High affinity isoform-selective TGFβ1 inhibitors, and their use

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 Applications No. 62 / 696,752 filed on 11 July 2018, No. 62 / 718,196 filed on 13 August 2018, No. 62 / 737,534 filed on 27 September 2018, No. 62 / 758,180 filed on 9 November 2018, No. 62 / 810,263 filed on 25 February 2019, and No. 62 / 827,552 filed on 1 April 2019, each titled "Affinity-Context-Independent TGFβ1 Inhibitors and Their Uses," the contents of which are expressly incorporated herein by reference as a whole.

[0002] Sequence List This application has been electronically filed in ASCII format and includes, in whole, a sequence listing incorporated herein for reference. The ASCII copy prepared on 11 July 2019 is named 127036-03520_SL.txt and is 261,252 bytes in size.

[0003] Transforming growth factor beta-1 (TGFβ1), along with two other structurally related isoforms, TGFβ2 and TGFβ3, each encoded by a separate gene, is a member of the TGFβ growth factor superfamily. These TGFβ isoforms function as pleomorphic cytokines that regulate cell proliferation, differentiation, immunomodulation (e.g., adaptive immune responses), and a variety of other 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 have shown diverse phenotypes, suggesting that each isoform plays a distinct role in vivo. This may be partially achieved by the differential expression patterns of the three isoforms.

[0004] Within the immune system, T cells are recognized as a primary direct target of TGFβ. TGFβ signaling is crucial in regulating the proliferation of effector cells and the differentiation of effector and regulatory T cells. For example, TGFβ is a potent suppressor of Th1 and Th2 effector T cells. 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 in the immune system, such as dendritic cells like Langerhans cells and natural killer (NK) cells, are also regulated by the TGFβ signaling pathway. TGFβ dysregulation has been linked to several medical conditions, including cancer, fibrosis, and immune disorders.

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

[0006] For these and other reasons, TGFβ is an attractive therapeutic target for treating immunodeficiencies, 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 serious side effects (see, for example, summarized in WO2017 / 156500). Thus, despite a body of direct and indirect evidence pointing to the involvement of TGFβ signaling in the progression of diseases such as cancer and fibrosis, no TGFβ treatment considered safe and effective has yet been commercially available.

[0007] Previously, the applicant described a class of monoclonal antibodies that function through a novel mechanism of action to modulate growth factor signaling (see, for example, WO2014 / 182676). These antibodies are designed to leverage the fact that TGFβ1 is expressed as a latent precursor protein complex consisting of a prodomain and a growth factor, and that an activation step is required to release the growth factor from the latent complex. Instead of taking conventional approaches that directly target the mature growth factor itself after activation (e.g., neutralizing antibodies), a novel class of inhibitory antibodies specifically targets the inactive precursor-proto [Disclosure of the Invention] [Problems that the invention aims to solve]

[0008] Using this method, we created a monoclonal antibody that binds to TGFβ1 in an isoform-selective manner and inhibits its activation step (i.e., the release of mature growth factors from the latent complex) (see WO2017 / 156500). The data presented therein support the view that isoform-specific inhibition of TGFβ (as a counter to general inhibition) may provide an improved safety profile for antagonizing TGFβ in vivo. Subsequently, with this in mind, the applicant sought to develop a TGFβ1 inhibitor that is i) isoform-specific and ii) broadly targets multiple TGFβ1 signaling complexes associated with different presenting molecules as a therapeutic agent for conditions driven by the multifaceted TGFβ1 effects and their dysregulation.

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

[0010] While the early studies cited above have demonstrated the usefulness of antibodies that can target known precursor TGFβ1 complexes and their inhibitory activity, improved isoform-selective TGFβ1 inhibitors with even higher in vivo efficacy are desirable. [Means for solving the problem]

[0011] This disclosure provides a novel class of high-affinity, isoform-selective antibodies capable of inhibiting TGFβ1 activation with high potency. These include antibodies (including immunoglobulins and antigen-binding fragments or portions thereof, as well as engineered molecules incorporating such fragments) capable of targeting multiple presenting molecule-precursor TGFβ1 complexes (referred to as “large latent complexes” or “LLCs”) with high affinity. These antibodies retain essential selectivity and safety profiles and have been shown to achieve improved in vivo efficacy with translationability to human conditions in multiple preclinical models. These characteristics of TGFβ1 inhibitors open up opportunities to develop safe and effective TGFβ1 therapeutics for treating diseases involving TGFβ1 dysregulation.

[0012] In constructing the precursor TGFβ1 antibodies of this disclosure, the following selection criteria were taken into consideration: 1) isoform selectivity, 2) high affinity for human LLC, e.g., LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1, 3) robust inhibitory efficacy, 4) favorable in vivo safety / toxicological profile, and 5) in vivo efficacy in preclinic models outlining human disease. Furthermore, when evaluating the efficacy of TGFβ1 inhibitors used as combination therapies (e.g., adjuvant therapy), the ability to achieve synergistic effects (as a counter to mere additive effects) should be considered. Based on these criteria, the inventors of this disclosure have identified a class and fragments of high-affinity monoclonal antibodies 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., KD in the range of nanomolar to sub-nanomolar). In preferred embodiments, such antibodies are unbiased across various precursor TGFβ1 complexes so that the antibody has equal affinity for all target complexes. Related compositions, therapeutic uses, preparations, formulations, processes, and methods are encompassed by the present invention.

[0013] Accordingly, in some embodiments, the present invention comprises a monoclonal antibody or its antigen-binding fragment that can be conjugated 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 conjugates 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 latent lasso or a portion thereof of the precursor TGFβ1. In some embodiments, the antibody or fragment further binds to portions of the growth factor domain, such as finger-1 and finger-2. For example, the antibody or fragment may bind to an epitope containing one or more amino acid residues of the latent lasso. Optionally, the epitope may further contain 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 inhibitors of the present invention are functional antibodies in that they have inhibitory activity against TGFβ1. The potency of such antibodies is isoform-specific, as measured by appropriate in vitro potency assays such as the cell-based reporter assays described herein. Therefore, the antibodies do not bind to or inhibit TGFβ2 or TGFβ3 counterparts.

[0016] The TGFβ1 inhibitors of the present invention can block the release of mature growth factors from the latent LLC complex. In some embodiments, the TGFβ1 inhibitors can inhibit integrin-dependent activation and / or protease-dependent activation of TGFβ1. In some embodiments, the protease is kallikrein, plasmin, or an MMP protease. In some embodiments, the TGFβ1 inhibitors block integrin-dependent TGFβ1 activation without blocking the binding of integrin to LLC.

[0017] In some embodiments, the TGFβ1 inhibitors of the present invention may function through a dual inhibitory mechanism of action against cell-associated LLCs (e.g., GARP-precursor TGFβ1 and LRRC33-precursor TGFβ1). In the first mechanism, such inhibitors block the activation step of membrane-immobilized GARP and / or LRRC33-associated TGFβ1. In the second mechanism, such inhibitors may induce antibody-dependent internal migration (and therefore removal) of LLCs from the cell surface upon target association, 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 inhibitor of the present invention is effective in reducing the phosphorylation of downstream effectors SMAD2 / 3 in vivo.

[0020] In some embodiments, the TGFβ1 inhibitors of the present invention are effective in treating TGFβ1-related indications. Such indications include, but are not intended to be mutually exclusive, diseases involving abnormal gene expression, diseases involving ECM dysregulation, diseases characterized by an increase in immunosuppressive cells (e.g., Treg, MDSC, and / or M2 macrophages), diseases involving mesenchymal transition, diseases involving proteases, and diseases related to abnormal stem cell proliferation and / or differentiation. In some embodiments, TGFβ1-related indications are proliferative disorders such as myeloproliferative disorders and cancers with solid tumors. In some embodiments, TGFβ1-related indications are fibrotic disorders such as organ fibrosis. Cancer may be advanced cancer, which includes 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 in disease sites such as the tumor microenvironment and fibrous microenvironment. In some embodiments, the immunosuppressive cell population may 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 is optionally immunosuppressive (e.g., immune elimination) in phenotype. In some embodiments, TGFβ1 inhibitors may achieve synergistic antitumor effects when used in combination with cancer treatments such as checkpoint blockade therapy, chemotherapy, and radiotherapy. Checkpoint blockade therapy may include, for example, anti-PD-(L)1 antibodies. In such combination therapies, the TGFβ1 inhibitor may overcome treatment resistance (e.g., primary resistance), thereby making the cancer more sensitive to cancer treatment. Therefore, TGFβ1 inhibitors may be used in the treatment of cancers, including immunosuppressive tumors, in subjects. Subjects may be i) primary nonresponders to cancer treatments such as checkpoint inhibitors, or ii) diagnosed with cancer for which at least one of the checkpoint inhibitors is approved as a treatment by the regulatory authority. The response rate (combined partial and complete responders among those treated) for cancers for which at least one checkpoint inhibitor is approved is less than 100%. Typically, response rates are approximately 10–60%. TGFβ1 inhibitors may increase response rates across patient populations. Furthermore, within the partial response group of primary responders, TGFβ1 inhibitors may provide improved clinical benefits. In some embodiments, within the primary responder group, TGFβ1 inhibitors may reduce the rate of acquired resistance to cancer treatment. Immunosuppressive tumors may be locally advanced cancers / tumors or metastatic cancers. In some embodiments, cancer treatment may include, for example, checkpoint inhibitor therapy, chemotherapy, and / or radiotherapy.

[0023] In some embodiments, the TGFβ1 inhibitor of the present invention is effective in achieving a life-extending effect in subjects having solid tumors that are locally advanced or metastatic cancers, at the discretion of the patient.

[0024] In some embodiments, the TGFβ1 inhibitors of the present invention are effective in achieving a persistent antitumor effect by inducing T cell memory function. Therefore, TGFβ1 inhibitors may reduce or delay disease recurrence.

[0025] In some embodiments, the TGFβ1 inhibitor of the present invention is effective in achieving an antitumor effect in tumors that predominantly express TGFB1 and / or TGFβ3. In some embodiments, tumors that co-express TGFβ1 and TGFβ3 are carcinomas.

[0026] In some embodiments, the TGFβ1 inhibitors of the present invention can overcome primary resistance of tumors to cancer treatment. In some embodiments, such tumors are infiltrated by 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 antitumor effector T cells.

[0027] In some embodiments, the TGFβ1 inhibitors of the present invention promote the infiltration of effector cells into the tumor. In some embodiments, effector cells can enter the tumor via the tumor's vascular structure. In some embodiments, the TGFβ1 inhibitors of the present invention promote the expansion (e.g., proliferation) of effector cells. This can be at least partially mediated 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 an antifibrotic effect on the bone marrow of a subject having 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 in achieving antifibrotic effects in vivo. The antifibrotic effect may include established fibrotic reversal, which may be partial or complete reversal.

[0030] In some embodiments, the TGFβ1 inhibitors of the present invention have been well tolerated in preclinical safety / toxicological studies when administered once weekly for at least four weeks at doses up to 100, 200, or 300 mg / kg. Such studies may be conducted in animal models known to be sensitive to TGFβ inhibition, such as rats and non-human primates. In some embodiments, the TGFβ1 inhibitors of the present invention do not cause observable toxicity associated with general inhibition of TGFβ, such as cardiovascular toxicity (e.g., valvular heart disease) and epithelial hyperplasia, as well as other toxicity known in the art.

[0031] In some embodiments, the TGFβ1 inhibitors of the present invention achieve a sufficient therapeutic window, such that the effective dose of the inhibitor shown in in vivo efficacy studies is considerably lower (at least 3 times, at least 6 times, or at least 10 times) than the dose or concentration that causes observable toxicity. In some embodiments, the therapeutically effective dose of the inhibitor is approximately 1 mg / kg to approximately 30 mg / kg per week. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 is a graph showing the inhibition of LTBP1-precursor TGFβ activation in the LN229 assay. [Figure 2] Figure 2 is a graph showing the inhibition of precursor TGFβ1 complex activation in the LN229 assay. [Figure 3] Figure 3 is a graph showing the inhibition of GARP-precursor TGFβ1 activation in the SW480β6 assay. [Figure 4] Figure 4 is a graph showing the inhibition of LRRC33-precursor TGFβ1 activation in the SW480β6 assay. [Figure 5A] Figure 5A shows the inhibitory effects of Ab3 and Ab6 on kallikrein-induced activation of TGFβ1 in vitro. [Figure 5B] Figure 5B shows the inhibitory effects of Ab3 and Ab6 on plasmin-induced activation of TGFβ1 in vitro. [Figure 6] Figure 6 provides a graph showing the rapid internal translocation of LRRC33-precursor TGFβ1 upon Ab6 binding in xenocellular cells transfused with LRRC33 and precursor TGFβ1. [Figure 7] Figure 7 provides two graphs showing the effects of Ab6 or Ab3 on the expression of collagen genes (Col1a1 and Col3a1) 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 8] Figure 8 provides two graphs showing the effects of Ab3 or Ab6 on the expression of the 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] Figure 9 summarizes the statistical significance of the changes in gene expression after treatment (vs. UUO+IgG) in the UUO model. [Figure 10] Figure 10 is a graph showing the percentage survival over time (days) in a Cloudman S91 melanoma model after administration of Ab3 at 30 mg / kg or 10 mg / kg in combination with anti-PD-1. 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 in combination with anti-PD-1, respectively, in a Cloudman S91 melanoma model. Anti-PD-1 monotherapy was used as a control. The dashed line represents animals that had to be sacrificed before reaching the 2000 mm3 endpoint due to tumor ulceration. [Figure 11B] Figure 11B provides two graphs showing the median tumor volume of Crowdmann 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 mm3 is shown by the upper dotted line, and the 25% threshold volume of 500 mm3 is shown by the lower dotted line. Responders were defined as those who achieved a tumor size of less than 25% of the endpoint volume. [Figure 11D] Figure 11D provides three graphs showing the change in tumor volume of S91 as a function of time in mice treated with a combination of anti-PD-1 and Ab6 at three dose levels (3, 10, and 30 mg / kg). A persistent antitumor effect is shown after treatment. [Figure 11E] Figure 11E provides a graph summarizing the data from Figure 11C, expressed as median tumor volume. [Figure 11F] Figure 11F provides a graph showing the survival of animals over time in each treatment group, as shown in Figure 11C. [Figure 12]Figure 12 is a graph showing the phosphorylation-to-total SMAD2 / 3 ratio (pSMAD / SMAD) in an MBT2 bladder cancer model. Animals were treated as follows: (1) anti-PD-1 antibody alone, (2) Ab5 (3 mg / kg) combined with anti-PD-1 antibody, (3) Ab5 (10 mg / kg) combined with anti-PD-1 antibody, (4) Ab3 (10 mg / kg) combined with anti-PD-1 antibody, and (5) Ab3 (30 mg / kg) combined 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 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. Change in tumor volume as a function of time is shown on a logarithmic scale (Figure 13A) and a linear scale (Figure 13B). The dashed line represents animals that had to be euthanized before reaching the 1200 mm3 endpoint due to tumor ulceration. [Figure 13C] Figure 13C provides a graph showing the median tumor volume as a function of time after administration of Ab3 (upper left) at 30 mg / kg or 10 mg / kg, or Ab6 (upper right) at 10 mg / kg or 3 mg / kg, in combination with anti-PD-1, in MBT2 syngeneic bladder cancer models. Anti-PD-1 alone, Ab3 alone, Ab6 alone, and IgG alone were used as controls. The median tumor volume at day 15 is summarized in the graph below. [Figure 13D] Figure 13D provides five graphs showing the effect of Ab6 in combination with anti-PD-1 in MBT2 syngeneic bladder cancer models. Responders are defined as those who achieved 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 the percentage survival over time (days) in MBT2 syngeneic bladder cancer models after administration of Ab3 at 10 mg / kg or Ab6 at 3 mg / kg or 10 mg / kg in combination with anti-PD-1. Anti-PD-1 monotherapy was used as a control. [Figure 15]Figure 15 provides a set of graphs showing changes in tumor growth (tumor volume mm3) measured over time (days) in a tumor re-induction study. Animals previously treated with anti-PD-1 / Ab3 or anti-PD-1 / Ab6 and whose tumors were removed (full responders achieving complete regression) were re-induced with MBT2 tumor cells. Naive, untreated animals were used as controls. The dashed line represents animals that had to be sacrificed before reaching the 1200 mm3 endpoint due to tumor ulceration. [Figure 16] Figure 16 is a heatmap showing that Ab5 Fab binding results in HDX protection in the regions of precursor TGFβ1 (regions 1 and 2). [Figure 17] Figure 17 illustrates the region of the precursor TGFβ1 complex that is protected from solvent exchange, as measured by HDX during Ab5 binding (see Figure 16). [Figure 18A] Figure 18A is a heatmap showing the protective effect of Ab6 Fab binding to precursor TGFβ1(C4S). Regions affected by antibody-antigen interactions are indicated by red squares (1, 2a, 2b, 2c, 3, 4, 5a, 5b, 6a, and 6b). [Figure 18B] Figure 18B shows HDX data superimposed on the crystal structure of TGFβ1. It shows the region 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 are located within the growth factor domain. [Figure 19B] Figure 19B shows various domains and motifs of precursor TGFβ1 compared to the three binding regions involved in Ab6 binding. Sequence alignments between the three isoforms are 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 control cells (by cancer type). Figure 20B shows the frequency of TGFβ isoform expression for each 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 for each cancer type. Figure 20D shows TGFβ isoform expression in a mouse syngeneic cancer cell model system. [Figure 20E] Figure 20E provides four gene expression panels showing that all of the presented molecules (LTBP1, LTBP3, GARP, and LRRC33) are highly expressed in most human cancer types. [Figure 20F] Figure 20F shows an analysis of TGFβ and related signaling pathway gene expression from the syngeneic mouse tumor models Cloudman S91, MBT-2, and EMT-6. [Figure 20G] Figure 20G provides three graphs comparing the protein expression of three TGFβ isoforms by ELISA in Cloudman S91, MBT-2, and EMT-6 tumor models. [Figure 20H] Figure 20H provides a graph comparing RNA expression levels by total tumor lysate qpCR of the presented molecule in Cloudman S91, MBT-2, and EMT-6 tumor models. [Figure 21A] Figure 21A shows microscopic cardiac findings from pan-TGFβ antibodies from a one-week toxicology study. Figure 21B shows microscopic cardiac findings from Ab3 compared to an ALK5 inhibitor or pan-TGFβ antibodies from a four-week rat toxicology study. Figure 21C shows microscopic findings from Ab6 compared to an ALK5 inhibitor or pan-TGFβ antibodies from a four-week rat toxicology study. [Figure 22]Figure 22 provides a graph showing the median tumor volume of S91 as a function of time. The combination arm represents four different isoform-selective, context-independent TGFβ1 inhibitors combined with anti-PD-1 treatment at two dose levels, respectively. [Figures 23A-23B] Figures 23A–23B provide representative immunohistochemical sections of S91 tumors stained with the CD8+ cell marker. Figure 23A is a tumor section from an animal treated with anti-PD-1 alone. Figure 23B is 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 is a tumor section from an animal treated with anti-PD-1 alone. Figure 24B is a tumor section from an animal treated with both anti-PD-1 and a representative context-independent TGFβ1 inhibitor. Figure 24C is a tumor section from an animal treated with anti-PD-1 and Ab3 (30 mg / kg), with anti-F4 / 80 used as a macrophage marker. Figure 24D is 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 log-2x 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] Figure 26A provides FACS data showing GARP CD3 / CD28-induced upregulation 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] Figure 27A shows the gating strategies for differentiating T cell subpopulations in MBT2 tumors. [Figure 27B]Figure 27B provides a set of graphs showing T cell subpopulations on day 13, expressed as a percentage of CD45+ cells. [Figure 28A] Figure 28A provides a gating strategy for separating bone marrow subpopulations in MBT2 tumors. [Figure 28B] Figure 28B provides a set of graphs showing a subpopulation of bone marrow cells on day 13. [Figure 28C] Figure 28C provides FACS data showing that tumor-associated macrophages in MBT-2 express cell surface LRRC33. [Figure 28D] Figure 28D shows that MBT-2 tumor-infiltrating MDSCs express LRRC33 on their cell surface. [Figures 29A-29C] Figures 29A–29C provide additional FACS data analysis demonstrating the effects 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 CD8-positive T cells within the tumor. [Figure 30E] Figure 30E provides a quantitative analysis of 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 immunohistochemical analysis of the effects of Ab6 and anti-PD-1 treatments on MBT2 tumors. In animals from the three treatment groups shown, tumor sections were visualized for phospho-SMAD3 (upper panel) or CD8 and CD31 (lower panel). [Figure 30G] Figure 30G provides data demonstrating that Ab6 and anti-PD-1, in combination, appear to initiate the recruitment of CD8+ T cells from CD31+ vessels and their invasion into MBT2 tumors. [Figure 31A-31D]Figures 31A–31D show the gene expressions of the 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 shown. [Figures 32A-32C] Figures 32A–32C show the gene expression of the effector function markers, Ifng (Figure 32A), Gzmb (Figure 32B), and Prf1 (Figure 32C), at day 10 and / or day 13, as shown. [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 on day 10 in MBT2 tumor samples. Each graph shows the multiplier of change in expression in three treatment groups: anti-PD-1 alone (left), Ab6 alone (center), and anti-PD-1 and Ab6 combination (right). [Figure 33A] Figure 33A shows the in vitro binding of Ab6 to the four large latent complexes shown, as measured by a solution equilibrium titration assay (MSD-SET). The measured KD values ​​(in picomolars) are shown on the right. [Figure 33B] Figure 33B illustrates a efficacy assay based on LN229 cells and provides a graph showing the concentration-dependent efficacy of Ab6 against the four large latent complexes shown. This also shows that Ab6 does not inhibit precursor TGFβ3. [Figure 34A] Figure 34A provides a set of nine graphs showing the effect of Ab6, 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 the percentage survival over time (days after the start of treatment) in EMT6 (Study 1). Treatment groups including both anti-PD-1 and Ab6 showed a significant survival benefit compared to anti-PD-1 alone. [Figure 34C] Figure 34C provides data showing the percentage survival over time (days after the start of treatment) in EMT6 (Study 2). Treatment groups including both anti-PD-1 and Ab6 showed a significant survival benefit compared to anti-PD-1 alone, and the antitumor effect was persistent after the end of treatment. [Figure 34D] Figure 34D shows the effect of the combination of anti-PD-1 and Ab6 on survival in an EMT6 breast cancer model. [Figure 35] Figure 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] Figure 36A provides a set of histological images showing silver staining of reticlin as a marker of the fibrous phenotype in the bone marrow in a mouse myeloproliferative disorder model. [Figure 36B] Figure 36B provides two graphs showing the effects of TGFβ1 inhibition in MPLW515L mice with a high disease burden from histopathological analysis of myelofibrosis and two separate replicate studies. [Figure 36C] Figure 36C provides a set of graphs showing hematological parameters in MPLW515L mice treated with Ab6 or control IgG. [Figure 36D] Figure 36D provides a set of graphs showing additional hematological parameters in MPLW515L mice treated with Ab6 or control IgG. [Figure 37A] Figure 37A provides a gene-group difference analysis (GSVA) ​​showing the correlation between TGFβ isoform expression and IPRES gene sets. [Figure 37B]Figure 37B provides a gene-group difference analysis (GSVA) ​​showing the correlation between TGFβ isoform expression and Plasari gene sets. TGFb1 isoform expression correlates with activation of the TGFβ pathway. The Plasari gene set of TGFβ-responsive genes is significantly and strongly correlated with the expression of TGFb1 RNA isoforms across many TCGA-annotated tumor species. The signature of the correlation between TGFB1 mRNA and TGFβ signaling. [Modes for carrying out the invention]

[0033] definition To facilitate understanding of this disclosure, certain terms are defined first. These definitions should be read in light of the remainder of this disclosure and in a manner understandable to 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 provided throughout the detailed description.

[0034] Advanced Cancer, Advanced Malignant Tumor: As used herein, the terms “advanced cancer” or “advanced malignant tumor” have the meanings understood in the relevant field, for example, in the context of diagnosing or treating subjects / patients with cancer, as understood by oncologists. Advanced malignant tumors with solid tumors may be locally advanced or metastatic. The term “locally advanced cancer” is used to describe cancer (e.g., a tumor) that has grown outside the organ in which it originated but has not yet spread to the distal parts of the body. Thus, this term includes cancer that has spread from where it originated to nearby tissues or lymph nodes. In contrast, “metastatic cancer” is cancer that has spread from the part of the body in which it originated (primary site) to other parts of the body (e.g., distal parts).

[0035] Affinity: Affinity is the strength of the binding between a molecule (such as an antibody) and its ligand (such as an antigen). Typically, this is expressed by the equilibrium dissociation constant (K). D ) is measured and reported. In the context of antibody-antigen interactions, KD This refers to the antibody dissociation rate ("off rate" or K オフ (How quickly the antibody dissociates from the antigen) Antibody association rate of the counter antibody ("ON rate" or K) オン This is the ratio of how quickly it binds to the antigen. For example, an antibody with affinity ≤5 nM is determined by a suitable in vitro binding assay, and a K of ≤5 nM is also determined. D It has a value (i.e., affinity of 5 nM or higher). The KD value of an antibody against its antigen can be measured using appropriate in vitro assays such as biolayer interferometry (BLI) and solution equilibrium titration (e.g., MSD-SET).

[0036] Antibody: The term “antibody” encompasses any naturally occurring, recombinant, modified, or manipulated immunoglobulin or immunoglobulin-like structure or its antigen-binding fragment or portion, or derivative thereof, as further described elsewhere herein. Therefore, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, including, for example, chimeric, humanized, fully human, and bispecific antibodies. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, although some examples, such as antibodies naturally occurring in camels, may contain fewer chains, including only heavy chains. Antibodies may originate from a single source or they may be “chimeric,” meaning that different parts of an antibody may originate from two different antibodies. Antibodies or their antigen-binding portions may 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 mimes”), chimeric antibodies, humanized antibodies, human antibodies, and antibody fusions (sometimes referred to herein as “antibody conjugates”). In some embodiments, this term also includes peptide bodies.

[0037] Antigen: The term "antigen" broadly includes any molecule that contains an antigenic determinant within a 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 of multiple components. An antigen provides an epitope, e.g., a molecule or a portion of a molecule, or a complex of molecules or portions of molecules, that can be conjugated by a selective binder, such as an antigen-binding protein (e.g., an antibody). Thus, a selective binder can specifically bind to an antigen formed by two or more components in the complex. In some embodiments, an antigen can be used in animals to produce an antibody that can bind to that antigen. An antigen may harbor one or more epitopes that can interact with different antigen-binding proteins, e.g., antibodies. In the context of this disclosure, a suitable antigen is a complex containing a precursor TGF dimer associated with a presenting molecule (e.g., a complex of multiple associated components). 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 complex is covalently bonded to the presenting molecule via a disulfide bond containing cysteine ​​residues located near the N-terminus of each precursor TGF monomer. This multi-complex formed by the precursor TGF dimers bound to the presenting molecule is generally called a large latent complex. For example, an antigen complex suitable for screening antibody or antigen-binding fragments contains a presenting molecule component of a large latent complex. Such a presenting molecule component may be a full-length presenting molecule or a fragment thereof. The minimum required portion of the presenting molecule typically contains at least 50 amino acids, but more preferably at least 100 amino acids of a presenting molecule polypeptide containing two cysteine ​​residues that can form a covalent bond with the precursor TGFβ1 dimer.

[0038] Antigen-binding moiety / fragment: As used herein, the terms “antigen-binding moiety” or “antigen-binding fragment” refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TGFβ1). Antigen-binding moieties include, but are not limited to, any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. In some embodiments, the antigen-binding moiety of an antibody may be derived from a complete antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, including manipulation and expression of DNA encoding the antibody’s variable domain and, optionally, its constant domain. Non-limiting examples of antigen-binding moieties include (i) a monovalent fragment consisting of a Fab fragment, VL, VH, CL, and CH1 domains; (ii) a bivalent fragment containing an F(ab')2 fragment and two Fab fragments linked by a disulfide bridge at a hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of a single arm of the 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) an dAb fragment (see, e.g., Ward et al., (1989), NATURE, 341:544-546); and (vii) minimal recognition units. Other forms of single-chain antibodies, such as diabodies, are also included. The term antigen-binding portion of an antibody includes a single-chain Fab fragment, also known as "scFab," which comprises an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following sequences in the direction from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL, and the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids.

[0039] Bias: In the context of this disclosure, the term “bias” means a distorted or uneven affinity of an antibody toward or toward a subset of antigens to which it can specifically bind. For example, an antibody is said to be biased if its affinity toward one antigen complex is not equivalent to its affinity toward another antigen complex. Context-independent antibodies according to this disclosure have equivalent affinity (i.e., unbiased) toward 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 a fragment thereof, can form an interface for antibody-antigen interaction. During antibody binding, the binding region is protected from surface exposure, which can be detected by appropriate techniques such as HDX-MS. Antibody-antigen interactions may be mediated through multiple (e.g., two or more) binding regions. Binding regions may contain antigenic determinants or epitopes.

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

[0042] Cancer: As used herein, the term “cancer” refers to a physiological condition in multicellular eukaryotes typically characterized by unregulated cell proliferation and malignant tumors. This term broadly encompasses tumors, solid and humoral malignancies, including hematological malignancies (e.g., leukemia, lymphoma, and myeloma), as well as myelofibrosis.

[0043] Cell-associated precursor TGFβ1: This term refers to membrane-bound (e.g., tethered to the cell surface) 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-presenting molecules expressed on the cell surface of certain cells. GARP-precursor TGFβ1 and LRRC33-precursor TGFβ1 complexes can be collectively referred to as “cell-associated” (or “cell surface”) precursor TGFβ1 complexes that mediate cell-associated (e.g., immune cell-associated) TGFβ1 activation / signaling. The 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 the cell membrane. The mean KD values ​​of antibodies (or fragments thereof) against the GARP-precursor TGFβ1 complex and the LRRC33-precursor TGFβ1 complex can be calculated to comprehensively 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 "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 the cell-tethered complex, cell-associated precursor TGFβ1 may be targets for internal translocation (e.g., endocytosis) and / or cell death, e.g., ADCC, ADCP, or ADC-mediated depletion of target cells expressing such cell surface complexes.

[0044] Checkpoint Inhibitors: In the context of this disclosure, checkpoint inhibitors mean immune checkpoint inhibitors and have the meanings understood in the art. Typically, the targets are receptor molecules on T cells or NK cells, or corresponding cell surface ligands on antigen-presenting cells (APCs) or tumor cells. Immune checkpoints are activated in immune cells to prevent inflammatory immunity that arises against "self." Therefore, altering the balance of the immune system through checkpoint inhibition should allow for their full activation, enabling the detection and elimination of cancer. The most well-known inhibitory receptors linked to the regulation of the immune response are cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), PD-L1, T cell immunoglobulin domain and mucin domain-3 (TIM3), lymphocyte activation gene 3 (LAG3), killer cell immunoglobulin-like receptor (KIR), glucocorticoid-induced tumor necrosis factor receptor (GITR), and V-domain immunoglobulin (Ig)-containing inhibitor of T cell activation (VISTA). Non-exclusive examples of checkpoint inhibitors include nivolumab, pembrolizumab, BMS-936559, atezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, IMP-321, BMS-986016, and lirirumab. Keytruda® is an example of a PD-1 inhibitor. Treatment using one or more immune checkpoint inhibitors may be called checkpoint blockade therapy (CBT).

[0045] Clinical Benefits: As used herein, the term “clinical benefits” is intended to include both the efficacy and safety of a treatment. Therefore, a treatment procedure that achieves desirable clinical benefits is both effective (e.g., achieving a therapeutically beneficial effect) and safe (e.g., having tolerable or acceptable levels of toxicity or adverse events).

[0046] Combination therapy: “Combination therapy” refers to a treatment regimen for a clinical indication that includes two or more therapeutic agents. Therefore, this term refers to a treatment regimen in which a first treatment, containing a first composition (e.g., an active ingredient), is administered to a patient in combination with a second treatment, containing a second composition (an active ingredient) intended to treat the same or overlapping disease or clinical condition. Both the first and second compositions may act on the same or distinct cellular targets. In the context of combination therapy, the phrase “in combination with” means that, in a subject receiving combination therapy, the therapeutic effect of the first treatment overlaps temporally and / or spatially with the therapeutic effect of the second treatment. Therefore, combination therapy may be formulated as a single formulation for simultaneous administration or as separate formulations for sequential administration of the treatments. When a subject treated with a first treatment for a disease is administered a second treatment for the same disease, the second treatment may be called an adjunct or auxiliary therapy.

[0047] Combinatri or Combinatrial Epitope: A combinatrial epitope is an epitope that is recognized and bound by a combinatrial antibody at a site (i.e., an antigenic determinant) formed by non-adjacent portions of one or more components of an antigen, which are close together and aggregate to form an epitope in three-dimensional structure. Therefore, the antibody of the present invention may bind to an epitope formed by two or more components (e.g., a portion or segment) of the precursor / latent TGFβ1 complex. A combinatrial epitope may include amino acid residues from a first component of the complex, and amino acid residues from a second component of the complex, and so on. Each component may be from a single protein or two or more proteins of the antigen complex. A combinatrial epitope is formed by structural contributions from two or more components (e.g., a portion or segment, e.g., amino acid residues) of the antigen or antigen complex.

[0048] Competitive or cross-competitive, cross-blocking: When used in the context of antigen-binding proteins (e.g., antibodies or their antigen-binding portions) competing for the same epitope, the term “competitive” means competition between antigen-binding proteins, as determined by an assay in which the antigen-binding protein under test prevents or inhibits (e.g., reduces) the specific binding of the 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 (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays, solid-phase direct biotin-avidin EIAs, solid-phase direct labeling assays, and solid-phase direct labeling sandwich assays. Typically, an excess of competing antigen-binding proteins inhibits (e.g., reduces) the specific binding of the reference antigen-binding protein to the common antigen by at least 40–45%, 45–50%, 50–55%, 55–60%, 60–65%, 65–70%, 70–75%, or more than 75%. In some cases, binding is inhibited by at least 80–85%, 85–90%, 90–95%, 95–97%, or more than 97%.

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

[0050] Antibody binning (sometimes called epitope binning or epitope mapping) can be performed to characterize and sort a set of monoclonal antibodies (e.g., a “library”) made against a target protein or protein complex (i.e., an antigen). Such antibodies against the same target are tested in pairs against all other antibodies in the library to assess whether the antibodies block each other’s binding to the antigen. A closely related binning profile indicates that antibodies have the same or closely related (e.g., overlapping) epitopes and are “binned” together. Binning provides a useful structure-function profile of antibodies that share similar binding regions within the same antigen, because the biological activity (e.g., intervention, potency) resulting from the binding of an antibody to its target is likely to carry over to another antibody in the same bin. Thus, among antibodies in the same epitope bin, those with higher affinity (lower KD) typically have higher potency.

[0051] Complementarity-Determining Regions: As used herein, the term "CDR" refers to the complementarity-determining regions within the antibody variable sequence. Three CDRs exist in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3 for each variable region. As used herein, a "CDR pair" refers to a group of three CDRs occurring within a single variable region capable of binding to an antigen. The precise 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, Maryland)) not only provides an obvious residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining three CDRs. These CDRs can be called Kabat CDRs. Chothia and collaborators (Chothia and Lesk (1987), J.Mol.Biol., 196:901-917 and Chothia et al., (1989), Nature, 342:877-883) described Kabat We found that certain sub-regions within the CDR adopt nearly identical peptide backbone conformations despite exhibiting high diversity at the amino acid sequence level. These sub-regions are named 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 called 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):732-745.Furthermore, other CDR boundary definitions may overlap with Kabat CDRs, even though they may not strictly adhere to one of the systems described herein; however, they may be shortened or lengthened in light of predictions or experimental findings that specific residues or groups of residues or even the entire CDR do not significantly affect antigen binding (see, for example, Lu X et al., MAbs., January 2019, 11(1):45–57). The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use CDRs defined by Kabat or Chothia.

[0052] Conformational epitopes: Conformational epitopes are epitopes that are recognized and bound by conformational antibodies in a three-dimensional conformation, but not in unfolded peptides with the same amino acid sequence. Conformational epitopes can also be called conformation-specific epitopes, conformation-dependent epitopes, or conformation-sensitive epitopes. The corresponding antibodies or fragments that specifically bind to such epitopes can be called conformation-specific antibodies, conformation-selective antibodies, or conformation-dependent antibodies. Binding of an antigen to a conformational epitope depends on the three-dimensional structure (conformation) of the antigen or antigen complex.

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

[0054] Context-biased: As used herein, “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 to or attached to) an interacting protein or a fragment thereof. Therefore, 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 its affinity for matrix-associated precursor TGFβ1 complexes (e.g., LTBP1-precursor TGFβ1 and LTBP3-precursor TGFβ1) is higher than its affinity for cell-associated precursor TGFβ1 complexes (e.g., GARP-precursor TGFβ1 and LRRC33-precursor TGFβ1). [Matrix-associated complex]: The relative affinity of a [cell-associated complex] is the mean K of the former, as illustrated herein. D The values ​​are taken, and the latter average K D It can be obtained by taking values ​​and calculating the ratio between them.

[0055] Context-Independent: According to this disclosure, “context-independent antibodies” that bind to precursor TGFβ1 have equivalent affinity across four known presentation molecule-precursor TGFβ1 complexes, namely LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1. The context-independent antibodies disclosed in this application can also be characterized as unbiased. Typically, context-independent antibodies exhibit equivalent affinity (i.e., bias of 5-fold or less) such that the relative ratio of measured KD values ​​between the matrix-associated complex and the cell-associated complex does not exceed 5, as measured by appropriate in vitro binding assays such as surface plasmon resonance, biolayer interferometry (BLI), and / or solution equilibrium titration (MSD-SET, etc.).

[0056] ECM-related TGFβ1 / precursor TGFβ1: This term refers to TGFβ1, which is a component of the extracellular matrix (e.g., accumulated therein), or its signaling complex (e.g., precursor / latent TGFβ1). TGFβ1 presented by LTBP1 or LTBP3 is ECM-related TGFβ1, namely, LTBP1-precursor TGFβ1 and LTBP3-precursor TGFβ1, respectively. LTBP is crucial for the proper accumulation and subsequent bioavailability of TGFβ in the ECM, where fibrillin (Fbn) and fibronectin (FN) are considered to be the main substrate proteins that mediate the association of LTBP with the ECM. Such matrix-related latent complexes are enriched in connective tissues, as well as in certain disease-related tissues such as tumor stroma and fibrotic tissues. The human counterparts of the presenting molecule or presenting molecule complex may be denoted by placing "h" in front of 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 dosage) 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 at dosages as low as 3 mg / kg and as high as 30 mg / kg in preclinical models. Thus, it can be said that the effective amount of Ab6 is 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 has regressed by a defined percentage (<25% etc.) of the endpoint tumor volume. For example, in a particular model, if the endpoint tumor volume is set at 2,000 mm 3 and assuming a <25% threshold, 500 mm 3Effective tumor control is achieved when the tumor size is reduced to less than [a certain level]. Therefore, effective tumor control encompasses complete regression. Clinically, effective tumor control includes partial response (PR) and complete response (CR) based on criteria understood in the art, such as RECIST 1.1 and the corresponding iRECIST. In some embodiments, effective tumor control in a clinical setting also includes stable disease, where the tumor is typically expected to grow at a certain rate, but such growth is prevented by treatment, if reduction 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 co-stimuli, 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 leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as 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). After activation, they rapidly divide and secrete small proteins called cytokines that regulate or support the active immune response. These cells can differentiate into one of several subtypes, including Th1, Th2, Th3, Th17, Th9, or TFh, which secrete different cytokines to promote different types of immune responses. Signaling from APCs directs T cells to specific subtypes: cytotoxic (killer). On the other hand, cytotoxic T cells (TC cells, CTLs, T killer cells, killer T cells) destroy virus-infected cells and cancer cells, and are also linked to 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 to antigens associated with MHC class I molecules present on the surface of all nucleated cells. Examples of cytotoxic effector cells (e.g., CD8+ cells) include perforin and granzyme B.

[0060] Epitope: The term “epitope” is sometimes also called an antigenic determinant and is a molecular determinant (e.g., polypeptide determinant) that can be specifically bound by a binder, immunoglobulin, or T cell receptor. Epitope determinants include surface groups of chemically active molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and in certain embodiments may have specific three-dimensional structural features and / or specific charge features. An epitope recognized by an antibody or an antigen-binding fragment of an antibody is a structural element of the antigen that interacts with the CDR (e.g., complementary site) of the antibody or fragment. An epitope may be formed by contributions from several amino acid residues that interact with the antibody's CDR to produce specificity. An antigenic fragment may 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, when antibodies cross-compete (one prevents the binding of the other or modulates its effect), antibodies are said to “bind to the same epitope.”

[0061] Equivalent Affinity: In the context of this disclosure, the term “equivalent affinity” means that the relative affinity of the antibody to the four complexes is uniform, either i) the antibody binds to the matrix-associated precursor TGFb1 complex and the cell-associated precursor TGFb1 complex with an affinity bias of less than 5 times, as measured by appropriate in vitro binding assays such as solution equilibrium titration (MSD-SET, etc.), biolayer interferometry (Octet®, etc.), or surface plasmon resonance (Biacore System, etc.), and / or ii) the lowest affinity (highest KD value) the antibody exhibits among the four antigen complexes is no more than 5 times lower than the average value calculated from the remaining three affinities, or the highest affinity (lowest KD value) the antibody exhibits among the four antigen complexes is no more than 5 times higher than the average value calculated from the remaining three affinities. Antibodies with equivalent affinity can achieve a more uniform inhibitory effect (hence "context-independent"), regardless of the specific presenting molecule associated with the precursor TGFβ1 complex. In a particularly preferred embodiment, the bias observed in the average affinity between the matrix-associated complex and the cell-associated complex is less than 3 times.

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

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

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

[0065] Finger-1 (of TGFβ1 growth factor): As used herein, "Finger-1" refers to 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 3D structure, the Finger-1 domain (partially shown as region "5a" in Figures 18 and 19) is in close proximity to latent lasso.

[0066] Finger-2 (of TGFβ1 growth factor): As used herein, "Finger-2" refers to 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 includes a "binding region 6" (i.e., "6a" and "6b"), as shown in Figures 18 and 19, which is spatially located in close proximity to the latent lasso.

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

[0068] High affinity: As used herein, the term "high affinity" refers to a K2 antibody with an affinity of ≤5nM, more preferably ≤1nM. D This refers to in vitro binding activity having a value. Therefore, the high affinity context-independent precursor TGFβ1 antibodies included herein by the present invention have a K value 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 Antibodies: 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 this disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDR, particularly in 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 mouse, have been transplanted onto a human framework sequence.

[0070] Humanized Antibodies: The term "humanized antibody" refers to an antibody that contains variable region sequences of heavy and light chains from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences has been modified to be more "human-like," i.e., more similar to the variable sequences of the human germline. One type of humanized antibody is a CDR-implanted antibody, in which a human CDR sequence is introduced into the non-human VH and VL sequences, replacing the corresponding non-human CDR sequence. Furthermore, "humanized antibody" refers to an antibody, or its variant, derivative, analogue, or fragment, that immunospecifically binds to a target antigen and contains an FR region having substantially the amino acid sequence of a human antibody and a CDR region having substantially the amino acid sequence of a non-human antibody. In the context of CDRs, the term "substantially" as used herein means 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 contains substantially all of at least one, typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin (i.e., donor antibody), and all or substantially all of the FR region is of the human immunoglobulin consensus sequence. In one embodiment, the humanized antibody also contains at least a portion of the immunoglobulin Fc region, typically of a human immunoglobulin. In some embodiments, the humanized antibody contains variable domains of the light chain and at least the 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 the humanized light chain. In some embodiments, the humanized antibody contains only the humanized heavy chain. In specific embodiments, the humanized antibody contains only the humanized variable domain of the light chain and / or the 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 investigate protein-protein interactions by measuring the degree of solvent accessibility. See, for example, Wei et al., (2014), Drug Discov Today, 19(1):95-102, "Hydrogen / deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications." The HDX-MS technique can be used to determine one or more regions of an antigen to which an antibody binds (i.e., "binding regions"). Such binding regions may contain or form epitopes.

[0072] Immune-excluded or immuno-excluded tumors: As used herein, tumors characterized as “immune-excluded” are those lacking or substantially lacking intratumor anti-tumor lymphocytes. For example, a tumor with poor T-cell infiltration may have T cells surrounding the tumor, e.g., on the periphery of the tumor mass and / or near the tumor’s vascular structure (“perivascular”), but nevertheless fail to effectively invade the tumor and exert cytotoxic function against cancer cells. In other situations, the tumor fails to elicit a potent immune response, with only a small number of T cells present near and within the tumor environment (so-called “cold” or “immune desert” tumors). In contrast to immune-excluded tumors, tumors with infiltrated 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 fraction of patients respond to CBT due to immune excludation that makes the tumor resistant to CBT.

[0073] Immunosuppression, immunosuppressiveness: This term refers to the ability to suppress immune cells such as T cells, NK cells, and B cells. The optimal criterion for evaluating immunosuppressive function is inhibition of T cell activity, which may include antigen-specific and non-specific suppression. Regulatory T cells (Tregs) and MDSCs can be considered immunosuppressive cells. M2-polarized macrophages (e.g., disease-specific macrophages such as TAM and FAM) can also be characterized as immunosuppressive.

[0074] Immunotherapy: Immunotherapy refers to the immune system's ability to quickly and specifically recognize previously encountered antigens and initiate a corresponding immune response. Generally, these are secondary, tertiary, and other subsequent immune responses to the same antigen. Immunotherapy is responsible for specialized T cells and B cells, so-called memory T cells and B cells, which are adaptive components of the immune system. Antigen-naive T cells expand and differentiate into memory and effector T cells after encountering their congener antigen in the context of MHC molecules on the surface of professional antigen-presenting cells (e.g., dendritic cells). The single unifying theme of all memory T cell subtypes is their longevity and their ability to rapidly expand into numerous effector T cells upon re-exposure to their congener antigen. This mechanism allows them to provide the immune system with a "memory" of previously encountered pathogens. Memory T cells can be either CD4+ or CD8+, and typically express CD45RO. In preclinical settings, immunotherapy can be tested in the tumor reinduction paradigm.

[0075] Isoform-Specific: The term "isoform-specific" refers to a drug's ability to distinguish one isoform from other structurally related isoforms with preference (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 isoforms of TGFβ. For example, an isoform-specific TGFβ1 antibody selectively binds to TGFβ1. A TGFβ1-specific inhibitor (antibody) preferentially targets (binds to and thereby inhibits) the TGFβ1 isoform with substantially higher affinity than TGFβ2 or TGFβ3. For example, selectivity in this context may refer to a difference of at least 500 to 1000 times in affinity, as measured by in vitro binding assays such as Octet® and Biacor. In some embodiments, selectivity means that when the inhibitor is used at a dose effective to inhibit TGFβ1 in vivo, TGFβ2 and TGFβ3 are not inhibited. For such inhibitors to be effective as therapeutic agents, the dosage required to achieve the desired effect (e.g., a therapeutically effective dose) 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, “isolated” antibody means an antibody that is substantially free from other antibodies having different antigen specificities. In some embodiments, the isolated antibody is substantially free from other unintended cellular material and / or chemicals.

[0077] Large Latent Complexes: In the context of this disclosure, the term “Large Latent Complex” (“LLC”) refers to a complex consisting of a precursor TGFβ1 dimer bound to a so-called presenting molecule. Therefore, Large Latent Complexes are presenting 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 forming the complex. For screening purposes, the presenting molecule used to form such LLCs does not need to be a full-length polypeptide. However, a protein moiety capable of forming a disulfide bond with the precursor TGFβ1 dimer complex via cysteine ​​residues near its N-terminal region is typically required.

[0078] Latent-related 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 latent lasso domain.

[0079] Latent Lasso: As used herein, “latent lasso,” sometimes also called a latent loop, is a domain within the prodomain of precursor TGFb1 adjacent to the alpha-1 helix and arm. In its unmutated form, the latent lasso of human precursor TGFβ1 contains the amino acid sequence LASPPSQGEVPPGPL (SEQ ID NO: 153), substantially corresponding to regions “2a” and “2b” shown in Figure 18A, and spanning region 1 identified in Figure 19A. As used herein, the term “extended latent lasso region” refers to the latent lasso combined with the motif immediately C-terminal to the prodomain, called the alpha-2 helix (α2-helix). The proline residue at the C-terminus of the latent lasso provides a “elbow-like” vertical “turn” that connects the lasso loop to the α2-helix. The extended latent lasso includes the regions shown as “2a,” “2b,” and “2c” in Figures 18 and 19. Certain high-affinity TGFβ1 activating inhibitors bind at least partially to a latent lasso or a portion thereof to confuse inhibitory activity (e.g., the ability to block activation), wherein optionally, the portion of the latent lasso is ASPPSQGEVPPGPL (SEQ ID NO: 266). In some embodiments, the antibodies of this disclosure bind to the precursor TGFβ1 complex at ASPPSQGEVPPGPL (SEQ ID NO: 266) or a portion thereof. Certain high-affinity TGFβ1 activating inhibitors bind at least partially to an extended latent lasso or a portion thereof to confuse inhibitory activity (e.g., the ability to block activation), wherein optionally, the portion of the extended latent lasso is KLRLASPPSQGEVPPGPLPEAVL (SEQ ID NO: 169).

[0080] Localized: In the context of this disclosure, the term “localized” (as in “localized tumor,” “disease-localized,” etc.) means an anatomically isolated or isolateable abnormality, such as a solid malignant tumor, as a contrast to a systemic disease. Certain leukemias may have both disease-localized components (e.g., bone marrow) and systemic components (e.g., circulating blood cells).

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

[0082] LTBP1-Progenitor TGFβ1 Complex: As used herein, the term “LTBP1-TGFβ1 complex” refers to a protein complex comprising a precursor protein form 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 includes LTBP1 covalently bonded to the precursor / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed using a cysteine ​​residue located near the N-terminus (e.g., amino acid position 4) of the precursor TGFβ1 dimer complex. In other embodiments, the LTBP1-TGFβ1 complex includes LTBP1 non-covalently bonded to the precursor / latent TGFβ1. In some embodiments, the LTBP1-TGFβ1 complex is a naturally occurring complex, such as the LTBP1-TGFβ1 complex in cells. The term “hLTBP1” refers to human LTBP1.

[0083] LTBP3-Progenitor TGFβ1 Complex: As used herein, the term “LTBP3-TGFβ1 complex” refers to a protein complex comprising a precursor protein form 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 bonded to the precursor / latent TGFβ1 via one or more disulfide bonds. In nature, such a covalent bond is formed using a cysteine ​​residue located near the N-terminus (e.g., amino acid position 4) of the precursor TGFβ1 dimer complex. In other embodiments, the LTBP3-TGFβ1 complex comprises LTBP1 non-covalently bonded to the precursor / latent TGFβ1. In some embodiments, the LTBP3-TGFβ1 complex is a naturally occurring complex, such as the LTBP3-TGFβ1 complex in cells. The term “hLTBP3” refers to human LTBP3.

[0084] M2 or M2-like macrophages: M2 macrophages represent a subset of activated or polarized macrophages, including disease-associated macrophages in both fibrous and tumor microenvironments. Typical cell surface markers for M2-polarized macrophages include CD206 and CD163 (i.e., CD206+ / CD163+). M2-polarized macrophages may also express cell surface LRRC33. Activation of M2 macrophages is primarily promoted by IL-4, IL-13, IL-10, and TGFβ. These secrete the same cytokines that activate them (IL-4, IL-13, IL-10, and TGFβ). These cells have high phagocytic capacity and produce ECM components, angiogenic and chemotactic factors. TGFβ release by macrophages can perpetuate myofibroblast activation and induce EMT and EndMT in diseased tissues such as fibrous tissue and tumor stroma. For example, M2 macrophages are essential for TGFβ-driven pulmonary fibrosis and are also abundant in several 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 the “ECM-associated” (or “matrix-associated”) precursor TGFβ1 complex, which mediates the activation / signaling of ECM-associated TGFβ1. This term also includes recombinant, purified LTBP1-precursor TGFβ1 and LTBP3-precursor TGFβ1 complexes in solution (e.g., in in vitro assays) that are not physically attached to the matrix or substrate.

[0086] Maximum Tolerable 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 of Ab6 in rats was the highest evaluated dose (100 mg / kg), which suggests that the MTD of Ab6 is >100 mg / kg based on a 4-week toxicological study. The NOAEL of Ab6 in non-human primates was the highest evaluated dose (300 mg / kg), which suggests that the MTD of Ab6 in non-human primates is >300 mg / kg based on a 4-week toxicological study.

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

[0088] Myelofibrosis: Also known as osteomyelofibrosis, "myelofibrosis" is a relatively rare myeloproliferative disorder (e.g., cancer) belonging to a group of diseases called myeloproliferative disorders. Myelofibrosis is classified in the Philadelphia chromosome-negative (-) section 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 called chronic idiopathic myelofibrosis (cIMF) (the terms idiopathic and primary mean that in these cases the disease is of unknown or spontaneous origin), as well as secondary forms of myelofibrosis, such as myelofibrosis that occurs secondarily in polycythemia vera (PV) or essential thrombocythemia (ET). Myelofibrosis is a form of myelometaplasia that refers to changes in the cell types in the hematopoietic tissue of the bone marrow, and the two terms are often used synonymously. The terms idiopathic myelometaplastic syndrome and myelofibrosis with myelometaplastic syndrome (MMM) are also used to refer to primary myelofibrosis. Myelofibrosis is characterized by mutations that cause upregulation or hyperactivation of the downstream JAK pathway.

[0089] Myelocytes: In hematopoiesis, myelocytes are blood cells arising from granulocytes, monocytes, erythrocytes, or platelet progenitor cells (common myelocyte precursors, i.e., CMP or CFU-GEMM), or, in a narrower sense also frequently used, from lymphocytes, i.e., from a lineage of myeloblasts (myelocytes, monocytes, and their daughters) distinct from lymphocytes, i.e., lymphocytes derived from common lymphoid progenitor cells that give rise to B cells and T cells. The specific types of myelocytes in both mouse and human, their general morphology, typical cell surface markers, and their immunosuppressive capabilities are summarized below. JPEG0007858715000001.jpg179166 JPEG0007858715000002.jpg122166

[0090] Myelo-derived suppressor cells (MDSCs) are a heterogeneous population of cells generated during various pathological conditions and are thought to represent the pathological conditions of activation of monocytes and relatively immature neutrophils. MDSCs include at least two categories of cells: i) “granulocytic” (G-MDSCs) or polymorphonuclear (PMN-MDSCs) that are phenotypic and morphologically similar to neutrophils, and ii) monocytic (M-MDSCs) that are phenotypic and morphologically similar to monocytes. MDSCs are characterized by distinctly different sets of genomic and biochemical features and can be identified 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 such 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 may include inhibition of antigen-nonspecific function and inhibition of antigen-specific function. MDSCs can express cell surface LRRC33 and / or LRRC33-precursor TGFβ1.

[0091] Myofibroblasts: Myofibroblasts are cells that possess specific phenotypes of fibroblasts and smooth muscle cells, and generally express vimentin, alpha-smooth muscle actin (α-SMA, human gene ACTA2), and paladins. In many pathological conditions involving dysregulation of the extracellular matrix (such as increased matrix rigidity), normal fibroblasts are dedifferentiated into myofibroblasts in a TGFβ-dependent manner. Abnormal overexpression of TGFβ is commonly seen in myofibroblast-driven pathology. TGFβ is known to promote myofibroblast differentiation, cell proliferation, and matrix formation. Myofibroblasts or myofibroblast-like cells in the fibrous microenvironment may be called fibrosis-associated fibroblasts (or "FAFs"), and myofibroblasts or myofibroblast-like cells in the tumor microenvironment may be called cancer-associated fibroblasts (or "CAFs").

[0092] Pan-TGFβ Inhibitors / Pan-Inhibition of TGFβ: 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. The term includes pan-TGFβ antibodies, which refer to any antibody capable of binding to each of the TGFβ isoforms, namely TGFβ1, TGFβ2, and TGFβ3. In some embodiments, pan-TGFβ antibodies bind to all three isoforms, namely TGFβ1, TGFβ2, and TGFβ3, and neutralize their activity. Antibody 1D11 (or its human analog fresolimmab (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 garnicertive (LY2157299 monohydrate), an antagonist of TGFβ receptor I kinase / ALK5, which mediates the signaling of all three TGFβ isoforms.

[0093] Perivascular (infiltration): The prefix "peri-" means "around," "surrounding," or "nearby," and therefore "perivascular" is literally interpreted as being around blood vessels. As used herein, in the context of tumor cell infiltration, the term "perivascular infiltration" refers to the mechanism by which tumor-infiltrating immune cells (e.g., lymphocytes) enter through the vascular structure of a solid tumor.

[0094] Efficacy: As used herein, "efficacy" refers to the activity of a drug, such as an inhibitory antibody (or fragment), with respect to the concentration or amount of the drug required to produce a defined effect. For example, an antibody that can produce a particular effect at a given dose is more potent than another antibody that requires twice the amount (dose) to produce an equivalent effect. Efficacy may be measured in cell-based assays, such as TGFβ activation / inhibition assays, which allow for the measurement of the degree of TGFβ activation, such as activation initiated by integrin binding, in the presence or absence of the test substance (e.g., inhibitory antibody) in a cell-based system. Typically, among those that can bind to the same or overlapping binding regions of an antigen (e.g., cross-blocking antibodies), higher affinity (lower K) is indicated. D Antibodies with a lower affinity (higher K) D It tends to show higher efficacy than antibodies with a specific value.

[0095] Presenting molecule: In the context of this disclosure, a presenting molecule is 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 (such as the ECM or the surface of an immune cell), thereby maintaining its latent state until an activation event occurs. Known presenting molecules for precursor TGFβ1 include LTBP1, LTBP3, GARP, and LRRC33, each of which can form a presenting molecule-precursor TGFβ1 complex (i.e., LLC), namely 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-related" (or "matrix-related") precursor TGFβ1 complexes that mediate ECM-related 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 be collectively referred to as "cell-related" (or "cell surface") precursor TGFβ1 complexes that mediate cell-related (e.g., immune cell-related) TGFβ1 signaling / activity.

[0096] Protection (from solvent exposure): In the context of HDX-MS-based evaluation 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 a solvent, thereby enabling proton exchange, is inversely correlated with the degree of binding / interaction. Therefore, since protein-protein interactions exclude accessibility by the surrounding solvent, when an antibody described herein binds to a region of an antigen, the binding region is "protected" from exposure to the solvent. Thus, the protected region is an indicator of the site of interaction. Typically, a suitable solvent is a physiological buffer.

[0097] Progenitor TGFβ1: As used herein, the term “progenitor TGFβ1” is intended to encompass the precursor form of the inactive TGFβ1 complex, which contains the prodomain sequence of TGFβ1 within the complex. Therefore, this term can include both the precursor and latent forms of TGFβ1. The expressions “progenitor / latent TGFβ1” can be used interchangeably. The “progenitor” form of TGFβ1 exists before proteolytic cleavage at the furin site. After cleavage, the resulting form is said to be the “latent” form of TGFβ1. The “latent” complex remains non-covalently associated until further activation is initiated, such as an integrin-driven activation event. The precursor TGFβ1 complex consists of a dimeric TGFβ1 precursor protein polypeptide linked by disulfide bonds. The latent dimeric complex is covalently linked to a single presenting molecule via a cysteine ​​residue at position 4 (Cys4). The adjective "latent" can be commonly / broadly used 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 tumor or tumor growth can be used as a measure of in vivo effectiveness. For example, in a preclinical setting, criteria for the effectiveness of a regression response treatment can be determined from the tumor volume of animals remaining in the study on the final day. The effectiveness of a treatment can also be determined from the incidence and magnitude of regression responses observed during the study. A treatment can induce partial regression (PR) or complete regression (CR) of the tumor in animals. Complete regression achieved in response to treatment (e.g., drug administration) may be called a “complete response,” and subjects that achieve a complete response may be called “complete responders.” Thus, a complete response excludes spontaneous complete regression. In some embodiments of preclinical tumor models, a PR response is defined as three consecutive measurements being less than 50% of the day 1 volume during the course of the study, and one or more of these three measurements being 13.5 mm. 3This is defined as the tumor volume being greater than or equal to 13.5 mm over three consecutive measurements during the course of the study. In some embodiments, the CR response is 13.5 mm over three consecutive measurements during the course of the study. 3 This is defined as a tumor volume less than 2,000 mm³. In preclinical models, animals with a complete response (CR) 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, tumor volume is reduced to <25% of the endpoint tumor volume in response to treatment. For example, in a particular model, the endpoint tumor volume is 2,000 mm³. 3 If the tumor is 500 mm 3 Effective tumor control is achieved when the threshold is reduced to below a certain level. Therefore, effective tumor control includes both complete regression and partial regression that reaches a lower threshold.

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

[0100] Resistance (to treatment): Resistance to a particular treatment (such as CBT) may be due to an innate characteristic of the disease, such as cancer ("primary resistance"), or to an acquired phenotype that develops over time after treatment ("acquired resistance"). Patients who do not respond to treatment (e.g., those who are unresponsive or poorly responsive to treatment) are said to have primary resistance to treatment. Patients who initially respond to treatment but later lose its effectiveness (e.g., progress or relapse despite continued treatment) are said to have acquired resistance to treatment.

[0101] The Response Evaluation Criteria in Solid Tumors (RECIST) and iRECIST: RECIST is a set of published rules that define when a tumor in a cancer patient improves ("response"), remains the same ("stabilization"), or worsens ("progression") during treatment. These criteria were published in February 2000 through an international collaboration including the European Organisation 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. Subsequently, a revised version of the RECIST guideline (RECIST v1.1) has been widely applied (see Eisenhauera et al., (2009), "New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1)," Eur J Cancer, 45: pp. 228-247, incorporated herein).

[0102] The response criteria are as follows: Complete response (CR): disappearance of all target lesions; Partial response (PR): at least 30% reduction in the total LD ​​of target lesions, with reference to baseline total LD; Stable disease (SD): no reduction sufficient to qualify as PR, nor an increase sufficient to qualify as PD, with reference to the lowest total LD ​​since the start of treatment; Progressive disease (PD): at least 20% increase in the total LD ​​of target lesions, with reference to the lowest total LD ​​recorded since the start of treatment or the appearance of one or more new lesions.

[0103] On the other hand, iRECIST provides a modified set of criteria that take immune-related responses into account (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 and may be revised from time to time as further data becomes available, and are well understood in the art.

[0104] Solid Tumors: The term “solid tumor” refers to a proliferative disorder that typically produces an abnormal growth or tissue mass that does not contain a cyst or fluid region. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Solid tumors include locally advanced solid tumors and advanced malignant tumors such as metastatic carcinomas. Solid tumors are typically composed of multiple cell types, including, but are not limited to, cancerous (malignant) cells, stromal cells such as CAFs, and invasive leukocytes such as macrophages, MDSCs, and lymphocytes. Solid tumors treated with isoform-selective inhibitors of TGFβ1, 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., be TGFβ3-positive). For example, certain tumors are TGFβ1 / 3 codominant. In some embodiments, such tumors are caused by epithelial cell carcinomas, e.g., 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, SET based on mesoscale discovery ("MSD"), or MSD-SET, is a useful form for determining the dissociation constant at equilibrium 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). Assays based on SET involve the K of antibodies with affinity of nanomolar or less (e.g., picomolar). D It is particularly useful for determining values.

[0106] Specific Binding: As used herein, the term “specific binding” or “specifically binding” means that the interaction between an antibody or its antigen-binding moiety and an antigen depends on the presence of a specific structure (e.g., an antigenic determinant or epitope). For example, an antibody or its antigen-binding moiety binds to a specific protein rather than to proteins in general. In some embodiments, an antibody or its antigen-binding moiety binds to a K target. D at least about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 If the value is M or less, it specifically binds to the target, for example, TGFβ1. In some embodiments, the terms used herein, “specific binding to the precursor TGFβ1 epitope,” “specific binding to the precursor TGFβ1 epitope,” “specific binding to precursor TGFβ1,” or “specific binding to precursor TGFβ1,” mean binding to precursor TGFβ1 and determined by a suitable in vitro binding assay such as surface plasmon resonance and biolayer interferometry (BLI) to a value of 1.0 × 10⁶. -8 Dissociation constants (K) less than or equal to M DThis refers to an antibody or its antigen-binding moiety having ). In one embodiment, the antibody or its antigen-binding moiety can specifically bind to both human and non-human (e.g., mouse) homologous species 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 or diagnosis. Appropriate subjects include, but are not limited to, vertebrates, but include mammals (e.g., humans and non-human mammals). When the subject is a human subject, the term "patient" may be used interchangeably. In a clinical context, the terms "patient population" or "patient subpopulation" are 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 specific conditions, responsiveness to treatment), medical history, health status, sex, age group, genetic criteria (e.g., carrier of specific mutations, polymorphism, gene duplication, DNA sequence repeats), and lifestyle factors (e.g., smoking, alcohol consumption, exercise).

[0108] Surface plasmon resonance (SPR): Surface plasmon resonance is an optical phenomenon that enables the real-time detection of unlabeled interacting molecules. Using SPR-based biosensors, such as those sold by Biacore, it is possible to measure the interactions of biomolecules, 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, velocity constant, and thermodynamics.

[0109] TGFβ1-related indications: “TGFβ1-related indications” means any disease or disorder and / or condition in which at least part of the pathogenesis and / or progression may be attributable to dysregulation of TGFβ1 signaling or its regulation. Certain TGFβ1-related disorders are predominantly driven by TGFβ1 isoforms. Subjects with TGFβ1-related indications may benefit from inhibition of 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 fibrosis of tissues involved in chronic inflammation), proliferative disorders (such as cancer, e.g., solid tumors and myelofibrosis), diseases associated with ECM dysregulation (such as conditions involved in matrix sclerosis and remodeling), diseases involved in 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 Inhibitors: The term "TGFβ inhibitor" refers to any agent capable of antagonizing the biological activity, signaling, or function of TGFβ growth factor (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 scavengers, and TGFβ activation inhibitors. Examples of TGFβ inhibitors include antibodies that can reduce the availability of latent precursor TGFβ that can be activated in a niche by inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADPC), as well as antibodies that can remove the precursor from the plasma membrane without depleting the cell itself by causing internal translocation of the cell surface complex containing latent precursor TGFβ. Internal translocation can be a suitable mechanism of action for LRRC33-containing protein complexes (such as human LRRC33-precursor TGFβ1) that results in a decrease in the level of cells expressing the LRRC33-containing protein complex on the cell surface.

[0111] The "TGFβ family" is a class within the TGFβ superfamily, and in humans, it includes three structurally similar members: TGFβ1, TGFβ2, and TGFβ3. These three growth factors are known to signal via the same receptor.

[0112] TGFβ1-positive cancer / tumor: As used herein, this term refers to cancer / tumors that have abnormal TGFβ1 expression (overexpression). Many human cancer / tumor species exhibit dominant expression of the TGFβ1 isoform (note that "TGFB" may be used to refer to the gene as a control of the protein). 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, the preclinical cancer / tumor model that outlines the human condition is a TGFβ1-positive cancer / tumor.

[0113] Therapeutic window: The term "therapeutic window" refers to the range of drug doses that produce a therapeutic response without causing significant / observable / unacceptable adverse effects (e.g., within the range of tolerable or acceptable adverse effects) in a subject. 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 dose of 10 mg / kg and exhibits tolerable or tolerable 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 below the effective dose is said to have "dose-restricted toxicity." Generally, the maximum tolerable 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 for 4 weeks at dosages ranging from approximately 3 to 30 mg / kg / week, and no observable toxicity associated with general inhibition of TGFβ has been shown at dosages of at least 100 or 300 mg / kg / week. Based on this, Ab6 exhibits a therapeutic window of at least 3.3 times and up to 100 times.

[0115] Toxicity: As used herein, “toxicity” or “multiple toxicity” means an undesirable in vivo effect in a subject (e.g., a patient) associated with a treatment administered to that subject (e.g., a patient), such as undesirable side effects and adverse events. “Tolerability” means the level of toxicity associated with a treatment or treatment regimen that can be moderately tolerated by a patient without discontinuing treatment due to toxicity. Typically, toxicology / toxicological 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 / toxicological studies may help determine the “no-observed-adverse-effect level (NOAEL)” and “maximum tolerable dose (MTD)” of the test substance, on which the treatment window may be estimated. Preferably, species that have been shown to be susceptible to a particular intervention should be selected as preclinical animal models for conducting safety / toxicity studies. In the case of TGFβ inhibition, suitable species include rats, dogs, and cynomolgus monkeys. Mice have been reported to be less sensitive to the pharmacological inhibition of TGFβ, and may not reveal potentially dangerous toxicity in other species, including humans; however, certain studies have reported toxicity observed in mice with pan-inhibition of TGFβ. For example, in the context of this disclosure, the NOAEL of Ab6 in rats is the highest dose evaluated (100 mg / kg), which suggests that the MTD is >100 mg / kg based on a 4-week toxicological study. The MTD of Ab6 in non-human primates is >300 mg / kg based on a 4-week toxicological study.

[0116] To determine NOAEL and MTD, species that have been shown to be susceptible to the specific intervention should preferably be selected as preclinical animal models for safety / toxicological studies. In the case of TGFβ inhibition, suitable species include, but are not limited to, rats, dogs, and cynomolgus monkeys. Mice have been reported to be less susceptible 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 specific quality or characteristic of a preclinical model or data that outlines human conditions. As used herein, a preclinical model outlining TGFβ1 indications typically shows dominant expression of TGFB1 (or TGFβ1) compared to TGFB2 (or TGFβ2) and TGFB3 (or TGFβ3). In combination therapy paradigms, for example, translatability may require the same underlying mechanism of action as the combination of actives intended for the model to be realized. As an example, many human tumors exhibit primary resistance to checkpoint blockade therapy (CBT) in immunosuppressed TGFβ1-positive tumors. A second therapy (such as a TGFβ1 inhibitor) may be used in combination to overcome resistance to CBT. In this scenario, an appropriate translatable preclinical model would include TGFβ1-positive tumors exhibiting primary resistance to checkpoint blockade therapy (CBT).

[0118] To treat / treatment: The term “to treat” or “treatment” includes therapeutic treatments, preventive treatments, and applications that reduce the risk of a subject developing a disability or other risk factor. Therefore, the term is intended to broadly mean producing therapeutic benefits in a patient, for example, by enhancing or boosting the body’s immunity, reducing or reversing immunosuppression, reducing, removing, or eradicating harmful cells or substances from the body, reducing the burden of disease (e.g., tumor load), preventing relapse or recurrence, extending the refractory period, and / or otherwise improving survival. The term includes therapeutic treatments, preventive treatments, and applications that reduce the risk of a subject developing a disability or other risk factor. Treatment does not require complete cure of the disability and includes embodiments that reduce symptoms or underlying risk factors. In the context of combination therapy, this term may also refer to i) the ability of a second therapy to reduce the effective dose of a first therapy, thereby reducing side effects and increasing tolerance; ii) the ability of a second therapy to make the patient more responsive to the first therapy; 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 medullary monocytes / macrophages recruited to the tumor site or tissue-resident macrophages derived from erythrocyte-bone marrow precursors. Differentiation of monocytes / macrophages into TAMs is influenced by several factors, including local chemical signaling such as cytokines, chemokines, growth factors, and other molecules acting as ligands, as well as intercellular 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 being more associated with a pro-tumor phenotype. In solid tumors, up to 50% of the tumor mass may correspond to macrophages, which are preferentially M2 polarized. Among tumor-associated monocytes 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) may express cell surface LRRC33 and / or LRRC33-precursor TGFβ1.

[0120] Tumor Microenvironment: The term "tumor microenvironment (TME)" refers to the localized disease niche in which a tumor (e.g., a solid tumor) resides in vivo. The TME may include disease-related molecular signatures (such as chemokines and cytokine sets), disease-related cell populations (such as TAMs, CAFs, and MDSCs), and disease-related ECM environments (modifications of ECM components and / or structures).

[0121] Variable Region: The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chain of an antibody, typically containing approximately 120–130 amino acids at the amino terminus of the heavy chain and approximately 100–110 amino acids at the amino terminus of the light chain. In certain embodiments, the variable regions of different antibodies can differ significantly in amino acid sequence, even between antibodies of the same species. Typically, the variable region of an antibody determines the specificity of a particular antibody to its target.

[0122] Unless otherwise specified in the operating examples, all figures representing quantities of ingredients or reaction conditions used herein should be understood to be modified in all cases by the term “approximately.” When used in relation to percentages, the term “approximately” may mean ±1%.

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

[0124] In this specification, the phrase "and / or" as used in the specification and claims should be understood to mean "either or both" of the elements thus coordinately combined; that is, the elements exist conjunctively in some cases and disjunctly in others. Other elements other than those specifically identified by the "and / or" clause may exist at their discretion, whether related to or unrelated to the specifically identified element, unless explicitly indicated otherwise. Thus, as a non-restrictive example, a reference to "A and / or B," when used in conjunction with an open word such as "comprising," may refer to A without B in one embodiment (optionally including elements other than B), B without A in another embodiment (optionally including elements other than A), and both A and B in yet another embodiment (optionally including other elements), and so on.

[0125] In this specification, the phrase “at least one” used in the specification and claims to refer 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 element list, but not necessarily including at least one of all elements specifically enumerated in the element list, nor excluding any combination of elements in the element list. This definition also permits the presence of elements other than those specifically identified in the element list to which the phrase “at least one” refers, whether related to or unrelated to the specifically identified elements, at the discretion of the user. Therefore, as a non-restrictive 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") could mean, in one embodiment, at least one A (optionally including multiple A's) and no B (and optionally including elements other than B); in another embodiment, at least one B (optionally including multiple B's) and no A (and optionally including elements other than A); and in yet another embodiment, at least one A (optionally including multiple A's) and at least one B (optionally including multiple B's) (and optionally including other elements).

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

[0127] The ranges provided herein are understood to be abbreviations for all values ​​within that range. For example, the range 1–50 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 the subsequent partial purification of soluble growth factors were first described in the late 1970s and early 1980s, marking the beginning of the TGFβ field approximately 40 years ago. To date, 33 gene products constituting the large TGFβ superfamily have been identified. The TGFβ superfamily can be classified into at least three subclasses based on structural similarity: TGFβ, growth and differentiation factors (GDFs), and bone morphogenesis proteins (BMPs). The TGFβ subclass consists of three highly conserved isoforms, namely TGFβ1, TGFβ2, and TGFβ3, which are encoded in humans by three separate genes.

[0129] TGFβ is thought to play a major role in a variety of processes, including inhibition of cell proliferation, extracellular matrix (ECM) remodeling, and immune homeostasis. The importance of TGFβ1 in T cell homeostasis has been demonstrated by the observation that TGFβ1- / - mice survive for only 3-4 weeks and succumb to multi-organ failure due to massive immune activation (Kulkarni, AB et al., Proc Natl Acad Sci USA, 1993, 90(2):770-744; Shull, MM et al., Nature, 1992, 359(6397):693-699). The roles of TGFβ2 and TGFβ3 are less clear. While the three TGFβ isoforms have distinctly different temporal and spatial expression patterns, they signal through the same receptors, TGFβRI and TGFβRII. However, in some cases, such as TGFβ2 signaling, type III receptors such as beta-glycans are also required (Feng, XH 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 TGFβRI / II initiates phosphorylation of SMAD transcription factors, leading to transcription of target genes such as Col1a1, Col3a1, ACTA2, and serpine 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 YEZhang, Nature, 2003, 425(6958): pp. 577-584; Holm, TM et al., Science, 2011, 332(6027): pp. 358-361).

[0130] The biological importance of the TGFβ pathway in humans has been validated by genetic diseases. Kamrachi-Engelmann disease is caused by an autosomal dominant mutation in the TGFB1 gene, leading to osteodysplasia and constitutive activation of TGFβ1 signaling (Janssens, K. et al., J Med Genet, 2006, 43(1):1-11). Patients with Loeys / Dietz syndrome have autosomal dominant mutations in components of the TGFβ signaling pathway, which cause aortic aneurysms, bilateral ocular dehiscence, and bifid uvula (Van Laer, L., H. Dietz, and B. Loeys, Adv Exp Med Biol, 2014, 802:95-105). Because dysregulation of the TGFβ pathway is linked to multiple diseases, several drugs targeting the TGFβ pathway have been developed and tested in patients, but with limited success.

[0131] Dysregulation of TGFβ signaling has been linked to a wide range of human diseases. In fact, in some pathological conditions, such dysregulation can be involved in multiple aspects of TGFβ function. Affected tissues such as fibrous and / or inflammatory tissues and tumors may create a local environment in which TGFβ activation can trigger disease exacerbation or progression, which may be at least partially mediated by interactions between multiple TGFβ-responsive cells, along with several other cytokines, chemokines, and growth factors that play a role in specific disease settings, in autocrine and / or paracrine manner.

[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, invasive 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 types of presenting molecules within the niche, such as LTBP1, LTBP3, LRRC33, and GARP.

[0133] Advances in immunotherapy are transforming the prospects for effective treatment of the ever-increasing number of cancer patients. Most notably, checkpoint blockade therapy (CBT) has become part of the growing standard treatment regimens for cancer. While significant and persistent responses to CBT have been observed across a growing number of cancer species, it is now clear that a significant proportion of tumors appear to be refractory to CBT, even at the initiation of treatment. Therefore, primary resistance is pointed out as a major challenge to enabling many patients' immune systems to target and eliminate tumor cells. Efforts are being made to understand and address the mechanisms underlying primary resistance to CBT in order to extend the effectiveness of treatment to a larger number of patients. However, this enthusiasm has been dampened by lackluster clinical trial results and failures when CBT is combined with drugs known to affect the same tumor species or modulate seemingly related components of the immune system. Possible reasons include the lack of clear mechanistic rationale for given combinations, often rooted in clinically derived data, and thus leading to uncertain and perplexing results in trials intended to augment approved monotherapy. It has become clear that the design of combination immunotherapies should be grounded in scientific evidence of their relevance to the underlying tumor and immune system biology.

[0134] In recent years, the phenomenon known as "immuno-exclusion" has been devised to explain tumor environments in which antitumor effector T cells (e.g., CD8+ T cells) are kept away from (and thus "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 CBTs. For example, transcriptional profiling and analysis of pre-treated melanoma biopsies have revealed enrichment of TGFβ-related pathways and biological processes in tumors unresponsive to anti-PD-1 CBTs. In immuno-exclusionary tumors, effector cells that would otherwise be able to attack cancer cells by recognizing cell surface tumor antigens are prevented from approaching the cancer cell sites. In this way, cancer cells evade immuno-oncological treatments such as checkpoint inhibitors that leverage and rely on the host immune system. Indeed, such tumors exhibit resistance to checkpoint inhibitors such as anti-PD-1 and anti-PD-L1 antibodies, presumably because target T cells are blocked from entering the tumor and therefore fail to exert their anti-cancer 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 pre-treated melanoma biopsies revealed enrichment of TGFβ-related pathways and biological processes in tumors unresponsive to anti-PD-1 CBT. More recently, similar analyses of tumors from patients with metastatic urothelial carcinoma revealed that the lack of response to PD-L1 blockade with atezolizumab was associated with the transcriptional signature of TGFβ signaling, particularly in tumors where CD8+ T cells appeared to be excluded from entering the tumor. The critical role of TGFβ signaling in mediating immune elimination and resulting in anti-PD-(L)1 resistance has been confirmed in an EMT-6 syngeneic mouse model of breast cancer. EMT-6 tumors show poor response to treatment with anti-PD-L1 antibodies, but combining this checkpoint inhibitor with 1D11, an antibody that blocks the activity of all TGFβ isoforms, resulted in a significant increase in the frequency of complete responses compared to treatment with the individual inhibitors. The synergistic antitumor activity is proposed to be due to alterations in the cancer-associated fibroblast (CAF) phenotype and disruption of the immunoexcluded phenotype, leading to the infiltration of activated CD8+ T cells into the tumor. Similar results were found in a mouse model of colorectal cancer and metastasis using a combination of anti-PD-L1 antibody and garnicertib, a small molecule inhibitor of type I TGFβ receptor ALK5 kinase. In summary, these findings suggest that inhibiting the TGFβ pathway in CBT-resistant tumors may be a promising approach to improve or increase the number of clinical responses to CBT. While recent studies have linked TGFβ pathway activation to primary CBT resistance, TGFβ signaling has long been associated with the pathogenesis of cancer. As a potent immunosuppressant, TGFβ inhibits antitumor T cell activity and promotes immunosuppressive macrophages. Malignant cells often develop resistance to TGFβ signaling as a mechanism to evade its growth and tumor-suppressive effects. TGFβ activates CAFs, inducing extracellular matrix generation and promoting tumor progression.Finally, TGFβ induces EMT, and therefore invasion of supporting tissues and tumor metastasis.

[0136] Mammals possess distinctly different genes that encode and express three TGFβ growth factors—TGFβ1, TGFβ2, and TGFβ3—all of which signal through the same heteromeric TGFβ receptor complex. Despite the common signaling pathway, each TGFβ isoform appears to have distinctly different 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, where the TGFβ prodomain, also called latent-associated peptide (LAP), is wrapped around the growth factor, keeping it in a latent, non-signaling state. Furthermore, latent TGFβ is co-expressed with latent TGFβ-binding protein, forming a large latent complex (LLC) via disulfide bonds. Association of latent TGFβ with latent TGFβ-binding protein-1 (LTBP1) or LTBP3 enables tethering to the extracellular matrix, while association with the transmembrane proteins GARP or LRRC33 enables anabolism on the surface of Treg or macrophages, respectively. In vivo, latent TGFβ1 and latent TGFβ3 are activated by a subset of αV integrin that binds to the consensus RGD sequence on LAP, initiating conformational changes and releasing growth factors. The mechanism by which latent TGFβ2 is activated is less clear, as it lacks the consensus RGD motif. TGFβ1 release via proteolytic cleavage of LAP is also linked to an activation mechanism, but its biological relevance is not as clear.

[0137] While the pathogenic role of TGFβ activation is clear in several pathological conditions, it is equally clear that therapeutic targeting of the TGFβ pathway has been challenging due to the pleiotropic effects resulting from broad and persistent pathway inhibition. For example, several studies have shown that small molecule-mediated inhibition of 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. Thus, these "pan-TGFβ" approaches that block all TGFβ signaling have a very narrow therapeutic window, which has proven to be an obstacle to treating several disease-related processes with very high, unmet medical demands. To date, there are no approved TGFβ-targeted therapies, and clinical trial results of such forms have been largely disappointing, likely due to the use of ineffective drug regimens that were necessary to accept safety concerns.

[0138] Safety concerns associated with broad-spectrum TGFβ inhibition, coupled with strong evidence of the critical role of this pathway in multiple disease processes, suggest that a better understanding of the specific roles played by one or more TGFβ family members in disease pathology could lead to viable therapeutic interventions. Regarding responses to TGFβ and CBT, we have observed widespread expression of TGFβ1 in many human tumors as described herein, suggesting that this family member may be a major driving factor in the pathway's contribution to primary resistance.

[0139] As mentioned above, growing evidence suggests that TGFβ may be a major player in inducing and / or maintaining immunosuppression in diseased tissue, including the immune-exclusionary tumor environment. Therefore, TGFβ inhibition may deblock immunosuppression, allowing effector T cells (particularly cytotoxic CD8+ T cells) to approach and kill target cancer cells. In addition to tumor invasion, TGFβ inhibition may also promote the expansion of CD8+ T cells. Such expansion can occur in lymph nodes and / or within tumors. While the exact underlying mechanisms of this process remain unclear, it is assumed that immunosuppression is at least partially mediated by the activation of immune cell-associated TGFβ1 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., Treg). Furthermore, Tregs suppress the proliferation of effector T cells (see, e.g., Figure 26B), thereby reducing the immune response. This process has been shown to be TGFβ1-dependent and is likely involved in GARP-related TGFβ1 signaling. Observations in both human and animal models have shown that increased Treg levels in TMEs are associated with poor prognosis in multiple cancers. In addition, the applicant has previously shown that M2-polarized macrophages exposed to tumor-derived factors such as M-CSF dramatically upregulate the cell surface expression of LRRC33, a TGFβ1-presenting molecule (see, e.g., PCT / US2018 / 031759). These so-called tumor-associated macrophages (or TAMs) are thought to contribute to the observed TGFβ1-dependent immunosuppression in TMEs and promote tumor growth.

[0140] Some solid tumors are characterized by having tumor stroma enriched with myofibroblasts or myofibroblast-like cells. These cells give rise to a collagenous matrix (such as fibrosis) that surrounds or envelops the tumor, which can be at least partially triggered by hyperactive TGFβ1 signaling. TGFβ1 activation is intended to be mediated via ECM-related presentation molecules, such as LTBP1 and LTBP3 in the tumor stroma.

[0141] The applicant has previously disclosed antibodies capable of inhibiting TGFβ1 activation in many of these biological contexts, which have shown promising effects both in vitro and in vivo (see, for example, PCT / US2018 / 012601). However, the challenges remained: i) to develop improved antibodies with fewer affinity types to various antigen complexes to ensure uniform inhibitory effects across various biological contexts or niches where disease-related TGFβ1 resides; and / or ii) to develop antibodies that offer even higher efficacy than previously described counterparts.

[0142] In the studies presented herein, it was assumed that improved antibodies should embody all or most of the following characteristics: 1) maintain selectivity for TGFβ1 to minimize undesirable toxicity associated with pan-inhibition ("isoform selectivity") (see, e.g., PCT / US2017 / 021972); 2) exhibit broad binding activity across various biological contexts or both matrix-related and cell-related categories ("context-independent"); 3) achieve more uniform or unbiased affinity across multiple antigen complexes ("homogeneity"); 4) exhibit potent binding activity for each antigen complex ("high affinity"); and 5) possess robust inhibitory activity for each context ("potency"). Furthermore, a preferred mechanism of action is to inhibit the activation step so that the inhibitor can target latent TGFβ1 complexes tethered in tissues to preemptively prevent downstream activation events and achieve a persistent effect, rather than directly targeting soluble / free growth factors ("persistence"). As will be further disclosed herein, the novel and improved TGFβ1 inhibitors of this disclosure are highly potent and selective inhibitors of latent TGFβ1 activation. The data presented herein demonstrate, among other things, that this mechanism of isoform-specific inhibition is sufficient to overcome primary resistance to anti-PD-1 in syngeneic mouse models, which well outline some of the features of primary resistance to CBT found in human cancer. Together with the improved preclinical safety profiles of such antibodies compared to "pan" TGFβ inhibitors, these efficacy data provide a rationale for exploring the therapeutic use of selective TGFβ1 inhibitors to broaden and enhance the clinical response to checkpoint blockade in cancer immunotherapy, and to address several additional TGFβ1-related indications.

[0143] Novel high-affinity isoform-selective antibody against precursor TGFβ1 General characteristics Improved high-affinity inhibitors of TGFβ1 are disclosed herein, characterized by having enhanced biding properties, increased inhibitory efficacy, and maintaining a desirable safety profile and isoform selectivity compared to previously disclosed TGFβ1 selective inhibitors. These TGFβ1 selective inhibitors of this 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 the antibodies according to this disclosure include increased affinity as measured at equilibrium. In some embodiments, the antibody has a KD of ≤1 nM to 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 to 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 to three 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 preferred embodiments, such antibodies have a KD of ≤1 nM to each of the human LLC complexes, namely hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1, as measured by MSD-SET. According to this disclosure, high-affinity antibodies may have KD values ​​for a particular antigen (e.g., an antigen complex) that are ≤1 nM at equilibrium, for example, ≤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 that can be measured in equilibrium, such as MSD-SET, to 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). In some embodiments, the antibody binds to each of the aforementioned LLC complexes with a KD of ≤5 nM, as measured by a method based on solution equilibrium titration. In some embodiments, the antibody binds to each of the aforementioned LLC complexes with a KD of ≤1 nM, as measured by a method based on solution equilibrium titration.

[0146] For therapeutic use to treat TGFβ1-related indications involving dysregulation of both the extracellular matrix and immune components, it is advantageous to select an antibody that has high affinity (e.g., KD of ≤1 nM) to at least one of the ECM-associated precursor TGFβ1 complexes (hLTBP1-precursor TGFβ1 and / or hLTBP3-precursor TGFβ1) and furthermore at least one of the cell-associated precursor TGFβ1 complexes (hGARP-precursor TGFβ1 and / or hLRRC33-precursor TGFβ1) in order to exert inhibitory effects in both contexts (e.g., in the ECM and attracted to immune cells). In some embodiments, the antibody has high affinity (e.g., KD of ≤1 nM) to both hLTBP1-precursor TGFβ1 and hLTBP3-precursor TGFβ1 and furthermore at least one of the cell-associated precursor TGFβ1 complexes (hGARP-precursor TGFβ1 or hLRRC33-precursor TGFβ1). In yet another embodiment, the antibody has high affinity (e.g., KD of ≤1 nM) for each of the aforementioned complexes (hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1). In a preferred embodiment, such an antibody has 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 this disclosure include high-affinity context-independent antibodies. Such antibodies can bind with equivalent affinity to four known presenting molecule-precursor TGFβ1 complexes, namely LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1. Equivalent affinity may mean that the lowest affinity (highest KD value) exhibited by the antibody among the four antigen complexes is no more than five times lower than the average calculated from the remaining three affinities, or that the highest affinity (lowest KD value) exhibited by the antibody among the four antigen complexes is no more than five times higher than the average calculated from the remaining three affinities. In some embodiments, such antibodies may be said to have equivalent affinity if the ratio of the average KD values ​​of two ECM-associated complexes to the average KD values ​​of two cell-associated complexes is 3 times or less.

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

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

[0150] Any one of the inhibitory antibodies incorporated herein may bind to each of the aforementioned large latent complexes (hLTBP1-proto-TGFβ1, hLTBP3-proto-TGFβ1, hGARP-proto-TGFβ1, and hLRRC33-proto-TGFβ1) via a binding region containing at least a portion of latent lasso within the prodomain of the precursor TGFβ1 complex. Such a binding region may further contain at least a portion of the growth factor domain. In a particularly preferred embodiment, such an antibody may bind to each of the LTBP1-proto-TGFβ1, LTBP3-proto-TGFβ1, GARP-proto-TGFβ1, and LRRC33-proto-TGFβ1 complexes at a K2 of ≤200 pM (e.g., ≤150 pM and ≤100 pM) via a binding region within the LLC complex containing at least a portion of 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 "opening" the cage-like prodomain structure, thereby causing the TGFβ1 growth factor to be released from its 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 the protease-dependent activation of TGFβ1. Conversely, antibodies that directly target one or more of the protease recognition or cleavage sites may not inhibit the integrin-dependent activation of TGFβ1. In contrast, in preferred embodiments of the present invention, high-affinity, context-independent antibodies can inhibit both the integrin-dependent and protease-dependent activation of TGFβ1.

[0152] High affinity binding to target human proteins is an essential feature of antibody therapeutics, but the ability to cross-react with additional species counterparts is advantageous. In particular, given that most preclinical pharmacological models are in rodents, species cross-reactivity with mouse / rat proteins provides a convenient tool as a surrogate antibody in preclinical studies. Therefore, in some embodiments, the high affinity antibodies of this disclosure favorably cross-react with other mammalian counterparts such as mouse, rat, and / or non-human primates.

[0153] Among the novel antibodies included in this disclosure, particularly preferred antibody classes and their characteristics are described below.

[0154] Preferred features In some embodiments, in addition to the core characteristics, preferred antibodies disclosed herein further satisfy one or more antibody criteria from categories 1 to 5 listed in Table 1 herein.

[0155] In some embodiments, additional required criteria for the antibody of the present invention are defined by its binding characteristics, such as the antibody's affinity for an antigen. In this context, “antigen” includes at least four protein complexes, namely human large latent complexes (LLCs) 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 the present disclosure, the antibody binds to each of these complexes, and typically K D Antibodies can bind with a specific affinity measured as a value. Category 1 and Category 2 antibodies fall within the scope of these embodiments. For the purpose of defining criteria based on binding characteristics (e.g., Categories 1 and 2), antibody affinity is determined in equilibrium state rather than by a kinetic assay (such as BLI).

[0156] In addition to or instead of the above, the additional required criteria for the antibodies of the present invention are defined by their amino acid sequence. Antibodies of categories 3 and 4 are defined by the antibody's CDR sequence, while antibodies of category 5 are defined by the variable domain sequences of their heavy and light chains. Table 1. Preferred features of the novel high-affinity TGFβ1 selective inhibitor of the present invention. JPEG0007858715000003.jpg240166 JPEG0007858715000004.jpg236166

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

[0158] Category 1 antibody The antibodies disclosed herein are high-affinity isoform-selective antibodies capable of specifically targeting large, latent complexes of human TGFβ1.

[0159] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to each of the following human LLCs at a KD of ≤200 pM, namely, the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complex, where affinity is measured in equilibrium using a suitable assay such as a solution equilibrium titration 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 the KD value of an antibody at equilibrium may be used, for example, including MSD-SET, which is detailed elsewhere in this specification. Non-limiting examples of antibodies disclosed herein that meet the antibody criteria for preferred antibodies of Category 1 include Ab6, Ab22, Ab24, Ab26, Ab29, Ab30, Ab31, Ab32, and Ab33.

[0161] Furthermore, the antibody can bind to the corresponding LLC of other species with high specificity and affinity. In preferred embodiments, the antibody exhibits species cross-reactivity to the mouse counterpart.

[0162] Category 2 antibodies The antibodies disclosed herein are high-affinity isoform-selective antibodies capable of specifically targeting large, latent complexes of human TGFβ1.

[0163] In another embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to each of the following human LLCs with a KD of ≤1 nM, namely, the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complex, where affinity is measured in equilibrium using a suitable assay such as a solution equilibrium titration assay, and the antibody or fragment binds to human LLC in a binding region containing 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). Using any suitable technique, it may be determined whether an antibody binds to human TGFβ1 LLC in a region containing at least a portion of the latent lasso. For example, a competitive assay utilizing the corresponding polypeptide may be performed. In some embodiments, the binding region can be determined by HD-X or X-ray crystallography.

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

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

[0166] Furthermore, the antibody can bind to the corresponding LLC of other species with high specificity and affinity. In preferred embodiments, the antibody exhibits species cross-reactivity to the mouse counterpart.

[0167] Category 3 antibodies The antibodies disclosed herein are high-affinity isoform-selective antibodies capable of specifically targeting large, latent complexes of human TGFβ1.

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

[0169] The antibody CDR-H2 has an amino acid sequence represented by YI(X1)(X2)(X3)A(X4)TIYYA(X5)SVKG (SEQ ID NO: 144). In some embodiments, in any combination, X1 may be S or H, X2 may be P or S, X3 may be S or D, X4 may be D or S, and / or X5 may be D or G. In some embodiments, if H-CDR2 contains at least one amino acid substitution, the X1 position may be replaced with S, the X2 position with P, the X3 position with D, the X4 position with S, and / or the X5 position with D.

[0170] The antibody CDR-H3 has an amino acid sequence represented by (X1)R(X2)(X3)(X4)D(X5)GDML(X6)P (SEQ ID NO: 145). In some embodiments, in any combination, X1 may be A or V, X2 may be G or A, X3 may be V or T, X4 may be L or W, X5 may be Y or M, and / or X6 may be M or D. In some embodiments, if H-CDR3 contains at least one amino acid substitution, the X1 position may be replaced with A, the X2 position with G, the X3 position with V, the X4 position with L, the X5 position with Y, and / or the X6 position with D.

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

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

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

[0174] Non-limiting examples of the antibodies disclosed herein that meet the antibody criteria of the preferred antibodies of Category 3 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 of the 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. Consensus CDR Sequences and Preferred Amino Acid Substitutions of Heavy and Light Chains JPEG0007858715000005.jpg139166

[0176] In some embodiments, a Category 3 antibody or antigen-binding fragment thereof specifically binds to each of the following human LLCs, i.e., hLTBP1-proTGFβ1, hLTBP3-proTGFβ1, hGARP-proTGFβ1, and hLRRC33-proTGFβ1 complexes, with a KD of ≤1 nM, where the affinity is measured at equilibrium using an appropriate assay such as an assay based on solution equilibrium titration.

[0177] In some embodiments, the antibody or fragment binds to human LLC in a binding region that includes at least a portion of the latent lattic. The latent lattic is a protein domain that forms part of the so-called "straightjacket" of the prodomain. In its native form, the latent lattic of the human proTGFβ1 polypeptide has the amino acid sequence LASPPSQGEVPPGPL (SEQ ID NO: 153). Any suitable technique can be used to determine whether the antibody binds to human TGFβ1 LLC in a region that includes at least a portion of the latent lattic. 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 crystallographic analysis.

[0178] In some embodiments, such antibodies or fragments may be conjugated to each of the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes at a KD of ≤500 pM (optionally ≤400 pM, ≤300 pM, ≤200 pM, or ≤100 pM) as measured by solution equilibrium titration, where the antibody or fragment conjugates to human LLC at a binding region containing at least a portion of latent lasso.

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

[0180] Furthermore, the antibody can bind to the corresponding LLC of other species with high specificity and affinity. In preferred embodiments, the antibody exhibits species cross-reactivity to the mouse counterpart.

[0181] This specification also includes cross-blocking antibodies or their antigen-binding fragments. In some embodiments, an antibody or fragment cross-blocks or cross-competes with one of the Category 3 antibodies, where the antibody has a KD of ≤1 nM to 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 to 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 to three 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 preferred embodiments, such antibodies have a KD of ≤1 nM to each of the human LLC complexes, namely hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1, as measured by MSD-SET. According to this disclosure, high-affinity antibodies may have KD values ​​for a particular antigen (e.g., an antigen complex) that are ≤1 nM at equilibrium, for example, ≤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 capable of specifically targeting large, latent complexes of human TGFβ1.

[0183] In a further embodiment, the present invention provides antibodies or antigen-binding fragments comprising H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, wherein H-CDR1 comprises FTFSSFSMD (SEQ ID NO: 107) or FTFSFSSMN (SEQ ID NO: 114), each optionally containing up to four amino acid changes (up to four, up to three, up to two, or one amino acid change), and H-CDR2 comprises YISPDASTIYYADSVKG (SEQ ID NO: 111), each optionally containing up to four amino acid changes (up to four, up to three, It may contain up to two amino acid changes, or one amino acid change; H-CDR3 contains ARGVLDYGDMLDP (SEQ ID NO: 110), and H-CDR3 may optionally contain up to three amino acid changes (up to three, up to two, or one amino acid change); L-CDR1 contains QASQDITNYLN (SEQ ID NO: 105), and optionally has one or two amino acid changes; L-CDR2 contains DASNLET (SEQ ID NO: 106), and optionally has one or two amino acid changes; and L-CDR3 contains QQADNHPPWT (SEQ ID NO: 12), and optionally has one or two amino acid changes.

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

[0185] Table 3 below summarizes the CDR sequences of Category 4 antibodies. In some embodiments, each CDR sequence may optionally contain one or more of the amino acid substitutions listed below. Table 3. CDR sequences and variants JPEG0007858715000006.jpg100166

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

[0187] In some embodiments, the antibody or fragment binds to human LLC via a binding region containing 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). Using any suitable technique, it may be determined whether the antibody binds to human TGFβ1 LLC via a region containing at least a portion of the latent lasso. For example, a competitive assay utilizing the corresponding polypeptide may be performed. In some embodiments, the binding region may be determined by HD-X or X-ray crystallography.

[0188] In some embodiments, such antibodies or fragments may be conjugated to each of the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes at a KD of ≤500 pM (optionally ≤400 pM, ≤300 pM, ≤200 pM, or ≤100 pM) as measured by solution equilibrium titration, where the antibody or fragment conjugates to human LLC at a binding region containing at least a portion of latent lasso.

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

[0190] Furthermore, the antibody can bind to the corresponding LLC of other species with high specificity and affinity. In preferred embodiments, the antibody exhibits species cross-reactivity to the mouse counterpart.

[0191] This specification also includes cross-blocking antibodies or their antigen-binding fragments. In some embodiments, an antibody or fragment cross-blocks or cross-competes with one of the Category 4 antibodies, where the antibody has a KD of ≤1 nM to 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 to 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 to three 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 preferred embodiments, such antibodies have a KD of ≤1 nM to each of the human LLC complexes, namely hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1, as measured by MSD-SET. According to this disclosure, high-affinity antibodies may have KD values ​​for a particular antigen (e.g., an antigen complex) that are ≤1 nM at equilibrium, for example, ≤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 capable of specifically targeting large, latent complexes of human TGFβ1.

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

[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 light 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 above VL sequence.

[0198] Non-limiting examples of the antibodies disclosed herein that meet the antibody criteria of the preferred antibodies of Category 5 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 its antigen-binding fragment specifically binds to each of the following human LLC complexes with a KD of ≤1 nM, namely, the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complex, where affinity is measured in equilibrium using a suitable assay such as a solution equilibrium titration assay.

[0200] In some embodiments, the antibody or fragment binds to human LLC via a binding region containing 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). Using any suitable technique, it may be determined whether the antibody binds to human TGFβ1 LLC via a region containing at least a portion of the latent lasso. For example, a competitive assay utilizing the corresponding polypeptide may be performed. In some embodiments, the binding region may be determined by HD-X or X-ray crystallography.

[0201] In some embodiments, such antibodies or fragments may be conjugated to each of the hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1 complexes at a KD of ≤500 pM (optionally ≤400 pM, ≤300 pM, ≤200 pM, or ≤100 pM) as measured by solution equilibrium titration, where the antibody or fragment conjugates to human LLC at a binding region containing at least a portion of latent lasso.

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

[0203] Furthermore, the antibody can bind to the corresponding LLC of other species with high specificity and affinity. In preferred embodiments, the antibody exhibits species cross-reactivity to the mouse counterpart.

[0204] This specification also includes cross-blocking antibodies or their antigen-binding fragments. In some embodiments, an antibody or fragment cross-blocks or cross-competes with one of the Category 5 antibodies, where the antibody has a KD of ≤1 nM to 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 to 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 to three 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 preferred embodiments, such antibodies have a KD of ≤1 nM to each of the human LLC complexes, namely hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1, as measured by MSD-SET. According to this disclosure, high-affinity antibodies may have KD values ​​for a particular antigen (e.g., an antigen complex) that are ≤1 nM at equilibrium, for example, ≤1 nM, ≤0.5 nM, ≤400 pM, ≤300 pM, ≤200 pM, and ≤100 pM.

[0205] Exemplary antibodies of the present invention Examples of exemplary antibodies useful for carrying out the present invention and corresponding nucleic acid sequences encoding such antibodies 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] Accordingly, the present invention provides isolated antibodies or antigen-binding fragments comprising six CDRs (e.g., H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3), where H-CDR1, H-CDR2, and H-CDR3 are selected from sets of H-CDRs of antibodies listed in Table 4, 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 includes H-CDR1 having the amino acid sequence FTFSSFSMD (SEQ ID NO: 107), H-CDR2 having the amino acid sequence YISPSADTIYYADSVKG (SEQ ID NO: 103), H-CDR-3 having the amino acid sequence ARGVLDYGDMLMP (SEQ ID NO: 6), L-CDR1 having the amino acid sequence QASQDITNYLN (SEQ ID NO: 105), L-CDR2 having the amino acid sequence DASNLET (SEQ ID NO: 106), and L-CDR3 having the amino acid sequence QQADNHPPWT (SEQ ID NO: 12). Table 4. Complementary antibody heavy chain determination regions determined using the numbering scheme described by Lu et al. JPEG0007858715000007.jpg130166 JPEG0007858715000008.jpg133166 Table 5. Complementarity determination regions of the light chain of exemplary antibodies determined using the Kabat numbering scheme or the Lu et al. numbering system JPEG0007858715000009.jpg24166

[0207] The determination of CDR sequences within an antibody depends on the specific numbering scheme used. Commonly used systems include, but are not limited to, the Kabat numbering system, the IMTG numbering system, the Chothia numbering system, and other numbering schemes such as those described by Lu et al. (Lu X et al., MAbs., January 2019, 11(1):45-57). For example, six CDR sequences of Ab6 defined by four different numbering systems are illustrated below. The CDR sequences of the antibodies of this disclosure can be defined using any CDR numbering system understood in the art. Table 6. Six CDRs of exemplary antibody (Ab6) based on four numbering schemes JPEG0007858715000010.jpg82166

[0208] Table 7 provides the amino acid sequences of the heavy chain variable domain and light chain variable domain of exemplary antibodies of this disclosure. Thus, in some embodiments, the high affinity isoform-selective TGFβ1 inhibitors of this disclosure have a heavy chain variable domain (V H ) and light chain variable domain (V L ) may be an antibody or an antigen-binding fragment thereof, where V H and V L The arrays are V listed in Table 7 below. H and V L It is selected from any one of the array sets. Table 7. Exemplary antibody heavy chain variable domains and light chain variable domains JPEG0007858715000011.jpg133166JPEG0007858715000012.jpg249166JPEG0007858715000013.jpg248166JPEG0007858715000014.jpg111166

[0209] Accordingly, the present invention provides an antibody or its antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain has at least 90% (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) sequence identity with 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 with 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 with SEQ ID NO: 13, and optionally, the light chain variable domain of the antibody or fragment has at least 90% sequence identity with SEQ ID NO: 15. In some embodiments, the heavy chain variable domain of the antibody or fragment has at least 95% sequence identity with SEQ ID NO: 13, and optionally, the light chain variable domain of the antibody or fragment has at least 95% sequence identity with SEQ ID NO: 15. In some embodiments, the heavy chain variable domain of the antibody or fragment has at least 98% sequence identity with SEQ ID NO: 13, and optionally, the light chain variable domain of the antibody or fragment has at least 98% sequence identity with SEQ ID NO: 15. In some embodiments, the heavy chain variable domain of the antibody or fragment has 100% sequence identity with SEQ ID NO: 13, and optionally, the light chain variable domain of the antibody or fragment has 100% sequence identity with SEQ ID NO: 15.

[0210] In some embodiments, an antibody or its antigen-binding moiety that specifically binds to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex includes 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 with the nucleic acid sequence described 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 with the nucleic acid sequence described in SEQ ID NO: 16. In some embodiments, an antibody or its antigen-binding moiety includes a heavy chain variable domain amino acid sequence encoded by the nucleic acid sequence described in SEQ ID NO: 14, and a light chain variable domain amino acid sequence encoded by the nucleic acid sequence described in SEQ ID NO: 16.

[0211] In some examples, any of the antibodies of this disclosure that specifically bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex includes any antibody (including its antigen-binding moiety) 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 contain one or more CDR sequences shown in Table 4, which 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 contain up to 3 amino acid changes compared to the sequences provided in Table 4. Such antibody variants containing up to 3 amino acid changes per CDR are encompassed by the present invention. In some embodiments, such mutant antibodies are produced by optimization steps such as affinity maturation. Table 7 provides the complete amino acid sequences of the heavy chain variable region and light chain variable region of the antibodies listed (e.g., Ab6), as well as the nucleic acid sequences encoding the heavy chain variable region and light chain variable region of specific antibodies: Ab3 - Heavy Chain Variable Region Amino Acid Sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVSSISGSGGATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSSGHWDFDYWGQGTLVTVSS(Sequence ID 95) Ab6-Heavy Chain Variable Region Amino Acid Sequence EVQLVESGGGLVQPGGSLRLSCTAS GFTFSSFS MDWVRQAPGKGLEWVSY ISPSADTI YYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYC ARGVLDYGDMLMP WGQGTLVTVSS (Sequence ID 13) Ab6 - Light chain variable region amino acid sequence DIQMTQSPSSLSASVGDRVTITCQAS QDITNY LNWYQQKPGKAPKLLIY DAS NLETGVPSRFSGSGSGTFTFTISSLQPEDIATYYC QQADNHPPWT FGGGTKVEIK(sequence number 15) Ab6-heavy chain amino acid sequence EVQLVESGGGLVQPGGSLRLSCTASGFTFS SFSMD WVRQAPGKGLEWVS YISPSADTIYYADSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR GVLDYGDMLMP WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(Sequence ID 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, 87:22, pp. 64-68, 1990, modified as described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:58, pp. 73-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol., 215:403-10, 1990. A BLAST protein search can be performed using the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the target protein molecule. If a gap exists between two sequences, gapped BLAST can be used, as described by Altschul et al., Nucleic Acids Res., 25(17):3389-3402, 1997. When using BLAST or gapped BLAST programs, the initial settings parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0213] In any of the antibody or antigen-binding fragments described herein, one or more conserved mutations can be introduced into the CDR or framework sequence at a location where the residue is unlikely to be involved in antibody-antigen interaction. In some embodiments, such conserved mutations can be introduced into the CDR or framework sequence at a location where, based on crystal structure, is unlikely to be involved in interaction with the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex. In some embodiments, possible interfaces (e.g., residues involved in antigen-antibody interaction) can be inferred from known structural information relating to another antigen that shares structural similarities.

[0214] As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or magnitude characteristics of the protein being substituted. Mutants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, such as Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 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 in the following groups: (a) M, I, L, V, (b) F, Y, W, (c) K, R, H, (d) A, G, (e) S, T, (f) Q, N, and (g) E, D.

[0215] In some embodiments, the antibodies provided herein include mutations that give the antibody desirable properties. For example, to avoid potential complications due to Fab arm exchange known to occur with native IgG4 mAbs, the antibodies provided herein may include a stabilizing "Adair" mutation that converts serine 228 (EU numbering, residue 241 in Kabat numbering) to proline, resulting in an IgG1-like (CPPCP (SEQ ID NO: 54)) hinge sequence (Angal et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol, 30, pp. 105-108, 1993). Thus, any of the antibodies may include a stabilizing "Adair" mutation or the amino acid sequence CPPP (SEQ ID NO: 54).

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

[0217] In addition to or instead of such antibodies, they may or may not include the framework region 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, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex are mouse antibodies and include the mouse framework region sequence.

[0218] In some embodiments, such antibodies have relatively high affinity, for example, 10 -9 Less than M, 10 -10 M, 10 -11The antibodies bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and LRRC33-TGFβ1 complex with a KD of M or lower. For example, such antibodies may bind to the GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex with affinities of 5 pM to 1 nM, e.g., 10 pM to 1 nM, e.g., 10 pM to 100 pM. The disclosure also includes antibodies or antigen-binding fragments having a KD value of 1 nM or less (e.g., ≤1 nM, ≤500 pM, ≤100 pM) 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. The affinity and binding kinetics of antibodies that specifically bind to GARP-TGFβ1, LTBP1-TGFβ1, LTBP3-TGFβ1, and / or LRRC33-TGFβ1 complexes 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, the inhibitors of cell-associated TGFβ1 (e.g., GARP-presented TGFβ1 and LRRC33-presented TGFβ1) according to the present invention include an antibody or fragment thereof that specifically binds to such complexes (e.g., GARP-precursor / latent TGFβ1 and LRRC33-precursor / latent TGFβ1) and initiates the internal translocation of the complex. This mechanism of action causes the 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 the amount of TGFβ1 available for activation. In some embodiments, such an antibody or fragment thereof binds to the target complex in a pH-dependent manner, such that binding occurs at neutral or physiological pH but the antibody dissociates from its antigen at acidic pH, or the dissociation rate is higher at acidic pH than at neutral pH. Such an antibody or fragment thereof may function as a recycling antibody.

[0220] Antibodies that compete with high-affinity isoform-specific inhibitory antibodies against TGFβ1 Aspects of this disclosure relate to antibodies that compete with or cross-compete with any of the antibodies provided herein. As used herein with respect to an antibody, “compete” means that the first antibody binds to an epitope (e.g., the epitopes of the GARP-precursor TGFβ1 complex, LTBP1-precursor TGFβ1 complex, LTBP3-precursor TGFβ1 complex, and LRRC33-precursor TGFβ1 complex) in a manner sufficiently similar to or overlapping with the binding of the second antibody, such that the result of 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. The alternative that the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody is possible, but not required. That is, the first antibody may inhibit the binding of the second antibody to its epitope even if the second antibody does not inhibit 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, higher, or lower degree, the antibodies are said to “cross-compete” with respect to the corresponding epitope binding. Both competing and cross-competing antibodies are within the scope of this disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), 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 term “cross-blocking” may be used interchangeably.

[0221] Two different monoclonal antibodies (or antigen-binding fragments) that bind to the same antigen may bind to the antigen simultaneously if their binding sites are sufficiently far apart in three-dimensional space so that their bindings do not interfere with the binding of the other. In contrast, two different monoclonal antibodies may have the same or overlapping antigen-binding regions, in which case the binding of the first antibody may prevent the second antibody from binding to the antigen, and 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 classifying multiple antibodies produced against the same antigen into different "bins" based on their relative cross-blocking activity. Thus, each "bin" represents a distinct binding region of the antigen. Antibodies in the same bin, by definition, cross-block each other. Binning can be tested using standard in vitro binding assays such as Biacor or Octet®, using standard test conditions, for example, according to the manufacturer's instructions (e.g., performing the binding assay at room temperature, approximately 20-25°C).

[0223] Aspects of this disclosure relate to antibodies that compete with or cross-compete with any of the specific antibodies provided herein or their antigen-binding moieties. In some embodiments, the antibody or its antigen-binding moiety binds to the same epitope as any of the antibodies provided herein, or near it. In some embodiments, the antibody or its antigen-binding moiety binds near the epitope if it binds to within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibodies provided herein or its antigen-binding moiety binds to within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues of the epitope to which any of the antibodies provided herein bind.

[0224] In another embodiment, regarding binding to any of the antigens provided herein (e.g., GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, and / or LRRC33-TGFβ1 complex), 10 -8 Equilibrium dissociation constant between antibodies and proteins of M or less, K D Antibodies or their antigen-binding moieties that compete or cross-compete with each other are provided herein. In other embodiments, the antibody is 10 for binding to any of the antigens provided herein. -12 M~10 -9 K in the range of M D They compete or cross-compete. In some embodiments, anti-TGFβ1 antibodies or their antigen-binding moieties that compete for binding to the antibodies or their antigen-binding moieties described herein are provided herein. In some embodiments, anti-TGFβ1 antibodies or their antigen-binding moieties that bind to the same epitope as the antibodies or their antigen-binding moieties described herein are provided herein.

[0225] Any of the antibodies provided herein can be characterized using any suitable method. For example, one method is to identify the epitope to which the antigen binds, or "epitope mapping." Many suitable methods exist for mapping and characterizing the location of epitopes on proteins, including, for example, analysis of the crystalline structure of antibody-antigen complexes, competitive 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, New York, 1999. In an additional example, epitope mapping can be used to determine the sequence to which an antibody binds. Epitopes can be linear epitopes, i.e., those contained in a single amino acid stretch, or conformational epitopes formed by three-dimensional interactions of amino acids, which do not necessarily have to be contained in a single stretch (a linear sequence of the primary structure). In some embodiments, the epitope is a TGFβ1 epitope that is only available for binding by the antibodies or their antigen-binding moieties described herein, when TGFβ1 is present in the GARP-precursor TGFβ1 complex, LTBP1-precursor TGFβ1 complex, LTBP3-precursor TGFβ1 complex, or LRRC33-precursor TGFβ1 complex. Peptides of various lengths (e.g., at least 4 to 6 amino acids) can be isolated or synthesized (e.g., by recombination) and used in antibody binding assays. In another example, the epitope to which the antibody binds can be determined in a systematic screening by using duplicate peptides derived from the target antigen sequence and determining antibody binding. According to gene fragment expression assays, an open reading frame encoding the target antigen is fragmented randomly or by a specific genetic structure, and the reactivity of the expressed antigen fragments with the antibody being tested is determined. The gene fragments can be generated, for example, by PCR, and then transcribed and translated into proteins in vitro in the presence of radioactive amino acids.Subsequently, binding of the antibody to the radiolabeled antigen fragment is determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified by using a large library of random peptide sequences displayed on the surface of phage particles (phage library). Alternatively, a defined library of duplicate peptide fragments can be tested for binding to the test antibody in a simple binding assay. In additional examples, antigen-binding domain mutagenesis, domain exchange experiments, and alanine scanning mutagenesis can be performed to identify residues necessary, sufficient, and / or required for epitope binding. For example, domain exchange experiments can be performed using mutants of the target antigen in which various fragments of the GARP-precursor TGFβ1 complex, LTBP1-precursor TGFβ1 complex, LTBP3-precursor TGFβ1 complex, and / or precursor LRRC33-TGFβ1 complex are replaced (exchanged) with sequences from closely related but antigenically distinct proteins, such as another member of the TGFβ protein family (e.g., GDF11).

[0226] Alternatively, a competitive assay 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. Competitive assays are well known to those skilled in the art.

[0227] In some embodiments, the present invention includes an antibody (e.g., an immunoglobulin, an antigen-binding fragment, etc.) that cross-blocks (cross-competes) any one of Category 1, Category 2, Category 3, Category 4, and / or Category 5 antibodies. Accordingly, in some embodiments, a pharmaceutical composition may be prepared by a process comprising the steps of selecting an antibody or antigen-binding fragment that cross-competes with a Category 1 antibody and incorporating the antibody into the pharmaceutical composition.

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

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

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

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

[0232] In some embodiments, a pharmaceutical composition may be prepared by a process that includes selecting an antibody or its antigen-binding fragment that cross-competes with an antibody selected from the group consisting of b4, Ab5, Ab6, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, and Ab34, and incorporating the antibody into the pharmaceutical composition.

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

[0234] Various modifications and transformations of antibodies Non-limiting variations, modifications, and features of any of the antibodies or antigen-binding fragments contained herein are briefly described below. Embodiments of related analytical methods are also provided.

[0235] Naturally occurring antibody structural units typically contain tetramers. Each such tetramer typically consists of two identical polypeptide chain pairs, each pair having one full-length "light" chain (approximately 25 kDa in certain embodiments) and one full-length "heavy" chain (approximately 50–70 kDa in certain embodiments). The amino-terminus of each chain typically contains a variable region of approximately 100–110 or more amino acids, which is typically responsible for antigen recognition. The carboxyl-terminus 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, defining the antibody isotype. Antibodies can be of any type (e.g., IgM, IgD, IgG, IgA, IgY, and IgE) and of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, and IgA2). Within the full-length light and heavy chains, the variable and constant regions are typically linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 more amino acids (see, for example, Fundamental Immunology, Chapter 7 (Paul, W. ed., 2nd edition, Raven Press, New York (1989)) (included as reference throughout)). The variable region of each light / heavy chain pair typically forms the antigen-binding site.

[0236] The variable regions typically exhibit the same overall structure as a relatively conserved framework region (FR), linked by three hypervariable regions, also known as complementarity-determining regions (CDRs). The CDRs from the two chains of each pair are typically aligned by the framework region, allowing for binding to specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chain variable regions typically contain the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. The amino acid assignments to each domain typically follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (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, the antibody may contain a few amino acid deletions from the carboxyl terminus of the heavy chain. In some embodiments, the antibody may contain a heavy chain having 1 to 5 amino acid deletions at the carboxyl terminus of the heavy chain. In certain embodiments, a definitive description of the CDR and identification of residues containing the antibody binding site are achieved by analyzing the structure of the antibody and / or the structure of the antibody-ligand complex. In certain embodiments, this can be achieved by any of the various techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, the CDR region can be identified or approximated using various analytical methods. 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-mature" antibody is an antibody that has one or more modifications in its one or more CDRs, resulting in improved affinity for the antigen compared to a parent antibody that does not possess those modifications. An example of an affinity-mature antibody is one that has nanomolar or even picomolar affinity (e.g., about 10) for the target antigen. -9 M~10 -12 K in the range of M D ) are present. Affinity-matured antibodies are produced by procedures known in the art. Marks et al. (1992), Bio / Technology, 10:779-783, describe affinity maturation by domain shuffling of VH and VL. Random mutagenesis of CDRs 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):3310-339; and Hawkins et al. (1992), J. Mol. Biol., 226:889-896. Selective mutagenesis at sites of mutagenesis, contact or high-frequency mutations using activity-enhancing amino acid residues has been described in U.S. Patent No. 6,914,128. Typically, a parent antibody and its affinity-matured offspring (e.g., derivatives) retain the same binding region within the antigen, but certain interactions at the molecular level may be altered due to changes in amino acid residues introduced during affinity maturation.

[0238] The term "CDR-transplanted antibody" refers to an antibody that contains variable region sequences of the heavy and light chains from one species, but in which one or more sequences of the VH and / or VL CDR regions are replaced with CDR sequences from another species. For example, an antibody that has variable regions of the heavy and light chains from a mouse, in which one or more of the mouse CDRs (e.g., CDR3) are replaced with human CDR sequences.

[0239] The term "chimeric antibody" refers to an antibody that contains variable region sequences of heavy and light chains from one species as well as a constant region sequence from another species. For example, an antibody having variable regions of mouse heavy and light chains linked to a human constant region.

[0240] As used herein, the terms "framework" or "framework sequence" refer to the remaining sequence of the variable region excluding the CDRs. Since the precise definition of a CDR sequence is determined by various systems, the meaning of the framework sequence is correspondingly subject to various interpretations. The six CDRs (CDR-L1, -L2, and -L3 on the light chain, and CDR-H1, -H2, and -H3 on the heavy chain) also divide the framework regions on the light and heavy chains into four subregions on each chain (FR1, FR2, FR3, and FR4), with CDR1 located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Unless a specific subregion is designated as FR1, FR2, FR3, or FR4, the framework region as referred to by others represents the combined FRs within 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 constitute the framework region.

[0241] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain framework region 1 (H-FR1) having the following amino acid sequence with one, two, or three optional 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 its antigen-binding fragment includes a heavy chain framework region 2 (H-FR2) having the following amino acid sequence with one, two, or three optional amino acid changes: WVRQAPGKGLEWVS (SEQ ID NO: 175).

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

[0244] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain framework region 4 (H-FR4) having the following amino acid sequence with one, two, or three optional amino acid changes: WGQGTLVTVSS (SEQ ID NO: 177).

[0245] In some embodiments, the antibody or its antigen-binding fragment includes a light chain framework region 1 (L-FR1) having the following amino acid sequence with one, two, or three optional amino acid changes: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 178).

[0246] In some embodiments, the antibody or its antigen-binding fragment includes a light chain framework region 2 (L-FR2) having the following amino acid sequence with one, two, or three optional amino acid changes: WYQQKPGKAPKLLIY (SEQ ID NO: 179).

[0247] In some embodiments, the antibody or its antigen-binding fragment includes a light chain framework region 3 (L-FR3) having the following amino acid sequence with one, two, or three optional amino acid changes: GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 180).

[0248] In some embodiments, the antibody or its antigen-binding fragment includes a light chain framework region 4 (L-FR4) having the following amino acid sequence with one, two, or three optional amino acid changes: FGGGTKVEIK (SEQ ID NO: 181).

[0249] In some embodiments, the antibody or its antigen-binding moiety includes a heavy chain immunoglobulin constant domain of a human IgM constant domain, human IgG constant domain, human IgG1 constant domain, human IgG2 constant domain, human IgG2A constant domain, human IgG2B constant domain, human IgG2 constant domain, human IgG3 constant domain, human IgG3 constant domain, human IgG4 constant domain, human IgA constant domain, human IgA1 constant domain, human IgA2 constant domain, human IgD constant domain, or human IgE constant domain. In some embodiments, the antibody or its antigen-binding moiety includes a heavy chain immunoglobulin constant domain of a human IgG1 constant domain or a human IgG4 constant domain. In some embodiments, the antibody or its antigen-binding moiety includes a heavy chain immunoglobulin constant domain of a human IgG4 constant domain. In some embodiments, the antibody or its antigen-binding moiety includes a heavy chain immunoglobulin constant domain of a human IgG4 constant domain having a Ser-to-Pro main chain substitution that produces an IgG1-like hinge and allows for the formation of interchain disulfide bonds.

[0250] In some embodiments, the antibody or its antigen-binding moiety further comprises a light chain immunoglobulin constant domain including 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, consisting of 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, an F(ab')2 fragment, an scFab fragment, or an scFv fragment.

[0254] As used herein, the terms “germline antibody gene” or “gene fragment” refer to an immunoglobulin sequence encoded by a non-lymphocyte cell that has not undergone the maturation process, resulting in genetic rearrangement and mutation of a particular immunoglobulin expression (see, for example, Shapiro et al., (2002), Crit. Rev. Immunol., 22(3): pp. 183–200; Marchalonis et al., (2001), Adv. Exp. Med. Biol., 484: pp. 13–30). One of the advantages provided by various embodiments of this disclosure stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals within a species, and therefore less likely to be recognized as exogenous sources when used therapeutically within that species.

[0255] As used herein, the term “neutralizing” refers to the biological activity of a counter-antigen (e.g., a target protein) when a binding protein specifically binds to the antigen. In one embodiment, a neutralizing binding protein binds to an antigen / target, such as a cytokine, kinase, growth factor, cell surface protein, soluble protein, phosphatase, or receptor ligand, and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, a neutralizing antibody against a growth factor specifically binds to a mature, soluble growth factor released from a latent complex, thereby interfering with 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, but is not limited to, any polypeptide that specifically binds to an antigen (e.g., TGFβ1), but also includes antibodies or their antigen-binding moieties, 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 containing it, can refer to an antibody preparation obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are highly specific and directed to a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which typically contain various antibodies directed to various determinants (epitopes), each mAb is directed to a single determinant on an antigen. The modifying phrase "monoclonal" should not be interpreted as requiring the production of the antibody by any particular method.

[0258] As used herein, the term "recombinant human antibody" refers to all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors translocated into host cells (as further described in Section II C below), and antibodies isolated from recombinant combinatorial human antibody libraries (as incorporated herein for reference by Hoogenboom, HR (1997), TIB Tech., 15:62-70, Azzazy, H. and Highsmith, WE (2002), Clin. Biochem., 35:425-445, Gavilondo, JV and Larrick, This is intended to include antibodies prepared, expressed, produced, or isolated by any other means involved in the splicing of human immunoglobulin gene sequences into other DNA sequences (see JW (2002), BioTechniques, 29:128-145; Hoogenboom, H. and Chames, P. (2000), Immunol.Today, 21:371-378), antibodies isolated from transgenic animals (e.g., mice) 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 involved in the 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 are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if transgenic animals for human Ig sequences are used), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but may not be naturally present in the in vivo human antibody germline repertoire.

[0259] As used herein, “Dual Variable Domain Immunoglobulin” or “DVD-Ig™” refers to a binding protein comprising a paired heavy-chain DVD polypeptide and a light-chain DVD polypeptide, wherein each paired heavy-chain and light-chain provides two antigen-binding sites. Each binding site contains a total of six CDRs per antigen-binding site. DVD-Ig™ typically has two arms linked to each other by at least partially dimerization of the CH3 domain, with each arm of DVD being bispecific and providing four binding sites to the immunoglobulin. DVD-Ig™ are provided in U.S. Patent Publications 2010 / 0260668 and 2009 / 0304693, each of which, including sequence listings, is incorporated herein by reference.

[0260] As used herein, “triply variable domain immunoglobulin” or “TVD-Ig” refers to a binding protein comprising a paired heavy-chain TVD-binding protein polypeptide and a light-chain TVD-binding protein polypeptide, wherein each paired heavy-chain and light-chain provides three antigen-binding sites. Each binding site contains a total of six CDRs per antigen-binding site. The TVD-binding protein may have two arms linked to each other at least partially by dimerization of the CH3 domain, and each arm of the TVD-binding protein is triply specific, providing six binding sites to the binding protein.

[0261] As used herein, “receptor antibody immunoglobulin” or “RAb-Ig” refers to a binding protein comprising a heavy-chain RAb polypeptide and a light-chain RAb polypeptide that together form a total of three antigen-binding sites. One antigen-binding site is formed by the pairing of heavy-chain and light-chain antibody-variable domains present in each of the heavy-chain 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-chain and light-chain RAb polypeptides contains a receptor sequence that independently binds to a ligand to provide second and third “antigen” binding sites. RAb-Ig typically has two arms linked to each other at least partially by dimerization of the CH3 domain, and each arm of RAb-Ig is tripspecific, providing six binding sites to the immunoglobulin. RAb-Ig is described in U.S. Patent Application Publication No. 2002 / 0127231 (its entire contents, including the sequence listing, are incorporated herein by reference).

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

[0263] As used herein, the term "bispecific antibody" refers to a full-length antibody that is distinct from bispecific semi-Ig binding proteins or bispecific binding proteins, and which can bind to two different antigens (or epitopes) at each of its two binding arms (a pair of HC / LC) (see PCT Publication WO02 / 02773). Therefore, a bispecific binding protein has two identical antigen-binding arms with identical 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 in which a binding protein (e.g., an antibody) associates with an antigen to form an antibody / antigen complex, for example, as known in the art. “K-on” is also known as the term “association rate constant” or “ka,” as used interchangeably herein. This value, which indicates the rate at which an antibody binds to its target antigen or the rate at which a complex is formed between the antibody and the antigen, is also expressed by the equation: antibody ("Ab") + antigen ("Ag") → Ab - Ag.

[0265] As used herein, the term “K-off” is intended to refer to the off-rate constant in the dissociation of a binding protein (e.g., an antibody) from an antibody / antigen complex known, for example, in the art. “K-off” is also known as the term “dissociation rate constant” or “kd,” as used herein interchangeably. This value represents 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 shown by the equation: Ab + Ag ← Ab - Ag.

[0266] The terms “equilibrium dissociation constant” or “K” are used herein for compatibility purposes. D The term "equilibrium dissociation constant" refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (k-off) by the association rate constant (k-on). The association rate constant, dissociation rate constant, and equilibrium dissociation constant are used to represent the binding affinity between binding proteins, such as antibodies and antigens. Methods for determining the rate constants of association and dissociation are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to test samples in physiological buffers at equilibrium. Other experimental techniques and instruments can be used, such as the BIAcore® (Biomolecular Interaction Analysis) assay (for example, instruments available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Furthermore, the KinExA® (Kinematic 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 its antigen-binding portion existing in crystalline form. A crystal is a solid state of matter, distinctly different from other states such as amorphous solid or liquid crystal states. A crystal consists of regularly 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 well understood in the art. The basic units, or constituent units, that are repeated in a crystal are called chiral units. The repetition of chiral units in an arrangement that conforms to a predetermined, well-defined crystallographic symmetry provides the “unit cell” of the crystal. The crystal is provided by the translation of lattice units by regular translation in all three dimensions. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd edition, pp. 201-2016, Oxford University Press, New York, NY (1999). The term “linker” is used to indicate a polypeptide that contains two or more amino acid residues linked by a peptide bond and is used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger, P. et al., (1993), Proc. Natl. Acad. Sci. USA, 90:6444-6448, and Poljak, RJ et al., (1994), Structure, 2:1121-1123).Examples of linkers, though not limited to these, include ASTKGPSVFPLAP (SEQ ID NO: 55), ASTKGP (SEQ ID NO: 56), TVAAPSVFIFPP (SEQ ID NO: 57), TVAAP (SEQ ID NO: 58), AKTTPKLEEGEFSEAR (SEQ ID NO: 59), AKTTPKLEEGEFSEARV (SEQ ID NO: 60), AKTTPKLGG (SEQ ID NO: 61), SAKTTPKLGG (SEQ ID NO: 62), SAKTTP (SEQ ID NO: 63), RADAAP (SEQ ID NO: 64), RADAAPTVS (SEQ ID NO: 65), RADAAAAGGPGS (SEQ ID NO: 66), RADAAAA(G4S)4 (SEQ ID NO: 67), SAKTTPKLEEGEFSEARV (SEQ ID NO: 68), ADAAPADAAP (SEQ ID NO: 66), RADAAAA(G4S)4 (SEQ ID NO: 67), SAKTTPKLEEGEFSEARV (SEQ ID NO: 68), ADAAP (SEQ ID NO: 65), RADAAP (SEQ ID NO: 66), RADAAAA(G4S)4 (SEQ ID NO: 67), SAKTTPKLEEGEFSEARV (SEQ ID NO: 68), ADAAP (SEQ ID NO: 65), RADAAP (SEQ ID NO: 65), RADAAP (SEQ ID NO: 66), RADAAAA(G4S)4 (SEQ ID NO: 67), SAKTTPKLEEGEFSEARV (SEQ Examples include (sequence number 69), ADAAPTVSIFPP (sequence number 70), QPKAAP (sequence number 71), QPKAAPSVTLFPP (sequence number 72), AKTTPP (sequence number 73), AKTTPPSVTPLAP (sequence number 74), AKTTAP (sequence number 75), AKTTAPSVYPLAP (sequence number 76), GGGGSGGGGSGGGGS (sequence number 77), GENKVEYAPALMALS (sequence number 78), GPAKELTPLKEAKVS (sequence number 79), GHEAAAVMQVQYPAS (sequence number 80), TVAAPSVFIFPPTVAAPSVFIFPP (sequence number 81), and ASTKGPSVFPLAPASTKGPSVFPLAP (sequence number 82).

[0268] "Label" and "detectable label" or "detectable moiety" mean a portion attached to a specific binding partner, such as an antibody or analyte, in order to make the reaction between members of a specific binding pair, such as an antibody and an analyte, detectable, and such a labeled specific binding partner, such as an antibody or analyte, is said to be "detectably labeled." Accordingly, as used herein, the term "labeled binding protein" means 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 detectable by visual or instrumental means, such as the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to a polypeptide that can be detected by labeled avidin (e.g., a fluorescent marker or streptavidin containing enzymatic activity that can be detected by optical or colorimetric methods). Examples of polypeptide labeling 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 153Magnetic agents such as Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, and lanthanidrine photopolymers), enzyme labels (e.g., horseradish peroxidase, luciferase, and alkaline phosphatase), chemiluminescent markers, biotinyl groups, pre-determined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, and epitope tags), and gadolinium chelates. Typical examples of labels commonly used in immunoassays include photoluminescent moieties, e.g., acridinium compounds, and fluorescent moieties, e.g., fluorescein. Other labels are described herein. From this perspective, a moiety itself does not need to be detectably labeled, but may become detectable when reacted with another moiety. The use of “detectably labeled” is intended to encompass the latter type of detectable label.

[0269] In some embodiments, the binding affinity between an antibody or its antigen-binding moiety and an antigen (e.g., a protein complex), such as a presenting molecule-precursor TGFβ1 complex, is determined using an Octet assay. In some embodiments, an Octet assay is an assay that determines one or more dynamic parameters that are indicators of binding between an antibody and an antigen. In some embodiments, the binding affinity between an antibody or its antigen-binding moiety and a presenting molecule-precursor TGFβ1 complex is determined using an Octet® system (ForteBio, Menlo Park, California). For example, the binding affinity of an antibody may be determined using a forteBio Octet QKe dip and read label-free assay system utilizing 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 presenting molecule-precursor TGFβ1 complex) are presented in a high concentration (50 μg / mL) solution to measure the binding interaction. In some embodiments, the binding affinity between an antibody or its antigen-binding moiety and a presenting molecule-precursor TGFβ1 complex is determined using a protocol outlined herein.

[0270] Characterization of novel, high-affinity, context-independent antibodies against precursor TGFβ1 Combined Profile The antibodies disclosed herein have enhanced binding activity. A class of high-affinity, context-independent antibodies capable of selectively inhibiting TGFβ1 activation is mentioned. Note that the term “context-independent” as used herein has a higher degree of rigor than its more general usage. According to this disclosure, this term implies a certain level of uniformity (i.e., unbiasedness) in the relative affinity that an antibody can exert on various antigen complexes. Therefore, the context-independent antibodies of the present invention target multiple TGFβ1 precursor complexes (e.g., presenting molecule-precursor TGFβ1 complexes) and, as measured by MSD-SET, exhibit equivalent affinity (i.e., a difference in relative affinity not exceeding 3x across complexes) to each such complex, with a K content of less than 10 nM, preferably less than 5 nM, more preferably less than 1 nM, and even more preferably less than 100 pM. D It can be bound by value. As shown below, many antibodies included in the present invention have K in the range of nanomolar or less. D It has a value.

[0271] Therefore, antibodies can specifically bind to each of the human presenting molecule-precursor TGFβ1 complexes (sometimes called "large latent complexes," which are ternary complexes consisting of a single presenting molecule coupled to a precursor TGFβ1 dimer), namely 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., an antigen complex) to evaluate or confirm the ability of an antibody to bind to the antigen complex in a suitable in vitro binding assay. Such assays are well known in the art, 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 are widely used in the art to measure the affinity and dynamics of antibodies to antigens. This is a label-free technique that analyzes biomolecular interactions based on optical interference. One protein, such as the antibody to be tested, can be immobilized on the tip of a biosensor. When the other protein in solution, such as the antigen, binds to the immobilized antibody, a shift in the interference pattern is caused, which can be measured in real time. This allows for monitoring of binding specificity, the rates of association and dissociation, and their concentration dependence. Therefore, BLI is a dynamic measure that reveals the dynamics of a system. Due to its ease of use and rapid results, BLI-based assays, such as the Octet® system (available from ForteBio / Molecular Devices, Fremont, California), are particularly advantageous when used as an initial screening method in the screening process to identify a pool of "binding agents" and separate them from a pool of "non-binding agents" or "weak binding agents."

[0273] BLI-based binding assays revealed that the novel antibodies were characterized as "context-balanced / context-independent" antibodies when their binding affinity was measured by Octet®. As seen in Table 8, which summarizes the BLI-based binding profiles of non-restrictive examples of the antibodies, these antibodies exhibited relatively uniform KD values ​​in the sub-nanomolar range across four target complexes, with relatively low matrix-versus-cell variance (bias not exceeding 5x) (see column (H)). This can be contrasted with the previously identified antibody Ab3, which is provided as a reference antibody and exhibits significantly higher relative affinity (27 + multiple of bias) for matrix-associated complexes than for cell-associated complexes.

[0274] Table 8 below provides non-limiting examples of high-affinity, context-independent precursor TGFβ1 antibodies encompassed by the present invention. The table provides representative results from in vitro binding assays measured by Octet®. Similar results can be 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 to have a clean toxicological profile. Columns (B), (D), (E), and (F) are K D This provides the affinity for each of the listed antibodies, as measured by [method / tool ​​name]. 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 mean K of (B) and (C) DThe values ​​are shown in the corresponding column (D), which comprehensively represent the antibody's affinity for the ECM or matrix-associated precursor TGFβ1 complex. Similarly, the mean K values ​​for (E) and (F) are shown. D The values ​​are shown in the corresponding column (G), which comprehensively represent the antibody's affinity for the cell surface or cell-associated precursor TGFβ1 complex. Finally, the mean K from columns (D) and (G) is shown. D The relative ratio between values ​​is expressed as a "multiple of bias" in column (H). Therefore, when comparing the binding priorities of antibodies to matrix-associated complexes and cell surface complexes, a higher number in column (H) indicates a greater bias for that particular antibody. This is one method of quantitatively representing and comparing the inherent bias of an antibody to its target complex. Such analysis can be useful in guiding the selection process of candidate antibodies for specific therapeutic use. Table 8. Non-limiting examples of context-independent TGFβ1 antibodies and K measured by BLI. D value JPEG0007858715000015.jpg59166

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

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

[0278] As shown, the precursor TGFβ1 antibody of this disclosure has particularly high affinity for matrix-associated precursor TGFβ1 complexes. In some embodiments, the mean K of matrix-associated complexes (i.e., LTBP1-precursor TGFβ1 and LTBP3-precursor TGFβ1) D The value is ≤1nM or ≤0.5nM.

[0279] As shown, the precursor TGFβ1 antibody of this disclosure has high affinity for cell-associated precursor TGFβ1 complexes. In some embodiments, the mean K of cell-associated complexes (i.e., GARP-precursor TGFβ1 and LRRC33-precursor TGFβ1) D The value is ≤2nM or ≤1nM.

[0280] The high-affinity precursor TGFβ1 antibody of this disclosure is characterized by its uniform (unbiased) affinity for all four antigen complexes (compared to, for example, Ab3). Of the four known presenting molecule-precursor TGFβ1 complexes described herein, any one antigen complex is K D This does not deviate significantly. In other words, compared to the previously described precursor TGFβ1 antibodies (including Ab3), a more uniform binding activity is achieved by this disclosure in that each of such antibodies exhibits equivalent affinity across the four antigen complexes. In some embodiments, the antibody or fragment is such that the difference (or range) in affinity of the antibody or fragment across the four precursor TGFβ1 antigen complexes is such that the lowest and highest K D It exhibits an unbiased or uniform binding profile characterized by a 5x or less difference between values. In some embodiments, the relative difference (or range) of affinity is 3x or less.

[0281] Table 8 further illustrates the concept of "uniformity" or lack of bias. The mean K between two matrix-associated and cell-associated complexes. D The values ​​were calculated individually (see columns (D) and (G)). Then, the average K of these was calculated. D The values ​​can be used to determine whether there is a bias in binding activity between matrix-related complexes and cell surface-related complexes (e.g., immune cells). As illustrated in Table 8, the bias is average K D The value can be expressed as a "multiple of difference". Compared to the previously described antibody Ab3, the high affinity context-independent precursor TGFβ1 antibodies included in this disclosure have an average K of more than 3 times between matrix and cell-related complexes. D It is remarkably unbiased in that it shows the difference in values ​​(compared to 25 + multiples of bias in Ab3).

[0282] Therefore, we provide a class or fragment of context-independent monoclonal antibodies that, as measured by biolayer interferometry or surface plasmon resonance, can bind with equivalent affinity to each of the following presenting molecule-precursor TGFβ1 complexes, namely LTBP1-precursor TGFβ1, LTBP3-precursor TGFβ1, GARP-precursor TGFβ1, and LRRC33-precursor TGFβ1, each with an affinity of ≤1 nM. Such antibodies, as measured by biolayer interferometry or surface plasmon resonance, specifically bind to each of the aforementioned complexes with an affinity of ≤5 nM, where the monoclonal antibody or fragment exhibits an affinity bias of no more than 3 times to 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) obtainable from BLI-based assays provide useful information, the applicants of this disclosure have considered that, based on the mechanism of action of activation inhibitors disclosed herein, namely antibodies that work by preventing activation of tethered (e.g., tissue-localized) inactive (e.g., latent) targets by binding to them, the binding characteristics measured at equilibrium may more accurately reflect their in vivo behavior and potency. To put this into words, for example, the fast "on" rate ("K") that would be reflected in the binding measurements obtained by BLI. オンAntibodies possessing the '' may provide relevant parameters for evaluating neutralizing antibodies (for example, antibodies that must directly target the active soluble growth factor itself and rapidly sequester it in order to function as an effective inhibitor). 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 via inhibition of the activation step, which is achieved by targeting the latent complex tethered to the tissue / cell as a countermeasure to the sequestering of the soluble post-activated growth factor. This is because TGFβ1 activation inhibitors target an inactive precursor localized to their respective tissues (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 thought to allow the inhibitor to achieve target saturation (e.g., equilibrium) in vivo without the need for rapid competition for endogenous receptors for transient growth factor molecules, which is required by conventional neutralizing inhibitors.

[0284] Taking these differences in mechanism of action into account, we performed further evaluation of binding properties by using a different in vitro binding assay method that allows for the determination of affinity under equilibrium conditions.

[0285] In light of this, assays that measure the binding affinity of such antibodies in equilibrium are intended to more accurately represent the in vivo target association pattern. Therefore, binding assays based on MSD-SET (or other suitable assays) can be performed, as illustrated 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 particularly useful form for determining the dissociation constant at equilibrium of high-affinity protein-protein interactions (see, e.g., Ducata et al., (2015), J Biomolecular Screening, 20(10):1256-1267). Assays based on SET are particularly useful for determining the KD value of antibodies with affinities of nanomolar or less (e.g., picomolar). Table 9. Non-limiting examples of high affinity context-independent TGFβ1 antibodies (hIgG4) and K measured by MSD-SET. D Value ("h" indicates the human complex) JPEG0007858715000016.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 disclosed herein 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., in the range of approximately nanomolar or less, e.g., having a KD of <1 nM). The high affinity, context-independent binding profile suggests that these antibodies may be advantageous for use in the treatment of TGFβ1-related indications involving dysregulation of both ECM-related and immune component-related conditions, such as cancer.

[0289] A binding assay based on solution equilibrium titration may use a protein complex containing one of the presenting molecules, such as those listed above, as the 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 during 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 and enable sensitive detection of very high affinity interactions. As shown in Table 9, in this disclosure, preferred high-affinity inhibitors of TGFβ1, as determined by SET-based assays, exhibit affinities in the sub-nanomolar (e.g., picomolar) range across all large latent complexes tested.

[0290] Therefore, we provide a class or fragment of context-independent monoclonal antibodies that, as measured by a solution equilibrium titration assay such as MSD-SET, can bind with equivalent affinity to each of the human presenting molecule-precursor TGFβ1 complexes, namely hLTBP1-precursor TGFβ1, hLTBP3-precursor TGFβ1, hGARP-precursor TGFβ1, and hLRRC33-precursor TGFβ1, each with a KD of ≤1 nM. Such antibodies, as measured by MSD-SET, specifically bind to each of the aforementioned complexes with a KD of ≤1 nM, where 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. In a preferred embodiment, such an antibody or fragment has a K content 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 It then binds to each of the aforementioned complexes. More preferably, such an antibody or fragment has a K concentration of 100 pM or less (i.e., ≤100 pM). DThe antibody or fragment then binds to each of the aforementioned complexes. In 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 appropriate in vitro binding assays known in the art, such as biolayer interference.

[0291] In a further preferred embodiment, such antibodies or fragments are also cross-reactive with mouse (e.g., rat and / or mouse) and / or non-human primate (e.g., cynomolgus monkey) counterparts. To give just one example, as illustrated in Table 10 and Example 9 below, Ab6 can bind with high affinity to each of several species of large latent complexes, including humans, mice, rats, and cynomolgus monkeys. Table 10. Non-limiting examples of high-affinity, context-independent TGFβ1 antibodies exhibiting heterogeneous reactivity as measured by MSD-SET ("h" indicates human, "m" indicates mouse). JPEG0007858715000017.jpg13166

[0292] efficacy The antibodies disclosed herein can be broadly characterized as “functional antibodies” for their ability to inhibit TGFβ1 signaling. As used herein, “functional antibodies” confer one or more biological activities thanks to their ability to bind to a target protein (e.g., an antigen) in a manner that modulates its function. Thus, functional antibodies broadly include those capable of modulating the activity / function of a target molecule (i.e., an antigen). Such modulating antibodies include inhibitory antibodies (or inhibitory antibodies) and activating antibodies. This disclosure focuses on antibodies capable of inhibiting biological processes mediated by TGFβ1 signaling, relevant to multiple contexts of TGFβ1. Inhibitors used to carry out the present invention, such as the antibodies described herein, are intended to be TGFβ1 selective and, when administered at therapeutically effective doses (dose at which sufficient efficacy is achieved within acceptable toxicity levels), do not target or interfere with TGFβ2 and TGFβ3. The novel antibodies of this disclosure have enhanced inhibitory activity (potency) compared to previously identified TGFβ1 activating inhibitors.

[0293] In some embodiments, the efficacy of the 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 xenocellular and primary cells can be used to perform cell-based efficacy assays. Cells expressing endogenous TGFβ1 and / or the desired presentation molecules such as LTBP1, LTBP3, GARP, and LRRC33 can be used. Alternatively, exogenous nucleic acids encoding the desired protein, such as TGFβ1, and / or the desired presentation molecules such as LTBP1, LTBP3, GARP, and LRRC33 can be introduced into such cells for expression, for example, by transfusion (e.g., stable or transient transfusion) or by infection based on a viral vector. In some embodiments, LN229 cells are used in such assays. Cells expressing TGFβ1 and the desired presentation molecule (e.g., LTBP1, LTBP3, GARP, or LRRC33) are grown naturally, and these "present" the large latent complex either on the cell surface (when associated with GARP or LRRC33) or by accumulating in the ECM (when associated with LTBP). Activation of TGFβ1 can be initiated by integrins expressed on the surface of other cells. The integrin-expressing cells may be the same cells or different cell types co-expressing the large latent complex. Reporter cells that take up TGFβ-responsive elements are added to the assay system. In this way, the degree of TGFβ activation can be measured by detecting the signal from the reporter cell (e.g., a TGFβ-responsive reporter gene such as luciferase coupled with a TGFβ-responsive promoter element) upon TGFβ activation. Using such cell-based assay systems, the inhibitory activity of an antibody can be determined by measuring the change (decrease) or difference in a reporter signal (e.g., luciferase activity measured by fluorescence readings) in the presence or absence of the test antibody. Such an assay is illustrated in Example 2 herein.

[0294] Thus, in some embodiments, the inhibitory potency (IC 50 ) of the novel antibodies of the disclosure calculated based on a cell-based reporter assay for measuring TGFβ1 activation (such as the LN229 cell assay described elsewhere herein) can be 5 nM or less when measured against each of the hLTBP1-pro-TGFβ1, hLTBP3-pro-TGFβ1, hGARP-pro-TGFβ1, and hLRRC33-pro-TGFβ1 complexes. In some embodiments, the antibody is titrated against each of the LLCs and has an IC 50 of 2 nM or less (i.e., ≤ 2 nM). In a preferred embodiment, the IC 50 of the antibody measured against each of the LLC complexes is 1 nM or less. In some embodiments, the antibody has an IC 50 of less than 1 nM against each of the hLTBP1-pro-TGFβ1, hLTBP3-pro-TGFβ1, hGARP-pro-TGFβ1, and hLRRC33-pro-TGFβ1 complexes. Table 11. Inhibitory Potency (IC 50 ) of Selected Antibodies Measured by Reporter Cell Assay JPEG0007858715000018.jpg66166

[0295] TGFβ1 activation can be initiated by an integrin-dependent or protease-dependent mechanism. The inhibitory activity (e.g., potency) of antibodies according to this disclosure can be evaluated for their ability to block TGFβ1 activation induced by one or both activation modes. 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 evaluated to measure the ability of an antibody to block protease-inducible activation of TGFβ1. Example 3 of this disclosure provides a non-limiting embodiment of such an assay. The results are summarized in Figures 5A and 5B. Thus, in some embodiments of the present invention, isoform-selective inhibitors according to this disclosure can inhibit both integrin-dependent 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 rigidity of the extracellular matrix (ECM), excessive or abnormal collagen deposition, or any combination thereof. Such conditions include, for example, fibrous disorders and cancer or myelofibrosis, including solid tumors (such as metastatic carcinomas).

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

[0297] Similarly, the relative potency of inhibitory antibodies can be determined by measuring pharmacodynamic (PD) effects. Commonly used measures of PD in the TGFβ signaling pathway include, but are not limited to, phosphorylation of SMAD2 / 3 and expression of downstream effector genes whose transcription is sensitive to TGFβ activation, such as those containing TGFβ-responsive promoter elements (e.g., Smad-binding elements). In some embodiments, the antibodies of this disclosure can completely block disease-inducible SMAD2 / 3 phosphorylation in preclinical fibrosis models when administered to animals at doses of 3 mg / kg or less. In some embodiments, the antibodies of this disclosure can significantly suppress fibrosis-inducible expression of a panel of marker genes, including Acta2, Col1a1, Col3a1, Fn1, Itga11, Lox, and Loxl2, when administered to animals at doses of 10 mg / kg or less in a UUO model of renal fibrosis.

[0298] Therefore, in some embodiments, the selection step for an antibody or its antigen-binding fragment for therapeutic use may include identifying an antibody or fragment that exhibits sufficient inhibitory efficacy. For example, the selection step may involve determining the efficacy of one or more test antibodies or fragments (e.g., IC). 50 The steps may include performing a cell-based TGFβ1 activation assay to measure the desired potency and selecting a candidate antibody or fragment thereof. In some embodiments, the IC of each human LLC 50 The concentration is 5 nM or less. The selected antibody or fragment can 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 the antibody-antigen interaction. As used herein, “binding region” refers to the interface region between an antibody and an antigen, determined by appropriate techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) to protect the binding region from solvent exposure when bound to a precursor TGFβ1 complex ("antigen") in a physiological solution by the antibody or fragment. Identification of the binding region is useful in gaining insight into the antigen-antibody interaction and the specific mechanism of action of the antibody. Identification of additional antibodies with similar or overlapping binding regions may be facilitated by cross-blocking experiments that enable epitope binning. Optionally, X-ray crystallography may be used to identify the precise amino acid residues of the epitope mediating the antigen-antibody interaction.

[0300] This field is familiar with HDX-MS, a technique widely used to explore the conformation of proteins in solution or protein-protein interactions. This method relies on exchanging hydrogen in the protein backbone amide with deuterium present in the solution. By measuring the hydrogen-deuterium exchange rate, information about protein dynamics and conformation can be obtained (see, for reference, 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): pp. 95-102). The application of this technique is based on the assumption that when antibody-antigen complexes are formed, the interface between binding partners absorbs solvent, which can reduce or hinder the exchange rate due to steric exclusion of the solvent.

[0301] This disclosure includes antibodies or antigen-binding fragments thereof that bind to human LLC in a region containing the latent lasso or a portion thereof ("binding region"). The latent lasso is a protein module within the prodomain. It is intended that numerous potent activating inhibitors may bind to this region of the precursor TGFβ1 complex in such a manner that antibody binding would "lock in" the growth factor, thereby preventing its release. Interestingly, this is the segment of the complex in which the butterfly-like elongated regions of the growth factor (corresponding to, for example, finger-1 and finger-2) interact closely with the cage-like structure of the prodomain.

[0302] As illustrated in Figure 18B, the latent lasso includes labeled regions 2a and 2b, which are part of the prodomain. Note that the region labeled 5a, directly adjacent to the latent lasso, corresponds to the so-called finger-1 within the growth factor domain, while the region labeled 6b, on the opposite side, is part of finger-2 within the growth factor domain. Based on this, it is not difficult to imagine that antibodies tightly wrapped around these regions can effectively prevent the dissociation of the precursor TGFβ1 complex, thereby blocking its activation.

[0303] The binding region of precursor TGFβ1 can be determined using HDX-MS techniques. In some embodiments, the portion of precursor TGFβ1 identified as important for binding to an antibody or fragment includes at least a portion of the prodomain and at least a portion of the growth factor domain. An antibody or fragment that binds to a first binding region ("Region 1" in Figure 19A) containing at least a portion of the latent lasso is preferred. More preferably, such an antibody or fragment further binds to a second binding region ("Region 2" in Figure 19A) containing at least a portion of the growth factor domain at finger-1 of the growth factor domain. Such an antibody or fragment may further bind to a third binding region ("Region 3" in Figure 19A) containing at least a portion of finger-2 of the growth factor domain.

[0304] Additional regions within precursor TGFβ1 may directly or indirectly contribute to the high-affinity interactions of these antibodies disclosed herein. Regions considered important for mediating high-affinity binding between antibodies and precursor TGFβ1 complexes (see Figure 18A) 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 sequences of human precursor TGFβ1).

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

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

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

[0308] In some embodiments, the high affinity antibodies of this 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, the high affinity antibodies of this 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, the high affinity antibodies of this 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 include at least one amino acid residue from sequences 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] Of particular note is that many of the binding regions identified in structural studies using four representative isoform-selective TGFβ1 antibodies have been found to overlap, pointing to specific regions within the precursor TGFβ1 complex that may be particularly important for maintaining the potential of the precursor TGFβ1 complex. Therefore, advantageously, an antibody or fragment thereof can be selected at least partially based on its binding regions, including the overlapping portions identified across the multiple inhibitors described herein. Examples of these overlapping portions of binding regions include SPPSQGEVPPGPLPEAVL (SEQ ID NO: 201), WKWIHEPKGYHANF (SEQ ID NO: 202), and PGPLPEAVL (SEQ ID NO: 203). Therefore, the high-affinity isoform-selective TGFβ1 inhibitors of this disclosure may bind to the precursor TGFβ1 complex (e.g., human LLC) via an epitope containing one or more amino acid residues of SPPSQGEVPPGPLPEAVL (SEQ ID NO: 201), WKWIHEPKGYHANF (SEQ ID NO: 202), and / or PGPLPEAVL (SEQ ID NO: 203).

[0313] Accordingly, any of the antibodies or antigen-binding fragments included in this disclosure, such as antibodies or fragments of categories 1 to 5 disclosed herein, may bind to one or more of the binding regions identified herein. Such antibodies may be used to treat TGFβ1 indications in subjects as described herein. Accordingly, the selection of an antibody or antigen-binding fragment suitable for therapeutic use according to this disclosure may involve identifying or selecting an antibody or fragment that binds to SPPSQGEVPPGPLPEAVL (SEQ ID NO: 201), WKWIHEPKGYHANF (SEQ ID NO: 202), PGPLPEAVL (SEQ ID NO: 203), or any part thereof.

[0314] Table 12 provides non-limiting examples of protein domains or motifs of human precursor TGFβ1 previously described (WO2014 / 182676). JPEG0007858715000019.jpg97166

[0315] Safety / Toxicology Conventional pan-inhibitors of TGFβ, capable of antagonizing multiple isoforms, are known to cause several toxicities, including cardiovascular toxicity (cardiac lesions, most notably valvular heart disease), which have been reported across multiple species, including dogs and rats. These include hyperplasia of the aortic valve, right AV valve, and left AV valve; inflammation of the aortic valve, left AV valve, and ascending aorta; hemorrhage of the ascending aorta, aortic valve, and left AV valve; and connective tissue degeneration of the ascending aorta (see, for example, 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 to all three TGFβ isoforms have been linked to specific epithelial toxicity observed across multiple species, some of which are summarized below. Table 13. Epithelial toxicity associated with TGFβ pan-inhibitors JPEG0007858715000020.jpg46166

[0317] Building upon the applicant's initial recognition (see PCT / US2017 / 021972) that the lack of isoform specificity in conventional TGFβ antagonists may be at the root of the toxicity associated with TGFβ inhibition, the inventors sought to achieve broad-spectrum TGFβ1 inhibition that would address a variety of diseases manifesting multifaceted TGFβ1 dysregulation while maintaining the safety / tolerability aspects of isoform-selective inhibitors.

[0318] In clinical settings, therapeutic benefit is achieved only when the minimum effective concentration (MEC) of a drug (e.g., a monoclonal antibody) is less than the minimum toxic concentration (MTC) of the drug. This has not been achieved with most, though not all, conventional TGFβ pan-inhibitors, which in practice appeared to cause dose-limiting toxicity. The applicant's previous research described isoform-selective inhibitors of TGFβ1 that showed significantly improved safety profiles compared to conventional pan-inhibitors such as small molecule receptor antagonists and neutralizing antibodies. WO2017 / 156500 disclosed isoform-selective inhibitors of TGFβ1 activation in which no test substance-related toxicity was observed when administered to rats for 4 weeks at doses up to 100 mg / kg per week, and the NOAEL of the antibody was established at the highest dose tested, i.e., 100 mg / kg. Subsequent research by the applicant also showed that antibodies with enhanced function also showed equivalent safety profiles. Here, one objective was to identify antibodies that have 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. While no test substance-related toxicity was noted with either isoform-selective antibody, the non-selective inhibitors, 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 a high dose level of 300 mg / kg when administered once weekly for 4 weeks in cynomolgus monkeys. This provides a therapeutic window of up to 100-fold, as Ab6 has been shown to be effective at a low dose of 3 mg / kg in several in vivo models. Importantly, this demonstrates that higher potency does not necessarily mean a higher risk of toxicity. Without wishing to be bound by any particular theory, it is intended that the highly selective nature of the antibodies disclosed herein is likely to explain the lack of observed toxicity.

[0320] Therefore, in some embodiments, the novel antibodies according to this disclosure have a maximum tolerated dose (MTD) of >100 mg / kg when administered once a week for at least 4 weeks. In some embodiments, the novel antibodies according to this disclosure have a no-observed-adverse-effect level (NOAEL) of up to 100 mg / kg when administered once a week for at least 4 weeks. Suitable animal models for use in conducting safety / toxicological 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 about one-third or less of the NOAEL. In particularly preferred embodiments, the minimum effective dose of the antibody is about one-sixth or less of the NOAEL. In some embodiments, the minimum effective dose of the antibody is about one-tenth or less of the NOAEL.

[0321] In some embodiments, the present invention encompasses isoform-selective antibodies capable of inhibiting TGFβ1 signaling in doses effective for treating TGFβ1-related indications without causing cardiovascular or known epithelial toxicity when administered to a subject. In some embodiments, the antibody has a minimum effective dose of about 3 to 10 mg / kg when administered weekly, bi-weekly, or monthly. Preferably, the antibody causes zero to minimal toxicity at doses at least 6 times the minimum effective dose (e.g., a 6x treatment window). More preferably, the antibody causes zero to minimal toxicity at doses at least 10 times the minimum effective dose (e.g., a 10x treatment window). Even more preferably, the antibody causes zero to minimal toxicity at doses at least 15 times the minimum effective dose (e.g., a 15x treatment window).

[0322] Accordingly, the selection of an antibody or antigen-binding fragment for therapeutic use may include selecting an antibody or antigen-binding fragment that meets one or more criteria from categories 1 to 5 described herein, conducting in vivo efficacy studies in a suitable preclinical model to determine an effective dose of the antibody or fragment, conducting in vivo safety / toxicological studies in a suitable model to determine a safe or toxic dose of the antibody (e.g., MTD, NOAEL, or any parameter understood in the art for evaluating safety / toxicity), and selecting an antibody or fragment that provides at least a 3x therapeutic window (preferably 6x, more preferably 10x, and even more preferably 15x). In preferred embodiments, the in vivo efficacy studies are conducted in two or more suitable preclinical models that outline human conditions. In some embodiments, such preclinical models may optionally include TGFβ1-positive cancers, which may include immunosuppressive tumors. Immunosuppressive tumors may be resistant to cancer treatments such as CBT, chemotherapy, and radiotherapy. In some embodiments, the preclinical model is selected from MBT-2, Crowdmann S91, and EMT6 tumor models.

[0323] The selected antibody or fragment may be used in the manufacture of a pharmaceutical composition containing the antibody or fragment. Such a pharmaceutical composition may be used in the treatment of TGFβ1 indications in a subject as described herein. For example, TGFβ1 indications may be proliferative disorders and / or fibrotic disorders.

[0324] Mechanism of action The antibody of the present invention, useful as a therapeutic agent, is a TGFβ1 inhibitory antibody. Furthermore, the antibody is an activation inhibitor; that is, rather than directly following the already activated growth factor, the antibody blocks the TGFβ1 activation step.

[0325] In a broad sense, the term “inhibitory antibody” refers to an antibody that antagonizes or neutralizes a target function, such as growth factor activity. Advantageously, preferred inhibitory antibodies of this disclosure can inhibit the release of mature growth factors from latent complexes, thereby reducing growth factor signaling. Examples of inhibitory antibodies include antibodies that target any epitope that, upon association with it, reduces the release or activity of growth factors. Such epitopes may be located on the prodomain of TGFβ protein (e.g., TGFβ1), growth factors, or other epitopes that, when bound by the antibody, result in reduced growth factor activity. Examples of inhibitory antibodies of the present invention include, but are not limited to, TGFβ1-inhibitory antibodies. In some embodiments, the inhibitory antibodies of this disclosure specifically bind to combinatorial epitopes, i.e., epitopes formed by two or more components / parts of an antigen or antigen complex. For example, combinatorial epitopes may be formed by contributions from multiple parts of a single protein, i.e., amino acid residues from multiple non-adjacent segments of the same protein. Alternatively, combinatorial epitopes may be formed by contributions from multiple protein components of an antigen complex. In some embodiments, the inhibitory antibodies of this disclosure specifically bind to conformational epitopes (or conformation-specific epitopes), such as epitopes sensitive to the three-dimensional structure (i.e., conformation) of an antigen or antigen complex.

[0326] Conventional methods for antagonizing TGFβ signaling have been: i) directly neutralizing mature growth factors after they have become active to deplete the free ligands available for receptor binding (e.g., those released from their latent precursor complex); ii) using soluble receptor fragments that can sequester free ligands (e.g., so-called ligand capture); or iii) targeting their cell surface receptors to block ligand-receptor interactions. Each of these conventional methods requires an antagonist to compete with the endogenous counterpart. Furthermore, the first two methods above (i and ii) target the active ligand, which is a transient species. Therefore, such antagonists must be able to kinetically compete with and win against the endogenous receptor within a short time window. The third method may offer a more permanent effect by comparison, but because many growth factors (e.g., up to 20) signal through the same receptor, it can lead to unintentional and unwanted inhibitory effects (and therefore, potentially toxicity).

[0327] To provide solutions to these shortcomings and to further enable higher selectivity and localized action, the preferred underlying mechanism of action of inhibitory antibodies, such as those described herein, acts upstream of TGFβ1 activation and ligand-receptor interaction. Therefore, suitable high-affinity TGFβ1 isoform-specific context-independent inhibitors for carrying out the present invention are intended to preferably target the inactive (e.g., latent) precursor TGFβ1 complex (e.g., a complex containing precursor / latent TGFβ1) before its activation in order to block the activation step at its source (e.g., in the disease microenvironment, e.g., in the TME). According to a preferred embodiment of the present invention, such inhibitors target both ECM-associated and cell surface-tethered precursor / latent TGFβ1 complexes with equivalent affinity, rather than free ligands transiently available for receptor binding.

[0328] The advantages of locally targeting tissue / cell-bound complexes as a control over soluble active species (i.e., mature growth factors after release from the source) have been 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 sites, they enhanced antitumor immunity and reduced treatment-related toxicity compared to untargeted counterparts.

[0329] The mechanism of action achieved by the antibodies disclosed herein may further contribute to enhanced efficacy persistence, as well as overall higher potency and safety.

[0330] Interestingly, these antibodies may exhibit additional inhibitory activity against cell-associated TGFβ1 (LRRC33-precursor TGFβ1 and GARP-precursor TGFβ1). The applicant found that LRRC33-binding antibodies tend to undergo internal translocation upon binding to cell surface LRRC33. It is unclear whether this internal translocation is actively induced by antibody binding or whether this phenomenon is due to the innate (e.g., passive) endocytotic activity of macrophages. However, the high-affinity isoform-selective TGFβ1 inhibitor, Ab6, can be rapidly translocated into cells translocated with LRRC33 and precursor TGFβ1, and the internal translocation rate achieved with Ab6 is significantly higher than that achieved with reference antibodies that recognize cell surface LRRC33 (Figure 6). Similar results have been obtained from primary human macrophages. These observations suggest that Ab6 induces internal translocation upon binding to its target, LRRC33-precursor TGFβ1, thereby removing the LRRC33-containing complex 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 factors from the latent complex, and ii) removing the LRRC33-precursor TGFβ1 complex from the cell surface via internal translocation. A similar inhibitory mechanism of action may be applicable to GARP-precursor TGFβ1.

[0331] In some embodiments, the antibody is a pH-sensitive antibody that binds to its antigen with higher affinity at a neutral pH (e.g., pH 7) than at an acidic pH (e.g., pH 5). Such an antibody 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., K at pH 5) オフ K at pH 7 オフThe ratio can be at least 1.2. Optionally, the ratio is at least 1.5. In some embodiments, the ratio is at least 2. Such pH-sensitive antibodies may be useful as recycling antibodies. Upon target association on the cell surface, the antibody may initiate antibody-dependent internal transport (and therefore removal) of the membrane-bound precursor TGFβ1 complex (associated with LRRC33 or GARP). Subsequently, in acidic intracellular compartments such as lysosomes, the antibody-antigen complex may dissociate, and the free antibody may be transported back to the extracellular domain.

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

[0333] Antigen complex and its components The novel antibodies described herein specifically bind to each of four known human large latent complexes (e.g., hLTBP1-progenitor TGFβ1, hLTBP3-progenitor TGFβ1, hGARP-progenitor TGFβ1, and hLRRC33-progenitor TGFβ1) that selectively inhibit TGFβ1 activation. Preferred antibodies further satisfy one or more criteria from categories 1 to 5 listed in Table 1.

[0334] Screening (e.g., identification and selection) of such antibodies typically involves the use of appropriate antigen complexes, which are usually produced by recombination. Useful protein components that may contain such antigen complexes include TGFβ isoforms and associated polypeptides, fragments, and variants, as well as presentation molecules (e.g., LTBP, GARP, LRRC33) and associated polypeptides, fragments, and variants. These components may be expressed, purified, and formed into protein complexes (such as large latent complexes), which can then be used in the antibody screening process. Screening may include positive selection, where a desired conjugate is selected from a pool or library of conjugates and non-conjugates, and negative selection, where undesirable conjugates are removed from the pool. Typically, to ensure that the selected conjugate has affinity for both such biological contexts, at least one matrix-related complex (e.g., LTBP1-precursor TGFβ1 and / or LTBP1-precursor TGFβ1) and at least one cell-related complex (e.g., GARP-precursor TGFβ1 and / or LRRC33-precursor TGFβ1) were included in the positive screening.

[0335] In some embodiments, TGFβ1 includes a naturally occurring mammalian amino acid sequence. In some embodiments, TGFβ1 includes a naturally occurring human amino acid sequence. In some embodiments, TGFβ1 includes a human, monkey, rat, or mouse amino acid sequence. In some embodiments, the antibody or antigen-binding moiety described herein does not specifically bind to TGFβ2. In some embodiments, the antibody or antigen-binding moiety described herein does not specifically bind to TGFβ3. In some embodiments, the antibody or antigen-binding moiety described herein does not specifically bind to TGFβ2 or TGFβ3. In some embodiments, the antibody or antigen-binding moiety described herein specifically binds to TGFβ1 including the amino acid sequence described in SEQ ID NO: 34. The amino acid sequences of TGFβ2 and TGFβ3 are described in SEQ ID NOs. 38 and 32, respectively. In some embodiments, the antibody or antigen-binding moiety described herein specifically binds to TGFβ1 including an amino acid sequence that does not exist naturally (also referred to herein as non-naturally occurring TGFβ1). For example, non-naturally occurring TGFβ1 may include one or more recombinant mutations compared to the naturally occurring TGFβ1 amino acid sequence. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequence includes the amino acid sequences described in SEQ ID NOs. 24-35 shown in Table 14. In some embodiments, the TGFβ1, TGFβ2, or TGFβ3 amino acid sequence includes the amino acid sequences described in SEQ ID NOs. 36-43 shown in Table 15.

[0336] TGFβ1(Prodomain+) Growth factor domain) LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRR ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS (Sequence ID 24)

[0337] TGFβ2(Prodomain+) Growth factor domain )SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKAR VPECRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKR ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS (Sequence No. 28)

[0338] TGFβ3(Prodomain+) Growth factor domain )SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNP SSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKR ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS(Sequence ID 32) Table 14. Exemplary amino acid sequences of TGFβ1, TGFβ2, and TGFβ3 JPEG0007858715000021.jpg246166JPEG0007858715000022.jpg247166JPEG0007858715000023.jpg244166 JPEG0007858715000024.jpg63166 Table 15. Exemplary non-human amino acid sequences JPEG0007858715000025.jpg252166JPEG0007858715000026.jpg246166JPEG0007858715000027.jpg247166JPEG00078587150 00028.jpg246166JPEG0007858715000029.jpg245166JPEG0007858715000030.jpg248166JPEG0007858715000031.jpg220166

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

[0340] The antibodies or antigen-binding moieties 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 protein or a fragment thereof that does not exist naturally. 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. Recombinant LTBP1 proteins 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, biotin labels, polyhistidine tags, myc tags, HA tags, and / or fluorescent tags. In some embodiments, the LTBP1 protein is a mammalian LTBP1 protein. In some embodiments, the LTBP1 protein is a human, monkey, mouse, or rat LTBP1 protein. In some embodiments, the LTBP1 protein contains the amino acid sequences described in SEQ ID NOs. 46 and 47 in Table 15. In some embodiments, the LTBP1 protein contains the amino acid sequence described in SEQ ID NOs. 50 in Table 17.

[0341] The antibodies or antigen-binding moieties 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 protein or a fragment thereof that does not exist naturally. 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, biotin labels, polyhistidine tags, myc tags, HA tags, and / or fluorescent tags. In some embodiments, the LTBP3 protein is a mammalian LTBP3 protein. In some embodiments, the LTBP3 protein is a human, monkey, mouse, or rat LTBP3 protein. In some embodiments, the LTBP3 protein contains the amino acid sequences described in SEQ ID NOs. 44 and 45 in Table 15. In some embodiments, the LTBP1 protein contains the amino acid sequence described in SEQ ID NO. 51 in Table 17.

[0342] The antibodies or antigen-binding moieties 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 protein or fragment thereof that does not exist naturally. In some embodiments, the GARP protein is a recombinant protein. Such GARP may be recombinant and is referred to herein as recombinant GARP. Some recombinant GARP may include one or more modifications, cleavages, and / or mutations compared to wild-type GARP. Recombinant GARP may be modified to be soluble. In some embodiments, the GARP protein includes a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the GARP protein does not include a leader sequence (i.e., the leader sequence is processed or cleaved). In other embodiments, recombinant GARP is modified to include one or more detectable labels. In further embodiments, such detectable labels may include, but are not limited to, biotin labels, polyhistidine tags, flag tags, myc tags, HA tags, and / or fluorescent tags. In some embodiments, the GARP protein is a mammalian GARP protein. In some embodiments, the GARP protein is a human, monkey, mouse, or rat GARP protein. In some embodiments, the GARP protein includes the amino acid sequences described in SEQ ID NOs. 48-49 in Table 15. In some embodiments, the GARP protein includes the amino acid sequences described in SEQ ID NOs. 52 and 53 in Table 18. In some embodiments, the antibodies or their antigen-binding moieties described herein do not bind to TGFβ1 in a context-dependent manner; for example, binding to TGFβ1 occurs only when the TGFβ1 molecule forms a complex with a specific presenting molecule such as GARP. Instead, the antibody and its antigen-binding moiety bind to TGFβ1 in a context-independent manner. In other words, the antibody or its antigen-binding moiety binds to TGFβ1 when bound to any presenting molecule, namely GARP, LTBP1, LTBP3, and / or LRCC33.

[0343] The antibodies or antigen-binding moieties 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 protein or fragment thereof that does not exist naturally. 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, cleavage, and / or mutations compared to wild-type LRRC33. Recombinant LRRC33 proteins may be modified to be soluble. For example, in some embodiments, the external domain of LRRC33 may be expressed with a C-terminal His tag to express a soluble LRRC33 protein (see sLRRC33, e.g., SEQ ID NO: 84). In some embodiments, the LRRC33 protein includes a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LRRC33 protein does not include 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, biotin labels, polyhistidine tags, flag tags, myc tags, HA tags, and / or fluorescent tags. In some embodiments, the LRRC33 protein is a mammalian LRRC33 protein. In some embodiments, the LRRC33 protein is a human, monkey, mouse, or rat LRRC33 protein. In some embodiments, the LRRC33 protein contains the amino acid sequences described in SEQ ID NOs. 83, 84, and 101 in Table 18. Table 17. Exemplary LTBP amino acid sequences JPEG0007858715000032.jpg206166JPEG0007858715000033.jpg183166 Table 18. Exemplary GARP and LRRC33 amino acid sequences JPEG0007858715000034.jpg69166JPEG0007858715000035.jpg248166JPEG0007858715000036.jpg249166JPEG0007858715000037.jpg64166

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

[0345] The pharmaceutical compositions of the present invention may be administered to patients to alleviate TGFβ-related indications (e.g., fibrosis, immunodeficiency, and / or cancer). “Acceptable” means that the carrier is compatible with (and preferably stabilizes) the active ingredient of the composition and is not harmful to the target being treated. Examples of pharmaceutically acceptable excipients (carriers), including buffers, 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., KE Hoover. For example, the pharmaceutical compositions described herein contain multiple antibodies that specifically bind to GARP-precursor TGFβ1 complexes, LTBP1-precursor TGFβ1 complexes, LTBP3-precursor TGFβ1 complexes, and LRRC33-precursor TGFβ1 complexes, the antibodies recognizing different epitopes / residues of the complexes.

[0346] The pharmaceutical composition used in this method may include pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy, 20th edition (2000), edited by Lippincott Williams and Wilkins, KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides; serum albumin, gelatin This may include proteins such as tin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

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

[0348] Therefore, molecules containing antibodies or antigen-binding fragments of such antibodies can be incorporated into pharmaceutical compositions suitable for human administration.

[0349] A pharmaceutical formulation may contain 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 so that the final concentration of the active biologic (e.g., monoclonal antibodies, manipulated binding molecules containing antigen-binding fragments, etc.) is approximately 2 mg / mL to approximately 200 mg / mL. For example, the final concentrations (weight / volume) of the formulations are approximately 2-200, 2-180, 2-160, 2-150, 2-120, 2-100, 2-80, 2-70, 2-60, 2-50, 2-40, 5-200, 5-180, 5-160, 5-150, 5-120, 5-100, 5-80, 5-70, 5-60, 5-50, 5-40, and 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 possible ranges are 150, 50-120, 50-100, 50-80, 50-70, 50-60, 60-200, 60-180, 60-160, 60-150, 60-120, 60-100, 60-80, 60-70, 70-200, 70-180, 70-160, 70-150, 70-120, 70-100, and 70-80 mg / mL. In some embodiments, the final concentration of the biological agent in the formulation is approximately 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 composition of the present invention is preferably formulated using a suitable buffer. Suitable buffers include, but are 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 a pharmaceutical composition may be approximately 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] The pharmaceutical compositions of this disclosure may include surfactants approved for use in pharmaceutical formulations, such as nonionic detergents. Examples of such surfactants include polysorbates such as polysorbate 20 (Tween-20), polysorbate 80 (Tween-80), and NP-40.

[0354] The pharmaceutical compositions of this disclosure may include stabilizers. In liquid protein preparations, stability can be enhanced by selecting pH buffer salts, and often amino acids can also be used. Aggregation after protein adsorption and unfolding is often due to interactions at the liquid / air interface or the liquid / solid interface (interface with the 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 this 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, disodium edetate, sodium chloride, potassium chloride, maltose, histidine acetate, sorbitol, pentetic acid, human serum albumin, pentetic acid.

[0356] In some embodiments, the pharmaceutical compositions of this disclosure may contain preservatives.

[0357] The pharmaceutical compositions of this 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 may be presented as a pre-filled liquid 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. Patents No. 4,485,045 and No. 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 lipid compositions comprising phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with a defined pore size to obtain liposomes with the desired diameter.

[0359] In some embodiments, liposomes with targeting properties are selected to preferentially deliver or localize a pharmaceutical composition to a specific tissue or cell type. For example, carriers based on specific nanoparticles with myelotargeting 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 include or be used in combination with adjuvants. The intention is that specific adjuvants can, for example, boost the target immune response to tumor antigens, thereby facilitating T effector function, DC differentiation from monocytes, and enhanced antigen uptake and presentation by APCs. 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), alpha-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 edition, Mack Publishing (2000).

[0362] In other examples, the pharmaceutical compositions described herein may be formulated in a sustained-release manner. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which are matrices in the form of molded articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent 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 (microspheres for injection consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose isobutyrate acetate, and poly-D-(-)-3-hydroxybutyric acid.

[0363] Pharmaceutical compositions used for in vivo administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane. Therapeutic antibody compositions are generally placed in containers with a sterile access port, such as intravenous injection solution bags or vials with a stopper that can be punctured by a subcutaneous needle.

[0364] The pharmaceutical compositions described herein may be in the form of tablets, pills, capsules, powders, granules, liquids or suspensions, or suppositories, for oral, parenteral, or rectal administration, or administration by inhalation or gas injection.

[0365] Suitable surfactants incl...

Claims

1. Each of the following antigen complexes: i) Human LTBP1-precursor TGFβ1, ii) Human LTBP3-precursor TGFβ1, iii) Human GARP-precursor TGFβ1, and iv) Human LRRC33-precursor TGFβ1 An antibody or antigen-binding fragment that binds to, It inhibits the activation of TGFβ1, A fully human or humanized antibody or its antigen-binding fragment, H-CDR1 as defined in Sequence ID No. 107, H-CDR2 as defined in Sequence ID No. 103, H-CDR3 as defined in Sequence ID No. 6, L-CDR1 described in Sequence ID No. 105, L-CDR2 described in Sequence ID No. 106, L-CDR3 described in Sequence ID No. 12 and Includes, The antibody or its antigen-binding fragment includes a Ser-to-Pro main chain substitution in the constant domain that generates an IgG1-like hinge. An antibody or its antigen-binding fragment.

2. The antibody or antigen-binding fragment according to claim 1, It includes 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 its antigen-binding fragment.

3. The antibody or antigen-binding fragment according to claim 1, A heavy chain variable domain sequence containing the amino acid sequence of SEQ ID NO: 13 and a light chain variable domain sequence containing the amino acid sequence of SEQ ID NO: 15 are included. An antibody or its antigen-binding fragment.

4. The antibody or antigen-binding fragment according to claim 1, It cross-reacts with human and mouse precursor TGFβ1 complexes. An antibody or its antigen-binding fragment.

5. A composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, and an excipient.

6. A composition according to claim 5 for the treatment of cancer in a subject, The aforementioned cancer is a solid tumor or a hematological cancer. composition.

7. The composition according to claim 6, The aforementioned solid tumors show poor response to cancer treatment; The aforementioned solid tumor is an immune-exclusionary 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 stroma enriched with cancer-associated fibroblasts (CAFs) and / or myofibroblasts. composition.

8. The composition according to claim 6, The subject has primary or acquired resistance to cancer treatment. composition.

9. The composition according to claim 6, The subject has cancer that is positive for TGFβ1 and TGFβ3, The subject has received, is receiving, or is a candidate for checkpoint inhibitor therapy (CBT), or the subject has not previously received CBT. composition.

10. The composition according to claim 6, The aforementioned cancer is a type of cancer characterized by a response rate of less than 25%, which is statistically low in response to CBT. composition.

11. The composition according to claim 6, The antibody or its antigen-binding fragment is administered to the subject in combination with at least one additional treatment. composition.

12. The composition according to claim 11, The aforementioned additional treatment is selected from the group consisting of cancer vaccines, therapy with modified immune cells, chemotherapy, radiotherapy, modulators of members of the TGFβ superfamily, myostatin inhibitors, GDF11 inhibitors, VEGF agonists, IGF1 agonists, FXR agonists, CCR2 inhibitors, CCR5 inhibitors, dual CCR2 / CCR5 inhibitors, CCR4 inhibitors, lysyl oxidase-like -2 inhibitors, ASK1 inhibitors, acetyl-CoA carboxylase (ACC) inhibitors, p38 kinase inhibitors, pirfenidone, nintedanib, M-CSF inhibitors, MAPK inhibitors, immune checkpoint inhibitors, IL-11 antagonists, IL-6 antagonists, indoleamine 2,3-dioxygenase (IDO) inhibitors, arginase inhibitors, tyrosine kinase inhibitors, Ser / Thr kinase inhibitors, and bispecific kinase inhibitors. composition.

13. The composition according to claim 11, The aforementioned additional treatment is an immune checkpoint inhibitor. composition.

14. The composition according to claim 13, The immune checkpoint inhibitor is selected from the group consisting of cytotoxic T lymphocyte antigen-4 (CTLA-4) antibody, programmed cell death protein 1 (PD-1) antibody, PD-L1 antibody, T cell immunoglobulin domain and mucin domain-3 (TIM3) antibody, lymphocyte activation gene 3 (LAG3) antibody, killer cell immunoglobulin-like receptor (KIR) antibody, glucocorticoid-induced tumor necrosis factor receptor (GITR) antibody, and T cell activation V-domain immunoglobulin (Ig) containing inhibitory factor (VISTA) antibody, or a combination thereof. composition.

15. The composition according to claim 14, The immune checkpoint inhibitor is selected from programmed cell death protein 1 (PD-1) antibody or PD-L1 antibody. composition.

16. The composition according to claim 6, The aforementioned cancers include melanoma, adjuvant-treated 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 lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), diastolic small cell lung cancer (ES-SCLC), lymphoma (classical Hodgkin and non-Hodgkin), mediastinal primary B-cell large cell lymphoma (PMBCL), T-cell lymphoma, diffuse large B-cell lymphoma, histiocytic sarcoma, follicular dendritic cell sarcoma, finger-shaped incarcerated dendritic cell sarcoma, myeloma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), small lymphocytic lymphoma (SLL), head and neck cancer, and urothelial carcinoma. Selected from the group consisting of Merkel cell carcinoma, Merkel cell cutaneous carcinoma, microsatellite instability-associated carcinoma (MSI-H), mismatch repair deficiency-associated carcinoma (dMMR), mesothelioma, gastric cancer, gastroesophageal junction carcinoma (GEJ), gastric adenocarcinoma, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), gastric cardia adenocarcinoma, kidney cancer, bile duct cancer, bile duct cancer, 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, gastric cancer, brain cancer, malignant glioma, glioblastoma, gliosarcoma, neuroblastoma, thyroid cancer, adrenocortical carcinoma, oral intraepithelial neoplasia, esophageal cancer, nasal cavity and paranasal sinus squamous cell carcinoma, nasopharyngeal carcinoma, salivary gland cancer, liver cancer, and hepatocellular carcinoma (HCC), composition.

17. A method for screening isoform-selective TGFβ1 inhibitors suitable for therapeutic use, i) Providing an antibody or an antigen-binding fragment thereof that specifically binds to each of the human LTBP1-precursor TGFβ1, human LTBP3-precursor TGFβ1, human GARP-precursor TGFβ1, and human LRRC33-precursor TGFβ1 complexes with a KD of ≤1 nM, ii) A step of conducting in vivo efficacy studies in preclinical animal models, Here, the aforementioned preclinical animal model is a syngeneic tumor model that reproduces the human pathology, The antibody or its antigen-binding fragment is administered to the preclinical animal model in combination with an immune checkpoint inhibitor at a dose of 1 to 30 mg / kg / week. The in vivo efficacy study includes measuring tumor volume in the animal model, In the aforementioned preclinical animal model, if the tumor volume is less than 25% of the endpoint tumor volume, the antibody or its antigen-binding fragment is selected as having achieved efficacy. iii) A step of conducting toxicological studies in a preclinical model known to be sensitive to TGFβ inhibition in order to determine the maximum tolerated dose and / or minimum toxic dose of the antibody or its antigen-binding fragment. Here, the toxicological study includes an evaluation of cardiovascular toxicity, including cardiac lesions, valvular heart disease, hyperplasia of the aortic valve, right AV valve or left AV valve, inflammation of the aortic valve, left AV valve or ascending aorta, bleeding of the ascending aorta, aortic valve or left AV valve, and / or connective tissue degeneration of the ascending aorta. The antibody or its antigen-binding fragment is administered to the animal model for at least four weeks at a dose of at least 30 to 100 mg / kg / week. When the maximum tolerated dose exceeds 30 mg / kg / week, the antibody or its antigen-binding fragment is selected as one that does not cause cardiovascular toxicity. And, iv) If the antibody or its antigen-binding fragment is selected in step (ii) as having achieved efficacy, and the antibody or its antigen-binding fragment is selected in step (iii) as lacking cardiovascular toxicity, the step of selecting the antibody or its antigen-binding fragment as a candidate therapeutic agent. including, method.

18. The method according to claim 17, If the maximum tolerated dose or minimum toxic dose is at least 100 mg / kg / week, the antibody or its antigen-binding fragment is selected as one that does not cause cardiovascular toxicity. method.

19. The method according to claim 17, The antibody or antigen-binding fragment is selected as a candidate therapeutic agent if the maximum tolerated dose or minimum toxic dose determined in step (iii) has a therapeutic window of at least three times compared to the dose of the antibody or antigen-binding fragment required to achieve efficacy in step (ii). method.

20. The method according to claim 17, The antibody or antigen-binding fragment is selected as a therapeutic candidate if the maximum tolerated dose or minimum toxic dose determined in step (iii) has a therapeutic window of at least six times compared to the dose of the antibody or antigen-binding fragment required to achieve efficacy in step (ii). method.

21. A method according to any one of claims 17 to 20, The aforementioned syngeneic tumor model that reproduces the human pathological condition includes overexpression of the TGFB1 gene or the TGFβ1 protein, method.

22. A method according to any one of claims 17 to 21, The aforementioned syngeneic tumor model that replicates the human pathology includes resistance to checkpoint inhibitor therapy. method.

23. A method according to any one of claims 17 to 22, The aforementioned syngeneic tumor models that reproduce the human pathology include the Cloudman S91 model, the MBT-2 model, or the EMT-6 tumor model. method.

24. A method according to any one of claims 17 to 23, The in vivo efficacy study conducted in the aforementioned preclinical model further includes one or more of the following: immunohistochemical analysis, measurement of tumor growth, regression of tumor volume, regression of tumor growth, incidence of regression response, or degree of regression response. method.

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