WO2022271917A1 - Transforming growth factor-beta ligand traps for the treatment of disease - Google Patents

Abstract

The present application relates to methods using Transforming Growth Factor-β (TGF-β) ligand traps. The TGF-β ligand traps described herein may be suitable for combination therapy with an immunotherapy, for treating a disease or disorder such as a cancer. The TGF-β ligand traps described herein may also be suitable for monotherapy for treating a disease or disorder such as a cancer. In particular, provided herein are methods and compositions for treating a disease or disorder such as a cancer by administering a TGF-β ligand trap in combination with an immune checkpoint inhibitor.

Description

TRANSFORMING GROWTH FACTOR-BETA LIGAND TRAPS

FOR THE TREATMENT OF DISEASE

PRIORITY

[0001] This application claims the benefit of priority to U.S. Serial No. 63/214,588 filed June 24, 2021, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] This application incorporates by reference a Sequence Listing submitted with this application as text file entitled ‘T4247-670-228_Sequence_Listing.txf’ created on June 17, 2022 and having a size of 32,722 bytes.

FIELD

[0003] The present invention relates to methods of using a Transforming Growth Factor-b (TGF-b) ligand trap, including combinations of a TGF-b ligand trap and an immunotherapy, for treating a disease or disorder such as a cancer.

1. BACKGROUND

[0004] Cancer can induce significant suppression of the immune system and escape from the immune surveillance mechanisms of the host. Dysregulation of host immune system is now considered one important hallmark of cancer (Hanahan etal. , Cell , 2011, 144, 646-674). The interactions between cancer and the host immune system have been extensively studied and many types of immunotherapies have been explored for cancer treatment. One class of immunotherapy is agents targeting specific immune checkpoint proteins that play critical roles in regulating T cell activation and proliferation. These proteins function as co-receptors on the surfaces of T cells and help regulate T cell responses following T cell activation (Wolchok et al ., Cancer ./., 2010, 16, 311-317). The two best characterized checkpoint proteins are cytotoxic T- lymphocyte antigen 4 (CTLA-4) and programmed death- 1 (PD-1), both serve as negative regulators of T cell activation.

[0005] While immunotherapy with checkpoint inhibitors have changed survival expectations for many cancer patients, not all patients respond to them and some stop responding over time (Jenkins et al, Br. J. Cancer, 2018, 118(1), 9-16). In investigating the mechanisms of resistance to immune checkpoint inhibitors, it has been found that tumors can evolve to evade both innate and adaptive arms of the immune system, thereby rendering immunotherapy with immune checkpoint inhibitors ineffective (Pitt etal. , Immunity , 2016, 44(6), 1255-1269; Restifo etal. , Nat. Rev. Cancer , 2016, 12(2), 121-126). There remains a need of effective combination therapies to overcome both innate and acquired resistance to immunotherapy with immune checkpoint inhibitors.

[0006] TGF-b ligand traps (e.g., M7824 (Knudson eta/., Oncoimmunology 7(5):el426519 (2018)) or AVID200 (Thwaites etal., Blood 130:2532 (2017))) are comprised of isolated TGF-b receptors that inhibit the binding of TGF-b ligand to its cognate receptor on cells. TGF-b ligand traps prevent TGF ligands from binding to TGF receptors, thereby preventing TGF^-mediated signaling. The TGF-b ligand can exist in three known isoforms, TGF-bI, TGF^2, and TGF^3. TGF-bI and TGF^3 have been identified as oncogenic isoforms while TGF^2 promotes normal cardiac function. Additionally, the role of TGF-b in metastasis has been described in O'Connor-McCourt et al., Cancer Research, 2018, 1759. Therefore, in the design of TGF-b ligand traps for use as cancer therapeutics, it is beneficial for the ligand trap to bind and sequester TGF-b isoforms TGF-bI and TGF^3 specifically while not binding to TGF^2. TGF- b ligand traps have been promising new immunotherapy that enhances desirable anti-tumor immunity.

2. SUMMARY

[0007] Provided herein are methods of preventing or treating a cancer in a subject comprising administering to a subject a TGF-b ligand trap and/or an additional therapeutic agent, for example nivolumab.

[0008] In certain embodiments, the cancer is resistant to treatment with nivolumab.

[0009] In certain embodiments, the TGF-b ligand trap is AVID200.

[0010] Also provided herein are methods of treating a cancer in a subject comprising administering to a subject a therapeutically effective amount of AVID200, wherein the cancer is relapsed or refractory.

[0011] In certain embodiments, the relapsed or refractory cancer is resistant to treatment with nivolumab. [0012] In certain embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), colorectal cancer, hepatocellular carcinoma, ovarian cancer, breast cancer, renal cell carcinoma (RCC), and pancreatic cancer.

[0013] In certain embodiments, the TGF-b ligand trap is a polypeptide comprising an amino acid sequence selected from any one of the amino acid sequences of SEQ ID NO: 1 to 5, or a fragment thereof, or an amino acid sequence that is at least 80%-99%, for example 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, identical to any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5. SEQ ID and SEQ ID NO: are used interchangeably here. In certain embodiments, the TGF-b ligand trap is a polypeptide comprising an amino acid sequence that is at least 80%-85%, 85%-90%, 90%-95%, or 95%-99% identical to any one of the amino acid sequences of SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or a fragment thereof.

[0014] In certain embodiments, the TGF-b ligand trap comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. In certain embodiments, the TGF-b ligand trap is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. In certain embodiments, the TGF-b ligand trap is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof.

[0015] In certain embodiments, the TGF-b ligand trap comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a fragment thereof. In certain embodiments, the TGF-b ligand trap is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a fragment thereof. In certain embodiments, the TGF-b ligand trap is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.

[0016] In certain embodiments, the TGF-b ligand trap comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. In certain embodiments, the TGF-b ligand trap is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a fragment thereof. In certain embodiments, the TGF-b ligand trap is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 or a fragment thereof.

[0017] In certain embodiments, the TGF-b ligand trap comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. In certain embodiments, the TGF-b ligand trap is a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a fragment thereof. In certain embodiments, the TGF-b ligand trap is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 or a fragment thereof.

[0018] In certain embodiments, the TGF-b ligand trap comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. In certain embodiments, the TGF-b ligand trap is a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or a fragment thereof. In certain embodiments, the TGF-b ligand trap is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 or a fragment thereof.

[0019] In various embodiments, the TGF-b ligand trap comprises a variant of any one of SEQ ID Nos: 1-5.

[0020] In certain embodiments, the nivolumab is present in a pharmaceutical composition that further comprises an excipient.

[0021] In certain embodiments, the TGF-b ligand trap is present in a pharmaceutical composition that further comprises an excipient.

3. DETAILED DESCRIPTION

[0022] Provided herein are immunotherapies for treating a disease or disorder such as a hyperproliferative malignancy, e.g ., a cancer. The immunotherapies provided herein include various methods and compositions. More specifically, provided herein are combination treatments with a TGF-b ligand trap (see Section 3.2) and an immune checkpoint inhibitor (see Section 3.3.1).

[0023] In certain embodiments, provided herein are methods and compositions for treating a disease or disorder such as a cancer using a TGF-b ligand trap, such as a polypeptide comprising an amino acid sequence selected from any one of the amino acid sequences of SEQ ID NO: 1 to 5 (see Table 1), or an amino acid sequence that is at least 95, 96, 97, 98, or 99% identical to any one of the amino acid sequences of SEQ ID NO: 1 to 5 or a fragment thereof, and an immune checkpoint inhibitor. Such an immune checkpoint inhibitor can inhibit, decrease, or interfere with the activity of a negative checkpoint regulator. Combination methods and for using a TGF-b ligand trap and an immune checkpoint inhibitor provided herein are described in more detail in Section 3.3 below.

3.1 Definitions

[0024] Techniques and procedures described or referenced herein include those that are generally well understood and/or commonly employed using conventional methodology by those skilled in the art. Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0025] The term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of a therapeutic compound, a combination of therapeutic compounds or pharmaceutical compositions thereof provided herein, which is sufficient to result in the desired outcome.

[0026] The terms “subject” and “patient” may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal. In specific embodiments, the subject is a human.

In one embodiment, the subject is a mammal, e.g ., a human, diagnosed with or suffering from a disease or disorder. In another embodiment, the subject is a mammal, e.g. , a human, at risk of developing a disease or disorder.

[0027] “Administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.

[0028] As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or disorder resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and/or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder. The term “treating” includes both managing and ameliorating the disease.

[0029] The terms “prevent,” “preventing,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s).

[0030] The terms “inhibition”, “inhibit”, “inhibiting” refer to a reduction in the activity or expression of a polypeptide or protein (e.g., TGFp i and TGFP3) or reduction or amelioration of a disease, disorder, or condition or a symptom thereof. Inhibiting as used here can include partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating protein or enzyme activity.

[0031] The term “immune checkpoint inhibitor” refers to a molecule that inhibits, decreases or interferes with the activity of a negative checkpoint regulator. In certain embodiments, immune checkpoint inhibitors for use with the methods and compositions disclosed herein can inhibit the activity of a negative checkpoint regulator directly, or decrease the expression of a negative checkpoint regulator, or interfere with the interaction of a negative checkpoint regulator and a binding partner ( e.g ., a ligand). Immune checkpoint inhibitors for use with the methods and compositions disclosed herein include a protein, a polypeptide, a peptide, an antisense oligonucleotide, an antibody, an antibody fragment, or an inhibitory RNA molecule that targets the expression of a negative checkpoint regulator.

[0032] A “negative checkpoint regulator” refers to a molecule that down-regulates immune responses (e.g., T-cell activation) by delivery of a negative signal to T-cells following their engagement by ligands or counter-receptors. Exemplary functions of a negative-checkpoint regulator are to prevent out-of-proportion immune activation, minimize collateral damage, and/or maintain peripheral self-tolerance. In certain embodiments, a negative checkpoint regulator is a ligand or receptor expressed by an antigen presenting cell. In certain embodiments, a negative checkpoint regulator is a ligand or receptor expressed by a T-cell. In certain embodiments, a negative checkpoint regulator is a ligand or receptor expressed by both an antigen presenting cell and a T-cell.

[0033] The term “antibody” also known as an immunoglobulin, as used herein, refers to a large (e.g., Y-shaped) protein that binds to an antigen and/or target. Antibodies are used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the antigen, because each tip of the “Y” of the antibody contains a site that is specific to a site on an antigen, allowing these two structures to bind with precision. An antibody (e.g., a multi-chain antibody) may consist of four polypeptide chains, two heavy chains and two light chains connected by interchain cysteine disulfide bonds. For example, antibodies (e.g., multi-chain antibodies) include human IgGl and human IgG4 which have four interchain disulfide bonds (e.g., two heavy chain-light chain interchain disulfide bonds and two hinge heavy chain-heavy chain interchain disulfide bonds), human IgG2 which has six interchain disulfide bonds (e.g., four heavy chain-light chain interchain disulfide bonds and two hinge heavy chain-heavy chain interchain disulfide bonds), and human IgG3 which has thirteen interchain disulfide bonds ( e.g ., eleven heavy chain-light chain interchain disulfide bonds and two hinge heavy chain-heavy chain interchain disulfide bonds).

[0034] The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, and are not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region.

[0035] “Antibody fragments” comprise only a portion of an intact antibody, wherein the portion retains at least one, two, three and as many as most or all of the functions normally associated with that portion when present in an intact antibody. In one aspect, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another aspect, an antibody fragment, such as an antibody fragment that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody. Such functions may include FcRn binding, antibody half life modulation, ADC function and complement binding. In another aspect, an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody. For example, such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

[0036] The term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g, naturally occurring mutations, that may be present in minor amounts.

[0037] The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0038] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0039] The term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

[0040] As used herein, numerical values are often presented in a range format throughout this document. The use of a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention unless the context clearly indicates otherwise. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges including integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document.

3.2 Transforming Growth Factor-b (TGF-b) Ligand Traps [0041] A Transforming Growth Factor-b (TGF-b) ligand trap that can be used with the methods and compositions disclosed herein can comprise one extracellular binding domain (ECD) of the TGF-b receptor. In certain embodiments, a TGF-b ligand trap can comprise more than one ECD of the TGF-b receptor such as the TGF-b ligand traps described in WO 2020/069372 (Elstar Therapeutics, Inc.) and US 2015/0225483 (Merck Patent GMBH). There are three known TGF-b receptor types, the TGF-b type I receptor is the signaling chain and does not bind the ligand; the TGF-b type II receptor binds ligands TGF-bI and TGF^3 with high affinity but only weakly binds TGF^2; and the TGF-b type III receptor functions as a co receptor. In certain embodiments, a TGF-b ligand trap that can be used with the methods and compositions disclosed herein comprises at least one ectodomain of a TGF-b type II receptor (TGF^RII) (Accession number P37173).

[0042] Wherein a TGF-b ligand trap comprises more than one TGF-b ECD, the domains can be joined to each other by a peptide linker, for example a short peptide linker. In certain embodiments, the linker can comprise multiples glycine residues such as the linker described in WO 2008/157367 (Genzyme Corporation). Alternatively, in certain embodiments, the TGF-b ECDs can be joined together by way of a natural linker. In certain embodiments, the TGF-b binding domain(s) of a TGF-b ligand trap can be fused or linked to an additional protein or domain. The additional protein can be the constant region of an immunoglobulin as is disclosed in WO 1998/48024 (Biogen Inc); WO 2011/109789 (The Johns Hopkins University); WO 2015/077540 (The Brigham and Womens Hospital, Inc.); US 20200002425 (Altor Biosciences Corporation); and US 10,316,076 (Acceleron Pharma, Inc.). A Fc domain fused to a TGF-b ligand trap can derived from IgGl, IgG2, IgG3, or IgG4.

[0043] A TGF-b ligand trap that can be used with the methods and compositions disclosed herein. The TGF-b ligand trap is in certain embodiments a polypeptide. In various embodiments, the polypeptide comprises from N-terminus to C-terminus (i) an amino acid sequence consisting of amino acids II to D272 of any one of SEQ ID NO: 1-5; and (ii) the Fc region of an antibody heavy chain. In certain embodiments, the Fc region is the Fc region of an IgGl antibody. In certain embodiments, the Fc region is the Fc region of an IgG2 antibody. In certain embodiments, the polypeptide further comprises a glycine rich linker fused to the N terminus of the antibody Fc portion of the sequence.

[0044] Exemplary TGF-b ligand traps that can be used with the methods and compositions disclosed herein are provided in Table 1. In certain embodiments, the TGF-b ligand trap is a polypeptide comprising an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 1 to 5. In some embodiments, the TGF-b ligand trap is a polypeptide comprising an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to a sequence selected from SEQ ID NO: 1 to 5. In some embodiments, the TGF-b ligand trap is a polypeptide comprising an amino acid sequence set forth in Table 1, or an amino acid sequence that is 95%, 96%, 97%,

98% or 99% identical to a sequence set forth in Table 1. In some embodiments, the TGF-b ligand trap is a polypeptide consisting of an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 1 to 5 (see, Table 1). The TGF-b ligand trap polypeptides of SEQ ID NO: 1-5 are comprised of isolated TGF-b receptors that were observed to inhibit the binding of TGF-b ligand to its cognate receptor on cells and inhibit in vivo tumor growth (WO2017/037634, Thwaites et al., Blood 130:2532 (2017) and FIGs. 1-5).

[0045] Table 1. Amino acid sequences of exemplary TGF-b ligand trap polypeptides.

[0046] In certain embodiments, the TGF-b ligand trap is a polypeptide having the N-terminus of any one of SEQ ID: 1-5 precisely as shown in Table 1. In other embodiments, the TGF-b ligand trap is a polypeptide having the C-terminus of any one of SEQ ID: 1-5 precisely as shown in Table 1. In some embodiments, the TGF-b ligand trap is a polypeptide having the N-terminus and C terminus of any one of SEQ ID: 1-5 precisely as shown in Table 1.

[0047] It is understood in the art that polypeptides can be post-translationally modified in a variety of ways. Examples of post-translational modifications commonly observed include, but are not limited to, phosphorylation, glycosylation, sialylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, formation of disulfide bonds, and cross-linking of amino acids. In certain embodiments, a TGF-b ligand trap that can be used with the methods and compositions disclosed herein is a polypeptide comprising an amino acid sequence selected from the amino acid sequence of SEQ ID NO:l to 5, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from SEQ ID NO: 1 to 5 as shown in Table 1, wherein the polypeptide further comprises one or more post-translational modifications.

[0048] A TGF-b ligand trap that can be used with the methods and compositions disclosed herein can be fused or linked to other immunomodulatory or targeting domains. These resulting polypeptides can be bifunctional such as those described in WO 2019241625 (Accel eron Pharma, Inc.); US 2015/0225483 (Merck Patent GMBH); or David etal. , Oncoimmunology , 2017; or multifunctional such as US 20200140547 (The Johns Hopkins University); or WO 2020/069372 (Elstar Therapeutics, Inc.). A TGF-b ligand trap that can be used with the methods and compositions disclosed herein binds to TGF-bI and TGF^3 with low pM potency. In certain embodiments, a TGF-b ligand trap binds to TGF-bI and TGF^3 with a binding affinity (KD) of 10 micromolar (mM), 5mM, 1 mM, 500 nanomolar (nM), 100 nM, 50 nM, 10 nM, 1 nM, 100 picomolar (pM), 50 pM, 10 pM, 1 pM, or lower. In certain embodiments, the KD of the TGF-b ligand trap against the TGF^2 isoform is 1 nM, 5nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 mM, or higher. To determine the binding affinity for a TGF-b ligand trap disclosed herein, various assays can be used including, but are not limited to, Enzyme-Linked Immunosorbent Assays (ELISAs) and/or surface plasmon resonance (SPR) methods such as the Biacore system.

[0049] Additionally, a TGF-b ligand trap can inhibit TGF-b ligand binding to its receptor on cells resulting in the neutralization of the biological activity of TGF-b. In certain embodiments, the IC5₀ of the TGF-b ligand trap against the TGF-bI or TGF^3 isoforms is 1 mM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 100 pM, 50 pM, 10 pM, 1 pM, or lower. Various assays can be used to determine the ability of the TGF-b ligand trap to neutralize TGF-b including, but not limited to, the in vitro bioassays described in WO 2008/157367 (Genzyme, Inc.). TGF-b isoforms can be determined using in vitro assays such as the Magnetic Luminex Performance Assay.

[0050] A TGF-b ligand trap that can be used with the methods and compositions disclosed herein can enhance the capacity of T-cells isolated from draining lymph nodes to specifically recognize and kill tumor cells. In certain embodiments, a TGF-b ligand trap will have high anti tumor T-cell activating potency. In a specific embodiment, a TGF-b ligand trap that can be used with the methods and compositions disclosed herein has a higher anti-tumor T-cell-activating potency than that of the pan-neutralizing TGF-b antibody, 1D11 (Ling et al., 2013, PLoS One. 8(1): e54499; Watanabe H et al. 2020 Sci Rep 10 (1):9211). In certain embodiments, a TGF-b ligand trap that can be used with the methods and compositions described herein can be AVID200.

3.3 Combination Therapies

[0051] Provided herein are methods and compositions relating to the combination of a Transforming Growth Factor-b (TGF-b) ligand trap (e.g, a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1 to 5, and AVID200) and an immunotherapy. In certain embodiments, an immunotherapy comprises administering an immune checkpoint inhibitor. Immune checkpoint inhibitors, including agents that can be used as inhibitors of PD-1 mediated signaling such as inhibitors of PD-1 are set forth in Section 3.3.1.

3.3.1 Immune Checkpoint Inhibitors

[0052] The clinical use of immune-oncology agents targeting cytotoxic T-lymphocyte- associated protein 4 (CTLA-4) and the programmed cell death receptor- 1 (PD-1) and its ligand PD-L1, have resulted in improvements over the standard of care in the treatment of many cancer types. While these checkpoint inhibitors have produced improved clinical responses in such certain cancers, durable clinical responses only occur in approximately 10-45% of patients. Moreover, a significant number of tumors are either resistant or become refractory.

[0053] The immune checkpoint inhibitor to be administered in combination with the TGF-b ligand trap (e.g., AVID200) can be any pharmaceutical agent that inhibits or blocks the activity of an inhibitory immune checkpoint molecule. In specific embodiments, the activity is binding to the natural binding partner of the immune checkpoint molecule. If the immune checkpoint molecule is a receptor, the activity can be ligand-binding activity. If the immune checkpoint molecule is a ligand, the activity can be receptor-binding activity.

[0054] In specific embodiments, the immune checkpoint inhibitor to be administered in combination with the TGF-b ligand trap (e.g., AVID200) is a negative checkpoint regulator that is involved in T-Cell activation. In certain, more specific embodiments, such a negative checkpoint regulator is Cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD80, CD86, Programmed cell death 1 (PD-1), Programmed cell death ligand 1 (PD-L1), Programmed cell death ligand 2 (PD-L2), Lymphocyte activation gene-3 (LAG-3; also known as CD223), Galectin-3, B and T lymphocyte attenuator (BTLA), T-cell membrane protein 3 (TIM3), Galectin-9 (GAL9), B7-H1, B7-H3, B7-H4, T-Cell immunoreceptor with Ig and ITIM domains (TIGIT/Vstm3/WUCAM/VSIG9), V-domain Ig suppressor of T-Cell activation (VISTA), Glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein, Herpes Virus Entry Mediator (HVEM), 0X40, CD27, CD28, CD137. CGEN-15001T, CGEN-15022, CGEN- 15027, CGEN-15049, CGEN-15052, or CGEN-15092. An overview such checkpoint regulators and drugs that target them is set forth in Table 2. In a specific embodiment, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, IDO, or TDO.

Table 2

[0055] In certain embodiments, the immune checkpoint inhibitor can be an antibody, a small molecule, or an oligonucleotide (such as an aptamer, an shRNA, miRNA, siRNA, or antisense DNA). In specific embodiments, the immune checkpoint inhibitor has been approved by Food and Drug Administration (FDA) in the United States or a foreign counterpart agency for the treatment of the cancer or a disease caused by the pathogen.

[0056] In specific embodiments, the immune checkpoint inhibitor is an antibody that binds to and inhibits the activity of the immune checkpoint. Antibodies that can be the immune checkpoint inhibitor include, but are not limited to, monoclonal antibodies (including Fc- optimized monoclonal antibodies), polyclonal antibodies, multispecific antibodies ( e.g ., bispecific antibodies), antibody fragments retaining antigen-binding activity, such as Fv, Fab, Fab', F(ab' diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), multispecific antibodies formed from antibody fragments, and fusion proteins containing antibody fragments. In a specific embodiment, the antibody is a monoclonal antibody. In various embodiments, the antibody is a humanized antibody.

[0057] In certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In a specific embodiment, the immune checkpoint inhibitor is a monoclonal antibody that binds to and inhibits the activity (e.g, ligand-binding activity) of PD-1.

[0058] In certain embodiments, the monoclonal antibody is selected from the group consisting of nivolumab, pidilizumab, MEDI0680, pembrolizumab, AMP-224, AMP-514, STI-A1110, TSR-042, AUR-012, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, and toripalimab.

[0059] In a specific embodiment, the monoclonal antibody is nivolumab, pidilizumab, MEDI0680, or pembrolizumab. In a further specific embodiment, the monoclonal antibody is nivolumab. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is AMP-224. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is pidilizumab. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is pembrolizumab. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is MEDI0680. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is STI-A1110. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is TSR-042. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-1 is AUR-012.

[0060] In certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In a specific embodiment, the immune checkpoint inhibitor is a monoclonal antibody that binds to and inhibits the activity ( e.g ., receptor-binding activity) of PD-L1.

[0061] In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of mpdl3280A, durvalumab, avelumab, BMS-936559, atezolizumab, RG7446, and STI-A1010.

[0062] In a specific embodiment, the monoclonal antibody is mpdl3280A, durvalumab, avelumab, BMS-936559, or atezolizumab. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-L1 is RG7446. In another specific embodiment, the immune checkpoint inhibitor that is an inhibitor of PD-L1 is STI-A1010.

[0063] In certain embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4 (for example, ipilimumab).

[0064] In certain embodiments, the immune checkpoint inhibitor is an inhibitor of LAG3 (for example, BMS-986016).

[0065] In certain embodiments, immune checkpoint inhibitors to be administered in combination with the TGF-b ligand trap (e.g., AVID200) include but are not limited to: OPDIVO^® (nivolumab); YERVOY^® (ipilimumab); relatilimab; linrodostat; EMPLICITI^® (elotuzumab); BMS-986258; BMS 986315; BMS-986207; BMS-986249; and BMS-986218. [0066] PD-1 inhibitors useful in the combinations described herein include any molecule capable of inhibiting, blocking, abrogating or interfering with the activity or expression of PD-1. In particular, an anti -PD-1 inhibitor can be a small molecule compound, a nucleic acid, a polypeptide, an antibody, a peptibody, a diabody, a minibody, a single-domain antibody or nanobody, a single-chain variable fragment (ScFv), or a functional fragment or variant thereof.

In one instance the PD-1 inhibitor is a small molecule compound (e.g., a compound having a molecule weight of less than about 1000 Da.) In other embodiments, useful PD-1 inhibitors in the combinations described herein include nucleic acids and polypeptides.

3.3.2 Nivolumab

[0067] In a specific embodiment, the anti-PD-1 antibody provided herein is nivolumab (Bristol Myers Squibb).

[0068] The heavy chain and light chain sequences of nivolumab are shown in Table 3.

Table 3:

Heavy chain

QVQLVESGGG WQPGRSLRL DCKASGITFS NSG HWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQ NSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS WTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKP KDTL ISRTP EVTCVWDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRWSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV HEALHNHYT QKSLSLSLGK

(SEQ ID NO: 6)

Light chain

EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA

RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP

SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT

LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

(SEQ ID NO: 7) [0069] The heavy chain variable region of nivolumab has an amino acid sequence of SEQ ID NO:9, comprising the CDR1 (SEQ ID NO: 23), CDR2 (SEQ ID NO:30) and CDR3 (SEQ ID NO:37) regions. The light chain variable region of nivolumab has an amino acid sequence of SEQ ID NO: 16, comprising the CDR1 (SEQ ID NO:44), CDR2 (SEQ ID NO: 51) and CDR3 (SEQ ID NO: 58) regions. The sequences of the heavy chain variable region, the light chain variable region, and CDR1, CDR2, and CDR3 regions of nivolumab are shown in Table 4 below.

Table 4

3.3.3 Combination Therapies

[0070] Provided herein are methods of preventing and/or treating a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject a TGF-b ligand trap (e.g., AVID200) and an immune checkpoint inhibitor described herein. In certain embodiments, provided herein are methods of preventing a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject a TGF-b ligand trap (e.g., AVID200) and an immune checkpoint inhibitor (e.g., nivolumab) described herein. In certain embodiments, provided herein are methods of treating a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject a TGF-b ligand trap (e.g., AVID200) and an immune checkpoint inhibitor described herein. In certain embodiments, a pharmaceutically effective amount of the TGF-b ligand trap (e.g., AVID200) is administered. In certain embodiments, the TGF-b ligand trap (e.g., AVID200) and the immune checkpoint inhibitor are concomitantly administered. In certain embodiments, the co-administration of the TGF-b ligand trap (e.g., AVID200) and the immune checkpoint inhibitor (e.g., nivolumab) is pharmaceutically effective to treat the disease or disorder (e.g., a cancer).

[0071] In certain embodiments, provided herein are methods of preventing and/or treating a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject a TGF-b ligand trap (e.g., AVID200) and an inhibitor of PD-1 mediated signaling described herein (e.g., an anti -PD-1 antibody). In certain embodiments, provided herein are methods of preventing a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject a TGF-b ligand trap (e.g., AVID200) and an inhibitor of PD-1 mediated signaling described herein (e.g., an anti -PD-1 antibody). In certain embodiments, provided herein are methods of treating a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject a TGF-b ligand trap (e.g., AVID200) and an inhibitor of PD-1 mediated signaling described herein (e.g., an anti -PD-1 antibody). In certain embodiments, a pharmaceutically effective amount of the TGF-b ligand trap (e.g., AVID200) is administered. In certain embodiments, TGF-b ligand trap (e.g., AVID200) and the inhibitor of PD-1 mediated signaling are concomitantly administered.

In certain embodiments, the co-administration of TGF-b ligand trap (e.g., AVID200) and the inhibitor of PD-1 mediated signaling is pharmaceutically effective to treat the disease or disorder (e.g., a cancer).

[0072] In certain embodiments, the inhibitor of PD-1 mediated signaling is an anti-PD- 1 inhibitor.

[0073] In certain embodiments, provided herein are methods of preventing and/or treating a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject a TGF-b ligand trap (e.g., AVID200) and an anti-PD-1 antibody described herein (e.g., nivolumab). In certain embodiments, provided herein are methods of preventing a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject a TGF-b ligand trap (e.g., AVID200) and an anti-PD-1 antibody described herein (e.g., nivolumab). In certain embodiments, provided herein are methods of treating a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject a TGF-b ligand trap (e.g., AVID200) and an anti-PD-1 antibody described herein (e.g., nivolumab). In certain embodiments, a pharmaceutically effective amount of the TGF-b ligand trap (e.g., AVID200) is administered. In certain embodiments, TGF-b ligand trap (e.g., AVID200) and the anti-PD-1 antibody are concomitantly administered. In certain embodiments, the co-administration of TGF-b ligand trap (e.g., AVID200) and the anti- PD-1 antibody described herein (e.g., nivolumab) is pharmaceutically effective to treat the disease or disorder (e.g., a cancer). In certain embodiments, the anti-PD-1 antibody is nivolumab.

[0074] In certain embodiments, provided herein are methods of preventing and/or treating a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject AVID200 and nivolumab. In certain embodiments, provided herein are methods of preventing a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject AVID200 and nivolumab. In certain embodiments, provided herein are methods of treating a diease or disorder (e.g., a cancer) in a subject comprising administering to a subject AVID200 and nivolumab. In certain embodiments, a pharmaceutically effective amount of AVID200 is administered. In certain embodiments, AVID200 and nivolumab are concomitantly administered. In certain embodiments, the co-administration of AVID200 and nivolumab is pharmaceutically effective to treat the disease or disorder (e.g., a cancer).

Indications

[0075] Diseases and disorders that can be treated or prevented using the methods and compositions described herein include the oncology indications listed below. Specifically, these indications can be treated with the combination therapies of the TGF-b ligand trap (see Section 3.2) and an immunotherapy (see Section 3.3.1) as well as the monotherapy of the TGF-b ligand trap described in herein (see Section 3.4).

[0076] In some embodiments, the disease or disorder to be treated with the methods and compositions disclosed herein is a disease of abnormal cell growth and/or dysregulated apoptosis. Examples of such diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and/or biliary duct) cancer, primary or secondary central nervous system tumor, primary or secondary brain tumor, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell orB-cell origin, melanoma, multiple myeloma, oral cancer, non-small-cell lung cancer, prostate cancer, small-cell lung cancer, cancer of the kidney and/or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system, primary central nervous system lymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma or a combination thereof.

[0077] In some embodiments, the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small- cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer.

[0078] In some embodiments, the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma. In some embodiments, the cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia. In a specific embodiment, the disease or disorder is myeloma. In a specific embodiment, the disease or disorder is myelodysplastic syndromes (MDS). In another specific embodiment, the disease or disorder is acute myeloid leukemia (AML). In another specific embodiment, the disease or disorder is chronic lymphocytic leukemia (CLL). In yet another specific embodiment, the myeloma is multiple myeloma (MM). [0079] In other embodiments, the disease or disorder is a solid tumor malignancy. In some embodiments, the solid tumor malignancy is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer.

[0080] In some embodiments, the solid tumor malignancy is an advanced non-CNS-primary solid tumor. In some embodiments, the solid tumor malignancy is selected from a group consisting of gastric/gastroesophageal junction (GEJ) cancer, bladder/urothelial cancer, and non small-cell lung cancer (NSCLC).

[0081] In certain embodiments, the disease or disorder is malignant solid tumor or myelofibrosis. In certain embodiments, the myelofibrosis is intermediate-2 or higher primary myelofibrosis (PMF), post-essential thrombocythemia or polycythemia-vera related MF (Post ET/PV MF). In other embodiments, the disease or disorder is advanced or metastatic malignancy.

[0082] In some embodiments, the disease or disorder is a cancer expressing TGF-b. For example, the cancer is associated with expression of TGF-bI and/or TGF^3. In some embodiments, the disease or disorder is a cancer associated with high or elevated expression levels of TGF-b compared to a normal patient not having the cancer. Examples of such disease include, but are not limited to, pancreatic cancer, sarcomas, mesothelioma, cervical cancer, myelofibrosis, NSCLC, UC, colorectal cancer, SCCHN, hepatocellular carcinoma (HCC), ovarian cancer, and breast cancer.

[0083] In some embodiments, the subject is a human. In some embodiments, the subject is a subject diagnosed with cancer, e.g ., a hematological malignancy.

Dosing and Regimen [0084] The amount of a prophylactic or therapeutic agent (the TGF-b ligand trap and the immune checkpoint inhibitor provided herein), or a composition provided herein that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and in some embodiments, should be decided according to the judgment of the practitioner and each patient’s circumstances.

[0085] The dose administered to a subject in the context of the present disclosure should be sufficient to affect a therapeutic response. One skilled in the art will recognize that dosage will depend upon a variety of factors including the potency of the specific compound, the age, condition and body weight of the patient, as well as the stage/severity of the disease. The dose will also be determined by the route (administration form) timing and frequency of administration.

[0086] The TGF-b ligand trap (e.g., AVID200) and the immune checkpoint inhibitor can be formulated in different pharmaceutical compositions and administered separately to the subject in need thereof. Alternatively, the TGF-b ligand trap (e.g., AVID200) and the immune checkpoint inhibitor are administered together in the same pharmaceutical composition.

[0087] In some embodiments, the TGF-b ligand trap (e.g., AVID200) and the immune checkpoint inhibitor are administered simultaneously. The term “simultaneously” means at the same time or within a short period of time, for example, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, or less than 12 hours.

[0088] In some embodiments, the TGF-b ligand trap (e.g., AVID200) and the immune checkpoint inhibitor are not administered simultaneously, and instead the two compounds are administered at different times. In various embodiments, the TGF-b ligand (e.g., AVID200) trap is administered prior to the administration of the immune checkpoint inhibitor (e.g., nivolumab). Alternatively, the immune checkpoint inhibitor is administered prior to the administration of the the TGF-b ligand trap. In some embodiments, the TGF-b ligand trap (e.g., AVID200) and the immune checkpoint inhibitor are administered at least once during a dosing period. A dosing period as used herein is meant a period of time, during which each therapeutic agent has been administered at least once. A dosing cycle can be about 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, or 30 days. In some embodiments, a dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In certain embodiments, a dosing period is a dosing cycle.

[0089] The prophylactic or therapeutic agent (the TGF-b ligand trap (e.g., AVID200) and the immune checkpoint inhibitor provided herein) can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g, continuous infusion over time or divided bolus doses over time). The agent can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity. Stable disease or lack is determined by methods known in the art such as evaluation of patient symptoms, physical examination, and visualization of the tumor that has been imaged using X-ray, CAT, PET, MRI scan, or other commonly accepted evaluation modalities.

[0090] The prophylactic or therapeutic agent (the TGF-b ligand trap (e.g., AVID200) and the immune checkpoint inhibitor provided herein) can be administered once daily (QD) or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID). In addition, the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g, in cycles (i.e., including days, weeks, or months of rest without drug). As used herein, the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time. The term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days. The term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g, daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days. [0091] In some embodiments, the frequency of administration is in the range of about a daily dose to about a monthly dose. In certain embodiments, administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks.

[0092] In certain embodiments, the prophylactic or therapeutic agent (the TGF-b ligand trap (e.g., AVID200) and the immune checkpoint inhibitor provided herein) is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks.

[0093] In some embodiments, AVID200 and the immune checkpoint inhibitor are administered for 1 to 10 cycles. In some embodiments, AVID200 and the immune checkpoint inhibitor are administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles. In some embodiments, AVID200 and the immune checkpoint inhibitor are administered for more than 10 cycles.

[0094] Regimens for administration of a combination described herein can be modified as necessary to include administration of the TGF-b ligand trap and/or the immune checkpoint inhibitor. Administration of such active agents, e.g., the TGF-b ligand trap and/or the immune checkpoint inhibitors, can be performed QD, QW, QM, BID, BIW, TIW, Q2W, Q3W, or Q4W, or in accordance with prescribing information for such immune checkpoint inhibitors as set forth, for example, in a package insert. In certain embodiments, the immune checkpoint inhibitor (e.g., nivolumab) is administered once every 1, 2, 3, or 4 weeks. In a specific embodiment, the immune checkpoint inhibitor (e.g., nivolumab) is administered once every week. In another specific embodiment, the immune checkpoint inhibitor (e.g., nivolumab) is administered once every 2 weeks. In another specific embodiment, the immune checkpoint inhibitor (e.g., nivolumab) is administered once every 3 weeks. In another specific embodiment, the immune checkpoint inhibitor (e.g., nivolumab) is administered once every 4 weeks. In certain embodiments, the TGF-b ligand trap (e.g., AVID200) is administered once every 1, 2, 3, or 4 weeks. In a specific embodiment, the TGF-b ligand trap (e.g., AVID200) is administered once every week. In a specific embodiment, the TGF-b ligand trap (e.g., AVID200) is administered once every 2 weeks. In another specific embodiment, the TGF-b ligand trap (e.g., AVID200) is administered once every 3 weeks. In another specific embodiment, the TGF-b ligand trap (e.g., AVID200) is administered once every 4 weeks.

[0095] In certain embodiments, the combination includes a TGF-b ligand trap (e.g., AVID200) administered at an amount of greater than about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg. In certain embodiments, the combination includes a TGF-b ligand trap (e.g., AVID200) administered at a dose of about 400 mg to about 1600 mg. For example, the dose is 400 mg, 800 mg, 1200 mg, and 1600 mg. In one embodiment, the combination described herein includes a TGF-b ligand trap (e.g., AVID200) administered at an amount greater than about: 100 mg/m², 200 mg/m², 300 mg/m², 400 mg/m², 500 mg/m², 600 mg/m², 700 mg/m², 800 mg/m², 900 mg/m², 1000 mg/m², 1500 mg/m², 2000 mg/m², 2500 mg/m², 3000 mg/m², 3500 mg/m², 4000 mg/m², 4500 mg/m², 5000 mg/m², 5500 mg/m², 6000 mg/m², 6500 mg/m², 7000 mg/m², 8000 mg/m², 9000 mg/m², or 10000 mg/m². In specific embodiments, the combination can include a TGF-b ligand trap (e.g., AVID200) administered at an amount of about 70 mg/m², about 180 mg/m², about 550 mg/m², or about 1100 mg/m².

[0096] In certain embodiments, the combination described herein includes an immune checkpoint inhibitor administered at an amount between 100 mg and 200 mg, between 200 mg and 300 mg, between 300 mg and 400 mg, between 400 mg and 500 mg, between 500 mg and 600 mg, between 600 mg and 700 mg, between 700 mg and 800 mg, between 800 mg and 900 mg, or between 900 mgand 1000 mg In specific embodiments, the combination described herein includes an immune checkpoint inhibitor (e.g., nivolumab) administered at an amount of about 240 mg, about 360 mg, about 400 mg, or about 480 mg. In a specific embodiment, the combination described herein includes an immune checkpoint inhibitor (e.g., nivolumab) administered at an amount of about 240 mg. In a specific embodiment, the combination described herein includes an immune checkpoint inhibitor (e.g., nivolumab) administered at an amount of about 480 mg.

[0097] In certain embodiments, the dosing of the immune checkpoint inhibitor in the combinations described herein is relative to the weight of the patient (/. ., mg/kg). In some embodiments, the immune checkpoint inhibitor is administered in an amount equivalent to about: 0.0001 mg/kg to about 200 mg/kg, 0.001 mg/kg to about 200 mg/kg, 0.01 mg/kg to about 200 mg/kg, 0.01 mg/kg to about 150 mg/kg, 0.01 mg/kg to about 100 mg/kg, 0.01 mg/kg to about 50 mg/kg, 0.01 mg/kg to about 25 mg/kg, 0.01 mg/kg to about 10 mg/kg, or 0.01 mg/kg to about 5 mg/kg, 0.05 mg/kg to about 200 mg/kg, 0.05 mg/kg to about 150 mg/kg, 0.05 mg/kg to about 100 mg/kg, 0.05 mg/kg to about 50 mg/kg, 0.05 mg/kg to about 25 mg/kg, 0.05 mg/kg to about 10 mg/kg, or 0.05 mg/kg to about 5 mg/kg, 0.5 mg/kg to about 200 mg/kg, 0.5 mg/kg to about 150 mg/kg, 0.5 mg/kg to about 100 mg/kg, 0.5 mg/kg to about 50 mg/kg, 0.5 mg/kg to about 25 mg/kg, 0.5 mg/kg to about 10 mg/kg, or 0.5 mg/kg to about 5 mg/kg.

[0098] In a specific embodiment, the immune checkpoint inhibitor (e.g., nivolumab) is administered at a dose of about0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, or about 3.0 mg/kg. In a specific embodiment, the immune checkpoint inhibitor (e.g., nivolumab) is administered at a dose of about 3.0 mg/kg.

[0099] In certain embodiments, the therapeutically effective amount of an anti -PD- 1 antibody (e.g., nivolumab) is determined as an amount provided in a package insert provided with the PD- 1 antibody (e.g, nivolumab). The term package insert refers to instructions customarily included in commercial packages of medicaments approved by the U.S. Food and Drug Administration (US FDA) or a similar regulatory agency of a country other than the USA (e.g., European Medicines Agency (EMA)), which contains information about, for example, the usage, dosage, administration, contraindications, and/or warnings concerning the use of such medicaments.

3.4 Monotherapy

[00100] In another aspect, provided herein are methods of treating a disease or disorder (e.g., a cancer and myelofibrosis) in a subject comprising administering to a subject a therapeutically effective amount of AVID200, wherein the disease or disorder (e.g., a cancer) is relapsed or refractory. In certain embodiments, the relapsed or refractory disease or disorder (e.g., a cancer) is resistant to treatment with an anti-PD-1 antibody (e.g., nivolumab). In certain embodiments, the subject was previously treated with an anti-PD-1 antibody (e.g., nivolumab).

[00101] In certain embodiments, the disease or disorder (e.g., a cancer) is a disease or disorder (e.g., a cancer) disclosed in Section 3.3.3, treated by the combination therapy disclosed herein. [00102] In certain embodiments, the relapsed or refractory disease or disorder (e.g., a cancer) is selected from the group consisting of non-small cell lung cancer (NSCLC), colorectal cancer, hepatocellular carcinoma, ovarian cancer, breast cancer, and pancreatic cancer [00103] In yet another aspect, provided herein are methods of treating relapsed or refractory non-small cell lung cancer (NSCLC) by administering a therapeutically effective amount of AVID200, wherein the relapsed or refractory NSCLC is resistant to treatment with an anti-PD-1 antibody (e.g., nivolumab). In certain embodiments, the subject was previously treated with an anti-PD-1 antibody (e.g., nivolumab). In some embodiments, the relapsed or refractory NSCLC is Stage IIA or Stage IIB. The relapsed or refractory NSCLC can be a Stage IIIA or Stage MB cancer. The NSCLC can be a Stage IV cancer. Staging of cancers as described herein is described by the American Joint Committee on Cancer TNM classification of malignant tumours cancer staging notation as is well understood in the art. [00104] In yet another aspect, provided herein are methods of treating relapsed or refractory colorectal cancer by administering a therapeutically effective amount of AVID200, wherein the relapsed or refractory colorectal cancer is resistant to treatment with an anti-PD-1 antibody (e.g., nivolumab). In certain embodiments, the subject was previously treated with an anti-PD-1 antibody (e.g., nivolumab). In some embodiments the colorectal cancer is a Stage I cancer. In another embodiment, the colorectal cancer is a Stage IIA, Stage IIB, or Stage IIC cancer. In still another embodiment, the colorectal cancer is a Stage IIIA, Stage IIIB, or Stage IIIC cancer. In yet another embodiment, the colorectal cancer is a Stage IVA or Stage IVB cancer. In certain instances the colorectal cancer is further characterized by the grade of the cancer. The colorectal cancer can be a Grade 1, Grade 2, Grade 3, or Grade 4 cancer in any of the stages provided herein.

[00105] In yet another aspect, provided herein are methods of treating relapsed or refractory hepatocellular carcinoma by administering a therapeutically effective amount of AVID200, wherein the relapsed or refractory hepatocellular carcinoma is resistant to treatment with an anti- PD-1 antibody (e.g., nivolumab). In certain embodiments, the subject was previously treated with an anti-PD-1 antibody (e.g., nivolumab). In some embodiments, the relapsed or refractory hepatocellular carcinoma is Stage II cancer. The relapsed or refractory hepatocellular carcinoma can be a Stage IIIA, Stage IIIB, or Stage IIIC cancer. The hepatocellular carcinoma can be a a Stage IVA or Stage IVB cancer.

[00106] In yet another aspect, provided herein are methods of treating relapsed or refractory melanoma by administering a therapeutically effective amount of AVID200, wherein the relapsed or refractory melanoma is resistant to treatment with an anti-PD-1 antibody (e.g., nivolumab). In certain embodiments, the subject was previously treated with an anti-PD-1 antibody (e.g., nivolumab). In some embodiments, the relapsed or refractory melanoma is a Stage IIA, IIB, or IIC cancer. The relapsed or refractory melanoma can be a Stage IIIA, Stage IIIB, or Stage IIIC cancer. The melanoma can be a Stage IV cancer.

[00107] In yet another aspect, provided herein are methods of treating relapsed or refractory ovarian cancer by administering a therapeutically effective amount of AVID200, wherein the relapsed or refractory ovarian cancer is resistant to treatment with an anti-PD-1 antibody (e.g., nivolumab). In certain embodiments, the subject was previously treated with an anti-PD-1 antibody (e.g., nivolumab). In some embodiments the ovarian cancer is a Stage I cancer as defined by the FIGO Ovarian Cancer Staging standards. The ovarian cancer can be a Stage IA, IB, or IC (e.g., IC1, IC2, or IC3) cancer. In another embodiment, the ovarian cancer is a Stage II cancer. The ovarian cancer can be a Stage IIA or IIB cancer.

[00108] In yet another aspect, provided herein are methods of treating relapsed or refractory breast cancer by administering a therapeutically effective amount of AVID200, wherein the relapsed or refractory breast cancer is resistant to treatment with an anti -PD- 1 antibody (e.g., nivolumab). In certain embodiments, the subject was previously treated with an anti -PD- 1 antibody (e.g., nivolumab). The breast cancer can be HER2 negative breast cancer. The breast cancer can be a HER2 positive breast cancer. The breast cancer can be triple-negative breast cancer. In some embodiments the breast cancer is a Stage IA or Stage IB cancer. In another embodiment, the breast cancer is a Stage IIA or Stage IIB cancer. In still another embodiment, the breast cancer is a Stage IIIA, Stage IIIB, or Stage IIIC cancer. In yet another embodiment, the breast cancer is a Stage IV cancer.

[00109] In yet another aspect, provided herein are methods of treating relapsed or refractory pancreatic cancer by administering a therapeutically effective amount of AVID200, wherein the relapsed or refractory pancreatic cancer is resistant to treatment with an anti-PD-1 antibody (e.g., nivolumab). In certain embodiments, the subject was previously treated with an anti-PD-1 antibody (e.g., nivolumab). In some embodiments, the pancreatic cancer is locally advanced, surgically resected or unresected pancreatic cancer or metastatic pancreatic adenocarcinoma. In some embodiments the pancreatic cancer is a Stage IA or Stage IB cancer. In another embodiment, the pancreatic cancer is a Stage IIA or Stage IIB cancer. In still another embodiment, the pancreatic cancer is a Stage III cancer. In yet another embodiment, the pancreatic cancer is a Stage IV cancer.

3.5 Pharmaceutical Compositions

[00110] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia. European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[00111] “Excipient” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof. The term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle.

[00112] In some embodiments, excipients are pharmaceutically acceptable excipients.

Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington’s Pharmaceutical Sciences (18th ed. 1990).

[00113] In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al, Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. In some embodiments, a pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[00114] The TGF-b ligand trap (e.g., AVID200) can be formulated with one or more pharmaceutically acceptable excipient. Similarly, the immune checkpoint inhibitor can be formulated in a pharmaceutical composition with one or more pharmaceutically acceptable excipients. In some embodiments, provided herein is a combination therapy comprising a first pharmaceutical composition comprising a TGF-b ligand trap provided herein (e.g., AVID200) and a first pharmaceutically acceptable excipient, and a second pharmaceutical composition comprising an immune checkpoint inhibitor (e.g., nivolumab) provided herein and a second pharmaceutically acceptable excipient. The first and the second pharmaceutically acceptable excipients can be the same or different. In some embodiments, a TGF-b ligand trap provided herein (e.g., AVID200) and an immune checkpoint inhibitor provided herein are formulated together in a single pharmaceutical composition. In various embodiments, the single pharmaceutical composition further comprises an excipient. In other embodiments, provided herein is a pharmaceutical composition comprising a TGF-b ligand trap provided herein (e.g., AVID200), an immune checkpoint inhibitor provided herein, and one or more pharmaceutically acceptable excipient.

[00115] The TGF-b ligand traps and/or the immune checkpoint inhibitors provided herein can be formulated into suitable pharmaceutical compositions for different routes of administration, such as injection (subcutaneous, intramuscular, intravenous, intraperitoneal, intraosseous, intracardiac, intraarticular, and intracavernous), sublingual and buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal. The compounds described above may be formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms. (7th ed. 1999)). [00116] In the compositions, effective concentrations of one or more compounds (i.e., the TGF- b ligand trap or the immune checkpoint inhibitor provided herein) or pharmaceutically acceptable salts are mixed with a suitable pharmaceutical excipient. In certain embodiments, the concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder provided herein (e.g., cancer, including solid cancer and blood borne cancer).

[00117] The active compound is in an amount sufficient to exert a therapeutically useful effect on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems and then extrapolated therefrom for dosages for humans. The concentration of active compound in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

[00118] It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[00119] The TGF-b ligand traps and/or the immune checkpoint inhibitors provided herein can be provided in forms convenient to or facilitate their administration to a patient. For example, in some embodiments, when the immune checkpoint inhibitor is an anti-PD-1 antibody as described herein, the PD-1 inhibitor can be formulated as a ready to use solution for parenteral administration. In other embodiments, the PD-1 inhibitor, including for example an anti-PD-1 antibody, can be formulated as a powder ( e.g ., lyophilized powder) that can be resuspended in a liquid suitable for parenteral administration. In one embodiment, the PD-1 antibody is formulated for intravenous administration. In another embodiment, the TGF-b ligand trap and the PD-1 inhibitor are both formulated for intravenous administration. In another embodiment, the TGF-b ligand trap and the PD-1 inhibitor are both formulated for intravenous infusion administration.

[00120] Combinations described herein can be provided as controlled release pharmaceutical products, which have a goal of improving drug therapy over that achieved by their non controlled counterparts. Controlled release formulations can extend activity of the drug, reduce dosage frequency, and increase subject compliance. In addition, controlled release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.

[00121] The combinations and pharmaceutical compositions described herein can be provided as part of a kit. Such kits can, for example, improve patient compliance or improve the accuracy or ease of preparation for administering the combination. The kit includes a TGF-b ligand trap as described herein. The kit also includes an anti-PD-1 inhibitor as described herein. The kit can include AMP-224. In some embodiments, the kit includes an anti-PD-1 antibody, as described herein, such as for example, nivolumab, pembrolizumab, pidilizumab, REGN2810, PDR 001, or MEDI0680. The kit can include a package insert or other information ( e.g ., prescribing information) useful for administration of the combination to a patient in need thereof, such as a cancer patient described herein.

[00122] Kits of the invention can include the combinations described herein (e.g., a TGF-b ligand trap and an anti-PD-1 antibody) having the same or different formulation. Each component of a combination described herein in a kit can be supplied in a separate, individual container. Alternatively or additionally, components of the combinations described herein can be supplied in a single container. In such instances, the container can be a container that is ready for administration to a patient in need thereof, such as for example, an IV bag, ampoule, or a syringe. In one embodiment, the PD-1 inhibitor can be supplied as, for example, a powder (e.g, lyophilized powder) or as a solution for parenteral administration. In certain embodiments, the PD-1 inhibitor is an anti-PD-1 antibody as described herein formulated for parenteral administration by, for example, intravenous administration.

[00123] The TGF-b ligand trap (e.g., AVID200) or the immune checkpoint inhibitor provided herein, or pharmaceutically acceptable salts thereof, may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. In one embodiment, liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable excipients. These may be prepared according to methods known to those skilled in the art.

[00124] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include, aspects that are not expressly included in the invention are nevertheless disclosed herein. [00125] From the foregoing, it will be appreciated that, although specific embodiments have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of what is provided herein. All of the references referred to above are incorporated herein by reference in their entireties.

Claims

WHAT IS CLAIMED:

1. A method of preventing or treating a cancer in a subject comprising administering to a subject a TGF-b ligand trap and nivolumab.

2. The method of claim 1, wherein the cancer is resistant to treatment with nivolumab.

3. The method of claim 1 or 2, wherein the TGF-b ligand trap is AVID200.

4. A method of treating a cancer in a subject comprising administering to a subject a therapeutically effective amount of AVID200, wherein the cancer is relapsed or refractory.

5. The method of claim 4, wherein the relapsed or refractory cancer is resistant to treatment with nivolumab.

6. The method of any one of claims 1-5, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), colorectal cancer, hepatocellular carcinoma, ovarian cancer, breast cancer, and pancreatic cancer.

7. The method of claim 1 or claim 2, wherein the TGF-b ligand trap is a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1 to 5, or a fragment thereof.

8. The method of claim 1 or claim 2, wherein the TGF-b ligand trap is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

9. The method of claim 1 or claim 2, wherein the TGF-b ligand trap is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

10. The method of claim 1 or claim 2, wherein the TGF-b ligand trap is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3.

11. The method of claim 1 or claim 2, wherein the TGF-b ligand trap is a polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

12. The method of claim 1 or claim 2, wherein the TGF-b ligand trap is a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

13. The method of any one of claims 1-12, wherein the nivolumab is present in a pharmaceutical composition that further comprises an excipient.

14. The method of any one of claims 1-13, wherein the TGF-b ligand trap is present in a pharmaceutical composition that further comprises an excipient.