii) Human LTBP3-proTGFβ1; iii) Human GARP-proTGFβ1; and iv) Human LRRC33-proTGFβ1; The antibody or its antigen-binding fragment comprises: H-CDR1 as shown in SEQ ID NO: 114; H-CDR2 as shown in SEQ ID NO: 103; H-CDR3 as shown in SEQ ID NO: 125; L-CDR1 as shown in SEQ ID NO: 105; L-CDR2 as shown in SEQ ID NO: 106; and The L-CDR3 shown in SEQ ID NO:

12.

13. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:

13. H ) and the light chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:

15. L ).

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein the V H Contains SEQ ID NO: 13 and the V L Includes SEQ ID NO:

15.

15. The antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:

129. H ) and the light chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:15 L ).

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein the V H Contains SEQ ID NO: 129 and the V L Includes SEQ ID NO:

15.

17. The antibody or antigen-binding fragment thereof according to claim 3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:

131. H ) and the light chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:15 L ).

18. The antibody or antigen-binding fragment thereof according to claim 17, wherein the V H Contains SEQ ID NO: 131 and the V L Includes SEQ ID NO:

15.

19. The antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:

133. H ) and the light chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:15 L ).

20. The antibody or antigen-binding fragment thereof according to claim 19, wherein the V H Contains SEQ ID NO: 133 and the V L Includes SEQ ID NO:

15.

21. The antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:

134. H ) and the light chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:15 L ).

22. The antibody or antigen-binding fragment thereof according to claim 21, wherein the V H Contains SEQ ID NO: 134 and the V L Includes SEQ ID NO:

15.

23. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:

137. H ) and the light chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:15 L ).

24. The antibody or antigen-binding fragment thereof according to claim 23, wherein the V H Contains SEQ ID NO: 137 and the V L Includes SEQ ID NO:

15.

25. The antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:

141. H ) and the light chain variable domain (V) having at least 90% sequence identity with SEQ ID NO:15 L ).

26. The antibody or antigen-binding fragment thereof according to claim 25, wherein the V H Contains SEQ ID NO: 141 and the V L Includes SEQ ID NO:

15.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1-26, wherein the antibody or antigen-binding fragment thereof binds each of the antigen complexes with a KD of ≤1 nM, wherein the KD is measured by biolayer interferometry.

28. The antibody or antigen-binding fragment thereof according to claim 27, wherein the antibody or antigen-binding fragment thereof binds each of the antigen complexes with a KD of ≤0.5 nM, wherein the KD is measured by biolayer interferometry.

29. The antibody or antigen-binding fragment thereof according to any one of claims 1-26, wherein the binding site is located within a portion of the growth factor domain shown in SEQ ID NO: 169 and SEQ ID NO:

148.

30. The antibody or antigen-binding fragment thereof according to any one of claims 1-26, wherein it is a human IgG4 or IgG1 subtype.

31. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-26, and an excipient.

32. Use of the antibody or antigen-binding fragment thereof according to claims 1-26 in the preparation of a medicament for treating a subject with TGFβ1-related cancer, myelofibrosis, and / or renal fibrosis, wherein the TGFβ1-related cancer is a solid tumor.

33. The use according to claim 32, wherein the subject has TGFβ1-related cancer.

34. The use according to claim 33, wherein the subject has primary or acquired resistance to the cancer therapy.

35. The use according to claim 34, wherein the cancer therapy is checkpoint inhibition therapy, chemotherapy, and / or radiation therapy.

36. The use according to claim 33, wherein the treatment comprises administering the composition of claim 31 in combination with other cancer therapies selected from: checkpoint inhibitors, chemotherapy, radiotherapy and cancer vaccines.

37. A method for preparing a pharmaceutical composition, comprising: i) Providing an antibody or antigen-binding fragment thereof according to any one of claims 1-26; ii) Formulate the antibody or its antigen-binding fragment into a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

Images