High-affinity, isoform-selective TGFβ1 inhibitors and use thereof

Abstract

To provide monoclonal antibodies and antigen-binding fragments thereof capable of selectively inhibiting TGFβ1 with high potency, and also to provide related compositions, methods and therapeutic use.SOLUTION: Provided is an antibody or an antigen-binding fragment thereof, binding each of the following antigen complexes with a KD of ≤10 nM, as measured by a solution equilibrium titration-based assay: i) human LTBP1-precursor TGFβ1; ii) human LTBP3-precursor TGFβ1; iii) human GARP-precursor TGFβ1; and iv) human LRRC33-precursor TGFβ1, the antibody or an antigen-binding fragment thereof being a fully human or humanized antibody or fragment thereof and optionally binding with a KD of ≤1 nM.SELECTED DRAWING: None

Description

[Background technology]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application Nos. 62 / 696,752, filed July 11, 2018; 62 / 718,196, filed August 13, 2018; 62 / 737,534, filed September 27, 2018; 62 / 758,180, filed November 9, 2018; 62 / 810,263, filed February 25, 2019; and 62 / 827,552, filed April 1, 2019, each of which is entitled "High-Affinity Context-Independent TGFβ1 Inhibitors and Uses Thereof," the contents of each of which are expressly incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on July 11, 2019, is titled 127036-03520_SL.txt and is 261,252 bytes in size.

[0003] Transforming growth factor beta 1 (TGFβ1) is a member of the TGFβ growth factor superfamily, along with two other structurally related isoforms, TGFβ2 and TGFβ3, each encoded by a separate gene. These TGFβ isoforms function as pleiotropic cytokines that regulate cell proliferation, differentiation, immunoregulation (e.g., adaptive immune response), and other diverse biological processes in both homeostatic and disease contexts. The three TGFβ isoforms signal through the same cell surface receptor and initiate similar canonical downstream signaling events, including the SMAD2 / 3 pathway. However, gene knockout studies in mice demonstrate diverse phenotypes, suggesting that each isoform plays a distinct role in vivo. This may be achieved in part by the differential expression patterns of the three isoforms.

[0004] Within the immune system, T cells are recognized as the primary direct target of TGFβ. TGFβ signaling is important in regulating effector cell proliferation and the differentiation of effector and regulatory T cells. For example, TGFβ is a potent suppressor of Th1 and Th2 effector T cells. The effector function of cytotoxic T cells has also been shown to be suppressed by TGFβ through multiple mechanisms. Furthermore, evidence indicates that other cell types of the immune system, such as dendritic cells (e.g., Langerhans cells) and natural killer (NK) cells, are also regulated by the TGFβ signaling pathway. Dysregulation of TGFβ has been linked to several pathologies, including cancer, fibrosis, and immune disorders.

[0005] Many biological processes in which the extracellular matrix plays a role are related to TGFβ signaling: TGFβ has been implicated in wound healing, tumor invasion and metastasis, and the progression of fibrosis, to name a few.

[0006] For these and other reasons, TGFβ is an attractive therapeutic target for treating immune disorders, various proliferative disorders, and fibrotic conditions. However, observations from preclinical studies, including in rats and dogs, have revealed significant toxicity associated with systemic inhibition of TGFβ in vivo. Furthermore, although several TGFβ inhibitors have been developed to date, most clinical programs targeting TGFβ have been discontinued due to the risk of significant side effects (e.g., summarized in WO 2017 / 156500). Therefore, despite a body of direct and indirect evidence implicating TGFβ signaling in the progression of diseases such as cancer and fibrosis, no TGFβ therapeutics currently available are considered safe and effective.

[0007] Previously, the present applicants have described a class of monoclonal antibodies that function via a novel mechanism of action to modulate growth factor signaling (see, e.g., WO 2014 / 182676 ). These antibodies were designed to exploit the fact that TGFβ1 is expressed as a latent precursor protein complex consisting of a prodomain and growth factor, requiring an activation step to release the growth factor from the latent complex. Instead of taking the traditional approach of directly targeting the mature growth factor itself after activation (e.g., neutralizing antibodies), this novel class of inhibitory antibodies specifically targets the inactive precursor protein complex itself to preemptively block the activation step upstream of the ligand-receptor interaction. It was reasoned that this unique mechanism of action should offer the advantage of achieving both spatial and temporal benefits by acting at its source, i.e., by targeting the latent precursor TGFβ1 (proTGFβ1) complex within the disease microenvironment before activation occurs. DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0008] Using this approach, monoclonal antibodies were generated that bind to TGFβ1 in an isoform-selective manner and inhibit its activation step (i.e., release of mature growth factor from the latent complex) (see WO2017 / 156500). The data presented therein support the view that isoform-specific inhibition of TGFβ (as opposed to pan-inhibition) may provide an improved safety profile for antagonizing TGFβ in vivo. Taking this into consideration, the applicant then sought to develop TGFβ1 inhibitors that are i) isoform-specific and ii) broadly capable of targeting multiple TGFβ1 signaling complexes associated with different presentation molecules as therapeutic agents for conditions driven by pleiotropic TGFβ1 effects and their dysregulation.

[0009] Such antibodies were subsequently described in PCT / US2018 / 012601 (filed January 5, 2018). Indeed, the isoform-specific inhibitors described therein targeted both ECM-associated TGFβ1 and immune cell-associated TGFβ1, thereby blocking multiple sources of TGFβ1 in multiple biological contexts while maintaining isoform specificity. Data from several in vivo models demonstrating the efficacy and safety of isoform-selective TGFβ1 activation inhibitors have been disclosed, demonstrating that such inhibitors are useful for treating diseases involving dysregulation of both ECM-associated TGFβ1 and immune cell-associated TGFβ1 in vivo.

[0010] While the earlier studies cited above have demonstrated the utility of antibodies capable of targeting each of the known precursor TGFβ1 complexes and inhibitory activities, improved isoform-selective TGFβ1 inhibitors with even greater in vivo potency are desirable. [Means for solving the problem]

[0011] The present disclosure provides a novel class of high-affinity, isoform-selective antibodies capable of inhibiting TGFβ1 activation with high efficacy. These include antibodies (including immunoglobulins and antigen-binding fragments or portions thereof, as well as engineered molecules incorporating such fragments) that can target multiple presentation molecule-precursor TGFβ1 complexes (referred to as "large latent complexes" or "LLCs") with high affinity. These antibodies retain the requisite selectivity and safety profile and have been shown to achieve improved in vivo efficacy in multiple preclinical models, with potential for translation to human conditions. These attributes of TGFβ1 inhibitors open up opportunities for developing safe and effective TGFβ1 therapeutic agents for treating diseases involving TGFβ1 dysregulation.

[0012] The following selection criteria were taken into consideration when generating the precursor TGFβ1 antibodies of the present disclosure: 1) isoform selectivity, 2) high affinity for human LLCs, e.g., LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1, 3) robust inhibitory potency, 4) favorable in vivo safety / toxicity profile, and 5) in vivo efficacy in preclinical models that recapitulate human disease. Furthermore, when evaluating the efficacy of TGFβ1 inhibitors used in combination therapy (e.g., add-on therapy), the ability to achieve synergistic effects (as a control for merely additive effects) should be considered. Based on these criteria, the inventors of the present disclosure have identified a class of high-affinity monoclonal antibodies and fragments thereof that can specifically target the precursor TGFβ1 complex and potently block TGFβ1 activation. In some embodiments, the novel antibodies disclosed herein exhibit high affinity across all target LLCs (e.g., KDs in the nanomolar to subnanomolar range). In preferred embodiments, such antibodies are unbiased across various precursor TGFβ1 complexes, such that the antibodies have equal affinity for all target complexes. Related compositions, therapeutic uses, preparations, formulations, processes, and methods are encompassed by the present invention.

[0013] Thus, in some embodiments, the invention includes a monoclonal antibody or antigen-binding fragment thereof capable of binding to each of the following human LLC complexes with a KD of ≦10 nM as measured by solution equilibrium titration: LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1. In some embodiments, the antibody binds to each of the human LTBP1-precursor TGFβ1 and LTBP3-precursor TGFβ1 complexes with a KD of ≦1 nM. Preferably, the antibody has a KD of ≦1 nM for each of the four human LLCs.

[0014] In some embodiments, the antibody or fragment binds to the Latency Lasso of precursor TGFβ1 or a portion thereof. In some embodiments, the antibody or fragment further binds to a portion of the growth factor domain, 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 Latency Lasso. Optionally, the epitope may further comprise one or more amino acid residues of the growth factor domain. Thus, such an epitope may be a combinatorial epitope. In preferred embodiments, the antibody does not bind to free TGFβ1 growth factor that is not associated with the precursor TGFβ1 complex.

[0015] The TGFβ1 inhibitor of the present invention is a functional antibody in that it has inhibitory activity against TGFβ1.The potency of such antibody is isoform-specific, as measured by suitable in vitro potency assay, such as the cell-based reporter assay described herein.Therefore, the antibody does not bind to or inhibit TGFβ2 or TGFβ3 counterparts.

[0016] The TGFβ1 inhibitor of the present invention can block the release of mature growth factors from latent 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 integrins to LLC.

[0017] In some embodiments, the TGFβ1 inhibitors of the present invention may function through a dual inhibitory mechanism of action on cell-associated LLC (e.g., GARP-precursor TGFβ1 and LRRC33-precursor TGFβ1). In the first mechanism, such inhibitors block the activation step of TGFβ1 associated with membrane-anchored GARP and / or LRRC33. In the second mechanism, such inhibitors may induce antibody-dependent internalization (and thus removal) of LLC from the cell surface upon target engagement, thereby reducing TGFβ1 signaling in the niche. In some embodiments, the antibody is a pH-sensitive antibody, characterized by binding to the precursor TGFβ1 complex with higher affinity at neutral pH than at acidic pH.

[0018] In some embodiments, the TGFβ1 inhibitors of the present invention are effective in reducing the expression of disease-related genes such as TGFB1, Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, Loxl2, CCL2, and Mmp2.

[0019] In some embodiments, the TGFβ1 inhibitors of the present invention are effective in reducing phosphorylation of the downstream effector SMAD2 / 3 in vivo.

[0020] In some embodiments, the TGFβ1 inhibitors of the present invention are effective for treating TGFβ1-related indications. Such indications include diseases involving aberrant gene expression, diseases involving ECM dysregulation, diseases characterized by an increase in immunosuppressive cells (e.g., Tregs, MDSCs, and / or M2 macrophages), diseases involving mesenchymal transition, diseases involving proteases, diseases associated with abnormal stem cell proliferation and / or differentiation, etc., although these disease categories are not intended to be mutually exclusive. In some embodiments, the TGFβ1-related indication is a proliferative disorder, such as a myeloproliferative disorder and cancer with a solid tumor. In some embodiments, the TGFβ1-related indication is a fibrotic disorder, such as organ fibrosis. The cancer may be an advanced cancer, including locally advanced tumors / cancers and metastatic cancers.

[0021] In some embodiments, the TGFβ1 inhibitors of the present invention can reduce the number of immunosuppressive cell populations at disease sites, such as tumor and fibrotic microenvironments. In some embodiments, the immunosuppressive cell populations can include M2-polarized macrophages and / or MDSCs.

[0022] In some embodiments, the TGFβ1 inhibitors of the present invention are effective in achieving tumor control (e.g., partial and complete responses), where the tumor optionally has an immunosuppressive (e.g., immune-negative) phenotype. In some embodiments, the TGFβ1 inhibitors can achieve synergistic anti-tumor effects when used in conjunction with cancer therapies such as checkpoint blockade therapy, chemotherapy, and radiation therapy. The checkpoint blockade therapy may include, for example, an anti-PD-(L)1 antibody. In such combination therapy, the TGFβ1 inhibitor may overcome treatment resistance (e.g., primary resistance), thereby making the cancer more sensitive to the cancer treatment. Thus, the TGFβ1 inhibitors can be used to treat cancers, including immunosuppressive tumors, in subjects. The subjects may be i) primary non-responders to cancer treatments such as checkpoint inhibitors, or ii) individuals diagnosed with a cancer for which at least one checkpoint inhibitor has been approved by a regulatory agency. The response rate (combined partial and complete responders among those treated) for the cancer for which at least one checkpoint inhibitor has been approved is less than 100%. Typically, response rates are about 10-60%. TGFβ1 inhibitors can increase response rates among patient populations. Furthermore, within the partial response group among primary responders, TGFβ1 inhibitors can provide improved clinical benefit. In some embodiments, within the primary responder group, TGFβ1 inhibitors can reduce the rate of acquired resistance to cancer therapy. The immunosuppressive tumor can be a locally advanced cancer / tumor or a metastatic cancer. In some embodiments, the cancer therapy can include, for example, checkpoint inhibitor therapy, chemotherapy, and / or radiation therapy.

[0023] In some embodiments, the TGFβ1 inhibitors of the present invention are effective to achieve a survival benefit in subjects with solid tumors, which are optionally locally advanced or metastatic cancers.

[0024] In some embodiments, the TGFβ1 inhibitors of the present invention are effective in achieving durable anti-tumor effects by inducing T cell memory function, and thus can reduce or delay disease recurrence.

[0025] In some embodiments, the TGFβ1 inhibitors of the present invention are effective in achieving anti-tumor effects in tumors that predominantly express TGFβ1 and / or TGFβ3. In some embodiments, the tumors that co-express TGFβ1 and TGFβ3 are carcinomas.

[0026] In some embodiments, the TGFβ1 inhibitors of the present invention can overcome primary tumor resistance to cancer treatment. 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). Upon treatment, there is a decrease in the number of tumor-associated immunosuppressive cells and a corresponding increase in the number of anti-tumor effector T cells.

[0027] In some embodiments, TGFβ1 inhibitors of the present invention promote the infiltration of effector cells into tumors. In some embodiments, effector cells may enter tumors via the tumor vasculature. In some embodiments, TGFβ1 inhibitors of the present invention promote the expansion (e.g., proliferation) of effector cells. This may be mediated, at least in part, by the inhibition of GARP-positive regulatory T cells.

[0028] In some embodiments, the TGFβ1 inhibitor of the present invention is effective for treating myelofibrosis.In some embodiments, the TGFβ1 inhibitor achieves the anti-fibrotic effect of the bone marrow of the subject with myelofibrosis.In some embodiments, the TGFβ1 inhibitor is effective for normalizing certain hematological parameters.

[0029] In some embodiments, the TGFβ1 inhibitors of the present invention are effective to achieve an anti-fibrotic effect in vivo. The anti-fibrotic effect may include reversal of established fibrosis, which may be partial or complete.

[0030] In some embodiments, the TGFβ1 inhibitors of the present invention have been well tolerated in preclinical safety / toxicology studies when administered at doses of up to 100, 200, or 300 mg / kg once a week for at least 4 weeks. Such studies can be carried out 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 observable toxicity associated with general inhibition of TGFβ, such as cardiovascular toxicity (e.g., valvular disease) and epithelial hyperplasia, and other toxicities known in the art.

[0031] In some embodiments, the TGFβ1 inhibitors of the present invention achieve a sufficient therapeutic window in that the effective amount of the inhibitor as shown by in vivo efficacy studies is significantly lower (e.g., at least 3-fold, at least 6-fold, or at least 10-fold) than the amount or concentration that causes observable toxicity. In some embodiments, the therapeutically effective amount of the inhibitor is from about 1 mg / kg to about 30 mg / kg per week. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 1 is a graph showing inhibition of LTBP1-precursor TGFβ activation in the LN229 assay. [Figure 2] FIG. 2 is a graph showing inhibition of activation of the pre-TGFβ1 complex in the LN229 assay. [Figure 3] FIG. 3 is a graph showing inhibition of activation of GARP-precursor TGFβ1 in the SW480β6 assay. [Figure 4] FIG. 4 is a graph showing inhibition of LRRC33-precursor TGFβ1 activation in the SW480β6 assay. [Figure 5A] FIG. 5A shows the inhibitory effects of Ab3 and Ab6 on kallikrein-induced activation of TGFβ1 in vitro. [Figure 5B] FIG. 5B shows the inhibitory effects of Ab3 and Ab6 on plasmin-induced activation of TGFβ1 in vitro. [Figure 6] FIG. 6 provides a graph showing rapid internalization of LRRC33-pre-TGFβ1 upon Ab6 binding in heterologous cells transfected with LRRC33 and pre-TGFβ1. [Figure 7] Figure 7 provides two graphs showing the effect 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. [Figure 8] Figure 8 provides two graphs showing the effect of Ab3 or Ab6 on the expression of Fn1 and Loxl2 genes in UUO mice. Mice were treated with 3, 10, or 30 mg / kg / week of Ab3 or 3 or 10 mg / kg / week of Ab6. IgG alone was used as a control. [Figure 9] FIG. 9 summarizes the statistical significance of changes in gene expression following treatment (vs. UUO+IgG) in the UUO model. [Figure 10] Figure 10 is a graph showing percent survival over time (days) after administration of Ab3 at 30 mg / kg or 10 mg / kg in combination with anti-PD-1 in the Cloudman S91 melanoma model. Anti-PD-1 alone and anti-SR-AB3 were used as controls. [Figure 11A]Figure 11A provides five graphs showing the change in tumor growth (tumor volume mm3), expressed as median tumor progression measured over time (days), after administration of Ab3 or Ab6 at 30 mg / kg or 10 mg / kg, respectively, in combination with anti-PD-1 in the Cloudman S91 melanoma model. Anti-PD-1 alone was used as a control. The dashed line represents animals that had to be sacrificed before reaching the 2000 mm3 endpoint criterion due to tumor ulceration. [Figure 11B] Figure 11B provides two graphs showing median tumor volume in Cloudman S91 as a function of time after administration of Ab3 (left) or Ab6 (right) at 30 mg / kg or 10 mg / kg in combination with anti-PD-1. Anti-PD-1 alone, Ab3 alone, Ab6 alone, and IgG alone were used as controls. [Figure 11C] Figure 11C provides six graphs showing the change 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 endpoint tumor volume of 2,000 mm is indicated by the upper dotted line, and the 25% threshold volume of 500 mm is indicated by the lower dotted line. Responders were defined as those achieving a tumor size less than 25% of the endpoint volume. [Figure 11D] Figure 11D provides three graphs showing the change in S91 tumor volume as a function of time in mice treated with the combination of anti-PD-1 and Ab6 at three dose levels (3, 10, and 30 mg / kg). A durable anti-tumor effect is demonstrated after treatment. [Figure 11E] FIG. 11E provides a graph summarizing the data expressed as median tumor volume from FIG. 11C. [Figure 11F] FIG. 11F provides a graph showing animal survival over time in each treatment group from FIG. 11C. [Figure 12]12 is a graph showing the phosphorylated to total SMAD2 / 3 ratio (pSMAD / SMAD) in an MBT2 bladder cancer model. Animals were treated with: (1) anti-PD-1 antibody alone, (2) Ab5 (3 mg / kg) in combination with anti-PD-1 antibody, (3) Ab5 (10 mg / kg) in combination with anti-PD-1 antibody, (4) Ab3 (10 mg / kg) in combination with anti-PD-1 antibody, and (5) Ab3 (30 mg / kg) in combination with anti-PD-1 antibody. [Figures 13A-13B] Figures 13A and 13B provide two sets of five graphs showing the change in MBT2 tumor growth (tumor volume in mm3) measured 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. Anti-PD-1 alone was used as a control. The change in tumor volume as a function of time is presented on a logarithmic scale (Figure 13A) and a linear scale (Figure 13B). The dashed line represents animals that had to be sacrificed before reaching the 1200 mm3 endpoint criterion due to tumor ulceration. [Figure 13C] Figure 13C provides 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 the MBT2 syngeneic bladder cancer model. Anti-PD-1 alone, Ab3 alone, Ab6 alone, and IgG alone were used as controls. The median tumor volumes at day 15 are summarized in the graph below. [Figure 13D] Figure 13D provides five graphs showing the effect of Ab6 in combination with anti-PD-1 in the MBT2 syngeneic bladder cancer model. Responders are defined as those achieving a tumor size of less than 25% of the endpoint volume at the end of the study. [Figure 14] Figure 14 is a graph showing percent 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. [Figure 15]Figure 15 provides a set of graphs showing the change in tumor growth (tumor volume in mm) measured over time (days) in a tumor rechallenge study. Animals previously treated with anti-PD-1 / Ab3 or anti-PD-1 / Ab6 and in which tumors were eliminated (complete responders that achieved complete regression) were rechallenged with MBT2 tumor cells. Naive, untreated animals served as controls. The dashed line represents animals that had to be sacrificed before reaching the 1200 mm endpoint criterion due to tumor ulceration. [Figure 16] FIG. 16 is a heat map showing that Ab5 Fab binding confers HDX protection in regions of precursor TGFβ1 (region 1 and region 2). [Figure 17] FIG. 17 illustrates the region of the precursor TGFβ1 complex that is protected from solvent exchange as measured by HDX upon Ab5 binding (see FIG. 16). [Figure 18A] Figure 18A is a heat map showing the protective effect of Ab6 Fab binding to precursor TGFβ1(C4S). Regions affected by antibody-antigen interaction are indicated by red boxes (1, 2a, 2b, 2c, 3, 4, 5a, 5b, 6a, and 6b). [Figure 18B] Figure 18B provides the HDX data overlaid onto the crystal structure of TGFβ1, showing the regions identified in Figure 18A. [Figure 19A] Figure 19A illustrates the identification of three binding regions (region 1, region 2, and region 3) after statistical analysis. Region 1 overlaps with the so-called "latent lasso" within the prodomain of precursor TGFβ1, while regions 2 and 3 lie within the growth factor domain. [Figure 19B] Figure 19B shows the various domains and motifs of precursor TGFβ1 compared to the three binding regions involved in Ab6 binding. A sequence alignment between the three isoforms is also provided. [Figures 20A-20D]Figures 20A-20D show the relative RNA expression of TGFβ isoforms in various tissues and cells. Figure 20A shows TGFβ isoform expression in various human cancer tissues versus normal controls (by cancer type). Figure 20B shows the frequency of TGFβ isoform expression by human cancer type based on analysis of over 10,000 samples from 33 tumor types. Figure 20C shows TGFβ isoform expression in individual tumor samples by cancer type. Figure 20D shows TGFβ isoform expression in a mouse syngeneic cancer cell model system. [Figure 20E] Figure 20E provides a four gene expression panel showing that all presentation molecules (LTBP1, LTBP3, GARP, and LRRC33) are highly expressed in most human cancer types. [Figure 20F] FIG. 20F provides an analysis of the expression of TGFβ and related signaling pathway genes from syngeneic mouse tumor models Cloudman S91, MBT-2, and EMT-6. [Figure 20G] FIG. 20G provides three graphs comparing protein expression of three TGFβ isoforms by ELISA in Cloudman S91, MBT-2, and EMT-6 tumor models. [Figure 20H] FIG. 20H provides a graph comparing RNA expression levels by whole tumor lysate qpCR of the presented molecules in Cloudman S91, MBT-2, and EMT-6 tumor models. [Figure 21A] Figure 21A shows microscopic cardiac findings from a pan-TGFβ antibody from a 1-week toxicology study, Figure 21B shows microscopic cardiac findings from Ab3 compared to an ALK5 inhibitor or a pan-TGFβ antibody from a 4-week rat toxicology study, and Figure 21C shows microscopic findings from Ab6 compared to an ALK5 inhibitor or a pan-TGFβ antibody from a 4-week rat toxicology study. [Figure 22]Figure 22 provides a graph showing median tumor volume of S91 as a function of time. The combination arms represent four different isoform-selective, context-independent TGFβ1 inhibitors, each combined with anti-PD-1 treatment, at two dose levels. [Figures 23A-23B] Figures 23A-23B provide representative immunohistochemical sections of S91 tumors stained with CD8+ cell markers. Figure 23A shows a tumor section from an animal treated with anti-PD-1 alone. Figure 23B shows a tumor section from an animal treated with both anti-PD-1 and a representative context-independent TGFβ1 inhibitor. [Figures 24A-24D] Figures 24A-24D provide representative immunohistochemical sections of S91 tumors stained with macrophage markers. Figure 24A shows a tumor section from an animal treated with anti-PD-1 alone. Figure 24B shows a tumor section from an animal treated with both anti-PD-1 and a representative context-independent TGFβ1 inhibitor. Figure 24C shows a tumor section from an animal treated with anti-PD-1 and Ab3 (30 mg / kg), using anti-F4 / 80 as a macrophage marker. Figure 24D shows a section using anti-CD163 as an M2 macrophage marker, showing that most cells are CD163-negative. [Figure 25] Figure 25 is a graph showing the log2 fold change in CD8+ T lymphocyte genes (CD8α, perforin, and granzyme B) after 1 week of treatment with anti-PD-1 / Ab3 in MBT2 tumors compared to animals treated with anti-PD-1 alone. [Figure 26A] FIG. 26A provides FACS data showing CD3 / CD28-induced upregulation of GARP in peripheral human regulatory T cells. [Figure 26B] Figure 26B is a graph showing the effect of Ab3 or Ab6 on Treg-mediated inhibition of Teff proliferation. IgG was used as a control. [Figure 27A] FIG. 27A shows the gating strategy for sorting T cell subpopulations in MBT2 tumors. [Figure 27B]FIG. 27B provides a set of graphs showing T cell subpopulations on day 13, expressed as percent of CD45+ cells. [Figure 28A] FIG. 28A provides the gating strategy for sorting myeloid subpopulations in MBT2 tumors. [Figure 28B] FIG. 28B provides a set of graphs showing bone marrow cell subpopulations on day 13. [Figure 28C] Figure 28C provides FACS data showing that tumor-associated macrophages in MBT-2 express cell surface LRRC33. [Figure 28D] FIG. 28D shows that MBT-2 tumor-infiltrating MDSCs express cell surface LRRC33. [Figures 29A-29C] Figures 29A-29C provide additional FACS data analyses showing the effect of Ab6 and anti-PD-1 treatment in MBT2 tumors. [Figures 30A-30D] Figures 30A-30D provide IHC images of representative MBT2 tumor sections showing intratumoral CD8-positive T cells. [Figure 30E] Figure 30E provides quantification of the IHC data from Figures 30A-30D, expressed as the percentage of CD8-positive cells in each treatment group. Necrotic areas of the sections were excluded from the analysis. [Figure 30F] Figure 30F provides an immunohistochemical analysis of the effects of Ab6 and anti-PD-1 treatment in MBT2 tumors. Tumor sections were visualized for phospho-SMAD3 (upper panel) or CD8 and CD31 (lower panel) in animals from the three treatment groups indicated. [Figure 30G] Figure 30G provides data demonstrating that Ab6 and anti-PD-1 in combination appear to trigger recruitment of CD8+ T cells from CD31+ blood vessels and infiltration into MBT2 tumors. [Figures 31A-31D]Figures 31A-31D provide gene expression of immune response markers Ptprc (Figure 31A), CD8a (Figure 31B), CD4 (Figure 31C), and Foxp3 (Figure 31D) collected from MBT2 tumors from the four treatment groups indicated. [Figures 32A-32C] Figures 32A-32C provide gene expression of effector function markers, Ifng (Figure 32A), Gzmb (Figure 32B), and Prf1 (Figure 32C), at days 10 and / or 13 as indicated. [Figure 32D] Figure 32D provides a set of graphs showing the expression of four gene markers (granzyme B, perforin, IFNγ, and Klrk1) measured by qpCR in MBT2 tumor samples at day 10. Each graph provides the fold change in expression in three treatment groups: anti-PD-1 alone (left), Ab6 alone (center), and the combination of anti-PD-1 and Ab6 (right). [Figure 33A] Figure 33A shows the in vitro binding of Ab6 to the four large latent complexes indicated, as measured by a solution equilibrium titration-based assay (MSD-SET). Measured K values ​​(in picomolar) are shown on the right. [Figure 33B] Figure 33B illustrates an LN229 cell-based potency assay and provides a graph showing the concentration-dependent potency of Ab6 against the four large latent complexes shown, which also demonstrates that Ab6 does not inhibit pro-TGFβ3. [Figure 34A] Figure 34A provides a set of nine graphs showing the effect of Ab6 in combination with or without anti-PD1 and / or anti-TGFβ3 on tumor growth / regression over time in EMT6 (Study 1). The upper dotted line in each graph represents the endpoint tumor volume of 2000 mm3, while the lower dotted line in each graph represents 25% of the endpoint volume (i.e., 500 mm3). [Figure 34B]Figure 34B provides a graph showing percent survival over time (days after treatment initiation) in EMT6 (Study 1). Treatment groups containing both anti-PD-1 and Ab6 demonstrated a significant survival benefit compared to anti-PD-1 alone. [Figure 34C] Figure 34C provides data showing percent survival over time (days after treatment initiation) in EMT6 (Study 2). Treatment groups containing both anti-PD-1 and Ab6 demonstrated a significant survival benefit compared to anti-PD-1 alone, and the anti-tumor effect was durable after treatment ended. [Figure 34D] Figure 34D provides the effect of combining anti-PD-1 with Ab6 on survival in an EMT6 breast cancer model. [Figure 35] FIG. 35 provides two graphs showing the relative expression of three TGFβ isoforms in EMT6 tumors, measured at the mRNA level (left) and the protein level (right). [Figure 36A] FIG. 36A provides a set of histological images showing silver staining for reticulin as a marker of the fibrotic phenotype of bone marrow in a murine myeloproliferative disease model. [Figure 36B] FIG. 36B provides two graphs showing histopathological analysis of myelofibrosis and the effect of TGFβ1 inhibition in MPLW515L mice with high disease burden from two separate repeat studies. [Figure 36C] FIG. 36C provides a set of graphs showing hematological parameters in MPLW515L mice treated with Ab6 or control IgG. [Figure 36D] FIG. 36D provides a set of graphs showing additional hematological parameters in MPLW515L mice treated with Ab6 or control IgG. [Figure 37A] FIG. 37A provides a gene set difference analysis (GSVA) ​​showing the correlation between TGFβ isoform expression and the IPRES gene set. [Figure 37B]Figure 37B provides a gene set differential analysis (GSVA) ​​showing the correlation between TGFβ isoform expression and the Plasari gene set. Expression of the TGFb1 isoform correlates with activation of the TGFβ pathway. The Plasari gene set of TGFβ-responsive genes significantly and strongly correlates with expression of TGFb1 RNA isoforms across many TCGA-annotated tumor types. Signature of correlation between TGFβ mRNA and TGFβ signaling DETAILED DESCRIPTION OF THE INVENTION

[0033] definition In order to make this disclosure more readily understandable, certain terms are first defined. These definitions should be read in light of the remainder of this disclosure and as understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Additional definitions are set forth throughout the detailed description.

[0034] Advanced cancer, advanced malignancy: As used herein, the term "advanced cancer" or "advanced malignancy" has the meaning understood in the relevant field, e.g., by an oncologist in the context of diagnosing or treating a subject / patient with cancer. Advanced malignancies, including solid tumors, may be locally advanced or metastatic. The term "locally advanced cancer" is used to describe a cancer (e.g., a tumor) that has grown outside the organ where it began but has not yet spread to distant parts of the body. Thus, this term includes cancer that has spread from the place where it began to nearby tissues or lymph nodes. In contrast, a "metastatic cancer" is a cancer that has spread from the part of the body where it began (the primary site) to other parts of the body (e.g., distant sites).

[0035] Affinity: Affinity is the strength of binding between a molecule (such as an antibody) and its ligand (such as an antigen). Typically, this is expressed as the equilibrium dissociation constant (K D ) is measured and reported. In the context of antibody-antigen interactions, KD is the antibody dissociation rate ("off rate" or K オフ the antibody association rate ("on-rate" or K オン For example, an antibody with an affinity of ≦5 nM has a K of ≦5 nM, as determined by a suitable in vitro binding assay. D The KD value of an antibody for its antigen can be measured using suitable in vitro assays such as biolayer interferometry (BLI) and solution equilibrium titration (e.g., MSD-SET).

[0036] Antibody: The term "antibody" includes any naturally occurring, recombinant, modified, or engineered immunoglobulin or immunoglobulin-like structure, or antigen-binding fragment or portion thereof, or derivatives thereof, as further described elsewhere herein. Thus, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, including, for example, chimeric, humanized, fully human, and bispecific antibodies. An intact antibody generally contains at least two full-length heavy chains and two full-length light chains, although in some cases it may contain fewer chains, such as antibodies naturally occurring in camels that contain only heavy chains. An antibody can be derived solely from a single source or can be "chimeric," i.e., different portions of the antibody can be derived from two different antibodies. Antibodies, or antigen-binding portions thereof, can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. As used herein, the term antibody includes monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, and antibody fusions (sometimes referred to herein as "antibody conjugates"). In some embodiments, the term also encompasses peptibodies.

[0037] Antigen: The term "antigen." The term "antigen" broadly includes any molecule that contains an antigenic determinant within the binding region to which an antibody or fragment specifically binds. An antigen can be a single unit molecule (such as a protein monomer or fragment) or a complex consisting of multiple components. An antigen provides an epitope, e.g., a molecule or portion of a molecule, or a complex of molecules or portions of molecules, that can be bound by a selective binding agent, such as an antigen-binding protein (including, e.g., an antibody). A selective binding agent can therefore specifically bind to an antigen formed by two or more components in a complex. In some embodiments, an antigen can be used in an animal to generate antibodies capable of binding to the antigen. An antigen can possess one or more epitopes that can interact with different antigen-binding proteins, e.g., antibodies. In the context of the present disclosure, a suitable antigen is a complex (e.g., a multimeric complex consisting of associated components) containing a precursor TGF dimer associated with a presentation molecule. Each monomer of the precursor TGF dimer contains a prodomain and a growth factor domain separated by a furin cleavage sequence. Two such monomers form a precursor TGF dimer complex (see Figure 19). This, in turn, is covalently linked to a presentation molecule via a disulfide bond involving a cysteine ​​residue present near the N-terminus of each precursor TGF monomer. This multicomplex formed by precursor TGF dimers bound to a presentation molecule is generally referred to as a large latent complex. For example, an antigen complex suitable for screening antibodies or antigen-binding fragments contains the presentation molecule component of the large latent complex. Such a presentation molecule component can be a full-length presentation molecule or a fragment thereof. The minimum required portion of a presentation molecule typically contains at least 50 amino acids, but more preferably contains at least 100 amino acids of the presentation molecule polypeptide, including two cysteine ​​residues capable of forming covalent bonds with the precursor TGFβ1 dimer.

[0038] Antigen-binding portion / fragment: As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TGFβ1). Antigen-binding portions include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. In some embodiments, an antigen-binding portion of an antibody may be derived from an intact antibody molecule using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Non-limiting examples of antigen-binding portions include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a single-chain Fv (scFv) molecule (see, e.g., Bird et al. (1988), SCIENCE, 242:423-426 and Huston et al. (1988), PROC. NAT'L. ACAD. SCI. USA, 85:5879-5883); (vi) a dAb fragment (see, e.g., Ward et al. (1989), NATURE, 341:544-546); and (vii) a minimal recognition unit. Other forms of single-chain antibodies, such as diabodies, are also encompassed. The term antigen-binding portion of an antibody includes a "single-chain Fab fragment," also known as "scFab," comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and linker have, from N-terminal to C-terminal, one of the following orders: 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.

[0039] Bias: In the context of the present disclosure, the term "bias" refers to skewed or unequal affinities toward or for a subset of antigens to which an antibody can specifically bind. For example, an antibody is said to be biased if its affinity for one antigen complex is not equivalent to its affinity for another antigen complex. A context-independent antibody according to the present disclosure has equivalent affinities (i.e., unbiased) for such antigen complexes.

[0040] Binding region: As used herein, a "binding region" is a portion of an antigen that, when bound to an antibody or fragment thereof, can form the interface of the antibody-antigen interaction. Upon antibody binding, the binding region is protected from surface exposure, which can be detected by an appropriate technique, such as HDX-MS. The antibody-antigen interaction can be mediated through multiple (e.g., two or more) binding regions. A binding region can comprise an antigenic determinant, or epitope.

[0041] Biolayer Interferometry (BLI): BLI is a label-free technique for optically measuring biomolecular interactions, for example, between a ligand immobilized on the apical surface of a biosensor and an analyte in solution. BLI offers the ability to monitor binding specificity, association and dissociation rates, or concentration with precision and accuracy. BLI platform instruments are commercially available, for example, from ForteBio and are commonly referred to as Octet® systems.

[0042] Cancer: As used herein, the term "cancer" refers to a physiological condition in multicellular eukaryotic organisms that is typically characterized by unregulated cell growth and malignant tumors. The term broadly encompasses tumors, solid and liquid malignancies, including blood cancers (e.g., leukemia, lymphoma, and myeloma), and myelofibrosis.

[0043] Cell-associated precursor TGFβ1: This term refers to membrane-bound (e.g., cell surface-tethered) TGFβ1 or its signaling complex (e.g., precursor / latent TGFβ1). Typically, such cells are immune cells. TGFβ1 presented by GARP or LRRC33 is cell-associated TGFβ1. GARP and LRRC33 are transmembrane presentation molecules expressed on the cell surface of certain cells. GARP-precursor TGFβ1 and LRRC33 can be collectively referred to as "cell-associated" (or "cell surface") precursor TGFβ1 complexes that mediate cellular precursor TGFβ1-associated (e.g., immune cell-associated) TGFβ1 activation / signaling. This term also includes recombinant, purified GARP-precursor TGFβ1 and LRRC33-precursor TGFβ1 complexes in solution (e.g., in vitro assays) that are not physically attached to a cell membrane. The average KD values ​​of antibodies (or fragments thereof) for GARP-precursor TGFβ1 complexes and LRRC33-precursor TGFβ1 complexes can be calculated to collectively represent the affinity of cell-associated (e.g., immune cell-associated) precursor TGFβ1 complexes. See, for example, column (G) of Table 8. Human counterparts of the presenting molecule or presenting molecule complex may be indicated by placing an "h" before the protein or protein complex, e.g., "hGARP," "hGARP-precursor TGFβ1," "hLRRC33," and "hLRRC33-precursor TGFβ1." In addition to blocking the release of active TGFβ1 growth factor from cell-tethered complexes, cell-associated precursor TGFβ1 can be targeted for internalization (e.g., endocytosis) and / or cell killing, e.g., ADCC-, ADCP-, or ADC-mediated depletion of target cells expressing such cell surface complexes.

[0044] Checkpoint inhibitor: In the context of this disclosure, checkpoint inhibitor refers to immune checkpoint inhibitor and has the meaning understood in the art. Typically, the target is a receptor molecule on T cells or NK cells, or a corresponding cell surface ligand on antigen-presenting cells (APCs) or tumor cells. Immune checkpoints are activated in immune cells to prevent inflammatory immunity from occurring against "self". Therefore, by changing the balance of the immune system through checkpoint inhibition, it should be fully activated, allowing it to detect and eliminate cancer. The most well-known inhibitory receptors implicated in the regulation of immune responses 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 V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA). Non-limiting examples of checkpoint inhibitors include nivolumab, pembrolizumab, BMS-936559, atezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, IMP-321, BMS-986016, and lirilumab. Keytruda® is an example of a PD-1 inhibitor. Treatment with one or more immune checkpoint inhibitors may be referred to as checkpoint blockade therapy (CBT).

[0045] Clinical benefit: As used herein, the term "clinical benefit" is intended to include both therapeutic efficacy and safety. Thus, a therapeutic treatment that achieves a desired clinical benefit is both effective (e.g., achieves a therapeutically beneficial effect) and safe (e.g., has a tolerable or acceptable level of toxicity or adverse events).

[0046] Combination therapy: "Combination therapy" refers to a therapeutic regimen for a clinical indication that includes two or more therapeutic agents. Thus, this term refers to a therapeutic regimen in which a first treatment containing a first composition (e.g., active ingredient) is administered to a patient in conjunction with a second treatment containing a second composition (active ingredient) intended to treat the same or an overlapping disease or clinical condition. The first and second compositions may act on the same or distinct cellular targets. In the context of combination therapy, the phrase "in conjunction with" means that the therapeutic effect of the first treatment overlaps in time and / or space with the therapeutic effect of the second treatment in a subject receiving the combination therapy. Thus, the combination therapy may be formulated as a single formulation for simultaneous administration or as separate formulations for sequential administration of the treatments. When a second treatment for treating the same disease is administered to a subject who has been treated with a first treatment for treating the same disease, the second treatment may be referred to as an additive or adjunctive therapy.

[0047] Combinatorial or combinatorial epitope: A combinatorial epitope is an epitope recognized and bound by a combinatorial antibody at a site (i.e., antigenic determinant) formed by non-adjacent portions of one or more components of an antigen that assemble together in close proximity in a three-dimensional structure to form the epitope. Thus, the antibodies of the present invention can bind to an epitope formed by two or more components (e.g., portions or segments) of the precursor / latent TGFβ1 complex. A combinatorial epitope can include amino acid residues from a first component of the complex and amino acid residues from a second component of the complex, etc. Each component can be from a single protein or two or more proteins of the antigen complex. A combinatorial epitope is formed by structural contributions from two or more components (e.g., portions or segments, e.g., amino acid residues) of an antigen or antigen complex.

[0048] Compete or cross-compete, cross-block: When used in the context of antigen-binding proteins (e.g., antibodies or antigen-binding portions thereof) that compete for the same epitope, the term "compete" refers to competition between the antigen-binding proteins as determined by an assay in which the antigen-binding protein being tested 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). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, including, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays, solid-phase direct biotin-avidin EIA, solid-phase direct label assays, and solid-phase direct label sandwich assays. Typically, when a competing antigen-binding protein is present in excess, it inhibits (e.g., reduces) specific binding between the reference antigen-binding protein and a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0049] In some embodiments, a first antibody or antigen-binding portion thereof and a second antibody or antigen-binding portion thereof "cross-block" each other with respect to the same antigen, as determined by, for example, Biacor or Octet®, using standard assay conditions, e.g., assaying for binding at room temperature, approximately 20-25°C, according to the manufacturer's instructions. In some embodiments, a first antibody or fragment thereof and a second antibody or fragment thereof may have the same epitope. In other embodiments, a first antibody or fragment thereof and a second antibody or fragment thereof may have overlapping, but non-identical, epitopes. In further embodiments, a first antibody or fragment thereof and a second antibody or fragment thereof may have separate (different) epitopes that are in close proximity in three-dimensional space such that antibody binding is cross-blocked by steric hindrance. "Cross-blocking" means that binding of a first antibody to an antigen prevents binding of a second antibody to the same antigen, and similarly, binding of a second antibody to an antigen prevents binding of the first antibody to the same antigen.

[0050] Antibody binning (sometimes called epitope binning or epitope mapping) can be performed to characterize and select a set (e.g., a "library") of monoclonal antibodies generated against a target protein or protein complex (i.e., an antigen). Such antibodies against the same target are tested in a pairwise manner against all other antibodies in the library to assess whether they block each other's binding to the antigen. Closely related binning profiles indicate that the antibodies have the same or closely related (e.g., overlapping) epitopes and are "binned" together. Because biological activity (e.g., intervention, efficacy) resulting from binding of an antibody to its target is likely to carry over to other antibodies in the same bin, binning provides a useful structure-function profile of antibodies that share similar binding regions within the same antigen. Thus, among antibodies within the same epitope bin, those with higher affinity (lower KD) typically have higher efficacy.

[0051] Complementarity-determining region: As used herein, the term "CDR" refers to a complementarity-determining region in an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single variable region that can bind to an antigen. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., (1987, 1991), Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk (1987), J. Mol. Biol., 196:901-917 and Chothia et al., (1989), Nature, 342:877-883) have used the Kabat CDRs to We have found that certain subregions within CDRs adopt nearly identical peptide backbone conformations despite their high degree of diversity at the amino acid sequence level. These subregions are designated L1, L2, and L3 or H1, H2, and H3, or L-CDR1, L-CDR2, and L-CDR3 or H-CDR1, H-CDR2, and H-CDR3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, and have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs are described by Padlan (1995) FASEB J. 9:133-139 and MacCallum (1996) J. Mol. Biol. 262(5):732-45.Still other CDR boundary definitions will overlap with the Kabat CDRs, although they may not strictly adhere to one of the systems described herein, but may be shorter or longer in light of predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding (see, e.g., Lu X et al., MAbs., 2019 January; 11(1):45-57). The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat- or Chothia-defined CDRs.

[0052] Conformational epitope: A conformational epitope is an epitope that is recognized and bound by a conformational antibody in a three-dimensional conformation, but not in an unfolded peptide of the same amino acid sequence. A conformational epitope may be called a conformation-specific epitope, a conformation-dependent epitope, or a conformation-sensitive epitope. A corresponding antibody or fragment thereof that specifically binds to such an epitope may be called a conformation-specific antibody, a conformation-selective antibody, or a conformation-dependent antibody. The binding of an antigen to a conformational epitope depends on the three-dimensional structure (conformation) of the antigen or antigen complex.

[0053] Constant region: Immunoglobulin constant domain refers to a heavy or light chain constant domain. The amino acid sequences of human IgG heavy and light chain constant domains are known in the art.

[0054] Context-biased: As used herein, a "context-biased antibody" refers to a type of conformational antibody that binds to an antigen with differential affinity when the antigen is associated with (i.e., bound or attached to) an interacting protein or fragment thereof. Thus, a context-biased antibody that specifically binds to an epitope within precursor TGFβ1 may bind with different affinities to LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1. For example, an antibody is said to be "matrix-biased" if it has higher affinity for matrix-associated precursor TGFβ1 complexes (e.g., LTBP1-precursor TGFβ1 and LTBP3-precursor TGFβ1) than for cell-associated precursor TGFβ1 complexes (e.g., GARP-precursor TGFβ1 and LRRC33-precursor TGFβ1). The relative affinity of a [matrix-associated complex]:[cell-associated complex] is determined by the average K D values ​​and the average of the latter K D It can be obtained by taking the values ​​of α and β and calculating the ratio of the two.

[0055] Context-independent: According to the present disclosure, a "context-independent antibody" that binds to precursor TGFβ1 has equivalent affinity across the four known presentation molecule-precursor TGFβ1 complexes, i.e., LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1. The context-independent antibodies disclosed in the present application may also be characterized as unbiased. Typically, context-independent antibodies exhibit equivalent affinity (i.e., the bias is 5-fold or less), such that the relative ratio of the measured KD values ​​between the matrix-associated complex and the cell-associated complex is no greater than 5, as measured by a suitable in vitro binding assay, such as surface plasmon resonance, biolayer interferometry (BLI), and / or solution equilibrium titration (e.g., MSD-SET).

[0056] ECM-associated TGFβ1 / precursor TGFβ1: This term refers to TGFβ1 or its signaling complexes (e.g., precursor / latent TGFβ1) that are components of (e.g., accumulated within) the extracellular matrix. TGFβ1 presented by LTBP1 or LTBP3 is ECM-associated TGFβ1, i.e., LTBP1-precursor TGFβ1 and LTBP3-precursor TGFβ1, respectively. LTBPs are crucial for the correct accumulation and subsequent bioavailability of TGFβ in the ECM, where fibrillin (Fbn) and fibronectin (FN) are thought to be the main matrix proteins responsible for the association of LTBPs with the ECM. Such matrix-associated latent complexes are enriched in connective tissues and certain disease-related tissues, such as tumor stroma and fibrous tissue. Human counterparts of presentation molecules or presentation molecule complexes may be indicated by placing an "h" before the protein or protein complex, for example, "hLTBP1," "hLTBP1-precursor TGFβ1," "hLTBP3," and "hLTBP3-precursor TGFβ1."

[0057] Effective amount: An "effective amount" (or therapeutically effective amount, or therapeutic dose) is a dosage or dosing regimen that achieves a statistically significant clinical benefit (e.g., efficacy) in a patient population. For example, Ab6 has been shown to be effective in preclinical models at doses as low as 3 mg / kg and as high as 30 mg / kg. Thus, an effective amount of Ab6 can be said to be about 3-30 mg / kg.

[0058] Effective tumor control: The term "effective tumor control" may be used to refer to the degree of tumor regression achieved in response to treatment, where, for example, the tumor regresses by a defined percentage (e.g., <25%) of the endpoint tumor volume. For example, in a particular model, an endpoint tumor volume of 2,000 mm 3 If set to , then assuming a threshold of <25%, 3Effective tumor control is achieved when the tumor density drops to less than 100%. Thus, effective tumor control encompasses complete regression. Clinically, effective tumor control includes partial response (PR) and complete response (CR) based on art-recognized criteria, such as RECIST 1.1 and the corresponding iRECIST. In some embodiments, effective tumor control in a clinical setting also includes stable disease, in which a tumor that is typically expected to grow at a certain rate is prevented from growing by treatment, even if shrinkage is not achieved.

[0059] Effector T cells: As used herein, effector T cells are T lymphocytes that respond immediately and aggressively to stimuli, such as costimulation, and include, but are not limited to, CD4+ T cells (also called T helper or Th cells) and CD8+ T cells (also called cytotoxic T cells). Th cells assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when peptide antigens are presented 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 support 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 toward specific subtypes: cytotoxic (killer). On the other hand, cytotoxic T cells (TC cells, CTLs, T killer cells, killer T cells) destroy virus-infected and cancer cells and have also been implicated in graft rejection. These cells express the CD8 glycoprotein on their surface and are therefore also known as CD8+ T cells. These cells recognize their targets by binding antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells. Cytotoxic effector cells (e.g., CD8+ cells) include, for example, perforin and granzyme B.

[0060] Epitope: The term "epitope," sometimes referred to as antigenic determinant, is a molecular determinant (e.g., a polypeptide determinant) capable of being specifically bound by a binding agent, immunoglobulin, or T-cell receptor. Epitope determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three-dimensional structural and / or charge characteristics. The epitope recognized by an antibody or antigen-binding fragment of an antibody is a structural element of the antigen that interacts with the CDR (e.g., complementary site) of the antibody or fragment. An epitope can be formed by contributions from several amino acid residues that interact with the CDR of the antibody to generate specificity. An antigenic fragment can contain multiple epitopes. In certain embodiments, an antibody specifically binds to an antigen when it recognizes its target antigen in a complex mixture of proteins and / or macromolecules. For example, antibodies are said to "bind the same epitope" if they cross-compete (one prevents or modulates the binding effect of the other).

[0061] Equivalent Affinity: In the context of the present disclosure, the term "equivalent affinities" is intended to mean that i) the antibody binds to the matrix-associated and cell-associated precursor TGFb1 complexes with an affinity bias of less than 5-fold, as measured by a suitable in vitro binding assay, such as solution equilibrium titration (such as MSD-SET), biolayer interferometry (such as Octet®), or surface plasmon resonance (such as Biacore System), and / or ii) the relative affinities of the antibody for the four complexes are uniform, in that either the lowest affinity (highest KD value) exhibited by the antibody among the four antigen complexes is no more than 5-fold lower than the average calculated from the affinities of the remaining three, or the highest affinity (lowest KD value) exhibited by the antibody among the four antigen complexes is no more than 5-fold higher than the average calculated from the affinities of the remaining three. Antibodies with equivalent affinities may achieve a more uniform inhibitory effect, regardless of the specific presenting molecule associated with the precursor TGFβ1 complex (hence "context independent"). In particularly preferred embodiments, the observed deviation in average affinity between matrix-associated and cell-associated complexes is no more than three-fold.

[0062] Extended Latent Lasso: As used herein, the term "extended latent lasso" refers to a portion of a prodomain comprising a latent lasso and an alpha-2 helix, e.g., LASPPSQGEVPPGPLPEAVLALYNSTR (SEQ ID NO: 154). In some embodiments, the extended latent lasso further comprises a portion of an alpha-1 helix, e.g., LVKRKRIEA (SEQ ID NO: 159) or a portion thereof.

[0063] Fibrosis: The term "fibrosis" or "fibrotic condition / disorder" refers to a process or manifestation characterized by the pathological accumulation of extracellular matrix (ECM) components, such as collagen, within a tissue or organ.

[0064] Fibrous microenvironment: The term "fibrotic microenvironment" refers to the localized disease niche within tissues where fibrosis occurs in vivo. The fibrotic microenvironment may contain disease-related molecular signatures (e.g., chemokines, cytokine sets), disease-related cell populations (e.g., activated macrophages, MDSCs), and disease-related ECM environments (altered ECM components and / or structure). The fibrotic microenvironment is thought to support the transition of fibroblasts to α-smooth muscle actin-positive myofibroblasts in a TGFβ-dependent manner. The fibrotic microenvironment may be further characterized by the infiltration of specific immune cells (e.g., macrophages and MDSCs).

[0065] Finger-1 (of TGFβ1 growth factor): As used herein, "finger-1" is a domain within the TGFβ1 growth factor domain. In its unmutated form, finger-1 of human precursor TGFβ1 contains the following amino acid sequence: CVRQLYIDFRKDLGWKWIHEPKGYHANFC (SEQ ID NO: 151). In the 3D structure, the finger-1 domain (a portion of which is shown as region "5a" in Figures 18 and 19) is in close proximity to the latent lasso.

[0066] Finger-2 (of TGFβ1 growth factor): As used herein, "finger-2" is a domain within the TGFβ1 growth factor domain. In its unmutated form, finger-2 of human precursor TGFβ1 contains the following amino acid sequence: CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 152). Finger-2 contains "binding region 6" (i.e., "6a" and "6b"), as illustrated in Figures 18 and 19, which is spatially positioned in close proximity to the latent lasso.

[0067] GARP-precursor TGFβ1 complex: As used herein, the term "GARP-TGFβ1 complex" refers to a protein complex comprising a precursor or latent form of transforming growth factor-β1 (TGFβ1) protein and glycoprotein A repeat dominant protein (GARP) or a fragment or variant thereof. In some embodiments, the precursor or latent form of TGFβ1 protein may be referred to as a "precursor / latent TGFβ1 protein." In some embodiments, a GARP-TGFβ1 complex comprises GARP covalently linked to precursor / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed using a cysteine ​​residue present near the N-terminus (e.g., amino acid position 4) of the precursor TGFβ1 dimeric complex. In other embodiments, a GARP-TGFβ1 complex comprises GARP non-covalently linked to precursor / latent TGFβ1. In some embodiments, the GARP-TGFβ1 complex is a naturally occurring complex, e.g., a GARP-TGFβ1 complex in a cell. The term "hGARP" refers to human GARP.

[0068] High affinity: As used herein, the term "high affinity," as in "high affinity pro-TGFβ1 antibody," refers to an antibody having a K of ≦5 nM, more preferably ≦1 nM. D Thus, high affinity, context-independent precursor TGFβ1 antibodies encompassed herein by the present invention have a K of ≦5 nM, more preferably ≦1 nM, for each of the following antigen complexes: LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1. D It has a value.

[0069] Human antibody: As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, particularly CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0070] Humanized antibody: The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences 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 make them more "human-like," i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Additionally, a "humanized antibody" is an antibody, or variant, derivative, analog, or fragment thereof, that immunospecifically binds to an antigen of interest and comprises FR regions having substantially the amino acid sequence of a human antibody and CDR regions having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of CDRs refers to a CDR having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the FR regions are of human immunoglobulin consensus sequences. In one embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin Fc region, typically that of a human immunoglobulin. In some embodiments, the humanized antibody contains the variable domains of a light chain and at least a heavy chain. The antibody may also contain the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In specific embodiments, the humanized antibody contains only a humanized variable domain of the light chain and / or a humanized heavy chain.

[0071] Hydrogen / Deuterium Exchange Mass Spectrometry (HDX-MS): HDX-MS is a well-known technique used to confirm proteins in solution and examine protein-protein interactions by measuring the degree of solvent accessibility. See, e.g., Wei et al. (2014), Drug Discovery Today, 19(1):95-102, "Hydrogen / Deuterium Exchange Mass Spectrometry for Probing Higher Order Structure of Protein Therapeutics: Methodology and Applications." HDX-MS techniques can be used to determine one or more regions of an antigen that are bound by an antibody (i.e., "binding regions"). Thus, such binding regions may contain or form epitopes.

[0072] Immune-excluded or immuno-excluded tumor: As used herein, a tumor characterized as "immune-excluded" lacks or substantially lacks intratumoral anti-tumor lymphocytes. For example, a tumor with poor T cell infiltration may have T cells surrounding the tumor, e.g., at the periphery of the tumor mass and / or near the tumor vasculature ("perivascular"), but nevertheless fail to effectively invade the tumor and exert cytotoxic function against cancer cells. In other situations, tumors fail to elicit a strong immune response, with few T cells present near and within the tumor environment (so-called "cold" or "immune desert" tumors). In contrast to immune-excluded tumors, tumors infiltrated with anti-tumor lymphocytes may be characterized as "hot" or "inflammatory" tumors. Such tumors tend to be more responsive and are therefore targets for immune checkpoint blockade therapy ("CBT"). However, typically, only a small proportion of patients respond to CBT due to immune exclusion, which renders tumors resistant to CBT.

[0073] 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 assessing immunosuppressive function is inhibition 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., macrophages localized in diseases such as TAMs and FAMs) can also be characterized as immunosuppressive.

[0074] Immune memory: Immune memory refers to the immune system's ability to rapidly and specifically recognize previously encountered antigens and mount a corresponding immune response. Generally, these are secondary, tertiary, and other subsequent immune responses to the same antigen. Immune memory is the source of specialized T and B cells, so-called memory T and B cells, which are the adaptive components of the immune system. Antigen-naive T cells expand and differentiate into memory and effector T cells after encountering their cognate antigen in the context of MHC molecules on the surface of professional antigen-presenting cells (e.g., dendritic cells). The only unifying theme among all memory T cell subtypes is their longevity and their ability to rapidly expand into large numbers of effector T cells upon re-exposure to their cognate antigen. By this mechanism, they provide the immune system with "memory" against previously encountered pathogens. Memory T cells can be either CD4+ or CD8+ and typically express CD45RO. In a preclinical setting, immune memory can be tested in a tumor rechallenge paradigm.

[0075] Isoform-specific: The term "isoform specificity" refers to the ability of an agent to preferentially discriminate one isoform over other structurally related isoforms (i.e., selectivity). An isoform-specific TGFβ inhibitor exerts its inhibitory activity against one isoform of TGFβ at a given concentration, but not against other TGFβ isoforms. For example, an isoform-specific TGFβ1 antibody selectively binds to TGFβ1. A TGFβ1-specific inhibitor (antibody) preferentially targets (binds to and thereby inhibits) the TGFβ1 isoform over TGFβ2 or TGFβ3 with substantially higher affinity. For example, selectivity in this context can refer to at least a 500- to 1000-fold difference in the respective affinities, as measured by in vitro binding assays such as Octet® and Biacor. In some embodiments, selectivity is such that TGFβ2 and TGFβ3 are not inhibited when the inhibitor is used at a dosage effective to inhibit TGFβ1 in vivo. For such inhibitors to be effective as therapeutic agents, the dosage to achieve the desired effect (e.g., a therapeutically effective amount) must fall within a window in which the inhibitor can effectively inhibit the TGFβ1 isoform without inhibiting TGFβ2 or TGFβ3.

[0076] Isolated: As used herein, an "isolated" antibody refers to an antibody that is substantially free of other antibodies having different antigen specificities. In some embodiments, an isolated antibody is substantially free of other unintended cellular material and / or chemicals.

[0077] Large latent complex: In the context of the present disclosure, the term "large latent complex" ("LLC") refers to a complex consisting of a precursor TGFβ1 dimer bound to a so-called presentation molecule. Thus, large latent complexes are presentation molecule-precursor TGFβ1 complexes such as LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1. Such complexes can be formed in vitro using recombinant purified components capable of complex formation. For screening purposes, the presentation molecule used to form such an LLC does not need to be a full-length polypeptide. However, a protein portion capable of forming a disulfide bond with the precursor TGFβ1 dimer complex via a cysteine ​​residue near its N-terminal region is typically required.

[0078] Latency-associated peptide (LAP): LAP is the so-called "prodomain" of the precursor TGFβ1. 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 alpha-1 helix and a latency-type lasso domain.

[0079] Latent Lasso: As used herein, the "latent lasso," sometimes referred to as the latency loop, is a domain flanked by the alpha-1 helix and arm in the prodomain of precursor TGFb1. In its unmutated form, the latent lasso of human precursor TGFβ1 comprises the amino acid sequence LASPPSQGEVPPGPL (SEQ ID NO: 153), substantially corresponding to regions "2a" and "2b" shown in FIG. 18A and spanning region 1 identified in FIG. 19A. As used herein, the term "extended latent lasso region" refers to the latent lasso together with the motif immediately C-terminal to it, called the alpha-2 helix (α2-helix) of the prodomain. A proline residue at the C-terminus of the latent lasso provides an "elbow"-like vertical "turn" that connects the lasso loop to the α2-helix. The extended latent lasso includes the regions shown in FIGS. 18 and 19 as "2a," "2b," and "2c." Particular high-affinity inhibitors of TGFβ1 activation confer inhibitory potency (e.g., the ability to block activation) by at least partially binding to latent Lasso or a portion thereof, where optionally, the latent Lasso portion is ASPPSQGEVPPGPL (SEQ ID NO: 266). In some embodiments, antibodies of the present disclosure bind to the precursor TGFβ1 complex at ASPPSQGEVPPGPL (SEQ ID NO: 266) or a portion thereof. Particular high-affinity inhibitors of TGFβ1 activation confer inhibitory potency (e.g., the ability to block activation) by at least partially binding to extended latent Lasso or a portion thereof, where optionally, the extended latent Lasso portion is KLRLASPPSQGEVPPGPLPEAVL (SEQ ID NO: 169).

[0080] Localized: In the context of this disclosure, the term "localized" (as in "localized tumor," "localized to disease," etc.) refers to an anatomically isolated or isolatable abnormality, e.g., a solid malignant tumor, as opposed to a systemic disease. A particular leukemia, for example, may have both a localized (e.g., bone marrow) and a systemic (e.g., circulating blood cells) component of the disease.

[0081] LRRC33-precursor TGFβ1 complex: As used herein, the term "LRRC33-TGFβ1 complex" refers to a complex between the precursor or latent form of transforming growth factor-β1 (TGFβ1) protein and leucine-rich repeat-containing protein 33 (LRRC33, also known as negative regulator of reactive oxygen species or NRROS) or a fragment or variant thereof. In some embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 covalently bound to precursor / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed using a cysteine ​​residue present near the N-terminus (e.g., amino acid position 4) of the precursor TGFβ1 dimeric complex. In other embodiments, the LRRC33-TGFβ1 complex comprises LRRC33 non-covalently bound to precursor / latent TGFβ1. In some embodiments, the LRRC33-TGFβ1 complex is a naturally occurring complex, e.g., a LRRC33-TGFβ1 complex in a cell. The term "hLRRC33" refers to human LRRC33. In vivo, LRRC33 on the cell surface and complexes containing LRRC33 can be internalized. LRRC33 is expressed on a subset of myeloid cells, including M2-polarized macrophages (such as TAMs) and MDSCs.

[0082] LTBP1-precursor TGFβ1 complex: As used herein, the term "LTBP1-TGFβ1 complex" refers to a protein complex comprising a precursor or latent form of the transforming growth factor-β1 (TGFβ1) protein and latent TGF-beta binding protein 1 (LTBP1) or a fragment or variant thereof. In some embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 covalently linked to precursor / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed using a cysteine ​​residue present near the N-terminus (e.g., amino acid position 4) of the precursor TGFβ1 dimeric complex. In other embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 non-covalently linked to precursor / latent TGFβ1. In some embodiments, the LTBP1-TGFβ1 complex is a naturally occurring complex, e.g., a LTBP1-TGFβ1 complex in a cell. The term "hLTBP1" refers to human LTBP1.

[0083] LTBP3-precursor TGFβ1 complex: As used herein, the term "LTBP3-TGFβ1 complex" refers to a protein complex comprising a precursor or latent form of the transforming growth factor-β1 (TGFβ1) protein and latent TGF-beta binding protein 3 (LTBP3) or a fragment or variant thereof. In some embodiments, the LTBP3-TGFβ1 complex comprises LTBP3 covalently bound to precursor / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed using a cysteine ​​residue present near the N-terminus (e.g., amino acid position 4) of the precursor TGFβ1 dimeric complex. In other embodiments, the LTBP3-TGFβ1 complex comprises LTBP1 non-covalently bound to precursor / latent TGFβ1. In some embodiments, the LTBP3-TGFβ1 complex is a naturally occurring complex, e.g., a LTBP3-TGFβ1 complex in cells. The term "hLTBP3" refers to human LTBP3.

[0084] M2 or M2-like macrophages: M2 macrophages represent a subset of activated or polarized macrophages and include disease-associated macrophages in both fibrotic and tumor microenvironments. Cell surface markers of M2-polarized macrophages typically include CD206 and CD163 (i.e., CD206+ / CD163+). M2-polarized macrophages may also express cell surface LRRC33. M2 macrophage activation is primarily driven 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 have a high phagocytic capacity and produce ECM components, angiogenic, and chemotactic factors. The release of TGFβ by macrophages can perpetuate myofibroblast activation and the induction of EMT and EndMT in diseased tissues, such as fibrotic tissues and tumor stroma. For example, M2 macrophages are essential for TGFβ-driven pulmonary fibrosis and are also enriched in some tumors.

[0085] Matrix-associated precursor TGFβ1: LTBP1 and LTBP3 are presentation molecules that are components of the extracellular matrix (ECM). LTBP1-precursor TGFβ1 and LTBP3-precursor TGFβ1 can be collectively referred to as "ECM-associated" (or "matrix-associated") precursor TGFβ1 complexes, which mediate ECM-associated TGFβ1 activation / signaling. This term also includes recombinant, purified LTBP1-precursor TGFβ1 and LTBP3-precursor TGFβ1 complexes in solution (e.g., in vitro assays) that are not physically attached to a matrix or substrate.

[0086] Maximum Tolerated Dose (MTD): The term MTD generally refers to the highest dose of a test substance (such as a TGFβ1 inhibitor) evaluated using the no-observed-adverse-effect level (NOAEL) in the context of safety / toxicology considerations. For example, the NOAEL for Ab6 in rats was the highest dose evaluated (100 mg / kg), suggesting that the MTD for Ab6 is >100 mg / kg based on a 4-week toxicology study. The NOAEL for Ab6 in non-human primates was the highest dose evaluated (300 mg / kg), suggesting that the MTD for Ab6 in non-human primates is >300 mg / kg based on a 4-week toxicology study.

[0087] Mesoscale Discovery: "Mesoscale discovery" or "MSD" is a type of immunoassay that uses electrochemiluminescence (ECL) as a detection technique. Typically, a high-binding carbon electrode is used to capture proteins (e.g., antibodies). The antibody can be incubated with a specific antigen, and the binding can be detected using a secondary antibody conjugated to an electrochemiluminescent label. Upon electrical signal, light intensity can be measured to quantify the analyte in the sample.

[0088] Myelofibrosis: Myelofibrosis, also known as osteomyelofibrosis, is a relatively rare myeloproliferative disorder (e.g., cancer) that belongs to a group of diseases called myeloproliferative disorders. Myelofibrosis is classified as the Philadelphia chromosome-negative (-) division of myeloproliferative neoplasms. Myelofibrosis is characterized by the proliferation of abnormal clones of hematopoietic stem cells in the bone marrow and other sites, resulting in fibrosis or replacement of the marrow with scar tissue. The term myelofibrosis encompasses primary myelofibrosis (PMF), also known as chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary refer to the disease in these cases as being of unknown or spontaneous origin), as well as secondary forms of myelofibrosis, such as myelofibrosis occurring secondary to polycythemia vera (PV) or essential thrombocythemia (ET). Myelofibrosis is a form of myeloid metaplasia, which refers to changes in cell types in the blood-forming tissues of the bone marrow; the two terms are often used interchangeably. The terms idiopathic myeloid metaplasia and myelofibrosis with myeloid metaplasia (MMM) are also used to refer to primary myelofibrosis, which is characterized by mutations that cause upregulation or hyperactivation of the downstream JAK pathway.

[0089] Myeloid cells: In hematopoiesis, myeloid cells are blood cells that arise from precursors of granulocytes, monocytes, erythrocytes, or platelets (common myeloid precursors, i.e., CMPs or CFU-GEMMs), or, in the narrower, also frequently used, from the myeloblast lineage (myelocytes, monocytes, and their daughter types), distinct from lymphocytes, i.e., lymphocytes derived from a common lymphoid precursor that gives rise to B cells and T cells. Specific myeloid cell types, their general morphology, typical cell surface markers, and their immunosuppressive capabilities in both mice and humans are summarized below. JPEG2024099673000001.jpg179166 JPEG2024099673000002.jpg122166

[0090] Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells generated during various pathological conditions and are thought to represent the pathological manifestation of monocyte and relatively immature neutrophil activation. MDSCs comprise at least two categories of cells: i) phenotypically and morphologically similar to neutrophils, termed "granulocytic" (G-MDSC) or polymorphonuclear (PMN-MDSC), and ii) phenotypically and morphologically similar to monocytes, termed "monocytic" (M-MDSC). MDSCs are characterized by distinct sets of genomic and biochemical features 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 may also express HLA-DR and / or arginase. In comparison, human M-MDSCs typically express the cell surface markers CD11b, CD33, and CD14. Furthermore, human M-MDSCs may also express HLA-DR. In addition to these cell surface markers, MDSCs are characterized by their ability to suppress immune cells such as T cells, NK cells, and B cells. The immunosuppressive function of MDSCs can include inhibition of antigen-nonspecific and antigen-specific functions. MDSCs can express cell surface LRRC33 and / or LRRC33-progenitor TGFβ1.

[0091] Myofibroblasts: Myofibroblasts are cells with a specific phenotype of fibroblasts and smooth muscle cells and generally express vimentin, alpha-smooth muscle actin (α-SMA, human gene ACTA2), and palladin. In many pathologies involving extracellular matrix dysregulation (e.g., increased matrix stiffness), normal fibroblasts dedifferentiate into myofibroblasts in a TGFβ-dependent manner. Abnormal overexpression of TGFβ is common in myofibroblast-driven pathologies. TGFβ is known to promote myofibroblast differentiation, cell proliferation, and matrix production. Myofibroblasts or myofibroblast-like cells within a fibrotic microenvironment may be referred to as fibrosis-associated fibroblasts (or "FAFs"), while myofibroblasts or myofibroblast-like cells within a tumor microenvironment may be referred to as cancer-associated fibroblasts (or "CAFs").

[0092] Pan-TGFβ inhibitor / pan-TGFβ inhibition: The term "pan-TGFβ inhibitor" refers to any agent capable of inhibiting or antagonizing all three isoforms of TGFβ. Such inhibitors may be small molecule inhibitors of TGFβ isoforms, such as those known in the art. This term includes pan-TGFβ antibodies, which refer to any antibody capable of binding to each of the TGFβ isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3. In some embodiments, the pan-TGFβ antibody binds to and neutralizes the activity of all three isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3. Antibody 1D11 (or the human analog fresolimumab (GC1008)) is a well-known example of a pan-TGFβ antibody that neutralizes all three isoforms of TGFβ. An example of a small molecule pan-TGFβ inhibitor is galunisertib (LY2157299 monohydrate), an antagonist of TGFβ receptor I kinase / ALK5, which mediates signaling of all three TGFβ isoforms.

[0093] Perivascular (infiltration): The prefix "peri-" means "around," "surrounding," or "near," and therefore "perivascular" is taken literally as around blood vessels. As used herein in the context of tumor cell infiltration, the term "perivascular infiltration" refers to the mechanism of entry of tumor-infiltrating immune cells (e.g., lymphocytes) through the vasculature of solid tumors.

[0094] Potency: As used herein, the term "potency" refers to the activity of a drug, such as an inhibitory antibody (or fragment) having inhibitory activity, in terms of the concentration or amount of drug to produce a defined effect. For example, an antibody that can produce a particular effect at a given dosage is more potent than another antibody that requires twice the amount (dosage) to produce an equivalent effect. Potency may be measured in a cell-based assay, such as a TGFβ activation / inhibition assay, whereby the degree of TGFβ activation, such as activation triggered by integrin binding, can be measured in a cell-based system in the presence or absence of a test substance (e.g., an inhibitory antibody). Typically, among those that can bind to the same or overlapping binding region of an antigen (e.g., cross-blocking antibodies), higher affinity (lower K D An antibody with a lower affinity (higher K D These antibodies tend to be more potent than antibodies with higher IgG values.

[0095] Presentation molecule: In the context of this disclosure, a presentation molecule refers to a protein that forms a covalent bond with a latent precursor protein (e.g., precursor TGFβ1) and tethers ("presents") the inactive complex to an extracellular niche (e.g., the ECM or the surface of an immune cell), thereby maintaining its latency until an activation event occurs. Known presentation molecules for precursor TGFβ1 include LTBP1, LTBP3, GARP, and LRRC33, each of which can form a presentation molecule-precursor TGFβ1 complex (i.e., LLC), i.e., LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1, respectively. In nature, LTBP1 and LTBP3 are components of the extracellular matrix (ECM), and therefore, LTBP1-precursor TGFβ1 and LTBP3-precursor TGFβ1 can be collectively referred to as "ECM-associated" (or "matrix-associated") precursor TGFβ1 complexes that mediate ECM-associated TGFβ1 signaling / activity. On the other hand, GARP and LRRC33 are transmembrane proteins expressed on the cell surface of certain cells, and therefore, GARP-precursor TGFβ1 and LRRC33-precursor TGFβ1 can collectively be referred to as "cell-associated" (or "cell surface") precursor TGFβ1 complexes that mediate cell-associated (e.g., immune cell-associated) TGFβ1 signaling / activity.

[0096] Protection (from solvent exposure): In the context of HDX-MS-based assessment of protein-protein interactions, such as antibody-antigen binding, the degree to which a protein (e.g., a region of a protein containing an epitope) is exposed to solvent, thereby allowing proton exchange to occur, is inversely correlated with the degree of binding / interaction. Thus, when an antibody described herein binds to a region of an antigen, the binding region is "protected" from exposure to solvent, because protein-protein interactions preclude the binding region from being accessible by the surrounding solvent. Thus, the protected region is indicative of the site of interaction. Typically, the appropriate solvent is a physiological buffer.

[0097] Precursor TGFβ1: As used herein, the term "precursor TGFβ1" is intended to encompass the precursor form of the inactive TGFβ1 complex, which contains the prodomain sequence of TGFβ1 within the complex. Thus, this term can include precursor and latent forms of TGFβ1. The expressions "precursor / latent TGFβ1" can be used interchangeably. The "precursor" form of TGFβ1 exists prior to proteolytic cleavage at the furin site. After cleavage, the resulting form is said to be the "latent" form of TGFβ1. The "latent" complex remains noncovalently associated until further activation, such as an integrin-driven activation event, is triggered. The precursor TGFβ1 complex consists of a dimeric TGFβ1 precursor protein polypeptide linked by a disulfide bond. The latent dimeric complex is covalently bound to a single presentation molecule via the cysteine ​​residue at position 4 (Cys4). The adjective "latent" can be used generally / broadly to describe the "inactive" state of TGFβ1, prior to integrin-mediated or other activation events. The precursor TGFβ1 polypeptide contains a prodomain (LAP) and a growth factor domain (SEQ ID NO: 146).

[0098] Regression (Tumor Regression): Regression of tumors or tumor growth can be used as a measure of in vivo efficacy. For example, in a preclinical setting, median tumor volume (MTV) and regression response treatment efficacy measures can be determined from the tumor volume of animals remaining in the study on the final day. Treatment efficacy can also be determined from the incidence and magnitude of regression responses observed during the study. Treatment can cause partial tumor regression (PR) or complete tumor regression (CR) in animals. Complete regression achieved in response to treatment (e.g., administration of a drug) may be referred to as a "complete response," and subjects achieving a complete response may be referred to as "complete responders." Thus, complete response excludes spontaneous complete regression. In some embodiments of preclinical tumor models, a PR response is defined as a tumor volume that is 50% or less of its day 1 volume for three consecutive measurements during the course of the study and is 13.5 mm or less for one or more of those three measurements. 3In some embodiments, a CR response is defined as a tumor volume greater than or equal to 13.5 mm for three consecutive measurements over the course of the study. 3 A CR response is defined as a tumor volume that is less than 25% of the endpoint tumor volume. In preclinical models, animals with a CR response at the end of the study may be further classified as tumor-free survivors (TFS). The term "effective tumor control" may be used to refer to the degree of tumor regression achieved in response to treatment, where, for example, the tumor volume has decreased to <25% of the endpoint tumor volume in response to treatment. For example, in certain models, an endpoint tumor volume of 2,000 mm 3 If the tumor is 500 mm 3 Effective tumor control is achieved when the tumor activity drops below 100%. Effective tumor control therefore encompasses complete regression and partial regression where a threshold reduction is reached.

[0099] Regulatory T cells: "Regulatory T cells" or Tregs are a type of immune cell characterized by expression of the biomarkers CD4, FOXP3, and CD25. Tregs, sometimes called suppressor T cells, represent a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T (Teff) cells. Tregs can arise in the thymus (so-called CD4+Foxp3+ "natural" Tregs) or can be cultured from naive CD4+ T cells in the periphery, for example, after exposure to TGFβ or retinoic acid. Tregs can express the cell surface GARP-progenitor TGFβ1.

[0100] Resistance (to treatment): Resistance to a particular treatment (e.g., CBT) may be due to an innate feature of a disease, such as cancer ("primary resistance"), or may be due to an acquired phenotype that develops over time after treatment ("acquired resistance"). Patients who do not show a therapeutic response to treatment (e.g., non-responders or poor responders to treatment) are said to have primary resistance to treatment. Patients who initially show a therapeutic response to treatment but later lose benefit (e.g., progress or relapse despite continued treatment) are said to have acquired resistance to treatment.

[0101] Response Evaluation Criteria in Solid Tumors (RECIST) and iRECIST: RECIST is a set of published rules that define when tumors in cancer patients improve ("response"), stay the same ("stabilize"), or worsen ("progress") during treatment. The criteria were published in February 2000 by an international collaboration including the European Organization for Research and Treatment of Cancer (EORTC), the National Cancer Institute of the United States, and the National Cancer Institute of Canada Clinical Trials Group. Since then, a revised version of the RECIST guidelines (RECIST v1.1) 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).

[0102] Response criteria were as follows: complete response (CR): disappearance of all target lesions; partial response (PR): at least a 30% reduction in the sum of the LD of target lesions, taking the baseline total LD ​​as reference; stable disease (SD): neither a sufficient reduction to qualify as a PR nor a sufficient increase to qualify as a PD, taking the smallest total LD ​​since the start of treatment as reference; progressive disease (PD): at least a 20% increase in the sum of the LD of target lesions, taking the smallest total LD ​​recorded since the start of treatment or the appearance of one or more new lesions.

[0103] iRECIST, on the other hand, provides a modified set of criteria that takes into account immune-related responses (see www.ncbi.nlm.nih.gov / pmc / articles / PMC5648544 / , the contents of which are incorporated herein by reference). The RECIST and iRECIST criteria are standardized, may be revised from time to time as additional data becomes available, and are well understood in the art.

[0104] Solid tumor: The term "solid tumor" refers to a proliferative disorder that results in an abnormal growth or tissue mass that usually does not contain cysts or liquid areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Solid tumors include locally advanced solid tumors and tumors of advanced malignancies, such as metastatic cancer. Solid tumors 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. Solid tumors treated with TGFβ1 isoform-selective inhibitors such as those described herein are typically TGFβ1-positive (TGFβ1+) tumors, which may contain multiple cell types that produce TGFβ1. In certain embodiments, TGFβ1+ tumors may also co-express TGFβ3 (i.e., are TGFβ3-positive). For example, certain tumors are TGFβ1 / 3 co-dominant. In some embodiments, such tumors are caused by cancer of epithelial cells, such as carcinomas.

[0105] Solution equilibrium titration (SET): SET is an assay that can measure the binding between two molecules (such as an antigen and an antibody that binds to the antigen) in solution at equilibrium. For example, mesoscale discovery ("MSD")-based SET, or MSD-SET, is a useful format for determining the equilibrium dissociation constant of particularly high-affinity protein-protein interactions, such as the binding of a picomolar affinity antibody to its antigen (see, e.g., Ducata et al., (2015), J Biomolecular Screening, 20(10):1256-1267). SET-based assays can be used to determine the K of antibodies with subnanomolar (e.g., picomolar) affinities. D This is particularly useful for determining values.

[0106] Specific binding: As used herein, the terms "specific binding" or "specifically binds" mean that the interaction of an antibody, or antigen-binding portion thereof, with an antigen is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope). For example, an antibody, or antigen-binding portion thereof, binds to a specific protein rather than to proteins in general. In some embodiments, an antibody, or antigen-binding portion thereof, binds to a target. D is at least about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less. In some embodiments, the terms "specific binding to an epitope of precursor TGFβ1," "specifically binds to an epitope of precursor TGFβ1," "specific binding to precursor TGFβ1," or "specifically binds to precursor TGFβ1" refer to binding to precursor TGFβ1 and having a binding affinity of 1.0×10 or less as determined by a suitable in vitro binding assay, such as surface plasmon resonance and biolayer interferometry (BLI). -8 The dissociation constant (K DIn one embodiment, the antibody or antigen-binding portion thereof is capable of specifically binding to both human and non-human (e.g., murine) orthologs of precursor TGFβ1.

[0107] Subject: In the context of therapeutic applications, the term "subject" refers to an individual receiving clinical care or intervention, such as treatment, diagnosis, etc. Suitable 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 term "patient population" or "patient subpopulation" is used to refer to a group of individuals that fall within a set of criteria, such as clinical criteria (e.g., disease presentation, disease stage, susceptibility to a particular condition, responsiveness to treatment, etc.), medical history, health status, sex, age group, genetic criteria (e.g., carrier of a particular mutation, polymorphism, gene duplication, DNA sequence repeat, etc.), and lifestyle factors (e.g., smoking, alcohol consumption, exercise, etc.).

[0108] 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 sold by Biacore, can be used to measure biomolecular interactions, including protein-protein interactions such as antigen-antibody binding. This technique is widely known in the art and is useful for determining parameters such as binding affinity, kinetic rate constants, and thermodynamics.

[0109] TGFβ1-related indications: "TGFβ1-related indications" refers to any disease or disorder and / or condition whose etiology and / or progression can be attributed, at least in part, to TGFβ1 signaling or its dysregulation. Certain TGFβ1-related disorders are predominantly driven by TGFβ1 isoforms. Subjects with TGFβ1-related indications may benefit from inhibiting TGFβ1 activity and / or levels. Certain TGFβ1-related indications are predominantly driven by TGFβ1 isoforms. TGFβ1-related indications include, but are not limited to, fibrotic conditions (such as organ fibrosis and tissue fibrosis involved in chronic inflammation), proliferative disorders (such as cancer, e.g., solid tumors and myelofibrosis), diseases associated with ECM dysregulation (such as conditions involving matrix stiffening 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.

[0110] TGFβ inhibitor: The term "TGFβ inhibitor" refers to any agent capable of antagonizing the biological activity, signal transduction, 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 capture agents, and TGFβ activation inhibitors. TGFβ inhibitors also include antibodies that can reduce the availability of latent precursor TGFβ, which can be activated in the niche, for example, by inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADPC), as well as antibodies that can remove precursors from the plasma membrane without depleting the cells themselves by causing internalization of cell surface complexes containing latent precursor TGFβ. Internalization may be a suitable mechanism of action of LRRC33-containing protein complexes (such as human LRRC33-precursor TGFβ1), resulting in a reduction in the level of cells expressing the LRRC33-containing protein complex on the cell surface.

[0111] The "TGF-β family" is a class within the TGF-β superfamily, which in humans contains three structurally similar members: TGF-β1, TGF-β2, and TGF-β3. The three growth factors are known to signal through the same receptor.

[0112] TGFβ1-positive cancer / tumor: As used herein, this term refers to a cancer / tumor with abnormal TGFβ1 expression (overexpression). Many human cancer / tumor types exhibit dominant expression of the TGFβ1 (note that "TGFB" may be used to refer to the gene as a protein reference) isoform. In some cases, such cancers / tumors may exhibit co-dominant expression of another isoform, such as TGFβ3. Some epithelial cancers (e.g., carcinomas) may co-express TGFβ1 and TGFβ3. Within the tumor environment of a TGFβ1-positive tumor, TGFβ1 may arise from multiple sources, including, for example, cancer cells, tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and the surrounding extracellular matrix (ECM). In the context of this disclosure, a preclinical cancer / tumor model that encapsulates the human condition is a TGFβ1-positive cancer / tumor.

[0113] Therapeutic window: The term "therapeutic window" refers to the range of dosages that produce a therapeutic response without causing significant / observable / unacceptable adverse effects in subjects (e.g., within the range of acceptable or tolerable adverse effects). The therapeutic window can be calculated as the ratio between the minimum effective concentration (MEC) and the minimum toxic concentration (MTC). For example, a TGFβ1 inhibitor that achieves in vivo efficacy at a dosage of 10 mg / kg and shows tolerable or acceptable toxicity at 100 mg / kg provides a therapeutic window of at least 10 times (e.g., 10×). In contrast, a pan-inhibitor of TGFβ that is effective at 10 mg / kg but causes adverse effects at doses below the effective dose is said to have "dose-limiting toxicity." Generally, the maximum tolerated dose (MTD) can set the upper limit of the therapeutic window.

[0114] For example, Ab6 has been shown to be effective in rats or non-human primates at doses ranging from approximately 3 to 30 mg / kg / week for 4 weeks, with no observable toxicity associated with pan-TGFβ inhibition at doses of at least 100 or 300 mg / kg / week. Based on this, Ab6 exhibits a therapeutic window of at least 3.3-fold and up to 100-fold.

[0115] Toxicity: As used herein, the term "toxicity" or "toxicities" refers to unwanted in vivo effects in a subject (e.g., a patient) associated with a treatment administered to the subject (e.g., a patient), such as unwanted side effects and adverse events. "Tolerability" refers to the level of toxicity associated with a treatment or treatment regimen that can be reasonably tolerated by a patient without discontinuing treatment due to toxicity. Typically, toxicity / toxicology studies are conducted in one or more preclinical models prior to clinical development to evaluate the safety profile of a drug candidate (e.g., a monoclonal antibody treatment). Toxicity / toxicology studies may help determine the "no observed adverse effect level (NOAEL)" and "maximum tolerated dose (MTD)" of a test substance, based on which a therapeutic window can be estimated. Preferably, a species shown to be sensitive to a particular intervention should be selected as the preclinical animal model in which safety / toxicity studies are conducted. In the case of TGFβ inhibition, suitable species include rats, dogs, and cynomolgus monkeys. Although mice have been reported to be less sensitive to pharmacological inhibition of TGFβ and may not demonstrate potentially dangerous toxicity in other species, including humans, certain studies have reported toxicity observed with pan-inhibition of TGFβ in mice. For example, in the context of this disclosure, the NOAEL for Ab6 in rats is the highest dose evaluated (100 mg / kg), suggesting an MTD of >100 mg / kg based on a 4-week toxicology study. The MTD for Ab6 in non-human primates is >300 mg / kg based on a 4-week toxicology study.

[0116] To determine NOAEL and MTD, preferably, species that have been shown to be sensitive to specific intervention should be selected as the preclinical animal model for safety / toxicology research.For TGFβ inhibition, suitable species include but are not limited to rats, dogs, and cynomolgus monkeys.Mice have been reported to be less sensitive to the pharmacological inhibition of TGFβ, and may not reveal potentially serious or dangerous toxicity in other species, including humans.

[0117] Translatability: In the context of drug discovery and clinical development, the term "translatability" or "translatable" refers to a particular quality or characteristic of a preclinical model or data that recapitulates the human condition. As used herein, preclinical models that recapitulate TGFβ1 indications typically exhibit dominant expression of TGFB1 (or TGFβ1) compared to TGFB2 (or TGFβ2) and TGFB3 (or TGFβ3). In a combination therapy paradigm, for example, translatability may require the same underlying mechanism of action as the intended combination of active agents to be realized in the model. As an example, many human tumors are immunoexcluded, and TGFβ1-positive tumors exhibit primary resistance to checkpoint blockade therapy (CBT). A second treatment (such as a TGFβ1 inhibitor) may be used in combination to overcome resistance to CBT. In this scenario, an appropriate translatable preclinical model includes a TGFβ1-positive tumor that exhibits primary resistance to checkpoint blockade therapy (CBT).

[0118] Treat / Treatment: The term "treat" or "treatment" includes therapeutic treatment, prophylactic treatment, and applications that reduce a subject's risk of developing a disorder or other risk factor. Thus, the term is intended broadly to mean causing a therapeutic benefit in a patient by, for example, enhancing or boosting the body's immunity, reducing or reversing immunosuppression, reducing, removing, or eradicating harmful cells or substances from the body, reducing disease burden (e.g., tumor burden), preventing recurrence or flare-ups, prolonging the period of refractory disease, and / or otherwise improving survival. The term includes therapeutic treatment, prophylactic treatment, and applications that reduce a subject's risk of developing a disorder or other risk factor. Treatment does not require a complete cure of the disorder, and encompasses embodiments that reduce symptoms or underlying risk factors. In the context of combination therapy, the term can also refer to i) the ability of a second therapeutic agent to reduce the effective dosage of a first therapeutic agent, thereby reducing side effects and increasing tolerability, ii) the ability of a second treatment to make a patient more responsive to the first treatment, and / or iii) the ability to achieve additive or synergistic clinical benefits.

[0119] Tumor-associated macrophages (TAMs): TAMs are polarized / activated macrophages with a pro-tumor phenotype (M2-like macrophages). TAMs can be either bone marrow-derived monocytes / macrophages recruited to the tumor site or tissue-resident macrophages derived from erythroid myeloid precursors. The differentiation of monocytes / macrophages into TAMs is influenced by several factors, including local chemical signals such as cytokines, chemokines, growth factors, and other molecules that act as ligands, as well as cell-cell interactions between monocytes / macrophages present in the niche (tumor microenvironment). Generally, monocytes / macrophages can be polarized into so-called "M1" or "M2" subtypes, the latter associated with a more tumor-promoting phenotype. In solid tumors, up to 50% of the tumor mass may correspond to macrophages, which are preferentially M2-polarized. Among 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 M2c and M2d subtypes) can express cell surface LRRC33 and / or LRRC33-progenitor TGFβ1.

[0120] Tumor microenvironment: The term "tumor microenvironment (TME)" refers to the local disease niche in which a tumor (e.g., a solid tumor) resides in vivo. The TME can include disease-associated molecular signatures (e.g., chemokines, cytokine suites), disease-associated cell populations (e.g., TAMs, CAFs, MDSCs), and disease-associated ECM milieu (altered ECM components and / or structure).

[0121] Variable region: The term "variable region" or "variable domain" refers to a portion of an antibody light chain and / or heavy chain, typically comprising about the amino-terminal 120-130 amino acids in the heavy chain and about the amino-terminal 100-110 amino acids in the light chain. In certain embodiments, the variable regions of different antibodies vary extensively in amino acid sequence, even among antibodies of the same species. Typically, the variable regions of an antibody determine the specificity of a particular antibody for its target.

[0122] Except in the operating examples or where otherwise specified, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%.

[0123] As used herein, the indefinite articles "a" and "an," as used in the specification and claims, unless expressly indicated otherwise, should be understood to mean "at least one."

[0124] As used herein, in the specification and claims, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements are present conjunctively in some instances and disjunctively in other instances. Other elements, whether related or unrelated to the elements specifically identified, may optionally be present, unless expressly stated otherwise. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so forth.

[0125] As used herein in the specification and claims, the phrase "at least one," when referring to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the 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") can refer in one embodiment to at least one A (optionally including multiple As) and no B (and optionally including elements other than B); in another embodiment to at least one B (optionally including multiple Bs) and no A (and optionally including elements other than A); in yet another embodiment to at least one A (optionally including multiple As) and at least one B (optionally including multiple Bs) (and optionally including other elements); and so forth.

[0126] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, by itself, imply any priority, precedence, or order of one claim element relative to another, or any chronological order in which method actions are performed, but is merely used as a label to distinguish one claim element having a particular name from another element having the same name (other than the use of ordinal terms) to identify the claim element.

[0127] Ranges provided herein are understood to be shorthand for all values ​​within that range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, such as 10-20, 1-10, 30-40, etc.

[0128] Transforming growth factor-beta (TGFβ) The activity of transforming growth factor-beta (TGFβ) and subsequent partial purification of the soluble growth factor were first described in the late 1970s and early 1980s, beginning the TGFβ field approximately 40 years ago. To date, 33 gene products that constitute the large TGFβ superfamily have been identified. The TGFβ superfamily can be divided into at least three subclasses based on structural similarity: TGFβ, growth differentiation factors (GDFs), and bone morphogenetic proteins (BMPs). The TGFβ subclass consists of three highly conserved isoforms, TGFβ1, TGFβ2, and TGFβ3, encoded by three separate genes in humans.

[0129] TGF-β is thought to play a key role in diverse processes, including the inhibition of cell proliferation, extracellular matrix (ECM) remodeling, and immune homeostasis. The importance of TGF-β1 in T cell homeostasis is demonstrated by the observation that TGF-β1- / - mice survive only 3–4 weeks and succumb to multiple organ failure due to extensive immune activation (Kulkarni, AB et al., Proc Natl Acad Sci USA, 1993, 90(2):770–774; Shull, MM et al., Nature, 1992, 359(6397):693–79). The roles of TGF-β2 and TGF-β3 are less clear. While the three TGF-β isoforms have distinct temporal and spatial expression patterns, they signal through the same receptors, TGF-βRI and TGF-βRII. However, in some cases, for example, TGF-β2 signaling, type III receptors such as betaglycan are also required (Feng, X.H. and R. Derynck, Annu Rev Cell Dev Biol, 2005, 21:659-93; Massague, J., Annu Rev Biochem, 1998, 67:753-91). Ligand-induced oligomerization of TGF-βRI / II triggers phosphorylation of SMAD transcription factors, leading to the transcription of target genes such as Col1a1, Col3a1, ACTA2, and serpin 1 (Massague, J., J. Seoane, and D. Wotton, Genes Dev, 2005, 19(23):2783-810). SMAD-independent TGFβ signaling pathways have also been described, for example, in cancer or aortic lesions in Marfan mice (Derynck, R. and YE Zhang, Nature, 2003, 425(6958):577-84; Holm, TM et al., Science, 2011, 332(6027):358-61).

[0130] The biological importance of the TGF-β pathway in humans is validated by genetic diseases. Kamurachi-Engelman disease results from an autosomal dominant mutation in the TGFB1 gene, leading to bone dysplasia and constitutive activation of TGF-β1 signaling (Janssens, K. et al., J Med Genet, 2006, 43(1):1-11). Patients with Loeys / Dietz syndrome carry autosomal dominant mutations in components of the TGF-β signaling pathway, which cause aortic aneurysms, hypertelorism, and a bifid uvula (Van Laer, L., H. Dietz, and B. Loeys, Adv Exp Med Biol, 2014, 802:95-105). Because dysregulation of the TGF-β pathway has been linked to multiple diseases, several drugs targeting the TGF-β pathway have been developed and tested in patients, but with limited success.

[0131] Dysregulation of TGFβ signaling has been implicated in a wide range of human diseases. Indeed, in some pathologies, such dysregulation may involve multiple aspects of TGFβ function. Diseased tissues, such as fibrotic and / or inflammatory tissues and tumors, may create local environments in which TGFβ activation can lead to disease exacerbation or progression, which may be mediated, at least in part, by interactions between multiple TGFβ-responsive cells in an autocrine and / or paracrine manner, along with several other cytokines, chemokines, and growth factors that play a role in specific disease settings.

[0132] For example, the tumor microenvironment (TME) contains multiple cell types that express TGFβ1, including cancer (i.e., malignant) cells as well as activated myofibroblast-like fibroblasts, stromal cells, infiltrating macrophages, MDSCs, and other immune cells. Thus, the TME represents a heterogeneous population of cells that express and / or are responsive to TGFβ1 but associate with multiple presentation molecules, e.g., LTBP1, LTBP3, LRRC33, and GARP, within the niche.

[0133] Advances in immunotherapy have transformed the effective treatment landscape for an ever-increasing number of cancer patients. Most notably, checkpoint blockade therapy (CBT) has now become part of standard treatment regimens for a growing number of cancers. While significant and durable responses to CBT have been observed across an ever-increasing number of cancer types, it is now clear that a significant proportion of tumors appear refractory to CBT even at the onset of treatment. Thus, primary resistance has been identified as a major challenge to enabling many patients' immune systems to target and eliminate tumor cells. To broaden the effectiveness of treatment in a larger number of patients, efforts are underway to understand and address the mechanisms underlying primary resistance to CBT. However, this enthusiasm has been tempered by disappointing clinical trial results and failures when CBT is combined with agents known to affect the same tumor type or modulate seemingly related components of the immune system. A possible reason is that a clear mechanistic rationale for a given combination is often not rooted in clinically derived data, thus leading to uncertain and perplexing results in clinical trials intended to augment approved single-agent therapies. It has become clear that the design of combination immunotherapies should be rooted in scientific evidence of the link between the underlying tumor and immune system biology.

[0134] Recently, a phenomenon called "immune exclusion" has been coined to describe tumor environments in which antitumor effector T cells (e.g., CD8+ T cells) are kept away (and therefore "excluded") by immunosuppressive local cues. More recently, several retrospective analyses of clinically derived tumors have linked activation of the TGFβ pathway to mediating primary resistance to CBT. For example, transcriptional profiling and analysis of pretreatment melanoma biopsies revealed enrichment of TGFβ-related pathways and biological processes in tumors unresponsive to anti-PD-1 CBT. In immune-excluded tumors, effector cells that would otherwise be able to attack cancer cells by recognizing cell-surface tumor antigens are prevented from accessing the site of cancer cells. In this way, cancer cells evade host immunity and immuno-oncological therapeutic agents, such as checkpoint inhibitors, that exploit and rely on such immunity. Indeed, such tumors are resistant to checkpoint blockade, such as anti-PD-1 and anti-PD-L1 antibodies, presumably because targeted T cells are blocked from entering the tumor and therefore fail to exert their anticancer effects.

[0135] Several retrospective analyses of clinically derived tumors have pointed to activation of the TGFβ pathway in mediating primary resistance to CBT. For example, transcriptional profiling and analysis of pretreatment melanoma biopsies revealed enrichment of TGFβ-related pathways and biological processes in tumors unresponsive to anti-PD-1 CBT. More recently, a similar analysis of tumors from patients with metastatic urothelial carcinoma revealed that the lack of response to PD-L1 blockade with atezolizumab was associated with a transcriptional signature of TGFβ signaling, particularly in tumors that appear to exclude CD8+ T cells from entering the tumor. The critical role of TGFβ signaling in mediating immune exclusion and resulting in anti-PD-(L)1 resistance has been confirmed in the EMT-6 syngeneic mouse model of breast cancer. EMT-6 tumors respond poorly to treatment with an anti-PD-L1 antibody, but combining this checkpoint inhibitor with 1D11, an antibody that blocks the activity of all TGFβ isoforms, significantly increased the frequency of complete responses compared with treatment with individual inhibitors. It has been proposed that the synergistic antitumor activity is due to the alteration of the cancer-associated fibroblast (CAF) phenotype and the disruption of the immune-excluded phenotype, leading to the infiltration of activated CD8+ T cells into the tumor. Similar results were found in a murine model of colorectal cancer and metastasis using a combination of an anti-PD-L1 antibody and galunisertib, a small molecule inhibitor of the type I TGFβ receptor ALK5 kinase. Collectively, these findings suggest that inhibition of the TGFβ pathway in CBT-resistant tumors may be a promising approach to improve or increase the number of clinical responses to CBT. While recent studies have linked activation of the TGFβ pathway to primary CBT resistance, TGFβ signaling has long been implicated in cancer pathogenesis. As a potent immunosuppressant, TGFβ interferes with antitumor T cell activity and promotes immunosuppressive macrophages. Malignant cells often become resistant to TGFβ signaling as a mechanism to evade its growth and tumor-suppressing effects. TGFβ activates CAFs, inducing extracellular matrix production and promoting tumor progression.Finally, TGFβ induces EMT and therefore supporting tissue invasion and tumor metastasis.

[0136] Mammals possess distinct genes encoding and expressing three TGFβ growth factors: TGFβ1, TGFβ2, and TGFβ3, all of which signal through the same heteromeric TGFβ receptor complex. Despite a common signaling pathway, each TGFβ isoform appears to have distinct biological functions, as evidenced by the phenotypes of non-overlapping TGFβ knockout mice. All three TGFβ isoforms are expressed as inactive prodomain-growth factor complexes, in which the TGFβ prodomain, also known as latency-associated peptide (LAP), wraps around the growth factor, keeping it in a latent, non-signaling state. Furthermore, latent TGFβ is coexpressed with latent TGFβ-binding protein, forming the large latent complex (LLC) through disulfide bonds. Association of latent TGFβ with latent TGFβ-binding protein-1 (LTBP1) or LTBP3 allows tethering to the extracellular matrix, while association with the transmembrane proteins GARP and LRRC33 allows for assimilation onto the surface of Tregs or macrophages, respectively. In vivo, latent TGF-β1 and latent TGF-β3 are activated by a subset of αV integrins that bind to a consensus RGD sequence on LAP and trigger a conformational change that releases growth factors. The mechanism by which latent TGF-β2 is activated is less clear because it lacks the consensus RGD motif. Proteolytic cleavage of LAP to release TGF-β1 has also been implicated in the activation mechanism, but its biological relevance is less clear.

[0137] While the pathogenic role of TGFβ activation is clear in several disease states, it is equally clear that therapeutic targeting of the TGFβ pathway has been challenging due to the pleiotropic effects resulting from broad and sustained pathway inhibition. For example, several studies have shown that small-molecule-mediated inhibition of the TGFβ type I receptor kinase ALK5 (TGFβR1) or blockade of all three highly related TGFβ growth factors using high-affinity antibodies resulted in severe valvular heart disease in mice, rats, and dogs. Therefore, these "pan" TGFβ approaches that block all TGFβ signaling have a very narrow therapeutic window, which has proven to be an obstacle to treating several disease-related processes with significant unmet medical needs. To date, no TGFβ-targeted therapies have been approved, and the results of clinical trials with such modalities have been largely disappointing, likely due to the use of what proved to be ineffective dosing regimens that were necessary to address safety concerns.

[0138] The safety concerns associated with broad TGFβ inhibition, combined with compelling evidence of the pathway's critical role in multiple disease processes, suggest that a better understanding of the specific role played by one or more TGFβ family members in disease pathology may provide viable avenues for therapeutic intervention. Regarding response to TGFβ and CBT, we herein observe widespread expression of TGFβ1 in many human tumors, suggesting that this family member may be a major driver of this pathway's contribution to primary resistance.

[0139] As mentioned above, growing evidence suggests that TGFβ may be a key player in generating and / or maintaining immunosuppression in diseased tissues, including immune-negative tumor environments. Thus, TGFβ inhibition may unblock immunosuppression, allowing effector T cells (especially cytotoxic CD8+ T cells) to approach and kill target cancer cells. In addition to tumor infiltration, TGFβ inhibition may also promote CD8+ T cell expansion. Such expansion may occur in lymph nodes and / or within tumors (intratumorally). While the precise mechanisms underlying this process remain to be elucidated, it is believed that immunosuppression is mediated, at least in part, by activation of immune cell-associated TGFβ1, which is involved in regulatory T cells and activated macrophages. It has been reported that TGFβ directly promotes Foxp3 expression in CD4+ T cells, thereby converting them to a regulatory (immunosuppressive) phenotype (i.e., Tregs). Furthermore, Tregs suppress the proliferation of effector T cells (see, e.g., Figure 26B), thereby dampening the immune response. This process has been shown to be TGFβ1-dependent and likely involves GARP-associated TGFβ1 signaling. Observations in both humans and animal models have shown that increased Tregs in the TME are associated with poor prognosis in multiple cancer types. Furthermore, applicants have previously shown that M2-polarized macrophages exposed to tumor-derived factors such as M-CSF dramatically upregulate the cell surface expression of LRRC33, a presenting molecule for TGFβ1 (see, e.g., PCT / US2018 / 031759). These so-called tumor-associated macrophages (or TAMs) are thought to contribute to the observed TGFβ1-dependent immune suppression in the TME and promote tumor growth.

[0140] Some solid tumors are characterized by having a tumor stroma enriched with myofibroblasts or myofibroblast-like cells. These cells produce a collagenous matrix (such as desmoplasia) that surrounds or encases the tumor, which may be at least partially caused by overactive TGFβ1 signaling. It is believed that the activation of TGFβ1 is mediated through ECM-related presentation molecules, such as LTBP1 and LTBP3, in the tumor stroma.

[0141] Applicants have previously disclosed antibodies capable of inhibiting TGFβ1 activation in many of these biological contexts that have shown promising effects both in vitro and in vivo (see, e.g., PCT / US2018 / 012601). However, the challenge remained to: i) develop improved antibodies that exhibit less typology of affinities for different antigen complexes to ensure uniform inhibitory effects across the various biological contexts or niches in which disease-associated TGFβ1 resides; and / or ii) develop antibodies that offer even greater potency than previously described counterparts.

[0142] In the work presented herein, it was envisioned that improved antibodies should embody all or most of the following features: 1) maintain selectivity for TGFβ1 to minimize unwanted toxicity associated with pan-inhibition ("isoform selectivity") (see, e.g., PCT / US2017 / 021972); 2) exhibit broad binding activity across various biological contexts or both matrix-associated and cell-associated categories ("context-independence"); 3) achieve more uniform or unbiased affinity across multiple antigen complexes ("homogeneity"); 4) exhibit potent binding activity for each of the antigen complexes ("high affinity"); and 5) possess robust inhibitory activity for each context ("potency"). Furthermore, a preferred mechanism of action would be to inhibit the activation step, allowing the inhibitor to target tissue-tethered latent TGFβ1 complexes to preemptively prevent downstream activation events and achieve a durable effect, rather than directly targeting soluble / free growth factor ("persistence"). As disclosed in further detail herein, the novel and improved TGFβ1 inhibitors of the present disclosure are highly potent and selective inhibitors of latent TGFβ1 activation. The data presented herein demonstrate, inter alia, that this mechanism of isoform-specific inhibition is sufficient to overcome primary resistance to anti-PD-1 in a syngeneic mouse model that closely recapitulates some of the features of primary resistance to CBT found in human cancers. These efficacy data, combined with the improved preclinical safety profile of such antibodies compared to "pan" TGFβ inhibitors, provide a rationale for exploring the therapeutic use of selective TGFβ1 inhibition to broaden and enhance clinical responses to checkpoint blockade in cancer immunotherapy, as well as to treat several additional TGFβ1-related indications.

[0143] Novel high-affinity isoform-selective antibodies to precursor TGF-.BETA.1 General characteristics Disclosed herein are improved, high-affinity inhibitors of TGFβ1, characterized by enhanced binding properties, increased inhibitory potency, and maintaining a desirable safety profile and isoform selectivity compared to earlier disclosed TGFβ1-selective inhibitors. These TGFβ1-selective inhibitors of the present disclosure are monoclonal antibodies (e.g., immunoglobulins, engineered immunoglobulin-like molecules, antigen-binding fragments, or portions thereof) that specifically bind to at least a portion of the prodomain (sometimes referred to as "LAP") of the latent precursor TGFβ1 complex and have isoform-selective inhibitory activity against TGFβ1 (see "Core Properties" in Table 1).

[0144] The enhanced binding properties of antibodies according to the present disclosure include increased affinity as measured at equilibrium. In some embodiments, the antibody has a KD of ≦1 nM for at least one of the human LLC complexes (hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and / or hLRRC33-precursor TGFβ1) as measured by MSD-SET. In some embodiments, such antibodies have a KD of ≦1 nM for two of the human LLC complexes selected from hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 as measured by MSD-SET. In some embodiments, such antibodies have a KD of ≦1 nM, as measured by MSD-SET, for three of the human LLC complexes selected from hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1. In preferred embodiments, such antibodies have a KD of ≦1 nM, as measured by MSD-SET, for each of the human LLC complexes, i.e., hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1. According to the present disclosure, high-affinity antibodies may have KD values ​​for a particular antigen (e.g., antigen complex) at equilibrium that are ≦1 nM, e.g., ≦1 nM, ≦0.5 nM, ≦400 pM, ≦300 pM, ≦200 pM, and ≦100 pM.

[0145] The present invention also includes antibodies or antigen-binding fragments thereof that can specifically bind to each of the human LLC complexes (hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1) 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), as measured at equilibrium, such as MSD-SET. In some embodiments, the antibody binds to each of the aforementioned LLC complexes with a KD of ≦5 nM, as measured by a solution equilibrium titration-based method. In some embodiments, the antibody binds to each of the aforementioned LLC complexes with a KD of ≦1 nM, as measured by a solution equilibrium titration-based method.

[0146] For therapeutic use to treat TGFβ1-related indications involving dysregulation of both extracellular matrix and immune components, it is advantageous to select an antibody with high affinity (e.g., a KD of ≦1 nM) for at least one of the ECM-associated precursor TGFβ1 complexes (hLTBP1-precursor TGFβ1 and / or hLTBP3-precursor TGFβ1) and also at least one of the cell-associated precursor TGFβ1 complexes (hGARP-precursor TGFβ1 and / or hLRRC33-precursor TGFβ1) in order to exert an inhibitory effect in both contexts (e.g., in the ECM and attracted to immune cells). In some embodiments, the antibody has high affinity (e.g., a KD of ≦1 nM) for both hLTBP1-precursor TGFβ1 and hLTBP3-precursor TGFβ1 and also at least one of the cell-associated precursor TGFβ1 complexes (hGARP-precursor TGFβ1 or hLRRC33-precursor TGFβ1). In still other embodiments, the antibody has high affinity (e.g., a KD of ≦1 nM) for each of the aforementioned complexes (hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1). In preferred embodiments, such antibodies have a KD of ≦200 pM, e.g., ≦100 pM, for each of the human complexes, as measured by a solution equilibrium titration-based method such as MSD-SET.

[0147] Embodiments of the present disclosure include high-affinity, context-independent antibodies. Such antibodies can bind with equivalent affinity to four known presentation molecule-precursor TGFβ1 complexes: LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1. Equivalent affinity can mean either that the lowest affinity (highest KD value) exhibited by the antibody among the four antigen complexes is no more than 5-fold lower than the average calculated from the affinities of the remaining three, or that the highest affinity (lowest KD value) exhibited by the antibody among the four antigen complexes is no more than 5-fold higher than the average calculated from the affinities of the remaining three. In some embodiments, such antibodies can be said to have equivalent affinity if the ratio of the average KD value of two ECM-associated complexes to the average KD value of two cell-associated complexes is no more than 3-fold.

[0148] Antibodies with equivalent affinities may achieve a more uniform (e.g., unbiased) inhibitory effect regardless of the specific presenting molecule associated with the precursor TGFβ1 complex (hence "context-independent"). In particularly preferred embodiments, the antibody is a high-affinity, context-independent antibody in that its affinity for each of four human LLCs is 1 nM or less (e.g., 200 pM or less) as measured by a solution equilibrium titration-based method, and the antibody has equivalent affinity for all four of the aforementioned human LLCs. For example, the observed bias in average affinity between the matrix-associated complex and the cell-associated complex is 3-fold or less.

[0149] In some embodiments, such antibodies specifically bind to each of the aforementioned complexes (hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1) 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) as measured by a solution equilibrium titration-based method such as MSD-SET.

[0150] Any one of the inhibitory antibodies encompassed herein may bind to each of the aforementioned large latent complexes (hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1) at a binding region comprising at least a portion of the latent Lasso within the prodomain of the precursor TGFβ1 complex. Such binding regions may further comprise at least a portion of the growth factor domain. In particularly preferred embodiments, such antibodies have a K of ≦200 pM (such as ≦150 pM and ≦100 pM) with each of the LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1 complexes at a binding region within the LLC complex comprising at least a portion of the latent Lasso and at least a portion of the growth factor domain. D Join by value.

[0151] In some embodiments, high-affinity, context-independent antibodies capable of selectively inhibiting TGFβ1 can inhibit activated TGFβ1 regardless of the mode of activation. For example, certain integrins are known to directly bind to the RGD motif within the prodomain of LLC, mechanically "pulling open" the cage-like prodomain structure, thereby releasing the TGFβ1 growth factor from the latent 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 and thereby interfere with integrin binding may not inhibit protease-dependent activation of TGFβ1. Conversely, antibodies that directly target one or more protease recognition or cleavage sites may not inhibit integrin-dependent activation of TGFβ1. In contrast, in preferred embodiments of the present invention, high-affinity, context-independent antibodies can inhibit integrin-dependent activation of TGFβ1 and protease-dependent activation of TGFβ1.

[0152] While high affinity binding to target human proteins is an essential feature of antibody therapeutics, the ability to cross-react with counterparts from additional species is advantageous. Particularly considering that most preclinical pharmacological models are in rodents, species cross-reactivity with murine / rat proteins provides a convenient tool as a surrogate antibody for preclinical research. Thus, in some embodiments, the high affinity antibodies of the present disclosure advantageously cross-react with counterparts from other mammals, such as mice, rats, and / or non-human primates.

[0153] Among the novel antibodies encompassed by the present disclosure, particularly preferred antibody classes and their characteristics are categorized and described below.

[0154] Favorable Features In some embodiments, in addition to the core property features, preferred antibodies disclosed herein further meet the criteria for antibodies in one or more of categories 1-5 set forth in Table 1 herein.

[0155] In some embodiments, an additional requirement for an antibody of the invention is defined by its binding characteristics, e.g., the affinity of the antibody for the antigen. "Antigen" in this context includes at least four protein complexes, namely the human large latent complex (LLC) of TGFβ1, which are referred to as the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes. According to the invention of this disclosure, antibodies bind to each of these complexes, typically in a K D The antibody can bind with a particular affinity, measured as a value. Category 1 and Category 2 antibodies fall within the scope of these embodiments. For purposes of defining criteria based on binding properties (e.g., Categories 1 and 2), the affinity of the antibody is determined at equilibrium rather than by a kinetic assay (such as BLI).

[0156] Additionally or alternatively, an additional requirement for the antibodies of the invention is that they are defined by their amino acid sequence: 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. Table 1. Favorable features of the novel high affinity TGFβ1 selective inhibitors of the present invention. JPEG2024099673000003.jpg240166 JPEG2024099673000004.jpg236166

[0157] Non-limiting embodiments of each category are provided below.

[0158] Category 1 antibodies The antibodies disclosed herein are high affinity, isoform-selective antibodies that can specifically target the large complex of the human latent form of TGFβ1.

[0159] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to each of the following human LLCs: hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes with a KD of ≦200 pM, wherein the affinity is measured at equilibrium using a suitable assay, such as a solution equilibrium titration-based assay.

[0160] Such antibodies or fragments may bind to each of the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes with a KD of ≦150 pM, as measured by solution equilibrium titration. More preferably, such antibodies or fragments may bind to each of the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes with a KD of ≦100 pM, as measured by solution equilibrium titration. Any suitable in vitro affinity assay capable of determining antibody KD values ​​at equilibrium may 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 antibody criteria for Category 1 preferred antibodies include Ab6, Ab22, Ab24, Ab26, Ab29, Ab30, Ab31, Ab32, and Ab33.

[0161] The antibodies may also bind with high specificity and affinity to the corresponding LLC of additional species. In preferred embodiments, the antibodies exhibit species cross-reactivity to their murine counterparts.

[0162] Category 2 antibodies The antibodies disclosed herein are high affinity, isoform-selective antibodies that can specifically target the large complex of the human latent form of TGFβ1.

[0163] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to each of the following human LLCs: hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes with a KD of ≦1 nM, where affinity is measured at equilibrium using a suitable assay, such as a solution equilibrium titration-based assay, and the antibody or fragment binds to human LLC at a binding region comprising at least a portion of the latent Lasso. Latent Lasso is a protein domain that forms part of the so-called "straight jacket" of the prodomain. In its native form, the latent Lasso of the human precursor TGFβ1 polypeptide has the amino acid sequence LASPPSQGEVPPGPL (SEQ ID NO: 153). Any suitable technique can be used to determine whether an antibody binds to human TGFβ1 LLC at a region comprising at least a portion of the latent Lasso. For example, a competitive assay utilizing the corresponding polypeptide can be performed. In some embodiments, the binding region may be determined by HD-X or X-ray crystallography.

[0164] In some embodiments, such antibodies or fragments may bind to each of the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes with a KD of ≦500 pM (optionally ≦400 pM, ≦300 pM, ≦200 pM, or ≦100 pM) as measured by solution equilibrium titration, wherein the antibody or fragment binds to human LLC at a binding region that includes at least a portion of latent Lasso. Non-limiting examples of antibodies disclosed herein that meet the antibody criteria for Category 2 preferred antibodies include Ab5 and Ab6.

[0165] In some embodiments, such antibodies may further bind to human LLC at an additional binding region comprising at least a portion of the growth factor domain within the pro-TGFβ1 complex. In some embodiments, the additional binding occurs only in the context of the latent complex, such that the antibody does not specifically bind to free growth factor that is not associated with the pro-domain complex. The additional binding region within the growth factor domain of LLC may comprise at least a portion of the protein domains referred to as "finger-1" and / or "finger-2." Thus, such antibodies may bind to a combinatorial epitope comprising at least one amino acid residue of latent Lasso and at least one amino acid residue of the growth factor domain.

[0166] The antibodies may also bind with high specificity and affinity to the corresponding LLC of additional species. In preferred embodiments, the antibodies exhibit species cross-reactivity to their murine counterparts.

[0167] Category 3 antibodies The antibodies disclosed herein are high affinity, isoform-selective antibodies that can specifically target the large complex of the human latent form of TGFβ1.

[0168] In a further aspect, the present invention provides an antibody or antigen-binding fragment thereof comprising H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, wherein CDR-H1 has the amino acid sequence represented by FTF(X1)(X2)(X3)(X4)M(X5) (SEQ ID NO: 143). In some embodiments, X1 can be S, G, or A, X2 can be S or F, X3 can be F or Y, X4 can be S or A, and / or X5 can be D, N, or Y, in any combination. In some embodiments, when H-CDR1 contains at least one amino acid substitution, position X1 can be replaced with S, position X2 can be replaced with S, position X3 can be replaced with F, position X4 can be replaced with S, and / or position X5 can be replaced with D.

[0169] The CDR-H2 of the antibody 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, when H-CDR2 contains at least one amino acid substitution, position X1 can be replaced with S, position X2 can be replaced with P, position X3 can be replaced with D, position X4 can be replaced with S, and / or position X5 can be replaced with D.

[0170] The CDR-H3 of the antibody 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, when H-CDR3 contains at least one amino acid substitution, position X1 can be replaced with A, position X2 can be replaced with G, position X3 can be replaced with V, position X4 can be replaced with L, position X5 can be replaced with Y, and / or position X6 can be replaced with D.

[0171] CDR-L1 has the amino acid sequence QASQDITNYLN (SEQ ID NO: 105), optionally with one or two amino acid changes.

[0172] CDR-L2 has the amino acid sequence DASNLET (SEQ ID NO: 106), optionally with one or two amino acid changes.

[0173] CDR-L3 has the amino acid sequence QQADNHPPWT (SEQ ID NO: 12), optionally with one or two amino acid changes.

[0174] Non-limiting examples of antibodies disclosed herein that meet the antibody criteria for Category 3 preferred antibodies include Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, and Ab34.

[0175] Table 2 below summarizes the CDR consensus sequences for Category 3 antibodies. In some embodiments, each of the CDR sequences may optionally contain one or more of the amino acid substitutions described below. Table 2. Heavy and light chain consensus CDR sequences and preferred amino acid substitutions JPEG2024099673000005.jpg139166

[0176] In some embodiments, a Category 3 antibody or antigen-binding fragment thereof specifically binds to each of the following human LLCs: hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes with a KD of ≦1 nM, where affinity is measured at equilibrium using a suitable assay, such as a solution equilibrium titration-based assay.

[0177] In some embodiments, the antibody or fragment binds to human LLC at a binding region comprising at least a portion of the latent Lasso. The latent Lasso is a protein domain that forms part of the so-called "straight jacket" of the prodomain. In its native form, the latent Lasso of the human precursor TGFβ1 polypeptide has the amino acid sequence LASPPSQGEVPPGPL (SEQ ID NO: 153). Any suitable technique can be used to determine whether an antibody binds to human TGFβ1 LLC at 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 embodiments, the binding region can be determined by HD-X or X-ray crystallography.

[0178] In some embodiments, such antibodies or fragments may bind to each of the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes with a KD of ≦500 pM (optionally ≦400 pM, ≦300 pM, ≦200 pM, or ≦100 pM) as measured by solution equilibrium titration, wherein the antibody or fragment binds to human LLC at a binding region that includes at least a portion of latent Lasso.

[0179] In some embodiments, such antibodies may further bind to human LLC at an additional binding region comprising at least a portion of the growth factor domain within the pro-TGFβ1 complex. In some embodiments, the additional binding occurs only in the context of the latent complex, such that the antibody does not specifically bind to free growth factor that is not associated with the pro-domain complex. The additional binding region within the growth factor domain of LLC may comprise at least a portion of the protein domains referred to as "finger-1" and / or "finger-2." Thus, such antibodies may bind to a combinatorial epitope comprising at least one amino acid residue of latent Lasso and at least one amino acid residue of the growth factor domain.

[0180] The antibodies may also bind with high specificity and affinity to the corresponding LLC of additional species. In preferred embodiments, the antibodies exhibit species cross-reactivity to their murine counterparts.

[0181] Also included herein are cross-blocking antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or fragment thereof cross-blocks or cross-competes with one of the Category 3 antibodies, wherein the antibody has a KD of ≦1 nM for at least one of the human LLC complexes (hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and / or hLRRC33-precursor TGFβ1) as measured by MSD-SET. In some embodiments, such an antibody has a KD of ≦1 nM for two of the human LLC complexes selected from hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 as measured by MSD-SET. In some embodiments, such antibodies have a KD of ≦1 nM, as measured by MSD-SET, for three of the human LLC complexes selected from hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1. In preferred embodiments, such antibodies have a KD of ≦1 nM, as measured by MSD-SET, for each of the human LLC complexes, i.e., hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1. According to the present disclosure, high-affinity antibodies may have KD values ​​for a particular antigen (e.g., antigen complex) at equilibrium that are ≦1 nM, e.g., ≦1 nM, ≦0.5 nM, ≦400 pM, ≦300 pM, ≦200 pM, and ≦100 pM.

[0182] Category 4 antibodies The antibodies disclosed herein are high affinity, isoform-selective antibodies that can specifically target the large complex of the human latent form of TGFβ1.

[0183] In a further aspect, the invention provides an antibody or 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 FTFSSFSMN (SEQ ID NO: 114), optionally each containing up to four amino acid changes (optionally up to four, up to three, up to two, or one amino acid change); and H-CDR2 comprises YISPDASTIYYADSVKG (SEQ ID NO: 111), optionally H-CDR2 contains up to four amino acid changes (optionally up to four, up to three, H-CDR1 comprises ARGVLDYGDMLDP (SEQ ID NO: 110), optionally with one or two amino acid changes; H-CDR3 comprises ARGVLDYGDMLDP (SEQ ID NO: 110), optionally with one or two amino acid changes; L-CDR1 comprises QASQDITNYLN (SEQ ID NO: 105), optionally with one or two amino acid changes; L-CDR2 comprises DASNLET (SEQ ID NO: 106), optionally with one or two amino acid changes; and L-CDR3 comprises QQADNHPPWT (SEQ ID NO: 12), optionally with one or two amino acid changes.

[0184] Non-limiting examples of antibodies disclosed herein that meet the antibody criteria for Category 4 preferred antibodies include Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34.

[0185] Table 3 below summarizes the CDR sequences of Category 4 antibodies. In some embodiments, each of the CDR sequences may optionally contain one or more of the amino acid substitutions described below. Table 3. CDR sequences and variants JPEG2024099673000006.jpg100166

[0186] In some embodiments, a Category 4 antibody or antigen-binding fragment thereof specifically binds to each of the following human LLCs: hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes with a KD of ≦1 nM, where affinity is measured at equilibrium using a suitable assay, such as a solution equilibrium titration-based assay.

[0187] In some embodiments, the antibody or fragment binds to human LLC at a binding region comprising at least a portion of the latent Lasso. The latent Lasso is a protein domain that forms part of the so-called "straight jacket" of the prodomain. In its native form, the latent Lasso of the human precursor TGFβ1 polypeptide has the amino acid sequence LASPPSQGEVPPGPL (SEQ ID NO: 153). Any suitable technique can be used to determine whether an antibody binds to human TGFβ1 LLC at 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 embodiments, the binding region can be determined by HD-X or X-ray crystallography.

[0188] In some embodiments, such antibodies or fragments may bind to each of the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes with a KD of ≦500 pM (optionally ≦400 pM, ≦300 pM, ≦200 pM, or ≦100 pM) as measured by solution equilibrium titration, wherein the antibody or fragment binds to human LLC at a binding region that includes at least a portion of latent Lasso.

[0189] In some embodiments, such antibodies may further bind to human LLC at an additional binding region comprising at least a portion of the growth factor domain within the pro-TGFβ1 complex. In some embodiments, the additional binding occurs only in the context of the latent complex, such that the antibody does not specifically bind to free growth factor that is not associated with the pro-domain complex. The additional binding region within the growth factor domain of LLC may comprise at least a portion of the protein domains referred to as "finger-1" and / or "finger-2." Thus, such antibodies may bind to a combinatorial epitope comprising at least one amino acid residue of latent Lasso and at least one amino acid residue of the growth factor domain.

[0190] The antibodies may also bind with high specificity and affinity to the corresponding LLC of additional species. In preferred embodiments, the antibodies exhibit species cross-reactivity to their murine counterparts.

[0191] Also included herein are cross-blocking antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or fragment thereof cross-blocks or cross-competes with one of the Category 4 antibodies, wherein the antibody has a KD of ≦1 nM for at least one of the human LLC complexes (hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and / or hLRRC33-precursor TGFβ1) as measured by MSD-SET. In some embodiments, such an antibody has a KD of ≦1 nM for two of the human LLC complexes selected from hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 as measured by MSD-SET. In some embodiments, such antibodies have a KD of ≦1 nM, as measured by MSD-SET, for three of the human LLC complexes selected from hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1. In preferred embodiments, such antibodies have a KD of ≦1 nM, as measured by MSD-SET, for each of the human LLC complexes, i.e., hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1. According to the present disclosure, high-affinity antibodies may have KD values ​​for a particular antigen (e.g., antigen complex) at equilibrium that are ≦1 nM, e.g., ≦1 nM, ≦0.5 nM, ≦400 pM, ≦300 pM, ≦200 pM, and ≦100 pM.

[0192] Category 5 antibodies The antibodies disclosed herein are high affinity, isoform-selective antibodies that can specifically target the large complex of the human latent form of TGFβ1.

[0193] In a further aspect, the present invention provides a heavy chain variable domain (V) having at least 90% sequence identity to EVQLVESGGGLVQPGGSLRLSCTASGFTFSSFSMDWVRQAPGKGLEWVSYISPSADTIYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARGVLDYGDMLMPWGQGTLVTVSS (SEQ ID NO: 13). H ) and a light chain variable domain (V) having at least 90% sequence identity to DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQADNHPPWTFGGGTKVEIK (SEQ ID NO: 15). L and

[0194] In some embodiments, the heavy chain variable domain of the antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the VH sequence set forth in SEQ ID NO:13.

[0195] In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to the above VH sequence.

[0196] In some embodiments, the heavy chain variable domain of the antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the VL sequence set forth in SEQ ID NO:15.

[0197] In some embodiments, the light chain variable domain of the antibody is at least 95% identical to the VL sequence above.

[0198] Non-limiting examples of antibodies disclosed herein that meet the antibody criteria for Category 5 preferred antibodies include Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34.

[0199] In some embodiments, a Category 5 antibody or antigen-binding fragment thereof specifically binds to each of the following human LLCs: hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes with a KD of ≦1 nM, where affinity is measured at equilibrium using a suitable assay, such as a solution equilibrium titration-based assay.

[0200] In some embodiments, the antibody or fragment binds to human LLC at a binding region comprising at least a portion of the latent Lasso. The latent Lasso is a protein domain that forms part of the so-called "straight jacket" of the prodomain. In its native form, the latent Lasso of the human precursor TGFβ1 polypeptide has the amino acid sequence LASPPSQGEVPPGPL (SEQ ID NO: 153). Any suitable technique can be used to determine whether an antibody binds to human TGFβ1 LLC at 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 embodiments, the binding region can be determined by HD-X or X-ray crystallography.

[0201] In some embodiments, such antibodies or fragments may bind to each of the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes with a KD of ≦500 pM (optionally ≦400 pM, ≦300 pM, ≦200 pM, or ≦100 pM) as measured by solution equilibrium titration, wherein the antibody or fragment binds to human LLC at a binding region that includes at least a portion of latent Lasso.

[0202] In some embodiments, such antibodies may further bind to human LLC at an additional binding region comprising at least a portion of the growth factor domain within the pro-TGFβ1 complex. In some embodiments, the additional binding occurs only in the context of the latent complex, such that the antibody does not specifically bind to free growth factor that is not associated with the pro-domain complex. The additional binding region within the growth factor domain of LLC may comprise at least a portion of the protein domains referred to as "finger-1" and / or "finger-2." Thus, such antibodies may bind to a combinatorial epitope comprising at least one amino acid residue of latent Lasso and at least one amino acid residue of the growth factor domain.

[0203] The antibodies may also bind with high specificity and affinity to the corresponding LLC of additional species. In preferred embodiments, the antibodies exhibit species cross-reactivity to their murine counterparts.

[0204] Also included herein are cross-blocking antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or fragment thereof cross-blocks or cross-competes with one of Category 5 antibodies, wherein the antibody has a KD of ≦1 nM for at least one of the human LLC complexes (hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and / or hLRRC33-precursor TGFβ1) as measured by MSD-SET. In some embodiments, such an antibody has a KD of ≦1 nM for two of the human LLC complexes selected from hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 as measured by MSD-SET. In some embodiments, such antibodies have a KD of ≦1 nM, as measured by MSD-SET, for three of the human LLC complexes selected from hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1. In preferred embodiments, such antibodies have a KD of ≦1 nM, as measured by MSD-SET, for each of the human LLC complexes, i.e., hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1. According to the present disclosure, high-affinity antibodies may have KD values ​​for a particular antigen (e.g., antigen complex) at equilibrium that are ≦1 nM, e.g., ≦1 nM, ≦0.5 nM, ≦400 pM, ≦300 pM, ≦200 pM, and ≦100 pM.

[0205] Exemplary Antibodies of the Invention Exemplary antibodies and corresponding nucleic acid sequences encoding such antibodies useful for practicing the present invention include one or more of the 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 the L-CDRs (L-CDR1, L-CDR2, and L-CDR3) provided in Table 5.

[0206] Thus, the present invention provides an isolated antibody or antigen-binding fragment thereof 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 the antibodies listed in Table 4, and 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 an H-CDR1 having the amino acid sequence FTFSSFSMD (SEQ ID NO: 107), an H-CDR2 having the amino acid sequence YISPSADTIYYADSVKG (SEQ ID NO: 103), and an H-CDR3 having the amino acid sequence ARGVLDYGDMLMP (SEQ ID NO: 6), an L-CDR1 having the amino acid sequence QASQDITNYLN (SEQ ID NO: 105), an L-CDR2 having the amino acid sequence DASNLET (SEQ ID NO: 106), and an L-CDR3 having the amino acid sequence QQADNHPPWT (SEQ ID NO: 12). Table 4. Complementarity determining regions of the heavy chains of exemplary antibodies determined using the numbering scheme described in Lu et al. JPEG2024099673000007.jpg130166JPEG2024099673000008.jpg133166Table 5. Complementarity Determining Regions of Exemplary Antibody Light Chains Determined Using the Kabat Numbering Scheme or the Lu et al. Numbering System JPEG2024099673000009.jpg24166

[0207] Determining the CDR sequences within an antibody depends on the particular 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 X et al., MAbs., January 2019, 11(1):45-57). For example, the six CDR sequences of Ab6 defined by four different numbering systems are illustrated below. Any CDR numbering system understood in the art may be used to define the CDR sequences of the antibodies of the present disclosure. Table 6. Six CDRs of an exemplary antibody (Ab6) based on four numbering schemes JPEG2024099673000010.jpg82166

[0208] The amino acid sequences of the heavy and light chain variable domains of exemplary antibodies of the disclosure are provided in Table 7. Thus, in some embodiments, the high affinity, isoform-selective TGFβ1 inhibitors of the disclosure comprise heavy chain variable domains (V H ) and the light chain variable domain (V L ), wherein V H and V L The sequences are listed in Table 7 below. H and V L The sequence is selected from any one of the set of sequences. Table 7. Exemplary antibody heavy and light chain variable domains JPEG2024099673000011.jpg133166JPEG2024099673000012.jpg249166JPEG2024099673000013.jpg248166JPEG2024099673000014.jpg111166

[0209] Accordingly, the present invention provides an antibody or 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 to any one of the sequences selected from the group consisting of Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34; The light chain variable domain has at least 90% identity to any one of the sequences selected from Ab4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34, and optionally the heavy chain variable domain may 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 to SEQ ID NO: 13, and optionally has at least 90% sequence identity to the light chain variable domain of the antibody or fragment SEQ ID NO: 15. In some embodiments, the heavy chain variable domain of the antibody or fragment has at least 95% sequence identity to SEQ ID NO: 13, and optionally has at least 95% sequence identity to the light chain variable domain of the antibody or fragment, SEQ ID NO: 15. In some embodiments, the heavy chain variable domain of the antibody or fragment has at least 98% sequence identity to SEQ ID NO: 13, and optionally has at least 98% sequence identity to the light chain variable domain of the antibody or fragment, SEQ ID NO: 15. In some embodiments, the heavy chain variable domain of the antibody or fragment has 100% sequence identity to SEQ ID NO: 13, and optionally the light chain variable domain of the antibody or fragment has 100% sequence identity to SEQ ID NO: 15.

[0210] In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex comprises a heavy chain variable domain amino acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 14, and a light chain variable domain amino acid sequence encoded by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 14, and a light chain variable domain amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 16.

[0211] In some examples, any of the antibodies of the present disclosure that specifically bind to a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, and / or an LRRC33-TGFβ1 complex include any antibody (including an antigen-binding portion thereof) having one or more CDR (e.g., CDRH or CDRL) sequences substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3. For example, an antibody may comprise one or more CDR sequences set forth in Table 4 that contain up to 5, 4, 3, 2, or 1 amino acid residue changes compared to the corresponding CDR region in any one of SEQ ID NOs: 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 contains up to three amino acid changes compared to the sequence provided in Table 4. Such antibody variants containing up to three amino acid changes per CDR are encompassed by the present invention. In some embodiments, such mutant antibodies are generated by an optimization process such as affinity maturation. The complete amino acid sequences of the heavy 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 and light chain variable regions of the particular antibodies, are provided below: Ab3-heavy chain variable region amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVSSISGSGGATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDFDYWGQGTLVTVSS (SEQ ID NO: 95) Ab6-heavy chain variable region amino acid sequence EVQLVESGGGLVQPGGSLRLSCTAS GFTFSSFS MDWVRQAPGKGLEWVSY ISPSADTI YYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYC ARGVLDYGDMLMP WGQGTLVTVSS (SEQ ID NO: 13) Ab6-light chain variable region amino acid sequence DIQMTQSPSSLSASVGDRVTITCQAS QDITNY LNWYQQKPGKAPKLLIY DAS NLETGVPSRFSGSGSGTFTFTISSLQPEDIATYYC QQADNHPPWT FGGGTKVEIK (SEQ ID NO: 15) Ab6-heavy chain amino acid sequence EVQLVESGGGLVQPGGSLRLSCTASGFTFS SFSMD WVRQAPGKGLEWVS YISPSADTIYYADSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR GVLDYGDMLMP WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 17) Ab6-heavy chain nucleic acid sequence Ab6-light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITC QASQDITNYLN WYQQKPGKAPKLLIY DASNLET GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQADNHPPWT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19) Ab6-light chain nucleic acid sequence (human kappa) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTACCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAGCAGGCCGACAATCACCCTCCTTGGACTTTTGGCGGAGGGACCAAGGTTGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 20)

[0212] In some embodiments, the "percent identity" of two amino acid sequences is determined using the algorithm of 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. Such an algorithm has been incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol., 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res., 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0213] In any of the antibodies or antigen-binding fragments described herein, one or more conservative mutations can be introduced into CDR or framework sequences at positions where the residues are unlikely to be involved in antibody-antigen interactions. In some embodiments, such conservative mutations can be introduced into CDR or framework sequences at positions where the residues are unlikely to be involved in interactions with the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex, as determined based on crystal structures. In some embodiments, potential interfaces (e.g., residues involved in antigen-antibody interactions) can be predicted from known structural information about other antigens that share structural similarity.

[0214] As used herein, a "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequences known to those skilled in the art, such as those found in compiled references of such methods, e.g., Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, or Current Protocols in Molecular Biology, edited by FMAusubel et al., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0215] In some embodiments, the antibodies provided herein contain mutations that confer desirable properties to the antibody. For example, to avoid potential complications due to Fab arm exchange, which is known to occur in native IgG4 mAbs, 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), which converts serine 228 (residue 241 in the EU numbering, Kabat numbering) to proline, resulting in an IgG1-like (CPPCP (SEQ ID NO: 54)) hinge sequence. Thus, any of the antibodies may contain the stabilizing "Adair" mutation or the amino acid sequence CPPCP (SEQ ID NO: 54).

[0216] The isoform-specific, context-independent inhibitors of TGFβ1 of the present disclosure may optionally comprise an antibody constant region or a portion thereof. For example, a VL domain may be attached at its C-terminus to a light chain constant domain such as Cκ or Cλ. Similarly, a VH domain or a portion thereof may be attached to all or part of a heavy chain such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody may comprise an appropriate constant region (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 91-3242, National Institutes of Health Publications, Bethesda, MD (1991)). Thus, antibodies within the scope of the present disclosure may comprise VH and VL domains or antigen-binding portions thereof combined with any appropriate constant region.

[0217] Additionally or alternatively, such antibodies may or may not comprise the framework regions of the antibodies of SEQ ID NOs: 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, and 15. In some embodiments, antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LRRC33-TGFβ1 complex are murine antibodies and comprise murine framework region sequences.

[0218] In some embodiments, such antibodies have relatively high affinity, e.g., 10 -9 Under M, 10 -10 M, 10 -11and LRRC33-TGFβ1 complex with an affinity of 5 pM to 1 nM, e.g., 10 pM to 1 nM, e.g., 10 pM to 100 pM. The present 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 less (e.g., 1 nM or less, 500 pM or less, 100 pM or less). The affinity and binding kinetics of antibodies that specifically bind to the GARP-TGFβ1 complex, the LTBP1-TGFβ1 complex, the LTBP3-TGFβ1 complex, and / or the LRRC33-TGFβ1 complex can be tested using any suitable method, including, but not limited to, biosensor-based techniques (e.g., OCTET® or BIACORE) and solution equilibrium titration-based techniques (e.g., MSD-SET).

[0219] In some embodiments, inhibitors of cell-associated TGFβ1 (e.g., GARP-presented TGFβ1 and LRRC33-presented TGFβ1) according to the present invention comprise antibodies or fragments thereof that specifically bind to such complexes (e.g., GARP-precursor / latent TGFβ1 and LRRC33-precursor / latent TGFβ1) and trigger internalization of the complexes. This mechanism of action causes removal or depletion of inactive TGFβ1 complexes (e.g., GARP-precursor TGFβ1 and LRRC33-precursor TGFβ1) from the cell surface (e.g., Tregs, macrophages, etc.), thus reducing TGFβ1 available for activation. In some embodiments, such antibodies or fragments thereof bind to target complexes 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 recycling antibodies.

[0220] High-affinity isoform-specific inhibitory antibodies and competing antibodies for TGF-.BETA.1 Embodiments of the present disclosure relate to antibodies that compete or cross-compete with any of the antibodies provided herein. As used herein, the term "compete" with respect to an antibody means that a first antibody binds to an epitope (e.g., an epitope of the GARP-precursor TGFβ1 complex, the LTBP1-precursor TGFβ1 complex, the LTBP3-precursor TGFβ1 complex, and the LRRC33-precursor TGFβ1 complex) in a manner sufficiently similar to or overlapping with the binding of a second antibody such that the binding of the first antibody to its epitope is detectably reduced in the presence of the second antibody 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 can, but need not, be detectably reduced in the presence of the first antibody. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its corresponding epitope. However, if each antibody detectably inhibits the binding of the other antibody to its epitope or ligand, whether to the same, a greater extent, or a lesser extent, the antibodies are said to "cross-compete" with each other for binding of their corresponding epitopes. Both competing and cross-competing antibodies are within the scope of the present disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), those skilled in the art will understand that such competing and / or cross-competing antibodies are encompassed and can be useful in the methods and / or compositions provided herein. The terms "cross-blocking" may be used interchangeably.

[0221] Two different monoclonal antibodies (or antigen-binding fragments) that bind to the same antigen may be able to simultaneously bind to the antigen if their binding sites are sufficiently separated in three-dimensional space so that the binding of each does not interfere with the binding of the other. In contrast, two different monoclonal antibodies may have binding regions for the same or overlapping antigens, in which 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 with respect to the same antigen.

[0222] Antibody "binning" experiments are useful for sorting multiple antibodies raised against the same antigen into various "bins" based on their relative cross-blocking activity. Each "bin" therefore represents a distinct binding region of the antigen. Antibodies in the same bin, by definition, cross-block each other. Binning can be tested by standard in vitro binding assays, such as Biacor or Octet®, using standard test conditions, e.g., according to the manufacturer's instructions (e.g., performing binding assays at room temperature, about 20-25°C).

[0223] Aspects of the present disclosure relate to antibodies that compete or cross-compete with any of the specific antibodies or antigen-binding portions thereof provided herein. In some embodiments, an antibody or antigen-binding portion thereof binds to the same epitope as any of the antibodies provided herein, or in the vicinity thereof. In some embodiments, an antibody or antigen-binding portion thereof binds in the vicinity of an epitope if it binds within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibodies provided herein, or antigen-binding portion thereof, binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the epitope bound by any of the antibodies provided herein.

[0224] In another embodiment, the antibody is selected from the group consisting of a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, and a LRRC33-TGFβ1 complex. -8 The equilibrium dissociation constant between the antibody and the protein, K, is less than or equal to M D Provided herein are antibodies or antigen-binding portions thereof that compete or cross-compete with any of the antigens provided herein. In other embodiments, the antibodies compete or cross-compete with any of the antigens provided herein. -12 M~10 -9 K in the M range D In some embodiments, provided herein are anti-TGFβ1 antibodies, or antigen-binding portions thereof, that compete for binding with the antibodies, or antigen-binding portions thereof, described herein. In some embodiments, provided herein are anti-TGFβ1 antibodies, or antigen-binding portions thereof, that bind to the same epitope as the antibodies, or antigen-binding portions thereof, described herein.

[0225] Any of the antibodies provided herein can be characterized using any suitable method. For example, one method is identifying the epitope to which an antigen binds, or "epitope mapping." There are many suitable methods for mapping and characterizing the location of epitopes on proteins, including, for example, analysis of the crystal structure of antibody-antigen complexes, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described in Chapter 11 of Harlow and Lane, "Using Antibodies, a Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In a further example, epitope mapping can be used to determine the sequence to which an antibody binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by three-dimensional interactions of amino acids that do not necessarily contain a single stretch (linear sequence of primary structure). In some embodiments, the epitope is a TGFβ1 epitope that is only available for binding by an antibody, or antigen-binding portion thereof, described herein when TGFβ1 is in a GARP-precursor TGFβ1 complex, an LTBP1-precursor TGFβ1 complex, an LTBP3-precursor TGFβ1 complex, or an LRRC33-precursor TGFβ1 complex. Peptides of various lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used in antibody binding assays. In another example, the epitope bound by an antibody can be determined in a systematic screen using overlapping peptides derived from the target antigen sequence and determining antibody binding. In a gene fragment expression assay, the open reading frame encoding the target antigen is fragmented randomly or by a specific genetic construct, and the reactivity of the expressed antigen fragment with the antibody being tested is determined. Gene fragments can be generated, for example, by PCR, followed by in vitro transcription and translation into protein in the presence of radioactive amino acids.Binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, defined libraries of overlapping peptide fragments can be tested for binding to test antibodies in simple binding assays. In additional examples, mutagenesis of antigen-binding domains, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues necessary, sufficient, and / or essential for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of the GARP-precursor TGFβ1 complex, the LTBP1-precursor TGFβ1 complex, the LTBP3-precursor TGFβ1 complex, and / or the precursor LRRC33-TGFβ1 complex are replaced (swapped) with sequences from a closely related but antigenically distinct protein, such as another member of the TGFβ protein family (e.g., GDF11).

[0226] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether one antibody binds to the same epitope as another antibody. Competition assays are well known to those skilled in the art.

[0227] In some embodiments, the present invention includes antibodies (e.g., immunoglobulins, antigen-binding fragments, etc.) that cross-block (cross-compete) with any one of Category 1, Category 2, Category 3, Category 4, and / or Category 5 antibodies. Thus, in some embodiments, a pharmaceutical composition may be made by a process comprising selecting an antibody or antigen-binding fragment thereof that cross-competes with a Category 1 antibody and formulating the antibody into a pharmaceutical composition.

[0228] In some embodiments, the pharmaceutical composition may be made by a process comprising selecting an antibody or antigen-binding fragment thereof that cross-competes with a Category 2 antibody and formulating the antibody into a pharmaceutical composition.

[0229] In some embodiments, the pharmaceutical composition may be made by a process comprising selecting an antibody or antigen-binding fragment thereof that cross-competes with a Category 3 antibody, and formulating the antibody into a pharmaceutical composition.

[0230] In some embodiments, the pharmaceutical composition may be made by a process comprising selecting an antibody or antigen-binding fragment thereof that cross-competes with a Category 4 antibody, and formulating the antibody into a pharmaceutical composition.

[0231] In some embodiments, the pharmaceutical composition may be made by a process comprising selecting an antibody or antigen-binding fragment thereof that cross-competes with a Category 5 antibody, and formulating the antibody into a pharmaceutical composition.

[0232] In some embodiments, the pharmaceutical composition may be made by a process comprising selecting an antibody or antigen-binding fragment thereof 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 antibody into a pharmaceutical composition.

[0233] Preferably, the antibody selected by the process is a high-affinity binder, characterized in that the antibody or antigen-binding fragment can bind to each of human LLCs (e.g., hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1) with a KD of ≦5 nM, as measured by solution equilibrium titration. In some embodiments, the antibody meets the criteria of one or more of Category 1, Category 2, Category 3, Category 4, and Category 5. Such cross-competing antibodies may be used in accordance with the present disclosure to treat TGFβ1-related indications in a subject.

[0234] Various antibody modifications and variations Non-limiting variations, modifications, and features of any of the antibodies or antigen-binding fragments thereof encompassed by the present disclosure are briefly described below. Embodiments of related analytical methods are also provided.

[0235] Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically consists of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (in certain embodiments, about 25 kDa) and one full-length "heavy" chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically contains a variable region of about 100-110 or more amino acids, which is typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region, which may be responsible for effector function. Human antibody light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, which defines the antibody's isotype. 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). Within full-length light and heavy chains, the variable and constant regions are typically joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids (see, e.g., Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, New York (1989)) (incorporated by reference in its entirety)). The variable regions of each light / heavy chain pair typically form the antigen-binding site.

[0236] Variable regions typically exhibit the same overall structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called CDRs, or complementarity-determining regions. The CDRs from the two chains of each pair are typically aligned by the framework regions, enabling binding to a specific epitope. From the N-terminus to the C-terminus, both light and heavy chain variable regions typically contain the following domains: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each domain typically follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), or Chothia and Lesk (1987), J. Mol. Biol., 196:901-917; Chothia et al. (1989), Nature, 342:878-883. Light chain CDRs may also be referred to as CDR-L1, CDR-L2, and CDR-L3, and heavy chain CDRs may also be referred to as CDR-H1, CDR-H2, and CDR-H3. In some embodiments, an antibody may comprise a deletion of a small number of amino acids from the carboxy terminus of the heavy chain. In some embodiments, an antibody comprises a heavy chain with a deletion of 1 to 5 amino acids at the carboxy terminus of the heavy chain. In certain embodiments, definitive delineation of CDRs and identification of the residues comprising the antibody binding site are achieved by analysis of the structure of the antibody and / or the structure of the antibody-ligand complex. In certain 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 approximate CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the definition described in Lu et al. (see above), and the contact definition.

[0237] An "affinity matured" antibody is an antibody with one or more alterations in one or more of its CDRs that result in an improvement in the affinity of the antibody for antigen compared to a parent antibody that does not possess those alterations. Exemplary affinity matured antibodies have nanomolar or even picomolar affinities (e.g., about 10 -9 M~10 -12 K in the M range D ). Affinity matured antibodies can be produced by procedures known in the art. Marks et al. (1992) Bio / Technology 10:779-783 describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al. (1994), Proc. Nat. Acad. Sci. USA, 91:3809-3813; Schier et al. (1995), Gene, 169:147-155; Yelton et al. (1995), J. Immunol., 155:1994-2004; Jackson et al. (1995), J. Immunol., 154(7):3310-9; and Hawkins et al. (1992), J. Mol. Biol., 226:889-896; selective mutagenesis using activity-enhancing amino acid residues, selective mutations at contact or hypermutation positions, is described in U.S. Pat. No. 6,914,128. Typically, a parent antibody and its affinity matured progeny (e.g., derivative) retain the same binding region within the antigen, but specific interactions at the molecular level may be altered due to altered amino acid residues introduced by affinity maturation.

[0238] The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species, but in which the sequences of one or more of the VH and / or VL CDR regions have been replaced with CDR sequences from another species, such as an antibody having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[0239] 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, for example, an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0240] As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of a variable region excluding the CDRs. Because the precise definition of a CDR sequence is determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 in the light chain and CDR-H1, -H2, and -H3 in the heavy chain) also divide the framework regions on the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, with CDR1 located between FR1 and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR4. Unless a specific subregion is designated as FR1, FR2, FR3, or FR4, the framework region referred to by others refers to the combined FRs in the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FRs refers to two or more of the four subregions that make up the framework region.

[0241] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 1 (H-FR1) having the following amino acid sequence, optionally with one, two, or three amino acid changes: EVQLVESGGGLVQPG G SLRLSC A ASG (SEQ ID NO: 174). For example, the Gly residue at position 16 may be replaced with Arg (R) and / or the Ala residue at position 23 may be replaced with Thr (T).

[0242] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 2 (H-FR2) having the following amino acid sequence: WVRQAPGKGLEWVS (SEQ ID NO: 175), optionally with 1, 2, or 3 amino acid changes.

[0243] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 3 (H-FR3) having the following amino acid sequence: RFTISRDNAKNSLYLQMNSLRAEDTAVYYC (SEQ ID NO: 176), optionally with one, two, or three amino acid changes. For example, the Ser residue at position 12 may be replaced with a Thr (T).

[0244] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region 4 (H-FR4) having the following amino acid sequence: WGQGTLVTVSS (SEQ ID NO: 177), optionally with 1, 2, or 3 amino acid changes.

[0245] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 1 (L-FR1) having the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 178), optionally with 1, 2, or 3 amino acid changes.

[0246] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 2 (L-FR2) having the following amino acid sequence: WYQQKPGKAPKLLIY (SEQ ID NO: 179), optionally with 1, 2, or 3 amino acid changes.

[0247] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 3 (L-FR3) having the following amino acid sequence: GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 180), optionally with 1, 2, or 3 amino acid changes.

[0248] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain framework region 4 (L-FR4) having the following amino acid sequence: FGGGTKVEIK (SEQ ID NO: 181), optionally with 1, 2, or 3 amino acid changes.

[0249] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain immunoglobulin constant domain of a human IgM constant domain, a human IgG constant domain, a human IgG1 constant domain, a human IgG2 constant domain, a human IgG2A constant domain, a human IgG2B constant domain, a human IgG2 constant domain, a human IgG3 constant domain, a human IgG3 constant domain, a human IgG4 constant domain, a human IgA constant domain, a human IgA1 constant domain, a human IgA2 constant domain, a human IgD constant domain, or a human IgE constant domain. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain immunoglobulin constant domain of a human IgG1 constant domain or a human IgG4 constant domain. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain immunoglobulin constant domain of a human IgG4 constant domain. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain immunoglobulin constant domain of a human IgG4 constant domain with a Ser to Pro backbone substitution that creates an IgG1-like hinge and allows interchain disulfide bond formation.

[0250] In some embodiments, the antibody or antigen-binding portion thereof further comprises a light chain immunoglobulin constant domain comprising a human Ig lambda constant domain or a human Ig kappa constant domain.

[0251] In some embodiments, the antibody is an IgG having four polypeptide chains: two heavy chains and two light chains.

[0252] In some embodiments, the antibody is a humanized antibody, a diabody, or a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody comprises a framework having a human germline amino acid sequence.

[0253] In some embodiments, the antigen-binding portion is a Fab fragment, a F(ab')2 fragment, an scFab fragment, or an scFv fragment.

[0254] As used herein, the term "germline antibody gene" or "gene fragment" refers to an immunoglobulin sequence encoded by a non-lymphoid cell that has not undergone the maturation process that results in gene rearrangement and mutation for expression of a specific immunoglobulin (see, e.g., Shapiro et al. (2002) Crit. Rev. Immunol., 22(3):183-200; Marchalonis et al. (2001) Adv. Exp. Med. Biol., 484:13-30). One of the advantages provided by various embodiments of the present disclosure stems from the recognition that germline antibody genes are more likely than mature antibody genes to preserve essential amino acid sequence structures characteristic of individuals within a species, and therefore are less likely to be recognized as foreign when used therapeutically in that species.

[0255] As used herein, the term "neutralizing" refers to the biological activity of an antigen (e.g., a target protein) that is counteracted when a binding protein specifically binds to the antigen. In one embodiment, a neutralizing binding protein binds to an antigen / target, e.g., 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 released from a latent complex, thereby preventing its ability to bind to its receptor and induce downstream signaling. In some embodiments, the mature growth factor is TGFβ1 or TGFβ3.

[0256] As used herein, the term "binding protein" includes any polypeptide that specifically binds to an antigen (e.g., TGFβ1), including, but not limited to, an antibody or antigen-binding portion thereof, DVD-Ig™, TVD-Ig, RAb-Ig, bispecific antibodies, and dual specific antibodies.

[0257] The term "monoclonal antibody" or "mAb," when used in the context of a composition comprising it, can refer to an antibody preparation obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method.

[0258] As used herein, the term "recombinant human antibody" refers to any human antibody that is prepared, expressed, generated, or isolated by recombinant means, such as an antibody expressed using a recombinant expression vector transfected into a host cell (described further in Section IIC below), an antibody isolated from a recombinant combinatorial human antibody library (see, for example, Hoogenboom, H.R. (1997) TIB Tech. 15:62-70; Azzazy, H. and Highsmith, W.E. (2002) Clin. Biochem. 35:425-445; Gavilondo, J.V. and Larrick, J.M. (2003) Clin. Biochem. 35:425-445, which are incorporated herein by reference). "Human immunoglobulin genes" is intended to include antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (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 means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies have been subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when animals transgenic for human Ig sequences are used) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0259] As used herein, the term "dual variable domain immunoglobulin" or "DVD-IgTM" refers to a binding protein comprising a paired heavy chain DVD polypeptide and a paired light chain DVD polypeptide, each paired heavy chain providing two antigen-binding sites. Each binding site comprises a total of six CDRs per antigen-binding site. DVD-IgTM typically has two arms connected to each other at least in part by dimerization of the CH3 domain, and each arm of the DVD is bispecific, providing the immunoglobulin with four binding sites. DVD-IgTM are provided in U.S. Patent Publication Nos. 2010 / 0260668 and 2009 / 0304693, each of which, including its sequence listing, is incorporated herein by reference.

[0260] As used herein, the term "triple variable domain immunoglobulin" or "TVD-Ig" refers to a binding protein comprising paired heavy and light chain TVD binding protein polypeptides, each paired heavy and light chain providing three antigen-binding sites. Each binding site comprises a total of six CDRs per antigen-binding site. The TVD binding protein may have two arms connected to each other at least in part by dimerization of the CH3 domains, and each arm of the TVD binding protein is trispecific, providing the binding protein with six binding sites.

[0261] As used herein, a "receptor antibody immunoglobulin" or "RAb-Ig" is a binding protein comprising a heavy chain RAb polypeptide and a light chain RAb polypeptide, which together form a total of three antigen-binding sites. One antigen-binding site is formed by pairing of the heavy and light chain antibody variable domains present in each of the heavy and light chain RAb polypeptides to form a single binding site, with a total of six CDRs providing the first antigen-binding site. Each of the heavy and light chain RAb polypeptides contains a receptor sequence that independently binds to a ligand to provide a second and third "antigen"-binding site. RAb-Ig typically has two arms connected to each other at least in part by dimerization of the CH3 domain, and each arm of RAb-Ig is trispecific, providing the 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, are incorporated herein by reference).

[0262] The term "bispecific antibody" as used herein is to be distinguished from "bispecific semi-Ig binding proteins" or "bispecific (semi-Ig) binding proteins" and can be produced by quadroma technology (see Milstein, C. and Cuello, AC (1983), Nature, 305(5934):537-540), by chemical conjugation of two different monoclonal antibodies (see Staerz, UD et al. (1985), Nature, 314(6012):628-631), or by introducing mutations in the Fc region that do not inhibit CH3-CH3 dimerization (see Holliger, P. et al. (1993), Proc. Natl. Acad. Sci. USA 90(14):6444-6448), refers to full-length antibodies generated by knob-into-hole or similar techniques, resulting in multiple different immunoglobulin species, only one of which is a functional bispecific antibody. According to molecular function, a bispecific antibody binds to one antigen (or epitope) on one of its two binding arms (a pair of HC / LC) and to a different antigen (or epitope) on its second arm (a different pair of HC / LC). By this definition, a bispecific antibody has two distinct antigen-binding arms (both in specificity and CDR sequence) and is monovalent for each antigen to which it binds.

[0263] As used herein, the term "bispecific antibody" is distinct from a bispecific half-Ig binding protein or a bispecific binding protein and refers to a full-length antibody that can bind to two different antigens (or epitopes) in each of its two binding arms (a pair of HC / LC) (see PCT Publication WO 02 / 02773). Thus, a bispecific binding protein has two identical antigen-binding arms with the same specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

[0264] As used herein, the term "K on " is intended to refer to the on rate constant for a binding protein (e.g., an antibody) to associate with an antigen to form, for example, an antibody / antigen complex as known in the art. "K on " is also known by the term "association rate constant" or "ka," which are used interchangeably herein. This value, which indicates the rate of binding of an antibody to its target antigen or the rate of complex formation between an antibody and an antigen, is also given by the equation: antibody ("Ab") + antigen ("Ag") → Ab - Ag.

[0265] As used herein, the term "Koff" is intended to refer to the off-rate constant for a binding protein (e.g., an antibody) to dissociate from an antibody / antigen complex, e.g., as known in the art. "Koff" is also known by the term "dissociation rate constant" or "kd," which are used interchangeably herein. This value indicates the rate of dissociation of an antibody from its target antigen, or the separation of the b-Ag complex into free antibody and antigen over time, as given by the equation: Ab+Ag←Ab-Ag.

[0266] The terms "equilibrium dissociation constant" or "K" are used interchangeably herein. D " refers to a value obtained in a titration measurement at equilibrium or by dividing the dissociation rate constant (koff) by the association rate constant (kon). Association rate constants, dissociation rate constants, and equilibrium dissociation constants are used to describe the binding affinity between a binding protein, e.g., an antibody, and an antigen. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques offers high sensitivity and the ability to test samples at equilibrium in physiological buffers. Other experimental techniques and instruments, such as the BIAcore® (Biomolecular Interaction Analysis) assay, can be used (e.g., instruments available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, the KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Idaho), can also be used.

[0267] As used herein, the terms "crystal" and "crystallized" refer to a binding protein (e.g., an antibody) or antigen-binding portion thereof that exists in crystalline form. A crystal is a form of the solid state of matter and is distinct from other forms, such as amorphous solid states or liquid crystalline states. Crystals consist of regular, repeating three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen / antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well understood in the art. The basic units, or building blocks, that are repeated in a crystal are called asymmetric units. The repetition of asymmetric units in an arrangement that follows a predetermined, well-defined crystallographic symmetry provides the "unit cell" of the crystal. Regular translation of the lattice units in all three dimensions provides a crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ed., pp. 201-16, Oxford University Press, New York, NY (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, e.g., 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), RADAAAAAGGPGS (SEQ ID NO:66), RADAAAA(G4S)4 (SEQ ID NO:67), SAKTTPKLEEGEFSEARV (SEQ ID NO:68), ADAAP (SEQ ID NO:69), ADAAP (SEQ ID NO:70), ADAAP (SEQ ID NO:71), ADAAP (SEQ ID NO:72), ADAAP (SEQ ID NO:73), ADAAP (SEQ ID NO:74), ADAAP (SEQ ID NO:75), ADAAP (SEQ ID NO:76), ADAAP (SEQ ID NO:77), ADAAP (SEQ ID NO:78), ADAAP (SEQ ID NO:79), ADAAP (SEQ ID NO:80), ADAAP (SEQ ID NO:81), ADAAP (SEQ ID NO:82), ADAAP (SEQ ID NO:83), ADAAP (SEQ ID NO:84), ADAAP (SEQ ID NO:85), ADAAP (SEQ ID NO:86), ADAAP (SEQ ID NO:87), ADAAP (SEQ ID NO:88), ADAAP (SEQ ID NO:89), ADAAP (SEQ ID NO:90), ADAAP (SEQ ID NO:91), ADAAP (S Sequence number 69), ADAATVSIFPP (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), GGGGSGGGSGGGGS (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).

[0268] "Label" and "detectable label" or "detectable moiety" refer to a moiety attached to a specific binding partner, such as an antibody or analyte, to render the reaction between members of a specific binding pair, such as, for example, an antibody and an analyte, detectable; a specific binding partner, e.g., an antibody or analyte, so labeled is said to be "detectably labeled." Thus, as used herein, the term "labeled binding protein" refers to a protein having an incorporated label that provides identification of the binding protein. In one embodiment, the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, e.g., the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to the polypeptide that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides 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 153Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, and lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, and alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, and epitope tags), and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include light-producing moieties, e.g., acridinium compounds, and fluorescence-producing moieties, e.g., fluorescein. Other labels are described herein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with yet another moiety. The use of "detectably labeled" is intended to encompass the latter type of detectable label.

[0269] In some embodiments, the binding affinity of an antibody, or antigen-binding portion thereof, to an antigen (e.g., a protein complex), such as a presentation molecule-precursor TGFβ1 complex, is determined using an Octet assay. In some embodiments, an Octet assay is an assay that determines one or more kinetic parameters indicative of binding between an antibody and an antigen. In some embodiments, the binding affinity of an antibody, or antigen-binding portion thereof, to a presentation molecule-precursor TGFβ1 complex is determined using the Octet® system (ForteBio, Menlo Park, CA). For example, the binding affinity of an antibody may be determined using the forteBio Octet QKe dip and read label-free assay system, which utilizes biolayer interferometry. In some embodiments, the antigen is immobilized on a biosensor (e.g., a streptavidin-coated biosensor), and the antibody and complex (e.g., a biotin-labeled presentation molecule-precursor TGFβ1 complex) are presented in a solution at a high concentration (50 μg / mL) to measure the binding interaction. In some embodiments, the binding affinity of an antibody, or antigen-binding portion thereof, to a presentation molecule-precursor TGFβ1 complex is determined using the protocol outlined herein.

[0270] Characterization of a novel high-affinity, context-independent antibody to precursor TGF-.BETA.1 Binding Profile The antibodies disclosed herein have enhanced binding activity. These include a class of high-affinity, context-independent antibodies that can selectively inhibit TGFβ1 activation. It should be noted that the term "context-independent" as used herein has a higher degree of rigor compared to its previous, more common usage. According to the present disclosure, this term confers a level of uniformity (i.e., unbiasedness) in the relative affinities that antibodies can exert toward various antigen complexes. Thus, the context-independent antibodies of the present invention target multiple TGFβ1 precursor complexes (e.g., presentation molecule-precursor TGFβ1 complexes) and have K values ​​of less than 10 nM, preferably less than 5 nM, more preferably less than 1 nM, and even more preferably less than 100 pM, with equivalent affinities to each such complex (i.e., no more than a three-fold difference in relative affinity across complexes), as measured, for example, by MSD-SET. D As presented below, many antibodies encompassed by the present invention can bind with K values ​​in the subnanomolar range. D It has a value.

[0271] Thus, the antibody can specifically bind to each of the human presentation molecule-precursor TGFβ1 complexes (sometimes referred to as "large latent complexes," which are ternary complexes consisting of a precursor TGFβ1 dimer coupled to a single presentation molecule), i.e., LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1. Typically, a recombinantly produced and purified protein complex is used as an antigen (e.g., antigen complex) to assess or confirm the ability of the antibody to bind to the antigen complex 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 (e.g., Octet®) and solution equilibrium titration-based assays (e.g., MSD-SET).

[0272] BLI-based binding assays have been widely used in the art to measure the affinity and kinetics of antibodies for antigens. This is a label-free technique that analyzes biomolecular interactions based on optical interference. One of the proteins, e.g., the antibody to be tested, can be immobilized on the tip of a biosensor. When the other protein in solution, e.g., an antigen, binds to the immobilized antibody, a shift in the interference pattern occurs, which can be measured in real time. This allows monitoring of binding specificity, association and dissociation rates, and concentration dependence. Therefore, BLI is a kinetic measure that reveals the dynamics of the system. Due to its ease of use and rapid results, BLI-based assays, such as the Octet® system (available from ForteBio / Molecular Devices, Fremont, CA), are particularly advantageous as an initial screening method to identify and separate pools of "binders" from pools of "non-binders" or "weak binders" in screening processes.

[0273] BLI-based binding assays revealed that the novel antibodies, when measured by Octet®, can be characterized as "context-balanced / context-independent" antibodies. As seen in Table 8, which summarizes the BLI-based binding profiles of non-limiting example antibodies, these antibodies exhibited relatively uniform K values ​​in the subnanomolar range across the four target complexes, with relatively low matrix-to-cell differences (no more than 5-fold bias) (see column (H)). This can be contrasted with the previously identified antibody Ab3, which served as a reference antibody, which exhibited significantly higher (27+ fold bias) relative affinity for matrix-associated complexes than for cell-associated complexes.

[0274] Table 8 below provides non-limiting examples of high affinity, context-independent pro-TGFβ1 antibodies encompassed by the present invention. The table provides representative results from an in vitro binding assay measured by Octet®. Similar results are obtained by SPR-based techniques (Biacore System).

[0275] Column (A) of the table lists monoclonal antibodies with distinct amino acid sequences. Ab3 (shown in bold) is a previously identified reference antibody (disclosed in PCT / US2018 / 012601) that has been shown to be potent in cell-based assays, effective in various animal models, and has a clean toxicology profile. Columns (B), (D), (E), and (F) list monoclonal antibodies with distinct amino acid sequences. D The affinity of each of the listed antibodies, as measured by K , is provided. For each antibody, column (B) shows the affinity for the recombinant human LTBP1-precursor TGFβ1 complex, column (C) shows the affinity for the recombinant human LTBP3-precursor TGFβ1 complex, column (E) shows the affinity for the recombinant human GARP-precursor TGFβ1 complex, and column (F) shows the affinity for the recombinant human LRRC33-precursor TGFβ1 complex. The average K for (B) and (C) is provided. DThe values ​​are shown in the corresponding (D) column, which represents the overall affinity of the antibody for the ECM or matrix-associated precursor-TGFβ1 complex. Similarly, the average K values ​​in (E) and (F) D The values ​​are shown in the corresponding column (G) and represent the overall affinity of the antibody for the cell surface or cell-associated precursor TGFβ1 complex. Finally, the average K values ​​from columns (D) and (G) are D The relative ratio between the values ​​is expressed as "fold bias" in column (H). Thus, when comparing the binding preferences of antibodies to matrix-associated complexes and cell surface complexes, the higher the number in column (H), the greater the bias present for that particular antibody. This is one way of quantitatively expressing and comparing the inherent bias of an antibody toward its target complex. Such analysis can be useful in guiding the selection process of candidate antibodies for specific therapeutic uses. Table 8. Non-limiting examples of context-independent TGFβ1 antibodies and their K values ​​measured by BLI D value JPEG2024099673000015.jpg59166

[0276] The present invention provides a class of high-affinity, context-independent antibodies, each of which can bind with equivalent affinity to each of the four known presentation molecule-precursor TGFβ1 complexes, i.e., LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1. In some embodiments, the antibodies bind with equivalent or higher affinity to each of the presentation molecule-precursor TGFβ1 complexes compared to the previously described reference antibody Ab3. According to the present invention, such antibodies bind with an affinity (K) of ≦5 nM to each of the aforementioned complexes, as measured by suitable in vitro binding assays, such as biolayer interferometry and surface plasmon resonance. DIn some embodiments, the antibody or fragment specifically binds to the human LTBP1-precursor TGFβ1 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-precursor TGFβ1 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-precursor TGFβ1 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-precursor TGFβ1 complex with an affinity of ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, or ≦0.5 nM.

[0277] In preferred embodiments, such antibodies are human and murine cross-reactive. Thus, in some embodiments, the antibody or fragment binds to the murine LTBP1-precursor TGFβ1 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 murine LTBP3-precursor TGFβ1 complex with an affinity of ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, or ≦0.5 nM. In some embodiments, the antibody or fragment binds to the murine GARP-precursor TGFβ1 complex with an affinity of ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, or ≦0.5 nM. In some embodiments, the antibody or fragment binds to the murine LRRC33-precursor TGFβ1 complex with an affinity of ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, or ≦0.5 nM.

[0278] As shown, the precursor TGFβ1 antibodies of the present disclosure have particularly high affinity for matrix-associated precursor TGFβ1 complexes. In some embodiments, the average K D The value is ≦1 nM or ≦0.5 nM.

[0279] As shown, the precursor TGFβ1 antibodies of the present disclosure have high affinity for cell-associated precursor TGFβ1 complexes. In some embodiments, the average K D The value is ≦2 nM or ≦1 nM.

[0280] The high affinity precursor TGFβ1 antibodies of the present disclosure are characterized by their uniform (unbiased) affinity for all four antigen complexes (e.g., compared to Ab3). Of the four known presentation molecule-precursor TGFβ1 complexes described herein, none of the antigen complexes exhibited K D In other words, a more uniform binding activity is achieved by the present disclosure compared to the precursor TGFβ1 antibodies (including Ab3) previously described, in that each of such antibodies exhibits equivalent affinity across the four antigen complexes. In some embodiments, the antibodies or fragments are designed such that the difference (or range) in affinity of the antibodies or fragments across the four precursor TGFβ1 antigen complexes is greater than or equal to the lowest and highest K D In some embodiments, the relative difference (or range) in affinity is 3-fold or less.

[0281] The concept of "uniformity" or lack of bias is further illustrated in Table 8. The average K between the two matrix-associated and cell-associated complexes D The K values ​​were then calculated (see columns (D) and (G)). D The values ​​can be used to determine whether there is a bias in binding activity between matrix-associated complexes versus cell surface (e.g., immune cell)-associated complexes. As illustrated in Table 8, the bias is the average K D Compared to the previously described antibody Ab3, the high affinity, context-independent pro-TGFβ1 antibodies encompassed by the present disclosure often have a 3-fold or less average K difference between matrix and cell-associated complexes. D It is remarkably unbiased in that it shows the difference in values ​​(compare this to the 25+ multiple bias in Ab3).

[0282] Thus, provided are classes of context-independent monoclonal antibodies or fragments capable of binding with equivalent affinity to each of the following presenting molecule-precursor TGFβ1 complexes: LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1, each with an affinity of ≦1 nM as measured by biolayer interferometry or surface plasmon resonance. Such antibodies specifically bind to each of the aforementioned complexes with an affinity of ≦5 nM as measured by biolayer interferometry or surface plasmon resonance, wherein the monoclonal antibody or fragment exhibits an affinity bias of no more than 3-fold for any one of the above complexes compared to the other, and the monoclonal antibody or fragment inhibits the release of mature TGFβ1 growth factor from each of the precursor TGFβ1 complexes, but not from the precursor TGFβ2 or precursor TGFβ3 complexes.

[0283] While the kinetics of binding profiles (e.g., "on" and "off" rates) that can be obtained from BLI-based assays provide useful information, applicants of the present disclosure contemplated that, based on the mechanism of action of the activation inhibitors disclosed herein, i.e., antibodies that work by binding to a tethered (e.g., tissue-localized) inactive (e.g., latent) target and thereby preventing it from being activated, binding properties measured at equilibrium may more accurately reflect their in vivo behavior and potency. To put this in perspective, as an example, fast "on" rates ("K") that would be reflected in binding measurements obtained by BLI may be more accurately reflected in the in vivo behavior and potency of the antibodies. オンAntibodies with a specific activity (e.g., a specific marker) may provide relevant parameters for evaluating neutralizing antibodies (e.g., antibodies that must directly target and rapidly sequester the active soluble growth factor itself in order for them to function as effective inhibitors). However, the same may not necessarily apply to antibodies that function as activation inhibitors, such as those disclosed herein. As described, the mechanism of action of the novel TGFβ1 inhibitors of the present invention is through inhibition of the activation step, which is achieved by targeting the tissue / cell-tethered latent complex as opposed to sequestration of the soluble post-activation growth factor. This is because TGFβ1 activation inhibitors target the inactive precursor localized in the respective tissue (e.g., within the ECM, on the surface of immune cells, etc.), thereby preemptively preventing the release of the mature growth factor from the complex. This mechanism of action is believed to allow the inhibitors to achieve target saturation (e.g., equilibrium) in vivo without the need to rapidly compete for endogenous receptors for transient growth factor molecules, as required by conventional neutralizing inhibitors.

[0284] Taking this difference in mechanism of action into account, further evaluation of the binding characteristics was carried out by using an alternative in vitro binding assay format that allows for the determination of affinity at equilibrium.

[0285] In light of this, it is contemplated that assays measuring the equilibrium binding affinity of such antibodies may more accurately represent the target association mode in vivo. Accordingly, MSD-SET-based binding assays (or other suitable assays) may be performed, as exemplified in Table 9 below.

[0286] Solution equilibrium titration ("SET") is an assay that can measure the binding between two molecules (such as an antigen and an antibody that binds to the antigen) in solution at equilibrium. For example, mesoscale discovery ("MSD")-based SET, or MSD-SET, is a useful format for determining the equilibrium dissociation constant of particularly high-affinity protein-protein interactions (see, e.g., Ducata et al., (2015), J. Biomolecular Screening, 20(10):1256-1267). SET-based assays are particularly useful for determining K values ​​of antibodies with subnanomolar (e.g., picomolar) affinities. Table 9. Non-limiting examples of high affinity context-independent TGFβ1 antibodies (hIgG4) and K measured by MSD-SET D Values ​​("h" indicates human complex) JPEG2024099673000016.jpg56166

[0287] 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, published as WO2017 / 156500, and Ab3 was described in PCT / US2018 / 012601, published as WO2018 / 129329.

[0288] As can be seen from the affinity data provided in Table 9, the binding activity of the novel antibodies according to the present disclosure is significantly higher than that of previously identified reference antibodies. Furthermore, the novel TGFβ1 antibodies are "context-independent" in that they bind with equivalent affinity to each of the human LLC complexes (e.g., having a KD in the subnanomolar range, e.g., <1 nM). The high-affinity, context-independent binding profile suggests that these antibodies may be advantageous for use in treating TGFβ1-related indications involving dysregulation of both ECM-associated and immune components, such as cancer.

[0289] Solution equilibrium titration-based binding assays can use a protein complex containing one of the presenting molecules listed above as an antigen (presenting molecule-TGFβ1 complex, or LLC). The test antibody forms an antigen-antibody complex in solution. The antigen-antibody reaction mixture is incubated to achieve equilibrium. The amount of antigen-antibody complex present in the assay reaction can be measured by appropriate means known in the art. Compared to BLI-based assays, SET-based assays are less affected by the on / off rate of the antigen-antibody complex, allowing for sensitive detection of very high affinity interactions. As shown in Table 9, in the present disclosure, preferred high-affinity inhibitors of TGFβ1, as determined by SET-based assays, exhibit affinities in the subnanomolar (e.g., picomolar) range across all large latent complexes tested.

[0290] Thus, provided is a class of context-independent monoclonal antibodies or fragments that can bind with equivalent affinity to each of the human presenting molecule-precursor TGFβ1 complexes, i.e., hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1, each of which has a KD of ≦1 nM or less, as measured by a solution equilibrium titration assay such as MSD-SET. Such antibodies specifically bind to each of the foregoing complexes with a KD of ≦1 nM, as measured by MSD-SET, wherein the monoclonal antibody or fragment inhibits release of mature TGFβ1 growth factor from each of the precursor TGFβ1 complexes, but not from the precursor TGFβ2 or precursor TGFβ3 complexes. In preferred embodiments, such antibodies or fragments have a K of 500 pM or less (i.e., ≦500 pM), 250 pM or less (i.e., ≦250 pM), or 200 pM or less (i.e., ≦200 pM). D Even more preferably, such antibodies or fragments bind to each of the aforementioned complexes with a K of 100 pM or less (i.e., ≦100 pM). DIn some embodiments, the antibody or fragment does not bind to free TGFβ1 growth factor that is not associated with the prodomain complex. This can be tested or confirmed by a suitable in vitro binding assay known in the art, such as biolayer interferometry.

[0291] In further preferred embodiments, such antibodies or fragments are also cross-reactive with their murine (e.g., rat and / or mouse) and / or non-human primate (e.g., cynomolgus monkey) counterparts. By way of example only, as illustrated in Table 10 and Example 9 below, Ab6 can bind with high affinity to each of the large latent complexes of multiple species, including human, murine, rat, and cynomolgus monkey. Table 10. Non-limiting examples of high affinity context-independent TGFβ1 antibodies with cross-species reactivity as measured by MSD-SET ("h" indicates human, "m" indicates murine) JPEG2024099673000017.jpg13166

[0292] efficacy The antibodies disclosed herein can be broadly characterized as "functional antibodies" for their ability to inhibit TGFβ1 signaling. As used herein, a "functional antibody" confers one or more biological activities by virtue of 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 that can modulate the activity / function of a target molecule (i.e., an antigen). Such modulating antibodies include inhibitory (or inhibitory) antibodies and activating antibodies. This disclosure focuses on antibodies that can inhibit biological processes mediated by TGFβ1 signaling associated with multiple contexts of TGFβ1. Inhibitors used to practice the present invention, such as the antibodies described herein, are intended to be selective for TGFβ1 and do not target or interfere with TGFβ2 and TGFβ3 when administered at therapeutically effective doses (doses that achieve sufficient efficacy within acceptable toxicity levels). The novel antibodies disclosed herein have enhanced inhibitory activity (potency) compared to previously identified TGFβ1 activation inhibitors.

[0293] In some embodiments, the potency of an inhibitory antibody can be measured in a suitable cell-based assay, such as the CAGA reporter cell assay described herein. Generally, cultured cells, such as heterologous cells and primary cells, can be used to perform cell-based potency assays. Cells expressing endogenous TGFβ1 and / or presentation molecules of interest, such as LTBP1, LTBP3, GARP, and LRRC33, can be used. Alternatively, exogenous nucleic acids encoding proteins of interest, such as TGFβ1 and / or presentation molecules of interest, such as LTBP1, LTBP3, GARP, and LRRC33, can be introduced into such cells for expression, for example, by transfection (e.g., stable or transient transfection) or infection based on a viral vector. In some embodiments, LN229 cells are used in such assays. Cells expressing TGFβ1 and a presentation molecule of interest (e.g., LTBP1, LTBP3, GARP, or LRRC33) are grown naturally, and they "present" the large latent complex either on the cell surface (if associated with GARP or LRRC33) or by accumulation within the ECM (if associated with LTBP). Activation of TGFβ1 can be triggered by an integrin expressed on the surface of another cell. The integrin-expressing cell can be the same cell or a different cell type that co-expresses the large latent complex. Reporter cells incorporating a TGFβ-responsive element are added to the assay system. In this way, the degree of TGFβ activation can be measured by detecting a signal from the reporter cell (e.g., a TGFβ-responsive reporter gene, such as luciferase, coupled to a TGFβ-responsive promoter element) upon TGFβ activation. Using such cell-based assay systems, the inhibitory activity of antibodies can be determined by measuring a change (decrease) or difference in reporter signal (e.g., luciferase activity measured by fluorescence readout) in the presence or absence of the test antibody. Such an assay is exemplified in Example 2 herein.

[0294] Thus, in some embodiments, the inhibitory potency (IC) of the novel antibodies of the present disclosure calculated based on a cell-based reporter assay for measuring activation of TGFβ1 (such as the LN229 cell assay described elsewhere herein) is 50 ) may be 5 nM or less as measured against each of the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes. In some embodiments, the antibody is titrated against each of the LLCs to have an IC of 2 nM or less (i.e., ≦2 nM). 50 In a preferred embodiment, the IC of the antibody measured against each of the LLC complexes 50 In some embodiments, the antibody has an IC of less than 1 nM for each of the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes. 50 It has. Table 11. Inhibitory potency (IC) of selected antibodies measured by reporter cell assay 50 ) JPEG2024099673000018.jpg66166

[0295] TGFβ1 activation can be initiated by an integrin-dependent mechanism or a protease-dependent mechanism. The inhibitory activity (e.g., potency) of an antibody according to the present disclosure can be assessed for its ability to block TGFβ1 activation induced by one or both modes of activation. The reporter cell assay described above is designed to measure the ability of an antibody to block or inhibit integrin-dependent activation of TGFβ1. Inhibitory potency can also be assessed to measure the ability of an antibody to block protease-induced activation of TGFβ1. Example 3 of the present disclosure provides a non-limiting example of such an assay. The results are summarized in Figures 5A and 5B. Thus, in some embodiments of the present invention, an isoform-selective inhibitor according to the present disclosure can inhibit integrin-dependent activation of TGFβ1 and protease-dependent activation of TGFβ1. Such inhibitors can be used to treat TGFβ1-related indications characterized by EDM dysregulation involving protease activity. For example, such TGFβ1-related indications may be associated with elevated myofibroblasts, increased stiffness of the ECM, excessive or abnormal collagen deposition, or any combination thereof. Such conditions include, for example, fibrotic disorders and cancer, including solid tumors (such as metastatic carcinomas), or myelofibrosis.

[0296] In some embodiments, efficacy can be evaluated as a measure of efficacy and / or pharmacodynamic effect in suitable in vivo model.For example, if a first antibody is effective at a certain concentration in an in vivo model, and a second antibody is equally effective at a lower concentration than the first antibody in the same in vivo model, then the second antibody can be said to be more potent than the first antibody.Depending on the specific indication of interest, such as cancer model and fibrosis model, any suitable disease model known in the art can be used to evaluate the relative efficacy of TGFβ1 inhibitors.Preferably, multiple doses or concentrations of each test antibody are included in such study.

[0297] Similarly, pharmacodynamic (PD) effects can be measured to determine the relative potency of inhibitory antibodies. Commonly used measures of PD of the TGFβ signaling pathway include, but are not limited to, SMAD2 / 3 phosphorylation and expression of downstream effector genes whose transcription is sensitive to TGFβ activation, such as those with TGFβ-responsive promoter elements (e.g., Smad-binding elements). In some embodiments, antibodies of the present disclosure can completely block disease-induced SMAD2 / 3 phosphorylation in preclinical fibrosis models when administered to animals at a dose of 3 mg / kg or less. In some embodiments, antibodies of the present disclosure can significantly suppress fibrosis-induced expression of a panel of marker genes, including Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, and Lox12, when administered to animals at a dose of 10 mg / kg or less in a UUO model of renal fibrosis.

[0298] Thus, in some embodiments, the process of selecting antibodies or antigen-binding fragments thereof for therapeutic use can involve identifying antibodies or fragments that exhibit sufficient inhibitory potency. For example, the selection process can involve determining the potency (e.g., IC) of one or more test antibodies or fragments thereof. 50 In some embodiments, the method may include performing a cell-based TGFβ1 activation assay to measure the IC of each of the human LLCs and selecting candidate antibodies or fragments thereof that exhibit the desired efficacy. 50 is 5 nM or less. Selected antibodies or fragments may then be used to treat TGFβ1-related indications as described herein.

[0299] bond area In the context of this disclosure, the "binding region" of an antigen provides the structural basis for antibody-antigen interaction. As used herein, "binding region" refers to the region of the interface between an antibody and an antigen, where the antibody or fragment protects the binding region from solvent exposure when bound to the precursor TGFβ1 complex ("antigen") in physiological solution, as determined by a suitable technique such as hydrogen-deuterium exchange mass spectrometry (HDX-MS). Identification of the binding region is useful in gaining insight into antigen-antibody interactions and the mechanism of action of specific antibodies. Identification of additional antibodies with similar or overlapping binding regions can be facilitated by cross-blocking experiments, which allow epitope binning. Optionally, X-ray crystallography can be used to identify the exact amino acid residues of the epitope that mediate antigen-antibody interaction.

[0300] The art is familiar with HDX-MS, a widely used technique for exploring protein conformation or protein-protein interactions in solution. This method relies on the exchange of hydrogens in protein backbone amides with deuterium present in solution. Measuring the rate of hydrogen-deuterium exchange can provide information about protein dynamics and conformation (reviewed in Wei et al., (2014), "Hydrogen / deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications," Drug Disco Today., 19(1):95-102, incorporated by reference). The application of this technique is based on the assumption that when an antibody-antigen complex is formed, the interface between the binding partners may occlude solvent, thereby reducing or preventing the exchange rate due to steric exclusion of the solvent.

[0301] The present disclosure includes antibodies or antigen-binding fragments thereof that bind to human LLC at a region ("binding region") comprising latent Lasso or a portion thereof. Latent Lasso is a protein module within the prodomain. It is contemplated that a number of potent activation inhibitors may bind to this region of the precursor TGFβ1 complex in such a way that antibody binding would "lock in" the growth factor, thereby preventing its release. Interestingly, this is the section of the complex where the butterfly-like, elongated regions of the growth factor (e.g., corresponding to finger-1 and finger-2) interact closely with the cage-like structure of the prodomain.

[0302] As illustrated in Figure 18B, the latent Lasso contains regions labeled 2a and 2b, which are part of the prodomain. Note that immediately adjacent to the latent Lasso, the region labeled 5a corresponds to so-called finger-1 in the growth factor domain, and the region around the other side, labeled 6b, is part of finger-2 in the growth factor domain. On this basis, it is not difficult to envision that antibodies that wrap tightly around these regions could effectively prevent the pre-TGFβ1 complex from dissociating, thereby blocking activation.

[0303] The HDX-MS technique can be used to determine the binding region of precursor TGFβ1. In some embodiments, the portion of precursor TGFβ1 identified as important for binding to the antibody or fragment comprises at least a portion of the prodomain and at least a portion of the growth factor domain. Antibodies or fragments that bind to a first binding region ("Region 1" in FIG. 19A) comprising at least a portion of the latent Lasso are preferred. More preferably, such antibodies or fragments further bind to a second binding region ("Region 2" in FIG. 19A) comprising at least a portion of the growth factor domain at finger-1 of the growth factor domain. Such antibodies or fragments may further bind to a third binding region ("Region 3" in FIG. 19A) comprising at least a portion of finger-2 of the growth factor domain.

[0304] Additional regions within precursor TGFβ1 may also directly or indirectly contribute to the high affinity interaction of these antibodies disclosed herein. Regions that have been deemed important for mediating high affinity binding of the antibody to the precursor TGFβ1 complex (see FIG. 18A) 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 native sequence of human precursor TGFβ1).

[0305] Among the regions that may contribute to antibody-antigen interactions, in some embodiments, high affinity antibodies of the present disclosure may bind to an epitope comprising at least one residue of the amino acid sequence KLRLASPPSQGEVPPGPLPEAVL ("Region 1") (SEQ ID NO: 169).

[0306] In some embodiments, high affinity antibodies of the present disclosure may bind to an epitope comprising at least one residue of the amino acid sequence RKDLGWKWIHEPKGYHANF (“Region 2”) (SEQ ID NO: 165).

[0307] In some embodiments, high affinity antibodies of the present disclosure may bind to an epitope comprising at least one residue of the amino acid sequence VGRKPKVEQL ("region 3") (SEQ ID NO: 168).

[0308] In some embodiments, a high affinity antibody of the present disclosure may 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).

[0309] In some embodiments, a high affinity antibody of the present disclosure may 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).

[0310] In some embodiments, a high affinity antibody of the present disclosure may 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).

[0311] In addition to contributions from regions 1, 2, and / or 3, such epitopes may further comprise at least one amino acid residue from a sequence selected from the group consisting of: 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).

[0312] It is noteworthy that many of the binding regions identified in structural studies using four representative isoform-selective TGFβ1 antibodies have been found to overlap, indicating specific regions within the precursor TGFβ1 complex that may be particularly important for maintaining the latency of the precursor TGFβ1 complex.Therefore, advantageously, antibodies or fragments thereof can be selected at least in part based on their binding regions, including overlapping portions identified across multiple inhibitors described herein.These overlapping portions of the binding regions include, for example, SPPSQGEVPPGPLPEAVL (SEQ ID NO: 201), WKWIHEPKGYHANF (SEQ ID NO: 202), and PGPLPEAVL (SEQ ID NO: 203). Thus, a high-affinity, isoform-selective TGFβ1 inhibitor according to the present disclosure may bind to a precursor TGFβ1 complex (e.g., human LLC) at an epitope comprising one or more amino acid residues of SPPSQGEVPPGPLPEAVL (SEQ ID NO: 201), WKWIHEPKGYHANF (SEQ ID NO: 202), and / or PGPLPEAVL (SEQ ID NO: 203).

[0313] Thus, any of the antibodies or antigen-binding fragments encompassed by the present disclosure, such as the antibodies or fragments of Categories 1-5 disclosed herein, can bind to one or more of the binding regions identified herein. Such antibodies can be used to treat TGFβ1 indications in a subject, as described herein. Thus, selecting an antibody or antigen-binding fragment thereof suitable for therapeutic use according to the present disclosure can include identifying or selecting an antibody or fragment thereof that binds to SPPSQGEVPPGPLPEAVL (SEQ ID NO: 201), WKWIHEPKGYHANF (SEQ ID NO: 202), PGPLPEAVL (SEQ ID NO: 203), or any portion thereof.

[0314] Non-limiting examples of protein domains or motifs of human precursor TGFβ1 that have been previously described (WO2014 / 182676) are provided in Table 12. JPEG2024099673000019.jpg97166

[0315] Safety / Toxicology Conventional pan-inhibitors of TGFβ, which can antagonize multiple isoforms, are known to cause several toxicities, including cardiovascular toxicity (heart lesions, most notably valvular heart disease), which has been reported across multiple species, including dogs and rats. These include hyperplasia in the aortic, right, and left AV valves, inflammation in the aortic, left, and ascending aorta, bleeding in the ascending aorta, aortic, and left AV valves, and connective tissue degeneration in the ascending aorta (see, e.g., Strauber et al., (2014), "Nonclinical safety evaluation of a Transforming Growth Factor β receptor I kinase inhibitor in Fischer 344 rats and beagle dogs," J. Clin. Pract, 4(3):1000196). See also Figure 21A.

[0316] Furthermore, neutralizing antibodies that bind all three TGFβ isoforms have been associated with specific epithelial toxicities observed across multiple species, some of which are summarized below. Table 13. Epithelial toxicity associated with pan-inhibitors of TGFβ JPEG2024099673000020.jpg46166

[0317] Given the earlier recognition by the applicants of the present disclosure (see PCT / US2017 / 021972) that the lack of isoform specificity of conventional TGFβ antagonists may underlie the source of toxicity associated with TGFβ inhibition, the inventors sought to further achieve broad-spectrum TGFβ1 inhibition that would treat a variety of diseases manifesting pleiotropic TGFβ1 dysregulation while maintaining the safety / tolerability profile of isoform-selective inhibitors.

[0318] In clinical settings, therapeutic benefit is achieved only if the minimum effective concentration (MEC) of a drug (e.g., a monoclonal antibody) is less than the drug's minimum toxic concentration (MTC). This has not been achieved with most, if not all, conventional pan-inhibitors of TGFβ, which appear to actually cause dose-limiting toxicity. Applicant's previous work described isoform-selective inhibitors of TGFβ1 that exhibited a significantly improved safety profile compared with conventional pan-inhibitors, such as small molecule receptor antagonists and neutralizing antibodies. WO 2017 / 156500 disclosed an isoform-selective inhibitor of TGFβ1 activation in which no test substance-related toxicity was observed when administered to rats at doses up to 100 mg / kg per week for four weeks, establishing the NOAEL for the antibody at the highest dose tested, i.e., 100 mg / kg. Applicant's subsequent work also demonstrated that antibodies with enhanced function also exhibited equivalent safety profiles. Here, one goal was to identify antibodies with even higher affinity and potency but at least the same or equivalent level of safety.

[0319] Results from a 4-week rat toxicology study are provided in Figures 21B and 21C. Two isoform-selective TGFβ1 inhibitors (Ab3 and Ab6) were tested in separate studies, along with a small molecule ALK5 inhibitor and a monoclonal neutralizing antibody as controls. No test article-related toxicity was noted with either isoform-selective antibody, while the non-selective inhibitor, as expected, caused a variety of adverse events consistent with published studies. Furthermore, Ab6 was shown to be safe (e.g., no observed adverse events) at dose levels as high as 300 mg / kg when dosed once weekly for 4 weeks in cynomolgus monkeys. Ab6 has been shown to be effective at doses as low as 3 mg / kg in several in vivo models, providing an up to 100-fold therapeutic window. Importantly, this demonstrates that high efficacy does not necessarily mean a higher risk of toxicity. Without wishing to be bound by any particular theory, it is contemplated that the highly selective nature of the antibodies disclosed herein likely explains the lack of observed toxicity.

[0320] Thus, in some embodiments, the novel antibodies disclosed herein have a maximum tolerated dose (MTD) of >100 mg / kg when dosed once weekly for at least four weeks. In some embodiments, the novel antibodies disclosed herein have a no observed adverse effect level (NOAEL) of up to 100 mg / kg when dosed once weekly for at least four weeks. Suitable animal models for use in conducting safety / toxicology studies of TGFβ inhibitors and TGFβ1 inhibitors include, but are not limited to, rats, dogs, cynomolgus monkeys, and mice. In preferred embodiments, the minimum effective dose of the antibody based on appropriate preclinical efficacy studies is less than the NOAEL. More preferably, the minimum effective dose of the antibody is no more than about one-third the NOAEL. In particularly preferred embodiments, the minimum effective dose of the antibody is no more than about one-sixth the NOAEL. In some embodiments, the minimum effective dose of the antibody is no more than about one-tenth the NOAEL.

[0321] In some embodiments, the present invention encompasses isoform-selective antibodies capable of inhibiting TGFβ1 signaling that, when administered to a subject, do not cause cardiovascular or known epithelial toxicity at doses effective for treating TGFβ1-related indications. In some embodiments, the antibodies have a minimum effective dose of about 3-10 mg / kg when administered weekly, biweekly, or monthly. Preferably, the antibodies cause no to minimal toxicity at doses that are at least six times the minimum effective dose (e.g., a six-fold therapeutic window). More preferably, the antibodies cause no to minimal toxicity at doses that are at least ten times the minimum effective dose (e.g., a ten-fold therapeutic window). Even more preferably, the antibodies cause no to minimal toxicity at doses that are at least 15 times the minimum effective dose (e.g., a 15-fold therapeutic window).

[0322] Thus, selecting an antibody or antigen-binding fragment thereof for therapeutic use may include selecting an antibody or antigen-binding fragment that meets one or more criteria of Categories 1-5 described herein, conducting in vivo efficacy studies in an appropriate preclinical model to determine the effective amount of the antibody or fragment, conducting in vivo safety / toxicology studies in an appropriate model to determine an amount of the antibody that is safe or toxic (e.g., MTD, NOAEL, or any art-recognized parameter for assessing safety / toxicity), and selecting an antibody or fragment that provides at least a 3-fold therapeutic window (preferably a 6-fold, more preferably a 10-fold, and even more preferably a 15-fold therapeutic window). In preferred embodiments, in vivo efficacy studies are conducted in two or more appropriate preclinical models that mimic the 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 treatments such as CBT, chemotherapy, and radiation therapy. In some embodiments, the preclinical model is selected from MBT-2, Cloudman S91, and EMT6 tumor models.

[0323] The selected antibody or fragment can be used to manufacture pharmaceutical compositions comprising the antibody or fragment. Such pharmaceutical compositions can be used to treat TGFβ1 indications in subjects as described herein. For example, TGFβ1 indications can be proliferative disorders and / or fibrotic disorders.

[0324] Mechanism of action The antibodies of the present invention useful as therapeutic agents are inhibitory antibodies of TGFβ1. Furthermore, the antibodies are activation inhibitors, i.e., they block the activation step of TGFβ1 rather than directly going after an already activated growth factor.

[0325] In a broad sense, the term "inhibitory antibody" refers to an antibody that antagonizes or neutralizes a target function, e.g., growth factor activity. Advantageously, preferred inhibitory antibodies of the present disclosure can inhibit the release of mature growth factors from latent complexes, thereby reducing growth factor signaling. Inhibitory antibodies include antibodies that target any epitope that reduces growth factor release or activity upon association with it. Such epitopes may be located on the prodomain of a TGFβ protein (e.g., TGFβ1), a growth factor, 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, TGFβ1-inhibitory antibodies. In some embodiments, inhibitory antibodies of the present disclosure specifically bind to combinatorial epitopes, i.e., epitopes formed by two or more components / portions of an antigen or antigen complex. For example, combinatorial epitopes may be formed by contributions from multiple portions of a single protein, i.e., amino acid residues from multiple non-adjacent segments of the same protein. Alternatively, a combinatorial epitope may be formed by contributions from multiple protein components of an antigen complex. In some embodiments, the inhibitory antibodies of the present disclosure specifically bind to a conformational epitope (or conformation-specific epitope), e.g., an epitope that is sensitive to the three-dimensional structure (i.e., conformation) of the antigen or antigen complex.

[0326] Traditional approaches to antagonizing TGFβ signaling have been to: i) directly neutralize mature growth factors after they have become active, depleting free ligand available for receptor binding (e.g., released from their latent precursor complexes); ii) use soluble receptor fragments that can sequester free ligand (e.g., so-called ligand capture); or iii) target the cell surface receptor to block the ligand-receptor interaction. Each of these conventional approaches requires the antagonist to compete with endogenous counterparts. Furthermore, the first two approaches (i and ii) target active ligands that are transient species. Therefore, such antagonists must be able to kinetically outcompete endogenous receptors within a short time window. The third approach may provide a more durable effect, but because many growth factors (e.g., up to 20) signal through the same receptor, it can result in unintended and unwanted inhibitory effects (and therefore, potentially toxicity).

[0327] To provide a solution to these shortcomings and further enable greater selectivity and localized action, the preferred mechanism of action underlying inhibitory antibodies such as those described herein acts upstream of TGFβ1 activation and ligand-receptor interaction. Therefore, it is contemplated that high-affinity, isoform-specific, context-independent inhibitors of TGFβ1 suitable for practicing the present invention should preferably target inactive (e.g., latent) precursor TGFβ1 complexes (e.g., complexes containing precursor / latent TGFβ1) prior to their activation, in order to block the activation step at its source (such as in the disease microenvironment, e.g., TME). According to preferred embodiments of the present invention, such inhibitors target both ECM-associated and cell-surface-tethered precursor / latent TGFβ1 complexes with equal affinity, rather than free ligand transiently available for receptor binding.

[0328] The advantage of local targeting of tissue / cell-tethered complexes at the source, as opposed to soluble active species (i.e., mature growth factors after release from the source), is further supported by recent studies. Ishihara et al. (Sci. Transl. Med., 11, eaau3259 (2019), "Targeted antibody and cytokine cancer immunotherapies through collagen affinity") reported that when systemically administered drugs were targeted to tumor sites by conjugating them with collagen-binding moieties, they were able to enhance antitumor immunity and reduce treatment-related toxicity compared to their non-targeted counterparts.

[0329] The mechanism of action achieved by the antibodies of the present disclosure may further contribute to enhanced durability of effect, as well as overall greater efficacy and safety.

[0330] Interestingly, these antibodies may exert additional inhibitory activity against cell-associated TGFβ1 (LRRC33-precursor TGFβ1 and GARP-precursor TGFβ1). We found that LRRC33-binding antibodies tend to be internalized upon binding to cell-surface LRRC33. It is unclear whether internalization is actively induced by antibody binding or whether this phenomenon results from the natural (e.g., passive) endocytic activity of macrophages. However, a high-affinity, isoform-selective TGFβ1 inhibitor, Ab6, can be rapidly internalized in cells transfected with LRRC33 and precursor TGFβ1, and the internalization rate achieved with Ab6 is significantly higher than that achieved with a reference antibody that recognizes cell-surface LRRC33 (Figure 6). Similar results have been obtained with primary human macrophages. These observations raise the possibility that Ab6, upon binding to its target, LRRC33-precursor TGFβ1, induces internalization, thereby removing LRRC33-containing complexes from the cell surface. In disease loci, this may reduce the availability of activatable, latent LRRC33-precursor TGFβ1 levels. Therefore, isoform-selective TGFβ1 inhibitors may inhibit the LRRC33 arm of TGFβ1 through two parallel mechanisms of action: i) blocking the release of mature growth factor from the latent complex, and ii) removing LRRC33-precursor TGFβ1 complexes from the cell surface via internalization. It is possible that a similar inhibitory mechanism of action may apply to GARP-precursor TGFβ1.

[0331] In some embodiments, the antibody is a pH-sensitive antibody, which binds to its antigen with higher affinity at neutral pH (e.g., a pH of about 7) than at acidic pH (e.g., a pH of about 5). Such antibodies may have 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., the K at pH 5) can be calculated. オフ / K at pH 7 オフ) 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 useful as recycling antibodies. Upon target engagement on the cell surface, the antibody can trigger antibody-dependent internalization (and therefore removal) of membrane-bound precursor TGFβ1 complexes (associated with LRRC33 or GARP). Subsequently, in acidic intracellular compartments such as lysosomes, the antibody-antigen complex dissociates and the free antibody can be transported back to the extracellular domain.

[0332] Thus, in some embodiments, selection of an antibody or antigen-binding fragment for therapeutic use may be based in part on the ability to induce antibody-dependent internalization and / or pH dependence of the antibody.

[0333] Antigen complexes and their components The novel antibodies of the present disclosure specifically bind to each of the four known human large latent complexes (e.g., hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1), selectively inhibiting activation of TGFβ1. Preferred antibodies further meet the criteria of one or more of categories 1-5 listed in Table 1.

[0334] Screening (e.g., identifying and selecting) such antibodies typically involves the use of appropriate recombinantly produced antigen complexes. Useful protein components that may comprise such antigen complexes are provided, including TGFβ isoforms and related polypeptides, fragments, and variants, and presentation molecules (e.g., LTBP, GARP, LRRC33) and related polypeptides, fragments, and variants. These components may be expressed and purified to form protein complexes (such as large latent complexes) that can be used in the antibody screening process. Screening may include positive selection, in which desirable binders are selected from a pool or library of binders and non-binders, and negative selection, in which undesirable binders are removed from the pool. Typically, positive screening included at least one matrix-associated complex (e.g., LTBP1-precursor TGFβ1 and / or LTBP1-precursor TGFβ1) and at least one cell-associated complex (e.g., GARP-precursor TGFβ1 and / or LRRC33-precursor TGFβ1) to ensure that the selected binders have affinity for both such biological contexts.

[0335] In some embodiments, TGFβ1 comprises a naturally occurring mammalian amino acid sequence. In some embodiments, TGFβ1 comprises a naturally occurring human amino acid sequence. In some embodiments, TGFβ1 comprises a human, monkey, rat, or mouse amino acid sequence. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not specifically bind to TGFβ2. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not specifically bind to TGFβ3. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not specifically bind to TGFβ2 or TGFβ3. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein specifically bind to TGFβ1 comprising the amino acid sequence set forth in SEQ ID NO: 34. The amino acid sequence of TGFβ2 and the TGFβ3 amino acid sequence are set forth in SEQ ID NOs: 38 and 32, respectively. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein specifically bind to TGFβ1 comprising a non-naturally occurring amino acid sequence (also referred to herein as non-naturally occurring TGFβ1). For example, non-naturally occurring TGFβ1 may include one or more recombinantly generated mutations compared to a naturally occurring TGFβ1 amino acid sequence. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequence comprises the amino acid sequence set forth in SEQ ID NOs: 24-35 shown in Table 14. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequence comprises the amino acid sequence set forth in SEQ ID NOs: 36-43 shown in Table 15.

[0336] TGFβ1 (prodomain + Growth Factor Domain) LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRR ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (SEQ ID NO: 24)

[0337] TGFβ2 (prodomain + Growth Factor Domain )SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKAR VPECRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKR ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS (SEQ ID NO: 28)

[0338] TGFβ3 (prodomain + Growth Factor Domain )SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNP SSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKR ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS(SEQ ID NO: 32) Table 14. Amino acid sequences of exemplary TGFβ1, TGFβ2, and TGFβ3 JPEG2024099673000021.jpg246166JPEG2024099673000022.jpg247166JPEG2024099673000023.jpg244166 JPEG2024099673000024.jpg63166 Table 15. Exemplary non-human amino acid sequences JPEG2024099673000025.jpg252166JPEG2024099673000026.jpg246166JPEG2024099673000027.jpg247166JPEG20240996730 00028.jpg246166JPEG2024099673000029.jpg245166JPEG2024099673000030.jpg248166JPEG2024099673000031.jpg220166

[0339] In some embodiments, an antigen-protein complex (e.g., an LTBP-TGFβ1 complex) can include one or more presentation molecules, such as LTBP proteins (e.g., LTBP1, LTBP2, LTBP3, and LTBP4), GARP proteins, LRRC33 proteins, or fragments thereof. Typically, the minimum required fragment suitable for practicing the embodiments disclosed herein includes at least 50 amino acids, preferably at least 100 amino acids, of a presentation molecule protein that includes at least two cysteine ​​residues capable of forming disulfide bonds with the precursor TGFβ1 complex. Specifically, these Cys residues form covalent bonds with cysteine ​​residues present near the N-terminus of each monomer of the precursor TGFβ1 complex. In the three-dimensional structure of the precursor TGFβ1 dimeric complex, the so-called "alpha-1 helices" at the N-terminus of each monomer are in close proximity to one another (see, e.g., Figure 18B, the two gray helices near the bottom of the structure), defining the distance between the two cysteine ​​residues (one from each helix) required for productive covalent bond formation with the corresponding cysteine ​​pair present in the display molecule (see, e.g., Cuende et al. (2015), Sci. Trans. Med., 7:284ra56). Therefore, when fragments of the display molecule are used to form LLCs in screening steps (e.g., immunization, library screening, identification, and selection), such fragments should contain cysteine ​​residues separated by the correct distance to allow proper disulfide bond formation with the precursor TGFβ1 complex in order to preserve the correct conformation of the resulting LLC. For example, LTBPs (eg, LTBP1, LTBP3, and TBP4) may contain a "cysteine-rich domain" to mediate covalent interactions with pro-TGFβ1.

[0340] The antibodies or antigen-binding portions thereof 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 include LTBP1, its alternatively spliced ​​variants, and / or fragments thereof. The recombinant LTBP1 protein may be modified to include one or more detectable labels. In some embodiments, the LTBP1 protein includes a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP1 protein does not include a leader sequence (i.e., the leader sequence is processed or cleaved). Such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a myc tag, an HA tag, and / or a fluorescent tag. In some embodiments, the LTBP1 protein is a mammalian LTBP1 protein. In some embodiments, the LTBP1 protein is a human, monkey, mouse, or rat LTBP1 protein. In some embodiments, the LTBP1 protein comprises the amino acid sequence set forth in SEQ ID NOs: 46 and 47 in Table 15. In some embodiments, the LTBP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 50 in Table 17.

[0341] The antibodies or antigen-binding portions thereof 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 include LTBP3, its alternatively spliced ​​variants, and / or fragments thereof. In some embodiments, the LTBP3 protein includes a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP3 protein does not include a leader sequence (i.e., the leader sequence is processed or cleaved). The recombinant LTBP3 protein may be modified to include one or more detectable labels. Such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a myc tag, an HA tag, and / or a fluorescent tag. 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 comprises the amino acid sequence set forth in SEQ ID NOs: 44 and 45 in Table 15. In some embodiments, the LTBP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 51 in Table 17.

[0342] The antibodies or antigen-binding portions thereof described herein can bind to the GARP-TGFβ1 complex. In some embodiments, the GARP protein is a naturally occurring protein or fragment thereof. In some embodiments, the GARP protein is a non-naturally occurring protein or fragment thereof. In some embodiments, the GARP protein is a recombinant protein. Such GARPs may 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 GARP. Recombinant GARPs may be modified to be soluble. In some embodiments, the GARP protein contains a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the GARP protein does not contain a leader sequence (i.e., the leader sequence is processed or cleaved). In other embodiments, the recombinant GARP is modified to contain one or more detectable labels. In further embodiments, such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a flag tag, a myc tag, an HA tag, and / or a fluorescent tag. 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 sequence set forth in SEQ ID NOS: 48-49 in Table 15. In some embodiments, the GARP protein comprises the amino acid sequence set forth in SEQ ID NOS: 52 and 53 in Table 18. In some embodiments, the antibodies, or antigen-binding portions thereof, described herein do not bind to TGFβ1 in a context-dependent manner; for example, binding to TGFβ1 occurs only when the TGFβ1 molecule is complexed with a specific presentation molecule, such as GARP. Instead, the antibodies, or antigen-binding portions thereof, bind to TGFβ1 in a context-independent manner. In other words, the antibodies, or antigen-binding portions thereof, bind to TGFβ1 when bound to any presentation molecule, i.e., GARP, LTBP1, LTBP3, and / or LRCC33.

[0343] The antibodies or antigen-binding portions thereof described herein can bind to the LRRC33-TGFβ1 complex. In some embodiments, the LRRC33 protein is a naturally occurring protein or fragment thereof. In some embodiments, the LRRC33 protein is a non-naturally occurring protein or fragment thereof. In some embodiments, the LRRC33 protein is a recombinant protein. Such LRRC33 may 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 may be modified to be soluble. For example, in some embodiments, the ectodomain of LRRC33 may be expressed with a C-terminal His tag to express a soluble LRRC33 protein (sLRRC33, see e.g., SEQ ID NO: 84). In some embodiments, the LRRC33 protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LRRC33 protein does not comprise a leader sequence (i.e., the leader sequence is processed or cleaved). In other embodiments, the recombinant LRRC33 protein is modified to include one or more detectable labels. In further embodiments, such detectable labels may include, but are not limited to, a biotin label, a polyhistidine tag, a flag tag, a myc tag, an HA tag, and / or a fluorescent tag. 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 sequence set forth in SEQ ID NOs: 83, 84, and 101 in Table 18. Table 17. Exemplary LTBP amino acid sequences JPEG2024099673000032.jpg206166JPEG2024099673000033.jpg183166 Table 18. Exemplary GARP and LRRC33 amino acid sequences JPEG2024099673000034.jpg69166JPEG2024099673000035.jpg248166JPEG2024099673000036.jpg249166JPEG2024099673000037.jpg64166

[0344] Pharmaceutical Compositions and Formulations The present invention further provides pharmaceutical compositions for use as medicaments suitable for administration in human and non-human subjects. One or more high-affinity, context-independent antibodies encompassed by the present invention can be formulated or mixed with a pharmaceutically acceptable carrier (excipient), including, for example, a buffer, to form a pharmaceutical composition. Such formulations can be used to treat diseases or disorders involving TGFβ signaling. In particularly preferred embodiments, such formulations can be used in immuno-oncology applications.

[0345] 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). By "acceptable," it is meant that the carrier is compatible with (and preferably capable of stabilizing) the active ingredients of the composition and not harmful to the subject being treated. Examples of pharmaceutically acceptable excipients (carriers), including buffering agents, will be apparent to those skilled in the art or have been previously described. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (2000), Lippincott Williams and Wilkins, eds. K.E. Hoover. In one example, the pharmaceutical composition described herein contains multiple antibodies that specifically bind to the GARP-precursor TGFβ1 complex, the LTBP1-precursor TGFβ1 complex, the LTBP3-precursor TGFβ1 complex, and the LRRC33-precursor TGFβ1 complex, wherein the antibodies recognize different epitopes / residues of the complexes.

[0346] The pharmaceutical compositions used in the present methods can contain pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy, 20th Edition (2000), Lippincott Williams and Wilkins, ed. K.E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); polypeptides of low molecular weight (less than about 10 residues); serum albumin, gelatin, etc. The excipient may include a protein such as a protein, such as a ribonucleotide, a protein such as a ribonucleotide, or an immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine; a monosaccharide, a disaccharide, and other carbohydrates, including glucose, mannose, or dextran; a chelating agent such as EDTA; a sugar such as sucrose, mannitol, trehalose, or sorbitol; a counterion that forms a salt, such as sodium; a metal complex (e.g., a Zn-protein complex); and / or a non-ionic surfactant such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0347] The present invention also includes a pharmaceutical composition comprising an antibody or fragment thereof according to the present invention and a pharmaceutically acceptable excipient.

[0348] Thus, antibodies or molecules comprising antigen-binding fragments of such antibodies can be formulated into pharmaceutical compositions suitable for human administration.

[0349] The pharmaceutical formulation may include one or more excipients. In some embodiments, the excipients may be selected from the list provided below: https: / / www.accessdata.fda.gov / scripts / cder / iig / index.Cfm?event=browseByLetter.page&Letter=A

[0350] Pharmaceutical compositions are typically formulated to provide a final concentration of active biologic (e.g., monoclonal antibodies, engineered binding molecules including antigen-binding fragments, etc.) of about 2 mg / mL to about 200 mg / mL. For example, the final concentration (weight / volume) of the formulation may be about 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, 10-20, 0, 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 ranges may be 150, 50-120, 50-100, 50-80, 50-70, 50-60, 60-200, 60-180, 60-160, 60-150, 60-120, 60-100, 60-80, 60-70, 70-200, 70-180, 70-160, 70-150, 70-120, 70-100, 70-80 mg / mL. In some embodiments, the final concentration of the biological agent 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.

[0351] The pharmaceutical compositions of the present invention are preferably formulated with a suitable buffer, including but not limited to, phosphate buffers, citrate buffers, and histidine buffers.

[0352] The final pH of the formulation is typically between pH 5.0 and 8.0. For example, the pH of the pharmaceutical composition can 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.

[0353] Pharmaceutical compositions of the present disclosure may include surfactants approved for use in pharmaceutical formulations, such as non-ionic detergents, including, for example, polysorbates such as polysorbate 20 (Tween-20), polysorbate 80 (Tween-80), and NP-40.

[0354] The pharmaceutical composition of the present disclosure may contain a stabilizer.In liquid protein preparations, stability can be enhanced by selecting pH buffer salts, and amino acids can often be used.After protein adsorption and unfolding, aggregation often occurs due to interactions at the liquid / air interface or the liquid / solid interface (interface with packaging material).Suitable stabilizers include, but are not limited to, sucrose, maltose, sorbitol, and certain amino acids such as histidine, glycine, methionine, and arginine.

[0355] The pharmaceutical compositions of the present disclosure may contain one or any combination of the following excipients: sodium phosphate, arginine, sucrose, sodium chloride, tromethamine, mannitol, benzyl alcohol, histidine, sucrose, polysorbate 80, sodium citrate, glycine, polysorbate 20, trehalose, poloxamer 188, methionine, trehalose, rh hyaluronidase, sodium succinate, potassium phosphate, edetate disodium, sodium chloride, potassium chloride, maltose, histidine acetate, sorbitol, pentetic acid, human serum albumin, pentetic acid.

[0356] In some embodiments, the pharmaceutical compositions of the present disclosure may contain a preservative.

[0357] The pharmaceutical compositions of the present disclosure are typically presented in liquid or lyophilized form. Typically, the product can be presented in a vial (e.g., a glass vial). Products available in syringes, pens, or autoinjectors can be presented as pre-filled liquids in these containers / closed systems.

[0358] In some examples, the pharmaceutical compositions described herein include liposomes containing antibodies that specifically bind to GARP-precursor TGFβ1 complexes, LTBP1-precursor TGFβ1 complexes, LTBP3-precursor TGFβ1 complexes, and LRRC33-precursor TGFβ1 complexes, which can be prepared by any suitable method, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985), Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980), and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be prepared by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0359] In some embodiments, liposomes with targeting properties are selected to preferentially deliver or localize pharmaceutical compositions to specific tissues or cell types. For example, certain nanoparticle-based carriers with bone marrow targeting properties may be used, such as lipid-based nanoparticles or liposomes. See, for example, Sou (2012), "Advanced drug carriers targeting bone marrow," ResearchGate publication, 232725109.

[0360] In some embodiments, the pharmaceutical compositions of the present invention may contain or be used in conjunction with an adjuvant. It is contemplated that certain adjuvants can, for example, boost a subject's immune response to tumor antigens, facilitate T effector function, DC differentiation from monocytes, enhanced antigen uptake and presentation by APCs, etc. Suitable adjuvants include, but are not limited to, retinoic acid-based adjuvants and 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 its derivatives.

[0361] The antibodies described herein may be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in microemulsions. Exemplary techniques have been previously described; see, for example, Remington, The Science and Practice of Pharmacy, 20th ed., Mack Publishing (2000).

[0362] In other examples, the pharmaceutical compositions described herein can be formulated in a sustained-release manner. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0363] Pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0364] The pharmaceutical compositions described herein may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral, or rectal administration, or administration by inhalation or insufflation.

[0365] Suitable surfactants include, inter alia, non-ionic agents such as polyoxyethylene sorbitans (e.g., Tween™ 20, 40, 60, 80, or 85) as well as other sorbitans (e.g., Span™ 20, 40, 60, 80, or 85). Compositions having surfactants conveniently contain 0.05-5% surfactant, and can be 0.1-2.5%. It will be appreciated that other ingredients, such as mannitol or other pharmaceutically acceptable vehicles, can be added if required.

[0366] Suitable emulsions can be prepared using commercially a...

Claims

1. Each of the following antigen complexes was detected by biolayer interferometry: i) human LTBP1-precursor TGFβ1; ii) human LTBP3-precursor TGFβ1; iii) human GARP-precursor TGFβ1, and iv) Human LRRC33-precursor TGFβ1 with a KD of ≦5 nM, Inhibits the activation of TGFβ1, a fully human or humanized antibody or antigen-binding fragment thereof; H-CDR1 as set forth in SEQ ID NO: 107; H-CDR2 as set forth in SEQ ID NO: 103; H-CDR3 as set forth in SEQ ID NO:6; L-CDR1 as set forth in SEQ ID NO: 105; L-CDR2 as set forth in SEQ ID NO: 106; L-CDR3 set forth in SEQ ID NO: 12; Including, The antibody or antigen-binding fragment thereof comprises in the constant domain a main chain substitution of Ser to Pro resulting in an IgG1-like hinge. An antibody or an antigen-binding fragment thereof.

2. 2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable domain sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 and a light chain variable domain sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15; An antibody or an antigen-binding fragment thereof.

3. 2. The antibody or antigen-binding fragment thereof of claim 1, A heavy chain variable domain sequence comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable domain sequence comprising the amino acid sequence of SEQ ID NO:

15. An antibody or an antigen-binding fragment thereof.

4. 2. The antibody or antigen-binding fragment thereof of claim 1, binds to each of the antigen complexes with a K of ≦1 nM as measured by biolayer interferometry; An antibody or an antigen-binding fragment thereof.

5. 2. The antibody or antigen-binding fragment thereof of claim 1, Cross-reacts with human and mouse precursor TGFβ1 complexes; An antibody or an antigen-binding fragment thereof.

6. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and an excipient.

7. 7. The composition of claim 6 for the treatment of cancer in a subject, comprising: the cancer is a solid tumor or a hematological cancer; composition.

8. 8. The composition of claim 7, the solid tumor is poorly responsive to cancer therapy; the solid tumor is an immune-excluded tumor; the solid tumor comprises regulatory T cells (Tregs), intratumoral M2 macrophages, and / or myeloid-derived suppressor cells (MDSCs); and / or the solid tumor comprises a stroma enriched with cancer-associated fibroblasts (CAFs) and / or myofibroblasts; composition.

9. 8. The composition of claim 7, the subject has primary or acquired resistance to a cancer treatment; composition.

10. 8. The composition of claim 7, the subject has a TGFβ1-positive and TGFβ3-positive cancer; the subject has received, is receiving, or is a candidate for receiving checkpoint inhibitor therapy (CBT), or the subject has not previously received CBT; composition.

11. 8. The composition of claim 7, The cancer is a cancer type with a statistically low response rate to CBT, characterized by a response rate of less than 25%. composition.

12. 8. The composition of claim 7, The antibody or antigen-binding fragment thereof is administered to the subject in combination with at least one additional treatment. composition.

13. 13. The composition of claim 12, the additional treatment is selected from the group consisting of a cancer vaccine, treatment with engineered immune cells, chemotherapy, radiation therapy, a modulator of a member of the TGFβ superfamily, a myostatin inhibitor, a GDF11 inhibitor, a VEGF agonist, an IGF1 agonist, an FXR agonist, a CCR2 inhibitor, a CCR5 inhibitor, a dual CCR2 / CCR5 inhibitor, a CCR4 inhibitor, a lysyl oxidase-like-2 inhibitor, an ASK1 inhibitor, an acetyl-CoA carboxylase (ACC) inhibitor, a p38 kinase inhibitor, pirfenidone, nintedanib, an M-CSF inhibitor, a MAPK inhibitor, an immune checkpoint inhibitor, an IL-11 antagonist, an IL-6 antagonist, an indoleamine 2,3-dioxygenase (IDO) inhibitor, an arginase inhibitor, a tyrosine kinase inhibitor, a Ser / Thr kinase inhibitor, and a dual specific kinase inhibitor; composition.

14. 13. The composition of claim 12, the additional treatment is an immune checkpoint inhibitor. composition.

15. 15. The composition of claim 14, the immune checkpoint inhibitor is selected from the group consisting of a cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibody, a programmed cell death protein 1 (PD-1) antibody, a PD-L1 antibody, a T-cell immunoglobulin domain and mucin domain-3 (TIM3) antibody, a lymphocyte activation gene 3 (LAG3) antibody, a killer cell immunoglobulin-like receptor (KIR) antibody, a glucocorticoid-induced tumor necrosis factor receptor (GITR) antibody, and a V-domain immunoglobulin (Ig)-containing inhibitor of T-cell activation (VISTA) antibody, or a combination thereof; composition.

16. 16. The composition of claim 15, The immune checkpoint inhibitor is selected from a programmed cell death protein 1 (PD-1) antibody or a PD-L1 antibody. composition.

17. 8. The composition of claim 7, The cancer is selected from the group consisting of melanoma, adjuvant melanoma, renal cell carcinoma (RCC), bladder cancer, colorectal cancer (CRC), colon cancer, rectal cancer, anal cancer, breast cancer, triple negative breast cancer (TNBC), HER2 negative breast cancer, BRCA mutated breast cancer, hematological malignancies, non-small cell carcinoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), extensive stage small cell lung cancer (ES-SCLC), lymphoma (classical Hodgkin and non-Hodgkin), primary mediastinal large B cell lymphoma (PMBCL), T cell lymphoma, diffuse large B cell lymphoma, histiocytic sarcoma, follicular dendritic cell sarcoma, interdigitating dendritic cell sarcoma, myeloma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), small lymphocytic lymphoma (SLL), head and neck cancer, urothelial carcinoma. , Merkel cell carcinoma, Merkel cell skin cancer, cancer with microsatellite instability (MSI-H), cancer with deficient mismatch repair (dMMR), mesothelioma, gastric cancer, gastroesophageal junction cancer (GEJ), gastric adenocarcinoma, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), gastric cardia adenocarcinoma, kidney cancer, biliary carcinoma, bile duct carcinoma, pancreatic cancer, prostate cancer, adenocarcinoma, squamous cell carcinoma, non-squamous cell carcinoma, cutaneous squamous cell carcinoma (CSCC), ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, peritoneal cancer, stomach cancer, brain cancer, malignant glioma, glioblastoma, gliosarcoma, neuroblastoma, thyroid cancer, adrenal cortical carcinoma, oral intraepithelial neoplasia, esophageal cancer, nasal and paranasal squamous cell carcinoma, nasopharyngeal carcinoma, salivary gland cancer, liver cancer, and hepatocellular carcinoma (HCC), composition.

18. 1. A method for screening for an isoform-selective TGFβ1 inhibitor suitable for therapeutic use, comprising: i) providing an antibody or antigen-binding fragment thereof that specifically binds to each of human LTBP1-precursor TGFβ1, human LTBP3-precursor TGFβ1, human GARP-precursor TGFβ1, and human LRRC33-precursor TGFβ1 complexes; ii) conducting an in vivo efficacy study to determine an amount of the antibody or antigen-binding fragment thereof effective to achieve efficacy in a preclinical animal model comprising TGFβ1-biased expression; iii) conducting toxicology studies in preclinical models known to be sensitive to TGFβ inhibition to determine the maximum tolerated dose and / or the minimum toxic dose of the antibody or antigen-binding fragment thereof; iv) selecting the antibody or antigen-binding fragment thereof as a candidate therapeutic agent if a sufficient therapeutic window is obtained based on steps (ii) and (iii); Including, method.

19. 20. The method of claim 18, the amount effective to achieve efficacy in step (ii) is between about 1 and 30 mg / kg / week; method.

20. 20. The method of claim 18, the maximum tolerated dose or minimum toxic dose in step (iii) is at least 30-100 mg / kg / week for at least 4 weeks; method.

21. 20. The method of claim 18, The therapeutic window is at least three-fold. method.

22. 22. The method of claim 21, The therapeutic window is at least 6-fold. method.

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