WO2021209458A1

WO2021209458A1 - Combination treatment of cancer

Info

Publication number: WO2021209458A1
Application number: PCT/EP2021/059577
Authority: WO
Inventors: Adam S. LAZORCHAK; Yan Lan
Original assignee: Ares Trading S.A.; Glaxosmithkline Intellectual Property (No. 4) Ltd.
Priority date: 2020-04-14
Filing date: 2021-04-13
Publication date: 2021-10-21

Abstract

The present invention relates to combination therapies useful for the treatment of cancer. In particular, the invention relates to the combined use of a PD-1 inhibitor, a TGFβ inhibitor, and a VEGF inhibitor to treat cancer.

Description

COMBINATION TREATMENT OF CANCER

FIELD OF INVENTION

The present invention relates to the treatment of cancer and to combinations useful in such treatment. In particular, the invention relates to a combination of compounds for inhibiting PD-1, TGFβ and VEGF for use in treating cancer.

BACKGROUND OF THE INVENTION

Cancer immune evasion is a major obstacle to effective anti-cancer therapeutic strategies. Two prominent pathways that cancers exploit for tumor survival and to escape immune surveillance are the vascular endothelial growth factor (VEGF) pathway and the programmed death ligand 1 (PD-L1)/programmed death 1 (PD-1) immune checkpoint pathway.

The VEGF family consist of 5 ligands (VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF) that bind to a family of 3 receptors (VEGF receptor type 1 , VEGF receptor type 2 and VEGF receptor type 3). The VEGF pathway plays a central role in angiogenesis in normal development, wound healing, but also in tumors. VEGF has been shown to be overexpressed by many different carcinomas and neoangiogenesis in tumors is believed to be crucial for supplying tumors with oxygen and nutrients, as well as for tumor metastasis. The inhibition of the VEGF pathway by agents like bevacizumab or tyrosine kinase inhibitors is a well-established therapy against many tumors.

A mechanism by which tumor cells are escaping immunosurveillance is the blockage of checkpoint receptors. Tumor cells oftentimes express surface proteins like PD-L1 which, by binding to the PD-1 checkpoint receptor on T cells, reduce T cell cytotoxicity. This mechanism is counteracted by checkpoint therapies like anti-PD-L1 antibodies, anti-PD-1 antibodies or, in a similar fashion, by anti-CTLA4 antibodies.

TGF-beta ( TGFβ) is a well-studied pleiotropic cytokine. It has been described as a driver of tumor progression by suppressing the host antitumor immune response and by the induction of angiogenesis, epithelial-mesenchymal transition (EMT) and metastasis.

WO 2015/118175 describes a bifunctional fusion protein composed of the extracellular domain of the tumor growth factor beta receptor type II (TGFβRII) to function as a TGF-β “trap” fused to a human lgG1 antibody blocking PD-L1. Specifically, the protein is a heterotetramer, consisting of the two immunoglobulin light chains of an anti-PD-L1 antibody, and two heavy chains each comprising a heavy chain of the anti-PD-L1 antibody genetically fused via a flexible glycine-serine linker to the extracellular domain of the human TGFβRII (see Fig. 1). This fusion molecule is designed to target both the PD-L1 pathway and the TGFβ pathway to counteract immunosuppression in the tumor microenvironment.

Though there have been many recent advances in the treatment of cancer, there remains a need for more effective and/or enhanced treatment of an individual suffering the effects of cancer. The methods herein that relate to combining therapeutic approaches for enhancing anti-tumor immunity address this need.

SUMMARY OF THE INVENTION

The present invention arises out of the discovery that a therapeutic benefit in the treatment of cancer can be achieved by combining compounds which inhibit PD-1 , TGFβ and VEGF.

Thus, in a first aspect, the present disclosure provides a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor for use in a method of treating a cancer in a subject, for use in inhibiting tumor growth or progression in a subject who has malignant tumors, for use in inhibiting metastasis of malignant cells in a subject, for use in decreasing the risk of metastasis development and/or metastasis growth in a subject, or for use in inducing tumor regression in a subject who has malignant cells, wherein the use comprises administering said compounds to the subject.

The present disclosure also provides a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor for the manufacture of a medicament for use in a method of treating a cancer in a subject, for use in inhibiting tumor growth or progression in a subject who has malignant tumors, for use in inhibiting metastasis of malignant cells in a subject, for use in decreasing the risk of metastasis development and/or metastasis growth in a subject, or for use in inducing tumor regression in a subject who has malignant cells, wherein the use comprises administering said compounds to the subject.

In another aspect, the present disclosure provides a method of treating a cancer in a subject, a method of inhibiting tumor growth or progression in a subject who has malignant tumors, a method of inhibiting metastasis of malignant cells in a subject, a method of decreasing the risk of metastasis development and/or metastasis growth in a subject, or a method of inducing tumor regression in a subject who has malignant cells, wherein the method comprises administering a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor to the subject. In a further aspect, the disclosure relates to a method for advertising treatment with a PD-1 inhibitor, a TGFβ inhibitor, and a VEGF inhibitor comprising promoting, to a target audience, the use of the combination for treating a subject with a cancer, e.g., based on PD- L1 expression in samples, such as tumor samples, taken from the subject. The PD-L1 expression can be determined by immunohistochemistry, e.g., using one or more primary anti-PD-L1 antibodies.

Provided herein is also a pharmaceutical composition comprising a PD-1 inhibitor, a TGFβ inhibitor, and a VEGF inhibitor and at least a pharmaceutically acceptable excipient or adjuvant. In one embodiment, the PD-1 inhibitor and TGFβ inhibitor are fused in such pharmaceutical composition. The PD-1 inhibitor, the TGFβ inhibitor and the VEGF inhibitor are provided in a single or separate unit dosage forms.

In a further aspect, the present disclosure relates to a kit comprising a PD-1 inhibitor, a TGFβ inhibitor, and a VEGF inhibitor and a package insert comprising instructions for using said compounds to treat or delay progression of a cancer in a subject. In a further aspect, the invention relates to a kit comprising a PD-1 inhibitor and a package insert comprising instructions for using the PD-1 inhibitor, a TGFβ inhibitor, and a VEGF inhibitor to treat or delay progression of a cancer in a subject. In a further aspect, the invention relates to a kit comprising a TGFβ inhibitor and a package insert comprising instructions for using the TGFβ inhibitor, a PD-1 inhibitor, and a VEGF inhibitor to treat or delay progression of a cancer in a subject. In a further aspect, the invention relates to a kit comprising a VEGF inhibitor and a package insert comprising instructions for using the VEGF inhibitor, a PD-1 inhibitor, and a TGFβ inhibitor to treat or delay progression of a cancer in a subject. In a further aspect, the invention relates to a kit comprising an anti-PD(L)1:TGFβRII fusion protein and a package insert comprising instructions for using the anti-PD(L)1:TGFβRII fusion protein and a VEGF inhibitor to treat or delay progression of a cancer in a subject.

The compounds of the kit may be comprised in one or more containers. The instructions can state that the medicaments are intended for use in treating a subject having a cancer that tests positive for PD-L1 expression by an immunohistochemical (IHC) assay.

In certain embodiments, the PD-1 inhibitor and the TGFβ inhibitor are fused. In one embodiment, the fusion molecule is an anti-PD(L)1:TGFβRII fusion protein. In one embodiment, the fusion molecule is an anti-PD-L1 :TGFβRII fusion protein. In one embodiment, the amino acid sequence of the anti-PD-L1 :TGFβRII fusion protein corresponds to the amino acid sequence of bintrafusp alfa. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the amino acid sequence of bintrafusp alfa. (A) SEQ ID NO: 8 represents the heavy chain sequence of bintrafusp alfa. The CDRs having the amino acid sequences of SEQ ID NOs: 1, 2 and 3 are underlined. (B) SEQ ID NO: 7 represents the light chain sequence of bintrafusp alfa. The CDRs having the amino acid sequences of SEQ ID NOs: 4, 5 and 6 are underlined.

Figure 2 shows an exemplary structure of an anti-PD-L1:TGFbRII fusion protein.

Figure 3. Bintrafusp alfa + Axitinib and bintrafusp alfa + Nintedanib combination therapies increased anti-tumor efficacy in the CT26 model relative to monotherapies. BALB/c mice were inoculated with CT26 cells (0.3 x 10⁶) in the left flank (day -9). At day 0, when average tumor volumes reached ~75 mm³, mice were treated (n = 10-20 mice/group) with isotype control (400 μg i.v.; days 0, 2, 4), bintrafusp alfa (492 μg i.v.; days 0, 2, 4), Axitinib (30 mg/kg p.o.; days 0-13, twice daily), Nintedanib (50 mg/kg p.o.; days 0-13, once daily), bintrafusp alfa + Axitinib, or bintrafusp alfa + Nintedanib. Tumor volumes were measured twice weekly and presented as mean ± SEM or as individual tumor volumes. P-values were calculated by two-way ANOVA with Tukey’s post-test.

Figure 4. Bintrafusp alfa + Lenvatinib and bintrafusp alfa + Regorafenib combination therapies increased anti-tumor efficacy in the CT26 model relative to monotherapies. BALB/c mice were inoculated with CT26 cells (1 x 10⁶) in the left flank (day -11). At day 0, when average tumor volumes reached -100 mm³, mice were treated (n = 10 mice/group) with isotype control (400 μg i.v.; days 0, 2, 6), bintrafusp alfa (492 μg i.v.; days 0, 2, 6), Lenvatinib (30 mg/kg p.o.; days 0-13), Regorafenib (10 mg/kg p.o.; days 0-13), bintrafusp alfa + Lenvatinib, or bintrafusp alfa + Regorafenib. Tumor volumes were measured twice weekly and presented as mean ± SEM or as individual tumor volumes. P-values were calculated by two-way ANOVA with Tukey’s post-test.

Figure 5. Bintrafusp alfa + Lenvatinib and bintrafusp alfa + Regorafenib combination therapies increased anti-tumor efficacy in the 4T1-Luc model relative to monotherapies. BALB/c mice were inoculated with 4T1-Luc cells (0.5 x 10⁶) orthotopically in the mammary fat pad (day -10). At day 0, when average tumor volumes reached -100 mm³, mice were treated (n = 10-20 mice/group) with isotype control (400 μg i.v.; days 0, 2, 4), bintrafusp alfa (492 μg i.v.; days 0, 2, 4), Lenvatinib (30 mg/kg p.o.; days 0-20), Regorafenib (10 mg/kg p.o.; days 0-20), bintrafusp alfa + Lenvatinib, or bintrafusp alfa + Regorafenib. Tumor volumes were measured twice weekly and presented as mean ± SEM or as individual tumor volumes. P-values were calculated by two-way ANOVA with Tukey’s post-test. Figure 6. Bintrafusp alfa and pazopanib combination therapy increased antitumor efficacy in the RENCA model. Jh mice, which are devoid of mature B lymphocytes, were inoculated with RENCA cells (0.8 x 10⁵) orthotopically in the left renal capsule (day -6). At day 0, mice were treated (n = 8 mice/group) with isotype control (400 μg i.v.; days 1, 5, 9), bintrafusp alfa (492 μg i.v.; days 1, 5, 9), pazopanib (30 mg/kg p.o.; days 0-9, twice daily), or bintrafusp alfa + pazopanib. At day 11 , mice were sacrificed, the weight of the left and right kidneys was measured, and net tumor volume was calculated. Mean ± standard error of the mean (SEM) are presented. P-values were calculated by unpaired t test with Welch’s correction.

Figure 7. Bintrafusp alfa and anti-VEGF-A (B20) combination therapy significantly inhibited tumor growth and prolonged survival in the 4T1 model. Jh mice were inoculated with 4T1 cells (5 x 10⁴) orthotopically in the right abdominal mammary fat pad (day -6). At day 0, when average tumor volumes reached ~50 mm³, mice were treated (n = 10 mice/group) with isotype control (400 μg i.v.; days 0, 3, 6, 10, 14), bintrafusp alfa (492 μg i.v.; days 0, 3, 6, 10, 14), anti-VEGF-A (B20) (5 mg/kg i.p.; days 0, 6, 14), or bintrafusp alfa + anti-VEGF-A (B20). Tumor volumes were measured twice weekly and presented as mean ± SEM. P-values were calculated by two-way ANOVA with Tukey’s post-test; **** p £ 0.0001 denotes a significant difference relative to combination therapy. For survival analysis, mice were sacrificed when tumor volumes reached ≈2000 mm³ and median survival times were calculated.

Figure 8. Bintrafusp alfa + anti-VEGF-A and bintrafusp alfa + Nintedanib combination therapies increased TIL populations in the MC38 model relative to monotherapies. MC38 tumor-bearing C57BL/6 mice were treated with: isotype control (400 μg i.v.; days 0, 3, 6), bintrafusp alfa (492 μg; i.v. days 0, 3, 6), anti-VEGF-A (B20) (100 μg i.v.; days 0, 3), Nintedanib (50 mg/kg; p.o.; QDx8), bintrafusp alfa + anti-VEGF-A, or bintrafusp alfa + Nintedanib. Mice were sacrificed and 5 tumors/group were harvested on day 9. Immune profiling by flow cytometry of tumors were performed. P-values were calculated by unpaired t-tests with Welch’s correction.

Figure 9. Bintrafusp alfa + anti-VEGF-A and bintrafusp alfa + Nintedanib combination therapies increased effector and central memory TILs in the MC38 model relative to monotherapies. MC38 tumor-bearing C57BL/6 mice were treated with: isotype control (400 μg i.v.; days 0, 3, 6), bintrafusp alfa (492 μg; i.v. days 0, 3, 6), anti-VEGF-A (B20) (100 μg i.v.; days 0, 3), Nintedanib (50 mg/kg; p.o.; QDx8), bintrafusp alfa + anti-VEGF-A, or bintrafusp alfa + Nintedanib. Mice were sacrificed and 5 tumors/group were harvested on day 9. Immune profiling by flow cytometry of tumors were performed. P-values were calculated by unpaired t-tests with Welch’s correction.

Figure 10. Bintrafusp alfa + anti-VEGF-A and bintrafusp alfa + Nintedanib combination therapies increased NK cells in the MC38 model relative to monotherapies. MC38 tumor-bearing C57BL/6 mice were treated with: isotype control (400 μg i.v.; days 0, 3, 6), bintrafusp alfa (492 μg; i.v. days 0, 3, 6), anti-VEGF-A (B20) (100 μg i.v.; days 0, 3), Nintedanib (50 mg/kg; p.o.; QDx8), bintrafusp alfa + anti-VEGF-A, or bintrafusp alfa + Nintedanib. Mice were sacrificed and 5 tumors/group were harvested on day 9. Immune profiling by flow cytometry of tumors were performed. P-values were calculated by unpaired t-tests with Welch’s correction.

DETAILED DESCRIPTION OF THE INVENTION

Each of the embodiments described herein can be combined with any other embodiment described herein not inconsistent with the embodiment with which it is combined. Furthermore, unless incompatible in a given context, wherever a compound is stipulated which is capable of ionization (e.g. protonation or deprotonation), the definition of said compound includes any pharmaceutically acceptable salts thereof. Accordingly, the phrase “or a pharmaceutically acceptable salt thereof’ is implicit in the description of all compounds described herein. Embodiments within an aspect as described below can be combined with any other embodiments not inconsistent within the same aspect or a different aspect. For instance, embodiments of any of the treatment methods of the present invention can be combined with any embodiments of the combination products of the present invention or pharmaceutical composition of the present invention, and vice versa. Likewise, any detail or feature given for the treatment methods of the present invention apply - if not inconsistent - to those of the combination products of the present invention and pharmaceutical compositions of the present invention, and vice versa.

The present invention may be understood more readily by reference to the detailed description above and below of the particular and preferred embodiments of the invention and the examples included herein. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. It is further to be understood that unless specifically defined herein, the terminology used herein is to be given its traditional meaning as known in the relevant art. So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Definitions

“A”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an antibody refers to one or more antibodies or at least one antibody. As such, the terms “a” (or “an”), “one or more”, and “at least one” are used interchangeably herein.

The term “about” when used to modify a numerically defined parameter refers to any minimal alteration in such parameter that does not change the overall effect, e.g., the efficacy of the agent in treatment of a disease or disorder. In some embodiments, the term “about” means that the parameter may vary by as much as 10% below or above the stated numerical value for that parameter.

“Administering” or “administration of” a drug to a patient (and grammatical equivalents of this phrase) refers to direct administration, which may be administration to a patient by a medical professional or may be self-administration, and/or indirect administration, which may be the act of prescribing a drug, e.g., a physician who instructs a patient to self-administer a drug or provides a patient with a prescription for a drug is administering the drug to the patient.

An “amino acid difference” refers to a substitution, a deletion or an insertion of an amino acid.

“Antibody” is an immunoglobulin (Ig) molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen-binding fragment or antibody fragment thereof that competes with the intact antibody for specific binding, as well as any protein comprising such antigen-binding fragment or antibody fragment thereof, including fusion proteins (e.g., antibody-drug conjugates, an antibody fused to a cytokine or an antibody fused to a cytokine receptor), antibody compositions with poly-epitopic specificity, and multi- specific antibodies (e.g., bispecific antibodies). The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intra-chain disulfide bridges. Each H chain has, at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and g chains and four CH domains for m and e isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a V_H and V_L together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8^th Edition, Sties et al. (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated a, d, e, g and m, respectively. The g and a classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: lgG1, lgG2A, lgG2B, lgG3, lgG4, lgA1, and lgK1.

"Antigen-binding fragment” of an antibody or “antibody fragment" comprises a portion of an intact antibody, which is still capable of antigen binding. Antigen-binding fragments include, for example, Fab, Fab’, F(ab’)2, Fd, and Fv fragments, domain antibodies (dAbs, e.g., shark and camelid antibodies), single-domain antibodies (sdAbs), fragments including CDRs, single chain variable fragment antibodies (scFv), single-chain antibody molecules, multi-specific antibodies formed from antibody fragments, maxibodies, nanobodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, linear antibodies (see e.g., U.S. Patent 5,641,870, Example 2; Zapata et al. (1995) Protein Eng. 8HO: 1057), and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e. , it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross- linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the C_H1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

“Anti-PD-L1 antibody” or “anti-PD-1 antibody” means an antibody, or an antigen binding fragment thereof, that specifically binds to PD-L1 or PD-1 respectively and blocks binding of PD-L1 to PD-1. In any of the treatment methods, medicaments and uses of the present invention in which a human subject is being treated, the anti-PD-L1 antibody specifically binds to human PD-L1 and blocks binding of human PD-L1 to human PD-1. In any of the treatment methods, medicaments and uses of the present invention in which a human subject is being treated, the anti-PD-1 antibody specifically binds to human PD-1 and blocks binding of human PD-L1 to human PD-1. The antibody may be a monoclonal antibody, human antibody, humanized antibody or chimeric antibody, and may include a human constant region. In some embodiments the human constant region is selected from the group consisting of lgG1, lgG2, lgG3 and lgG4 constant regions, and in some embodiments, the human constant region is an lgG1 or lgG4 constant region. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab')2, scFv and Fv fragments.

“Anti-PD(L)1 antibody” refers to an anti-PD-L1 antibody or an anti-PD-1 antibody.

“Anti-VEGF ligand antibody” or “anti-VEGF receptor antibody” means an antibody, or an antigen-binding fragment thereof, that specifically binds to a VEGF ligand or a VEGF receptor respectively and blocks binding of a VEGF ligand to a VEGF receptor. In any of the treatment methods, medicaments and uses of the present invention in which a human subject is being treated, the anti-VEGF ligand antibody specifically binds to human VEGF ligand and blocks binding of human VEGF ligand to human VEGF receptor. In any of the treatment methods, medicaments and uses of the present invention in which a human subject is being treated, the anti-VEGF receptor antibody specifically binds to human VEGF receptor and blocks binding of human VEGF ligand to human VEGF receptor. The antibody may be a monoclonal antibody, human antibody, humanized antibody or chimeric antibody, and may include a human constant region. In some embodiments the human constant region is selected from the group consisting of lgG1, lgG2, lgG3 and lgG4 constant regions, and in some embodiments, the human constant region is an lgG1 or lgG4 constant region. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab')2, scFv and Fv fragments.

“Bintrafusp alfa”, also known as M7824, is well understood in the art. Bintrafusp alfa is an anti-PD-L1 :TGFβRII fusion protein and described under the CAS Registry Number 1918149-01-5. It is also described in WO 2015/118175 and further elaborated in Lan et al. (Lan et al., “Enhanced preclinical antitumor activity of M7824, a bifunctional fusion protein simultaneously targeting PD-L1 and TGF-β”, Sci. Transl. Med. 10, 2018, p.1-15). In particular, bintrafusp alfa is a fully human lgG1 monoclonal antibody against human PD-L1 fused to the extracellular domain of human TGF-β receptor II (TGFβRII). As such, bintrafusp alfa is a bifunctional fusion protein that simultaneously blocks PD-L1 and TGF-β pathways.

In particular, WO 2015/118175 describes bintrafusp alfa on page 34 in Example 1 thereof as follows (bintrafusp alfa is referred to in this passage as “ anti-PD-L1 /TGFβ Trap”): “Anti-PD- L1/TGFβ Trap is an anti-PD-L1 antibody-TGFβ Receptor II fusion protein. The light chain of the molecule is identical to the light chain of the anti-PD-L1 antibody (SEQ ID NO: 1). The heavy chain of the molecule (SEQ ID NO:3) is a fusion protein comprising the heavy chain of the anti-PD-L1 antibody (SEQ ID NO: 2) genetically fused to via a flexible (Gly4Ser)4Gly linker (SEQ ID NO: 11) to the N-terminus of the soluble TGFβ Receptor II (SEQ ID NO: 10). At the fusion junction, the C-terminal lysine residue of the antibody heavy chain was mutated to alanine to reduce proteolytic cleavage.”

“Biomarker” generally refers to biological molecules, and quantitative and qualitative measurements of the same, that are indicative of a disease state. “Prognostic biomarkers” correlate with disease outcome, independent of therapy. For example, tumor hypoxia is a negative prognostic marker - the higher the tumor hypoxia, the higher the likelihood that the outcome of the disease will be negative. “Predictive biomarkers” indicate whether a patient is likely to respond positively to a particular therapy, e.g., HER2 profiling is commonly used in breast cancer patients to determine if those patients are likely to respond to Herceptin (trastuzumab, Genentech). “Response biomarkers” provide a measure of the response to a therapy and so provide an indication of whether a therapy is working. For example, decreasing levels of prostate-specific antigen generally indicate that anti-cancer therapy for a prostate cancer patient is working. When a marker is used as a basis for identifying or selecting a patient for a treatment described herein, the marker can be measured before and/or during treatment, and the values obtained are used by a clinician in assessing any of the following: (a) probable or likely suitability of an individual to initially receive treatment(s); (b) probable or likely unsuitability of an individual to initially receive treatment(s); (c) responsiveness to treatment; (d) probable or likely suitability of an individual to continue to receive treatment(s); (e) probable or likely unsuitability of an individual to continue to receive treatment(s); (f) adjusting dosage; (g) predicting likelihood of clinical benefits; or (h) toxicity. As would be well understood by one in the art, measurement of a biomarker in a clinical setting is a clear indication that this parameter was used as a basis for initiating, continuing, adjusting and/or ceasing administration of the treatments described herein.

By “cancer” is meant a collection of cells multiplying in an abnormal manner. As used herein, the term “cancer” refers to all types of cancer, neoplasm, malignant or benign tumors found in mammals, including leukemia, carcinomas, and sarcomas. Exemplary cancers include breast cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, lung cancer, pancreatic cancer, glioblastoma. Additional examples include cancer of the brain, lung cancer, non-small cell lung cancer, melanoma, sarcomas, prostate cancer, cervix cancer, stomach cancer, head and neck cancers, uterus cancer, mesothelioma, metastatic bone cancer, medulloblastoma, Hodgkin’s Disease, Non-Hodgkin’s Lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primary macrobulinemia, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, and neoplasms of the endocrine and exocrine pancreas.

“CDRs” are the complementarity determining region amino acid sequences of an antibody, antibody fragment or antigen-binding fragment. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin.

“Clinical outcome”, “clinical parameter”, “clinical response”, or “clinical endpoint” refers to any clinical observation or measurement relating to a patient’s reaction to a therapy. Non-limiting examples of clinical outcomes include tumor response (TR), overall survival (OS), progression free survival (PFS), disease free survival, time to tumor recurrence (TTR), time to tumor progression (TTP), relative risk (RR), toxicity, or side effect.

“Combination” as used herein refers to the provision of a first active modality in addition to one or more further active modalities (wherein one or more active modalities may be fused). Contemplated within the scope of the combinations described herein, are any regimen of combination modalities or partners (i.e., active compounds, components or agents), such as a combination of a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor, encompassed in single or multiple compounds and compositions. It is understood that any modalities within a single composition, formulation or unit dosage form (i.e., a fixed-dose combination) must have the identical dose regimen and route of delivery. It is not intended to imply that the modalities must be formulated for delivery together (e.g., in the same composition, formulation or unit dosage form). The combined modalities can be manufactured and/or formulated by the same or different manufacturers. The combination partners may thus be, e.g., entirely separate pharmaceutical dosage forms or pharmaceutical compositions that are also sold independently of each other. In some embodiments, the TGFβ inhibitor is fused to the PD-1 inhibitor and therefore encompassed within a single composition and having an identical dose regimen and route of delivery.

"Combination therapy", “in combination with” or “in conjunction with” as used herein denotes any form of concurrent, parallel, simultaneous, sequential or intermittent treatment with at least two distinct treatment modalities (i.e., compounds, components, targeted agents or therapeutic agents). As such, the terms refer to administration of one treatment modality before, during, or after administration of the other treatment modality to the subject. The modalities in combination can be administered in any order. The therapeutically active modalities are administered together (e.g., simultaneously in the same or separate compositions, formulations or unit dosage forms) or separately (e.g., on the same day or on different days and in any order as according to an appropriate dosing protocol for the separate compositions, formulations or unit dosage forms) in a manner and dosing regimen prescribed by a medical care taker or according to a regulatory agency. In general, each treatment modality will be administered at a dose and/or on a time schedule determined for that treatment modality. Optionally, four or more modalities may be used in a combination therapy. Additionally, the combination therapies provided herein may be used in conjunction with other types of treatment. For example, other anti-cancer treatment may be selected from the group consisting of chemotherapy, surgery, radiotherapy (radiation) and/or hormone therapy, amongst other treatments associated with the current standard of care for the subject.

"Complete response" or "complete remission" refers to the disappearance of all signs of cancer in response to treatment. This does not always mean the cancer has been cured.

“Comprising”, as used herein, is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of”, when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method. “Consisting of’ shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).

“Dose” and “dosage” refer to a specific amount of active or therapeutic agents for administration. Such amounts are included in a “dosage form,” which refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active agent calculated to produce the desired onset, tolerability, and therapeutic effects, in association with one or more suitable pharmaceutical excipients such as carriers.

“Fc” is a fragment comprising the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

The term “fusion molecule” is well understood in the art and it will be appreciated that the molecule comprising a fused PD-1 inhibitor and TGFβ inhibitor as referred to herein includes an lg: TGFβ fusion protein, such as an anti-PD-1:TGFbR fusion protein or an anti- PD-L 1:TGFβR fusion protein. An Ig:TGFβR fusion protein is an antibody (in some embodiments, a monoclonal antibody, e.g., in homodimeric form) or an antigen-binding fragment thereof fused to a TGF-β receptor. The nomenclature anti-PD-L1 :TGFβRII fusion protein indicates an anti-PD-L1 antibody, or an antigen-binding fragment thereof, fused to a TGF-β receptor II or a fragment of the extracellular domain thereof that is capable of binding TGF-β. The nomenclature anti-PD-1 :TGFβRII fusion protein indicates an anti-PD-1 antibody, or an antigen-binding fragment thereof, fused to a TGF-β receptor II or a fragment of the extracellular domain thereof that is capable of binding TGF-β. The nomenclature anti- PD(L)1:TGFβRII fusion protein, indicates an anti-PD-1 antibody or an antigen-binding fragment thereof, or an anti-PD-L1 antibody or an antigen-binding fragment thereof, fused to a TGF-β receptor II or a fragment of the extracellular domain thereof that is capable of binding TGF-β.

"Fv" is the minimum antibody fragment, which contains a complete antigen- recognition and antigen-binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

"Human antibody" is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen- binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries (see e.g., Hoogenboom and Winter (1991), JMB 227: 381; Marks et al. (1991) JMB 222: 581). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, page 77; Boerner et al. (1991), J. Immunol 147(1): 86; van Dijk and van de Winkel (2001) Curr. Opin. Pharmacol 5: 368). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled, e.g., immunized xenomice (see e.g., U.S. Pat. Nos. 6,075,181; and 6,150,584 regarding XENOMOUSE technology). See also, for example, Li et al. (2006)

PNAS USA, 103: 3557, regarding human antibodies generated via a human B-cell hybridoma technology.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity and/or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR are typically no more than 6 in the H chain, and no more than 3 in the L chain. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see e.g., Jones et al. (1986) Nature 321: 522; Riechmann et al. (1988), Nature 332: 323; Presta (1992) Curr. Op. Struct. Biol. 2: 593; Vaswani and Hamilton (1998), Ann. Allergy, Asthma & Immunol. 1: 105; Harris (1995) Biochem. Soc. Transactions 23: 1035; Hurle and Gross (1994) Curr. Op. Biotech. 5: 428; and U.S. Pat. Nos. 6,982,321 and 7,087,409.

"Infusion" or "infusing" refers to the introduction of a drug-containing solution into the body through a vein for therapeutic purposes. Generally, this is achieved via an intravenous (IV) bag.

"Metastatic" cancer refers to cancer which has spread from one part of the body (e.g., the lung) to another part of the body.

"Monoclonal antibody", as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. , the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations and amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture and uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein (1975) Nature 256: 495; Hongo et al. (1995) Hybridoma 14 (3): 253; Harlow et al. (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2^nd ed.; Hammerling et al. (1981) In: Monoclonal Antibodies and T-Cell Hybridomas 563 (Elsevier, N.Y.), recombinant DNA methods (see e.g., U.S. Patent No. 4,816,567), phage-display technologies (see e.g., Clackson et al.

(1991) Nature 352: 624; Marks et al. (1992) JMB 222: 581; Sidhu et al. (2004) JMB 338(2): 299; Lee et al. (2004) JMB 340(5): 1073; Fellouse (2004) PNAS USA 101(34): 12467; and Lee et al. (2004) J. Immunol. Methods 284(1-2): 119), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741 ; Jakobovits et al. (1993) PNAS USA 90: 2551; Jakobovits et al. (1993) Nature 362: 255; Bruggemann et al. (1993) Year in Immunol. 7: 33; U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al. (1992) Bio/Technology 10: 779; Lonberg et al. (1994) Nature 368: 856; Morrison (1994) Nature 368: 812; Fishwild et al. (1996) Nature Biotechnol. 14: 845; Neuberger (1996), Nature Biotechnol. 14: 826; and Lonberg and Huszar (1995), Intern. Rev. Immunol. 13: 65-93). The monoclonal antibodies herein specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is (are) identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see e.g., U.S. Patent No. 4,816,567; Morrison et al. (1984) PNAS USA, 81: 6851).

"Objective response" refers to a measurable response, including complete response (CR) or partial response (PR).

"Partial response" refers to a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment.

“Patient” and “subject” are used interchangeably herein to refer to a mammal in need of treatment for a cancer. Generally, the patient is a human diagnosed or at risk for suffering from one or more symptoms of a cancer. In certain embodiments a “patient” or “subject” may refer to a non-human mammal, such as a non-human primate, a dog, cat, rabbit, pig, mouse, or rat, or animals used, e.g., in screening, characterizing, and evaluating drugs and therapies.

“PD-1 inhibitor” as used herein refers to a molecule that inhibits the PD-1 pathway, e.g., by inhibiting the interaction of PD-1 axis binding partners, such as between the PD-1 receptor and the PD-L1 and/or PD-L2 ligand. Possible effects of such inhibition include the removal of immunosuppression resulting from signaling on the PD-1 signaling axis. Inhibition in this context need not be complete or 100%. Instead, inhibition means reducing, decreasing or abrogating binding between PD-1 and one or more of its ligands and/or reducing, decreasing or abrogating signaling through the PD-1 receptor. In some embodiments, the PD-1 inhibitor binds to PD-L1 or PD-1 to inhibit the interaction between these molecules, such as an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the PD-1 inhibitor is a PD-L1 antibody and such antibody may be fused to the TGFβ inhibitor, e.g., as an anti-PD(L)1:TGFβRII fusion protein.

“PD-L1 expression” as used herein means any detectable level of expression of PD- L1 protein on the cell surface or of PD-L1 mRNA within a cell or tissue. PD-L1 protein expression may be detected with a diagnostic PD-L1 antibody in an IHC assay of a tumor tissue section or by flow cytometry. Alternatively, PD-L1 protein expression by tumor cells may be detected by PET imaging, using a binding agent (e.g., antibody fragment, affibody and the like) that specifically binds to PD-L1. Techniques for detecting and measuring PD-L1 mRNA expression include RT-PCR and real-time quantitative RT-PCR.

A “PD-L1 positive” or “PD-L1 high” cancer is one comprising cells, which have PD-L1 present at their cell surface, and/or one producing sufficient levels of PD-L1 at the surface of cells thereof, such that an anti-PD-L1 antibody has a therapeutic effect, mediated by the binding of the said anti-PD-L1 antibody to PD-L1. Methods of detecting a biomarker, such as PD-L1 for example, on a cancer or tumor, are routine in the art and are contemplated herein. Non-limiting examples include immunohistochemistry (IHC), immunofluorescence and fluorescence activated cell sorting (FACS). Several approaches have been described for quantifying PD-L1 protein expression in IHC assays of tumor tissue sections. The ratio of PD-L1 positive cells is oftentimes expressed as a Tumor Proportion Score (TPS) or a Combined Positive Score (CPS). The TPS describes the percentage of viable tumor cells with partial or complete membrane staining (e.g., staining for PD-L1). The CPS is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. For instance, in some embodiments, “PD-L1 high” refers to ³ 80% PD-L1 positive tumor cells as determined by the PD-L1 Dako IHC 73- 10 assay, or tumor proportion score (TPS) ³ 50% as determined by the Dako IHC 22C3 PharmDx assay. Both IHC 73-10 and IHC 22C3 assays select a similar patient population at their respective cutoffs. In certain embodiments, Ventana PD-L1 (SP263) assay, which has high concordance with 22C3 PharmDx assay (see Sughayer et al., Appl. Immunohistochem. Mol. Morphol., (2018)), can also be used for determining the PD-L1 expression level. Another assay for determining PD-L1 expression in cancers is the Ventana PD-L1 (SP142) assay. In some embodiments, a cancer is counted as PD-L1 positive if at least 1%, at least 5%, at least 25%, at least 50%, at least 75% or at least 80% of the tumor cells show PD-L1 expression.

"Percent (%) sequence identity" with respect to a peptide or polypeptide sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2 or ALIGN software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

"Pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith. "Pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.

"Recurrent" cancer is one which has regrown, either at the initial site or at a distant site, after a response to initial therapy, such as surgery. A locally “recurrent" cancer is cancer that returns after treatment in the same place as a previously treated cancer.

“Reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) refers to decreasing the severity or frequency of the symptom(s), or elimination of the symptom(s).

"Single-chain Fv", also abbreviated as "sFv" or "scFv", are antibody fragments that comprise the V_H and V_L antibody domains connected into a single polypeptide chain. In some embodiments, the sFv polypeptide further comprises a polypeptide linker between the V_H and V_L domains which enables the sFv to form the desired structure for antigen binding. For a review of the sFv, see e.g., Pluckthun (1994), In: The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269.

By “substantially identical” is meant (1) a query amino acid sequence exhibiting at least 75%, 85%, 90%, 95%, 99% or 100% amino acid sequence identity to a subject amino acid sequence or (2) a query amino acid sequence that differs in not more than 20%, 30%, 20%, 10%, 5%, 1% or 0% of its amino acid positions from the amino acid sequence of a subject amino acid sequence and wherein a difference in an amino acid position is any of a substitution, deletion or insertion of an amino acid.

“Systemic” treatment is a treatment, in which the drug substance travels through the bloodstream, reaching and affecting cells all over the body.

“TGFβ inhibitor” as used herein refers to a molecule that inhibits the TGFβ pathway, e.g., by inhibiting the interaction between a TGFβ and a TGFβ receptor (TGFβR). Possible effects of such inhibition include the removal of immunosuppression resulting from signaling on the TGFβ signaling axis. Inhibition in this context need not be complete or 100%. Instead, inhibition means reducing, decreasing or abrogating binding between TGF-β and the TGFβR and/or reducing, decreasing or abrogating signaling through the TGFβR. In some embodiments, the TGFβ inhibitor binds to TGFβ or a TGFβR to inhibit the interaction between these molecules. In some embodiments, the TGFβ inhibitor comprises the extracellular domain of a TGFβRII, or a fragment of TGFβRII capable of binding TGFβ. In some embodiments, such TGFβ inhibitor is fused to the PD-1 inhibitor, e.g., as an anti- PD(L )1:TGFβRII fusion protein.

The term “TGF-β receptor” (TGFβR), as well as “TGF-β receptor I” (abbreviated as TGFβRI or TGFβR1) or “TGF-β receptor II” (abbreviated as TGFβRII or TGFβR2), are well known in the art. For the purposes of this disclosure, reference to such receptor includes the full receptor and fragments that are capable of binding TGF-β. In some embodiments, it is the extracellular domain of the receptor or a fragment of the extracellular domain that is capable of binding TGF-β. In some embodiments, the fragment of TGFβRII is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

“Therapeutically effective amount” of a PD-1 inhibitor, a TGFβ inhibitor, or a VEGF inhibitor, in each case of the invention, refers to an amount effective, at dosages and for periods of time necessary, that, when administered to a patient with a cancer, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation, or elimination of one or more manifestations of the cancer in the patient, or any other clinical result in the course of treating a cancer patient. A therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. Such therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a PD-1 inhibitor, a TGFβ inhibitor, or a VEGF inhibitor to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of a PD-1 inhibitor, a TGFβ inhibitor, or a VEGF inhibitor are outweighed by the therapeutically beneficial effects.

“Treating” or “treatment of” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation, amelioration of one or more symptoms of a cancer; diminishment of extent of disease; delay or slowing of disease progression; amelioration, palliation, or stabilization of the disease state; or other beneficial results. It is to be appreciated that references to “treating” or “treatment” include prophylaxis as well as the alleviation of established symptoms of a condition. “Treating” or “treatment” of a state, disorder or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e. , arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

“Tyrosine kinase inhibitor” refers to an agent inhibiting tyrosine kinases. In some embodiments, the TKI is a small molecule. In some embodiments, the TKI inhibits one or more receptor tyrosine kinase, such as the tyrosine kinase of a VEGF receptor. In some embodiments, the TKI is a VEGF inhibitor. Inhibition in this context need not be complete or 100%. Instead, inhibition means reducing, decreasing or abrogating the activity of a tyrosine kinase.

"Unit dosage form" as used herein refers to a physically discrete unit of therapeutic formulation appropriate for the subject to be treated. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular subject or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of specific active agent employed; specific composition employed; age, body weight, general health, sex and diet of the subject; time of administration, and rate of excretion of the specific active agent employed; duration of the treatment; drugs and/or additional therapies used in combination or coincidental with specific compound(s) employed, and like factors well known in the medical arts. "Variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.

“VEGF inhibitor” refers to a compound that inhibits the VEGF pathway, e.g., by inhibiting the interaction between a VEGF ligand and a VEGF receptor or by inhibiting the signaling of VEGF, such as inhibiting the tyrosine kinase activity of one or more VEGF receptors. In some embodiments, the VEGF inhibitor is a molecule which binds to a VEGF ligand or a VEGF receptor and prevents their interaction, such as an anti-VEGF ligand antibody or anti-VEGF receptor antibody. In some embodiments, the VEGF inhibitor inhibits the interaction between VEGF-A and a VEGF receptor, such as an antibody against VEGF- A, such as ranibizumab or bevacizumab. In some embodiments, the VEGF inhibitor is a bispecific binding molecule comprising a binding domain against a VEGF ligand and a binding domain against Ang2, such as a bispecific binding molecule comprising a VEGF- binding immunoglobulin single variable domain and an Ang2-binding immunoglobulin single variable domain. In some embodiments, such bispecific binding molecule is one of the binding molecules disclosed in W02012/131078 or a molecule having an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 which sequences are disclosed in WO2018/220169. In some embodiments, the VEGF inhibitor inhibits the interaction between the VEGF receptor type 2 and VEGF ligand, such as an antibody against this receptor, such as ramucirumab. In some embodiments, the VEGF inhibitor is a molecule that binds to and traps circulating VEGF ligand to prevent its interaction with a VEGF receptor, such as a soluble VEGF receptor, e.g., aflibercept. In some embodiments, the VEGF inhibitor is bevacizumab. In some embodiments, the VEGF inhibitor is ramucirumab. In some embodiments, the VEGF inhibitor is a TKI of one or more VEGF receptors, such as anlotinib, axitinib, bosutinib, lenvatinib, nintedanib, pazopanib, regorafenib, cediranib, linifanib, motesanib, fruquintinib, sorafenib, sunitinib, ponatinib and cabozantinib. In some embodiments, the VEGF inhibitor is nintedanib. In some embodiments, the VEGF inhibitor is lenvatinib. In some embodiments, the VEGF inhibitor is fruquintinib. In some embodiments, the VEGF inhibitor is regorafenib. In some embodiments, the TKI selectively inhibits the VEGF pathway, e.g., its inhibitory activity on the tyrosine kinase of one or more VEGF receptors is substantially higher than on tyrosine kinases outside the VEGF pathway. In some embodiments, the TKI primarily or selectively inhibits the VEGF pathway. In some embodiments, the IC50 value of the TKI is below 1000 mM, below 100 pM, below 1 pM or below 100 nM. Possible effects of the inhibition of the VEGF pathway include the removal of immunosuppression and/or the inhibition of tumor growth and metastasis. Inhibition in this context need not be complete or 100%. Instead, inhibition means reducing, decreasing or abrogating the activity of the VEGF pathway.

The terms “VEGF ligand” and “VEGF receptor” are well known in the art. In some embodiments, VEGF ligand is selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF. In some embodiments, the VEGF ligand is VEGF-A. In some embodiments, the VEGF receptor is selected from the group consisting of VEGF receptor type 1 , VEGF receptor type 2 and VEGF receptor type 3. In some embodiments, the VEGF receptor is VEGF receptor type 2. In some embodiments, the VEGF ligand or VEGF receptor are a human VEGF ligand or human VEGF receptor, respectively.

As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member.

Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 1 to about 5” should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4, and from 3-5, etc., as well as 1, 2, 3, 4, and 5, individually. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

Descriptive Embodiments

Therapeutic combination and method of use thereof

The present invention arose in part from the surprising discovery of a combination benefit for a PD-1 inhibitor, a TGFβ inhibitor, and a VEGF inhibitor. Treatment schedule and doses were designed to reveal potential synergies. Pre-clinical data demonstrated a synergy of the VEGF inhibitor when combined with the PD-1 inhibitor and the TGFβ inhibitor. Thus, in one aspect, the present invention provides a PD-1 inhibitor, a TGFβ inhibitor, and a VEGF inhibitor for use in a method for treating a cancer in a subject comprising administering the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor to the subject; as well as a method for treating a cancer in a subject comprising administering a PD-1 inhibitor, a TGFβ inhibitor, and a VEGF inhibitor to the subject; as well as the use of a PD-1 inhibitor, a TGFβ inhibitor, and a VEGF inhibitor in the manufacture of a medicament for treating a cancer in a subject. It shall be understood that a therapeutically effective amount of the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor is applied in each method of treatment. In some embodiments, the PD-1 inhibitor is an anti-PD(L)1 antibody and the TGFβ inhibitor is a TGFβRII or an anti-TGFβ antibody. In some embodiments, the PD-1 inhibitor is fused to the TGFβ inhibitor. For instance, the PD-1 inhibitor and TGFβ inhibitor may be comprised in an anti-PD(L)1:TGFβRII fusion protein, such as an anti-PD(L)1:TGFβRII fusion protein or an anti-PD-1:GFβRII fIusion protein. In some embodiments, the fusion molecule is an anti-PD-1:GFβRII fusion protein, e.g., an anti-PD-L1 :TGFβRII fusion protein wherein the light chain sequences and the heavy chain sequences correspond to SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

In some embodiments, the present invention provides a PD-1 inhibitor, a TGFβ inhibitor, and a TKI for use in a method for treating a cancer in a subject comprising administering the PD-1 inhibitor, TGFβ inhibitor, and TKI to the subject; as well as a method for treating a cancer in a subject comprising administering a PD-1 inhibitor, a TGFβ inhibitor, and a TKI to the subject; as well as the use of a PD-1 inhibitor, a TGFβ inhibitor, and a TKI in the manufacture of a medicament for treating a cancer in a subject. It shall be understood that a therapeutically effective amount of the PD-1 inhibitor, TGFβ inhibitor, and TKI is applied in each method of treatment. In some embodiments, the PD-1 inhibitor is an anti- PD(L)1 antibody and the TGFβ inhibitor is a TGFβRII or an anti-TGFβ antibody. In some embodiments, the PD-1 inhibitor is fused to the TGFβ inhibitor. For instance, the PD-1 inhibitor and TGFβ inhibitor may be comprised in an anti-PD(L)1 :TGFβRII fusion protein, such as an anti-PD-L1 :TGFβRII fusion protein or an anti-PD-1:GFβRII fusion protein. In some embodiments, the fusion molecule is an anti-PD(L)1:TGFβRII fusion protein, e.g., an anti-PD-L1 :TGFβRII fusion protein wherein the light chain sequences and the heavy chain sequences correspond to SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

In some embodiments, the present invention provides a PD-1 inhibitor, a TGFβ inhibitor, and an anti-VEGF ligand or anti-VEGF receptor antibody for use in a method for treating a cancer in a subject comprising administering the PD-1 inhibitor, TGFβ inhibitor, and anti-VEGF ligand or anti-VEGF receptor antibody to the subject; as well as a method for treating a cancer in a subject comprising administering a PD-1 inhibitor, a TGFβ inhibitor, and an anti-VEGF ligand or anti-VEGF receptor antibody to the subject; as well as the use of a PD-1 inhibitor, a TGFβ inhibitor, and an anti-VEGF ligand or anti-VEGF receptor antibody in the manufacture of a medicament for treating a cancer in a subject. It shall be understood that a therapeutically effective amount of the PD-1 inhibitor, TGFβ inhibitor, and anti-VEGF ligand or anti-VEGF receptor antibody is applied in each method of treatment. In some embodiments, the PD-1 inhibitor is an anti-PD(L)1 antibody and the TGFβ inhibitor is a TGFβRII or an anti-TGFβ antibody. In some embodiments, the PD-1 inhibitor is fused to the TGFβ inhibitor. For instance, the PD-1 inhibitor and TGFβ inhibitor may be comprised in an anti-PD(L)1:TGFβRII fusion protein, such as an anti-PD(L)1:TGFβRII fusion protein or an anti-PD-1 :TGFβRII fusion protein. In some embodiments, the fusion molecule is an anti-PD- L 1:TGFβRII fusion protein, e.g., an anti-PD(L)1:TGFβRII fusion protein wherein the light chain sequences and the heavy chain sequences correspond to SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

The PD-1 inhibitor may inhibit the interaction between PD-1 and at least one of its ligands, such as PD-L1 or PD-L2, and thereby inhibit the PD-1 pathway, e.g., the immunosuppressive signal of PD-1. The PD-1 inhibitor may bind to PD-1 or one of its ligands, such as PD-L1. In one embodiment, the PD-1 inhibitor inhibits the interaction between PD-1 and PD-L1. In some embodiments, the PD-1 inhibitor is an anti-PD(L)1 antibody, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, capable of inhibiting the interaction between PD-1 and PD-L1. In some embodiments, the anti-PD-1 antibody or anti- PD-L1 antibody is selected from the group consisting of pembrolizumab, nivolumab, avelumab, atezolizumab, durvalumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, cemiplimab, and an antibody wherein the light chain sequences and the heavy chain sequences of the antibody correspond to SEQ ID NO: 7 and SEQ ID NO: 16, or to SEQ ID NO: 15 and SEQ ID NO: 14, respectively, or an antibody that competes for binding with any of the antibodies of this group. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody is one that is still capable of binding to PD-1 or PD-L1 and which amino acid sequence is substantially identical, e.g., has at least 90% sequence identity, to the sequence of one of the antibodies selected from the group consisting of pembrolizumab, nivolumab, avelumab, atezolizumab, durvalumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, cemiplimab, and an antibody wherein the light chain sequences and the heavy chain sequences of the antibody correspond to SEQ ID NO: 7 and SEQ ID NO: 16, or to SEQ ID NO: 15 and SEQ ID NO: 14. In some embodiments, the PD-1 inhibitor is an anti-PD-L1 antibody capable of inhibiting the interaction between PD-1 and PD-L1. In some embodiments, the anti-PD-L1 antibody comprises a heavy chain, which comprises three CDRs having amino acid sequences of SEQ ID NO: 19 (CDRH1), SEQ ID NO: 20 (CDRH2) and SEQ ID NO: 21 (CDRH3), and a light chain, which comprises three CDRs having amino acid sequences of SEQ ID NO: 22 (CDRL1), SEQ ID NO: 23 (CDRL2) and SEQ ID NO: 24 (CDRL3). In some embodiments, the anti-PD-L1 antibody comprises a heavy chain, which comprises three CDRs having amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2) and SEQ ID NO: 3 (CDRH3), and a light chain, which comprises three CDRs having amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2) and SEQ ID NO: 6 (CDRL3). In some embodiments, the light chain variable region and the heavy chain variable region of the anti-PD-L1 antibody comprise SEQ ID NO: 25 and SEQ ID NO: 26, respectively. In some embodiments, the light chain sequences and the heavy chain sequences of the anti-PD-L1 antibody correspond to SEQ ID NO: 7 and SEQ ID NO: 16, or to SEQ ID NO: 15 and SEQ ID NO: 14, respectively.

In some embodiments, the PD-1 inhibitor is an anti-PD-L1 antibody, wherein each of the light and heavy chain sequences have greater than or equal to 80% sequence identity, such as greater than or equal to 90% sequence identity, greater than or equal to 95% sequence identity, greater than or equal to 99% sequence identity, or 100% sequence identity with the amino acid sequence of the heavy and light chains of the antibody moiety of bintrafusp alfa and wherein the PD-1 inhibitor is still capable of binding to PD-L1. In some embodiments, the PD-1 inhibitor is an anti-PD-L1 antibody, wherein each of the light and heavy chain sequences have greater than or equal to 80% sequence identity, such as greater than or equal to 90% sequence identity, greater than or equal to 95% sequence identity, greater than or equal to 99% sequence identity, or 100% sequence identity with the amino acid sequence of the heavy and light chains of the antibody moiety of bintrafusp alfa and wherein the CDRs are fully identical with the CDRs of bintrafusp. In some embodiments, the PD-1 inhibitor is an anti-PD-L1 antibody with an amino acid sequence with not more than 50, not more than 40, or not more than 25 amino acid residues different from each of the heavy and light chain sequences of the antibody moiety of bintrafusp alfa and wherein the PD-1 inhibitor is still capable of binding to PD-L1. In some embodiments, the PD-1 inhibitor is an anti-PD-L1 antibody with an amino acid sequence with not more than 50, not more than 40, not more than 25, or not more than 10 amino acid residues different from each of the heavy and light chain sequences of the antibody moiety of bintrafusp alfa and wherein the CDRs are fully identical with the CDRs of bintrafusp alfa. In some embodiments, the TGFβ inhibitor is capable of inhibiting the interaction between TGFβ and a TGFβ receptor; such as a TGFβ receptor, a TGFβ ligand- or receptor- blocking antibody, a small molecule inhibiting the interaction between TGFβ binding partners, and an inactive mutant TGFβ ligand that binds to the TGFβ receptor and competes for binding with endogenous TGFβ. In some embodiments, the TGFβ inhibitor is a soluble TGFβ receptor (e.g., a soluble TGFβ receptor II or III) or a fragment thereof capable of binding TGFβ. In some embodiments, the TGFβ inhibitor is an extracellular domain of human TGFβ receptor II (TGFβRII), or fragment thereof capable of binding TGFβ. In some embodiments, the TGFβRII corresponds to the wild-type human TGF-β Receptor Type 2 Isoform A sequence (e.g. the amino acid sequence of NCBI Reference Sequence (RefSeq) Accession No. NP_001020018 (SEQ ID NO: 9)), or the wild-type human TGF-β Receptor Type 2 Isoform B sequence (e.g., the amino acid sequence of NCBI RefSeq Accession No. NP_003233 (SEQ ID NO: 10)). In some embodiments, the TGFβ inhibitor comprises or consists of a sequence corresponding to SEQ ID NO: 11 or a fragment thereof capable of binding TGFβ. For instance, the TGFβ inhibitor may correspond to the full-length sequence of SEQ ID NO: 11. Alternatively, it may have an N-terminal deletion. For instance, the N- terminal 26 or less amino acids of SEQ ID NO: 11 may be deleted, such as 14-21 or 14-26 N-terminal amino acids. In some embodiments, the N-terminal 14, 19 or 21 amino acids of SEQ ID NO: 11 are deleted. In some embodiments, the TGFβ inhibitor comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. In some embodiments, the TGFβ inhibitor is a protein that is substantially identical, e.g., has at least 90% sequence identity, to the amino acid sequence of any one of SEQ ID NO: 11 , SEQ ID NO: 12 and SEQ ID NO: 13 and is capable of binding TGF$. In another embodiment, the TGFβ inhibitor is a protein that is substantially identical, e.g., has at least 90% sequence identity, to the amino acid sequence of SEQ ID NO: 11 and is capable of binding TGFβ. In one embodiment, the TGFβ inhibitor is a protein with an amino acid sequence that does not differ in more than 25 amino acids from SEQ ID NO: 11 and is capable of binding TGFβ.

In some embodiments, the TGFβ inhibitor is a protein that is substantially identical, e.g., has at least 90% sequence identity, to the amino acid sequence of the TGFβR of bintrafusp alfa and is still capable of binding TGFβ. In some embodiments, the TGFβ inhibitor is a protein with an amino acid sequence with not more than 50, not more than 40, or not more than 25 amino acid residues different from the TGFβR of bintrafusp alfa that is still capable of binding TGFβ. In some embodiments, the TGFβ inhibitor has 100-160 amino acid residues or 110-140 amino acid residues. In some embodiments, the amino acid sequence of the TGFβ inhibitor is selected from the group consisting of a sequence corresponding to positions 1-136 of the TGFβR of bintrafusp alfa, a sequence corresponding to positions 20-136 of the TGFβR of bintrafusp alfa and a sequence corresponding to positions 22-136 of the TGFβR of bintrafusp alfa.

In some embodiments, the TGFβ inhibitor is selected from the group consisting of lerdelimumab, XPA681, XPA089, LY2382770, LY3022859, 1D11, 2G7, AP11014, A-80-01, LY364947, LY550410, LY580276, LY566578, SB-505124, SD-093, SD-208, SB-431542, ISTH0036, ISTH0047, galunisertib (LY2157299 monohydrate, a small molecule kinase inhibitor of TGF^RI), LY3200882 (a small molecule kinase inhibitor TGF-βRI disclosed by Pei et al. (2017) CANCER RES 77(13 Suppl):Abstract 955), metelimumab (an antibody targeting TGF-β1, see Colak et al. (2017) TRENDS CANCER 3(1):56-71), fresolimumab (GC-1008; an antibody targeting TGF-β1 and TGF-β2), XOMA 089 (an antibody targeting TGF-β1 and TGF^2; see Mirza et al. (2014) INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE 55:1121), AVID200 (a TGF-β1 and TGF-β3 trap, see Thwaites et al. (2017) BLOOD 130:2532), Trabedersen/AP12009 (a TGF-β2 antisense oligonucleotide, see Jaschinski et al. (2011) CURR PHARM BIOTECHNOL. 12(12):2203-13), Belagen- pumatucel-L (a tumor cell vaccine targeting TGF-β2, see, e.g., Giaccone et al. (2015) EUR J CANCER 51(16):2321-9), TGB-b pathway targeting agents described in Colak et al. (2017), supra, including Ki26894, SD208, SM16, IMC-TR1, PF-03446962, TEW-7197, and GW788388.

In some embodiments, the PD-1 inhibitor and the TGFβ inhibitor are fused, e.g., as an anti-PD(L)1:TGFbRII fusion protein. In some embodiments, the fusion molecule is an anti-PD-1:TGFbRII fusion protein or an anti-PD-L1:TGFbRII fusion protein. In some embodiments, the anti-PD(L)1:TGFbRII fusion protein is one of the anti-PD(L)1:TGFbRII fusion proteins disclosed in WO 2015/118175, WO 2018/205985, WO 2020/014285 or WO 2020/006509. In some embodiments, the N-terminal end of the sequence of the TGFbRII or the fragment thereof is fused to the C-terminal end of each heavy chain sequence of the antibody or fragment thereof. In some embodiments, the antibody or the fragment thereof and the extracellular domain of TGFbRII or the fragment thereof are genetically fused via a linker sequence. In some embodiments, the linker sequence is a short, flexible peptide. In one embodiment, the linker sequence is (G4S)_XG, wherein x is 3-6, such as 4-5 or 4.

An exemplary anti-PD-L1:TGFbRII fusion protein is shown in Figure 2. The depicted heterotetramer consists of the two light chain sequences of the anti-PD-L1 antibody, and two sequences each comprising a heavy chain sequence of the anti-PD-L1 antibody which C- terminus is genetically fused via a linker sequence to the N-terminus of the extracellular domain of the TGFbRII or the fragment thereof. In one embodiment, the extracellular domain of TGFβRII or the fragment thereof of the anti-PD(L)1:TGFβRII fusion protein has an amino acid sequence that does not differ in more than 25 amino acids from SEQ ID NO: 11 and is capable of binding TGFβ. In some embodiments, the anti-PD-L1:TGFβRII fusion protein is one of the anti-PD-L1:TGFβRII fusion proteins disclosed in WO 2015/118175, WO 2018/205985 or WO 2020/006509. For instance, the anti-PD-L1:TGFβRII fusion protein may comprise the light chain sequences and heavy chain sequences of SEQ ID NO: 1 and SEQ ID NO: 3 of WO 2015/118175, respectively. In another embodiment, the anti-PD-L1:TGFβRII fusion protein is one of the constructs listed in Table 2 of WO 2018/205985, such as construct 9 or 15 thereof. In other embodiments, the antibody having the heavy chain sequences of SEQ ID NO: 11 and the light chain sequences of SEQ ID NO: 12 of WO 2018/205985 is fused via a linking sequence (G4S)xG, wherein x is 4-5, to the TGFβRII extracellular domain sequence of SEQ ID NO: 14 (wherein “x” of the linker sequence is 4) or SEQ ID NO: 15 (wherein “x” of the linker sequence is 5) of WO 2018/205985. In another embodiment, the anti-PD-L1:TGFβRII fusion protein is SHR1701. In a further embodiment, the anti-PD-L1:TGFβRII fusion protein is one of the fusion molecules disclosed in WO 2020/006509. In one embodiment, the anti-PD-L1:TGFβRII fusion protein is Bi-PLB-1, Bi-PLB-2 or Bi-PLB-1.2 disclosed in WO 2020/006509. In one embodiment, the anti-PD(L)1:TGFβRII fusion protein is Bi-PLB-1.2 disclosed in WO 2020/006509. In one embodiment, the anti-PD-L1:TGFβRII fusion protein comprises SEQ ID NO:128 and SEQ ID NO:95 disclosed in WO 2020/006509. In some embodiments, the amino acid sequence of the light chain sequences and the heavy chain sequences of the anti-PD-L1:TGFβRII fusion protein respectively correspond to the light chain sequences and the heavy chain sequences selected from the group consisting of: (1) SEQ ID NO: 7 and SEQ ID NO: 8, (2) SEQ ID NO: 15 and SEQ ID NO: 17, (3) SEQ ID NO: 15 and SEQ ID NO: 18 of the present disclosure and (4) SEQ ID NO:128 and SEQ ID NO:95 disclosed in WO 2020/006509. In some embodiments, the anti-PD-L1:TGFβRII fusion protein is still capable of binding PD-L1 and TGFβ and the amino acid sequence of its light chain sequences and heavy chain sequences are respectively substantially identical, e.g., have at least 90% sequence identity, to the light chain sequences and the heavy chain sequences selected from the group consisting of: (1) SEQ ID NO: 7 and SEQ ID NO: 8, (2) SEQ ID NO: 15 and SEQ ID NO: 17, (3) SEQ ID NO: 15 and SEQ ID NO: 18 of the present disclosure and (4) SEQ ID NO: 128 and SEQ ID NO:95 disclosed in WO 2020/006509. In some embodiments, the amino acid sequence of the light chain sequences and the heavy chain sequences of the PD-1 inhibitor of the anti-PD-L1:TGFβRII fusion protein are respectively not more than 50, not more than 40, not more than 25, or not more than 10 amino acid residues different from the light chain sequences and the heavy chain sequences of the antibody moiety of bintrafusp alfa and the CDRs are fully identical with the CDRs of bintrafusp alfa and/or the PD-1 inhibitor is still capable of binding to PD-L1. In some embodiments, the amino acid sequence of the anti-PD-L1:TGFβRII fusion protein is substantially identical, e.g., has at least 90% sequence identity, to the amino acid sequence of bintrafusp alfa and is capable of binding to PD-L1 and TGF-β. In some embodiments, the amino acid sequence of the anti-PD-L1:TGFβRII fusion protein corresponds to the amino acid sequence of bintrafusp alfa. In some embodiments, the anti-PD-L1:TGFβRII fusion protein is bintrafusp alfa.

In a particular embodiment, the anti-PD-1:TGFβRII fusion protein is one of the fusion molecules disclosed in WO 2020/014285 that binds both PD-1 and TGF-β, e.g. as depicted in Figure 4 therein or as described in Example 1, including those identified in Tables 2-9, as specified in table 16, therein, and in particular a fusion protein that binds both PD-1 and TGF-β and comprising a sequence that is substantially identical, e.g., has at least 90% sequence identity, to SEQ ID NO: 15 or SEQ ID NO:296 and a sequence that is substantially identical, e.g., has at least 90% sequence identity, to SEQ ID NO:16, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:294 or SEQ ID NO:295 therein. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:15 and SEQ ID NO:16 of WO 2020/014285. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:15 and SEQ ID NO:143 of WO 2020/014285. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:15 and SEQ ID NO:144 of WO 2020/014285. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:15 and SEQ ID NO:145 of WO 2020/014285. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:15 and SEQ ID NO:294 of WO 2020/014285. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:15 and SEQ ID NO:295 of WO 2020/014285. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:296 and SEQ ID NO: 16 of WO 2020/014285. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:296 and SEQ ID NO:143 of WO 2020/014285. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:296 and SEQ ID NO:144 of WO 2020/014285. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:296 and SEQ ID NO:145 of WO 2020/014285. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:296 and SEQ ID NO:294 of WO 2020/014285. In an embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:296 and SEQ ID NO:295 of WO 2020/014285. In a further embodiment, the anti-PD-1:TGFβRII fusion protein is one of the fusion molecules disclosed in WO 2020/006509. In one embodiment, the anti-PD-1:TGFβRII fusion protein is Bi-PB-1, Bi-PB-2 or Bi-PB-1.2 disclosed in WO 2020/006509. In one embodiment, the anti-PD-1:TGFβRII fusion protein is Bi-PB-1.2 disclosed in WO 2020/006509. In one embodiment, the anti-PD-1:TGFβRII fusion protein comprises SEQ ID NO:108 and SEQ ID NO:93 disclosed in WO 2020/006509.

In some embodiments, the VEGF inhibitor inhibits the interaction between a VEGF ligand and a VEGF receptor, thereby inhibiting the VEGF pathway. For instance, the VEGF inhibitor may bind to a VEGF ligand, such as VEGF-A, and inhibit its interaction to one or more of its receptors, such as the VEGF receptor type 1 and/or the VEGF receptor type 2 in the case of VEGF-A. Similarly, the VEGF inhibitor may bind to a VEGF receptor, such as VEGF receptor type 2, to inhibit its interaction to one or more of its ligands. In one embodiment, the VEGF inhibitor that binds to a VEGF ligand or VEGF receptor is an antibody. In one embodiment, the VEGF inhibitor that binds to a VEGF ligand is a soluble VEGF receptor. In one embodiment, the anti-VEGF ligand antibody is ranibizumab or bevacizumab. In one embodiment, the VEGF inhibitor is a molecule having an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 which sequences are disclosed in WO2018/220169. In one embodiment, the anti- VEGF receptor antibody is ramucirumab. In one embodiment, the VEGF inhibitor that binds to a VEGF ligand is a soluble VEGF receptor or a fragment thereof capable of binding to a VEGF ligand. In one embodiment, the VEGF inhibitor that binds to a VEGF ligand is a soluble VEGF receptor type 1 and/or VEGF receptor type 2 or a fragment of such soluble VEGF receptor capable of binding to a VEGF ligand. In one embodiment, the VEGF inhibitor is aflibercept. In some embodiments, the VEGF inhibitor is a TKI that inhibits the tyrosine kinase activity of a VEGF receptor, such as a small molecule selected from the group consisting of anlotinib, axitinib, bosutinib, lenvatinib, nintedanib, pazopanib, regorafenib, cediranib, linifanib, motesanib, fruquintinib, sorafenib, sunitinib, ponatinib and cabozantinib.

In one embodiment, the therapeutic combination of the invention is used in the treatment of a human subject. The main expected benefit in the treatment with the therapeutic combination is a gain in risk/benefit ratio for these human patients. The administration of the combinations of the invention may be advantageous over the individual therapeutic agents in that the combinations may provide one or more of the following improved properties when compared to the individual administration of a single therapeutic agent alone: i) a greater anticancer effect than the most active single agent, ii) synergistic or highly synergistic anticancer activity, iii) a dosing protocol that provides enhanced anticancer activity with reduced side effect profile, iv) a reduction in the toxic effect profile, v) an increase in the therapeutic window, and/or vi) an increase in the bioavailability of one or both of the therapeutic agents. In certain embodiments, the invention provides for the treatment of diseases, disorders, and conditions characterized by excessive or abnormal cell proliferation. Such diseases include a proliferative or hyperprol iterative disease. Examples of proliferative and hyperproliferative diseases include cancer and myeloproliferative disorders.

In another embodiment, the cancer is selected from carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More particular examples of such cancers include squamous cell carcinoma, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, biliary tract cancer, and head and neck cancer. The disease or medical disorder in question may be selected from any of those disclosed in WO2015118175, WO2018029367, W02018208720, PCT/US18/12604, PCT/US19/47734, PCT/US19/40129, PCT/US19/36725, PCT/US 19/732271, PCT/US19/38600,

PCT/EP2019/061558.

In various embodiments, the method of the invention is employed as a first, second, third or later line of treatment. A line of treatment refers to a place in the order of treatment with different medications or other therapies received by a patient. First line therapy regimens are treatments given first, whereas second or third line therapy is given after the first line therapy or after the second line therapy, respectively. Therefore, first line therapy is the first treatment for a disease or condition. In patients with cancer, first line therapy, sometimes referred to as primary therapy or primary treatment, can be surgery, chemotherapy, radiation therapy, or a combination of these therapies. Typically, a patient is given a subsequent chemotherapy regimen (second or third line therapy), either because the patient did not show a positive clinical outcome or only showed a sub-clinical response to a first or second line therapy or showed a positive clinical response but later experienced a relapse, sometimes with disease now resistant to the earlier therapy that elicited the earlier positive response.

In some embodiments, the therapeutic combination of the invention is applied in a later line of treatment, particularly a second line or higher treatment of the cancer. There is no limitation to the prior number of therapies provided that the subject underwent at least one round of prior cancer therapy. The round of prior cancer therapy refers to a defined schedule/phase for treating a subject with, e.g., one or more chemotherapeutic agents, radiotherapy or chemoradiotherapy, and the subject failed with such previous treatment, which was either completed or terminated ahead of schedule. One reason could be that the cancer was resistant or became resistant to prior therapy. The current standard of care (SoC) for treating cancer patients often involves the administration of toxic and old chemotherapy regimens. Such SoC is associated with high risks of strong adverse events that are likely to interfere with the quality of life (such as secondary cancers). In one embodiment, the combined administration of the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor may be as effective and better tolerated than the SoC in patients with cancer. As the modes of action of the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor are different, it is thought that the likelihood that administration of the therapeutic treatment of the invention may lead to enhanced immune-related adverse events (irAE) is small.

In one embodiment, the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor are administered in a second line or higher treatment of a cancer selected from the group of pre treated relapsing metastatic NSCLC, unresectable locally advanced NSCLC, pre-treated SCLC ED, SCLC unsuitable for systemic treatment, pre-treated relapsing (recurrent) or metastatic SCCHN, recurrent SCCHN eligible for re-irradiation, and pre-treated microsatellite status instable low (MSI-L) or microsatellite status stable (MSS) metastatic colorectal cancer (mCRC). SCLC and SCCHN are particularly systemically pre-treated. MSI-L/MSS mCRC occurs in 85% of all mCRC.

In one embodiment, the cancer exhibits microsatellite instability (MSI). Microsatellite instability ("MSI") is or comprises a change that in the DNA of certain cells (such as tumor cells) in which the number of repeats of microsatellites (short, repeated sequences of DNA) is different than the number of repeats that was contained in the DNA from which it was inherited. Microsatellite instability arises from a failure to repair replication-associated errors due to a defective DNA mismatch repair (MMR) system. This failure allows persistence of mismatch mutations all over the genome, but especially in regions of repetitive DNA known as microsatellites, leading to increased mutational load. It has been demonstrated that at least some tumors characterized by MSI-H have improved responses to certain anti-PD-1 agents (Le et al. (2015) N. Engl. J. Med. 372(26):2509-2520; Westdorp et al. (2016) Cancer Immunol. Immunother. 65(10): 1249-1259).

In some embodiments, a cancer has a microsatellite instability status of high microsatellite instability (e.g. MSI-H status). In some embodiments, a cancer has a microsatellite instability status of low microsatellite instability (e.g. MSI-L status). In some embodiments, a cancer has a microsatellite instability status of microsatellite stable (e.g.

MSS status). In some embodiments microsatellite instability status is assessed by a next generation sequencing (NGS)-based assay, an immunohistochemistry (IHC)-based assay, and/or a PCR-based assay. In some embodiments, microsatellite instability is detected by NGS. In some embodiments, microsatellite instability is detected by IHC. In some embodiments, microsatellite instability is detected by PCR.

In some embodiments, the cancer is associated with a high tumor mutation burden (TMB). In some embodiments, the cancer is associated with high TMB and MSI-H. In some embodiments, the cancer is associated with high TMB and MSI-L or MSS. In some embodiments, the cancer is endometrial cancer associated with high TMB. In some related embodiments, the endometrial cancer is associated with high TMB and MSI-H. In some related embodiments, the endometrial cancer is associated with high TMB and MSI-L or MSS.

In some embodiments, a cancer is a mismatch repair deficient (dMMR) cancer. Microsatellite instability may arise from a failure to repair replication-associated errors due to a defective DNA mismatch repair (MMR) system. This failure allows persistence of mismatch mutations all over the genome, but especially in regions of repetitive DNA known as microsatellites, leading to increased mutational load that may improve responses to certain therapeutic agents.

In some embodiments, a cancer is a hypermutated cancer. In some embodiments, a cancer harbors a mutation in polymerase epsilon (POLE). In some embodiments, a cancer harbors a mutation in polymerase delta (POLD).

In some embodiments, a cancer is endometrial cancer (e.g. MSI-H or MSS/MSI-L endometrial cancer). In some embodiments, a cancer is a MSI-H cancer comprising a mutation in POLE or POLD (e.g. a MSI-H non-endometrial cancer comprising a mutation in POLE or POLD).

In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a recurrent cancer (e.g. a recurrent gynecological cancer such as recurrent epithelial ovarian cancer, recurrent fallopian tube cancer, recurrent primary peritoneal cancer, or recurrent endometrial cancer). In one embodiment, the cancer is recurrent or advanced.

In one embodiment, the cancer is selected from: appendiceal cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer (in particular esophageal squamous cell carcinoma), fallopian tube cancer, gastric cancer, glioma (such as diffuse intrinsic pontine glioma), head and neck cancer (in particular head and neck squamous cell carcinoma and oropharyngeal cancer), leukemia (in particular acute lymphoblastic leukemia, acute myeloid leukemia) lung cancer (in particular non-small cell lung cancer), lymphoma (in particular Hodgkin’s lymphoma, non-Hodgkin’s lymphoma), melanoma, mesothelioma (in particular malignant pleural mesothelioma), Merkel cell carcinoma, neuroblastoma, oral cancer, osteosarcoma, ovarian cancer, prostate cancer, renal cancer, salivary gland tumor, sarcoma (in particular Ewing's sarcoma or rhabdomyosarcoma) squamous cell carcinoma, soft tissue sarcoma, thymoma, thyroid cancer, urothelial cancer, uterine cancer, vaginal cancer, vulvar cancer or Wilms tumor. In a further embodiment, the cancer is selected from: appendiceal cancer, bladder cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, melanoma, mesothelioma, non-small-cell lung cancer, prostate cancer and urothelial cancer. In a further embodiment, the cancer is selected from cervical cancer, endometrial cancer, head and neck cancer (in particular head and neck squamous cell carcinoma and oropharyngeal cancer), lung cancer (in particular non-small cell lung cancer), lymphoma (in particular non- Hodgkin’s lymphoma), melanoma, oral cancer, thyroid cancer, urothelial cancer or uterine cancer. In another embodiment, the cancer is selected from head and neck cancer (in particular head and neck squamous cell carcinoma and oropharyngeal cancer), lung cancer (in particular non-small cell lung cancer), urothelial cancer, melanoma or cervical cancer.

In one embodiment, the human has a solid tumor. In one embodiment, the solid tumor is advanced solid tumor. In one embodiment, the cancer is selected from head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN or HNSCC), gastric cancer, melanoma, renal cell carcinoma (RCC), esophageal cancer, non-small cell lung carcinoma, prostate cancer, colorectal cancer, ovarian cancer and pancreatic cancer. In one embodiment, the cancer is selected from the group consisting of: colorectal cancer, cervical cancer, bladder cancer, urothelial cancer, head and neck cancer, melanoma, mesothelioma, non-small cell lung carcinoma, prostate cancer, esophageal cancer, and esophageal squamous cell carcinoma. In one aspect the human has one or more of the following: SCCHN, colorectal cancer, esophageal cancer, cervical cancer, bladder cancer, breast cancer, head and neck cancer, ovarian cancer, melanoma, renal cell carcinoma (RCC), esophageal squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma (e.g. pleural malignant mesothelioma), and prostate cancer.

In another aspect the human has a liquid tumor such as diffuse large B cell lymphoma (DLBCL), multiple myeloma, chronic lymphoblastic leukemia, follicular lymphoma, acute myeloid leukemia and chronic myelogenous leukemia. In one embodiment, the cancer is head and neck cancer. In one embodiment, the cancer is HNSCC. Squamous cell carcinoma is a cancer that arises from particular cells called squamous cells. Squamous cells are found in the outer layer of skin and in the mucous membranes, which are the moist tissues that line body cavities such as the airways and intestines. Head and neck squamous cell carcinoma (HNSCC) develops in the mucous membranes of the mouth, nose, and throat. HNSCC is also known as SCCHN and squamous cell carcinoma of the head and neck.

HNSCC can occur in the mouth (oral cavity), the middle part of the throat near the mouth (oropharynx), the space behind the nose (nasal cavity and paranasal sinuses), the upper part of the throat near the nasal cavity (nasopharynx), the voice box (larynx), or the lower part of the throat near the larynx (hypopharynx). Depending on the location, the cancer can cause abnormal patches or open sores (ulcers) in the mouth and throat, unusual bleeding or pain in the mouth, sinus congestion that does not clear, sore throat, earache, pain when swallowing or difficulty swallowing, a hoarse voice, difficulty breathing, or enlarged lymph nodes.

HNSCC can metastasize to other parts of the body, such as the lymph nodes, lungs or liver.

Tobacco use and alcohol consumption are the two most important risk factors for the development of HNSCC, and their contributions to risk are synergistic. In addition, the human papillomavirus (HPV), especially HPV-16, is now a well-established independent risk factor. Patients with HNSCC have a relatively poor prognosis. Recurrent/metastatic (R/M) HNSCC is especially challenging, regardless of human papillomavirus (HPV) status, and currently, few effective treatment options are available in the art. HPV-negative HNSCC is associated with a locoregional relapse rate of 19-35% and a distant metastatic rate of 14- 22% following standard of care, compared with rates of 9-18% and 5-12%, respectively, for HPV-positive HNSCC. The median overall survival for patients with R/M disease is 10-13 months in the setting of first line chemotherapy and 6 months in the second line setting. The current standard of care is platinum-based doublet chemotherapy with or without cetuximab. Second line standard of care options include cetuximab, methotrexate, and taxanes. All of these chemotherapeutic agents are associated with significant side effects, and only 10- 13% of patients respond to treatment. HNSCC regressions from existing systemic therapies are transient and do not add significantly increased longevity, and virtually all patients succumb to their malignancy. In one embodiment, the cancer is head and neck cancer. In one embodiment the cancer is head and neck squamous cell carcinoma (HNSCC). In one embodiment, the cancer is recurrent/metastatic (R/M) HNSCC. In one embodiment, the cancer is recurring/refractory (R/R) HNSCC. In one embodiment, the cancer is HPV-negative or HPV- positive HNSCC. In one embodiment, the cancer is a locally advanced HNSCC. In one embodiment, the cancer is HNSCC, such as (R/M) HNSCC, in PD-L1 positive patients having a CPS of ≥1% or a TPS ≥50%. The CPS or TPS is as determined by an FDA- or EMA-approved test, such as the Dako IHC 22C3 PharmDx assay. In one embodiment, the cancer is HNSCC in PD-1 inhibitor experienced or PD-1 inhibitor naive patients. In one embodiment, the cancer is HNSCC in PD-1 inhibitor experienced or PD-1 inhibitor naive patients.

In one embodiment, the head and neck cancer is oropharyngeal cancer. In one embodiment, the head and neck cancer is an oral cancer (i.e. a mouth cancer).

In one embodiment, the cancer is lung cancer. In some embodiments, the lung cancer is a squamous cell carcinoma of the lung. In some embodiments, the lung cancer is small cell lung cancer (SCLC). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), such as squamous NSCLC. In some embodiments, the lung cancer is an ALK-translocated lung cancer (e.g. ALK-translocated NSCLC). In some embodiments, the cancer is NSCLC with an identified ALK translocation. In some embodiments, the lung cancer is an EGFR-mutant lung cancer (e.g. EGFR- mutant NSCLC). In some embodiments, the cancer is NSCLC with an identified EGFR mutation. In one embodiment, the cancer is NSCLC in PD-L1 positive patients having a TPS >1% or a TPS ≥50%. The TPS is as determined by an FDA- or EMA-approved test, such as the Dako IHC 22C3 PharmDx assay or the VENTANA PD-L1 (SP263) assay.

In one embodiment, the cancer is melanoma. In some embodiments, the melanoma is an advanced melanoma. In some embodiments, the melanoma is a metastatic melanoma. In some embodiments, the melanoma is a MSI-H melanoma. In some embodiments, the melanoma is a MSS melanoma. In some embodiments, the melanoma is a POLE-mutant melanoma. In some embodiments, the melanoma is a POLD-mutant melanoma. In some embodiments, the melanoma is a high TMB melanoma.

In one embodiment, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is an advanced colorectal cancer. In some embodiments, the colorectal cancer is a metastatic colorectal cancer. In some embodiments, the colorectal cancer is a MSI-H colorectal cancer. In some embodiments, the colorectal cancer is a MSS colorectal cancer. In some embodiments, the colorectal cancer is a POLE-mutant colorectal cancer. In some embodiments, the colorectal cancer is a POLD-mutant colorectal cancer. In some embodiments, the colorectal cancer is a high TMB colorectal cancer.

In some embodiments, the cancer is a gynecologic cancer (i.e. a cancer of the female reproductive system such as ovarian cancer, fallopian tube cancer, cervical cancer, vaginal cancer, vulvar cancer, uterine cancer, or primary peritoneal cancer, or breast cancer). In some embodiments, cancers of the female reproductive system include, but are not limited to, ovarian cancer, cancer of the fallopian tube(s), peritoneal cancer, and breast cancer.

In some embodiments, the cancer is ovarian cancer (e.g. serous or clear cell ovarian cancer). In some embodiments, the cancer is fallopian tube cancer (e.g. serous or clear cell fallopian tube cancer). In some embodiments, the cancer is primary peritoneal cancer (e.g. serous or clear cell primary peritoneal cancer).

In some embodiments, the ovarian cancer is an epithelial carcinoma. Epithelial carcinomas make up 85% to 90% of ovarian cancers. While historically considered to start on the surface of the ovary, new evidence suggests at least some ovarian cancer begins in special cells in a part of the fallopian tube. The fallopian tubes are small ducts that link a woman's ovaries to her uterus that are a part of a woman's reproductive system. In a normal female reproductive system, there are two fallopian tubes, one located on each side of the uterus. Cancer cells that begin in the fallopian tube may go to the surface of the ovary early on. The term “ovarian cancer” is often used to describe epithelial cancers that begin in the ovary, in the fallopian tube, and from the lining of the abdominal cavity, call the peritoneum. In some embodiments, the cancer is or comprises a germ cell tumor. Germ cell tumors are a type of ovarian cancer develops in the egg- producing cells of the ovaries. In some embodiments, a cancer is or comprises a stromal tumor. Stromal tumors develop in the connective tissue cells that hold the ovaries together, which sometimes is the tissue that makes female hormones called estrogen. In some embodiments, the cancer is or comprises a granulosa cell tumor. Granulosa cell tumors may secrete estrogen resulting in unusual vaginal bleeding at the time of diagnosis. In some embodiments, a gynecologic cancer is associated with homologous recombination repair deficiency/homologous repair deficiency (HRD) and/or BRCA1/2 mutation(s). In some embodiments, a gynecologic cancer is platinum-sensitive. In some embodiments, a gynecologic cancer has responded to a platinum-based therapy. In some embodiments, a gynecologic cancer has developed resistance to a platinum-based therapy. In some embodiments, a gynecologic cancer has at one time shown a partial or complete response to platinum-based therapy (e.g. a partial or complete response to the last platinum-based therapy or to the penultimate platinum-based therapy). In some embodiments, a gynecologic cancer is now resistant to platinum-based therapy.

In some embodiments, the cancer is breast cancer. Usually breast cancer either begins in the cells of the milk producing glands, known as the lobules, or in the ducts. Less commonly breast cancer can begin in the stromal tissues. These include the fatty and fibrous connective tissues of the breast. Over time the breast cancer cells can invade nearby tissues such the underarm lymph nodes or the lungs in a process known as metastasis. The stage of a breast cancer, the size of the tumor and its rate of growth are all factors which determine the type of treatment that is offered. Treatment options include surgery to remove the tumor, drug treatment which includes chemotherapy and hormonal therapy, radiation therapy and immunotherapy. The prognosis and survival rate varies widely; the five year relative survival rates vary from 98% to 23% depending on the type of breast cancer that occurs. Breast cancer is the second most common cancer in the world with approximately 1.7 million new cases in 2012 and the fifth most common cause of death from cancer, with approximately 521,000 deaths. Of these cases, approximately 15% are triple-negative, which do not express the estrogen receptor, progesterone receptor (PR) or HER2. In some embodiments, triple negative breast cancer (TNBC) is characterized as breast cancer cells that are estrogen receptor expression negative (<1% of cells), progesterone receptor expression negative (<1% of cells), and HER2-negative. In one embodiment, the cancer is TNBC in PD-L1 positive patients having PD-L1 expressing tumor-infiltrating immune cells (IC) of ≥1%. The IC is as determined by an FDA- or EMA-approved test, such as the Ventana PD-L1 (SP142) assay.

In some embodiments, the cancer is estrogen receptor(ER)-positive breast cancer, ER-negative breast cancer, PR-positive breast cancer, PR-negative breast cancer, HER2- positive breast cancer, HER2-negative breast cancer, BRCA1/2-positive breast cancer, BRCA1/2-negative cancer, or TNBC. In some embodiments, the breast cancer is a metastatic breast cancer. In some embodiments, the breast cancer is an advanced breast cancer. In some embodiments, the cancer is a stage II, stage III or stage IV breast cancer. In some embodiments, the cancer is a stage IV breast cancer. In some embodiments, the breast cancer is a triple negative breast cancer.

In one embodiment, the cancer is endometrial cancer. Endometrial carcinoma is the most common cancer of the female genital, tract accounting for 10-20 per 100,000 person- years. The annual number of new cases of endometrial cancer (EC) is estimated at about 325 thousand worldwide. Further, EC is the most commonly occurring cancer in post- menopausal women. About 53% of endometrial cancer cases occur in developed countries. In 2015, approximately 55,000 cases of EC were diagnosed in the U.S. and no targeted therapies are currently approved for use in EC. There is a need for agents and regimens that improve survival for advanced and recurrent EC in 1L and 2L settings. Approximately 10,170 people are predicted to die from EC in the U.S. in 2016. The most common histologic form is endometrioid adenocarcinoma, representing about 75-80% of diagnosed cases. Other histologic forms include uterine papillary serous (less than 10%), clear cell 4%, mucinous 1%, squamous less than 1% and mixed about 10%.

From the pathogenetic point of view, EC falls into two different types, so-called types I and II. Type I tumors are low-grade and estrogen-related endometrioid carcinomas (EEC) while type II are non-endometrioid (NEEC) (mainly serous and clear cell) carcinomas. The World Health Organization has updated the pathologic classification of EC, recognizing nine different subtypes of EC, but EEC and serous carcinoma (SC) account for the vast majority of cases. EECs are estrogen-related carcinomas, which occur in perimenopausal patients, and are preceded by precursor lesions (endometrial hyperplasia/endometrioid intraepithelial neoplasia). Microscopically, low-grade EEC (EEC 1-2) contains tubular glands, somewhat resembling the proliferative endometrium, with architectural complexity with fusion of the glands and cribriform pattern. High-grade EEC shows solid pattern of growth. In contrast, SC occurs in postmenopausal patients in absence of hyperestrogenism. At the microscope, SC shows thick, fibrotic or edematous papillae with prominent stratification of tumor cells, cellular budding, and anaplastic cells with large, eosinophilic cytoplasms. The vast majority of EEC are low grade tumors (grades 1 and 2), and are associated with good prognosis when they are restricted to the uterus. Grade 3 EEC (EEC3) is an aggressive tumor, with increased frequency of lymph node metastasis. SCs are very aggressive, unrelated to estrogen stimulation, mainly occurring in older women. EEC 3 and SC are considered high- grade tumors. SC and EEC3 have been compared using the surveillance, epidemiology and End Results (SEER) program data from 1988 to 2001. They represented 10% and 15% of EC respectively, but accounted for 39% and 27% of cancer death respectively. Endometrial cancers can also be classified into four molecular subgroups: (1) ultramutated/POLE-mutant; (2) hypermutated MSI+ (e.g., MSI-H or MSI-L); (3) copy number low/micro satellite stable (MSS); and (4) copy number high/serous -like. Approximately 28% of cases are MSI-high. (Murali, Lancet Oncol. (2014). In some embodiments, the patient has a mismatch repair deficient subset of 2L endometrial cancer. In some embodiments, the endometrial cancer is metastatic endometrial cancer. In some embodiments, the patient has a MSS endometrial cancer. In some embodiments, the patient has a MSI-H endometrial cancer. In one embodiment, the cancer is cervical cancer. In some embodiments, the cervical cancer is an advanced cervical cancer. In some embodiments, the cervical cancer is a metastatic cervical cancer. In some embodiments, the cervical cancer is a MSI-H cervical cancer. In some embodiments, the cervical cancer is a MSS cervical cancer. In some embodiments, the cervical cancer is a POLE-mutant cervical cancer. In some embodiments, the cervical cancer is a POLD-mutant cervical cancer. In some embodiments, the cervical cancer is a high TMB cervical cancer. In one embodiment, the cancer is cervical cancer in PD-L1 positive patients having a CPS ≥1%. The CPS is as determined by an FDA- or EMA- approved test, such as the Dako IHC 22C3 PharmDx assay.

In one embodiment, the cancer is uterine cancer. In some embodiments, the uterine cancer is an advanced uterine cancer. In some embodiments, the uterine cancer is a metastatic uterine cancer. In some embodiments, the uterine cancer is a MSI-H uterine cancer. In some embodiments, the uterine cancer is a MSS uterine cancer. In some embodiments, the uterine cancer is a POLE-mutant uterine cancer. In some embodiments, the uterine cancer is a POLD-mutant uterine cancer. In some embodiments, the uterine cancer is a high TMB uterine cancer.

In one embodiment, the cancer is urothelial cancer. In some embodiments, the urothelial cancer is an advanced urothelial cancer. In some embodiments, the urothelial cancer is a metastatic urothelial cancer. In some embodiments, the urothelial cancer is a MSI-H urothelial cancer. In some embodiments, the urothelial cancer is a MSS urothelial cancer. In some embodiments, the urothelial cancer is a POLE-mutant urothelial cancer. In some embodiments, the urothelial cancer is a POLD-mutant urothelial cancer. In some embodiments, the urothelial cancer is a high TMB urothelial cancer. In one embodiment, the cancer is urothelial carcinoma in PD-L1 positive patients having a CPS ³10%. The CPS is as determined by an FDA- or EMA-approved test, such as the Dako IHC 22C3 PharmDx assay. In one embodiment, the cancer is urothelial carcinoma in PD-L1 positive patients having PD- L1 expressing tumor-infiltrating immune cells (IC) of ≥5%. The IC is as determined by an FDA- or EMA-approved test, such as the Ventana PD-L1 (SP142) assay.

In one embodiment, the cancer is thyroid cancer. In some embodiments, the thyroid cancer is an advanced thyroid cancer. In some embodiments, the thyroid cancer is a metastatic thyroid cancer. In some embodiments, the thyroid cancer is a MSI-H thyroid cancer. In some embodiments, the thyroid cancer is a MSS thyroid cancer. In some embodiments, the thyroid cancer is a POLE-mutant thyroid cancer. In some embodiments, the thyroid cancer is a POLD-mutant thyroid cancer. In some embodiments, the thyroid cancer is a high TMB thyroid cancer. Tumors may be a hematopoietic (or hematologic or hematological or blood-related) cancer, for example, cancers derived from blood cells or immune cells, which may be referred to as “liquid tumors”. Specific examples of clinical conditions based on hematologic tumors include leukemias such as chronic myelocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia; plasma cell malignancies such as multiple myeloma, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS) and Waldenstrom’s macroglobulinemia; lymphomas such as non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, and the like.

In one embodiment, the cancer is a gastric cancer (GC) or a gastroesophageal junction cancer (GEJ). In one embodiment, the cancer is GC or GEJ in PD-L1 positive patients having a CPS ≥1%. The CPS is as determined by an FDA- or EMA-approved test, such as the Dako IHC 22C3 PharmDx assay.

In one embodiment, the cancer is esophageal squamous cell carcinoma (ESCC). In one embodiment, the cancer is ESCC in PD-L1 positive patients having a CPS ≥10%. The CPS is as determined by an FDA- or EMA-approved test, such as the Dako IHC 22C3 PharmDx assay.

The cancer may be any cancer in which an abnormal number of blast cells or unwanted cell proliferation is present or that is diagnosed as a hematological cancer, including both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myeloblastic) leukemia (undifferentiated or differentiated), acute promyeloid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia and megakaryocytic (or megakaryoblastic) leukemia. These leukemias may be referred together as acute myeloid (or myelocytic or myelogenous) leukemia. Myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic myelogenous (or myeloid or myelocytic) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polcythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MDS), which may be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBT); as well as myelofibrosis (MFS) with or without agnogenic myeloid metaplasia.

In one embodiment, the cancer is non-Hodgkin’s lymphoma. Hematopoietic cancers also include lymphoid malignancies, which may affect the lymph nodes, spleens, bone marrow, peripheral blood, and/or extranodal sites. Lymphoid cancers include B-cell malignancies, which include, but are not limited to, B-cell non-Hodgkin’s lymphomas (B- NHLs). B-NHLs may be indolent (or low-grade), intermediate-grade (or aggressive) or high- grade (very aggressive). Indolent B cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL) including nodal MZL, extranodal MZL, splenic MZL and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-associated-lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-NHLs include mantle cell lymphoma (MCL) with or without leukemic involvement, diffuse large B cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B- NHLs include Burkitt’s lymphoma (BL), Burkitt-like lymphoma, small non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV associated (or AIDS related) lymphomas, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom’s macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic or lymphoblastic) leukemia, and Castleman’s disease. NHL may also include T-cell non- Hodgkin’s lymphomas (T-NHLs), which include, but are not limited to T-cell non-Hodgkin’s lymphoma not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid disorder (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma/delta lymphoma, cutaneous T cell lymphoma, mycosis fungoides, and Sezary syndrome.

Hematopoietic cancers also include Hodgkin’s lymphoma (or disease) including classical Hodgkin’s lymphoma, nodular sclerosing Hodgkin’s lymphoma, mixed cellularity Hodgkin’s lymphoma, lymphocyte predominant (LP) Hodgkin’s lymphoma, nodular LP Hodgkin’s lymphoma, and lymphocyte depleted Hodgkin’s lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers such as multiple myeloma (MM) including smoldering MM, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom’s Macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic cancers may also include other cancers of additional hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes and natural killer cells. Tissues which include hematopoietic cells referred herein to as "hematopoietic cell tissues" include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues associated with mucosa (such as the gut-associated lymphoid tissues), tonsils, Peyer's patches and appendix, and lymphoid tissues associated with other mucosa, for example, the bronchial linings.

In one embodiment, the treatment is first line or second line treatment of HNSCC. In one embodiment, the treatment is first line or second line treatment of recurrent/metastatic HNSCC. In one embodiment the treatment is first line treatment of recurrent/metastatic (1 L R/M) HNSCC. In one embodiment, the treatment is first line treatment of 1L R/M HNSCC that is PD-L1 positive. In one embodiment the treatment is second line treatment of recurrent/metastatic (2L R/M) HNSCC.

In one embodiment, the treatment is first line, second line, third line, fourth line or fifth line treatment of PD-1/PD-L1-naÏve HNSCC. In one embodiment, the treatment first line, second line, third line, fourth line or fifth line treatment of PD-1/PD-L1 experienced HNSCC.

In some embodiments, the treatment of cancer is first line treatment of cancer. In one embodiment, the treatment of cancer is second line treatment of cancer. In some embodiments, the treatment is third line treatment of cancer. In some embodiments, the treatment is fourth line treatment of cancer. In some embodiments, the treatment is fifth line treatment of cancer. In some embodiments, prior treatment to said second line, third line, fourth line or fifth line treatment of cancer comprises one or more of radiotherapy, chemotherapy, surgery or radiochemotherapy.

In one embodiment, the prior treatment comprises treatment with diterpenoids, such as paclitaxel, nab-paclitaxel or docetaxel; vinca alkaloids, such as vinblastine, vincristine, or vinorelbine; platinum coordination complexes, such as cisplatin or carboplatin; nitrogen mustards such as cyclophosphamide, melphalan, or chlorambucil; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; triazenes such as dacarbazine; actinomycins such as dactinomycin; anthrocyclins such as daunorubicin or doxorubicin; bleomycins; epipodophyllotoxins such as etoposide orteniposide; antimetabolite anti-neoplastic agents such as fluorouracil, methotrexate, cytarabine, mecaptopurine, thioguanine, or gemcitabine; methotrexate; camptothecins such as irinotecan or topotecan; rituximab; ofatumumab; trastuzumab; cetuximab; bexarotene; sorafenib; erbB inhibitors such as lapatinib, erlotinib or gefitinib; pertuzumab; ipilimumab; nivolumab; FOLFOX; capecitabine; FOLFIRI; bevacizumab; atezolizumab; selicrelumab; obinotuzumab or any combinations thereof. In one embodiment, prior treatment to said second line treatment, third line, fourth line or fifth line treatment of cancer comprises ipilimumab and nivolumab. In one embodiment, prior treatment to said second line treatment, third line, fourth line or fifth line treatment of cancer comprises FOLFOX, capecitabine, FOLFIRI/bevacizumab and atezolizumab/selicrelumab. In one embodiment, prior treatment to said second line treatment, third line, fourth line or fifth line treatment of cancer comprises carboplatin/Nab-paclitaxel. In one embodiment, prior treatment to said second line treatment, third line, fourth line or fifth line treatment of cancer comprises nivolumab and electrochemotherapy. In one embodiment, prior treatment to said second line treatment, third line, fourth line or fifth line treatment of cancer comprises radiotherapy, cisplatin and carboplatin/paclitaxel.

In one embodiment, the treatment is first line or second line treatment of head and neck cancer (in particular head and neck squamous cell carcinoma and oropharyngeal cancer). In one embodiment, the treatment is first line or second line treatment of recurrent/metastatic HNSCC. In one embodiment the treatment is first line treatment of recurrent/metastatic (1L R/M) HNSCC. In one embodiment, the treatment is first line treatment of 1 L R/M HNSCC that is PD-L1 positive. In one embodiment the treatment is second line treatment of recurrent/metastatic (2L R/M) HNSCC.

In one embodiment, the treatment is first line, second line, third line, fourth line or fifth line treatment of PD-1/PD-L1-naive HNSCC. In one embodiment, the treatment is first line, second line, third line, fourth line or fifth line treatment of PD-1/PD-L1 experienced HNSCC.

In some embodiments, the treatment results in one or more of increased tumor infiltrating lymphocytes including cytotoxic T cells, helper T cell and NK cells, increased T cells, increased granzyme B-positive cells, reduced proliferating tumor cells and increased activated T cells as compared to levels prior to treatment (e.g. baseline level). Activated T cells may be observed by greater OX40 and human leukocyte antigen DR expression. In some embodiments, treatment results in upregulation of PD-1 and/or PD-L1 as compared to levels prior to treatment (e.g. baseline level).

In one embodiment, the methods of the present invention further comprise administering at least one neo-plastic agent or cancer adjuvant to said human. The methods of the present invention may also be employed with other therapeutic methods of cancer treatment.

Typically, any anti-neoplastic agent or cancer adjuvant that has activity versus a tumor, such as a susceptible tumor being treated may be co-administered in the treatment of cancer in the present invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita, T.S. Lawrence, and S.A. Rosenberg (editors), 10th edition (December 5, 2014), Lippincott Williams & Wilkins Publishers. In one embodiment, the human has previously been treated with one or more different cancer treatment modalities. In some embodiments, at least some of the patients in the cancer patient population have previously been treated with one or more therapies, such as surgery, radiotherapy, chemotherapy or immunotherapy. In some embodiments, at least some of the patients in the cancer patient population have previously been treated with chemotherapy (e.g. platinum-based chemotherapy). For example, a patient who has received two lines of cancer treatment can be identified as a 2L cancer patient (e.g. a 2L NSCLC patient). In some embodiments, a patient has received two lines or more lines of cancer treatment (e.g. a 2L+ cancer patient such as a 2L+ endometrial cancer patient). In some embodiments, a patient has not been previously treated with an antibody therapy, such as an anti-PD-1 therapy. In some embodiments, a patient previously received at least one line of cancer treatment (e.g. a patient previously received at least one line or at least two lines of cancer treatment). In some embodiments, a patient previously received at least one line of treatment for metastatic cancer (e.g. a patient previously received one or two lines of treatment for metastatic cancer). In some embodiments, a subject is resistant to treatment with a PD-1 inhibitor. In some embodiments, a subject is refractory to treatment with a PD-1 inhibitor. In some embodiments, a method described herein sensitizes the subject to treatment with a PD-1 inhibitor.

In certain embodiments, the cancer to be treated is PD-L1 positive. For example, in certain embodiments, the cancer to be treated exhibits PD-L1+ expression (e.g., high PD-L1 expression). Methods of detecting a biomarker, such as PD-L1 for example, on a cancer or tumor, are routine in the art and are contemplated herein. Non-limiting examples include immunohistochemistry, immunofluorescence and fluorescence activated cell sorting (FACS). In some embodiments, subjects or patients with PD-L1 high cancer are treated by intravenously administering anti-PD(L)1:TGFβRII fusion protein at a dose of about 1200 mg Q2W. In some embodiments, subjects or patients with PD-L1 high cancer are treated by intravenously administering anti-PD(L)1:TGFβRII fusion protein at a dose of about 1800 mg Q3W. In some embodiments, subjects or patients with PD-L1 high cancer are treated by intravenously administering anti-PD(L)1:TGFβRII fusion protein at a dose of about 2100 mg Q3W. In some embodiments, subjects or patients with PD-L1 high cancer are treated by intravenously administering anti-PD(L)1:TGFβRII fusion protein at a dose of about 2400 mg Q3W. In some embodiments, subjects or patients with PD-L1 high cancer are treated by intravenously administering anti-PD(L)1:TGFβRII fusion protein at a dose of about 15 mg/kg Q3W. In certain embodiments, the dosing regimen comprises administering the anti-PD(L)1:TGFβRII fusion protein, such as one having the amino acid sequence of bintrafusp alfa, at a dose of about 0.01 - 3000 mg (e.g. a dose about 0.01 mg; a dose about 0.08 mg; a dose about 0.1 mg; a dose about 0.24 mg; a dose about 0.8 mg; a dose about 1 mg; a dose about 2.4 mg; a dose about 8 mg; a dose about 10 mg; a dose about 20 mg; a dose about 24 mg; a dose about 30 mg; a dose about 40 mg; a dose about 48 mg; a dose about 50 mg; a dose about 60 mg; a dose about 70 mg; a dose about 80 mg; a dose about 90 mg; a dose about 100 mg; a dose about 160 mg; a dose about 200 mg; a dose about 240 mg; a dose about 300 mg; a dose about 400 mg; a dose about 500 mg; a dose about 600 mg; a dose about 700 mg; a dose about 800 mg; a dose about 900 mg; a dose about 1000 mg; a dose about 1100 mg; a dose about 1200 mg; a dose about 1300 mg; a dose about 1400 mg; a dose about 1500 mg; a dose about 1600 mg; a dose about 1700 mg; a dose about 1800 mg; a dose about 1900 mg; a dose about 2000 mg; a dose about 2100 mg; a dose about 2200 mg; a dose about 2300 mg; a dose about 2400 mg; a dose about 2500 mg; a dose about 2600 mg; a dose about 2700 mg; a dose about 2800 mg; a dose about 2900 mg; or a dose about 3000 mg). In some embodiments, the dose is a dose of about 500 mg. In some embodiments, the dose is about 1200 mg. In some embodiments, the dose is about 2400 mg. In some embodiments, the dose of the anti-PD(L)1:TGFβRII fusion protein, such as one having the amino acid sequence of bintrafusp alfa, is about 0.001-100 mg/kg (e.g., a dose about 0.001 mg/kg; a dose about 0.003 mg/kg; a dose about 0.01 mg/kg; a dose about 0.03 mg/kg; a dose about 0.1 mg/kg; a dose about 0.3 mg/kg; a dose about 1 mg/kg; a dose about 2 mg/kg; a dose about 3 mg/kg; a dose about 10 mg/kg; a dose about 15 mg/kg; or a dose about 30 mg/kg).

All fixed doses disclosed herein are considered comparable to the body-weight dosing based on a reference body weight of 80 kg. Accordingly, when reference is made to a fixed dose of 2400 mg, a body-weight dose of 30 mg/kg is likewise disclosed therewith.

In some embodiments, the anti-PD(L)1:TGFβRII fusion protein light chain and heavy chain sequences correspond to SEQ ID NO: 15 and SEQ ID NO: 17 or SEQ ID NO: 15 and SEQ ID NO: 18 respectively and the dose of the anti-PD(L)1:TGFβRII fusion protein is 30 mg/kg.

In one embodiment, the anti-PD(L)1:TGFβRII fusion protein, such as one having the amino acid sequence of bintrafusp alfa, is administered once every 2-6 weeks (e.g. 2, 3 or 4 weeks, in particular 3 weeks). In one embodiment, the anti-PD(L)1:TGFβRII fusion protein, such as one having the amino acid sequence of bintrafusp alfa, is administered for once every two weeks (“Q2W”). In one embodiment, the anti-PD(L)1:TGFβRII fusion protein, such as one having the amino acid sequence of bintrafusp alfa, is administered for once every three weeks (“Q3W”). In one embodiment, the anti-PD(L)1:TGFβRII fusion protein, such as one having the amino acid sequence of bintrafusp alfa, is administered for once every 6 weeks (“Q6W”). In one embodiment, the anti-PD(L)1:TGFβRII fusion protein, such as one having the amino acid sequence of bintrafusp alfa, is administered for Q3W for 2-6 dosing cycles (e.g. the first 3, 4, or 5 dosing cycles, in particular, the first 4 dosing cycles).

In some embodiments, the anti-PD(L)1:TGFβRII fusion protein light chain and heavy chain sequences correspond to SEQ ID NO: 15 and SEQ ID NO: 17 or SEQ ID NO: 15 and SEQ ID NO: 18 respectively and the anti-PD(L)1:TGFβRII fusion protein is administered Q3W.

In certain embodiments, about 1200 mg of the anti-PD(L)1:TGFβRII fusion protein, such as one having the amino acid sequence of bintrafusp alfa, is administered to a subject Q2W. In certain embodiments, about 2400 mg of the anti-PD(L)1:TGFβRII fusion protein, such as one having the amino acid sequence of bintrafusp alfa, is administered to a subject Q3W.

In some embodiments, the anti-PD(L)1:TGFβRII fusion protein light chain and heavy chain sequences correspond to SEQ ID NO: 15 and SEQ ID NO: 17 or SEQ ID NO: 15 and SEQ ID NO: 18 respectively and the anti-PD(L)1:TGFβRII fusion protein is administered at a dose of 30 mg/kg Q3W.

In some embodiments, the dosing regimen comprises administering the VEGF inhibitor at a dose of about 0.01 - 5000 mg (e.g. a dose about 0.01 mg; a dose about 0.08 mg; a dose about 0.1 mg; a dose about 0.24 mg; a dose about 0.8 mg; a dose about 1 mg; a dose about 2.4 mg; a dose about 8 mg; a dose about 10 mg; a dose about 20 mg; a dose about 24 mg; a dose about 30 mg; a dose about 40 mg; a dose about 48 mg; a dose about 50 mg; a dose about 60 mg; a dose about 70 mg; a dose about 80 mg; a dose about 90 mg; a dose about 100 mg; a dose about 160 mg; a dose about 200 mg; a dose about 240 mg; a dose about 300 mg; a dose about 400 mg; a dose about 500 mg; a dose about 600 mg; a dose about 700 mg; a dose about 800 mg; a dose about 900 mg; a dose about 1000 mg; a dose about 1100 mg; a dose about 1200 mg; a dose about 1300 mg; a dose about 1400 mg; a dose about 1500 mg; a dose about 1600 mg; a dose about 1700 mg; a dose about 1800 mg; a dose about 1900 mg; a dose about 2000 mg; a dose about 2100 mg; a dose about 2200 mg; a dose about 2300 mg; a dose about 2400 mg; a dose about 2500 mg; a dose about 2600 mg; a dose about 2700 mg; a dose about 2800 mg; a dose about 2900 mg; a dose about 3000 mg; a dose about 3100 mg; a dose about 3200 mg; a dose about 3300 mg; a dose about 3400 mg; a dose about 3500 mg; a dose about 3600 mg; a dose about 3700 mg; a dose about 3800 mg; a dose about 3900 mg; a dose about 4000 mg; a dose about 4100 mg; a dose about 4200 mg; a dose about 4300 mg; a dose about 4400 mg; a dose about 4500 mg; a dose about 4600 mg; a dose about 4700 mg; a dose about 4800 mg; a dose about 4900 mg; or a dose about 5000 mg). In some embodiments, the dose of the VEGF inhibitor is about 0.001-100 mg/kg (e.g., a dose about 0.001 mg/kg; a dose about 0.003 mg/kg; a dose about 0.01 mg/kg; a dose about 0.03 mg/kg; a dose about 0.1 mg/kg; a dose about 0.3 mg/kg; a dose about 1 mg/kg; a dose about 2 mg/kg; a dose about 3 mg/kg; a dose about 10 mg/kg; a dose about 15 mg/kg; or a dose about 30 mg/kg). In one embodiment, such doses of the VEGF inhibitor are, in case of a TKI, administered orally or, in case of an anti-VEGF ligand or anti-VEGF receptor antibody or a soluble VEGF receptor, intravenously.

In one embodiment, the VEGF inhibitor, e.g., a TKI, is administered one, two, three or four times a day. In one embodiment, the VEGF inhibitor, e.g., a TKI, is administered once daily (“QD”), particularly continuously. In one embodiment, the VEGF inhibitor, e.g., a TKI, is administered twice daily (“BID”), particularly continuously. In one embodiment, the VEGF inhibitor, e.g., a TKI, is administered three times per day (“TID”), particularly continuously. In one embodiment, the VEGF inhibitor, e.g., a TKI, is administered four times per day (“QID”), particularly continuously.

In one embodiment, the VEGF inhibitor, e.g., an anti-VEGF ligand or anti-VEGF receptor antibody or a soluble VEGF receptor, is administered once every 2-6 weeks (e.g. 2, 3 or 4 weeks, in particular 3 weeks). In one embodiment, the VEGF inhibitor, e.g., an anti- VEGF ligand or anti-VEGF receptor antibody or a soluble VEGF receptor, is administered for once every two weeks (“Q2W”). In one embodiment, the VEGF inhibitor, e.g., an anti-VEGF ligand or anti-VEGF receptor antibody or a soluble VEGF receptor, is administered for once every three weeks (“Q3W”). In one embodiment, the VEGF inhibitor, e.g., an anti-VEGF ligand or anti-VEGF receptor antibody or a soluble VEGF receptor, is administered for once every 6 weeks (“Q6W”). In one embodiment, the VEGF inhibitor, e.g., an anti-VEGF ligand or anti-VEGF receptor antibody or a soluble VEGF receptor, is administered for Q3Wfor 2-6 dosing cycles (e.g. the first 3, 4, or 5 dosing cycles, in particular, the first 4 dosing cycles).

In some embodiments, bevacizumab is administered Q2W or Q3W. In some embodiments, bevacizumab is administered at a dose of 5, 10 or 15 mg/kg. In some embodiments, bevacizumab is administered at a dose of 5, 10 or 15 mg/kg Q2W or Q3W. In some embodiments, ramucirumab is administered Q2W. In some embodiments, ramucirumab is administered at a dose of 8 mg/kg. In some embodiments, ramucirumab is administered at a dose of 8 mg/kg Q2W. In some embodiments, lenvatinib is administered QD. In some embodiments, lenvatinib is administered at a dose of 8, 12, 18 or 24 mg. In some embodiments, lenvatinib is administered at a dose of 8, 12, 18 or 24 mg QD. In some embodiments, regorafenib is administered QD. In some embodiments, regorafenib is administered at a dose of 160 mg. In some embodiments, regorafenib is administered at a dose of 160 mg QD. In some embodiments, axitinib is administered BID. In some embodiments, axitinib is administered at a dose of 5 mg. In some embodiments, axitinib is administered at a dose of 5 mg BID. In some embodiments, nintedanib is administered BID. In some embodiments, nintedanib is administered at a dose of 100, 150 or 200 mg. In some embodiments, nintedanib is administered at a dose of 100, 150 or 200 mg BID. In some embodiments, nintedanib is administered at a dose of 100, 150 or 200 mg BID and bintrafusp alfa is administered at a dose of 2400 mg Q3W. In some embodiments, fruquintinib is administered QD. In some embodiments, fruquintinib is administered at a dose of 5 mg. In some embodiments, fruquintinib is administered at a dose of 5 mg QD. In some embodiments, pazopanib is administered QD. In some embodiments, pazopanib is administered at a dose of 200 or 800 mg. In some embodiments, pazopanib is administered at a dose of 200 or 800 mg QD. In some embodiments, cediranib is administered QD. In some embodiments, cediranib is administered at a dose of 20 or 45 mg. In some embodiments, cediranib is administered at a dose of 20 or 45 mg QD. In some embodiments, anlotinib is administered QD. In some embodiments, anlotinib is administered at a dose of 12 mg. In some embodiments, anlotinib is administered at a dose of 12 mg QD. In some embodiments, bosutinib is administered QD. In some embodiments, bosutinib is administered at a dose of 500 or 600 mg. In some embodiments, bosutinib is administered at a dose of 500 or 600 mg QD. In some embodiments, linifanib is administered QD. In some embodiments, motesanib is administered QD. In some embodiments, motesanib is administered at a dose of 125 mg. In some embodiments, motesanib is administered at a dose of 125 mg QD. In some embodiments, sorafenib is administered BID. In some embodiments, sorafenib is administered at a dose of 400 mg. In some embodiments, sorafenib is administered at a dose of 400 mg BID. In some embodiments, sunitinib is administered QD. In some embodiments, sunitinib is administered at a dose of 37.5 or 50 mg. In some embodiments, sunitinib is administered at a dose of 37.5 or 50 mg QD. In some embodiments, ponatinib is administered QD. In some embodiments, ponatinib is administered at a dose of 45 mg. In some embodiments, ponatinib is administered at a dose of 45 mg QD. In some embodiments, cabozantinib is administered QD. In some embodiments, cabozantinib is administered at a dose of 60 mg. In some embodiments, cabozantinib is administered at a dose of 60 mg QD. Concurrent treatment, in addition to the treatment with the combination of the invention and considered necessary for the patient’s well-being, may be given at discretion of the treating physician. In some embodiments, the present invention provides methods of treating, stabilizing or decreasing the severity or progression of one or more diseases or disorders described herein comprising administering to a patient in need thereof a PD-1 inhibitor, a TGFβ inhibitor, and a VEGF inhibitor in combination with an additional therapy, such as chemotherapy, radiotherapy or chemoradiotherapy.

In one embodiment, diterpenoids, such as paclitaxel, nab-paclitaxel or docetaxel; vinca alkaloids, such as vinblastine, vincristine, or vinorelbine; platinum coordination complexes, such as cisplatin or carboplatin; nitrogen mustards such as cyclophosphamide, melphalan, or chlorambucil; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; triazenes such as dacarbazine; actinomycins such as dactinomycin; anthrocyclins such as daunorubicin or doxorubicin; bleomycins; epipodophyllotoxins such as etoposide or teniposide; antimetabolite anti-neoplastic agents such as fluorouracil, pemetrexed, methotrexate, cytarabine, mecaptopurine, thioguanine, or gemcitabine; methotrexate; camptothecins such as irinotecan or topotecan; rituximab; ofatumumab; trastuzumab; cetuximab; bexarotene; sorafenib; erbB inhibitors such as lapatinib, erlotinib or gefitinib; pertuzumab; ipilimumab; tremelimumab; nivolumab; pembrolizumab; FOLFOX; capecitabine; FOLFIRI; bevacizumab; atezolizumab; selicrelumab; obinotuzumab or any combinations thereof is/are further administered concurrently or sequentially with the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor.

In one embodiment, chemotherapy is further administered concurrently or sequentially with the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor. In one embodiment, chemotherapy is further administered concurrently or sequentially with the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor. In one embodiment, the chemotherapy is platinum- based chemotherapy. In one embodiment, the chemotherapy is platinum-based chemotherapy and fluorouracil. In one embodiment, the platinum-based chemotherapy is paclitaxel, nab-paclitaxel, docetaxel, cisplatin, carboplatin or any combination thereof. In one embodiment, the platinum-based chemotherapy is fluorouracil, cisplatin, carboplatin or any combination thereof. In one embodiment, chemotherapy is a platinum doublet of cisplatin or carboplatin with any one of pemetrexed, paclitaxel, gemcitabine, or fluorouracil. In one embodiment chemotherapy is further administered concurrently or sequentially with the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor to PD-1 inhibitor naive patients.

In one embodiment, the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor are administered concurrently or sequentially to PD-L1 positive patients. In one embodiment, radiotherapy is further administered concurrently or sequentially with the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor. In some embodiments, the radiotherapy is selected from the group consisting of systemic radiation therapy, external beam radiation therapy, image-guided radiation therapy, tomotherapy, stereotactic radio surgery, stereotactic body radiation therapy, and proton therapy. In some embodiments, the radiotherapy comprises external-beam radiation therapy, internal radiation therapy (brachytherapy), or systemic radiation therapy. See, e.g., Amini et al. , Radiat Oncol. “Stereotactic body radiation therapy (SBRT) for lung cancer patients previously treated with conventional radiotherapy: a review” 9:210 (2014); Baker et al., Radiat Oncol. “A critical review of recent developments in radiotherapy for non-small cell lung cancer” 11 (1): 115 (2016); Ko et al., Clin Cancer Res “The Integration of Radiotherapy with Immunotherapy for the Treatment of Non-Small Cell Lung Cancer” (24) (23) 5792-5806; and, Yamoah et al., Int J Radiat Oncol Biol Phys “Radiotherapy Intensification for Solid Tumors: A Systematic Review of Randomized Trials” 93(4): 737-745 (2015).

In some embodiments, the radiotherapy comprises external-beam radiation therapy, and the external bean radiation therapy comprises intensity-modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), tomotherapy, stereotactic radiosurgery, stereotactic body radiation therapy, proton therapy, or other charged particle beams.

In some embodiments, the radiotherapy comprises stereotactic body radiation therapy.

The PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor are administered using any amount and any route of administration effective for treating or decreasing the severity of a disorder provided above. The exact amount required may vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular agent, its mode of administration, and the like.

In some embodiments, the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor are administered simultaneously, separately or sequentially and in any order. The PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor are administered to the patient in any order (i.e., simultaneously or sequentially) and the compounds may be in separate compositions, formulations or unit dosage forms, or together in a single composition, formulation or unit dosage form. In one embodiment, the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor are administered simultaneously or sequentially in any order, in jointly therapeutically effective amounts (for example in synergistically effective amounts), e.g. in daily or intermittently dosages corresponding to the amounts described herein. The individual combination partners of the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor may be administered separately at different times during the course of therapy or concurrently. Typically, in such combination therapies, individual compounds are formulated into separate pharmaceutical compositions or medicaments. When the compounds are separately formulated, the individual compounds can be administered simultaneously or sequentially, optionally via different routes. Optionally, the treatment regimens for each of the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor have different but overlapping delivery regimens, e.g., daily, twice daily, vs. a single administration, or weekly. In certain embodiments, the PD-1 inhibitor, TGFβ inhibitor and VEGF inhibitor are administered simultaneously in the same composition comprising the PD-1 inhibitor, TGFβ inhibitor and VEGF inhibitor. In certain embodiments, the PD-1 inhibitor, TGFβ inhibitor and VEGF inhibitor are administered simultaneously in separate compositions, i.e., wherein the PD-1 inhibitor, TGFβ inhibitor and VEGF inhibitor are administered simultaneously each in a separate unit dosage form. In some embodiments, the PD-1 inhibitor and TGFβ inhibitor are fused and administered in a separate unit dosage form from the VEGF inhibitor and the PD-1 inhibitor and TGFβ inhibitor are administered simultaneously or sequentially in any order with the VEGF inhibitor. It will be appreciated that the PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor are administered on the same day or on different days and in any order as according to an appropriate dosing protocol. The instant invention is therefore to be understood as embracing all such regimens of simultaneous or alternating treatment and the term “administering” is to be interpreted accordingly. In one embodiment, the PD-1 inhibitor and the TGFβ inhibitor are administered Q2W or Q3W and the VEGF inhibitor is administered Q2W or Q3W in case of an anti-VEGF ligand or anti-VEGF receptor antibody or QD or BID in case of a TKI. In one embodiment, the PD-1 inhibitor and the TGFβ inhibitor are administered Q2W or Q3W and the TKI QD or BID.

In some embodiments, the anti-PD(L)1:TGFβRII fusion protein and the VEGF inhibitor are administered simultaneously, separately or sequentially and in any order. The anti-PD(L)1:TGFβRII fusion protein and the VEGF inhibitor are administered to the patient in any order (i.e. , simultaneously or sequentially) in separate compositions, formulations or unit dosage forms, or together in a single composition, formulation or unit dosage form. In one embodiment, the anti-PD(L)1:TGFβRII fusion protein and the VEGF inhibitor are administered simultaneously or sequentially in any order, in jointly therapeutically effective amounts (for example in synergistically effective amounts), e.g. in daily or intermittently dosages corresponding to the amounts described herein. The individual combination partners of the anti-PD(L)1:TGFβRII fusion protein and the VEGF inhibitor may be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. Typically, in such combination therapies, the individual compounds are formulated into separate pharmaceutical compositions or medicaments. When separately formulated, the individual compounds can be administered simultaneously or sequentially, optionally via different routes. Optionally, the treatment regimens for each of the anti-PD(L)1:TGFβRII fusion protein and the VEGF inhibitor have different but overlapping delivery regimens, e.g., daily, twice daily, vs. a single administration, or weekly. The anti-PD(L)1:TGFβRII fusion protein may be delivered prior to, substantially simultaneously with, or after the VEGF inhibitor. In certain embodiments, the anti-PD(L)1:TGFβRII fusion protein is administered simultaneously in the same composition comprising the anti-PD(L)1:TGFβRII fusion protein and the VEGF inhibitor. In certain embodiments, the anti-PD(L)1:TGFβRII fusion protein and the VEGF inhibitor are administered simultaneously in separate compositions, i.e., wherein the anti-PD(L)1:TGFβRII fusion protein and the VEGF inhibitor are administered simultaneously each in a separate unit dosage form. It will be appreciated that the anti-PD(L)1:TGFβRII fusion protein and the VEGF inhibitor are administered on the same day or on different days and in any order as according to an appropriate dosing protocol. In one embodiment, the anti-PD(L)1:TGFβRII fusion protein is administered Q2Wor Q3W, e.g., by intravenous infusion or injection, and the VEGF inhibitor is administered by intravenous infusion or injection Q2W or Q3W in case of an anti-VEGF ligand or anti-VEGF receptor antibody or orally QD or BID in case of a TKI. In one embodiment, the anti-PD(L)1:TGFβRII fusion protein is administered 1200 mg Q2Wor 2400 mg Q3W, e.g., by intravenous infusion or injection, and the VEGF inhibitor is administered by intravenous infusion or injection Q2W or Q3W in case of an anti-VEGF ligand or anti-VEGF receptor antibody or orally QD or BID in case of a TKI at one of the doses indicated for the VEGF inhibitor above. In one embodiment, the anti-PD(L)1:TGFβRII fusion protein is administered 1200 mg Q2Wor 2400 mg Q3W, e.g., by intravenous infusion or injection, and the TKI orally QD or BID at one of the doses indicated above.

In some embodiments, one or more of the PD-1 inhibitor, TGFβ inhibitor and VEGF inhibitor are administered to a patient in need of treatment at a first dose at a first interval for a first period and at a second dose at a second interval for a second period. Such first and second period could be the lead phase and maintenance phase of treatment. There may be a rest period between the first and second periods during which one or more of the PD-1 inhibitor, TGFβ inhibitor and VEGF inhibitor in the combination is/are not administered to the patient. In some embodiments, there is a rest period between the first period and second period. In some embodiments, the rest period is between 1 day and 30 days. In some embodiments, the rest period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. In some embodiments, the rest period is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks or 15 weeks.

In some embodiments, the first dose and second dose are the same. In some embodiments, the first dose and second dose are different.

In some embodiments, the first dose and the second dose of the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, are about 1200 mg. In some embodiments, the first dose and the second dose of the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, are about 2400 mg. In some embodiments, the first dose of the anti-PD(L)1 :TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is about 1200 mg and the second dose is about 2400 mg. In some embodiments, the first dose of the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is about 2400 mg and the second dose is about 1200 mg.

In some embodiments, the first interval and second interval are the same. In some embodiments, the first interval and the second interval are Q2W. In some embodiments, the first interval and the second interval are Q3W. In some embodiments, the first interval and the second interval are Q6W. In some embodiments, the first interval and the second interval are different. In some embodiments, the first interval is Q2W and the second interval is Q3W. In some embodiments, the first interval is Q3W and the second interval is Q6W.

In some embodiments, the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is administered at the first dose of 1200 mg Q2W for the first period of 2-6 dosing cycles (e.g. the first 3, 4, or 5 dosing cycles, in particular, the first 4 dosing cycles), and at the second dose of 2400 mg Q3W until therapy is discontinued (e.g. due to disease progression, an adverse event, or as determined by a physician). In some embodiments, the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is administered at the first dose of 1200 mg Q2Wfor the first three dosing cycles, and at the second dose of 2400 mg Q3W or more until therapy is discontinued (e.g. due to disease progression, an adverse event, or as determined by a physician). In some embodiments, the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is administered at the first dose of 1200 mg Q2W for the first four dosing cycles, and at the second dose of 2400 mg Q3W or more until therapy is discontinued (e.g. due to disease progression, an adverse event, or as determined by a physician). In some embodiments, the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is administered at the first dose of 1200 mg Q2W for the first five dosing cycles, and at the second dose of 2400 mg Q3W or more until therapy is discontinued (e.g. due to disease progression, an adverse event, or as determined by a physician).

It will be understood that there can be a first treatment with one or two compounds of the VEGF inhibitor, PD-1 inhibitor and TGFβ inhibitor, followed by the treatment with all three compounds. Between first administration to the patient of a VEGF inhibitor, a PD-1 inhibitor, a TGFβ inhibitor or a fused PD-1 inhibitor and TGFβ inhibitor as a monotherapy and the administration of the PD-1 inhibitor, TGFβ inhibitor and VEGF inhibitor as a combination therapy as described herein, a period of no treatment or no administration may be performed, such as for a defined number of cycles. For example, after first administration with a monotherapy, the patient may be administered no treatment for 1 cycle or 2 cycles of 3 weeks, 6 weeks or 12 weeks before being administered a combination therapy as described herein. Thus, in one embodiment, the patient is first administered a VEGF inhibitor as a monotherapy as described herein, then administered no treatment for 1 cycle or 2 cycles of 3 weeks, 6 weeks or 12 weeks, before the patient is administered a VEGF inhibitor with a PD-1 inhibitor and a TGFβ inhibitor as a combination therapy as described herein. In one embodiment, the patient is first administered a PD-1 inhibitor and/or a TGFβ inhibitor as a monotherapy as described herein, then administered no treatment for 1 cycle or 2 cycles of 3 weeks, 6 weeks or 12 weeks, before the patient is administered a PD-1 inhibitor, a TGFβ inhibitor with a VEGF inhibitor as a combination therapy as described herein.

Compositions of the present invention are administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intraperitoneally, subcutaneously or intravenously. In one embodiment, the compositions are administered by intravenous infusion or injection. In another embodiment, the compositions are administered by intramuscular or subcutaneous injection. In one embodiment, the anti-PD(L)1:TGFβRII fusion protein is administered by intravenous infusion or injection. In another embodiment, the anti-PD(L)1:TGFβRII fusion protein is administered by intramuscular or subcutaneous injection. In one embodiment, the VEGF inhibitor is a TKI and administered orally. In one embodiment, the VEGF inhibitor is an anti- VEGF ligand or anti-VEGF receptor antibody and administered by intravenous infusion or injection. In one embodiment, the anti-PD(L)1:TGFβRII fusion protein is administered by intravenous infusion or injection and the VEGF inhibitor is an anti-VEGF ligand or anti-VEGF receptor antibody and administered by intravenous infusion or injection. In one embodiment, the anti-PD(L)1:TGFβRII fusion protein is administered by intravenous infusion or injection and the VEGF inhibitor is a TKI and administered orally.

In some embodiments, the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is administered intravenously (e.g., as an intravenous infusion) or subcutaneously. In some embodiments, the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is administered as an intravenous infusion. In some embodiments, the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is administered intravenously at a dose of about 1200 mg or about 2400 mg. In some embodiments, the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is administered intravenously at a dose of about 1200 mg Q2W. In some embodiments, the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is administered intravenously at a dose of about 2400 mg Q3W. In some embodiments, the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, is administered intravenously at a dose of about 15 mg/kg Q3W.

In some embodiments, the VEGF inhibitor is a TKI and administered orally at one of the doses described above. In some embodiments, the VEGF inhibitor is an anti-VEGF ligand or anti-VEGF receptor antibody and administered intravenously at one of the doses described above. In some embodiments, the VEGF inhibitor is a TKI and administered orally at one of the doses described above QD or BID. In some embodiments, the VEGF inhibitor is an anti-VEGF ligand or anti-VEGF receptor antibody and administered intravenously at one of the doses described above Q2W or Q3W.

In some embodiments, the TKI is administered orally at one of the doses described above. In some embodiments, the TKI is administered orally at one of the doses described above QD or BID.

In some embodiments, the anti-VEGF ligand or anti-VEGF receptor antibody is administered intravenously at one of the doses described above. In some embodiments, the anti-VEGF ligand or anti-VEGF receptor antibody is administered intravenously at one of the doses described above Q2W or Q3W.

In some embodiments, the patient is first administered the anti-PD(L)1 :TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, at a dose of about 1200 mg as a monotherapy regimen and then the anti-PD(L)1:TGFβRII fusion protein at a dose of about 1200 mg, with the VEGF inhibitor as a combination therapy regimen. In some embodiments, the patient is first administered the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, at a dose of about 2400 mg as a monotherapy regimen and then the anti-PD(L)1 :TGFβRII fusion protein at a dose of about 2400 mg, with the VEGF inhibitor as a combination therapy regimen. In some embodiments, the patient is first administered the VEGF inhibitor as a monotherapy regimen and then the VEGF inhibitor with the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, at a dose of about 1200 mg, as a combination therapy regimen. In some embodiments, the patient is first administered the VEGF inhibitor as a monotherapy regimen and then the VEGF inhibitor with the anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, at a dose of about 2400 mg, as a combination therapy regimen.

In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the PD-1 inhibitor and the TGFβ inhibitor prior to first receipt of the VEGF inhibitor; and (b) under the direction or control of a physician, the subject receiving the VEGF inhibitor. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the VEGF inhibitor prior to first receipt of the PD-1 inhibitor and the TGFβ inhibitor; and (b) under the direction or control of a physician, the subject receiving the PD-1 inhibitor and TGFβ inhibitor. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the PD-1 inhibitor prior to first receipt of the TGFβ inhibitor and the VEGF inhibitor; and (b) under the direction or control of a physician, the subject receiving the TGFβ inhibitor and the VEGF inhibitor. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the TGFβ inhibitor and the VEGF inhibitor prior to first receipt of the PD-1 inhibitor; and (b) under the direction or control of a physician, the subject receiving the PD-1 inhibitor. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the TGFβ inhibitor prior to first receipt of the PD-1 inhibitor and the VEGF inhibitor; and (b) under the direction or control of a physician, the subject receiving the PD-1 inhibitor and the VEGF inhibitor. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the PD-1 inhibitor and the VEGF inhibitor prior to first receipt of the TGFβ inhibitor; and (b) under the direction or control of a physician, the subject receiving the TGFβ inhibitor. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the anti-PD(L)1 antibody and the TGFβRII or anti-TGFβ antibody prior to first receipt of the VEGF inhibitor; and (b) under the direction or control of a physician, the subject receiving the VEGF inhibitor. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the VEGF inhibitor prior to first receipt of the anti-PD(L)1 antibody and the TGFβRII or anti-TGFβ antibody; and (b) under the direction or control of a physician, the subject receiving the anti-PD(L)1 antibody and the TGFβRII or anti-TGFβ antibody. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the anti-PD(L)1 antibody prior to first receipt of the TGFβRII or anti-TGFβ antibody and the VEGF inhibitor; and (b) under the direction or control of a physician, the subject receiving the TGFβRII or anti-TGFβ antibody and the VEGF inhibitor. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the TGFβRII or anti-TGFβ antibody and the VEGF inhibitor prior to first receipt of the anti- PD(L)1 antibody; and (b) under the direction or control of a physician, the subject receiving the anti-PD(L)1 antibody. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the TGFβRII or anti-TGFβ antibody prior to first receipt of the anti-PD(L)1 antibody and the VEGF inhibitor; and (b) under the direction or control of a physician, the subject receiving the anti- PD(L)1 antibody and the VEGF inhibitor. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving the anti-PD(L)1 antibody and the VEGF inhibitor prior to first receipt of the TGFβRII or anti- TGFβ antibody; and (b) under the direction or control of a physician, the subject receiving the TGFβRII or anti-TGFβ antibody.

In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving an anti-PD(L)1:TGFβRII fusion protein, e.g., having the amino acid sequence of bintrafusp alfa, prior to first receipt of an VEGF inhibitor; and (b) under the direction or control of a physician, the subject receiving the VEGF inhibitor. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving an VEGF inhibitor prior to first receipt of an anti-PD(L)1:TGFβRII fusion protein, e.g., having the amino acid sequence of bintrafusp alfa, (b) under the direction or control of a physician, the subject receiving the anti-PD(L)1:TGFβRII fusion protein. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving an anti-PD(L)1:TGFβRII fusion protein, e.g., having the amino acid sequence of bintrafusp alfa, prior to first receipt of an VEGF inhibitor; and (b) under the direction or control of a physician, the subject receiving the VEGF inhibitor. In some embodiments, the combination regimen comprises the steps of: (a) under the direction or control of a physician, the subject receiving an VEGF inhibitor prior to first receipt of an anti-PD(L)1:TGFβRII fusion protein, e.g., having the amino acid sequence of bintrafusp alfa, (b) under the direction or control of a physician, the subject receiving the anti-PD(L)1:TGFβRII fusion protein.

Also provided is a combination comprising a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor. Also provided is a combination comprising an anti-PD(L)1 antibody, a TGFβRII or anti-TGFβ antibody, and a VEGF inhibitor. Also provided is a combination comprising a VEGF inhibitor and a fused PD-1 inhibitor and TGFβ inhibitor. Also provided is a combination comprising an anti-PD(L)1:TGFβRII fusion protein and an VEGF inhibitor. In some embodiments, any of said combinations is for use as a medicament or for use in the treatment of cancer.

It shall be understood that, in the various embodiments described above, the PD-1 inhibitor and the TGFβ inhibitor can be fused, e.g., as an anti-PD(L)1:TGFβRII fusion protein or an anti-PD-1:TGFβRI fIusion protein.

Pharmaceutical formulations and kits

The PD-1 inhibitor, TGFβ inhibitor, and VEGF inhibitor described herein may also be in the form of pharmaceutical formulations or kits.

In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising a PD-1 inhibitor. In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising a TGFβ inhibitor. In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising a fused PD-1 inhibitor and TGFβ inhibitor. In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising anti-PD(L)1:TGFβRII fusion protein. In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising anti-PD(L)1:TGFβRII fusion protein having the amino acid sequence of bintrafusp alfa. In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising a VEGF inhibitor. In some embodiments, the present invention provides a pharmaceutically acceptable composition of a chemotherapeutic agent. In some embodiments, the present invention provides a pharmaceutical composition comprising a PD-1 inhibitor and a TGFβ inhibitor. In some embodiments, the present invention provides a pharmaceutical composition comprising a TGFβ inhibitor and a VEGF inhibitor. In some embodiments, the present invention provides a pharmaceutical composition comprising a PD-1 inhibitor and a VEGF inhibitor. In some embodiments, the present invention provides a pharmaceutical composition comprising a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor. In some embodiments, the present invention provides a pharmaceutical composition comprising a VEGF inhibitor and a fused PD-1 inhibitor and TGFβ inhibitor. In some embodiments, the present invention provides a pharmaceutical composition comprising an anti- Rϋ(I_)1:TGFβRII fusion protein and an VEGF inhibitor. In some embodiments, the present invention provides a pharmaceutical composition comprising an anti-PD(L)1:TGFβRII fusion protein having the amino acid sequence of bintrafusp alfa and an VEGF inhibitor. The pharmaceutically acceptable composition may comprise at least a further pharmaceutically acceptable excipient or adjuvant, such as a pharmaceutically acceptable carrier.

In some embodiments, a composition comprising the fused PD-1 inhibitor and TGFβ inhibitor, e.g., an anti-PD(L)1:TGFβRII fusion protein, is separate from a composition comprising a VEGF inhibitor. In some embodiments, the PD-1 inhibitor and TGFβ inhibitor are fused e.g., as an anti-PD(L)1:TGFβRII fusion protein, and present with a VEGF inhibitor in the same composition.

Examples of such pharmaceutically acceptable compositions are described further below and herein.

The compositions of the present invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories.

Pharmaceutically acceptable carriers, adjuvants or vehicles that are used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may additionally contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of the combination compounds described herein, it is often desirable to slow absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered combination compounds described herein, is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of combination compounds described herein in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration can be suppositories, which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Dosage forms for oral administration include capsules, tablets, pills, powders, and granules, aqueous suspensions or solutions. In solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hardfilled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

The combination compounds described herein can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms, the combination compounds described herein may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of the combination compounds described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Exemplary carriers for topical administration of compounds of this are mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, provided pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

Pharmaceutically acceptable compositions of this invention are optionally administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

In a further aspect, the invention relates to a kit comprising a PD-1 inhibitor and a package insert comprising instructions for using the PD-1 inhibitor in combination with a VEGF inhibitor, and a TGFβ inhibitor to treat or delay progression of a cancer in a subject. Also provided is a kit comprising a VEGF inhibitor and a package insert comprising instructions for using the VEGF inhibitor in combination with a PD-1 inhibitor, and a TGFβ inhibitor to treat or delay progression of a cancer in a subject. Also provided is a kit comprising a TGFβ inhibitor and a package insert comprising instructions for using the TGFβ inhibitor in combination with a PD-1 inhibitor, and a VEGF inhibitor to treat or delay progression of a cancer in a subject. Also provided is a kit comprising an anti-PD-L1 antibody and a package insert comprising instructions for using the anti-PD-L1 antibody in combination with an VEGF inhibitor, and a TGFβRII or anti-TGFβ antibody to treat or delay progression of a cancer in a subject. Also provided is a kit comprising an VEGF inhibitor and a package insert comprising instructions for using the VEGF inhibitor in combination with an anti-PD-L1 antibody, and a TGFβRII or anti-TGFβ antibody to treat or delay progression of a cancer in a subject. Also provided is a kit comprising a TGFβRII or anti-TGFβ antibody and a package insert comprising instructions for using the TGFβRII or anti-TGFβ antibody in combination with an anti-PD-L1 antibody, and a VEGF inhibitor to treat or delay progression of a cancer in a subject. Also provided is a kit comprising a PD-1 inhibitor and a TGFβ inhibitor, and a package insert comprising instructions for using the PD-1 inhibitor and the TGFβ inhibitor in combination with a VEGF inhibitor to treat or delay progression of a cancer in a subject. Also provided is a kit comprising an anti-PD-L1 antibody and a TGFβRII or anti- TGFβ antibody, and a package insert comprising instructions for using the anti-PD-L1 antibody and the TGFβRII or anti-TGFβ antibody in combination with a VEGF inhibitor to treat or delay progression of a cancer in a subject. Also provided is a kit comprising an anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, and a package insert comprising instructions for using the anti-PD(L)1:TGFβRII fusion protein in combination with a VEGF inhibitor to treat or delay progression of a cancer in a subject. Also provided is a kit comprising a PD-1 inhibitor and a VEGF inhibitor, and a package insert comprising instructions for using the PD-1 inhibitor and the VEGF inhibitor in combination with a TGFβ inhibitor to treat or delay progression of a cancer in a subject. Also provided is a kit comprising a TGFβ inhibitor and a VEGF inhibitor, and a package insert comprising instructions for using the TGFβ inhibitor and the VEGF inhibitor in combination with a PD-1 inhibitor to treat or delay progression of a cancer in a subject. Also provided is a kit comprising an anti-PD-L1 antibody and a VEGF inhibitor, and a package insert comprising instructions for using the anti-PD-L1 antibody and the VEGF inhibitor in combination with a TGFβRII or anti-TGFβ antibody to treat or delay progression of a cancer in a subject. Also provided is a kit comprising a TGFβRII or anti-TGFβ antibody and a VEGF inhibitor, and a package insert comprising instructions for using the TGFβRII or anti-TGFβ antibody and the VEGF inhibitor in combination with an anti-PD-L1 antibody to treat or delay progression of a cancer in a subject. Also provided is a kit comprising a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor, and a package insert comprising instructions for using the PD-1 inhibitor, TGFβ inhibitor and VEGF inhibitor to treat or delay progression of a cancer in a subject. Also provided is a kit comprising an anti-PD-L1 antibody, a TGFβRII or anti-TGFβ antibody and a VEGF inhibitor, and a package insert comprising instructions for using the anti-PD-L1 antibody, TGFβRII or anti-TGFβ antibody and VEGF inhibitor to treat or delay progression of a cancer in a subject. Also provided is a kit comprising an anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, and a VEGF inhibitor and a package insert comprising instructions for using the anti-PD(L)1:TGFβRII fusion protein and the VEGF inhibitor to treat or delay progression of a cancer in a subject. The kit can comprise a first container, a second container, a third container and a package insert, wherein the first container comprises at least one dose of the PD-1 inhibitor, the second container comprises at least one dose of the VEGF inhibitor, the third container comprises at least one dose of the TGFβ inhibitor and the package insert comprises instructions for treating a subject for cancer using the three compounds. In some embodiments, the kit comprises a first container, a second container and a package insert, wherein the first container comprises at least one dose of an anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, the second container comprises at least one dose of a VEGF inhibitor and the package insert comprises instructions for treating a subject for cancer using the two compounds. The first, second and third containers may be comprised of the same or different shape (e.g., vials, syringes and bottles) and/or material (e.g., plastic or glass). The kit may further comprise other materials that may be useful in administering the medicaments, such as diluents, filters, IV bags and lines, needles and syringes. The instructions can state that the medicaments are intended for use in treating a subject having a cancer that tests positive for PD-L1, e.g., by means of an immunohistochemical (IHC) assay, FACS or LC/MS/MS.

Further diagnostic, predictive, prognostic and/or therapeutic methods

The disclosure further provides diagnostic, predictive, prognostic and/or therapeutic methods using the combination compounds described herein. Such methods are based, at least in part, on determination of the identity of the expression level of a marker of interest. In particular, the amount of human PD-L1 in a cancer patient sample can be used to predict whether the patient is likely to respond favorably to cancer therapy utilizing the therapeutic combination of the invention.

Any suitable sample can be used for the method. Non-limiting examples of such include one or more of a serum sample, plasma sample, whole blood, pancreatic juice sample, tissue sample, tumor lysate or a tumor sample, which can be an isolated from a needle biopsy, core biopsy and needle aspirate. For example, tissue, plasma or serum samples are taken from the patient before treatment and optionally on treatment with the therapeutic combination of the invention. The expression levels obtained on treatment are compared with the values obtained before starting treatment of the patient. The information obtained may be prognostic in that it can indicate whether a patient has responded favorably or unfavorably to cancer therapy.

It is to be understood that information obtained using the diagnostic assays described herein may be used alone or in combination with other information, such as, but not limited to, expression levels of other genes, clinical chemical parameters, histopathological parameters, or age, gender and weight of the subject. When used alone, the information obtained using the diagnostic assays described herein is useful in determining or identifying the clinical outcome of a treatment, selecting a patient for a treatment, or treating a patient, etc. When used in combination with other information, on the other hand, the information obtained using the diagnostic assays described herein is useful in aiding in the determination or identification of clinical outcome of a treatment, aiding in the selection of a patient for a treatment, or aiding in the treatment of a patient, and the like. In a particular aspect, the expression level can be used in a diagnostic panel each of which contributes to the final diagnosis, prognosis, or treatment selected for a patient.

Any suitable method can be used to measure the PD-L1 protein, DNA, RNA, or other suitable read-outs for PD-L1 levels, respectively, examples of which are described herein and/or are well known to the skilled artisan.

In some embodiments, determining the PD-L1 level comprises determining the PD- L1 expression. In some embodiments, the PD-L1 level is determined by the PD-L1 protein concentration in a patient sample, e.g., with PD-L1 specific ligands, such as antibodies or specific binding partners. The binding event can, e.g., be detected by competitive or non competitive methods, including the use of a labeled ligand or PD-L1 specific moieties, e.g., antibodies, or labeled competitive moieties, including a labeled PD-L1 standard, which compete with marker proteins for the binding event. If the marker specific ligand is capable of forming a complex with PD-L1, the complex formation can indicate PD-L1 expression in the sample. In various embodiments, the biomarker protein level is determined by a method comprising quantitative western blot, multiple immunoassay formats, ELISA, immunohistochemistry, histochemistry, or use of FACS analysis of tumor lysates, immunofluorescence staining, a bead-based suspension immunoassay, Luminex technology, or a proximity ligation assay. In one embodiment, the PD-L1 expression is determined by immunohistochemistry using one or more primary anti-PD-L1 antibodies.

In another embodiment, the biomarker RNA level is determined by a method comprising microarray chips, RT-PCR, qRT-PCR, multiplex qPCR or in-situ hybridization. In one embodiment of the invention, a DNA or RNA array comprises an arrangement of poly nucleotides presented by or hybridizing to the PD-L1 gene immobilized on a solid surface. For example, to the extent of determining the PD-L1 mRNA, the mRNA of the sample can be isolated, if necessary, after adequate sample preparation steps, e.g., tissue homogenization, and hybridized with marker specific probes, in particular on a microarray platform with or without amplification, or primers for PCR-based detection methods, e.g., PCR extension labeling with probes specific for a portion of the marker mRNA.

Several approaches have been described for quantifying PD-L1 protein expression in IHC assays of tumor tissue sections (Thompson et al. (2004) PNAS 101(49): 17174; Thompson et al. (2006) Cancer Res. 66: 3381; Gadiot et al. (2012) Cancer 117: 2192;

Taube et al. (2012) Sci Transl Med 4, 127ra37; and Toplian et al. (2012) New Eng. J Med. 366 (26): 2443). One approach employs a simple binary end-point of positive or negative for PD-L1 expression, with a positive result defined in terms of the percentage of tumor cells that exhibit histologic evidence of cell-surface membrane staining.

The level of PD-L1 mRNA expression may be compared to the mRNA expression levels of one or more reference genes that are frequently used in quantitative RT-PCR, such as ubiquitin C. In some embodiments, a level of PD-L1 expression (protein and/or mRNA) by malignant cells and/or by infiltrating immune cells within a tumor is determined to be “overexpressed” or “elevated” based on comparison with the level of PD-L1 expression (protein and/or mRNA) by an appropriate control. For example, a control PD-L1 protein or mRNA expression level may be the level quantified in non-malignant cells of the same type or in a section from a matched normal tissue.

In one embodiment, the efficacy of the therapeutic combination of the invention is predicted by means of PD-L1 expression in tumor samples. This disclosure also provides a kit for determining if the combination of the invention is suitable for therapeutic treatment of a cancer patient, comprising means for determining a protein level of PD-L1, or the expression level of its RNA, in a sample isolated from the patient and instructions for use. In another aspect, the kit further comprises a PD-1 inhibitor, a TGFβ inhibitor, and a VEGF inhibitor for therapy. In one aspect of the invention, the determination of a high PD-L1 level indicates increased PFS or OS when the patient is treated with the therapeutic combination of the invention. In one embodiment of the kit, the means for determining the PD-L1 protein level are antibodies with specific binding to PD-L1.

In still another aspect, the invention provides a method for advertising a PD-1 inhibitor in combination with a TGFβ inhibitor and a VEGF inhibitor, comprising promoting, to a target audience, the use of the combination for treating a subject with a cancer, optionally, based on PD-L1 expression in samples taken from the subject. In still another aspect, the invention provides a method for advertising a VEGF inhibitor in combination with a PD-1 inhibitor and a TGFβ inhibitor, wherein the PD-1 inhibitor and TGFβ inhibitor are can be fused, comprising promoting, to a target audience, the use of the combination for treating a subject with a cancer, optionally, based on PD-L1 expression in samples taken from the subject. In still another aspect, the invention provides a method for advertising a TGFβ inhibitor in combination with a PD-1 inhibitor and a VEGF inhibitor, comprising promoting, to a target audience, the use of the combination for treating a subject with a cancer, optionally, based on PD-L1 expression in samples taken from the subject. In still another aspect, the invention provides a method for advertising an anti-PD(L)1:TGFβRII fusion protein, e.g., one having the amino acid sequence of bintrafusp alfa, in combination with an VEGF inhibitor, comprising promoting, to a target audience, the use of the combination for treating a subject with a cancer, optionally, based on PD-L1 expression in samples taken from the subject. In still another aspect, the invention provides a method for advertising a combination comprising a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor, comprising promoting, to a target audience, the use of the combination for treating a subject with a cancer, optionally, based on PD-L1 expression in samples taken from the subject. Promotion may be conducted by any means available. In some embodiments, the promotion is by a package insert accompanying a commercial formulation of the therapeutic combination of the invention. The promotion may also be by a package insert accompanying a commercial formulation of the PD-1 inhibitor, TGFβ inhibitor, VEGF inhibitor or another medicament (when treatment is a therapy with the therapeutic combination of the invention and a further medicament). In some embodiments, the promotion is by a package insert where the package insert provides instructions to receive therapy with the therapeutic combination of the invention after measuring PD-L1 expression levels, and in some embodiments, in combination with another medicament. In some embodiments, the promotion is followed by the treatment of the patient with the therapeutic combination of the invention with or without another medicament. In some embodiments, the package insert indicates that the therapeutic combination of the invention is to be used to treat the patient if the patient's cancer sample is characterized by high PD-L1 biomarker levels. In some embodiments, the package insert indicates that the therapeutic combination of the invention is not to be used to treat the patient if the patient's cancer sample expresses low PD-L1 biomarker levels. In some embodiments, a high PD-L1 biomarker level means a measured PD-L1 level that correlates with a likelihood of increased PFS and/or OS when the patient is treated with the therapeutic combination of the invention, and vice versa. In some embodiments, the PFS and/or OS is decreased relative to a patient who is not treated with the therapeutic combination of the invention. In some embodiments, the promotion is by a package insert where the package insert provides instructions to receive therapy with an anti-PD(L)1:TGFβRII fusion protein in combination with an VEGF inhibitor after first measuring PD-L1 expression levels. In some embodiments, the promotion is followed by the treatment of the patient with an anti-PD(L)1:TGFβRII fusion protein in combination with an VEGF inhibitor with or without another medicament.

Further embodiments of the present disclosure:

1. A PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor for use in a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor, the TGFβ inhibitor and the VEGF inhibitor to the subject.

2. A PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor for use in a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor, the TGFβ inhibitor and the VEGF inhibitor to the subject; and wherein the PD-1 inhibitor is an anti-PD(L)1 antibody, the TGFβ inhibitor is a TGFβRII or anti-TGFβ antibody and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI.

3. A PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor for use in a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor, the TGFβ inhibitor and the VEGF inhibitor to the subject; and wherein the PD-1 inhibitor and the TGFβ inhibitor are fused as an anti- PD(L)1:TGFβRII fusion protein and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI.

4. A PD-1 inhibitor for use in a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor to the subject in combination with a TGFβ inhibitor and a VEGF inhibitor.

5. A TGFβ inhibitor for use in a method of treating a cancer in a subject, wherein the method comprises administering the TGFβ inhibitor to the subject in combination with a PD-1 inhibitor and a VEGF inhibitor.

6. A VEGF inhibitor for use in a method of treating a cancer in a subject, wherein the method comprises administering the VEGF inhibitor to the subject in combination with a PD-1 inhibitor and a TGFβ inhibitor.

7. A PD-1 inhibitor and a TGFβ inhibitor for use in a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor and the TGFβ inhibitor to the subject in combination with a VEGF inhibitor; and wherein the PD-1 inhibitor and the TGFβ inhibitor are fused.

8. A method of treating a cancer in a subject, wherein the method comprises administering a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor to the subject.

9. A method of treating a cancer in a subject, wherein the method comprises administering a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor to the subject; and wherein the PD-1 inhibitor is an anti-PD(L)1 antibody, the TGFβ inhibitor is a TGFβRII or anti-TGFβ antibody and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI.

10. A method of treating a cancer in a subject, wherein the method comprises administering a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor to the subject; and wherein the PD-1 inhibitor and the TGFβ inhibitor are fused as an anti- PD(L )1:TGFβRII fusion protein and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI.

11. Use of a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor for the manufacture of a medicament for a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor, the TGFβ inhibitor and the VEGF inhibitor to the subject.

12. Use of a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor for the manufacture of a medicament for a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor, the TGFβ inhibitor and the VEGF inhibitor to the subject; and wherein the PD-1 inhibitor is an anti-PD(L)1 antibody, the TGFβ inhibitor is a TGFβRII or anti-TGFβ antibody and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI.

13. Use of a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor for the manufacture of a medicament for a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor, the TGFβ inhibitor and the VEGF inhibitor to the subject; and wherein the PD-1 inhibitor and the TGFβ inhibitor are fused as an anti- PD(L)1:TGFβRII fusion protein and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI.

14. Use of a PD-1 inhibitor for the manufacture of a medicament for a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor to the subject in combination with a TGFβ inhibitor and a VEGF inhibitor.

15. Use of a TGFβ inhibitor for the manufacture of a medicament for a method of treating a cancer in a subject, wherein the method comprises administering the TGFβ inhibitor to the subject in combination with a PD-1 inhibitor and a VEGF inhibitor.

16. Use of a VEGF inhibitor for the manufacture of a medicament for a method of treating a cancer in a subject, wherein the method comprises administering the VEGF inhibitor to the subject in combination with a PD-1 inhibitor and a TGFβ inhibitor.

17. Use of a PD-1 inhibitor and a TGFβ inhibitor for the manufacture of a medicament for a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor and the TGFβ inhibitor to the subject in combination with a VEGF inhibitor; and wherein the PD-1 inhibitor and the TGFβ inhibitor are fused. 18. The compounds for use, method of treatment or use according to any one of items 1 to 17, wherein the PD-1 inhibitor is capable of inhibiting the interaction between PD-1 and PD-L1.

19. The compounds for use, method of treatment or use according to item 18, wherein the PD-1 inhibitor is an anti-PD(L)1 antibody.

20. The compounds for use, method of treatment or use according to item 19, wherein the PD-1 inhibitor is an anti-PD-L1 antibody.

21. The compounds for use, method of treatment or use according to item 20, wherein the anti-PD-L1 antibody comprises a heavy chain sequence, which comprises a CDRH1 having the sequence of SEQ ID NO: 1, a CDRH2 having the sequence of SEQ ID NO: 2 and a CDRH3 having the sequence of SEQ ID NO: 3, and a light chain sequence, which comprises a CDRL1 having the sequence of SEQ ID NO: 4, a CDRL2 having the sequence of SEQ ID NO: 5 and a CDRL3 having the sequence of SEQ ID NO: 6.

22. The compounds for use, method of treatment or use according to any one of items 1 to

21, wherein the TGFβ inhibitor is capable of inhibiting the interaction between a TGFβ and a TGFβ receptor.

23. The compounds for use, method of treatment or use according to any one of items 1 to

22, wherein the TGFβ inhibitor is a TGFβ receptor or a fragment thereof capable of binding TGFβ.

24. The compounds for use, method of treatment or use according to item 23, wherein the TGFβ receptor is TGFβ receptor II or a fragment thereof capable of binding TGFβ.

25. The compounds for use, method of treatment or use according to item 24, wherein the TGFβ receptor is an extracellular domain of TGFβ receptor II or a fragment thereof capable of binding TGFβ.

26. The compounds for use, method of treatment or use according to any one of items 1 to

25, wherein the TGFβ inhibitor has at least 80%, 90%, 95%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NO: 11 , SEQ ID NO: 12 and SEQ ID NO: 13 and is capable of binding TGFβ.

27. The compounds for use, method of treatment or use according to any one of items 1 to

26, wherein the TGFβ inhibitor has at least 80%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 11 and is capable of binding TGFβ. 28. The compounds for use, method of treatment or use according to any one of items 1 to 25, wherein the TGFβ inhibitor comprises the sequence of any one of SEQ ID NO: 11 , SEQ ID NO: 12 and SEQ ID NO: 13.

29. The compounds for use, method of treatment or use according to item 28, wherein the TGFβ inhibitor comprises the sequence of SEQ ID NO: 11.

30. The compounds for use, method of treatment or use according to any one of items 1 to

29, wherein the PD-1 inhibitor and the TGFβ inhibitor are fused.

31. The compounds for use, method of treatment or use according to any one of items 1 to

30, wherein the PD-1 inhibitor and the TGFβ inhibitor are fused in a molecule comprising (a) an antibody or a fragment thereof capable of binding PD-L1 or PD-1 and inhibiting the interaction between PD-1 and PD-L1 and (b) the extracellular domain of TGFβRII or a fragment thereof capable of binding TGFβ and inhibiting the interaction between TGFβ and a TGFβ receptor.

32. The compounds for use, method of treatment or use according to item 31 , wherein the fusion molecule is one of the respective fusion molecules disclosed in WO 2015/118175 or WO 2018/205985.

33. The compounds for use, method of treatment or use according to item 31 , wherein the extracellular domain of the TGFβRII or the fragment thereof is fused to each of the heavy chain sequences of the antibody or the fragment thereof.

34. The compounds for use, method of treatment or use according to item 33, wherein the fusion between the extracellular domains of TGFβRII or fragments thereof and the heavy chain sequences of the antibody or the fragment thereof occurs via a linker sequence.

35. The compounds for use, method of treatment or use according to item 34, wherein the amino acid sequence of the light chain sequences and the sequences comprising the heavy chain sequence and the extracellular domain of TGFβRII or the fragment thereof respectively correspond to the sequences selected from the group consisting of: (1) SEQ ID NO: 7 and SEQ ID NO: 8, (2) SEQ ID NO: 15 and SEQ ID NO: 17, and (3) SEQ ID NO: 15 and SEQ ID NO: 18.

36. The compounds for use, method of treatment or use according to any one of items 1 to 35, wherein the PD-1 inhibitor and the TGFβ inhibitor are fused and the fusion protein has at least 80%, 90%, 95% or 100% sequence identity to the amino acid sequence of bintrafusp alfa. The compounds for use, method of treatment or use according to any one of items 1 to 35, wherein the PD-1 inhibitor and the TGFβ inhibitor are fused and the fusion protein is bintrafusp alfa. The compounds for use, method of treatment or use according to any one of items 1 to 37, wherein the VEGF inhibitor is capable of inhibiting the interaction between a VEGF ligand and a VEGF receptor. The compounds for use, method of treatment or use according to item 38, wherein the VEGF inhibitor is an anti-VEGF ligand or anti-VEGF receptor antibody. The compounds for use, method of treatment or use according to item 39, wherein the VEGF inhibitor is selected from the group consisting of ranibizumab, bevacizumab, ramucirumab and a molecule having an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 which sequences are disclosed in WO2018/220169. The compounds for use, method of treatment or use according to any one of items 1 to 37, wherein the VEGF inhibitor inhibits signaling of the VEGF pathway. The compounds for use, method of treatment or use according to any one of items 1 to 37, wherein the VEGF inhibitor is a tyrosine kinase inhibitor of a VEGF receptor. The compounds for use, method of treatment or use according to item 42, wherein the tyrosine kinase inhibitor is selected from the group consisting of anlotinib, axitinib, bosutinib, lenvatinib, nintedanib, pazopanib, regorafenib, cediranib, linifanib, motesanib, fruquintinib, sorafenib, sunitinib, ponatinib and cabozantinib. A VEGF inhibitor for use in a method of treating a cancer in a subject, wherein the method comprises administering the VEGF inhibitor to the subject in combination with a PD-1 inhibitor and a TGFβ inhibitor; wherein the PD-1 and the TGFβ inhibitor are fused and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of bintrafusp alfa; and wherein the VEGF inhibitor is an anti-VEGF ligand or anti-VEGF receptor antibody or a TKI. 45. A PD-1 inhibitor and a TGFβ inhibitor for use in a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor and the TGFβ inhibitor to the subject in combination with a VEGF inhibitor; and wherein the PD-1 and the TGFβ inhibitor are fused and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of bintrafusp alfa; and wherein the VEGF inhibitor is an anti-VEGF ligand or anti-VEGF receptor antibody or a TKI.

46. A method of treating a cancer in a subject, wherein the method comprises administering a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor to the subject; wherein the PD-1 and the TGFβ inhibitor are fused and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of bintrafusp alfa; and wherein the VEGF inhibitor is an anti-VEGF ligand or anti-VEGF receptor antibody or a TKI.

47. Use of a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor for the manufacture of a medicament for a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor, the TGFβ inhibitor and the VEGF inhibitor to the subject; wherein the PD-1 and the TGFβ inhibitor are fused and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of bintrafusp alfa; and wherein the VEGF inhibitor is an anti-VEGF ligand or anti-VEGF receptor antibody or a TKI.

48. Use of a VEGF inhibitor for the manufacture of a medicament for a method of treating a cancer in a subject, wherein the method comprises administering the VEGF inhibitor to the subject in combination with a PD-1 inhibitor and a TGFβ inhibitor; wherein the PD-1 and the TGFβ inhibitor are fused and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of bintrafusp alfa; and wherein the VEGF inhibitor is an anti-VEGF ligand or anti-VEGF receptor antibody or a TKI. 49. Use of a PD-1 inhibitor and a TGFβ inhibitor for the manufacture of a medicament for a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor and the TGFβ inhibitor to the subject in combination with a VEGF inhibitor; wherein the PD-1 and the TGFβ inhibitor are fused and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of bintrafusp alfa; and wherein the VEGF inhibitor is an anti-VEGF ligand or anti-VEGF receptor antibody or a TKI.

50. The compounds for use, method of treatment or use according to any one of items 1 to

49, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, leukemia, blastoma, and sarcoma.

51. The compounds for use, method of treatment or use according to any one of items 1 to

50, wherein the cancer is selected from the group consisting of squamous cell carcinoma, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, biliary tract cancer, and head and neck cancer.

52. The compounds for use, method of treatment or use according to any one of items 1 to

51 , wherein the PD-1 inhibitor, TGFβ inhibitor and VEGF inhibitor are administered in a first line treatment of the cancer.

53. The compounds for use, method of treatment or use according to any one of items 1 to 51 , wherein the subject underwent at least one round of prior cancer therapy.

54. The compounds for use, method of treatment or use according item 53, wherein the cancer was resistant or became resistant to prior therapy.

55. The compounds for use, method of treatment or use according to any one of items 1 to 51 , wherein the PD-1 inhibitor, TGFβ inhibitor and VEGF inhibitor are administered in a second line or higher treatment of the cancer. 56. The compounds for use, method of treatment or use according to item 55, wherein the cancer is selected from the group consisting of pre-treated relapsing metastatic NSCLC, unresectable locally advanced NSCLC, pre-treated SCLC ED, SCLC unsuitable for systemic treatment, pre-treated relapsing or metastatic SCCHN, recurrent SCCHN eligible for re-irradiation, and pre-treated microsatellite status instable low (MSI-L) or microsatellite status stable (MSS) metastatic colorectal cancer (mCRC).

57. The compounds for use, method of treatment or use according to any one of items 1 to

56, wherein the PD-L1 inhibitor and the TGFβ inhibitor are fused and administered intravenously.

58. The compounds for use, method of treatment or use according to any one of items 1 to

57, wherein the PD-L1 inhibitor and the TGFβ inhibitor are fused and administered at a dose of about 1200 mg or about 2400 mg.

59. The compounds for use, method of treatment or use according to any one of items 1 to

58, wherein the PD-L1 inhibitor and the TGFβ inhibitor are fused and administered Q2W with a dose of about 1200 mg, or Q3Wwith a dose of about 2400 mg.

60. The compounds for use, method of treatment or use according to any one of items 1 to

59, wherein the VEGF inhibitor is a TKI and administered orally or an anti-VEGF ligand or anti-VEGF receptor antibody and administered intravenously.

61. The compounds for use, method of treatment or use according to any one of items 1 to

60, wherein the VEGF inhibitor is a TKI and administered at a dose of about 50-5000 mg or an anti-VEGF ligand or anti-VEGF receptor antibody and administered at a dose of 1- 100 mg/kg.

62. The compounds for use, method of treatment or use according to any one of items 1 to

61 , wherein the VEGF inhibitor is a TKI and administered once or more per day or an anti-VEGF ligand or anti-VEGF receptor antibody and administered once every 2-6 weeks.

63. The compounds for use, method of treatment or use according to any one of items 1 to

62, wherein the method comprises a lead phase, optionally followed by a maintenance phase after completion of the lead phase.

64. The compounds for use, method of treatment or use according to item 63, wherein the compounds are administered concurrently in either the lead or maintenance phase and optionally non-concurrently in the other phase, or the compounds are administered non- concurrently in the lead and maintenance phase, or two of the compounds are administered concurrently and the others non-concurrently in the lead and maintenance phase. The compounds for use, method of treatment or use according to item 64, wherein the concurrent administration occurs sequentially in either order or substantially simultaneously. The compounds for use, method of treatment or use according to any one of items 63 to

65, wherein the PD-1 inhibitor and TGFβ inhibitor are fused and the maintenance phase comprises administration of the fused PD-1 inhibitor and TGFβ inhibitor alone or concurrently with the VEGF inhibitor. The compounds for use, method of treatment or use according to any one of items 63 to

66, wherein the lead phase comprises the concurrent administration of the PD-1 inhibitor, TGFβ inhibitor and VEGF inhibitor. The compounds for use, method of treatment or use according to any one of items 1 to

67, wherein the cancer is selected based on PD-L1 expression in samples taken from the subject. A pharmaceutical composition comprising a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor and at least a pharmaceutically acceptable excipient or adjuvant. A pharmaceutical composition comprising a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor and at least a pharmaceutically acceptable excipient or adjuvant; wherein the PD-1 inhibitor is an anti-PD(L)1 antibody, the TGFβ inhibitor is a TGFβRII or anti-TGFβ antibody and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI. A pharmaceutical composition comprising a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor and at least a pharmaceutically acceptable excipient or adjuvant; wherein the PD-1 inhibitor and the TGFβ inhibitor are fused as an anti- Rϋ(I_)1:TGFβRII fusion protein and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI. A pharmaceutical composition comprising a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor and at least a pharmaceutically acceptable excipient or adjuvant; wherein the PD-1 inhibitor and the TGFβ inhibitor are fused as an anti- PD(L)1:TGFβRII fusion protein having the amino acid sequence of bintrafusp alfa and the VEGF inhibitor is an anti-VEGF ligand or anti-VEGF receptor antibody or a TKI.

73. The pharmaceutical composition according to any one of items 69 to 72 for use in therapy, e.g., for use in treating cancer.

74. A kit comprising a PD-1 inhibitor and a package insert comprising instructions for using the PD-1 inhibitor in combination with a VEGF inhibitor and a TGFβ inhibitor to treat or delay progression of a cancer in a subject.

75. A kit comprising a VEGF inhibitor and a package insert comprising instructions for using the VEGF inhibitor in combination with a PD-1 inhibitor and a TGFβ inhibitor to treat or delay progression of a cancer in a subject.

76. A kit comprising a TGFβ inhibitor and a package insert comprising instructions for using the TGFβ inhibitor in combination with a PD-1 inhibitor and a VEGF inhibitor to treat or delay progression of a cancer in a subject.

77. A kit comprising a PD-1 inhibitor and a package insert comprising instructions for using the PD-1 inhibitor in combination with a VEGF inhibitor and a TGFβ inhibitor to treat or delay progression of a cancer in a subject; wherein the PD-1 inhibitor is an anti-PD(L)1 antibody, the TGFβ inhibitor is a TGFβRII or anti-TGFβ antibody and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI.

78. A kit comprising a VEGF inhibitor and a package insert comprising instructions for using the VEGF inhibitor in combination with a PD-1 inhibitor and a TGFβ inhibitor to treat or delay progression of a cancer in a subject; wherein the PD-1 inhibitor is an anti-PD(L)1 antibody, the TGFβ inhibitor is a TGFβRII or anti-TGFβ antibody and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI.

79. A kit comprising a TGFβ inhibitor and a package insert comprising instructions for using the TGFβ inhibitor in combination with a PD-1 inhibitor and a VEGF inhibitor to treat or delay progression of a cancer in a subject; wherein the PD-1 inhibitor is an anti-PD(L)1 antibody, the TGFβ inhibitor is a TGFβRII or anti-TGFβ antibody and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI.

80. A kit comprising a PD-1 inhibitor, a TGFβ inhibitor and a package insert comprising instructions for using the PD-1 inhibitor and the TGFβ inhibitor in combination with a VEGF inhibitor to treat or delay progression of a cancer in a subject; wherein the PD-1 inhibitor and the TGFβ inhibitor are fused as an anti- PD(L)1:TGFβRII fusion protein and the VEGF inhibitor is an anti-VEGF ligand or anti- VEGF receptor antibody or a TKI.

81. The kit according to any one of items 74 to 80, wherein the instructions state that the medicaments are intended for use in treating a subject having a cancer that tests positive for PD-L1 expression.

82. A method for advertising a PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor comprising promoting, to a target audience, the use of the combination for treating a subject with a cancer, such as a cancer selected based on PD-L1 expression in samples taken from the subject.

All the references cited herein are incorporated by reference in the disclosure of the invention hereby.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable examples are described below. Within the examples, standard reagents and buffers that are free from contaminating activities (whenever practical) are used. The examples are particularly to be construed such that they are not limited to the explicitly demonstrated combinations of features, but the exemplified features may be unrestrictedly combined again provided that the technical problem of the invention is solved. Similarly, the features of any claim can be combined with the features of one or more other claims. The present invention having been described in summary and in detail, is illustrated and not limited by the following examples.

Examples

Example 1 : Combined treatment with a PD-1 inhibitor, a TGF3 inhibitor and a VEGF inhibitor in a mouse tumor model The combination of different PD-1 , TGFβ and VEGF inhibitors were tested in mouse tumor models.

Results

CT26 colon cancer model

In a subcutaneous CT26 colon cancer model, tumor growth inhibition (TGI) was induced by bintrafusp alfa (19.7%, day 20), Axitinib (30.8%, day 20), and Nintedanib (20.6%, day 20) monotherapies relative to isotype control (Figure 3). However, bintrafusp alfa and Axitinib combination therapy further induced TGI (73.0%, day 20) relative to bintrafusp alfa (p < 0.0001, day 10) or Axitinib (p < 0.0001, day 20), and bintrafusp alfa and Nintedanib combination therapy further induced TGI (57.9%, day 20) relative to bintrafusp alfa (p < 0.0001 , day 20) or Nintedanib (p < 0.0001 , day 20) (Figure 3).

In a subcutaneous CT26 colon cancer model, tumor growth inhibition (TGI) was induced by bintrafusp alfa (22.8%, day 13), Lenvatinib (39.6%, day 13), and Regorafenib (78.8%, day 13) monotherapies relative to isotype control (Figure 4). However, bintrafusp alfa and Lenvatinib combination therapy further induced TGI (51.3%, day 13) and decreased average tumor volumes relative to bintrafusp alfa (p < 0.0001, day 17) or Lenvatinib (p = 0.0013, day 17) and bintrafusp alfa and Regorafenib combination therapy further induced TGI (87.0%, day 13) and decreased average tumor volumes relative to bintrafusp alfa (p < 0.0001, day 17) or Regorafenib (p < 0.0001, day 21) (Figure 4).

4T1-Luc breast cancer model

In an orthotopic 4T1-Luc breast cancer model, tumor growth inhibition (TGI) was induced by bintrafusp alfa (36.2%, day 18), Lenvatinib (54.1%, day 18), and Regorafenib (57.3%, day 18) monotherapies relative to isotype control (Figure 5). However, bintrafusp alfa and Lenvatinib combination therapy further induced TGI (69.9%, day 18) relative to bintrafusp alfa (p < 0.0001, day 18) or Lenvatinib (p = 0.0139, day 18) and bintrafusp alfa and Regorafenib combination therapy further induced TGI (78.5%, day 18) relative to bintrafusp alfa (p < 0.0001, day 18) or Regorafenib (p = 0.0002, day 18) (Figure 5).

Renal carcinoma model

In an orthotopic renal carcinoma (RENCA) model, the combination of bintrafusp alfa and pazopanib decreased tumor weight more effectively than pazopanib monotherapy (p = 0.0145) and trended toward a significant decrease relative to bintrafusp alfa monotherapy (p = 0.0786) (Figure 6). 4T 1 breast cancer model

In an orthotopic 4T1 breast cancer model, tumor growth inhibition (TGI) was induced by both bintrafusp alfa (38.0%, p < 0.0001 ; day 20) and anti-VEGF-A (52.7%, p < 0.0001; day 20) monotherapies relative to isotype control (Figure 7). However, bintrafusp alfa and anti-VEGF-A combination therapy further induced TGI (68.0%) relative to bintrafusp alfa (p < 0.0001; day 20) or anti-VEGF-A (p < 0.0001; day 20) and significantly prolonged median survival (34 days) relative to isotype control (20 days; p < 0.0001), bintrafusp alfa monotherapy (23 days; p < 0.0001), or anti-VEGF-A monotherapy (27 days; p = 0.0002) (Figure 7).

Materials and Methods

Cell Lines

CT26, RENCA, and 4T1 cells were obtained from American Type Culture Collection (ATCC). 4T1-Luc2-1A4 (4T1-Luc) cells were obtained from PerkinElmer. CT26, 4T1 , and 4T1-Luc cells were cultured in RPMI Medium 1640 containing 2nM L-glutamine, 10mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate, and supplemented with 10% fetal bovine serum (FBS). CT26 and 4T1-Luc cells were cultured with the addition of 1% penicillin/streptomycin/L-glutamine. RENCA cells were cultured in Dulbecco’s Modified Eagle’s medium (DM EM) supplemented with 10% FBS.

Cells were cultured under aseptic conditions and incubated at 37°C with 5% CO2. Cells were passaged before in vivo implantation and adherent cells were harvested with TrypLE Express.

Mice

BALB/c mice were obtained from Shanghai Lingchang Biological Technology Co.,

Ltd, Charles River Laboratories, or Envigo, and Jh (lgh-J^tm1Dhu) BALB/c mice were obtained from Taconic. All mice used for experiments were 6-12-week old females. Mice were housed with ad libitum access to food and water in pathogen-free facilities.

Murine Tumor Models

CT26 tumor model

For the Axitinib and Nintedanib efficacy study, 0.3 x 10⁶ CT26 cells were inoculated subcutaneously in the flank of BALB/c mice on day -9. Treatment was initiated on day 0 when average tumor volumes reached ~75 mm³. Mice were sacrificed when tumor volumes reached ≃2000 mm³.

For the Lenvatinib and Regorafenib efficacy study, 1 x 10⁶ CT26 cells were inoculated subcutaneously in the flank of BALB/c mice on day -11. Treatment was initiated on day 0 when average tumor volumes reached -100 mm³. Mice were sacrificed when tumor volumes reached «2000 mm³.

4T1-Luc tumor model

For the efficacy study, 0.5 x 10⁶4T1-Luc cells were inoculated orthotopically in the mammary fat pad of BALB/c mice on day -10. Treatment was initiated on day 0 when average tumor volumes reached -100 mm³. Mice were sacrificed when tumor volumes reached «2000 mm³. Procedures were performed at Covance.

4T1 tumor model

For the efficacy study, 5 x 10⁴4T1 cells were inoculated orthotopically in the right abdominal mammary fat pad of Jh mice on day -6. Treatment was initiated on day 0 when average tumor volumes reached -50 mm³. For survival analysis, mice were sacrificed when tumor volumes reached «2000 mm³ and median survival times were calculated.

RENCA tumor model

For the efficacy study, 0.8 x 10⁵ RENCA cells were inoculated orthotopically in the left renal capsule of Jh mice on day -6. Treatment was initiated on day 0 and mice were sacrificed on day 11.

Treatment

Mice were randomized into treatment groups according to tumor volume on the day of treatment initiation (day 0). Exact dose and treatment schedules for each experiment are listed in the brief description of the drawings.

In tumor-bearing mice, bintrafusp alfa (492 μg) or isotype control (400 μg), which is a mutated version of anti-PD-L1 that completely lacks PD-L1 binding, were administered with an intravenous (i.v.) injection in 0.2 mL PBS.

Axitinib was administered per oral (p.o.) at 30 mg/kg, twice daily for 14 days. Nintedanib was administered p.o. at 50 mg/kg, once daily for 14 days. The Axitinib vehicle (0.5% hydroxy-propyl-methylcellulose / 0.1% Tween-80 in sterile water) and the Nintedanib vehicle (0.5% Natrosol in sterile water) were also administered p.o. (10 mI/g) as a control in the isotype control and bintrafusp alfa monotherapy treatment groups. Lenvatinib was administered p.o. at 30 mg/kg, once daily for 14 or 21 days. Regorafenib was administered per oral (p.o.) at 10 mg/kg, once daily for 14 or 21 days. The Lenvatinib vehicle (0.5% methylcellulose in sterile water) and the Regorafenib vehicle (2% DMSO, 30% Peg300, 5% Tween80, and 43% sterile water) were also administered p.o. (200 mI) as a control in the isotype control and bintrafusp alfa monotherapy treatment groups.

Pazopanib was administered p.o. at 30 mg/kg, two times per day.

Anti-VEGF-A (B20) is an lgG2a antibody that neutralizes both human and murine vascular endothelial growth factor (VEGF-A). In tumor-bearing mice, anti-VEGF-A or a control Rat lgG2a antibody were administered with intraperitoneal (i.p.) injection in 0.2 mL PBS.

Tumor Growth and Survival

Tumor sizes were measured twice per week with digital calipers and recorded automatically using WinWedge software. Tumor volumes were calculated with the following formula: tumor volume (mm³) = tumor length x width x height x 0.5236. Tumor growth inhibition (TGI) was calculated with the following formula: TGI (%) = [1 - (Ti-T0)/(Vi-V0)] x 100, where Ti is the average tumor volume (mm³) of a treatment group on a given day, TO is the average tumor volume of the treatment group on the first day of treatment, Vi is the average tumor volume of the vehicle control group on the same day as Ti, and V0 is the average tumor volume of the vehicle control group on the first day of treatment. To compare the percentage survival between different treatment groups, Kaplan-Meier survival curves were generated. Body weight was measured twice weekly and mice were sacrificed when their tumor volume exceeded ~2,000 mm³.

Net tumor weight in the inoculated left kidney was measured by calculating the difference between the right and left kidneys.

Statistical analyses

Statistical analyses were performed using GraphPad Prism Software, version 8.0.1. Tumor volume data are presented graphically as mean ± standard error of the mean (SEM) by symbols or as individual mice by lines. To assess differences in tumor volumes between treatment groups, a two-way analysis of variance (ANOVA) was performed followed by Tukey’s multiple comparison test. A Kaplan-Meier plot was generated to show survival by treatment group and significance was assessed by log-rank (Mantel-Cox) test.

Net tumor weight data are presented graphically as mean ± SEM by bar graphs and error bars. To assess differences in tumor weights between treatment groups, unpaired t tests with Welch’s correction were performed.

Example 2: Combined treatment with a PD-1 inhibitor, a TGF3 inhibitor and a VEGF inhibitor of MC38 tumor-bearing mice

Flow cytometry analysis was performed to further evaluate MC38 tumor-bearing mice treated with PD-1, TGFβ and VEGF inhibitors.

Results

In MC38 tumor-bearing mice, we observed decreased tumor volumes with bintrafusp alfa + anti-VEGF-A and bintrafusp alfa + Nintedanib combination therapies relative to isotype control and monotherapies.

Bintrafusp alfa and anti-VEGF-A combination therapy significantly increased the absolute number of CD3⁺, CD4⁺, and CD8⁺ tumor-infiltrating lymphocytes (TILs) relative to bintrafusp alfa or anti-VEGF-A monotherapies. Bintrafusp alfa and Ninedanib combination therapy also increased CD3⁺ and CD4⁺ TILs relative to bintrafusp alfa or Ninedanib monotherapies (Figure 8). Proliferating CD4⁺ TILs were significantly increased with bintrafusp alfa + anti-VEGF-A combination therapy relative to anti-VEGF-A monotherapy and with bintrafusp alfa + Ninedanib combination therapy relative to Nintedanib monotherapy, and CD4⁺/PD-1⁺ TILs were significantly increased with both combination therapies relative to respective monotherapies. Bintrafusp alfa + anti-VEGF-A combination therapy significantly increased proliferating CD8⁺ TILs relative to anti-VEGF-A monotherapy and CD8⁺/PD-1⁺ and CD8VP15E Pentamer⁺ TILs relative to bintrafusp alfa or anti-VEGF-A monotherapy. Proliferating CD8VP15E Pentamer⁺ TILs showed a trend toward increasing with bintrafusp alfa + anti-VEGF-A combination therapy relative to monotherapies (Figure 8).

Bintrafusp alfa + anti-VEGF-A combination therapy significantly increased the absolute number of CD4⁺ and CD8⁺ effector memory (EM) TILS relative to bintrafusp alfa or anti-VEGF-A monotherapy, and bintrafusp alfa + Nintedanib combination therapy significantly increased CD4⁺EM TILS relative to bintrafusp alfa or Nintedanib monotherapy (Figure 9). The absolute number of CD4⁺ central memory (CM) TILS were significantly increased with bintrafusp alfa + anti-VEGF-A combination therapy relative to anti-VEGF-A monotherapy, and with bintrafusp alfa + Nintedanib combination therapy relative to bintrafusp alfa or Nintedanib monotherapy. Bintrafusp alfa + Nintedanib combination therapy also significantly increased CD8⁺CM TILS relative to bintrafusp alfa monotherapy (Figure 9).

The number of NK cells trended toward increasing with both combination therapies relative to monotherapies. However, bintrafusp alfa + Nintedanib combination therapy significantly increased proliferating NK (NK1.17Ki-67⁺) cells relative to Nintedanib monotherapy, and increased PD-17Ki-67⁺ cells relative to bintrafusp alfa or Nintedanib monotherapy. Bintrafusp alfa + anti-VEGF-A combination therapy also increased PD-17Ki- 67⁺ cells relative to bintrafusp alfa or anti-VEGF-A monotherapies (Figure 10).

Materials and Methods

Cell Lines

The MC38 murine colon carcinoma cell line was a gift from the Scripps Research Institute. MC38 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Cells were cultured under aseptic conditions and incubated at 37°C with 5% C02. Cells were passaged before in vivo implantation and adherent cells were harvested with TrypLE Express.

Mice

C57BL/6 mice were obtained from Charles River Laboratories. All mice used for experiments were 7-8-week-old females. Mice were housed with ad libitum access to food and water in pathogen-free facilities. All procedures were performed in accordance with institutional protocols approved by the Institutional Animal Care and Use Committees (IACUC).

MC38 Murine Tumor Model

For the flow cytometry study, 1 x 10⁶ MC38 cells were inoculated subcutaneously in the flank of C57BL/6 mice on Day -13. T reatment was initiated on day 0 when average tumor volumes reached -100 mm³. MC38 tumor-bearing C57BL/6 mice (8 mice/group) were treated with: isotype control (400 μg i.v.; days 0, 3, 6), bintrafusp alfa (492 μg; i.v. days 0, 3, 6), anti-VEGF-A (B20) (100 μg i.v.; days 0, 3), Nintedanib (50 mg/kg; p.o.; QDx8), bintrafusp alfa + anti-VEGF-A, or bintrafusp alfa + Nintedanib. Mice were sacrificed and tumors were harvested on day 9. Flow Cytometry Analysis

Immune profiling by flow cytometry of tumors were performed. Single cell suspensions of tumors were obtained by mechanical disruption of tissue by cutting tumors into 1-2 mm pieces with a blade and gently crushing the tumor pieces with two frosted glass slides, followed by filtration through a 70-μm nylon cell strainer. Cells were resuspended in serum-containing media. For tumor cell analyses, trypan blue was used to distinguish viable cells prior to staining, and 1 ^c 10⁶ cells were used for flow cytometry analysis. Antibody staining of tumor cell suspensions for flow cytometry analysis was performed following the antibody manufacturer’s recommendations.

Fluorophore-conjugated antibodies to Ki-67 (MOPC-21), Gr-1 (RB6-8C5), and l-Ab (AF6- 120.1) were purchased from BD Biosciences, and Pentamer p15E (H-2Kb and KSPWFTTL) was purchased from Prolmmune. Antibodies to CD3 (17A2), CD8a (53-6.7), CD4 (RM4-5), CD25(PC61), NK1.1 (PK136), CD11b (M1/70), CD11c (N418), Ly-6G (1A8), Ly-6C (HK1.4), F4/80 (BM8), CD44 (IM7), CD80 (16-10A1), CD86 (GL-1), CD279 (PD- 1)(29F.1A12) and CD45 (30-F11) were purchased from Biolegend. Viability Dye eFluor 455 (UV) and antibodies against CD62L (MEL-14), FoxP3(FJk-16s) were purchased from eBiosciences.

Cells were blocked with anti-CD16/CD32 (Purified Anti-Mu CD16/CD32 (Fcglll/ll Receptor)(2.4G2), BD Biosciences), incubated with surface marker antibodies for 30 minutes on ice, stained with fixable viability dye for 30 min at 4°C, and then permeabilized with BD Cytofix/Cytoperm buffer (BD Fixation and Permeabilization Solution, BD Bioscience) before intracellular labeling antibodies were added for an overnight incubation at 4°C. Cells were suspended in FACS staining wash buffer (2% FBS-PBS , FBS, Corning Cellgro, PBS, gibco) and were analyzed on a BD LSR II flow cytometer according to the manufacturer’s instructions. Flow cytometry analysis was performed using BD FACSDiva V8 software. Cellular events were first gated by forward and side scatter characteristics and then by viability. Tumor cells were gated on immune cell subpopulations. For quantification, the proportion of each immune subpopulation (relative to CD45+ cells) was determined by flow cytometry. This proportion was then multiplied by the total number of leukocytes in the tumor, which had been determined before flow cytometry by counting with a hemocytometer (distinguished from tumor cells by cell size and morphology). To determine cell density, cell counts were normalized to tumor weight (cell number per 100 mg tumor).

Statistical analysis Statistical analyses were performed using GraphPad Prism Software, version 8.0.1. To assess differences between treatment groups in the flow cytometry plots, unpaired t-tests with Welch’s correction were performed. To assess differences between treatment groups in the tumor volume plot, a 2-way ANOVA was performed with Tukey’s multiple comparison test. * p ≤ 0.05, ** p ≤ 0.01 , *** p ≤ 0.001 , and **** p ≤ 0.0001 denote significant differences between treatment groups.

SEQUENCE LISTINGS

Claims

2. The compounds for use according to claim 1, wherein the PD-1 inhibitor is an anti- PD(L)1 antibody, or a fragment thereof capable of binding PD-L1 or PD-1 , the TGFβ inhibitor is a TGFβRII, or a fragment thereof capable of binding TGF-β, or an anti-TGFβ antibody, or a fragment thereof capable of binding TGFβ, and the VEGF inhibitor is an anti-VEGF ligand antibody, or a fragment thereof capable of binding VEGF, or an anti- VEGF receptor antibody, or a fragment thereof capable of binding VEGF receptor, or a tyrosine kinase inhibitor.

3. The compounds for use according to claim 1 or 2, wherein the PD-1 inhibitor is an anti- PD-L1 antibody, or fragment thereof capable of binding PD-L1, and comprises a heavy chain sequence, which comprises a CDRH1 having the sequence of SEQ ID NO: 1, a CDRH2 having the sequence of SEQ ID NO: 2 and a CDRH3 having the sequence of SEQ ID NO: 3, and a light chain sequence, which comprises a CDRL1 having the sequence of SEQ ID NO: 4, a CDRL2 having the sequence of SEQ ID NO: 5 and a CDRL3 having the sequence of SEQ ID NO: 6; or wherein the PD-1 inhibitor is an anti-PD-L1 antibody, or fragment thereof capable of binding PD-L1, and comprises a heavy chain sequence, which comprises a CDRH1 having the sequence of SEQ ID NO: 19, a CDRH2 having the sequence of SEQ ID NO: 20 and a CDRH3 having the sequence of SEQ ID NO: 21 , and a light chain sequence, which comprises a CDRL1 having the sequence of SEQ ID NO: 22, a CDRL2 having the sequence of SEQ ID NO: 23 and a CDRL3 having the sequence of SEQ ID NO: 24.

4. The compounds for use according to any one of claims 1 to 3, wherein the TGFβ inhibitor is an extracellular domain of TGFβRII or a fragment thereof capable of binding TGFβ.

5. The compounds for use according to any one of claims 1 to 4, wherein the PD-1 inhibitor and the TGFβ inhibitor are fused as an anti-PD(L)1:TGFβRII fusion protein.

6. The compounds for use according to claim 5, wherein the light chain sequences and the heavy chain sequences of the anti-PD(L)1:TGFβRII fusion protein have at least 90% sequence identity to the light chain sequence and the heavy chain sequence selected from the group consisting of: (1) SEQ ID NO: 7 and SEQ ID NO: 8, (2) SEQ ID NO: 15 and SEQ ID NO: 17, and (3) SEQ ID NO: 15 and SEQ ID NO: 18.

7. The compounds for use according to claim 5, wherein the amino acid sequence of the anti-PD(L)1:TGFβRII fusion protein corresponds to the amino acid sequence of bintrafusp alfa.

8. The compounds for use according to any one of claims 5 to 7, wherein the anti- PD(L)1:TGFβRII fusion protein is administered at a dose of 1200 mg Q2Wor at a dose of 2400 mg Q3W.

9. The compounds for use according to any one of claims 1 to 8, wherein the VEGF inhibitor is an anti-VEGF ligand antibody, or a fragment thereof capable of binding VEGF, or an anti-VEGF receptor antibody, or a fragment thereof capable of binding VEGF receptor.

10. The compounds for use according to claim 9, wherein the VEGF inhibitor is selected from the group consisting of bevacizumab, ranibizumab, ramucirumab and a molecule having an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 which sequences are disclosed in WO2018/220169.

11. The compounds for use according to claim 9 or 10, wherein the VEGF inhibitor is administered Q2W or Q3W at a dose of 5-100 mg/kg.

12. The compounds for use according to any one of claims 1 to 8, wherein the VEGF inhibitor is a tyrosine kinase inhibitor.

13. The compounds for use according to claim 12, wherein the tyrosine kinase inhibitor is nintedanib.

14. The compounds for use according to claim 12 or 13, wherein the tyrosine kinase inhibitor is administered once or more per day at a dose of 50-5000 mg.

15. A PD-1 inhibitor, a TGFβ inhibitor and a VEGF inhibitor for use in a method of treating a cancer in a subject, wherein the method comprises administering the PD-1 inhibitor, the TGFβ inhibitor and the VEGF inhibitor to the subject; and wherein the PD-1 inhibitor and TGFβ inhibitor are fused in a molecule having the amino acid sequence of bintrafusp alfa and the VEGF inhibitor is an anti-VEGF ligand antibody, or a fragment thereof capable of binding VEGF, or an anti-VEGF receptor antibody, or a fragment thereof capable of binding VEGF receptor, or a TKI.