Combination inhibition of PD-1, TGFβ, and TIGIT for cancer treatment

Abstract

This invention relates to combination therapies useful for treating cancer. [Solution] In particular, the present invention relates to the use of a combination of a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor for the treatment of cancer.

Description

[Technical Field]

[0001] This invention relates to the treatment of cancer and combinations useful in such treatment. In particular, this invention relates to combinations of compounds for inhibiting PD-1, TGFβ, and TIGIT for use in the treatment of cancer. [Background technology]

[0002] Effective treatment of hyperproliferative disorders (including cancer) is an ongoing goal in the field of oncology. Generally, cancer arises from dysregulation of normal processes that control cell division, differentiation, and apoptosis (programmed cell death), and is characterized by the proliferation of malignant cells with the potential for uncontrolled growth, local development, and systemic metastasis. Dysregulation of normal processes includes abnormalities in signaling pathways and responses to factors different from those seen in normal cells.

[0003] Immunotherapy is one approach to treating hyperproliferative disorders. One major hurdle that researchers and clinicians have encountered in developing various types of cancer immunotherapies has been disrupting tolerance to autoantigens (cancer) and inducing a robust antitumor response that leads to tumor regression. Unlike the traditional development of small and large molecule drugs that target tumors, cancer immunotherapy can target immune system target cells, in particular, which have the potential to generate a memory pool of effector cells, inducing more sustained effects and minimizing recurrence.

[0004] While there have been many advances in cancer treatment recently, there is still a need for more effective and / or enhanced treatments for individuals suffering from the effects of cancer. The methods described herein concerning the enhancement of anti-tumor immunity by combining multiple therapeutic approaches address this need. [Overview of the project]

[0005] This invention arises from the discovery that therapeutic effects in cancer treatment can be achieved by combining compounds that inhibit PD-1, TGFβ, and TIGIT.

[0006] Accordingly, in a first aspect, the Disclosure provides PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors for use in methods of treating cancer in a subject, for use in inhibiting the growth or progression of tumors in a subject having a malignant tumor, for use in inhibiting the metastasis of malignant cells in a subject, for use in reducing the risk of metastasis development and / or metastatic growth in a subject, and for use in inducing tumor regression in a subject having malignant cells, wherein such use includes administering the compound to the subject.

[0007] This disclosure also provides PD-1 inhibitors, TGFβ inhibitors and TIGIT inhibitors for the manufacture of pharmaceuticals for use in methods of treating cancer in subjects, for use in inhibiting tumor growth or progression in subjects having malignant tumors, for use in inhibiting metastasis of malignant cells in subjects, for use in reducing the risk of metastasis development and / or metastatic growth in subjects, or for use in inducing tumor regression in subjects having malignant cells, wherein such use includes administering the compounds to subjects.

[0008] In another embodiment, the Disclosure provides a method for treating cancer in a subject, a method for inhibiting the growth or progression of a tumor in a subject having a malignant tumor, a method for inhibiting the metastasis of malignant cells in a subject, a method for reducing the risk of metastasis development and / or metastatic growth in a subject, or a method for inducing tumor regression in a subject having malignant cells, wherein the method comprises administering a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor to the subject.

[0009] In a further embodiment, the disclosure also relates to a method for promoting a therapy comprising a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor, which includes promoting to a target audience the use of the combination thereof for treating a subject having cancer based, for example, on the expression of PD-L1 in a sample taken from the subject, e.g., a tumor sample. PD-L1 expression can be determined, for example, by an immunohistochemical method using one or more primary anti-PD-L1 antibodies.

[0010] This specification also provides pharmaceutical compositions comprising a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor, and at least a pharmaceutically acceptable excipient or adjuvant. In one embodiment, the PD-1 inhibitor and the TGFβ inhibitor are fused in such a pharmaceutical composition. The PD-1 inhibitor, the TGFβ inhibitor, and the TIGIT inhibitor are provided as single or separate unit dosage forms.

[0011] In a further embodiment, the present invention relates to a kit comprising a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor, and a package insert containing instructions for using these compounds to treat or delay the progression of cancer in a subject. In a further embodiment, the present invention relates to a kit comprising a PD-1 inhibitor and a package insert containing instructions for using the PD-1 inhibitor, TIGIT inhibitor, and TGFβ inhibitor to treat or delay the progression of cancer in a subject. In a further embodiment, the present invention relates to a kit comprising a TGFβ inhibitor and a package insert containing instructions for using the TGFβ inhibitor, PD-1 inhibitor, and TIGIT inhibitor to treat or delay the progression of cancer in a subject. In a further embodiment, the present invention relates to a kit comprising a TIGIT inhibitor and a package insert containing instructions for using the TIGIT inhibitor, PD-1 inhibitor, and TGFβ inhibitor to treat or delay the progression of cancer in a subject. In a further embodiment, the present invention relates to an anti-PD-L1:TGFβRII fusion protein and a kit comprising a package insert containing instructions for using the anti-PD-L1:TGFβRII fusion protein and a TIGIT inhibitor to treat or delay the progression of cancer in a subject. The compounds of the kit may be contained in one or more containers. The instructions may state that these pharmaceuticals are intended for use in the treatment of a subject having cancer that is tested to be positive for PD-L1 expression by an immunohistochemical (IHC) assay.

[0012] In one embodiment, a PD-1 inhibitor and a TGFβ inhibitor are fused. 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 vintrafusp alfa. [Brief explanation of the drawing]

[0013] [Figure 1]Figure 1 shows the amino acid sequence of bintrafusp alpha. (A) Sequence ID 8 represents the heavy chain sequence of bintrafusp alpha. CDRs with amino acid sequences of Sequence IDs 1, 2, and 3 are underlined. (B) Sequence ID 7 represents the light chain sequence of bintrafusp alpha. CDRs with amino acid sequences of Sequence IDs 4, 5, and 6 are underlined.

[0014] [Figure 2] Figure 2 shows a typical structure of an anti-PD-L1:TGFβRII fusion protein.

[0015] [Figure 3]Figure 3: (A - B) Activation of immune cells was evaluated by measuring IFN-γ in the supernatants of co-cultured PBMCs from two different human donors 2 days after treatment with anti-TIGIT antibody H03-12 with or without vintafusp alpha in an allogeneic two-way MLR assay. (A) Co-cultured cells were treated with serial dilutions of H03-12 or an inert anti-PD-L1 isotype control (the sequences of the light and heavy chains of the inert anti-PD-L1 isotype control are reflected in SEQ ID NO: 41 and SEQ ID NO: 42, respectively). Results from 7 assays with 7 different donor pairs were pooled and plotted as fold change relative to a 1 ng / mL isotype control (set to 1). (B) Co-cultured cells were treated with serial dilutions of vintafusp alpha combined with 5 μg / mL of an inert anti-PD-L1 isotype control or H-0312. Results from 6 assays were pooled and plotted as fold change relative to a 1 ng / mL inert anti-PD-L1 isotype control and vintafusp alpha (set to 1). Non-linear regression analysis was performed and mean ± SEM is shown. Data were analyzed using two-way ANOVA. (C - D) Activation of T cells was evaluated by measuring IFN-γ in the supernatants of co-cultured irradiated MDA-MB-231 cells and human T cells 2 days after treatment with H03-12 in an allogeneic one-way MLR assay. (C) Co-cultured cells were treated with serial dilutions of H03-12 or an inert anti-PD-L1 isotype control. H03-12 increased allo-antigen specific T cell activation in a dose-dependent manner with an EC50 of 136.9 ± 114.6 ng / mL (0.917 ± 0.768 nM). Results from 2 assays were pooled and plotted as fold change relative to a 1 ng / mL isotype control (set to 1). (D) Co-cultured cells were treated with serial dilutions of vintafusp alpha combined with 5 μg / mL of an inert anti-PD-L1 isotype control or H03-12. Non-linear regression analysis was performed and mean ± SEM is shown. Data were analyzed using an unpaired Student's t-test.

[0016] [Figure 4-1] Figure 4: (A) Female Balb / c mice were inoculated with 1 × 10⁶ CTA-KSA tumor cells in the right flank and treated with an inactive anti-PD-L1 isotype control (20 mg / Kg iv, days 0, 3, 6) or vintrafusp alfa (24.6 mg / Kg iv, days 0, 2, 4) when the mean tumor volume reached approximately 200 mm³. The mean tumor volume was measured by SEM. (B) Female Balb / c mice were inoculated with 2 × 10⁶ CTA-KSA tumor cells in the right flank and treated with an inactive anti-PD-L1 isotype control (400 μg iv, days 0, 3, 6) or vintrafusp alfa (24.6 mg / Kg, days 0, 3, 6) when the mean tumor volume reached approximately 400 mm³. TIGIT expression in CD4+ T cells, CD8+ T cells, NK cells, and Treg cells from the spleen and tumors was analyzed by flow cytometry. P-values ​​for the effect graphs were calculated using two-way ANOVA with Bonferroni's post-hoc test, and P-values ​​for the flow cytometry data were calculated using Student's t-test (where **P<0.01, ***P<0.001, and ****P<0.0001). [Figure 4-2]Figure 4: (A) 1 × 106 CTA-KSA tumor cells were inoculated into the right flank of female Balb / c mice. When the average tumor volume reached approximately 200 mm3, they were treated with an inert anti-PD-L1 isotype control (20 mg / Kg iv, on days 0, 3, 6) or vintafusp alpha (24.6 mg / Kg iv, on days 0, 2, 4). The average tumor volume was measured by SEM. (B) 2 × 106 CTA-KSA tumor cells were inoculated into the right flank of female Balb / c mice. When the average tumor volume reached approximately 400 mm3, they were treated with an inert anti-PD-L1 isotype control (400 μg iv, on days 0, 3, 6) or vintafusp alpha (24.6 mg / Kg, on days 0, 3, 6). The expression of TIGIT in CD4+ T cells, CD8+ T cells, NK cells, and Tregs in the spleen and tumor was analyzed by flow cytometry. The P value for the graph of the effect was calculated by two-way ANOVA using Bonferroni's post hoc test analysis, and the P value for the flow cytometry data was calculated by Student's t-test (where **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0017] [Figure 5] Figure 5: 1 × 106 CT26-KSA tumor cells were inoculated into the right flank of female BALB / c mice. When the average tumor volume reached approximately 250 mm3, they were treated with anti-muTIGIT antibody 18G10 (0.2 mg / kg ip, on days 0, 7, 14), vintafusp alpha (24.6 mg / kg iv, on days 0, 2, 4), or 18G10 + vintafusp alpha. Anti-HEL-muIgG2a isotype control (0.2 mg / kg ip, on days 0, 7, 14) and an inert anti-PD-L1 isotype control (20 mg / kg iv, on days 0, 2, 4) were used as isotype controls (the light and heavy chain sequences of anti-HEL-muIgG2a are reflected in SEQ ID NO: 45 and SEQ ID NO: 46, respectively). (A) Average tumor volume and SEM; (B) Survival rate; (C) Individual tumor volume. The P value was calculated by two-way ANOVA using Tukey's post hoc test analysis (where *P < 0.05, ***P < 0.001, ****P < 0.0001).

[0018] [Figure 6] Figure 6: Female C57BL / 6 mice were inoculated with 1 × 10⁶ MC38 tumor cells in the right flank and treated with anti-muTIGIT antibody 18G10 (5 mg / kg ip, days 0, 7, 14), vintrafusp alfa (24.6 mg / kg iv, days 0, 2, 4), or 18G10 + vintrafusp alfa when the mean tumor volume reached approximately 50 mm³. Anti-HEL-muIgG2a isotype control (5 mg / kg ip, days 0, 7, 14) and inactive anti-PD-L1 isotype control (20 mg / kg iv, days 0, 2, 4) were used as isotype controls. (A) Mean tumor volume and SEM; (B) Survival rate; (C) Individual tumor volume. P values ​​were calculated by two-way ANOVA with Tukey's post-hoc test (where *P<0.05, ****P<0.0001).

[0019] [Figure 7-1] Figure 7: Female B-huTIGIT knock-in mice were inoculated with 1 × 10⁶ MC38 cells in the right flank and treated with H03-12-muIgG2c (25 mg / kg ip, days 0, 7, 14), trap control (24.6 mg / kg iv, days 0, 2, 4), anti-PD-L1 (20 mg / kg iv, days 0, 2, 4), or vintrafusp alfa (24.6 mg / kg iv, days 0, 2, 4), or a combination thereof, when the mean tumor volume reached approximately 50–100 mm³. Anti-HEL-muIgG2c (25 mg / kg ip, days 0, 7, 14) and inactive anti-PD-L1 (20 mg / kg iv, days 0, 2, 4) were used as isotype controls (the light and heavy chain sequences of anti-HEL-muIgG2c are reflected in SEQ ID NOs. 43 and 44, respectively). (A) Mean tumor volume and SEM; (B) Survival rate; (C) Individual tumor volume. Tukey's multiple comparison post-hoc test analysis was performed following a two-way ANOVA (however, no significant difference was found for ns, **P<0.01, ***P<0.001, ****P<0.0001). [Figure 7-2]Figure 7: Female B-huTIGIT knock-in mice were inoculated with 1 × 10⁶ MC38 cells in the right flank and treated with H03-12-muIgG2c (25 mg / kg ip, days 0, 7, 14), trap control (24.6 mg / kg iv, days 0, 2, 4), anti-PD-L1 (20 mg / kg iv, days 0, 2, 4), or vintrafusp alfa (24.6 mg / kg iv, days 0, 2, 4), or a combination thereof, when the mean tumor volume reached approximately 50–100 mm³. Anti-HEL-muIgG2c (25 mg / kg ip, days 0, 7, 14) and inactive anti-PD-L1 (20 mg / kg iv, days 0, 2, 4) were used as isotype controls (the light and heavy chain sequences of anti-HEL-muIgG2c are reflected in SEQ ID NOs. 43 and 44, respectively). (A) Mean tumor volume and SEM; (B) Survival rate; (C) Individual tumor volume. Tukey's multiple comparison post-hoc test analysis was performed following a two-way ANOVA (however, no significant difference was found for ns, **P<0.01, ***P<0.001, ****P<0.0001). [Figure 7-3] Figure 7: Female B-huTIGIT knock-in mice were inoculated with 1 × 10⁶ MC38 cells in the right flank and treated with H03-12-muIgG2c (25 mg / kg ip, days 0, 7, 14), trap control (24.6 mg / kg iv, days 0, 2, 4), anti-PD-L1 (20 mg / kg iv, days 0, 2, 4), or vintrafusp alfa (24.6 mg / kg iv, days 0, 2, 4), or a combination thereof, when the mean tumor volume reached approximately 50–100 mm³. Anti-HEL-muIgG2c (25 mg / kg ip, days 0, 7, 14) and inactive anti-PD-L1 (20 mg / kg iv, days 0, 2, 4) were used as isotype controls (the light and heavy chain sequences of anti-HEL-muIgG2c are reflected in SEQ ID NOs. 43 and 44, respectively). (A) Mean tumor volume and SEM; (B) Survival rate; (C) Individual tumor volume. Tukey's multiple comparison post-hoc test analysis was performed following a two-way ANOVA (however, no significant difference was found for ns, **P<0.01, ***P<0.001, ****P<0.0001).

[0020] [Figure 8-1] Figure 8: Female B-huTIGIT knock-in mice were sc-inoculated with 3 × 10⁵ MC38 cells in the flank. When the mean tumor volume reached approximately 450 mm³, the mice were treated with anti-HEL muIgG2c isotype control (25 mg / kg ip, days 0 and 6), inactive anti-PD-L1 (20 mg / kg iv, days 0, 2 and 4), H03-12-muIgG2c (25 mg / kg ip, days 0 and 6), trap control (24.6 mg / kg iv, days 0, 2 and 4), anti-PD-L1 (20 mg / kg iv, days 0, 2 and 4), vintrafusp alfa (24.6 mg / kg iv, days 0, 2 and 4), or a dual combination of each. Tumor samples were collected 7 days post-treatment. Expression of various immune cell population markers was measured using flow cytometry. Marker expression per 100 mg of tumor is shown along with SEM. [Figure 8-2] Figure 8: Female B-huTIGIT knock-in mice were sc-inoculated with 3 × 10⁵ MC38 cells in the flank. When the mean tumor volume reached approximately 450 mm³, the mice were treated with anti-HEL muIgG2c isotype control (25 mg / kg ip, days 0 and 6), inactive anti-PD-L1 (20 mg / kg iv, days 0, 2 and 4), H03-12-muIgG2c (25 mg / kg ip, days 0 and 6), trap control (24.6 mg / kg iv, days 0, 2 and 4), anti-PD-L1 (20 mg / kg iv, days 0, 2 and 4), vintrafusp alfa (24.6 mg / kg iv, days 0, 2 and 4), or a dual combination of each. Tumor samples were collected 7 days post-treatment. Expression of various immune cell population markers was measured using flow cytometry. Marker expression per 100 mg of tumor is shown along with SEM.

[0021] [Figure 9]Figure 9: Female B-huTIGIT knock-in mice were sc-inoculated with 3 × 10⁵ MC38 cells in the flank. When the mean tumor volume reached approximately 450 mm³, the mice were treated with anti-HEL muIgG2c isotype control (25 mg / kg ip, days 0 and 6), inactive anti-PD-L1 (20 mg / kg iv, days 0, 2 and 4), H03-12-muIgG2c (25 mg / kg ip, days 0 and 6), trap control (24.6 mg / kg iv, days 0, 2 and 4), anti-PD-L1 (20 mg / kg iv, days 0, 2 and 4), vintrafusp alfa (24.6 mg / kg iv, days 0, 2 and 4), or a dual combination of each. Tumor samples were collected 7 days post-treatment. Expression of various immune cell population markers was measured using flow cytometry. Marker expression per 100 mg of tumor is shown along with SEM.

[0022] [Figure 10] Figure 10: Naive B-huTIGIT knock-in mice (n=10) and MC38 tumor-bearing B-huTIGIT knock-in mice that showed complete tumor regression for more than 3 months after the last dose of H03-12-muIgG2c + vintrafusp alfa combination therapy (n=4) were inoculated with 1 × 10⁶ MC38 cells sc-in on the flank opposite to the initial tumor growth. (A) Mean tumor volume and SEM, tumor uptake rate, and (B) Individual tumor volumes from naive and cured mice are shown. [Modes for carrying out the invention]

[0023] definition The words "one" and "that" imply the plural form unless the context clearly indicates otherwise. Therefore, for example, a reference to "one antibody" means one or more antibodies, or at least one antibody. Thus, the terms "one," "one or more," and "at least one" are used interchangeably in this specification.

[0024] The term "approximately" when used to modify a numerically defined parameter means that the difference in that parameter is minimal and does not alter the overall effect, or the efficacy of the drug in treating a disease or disorder. For example, a dose of approximately 10 mg / kg may vary between 9 mg / kg and 11 mg / kg. In some embodiments, it means that many of the numerical values ​​stated for that parameter may vary by no more than 10% above or below.

[0025] "Administering" a drug to a patient or "giving" a drug to a patient (and grammatically equivalent expressions) can mean direct administration (administration by a medical professional to the patient, or self-administration), and / or indirect administration (the act of prescribing a drug). For example, a doctor who instructs a patient to self-administer a drug or provides a patient with a prescription is administering a drug to that patient.

[0026] "Amino acid differences" refer to amino acid substitutions, deletions, or insertions.

[0027] "Antibody" means an immunoglobulin molecule (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) that can specifically bind to a target through at least one antigen recognition site located within the variable region of an immunoglobulin (Ig) molecule. In this specification, the term "antibody" includes not only complete polyclonal antibodies or monoclonal antibodies, but also, unless otherwise specified, any antigen-binding fragment or antibody fragment thereof that competes with the complete antibody for specific binding, as well as any protein containing the antigen-binding fragment or antibody fragment thereof (including fusion proteins (such as antibody-drug conjugates, antibodies fused to cytokines, or antibodies fused to cytokine receptors), antibody compositions with polyepitope specificity, and multispecific antibodies (such as bispecific antibodies)). The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five of these basic heterotetrameric units plus an additional polypeptide called the J chain and contain antigen-binding sites, while IgA antibodies contain two to five basic four-chain units that can combine with the J chain and polymerize to form a multivalent aggregate. In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the 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 isotype of the H chain. Each H chain and L chain also have regularly spaced intra-chain disulfide bridges. Each H chain has a variable domain (V H ) at the N-terminus, followed by three constant domains (C H ) for each of the α and γ chains, and four C H domains for the μ and ε isotypes. Each L chain has a variable domain (V L ) at the N-terminus, followed by a constant domain located at the other end. V L aligns with V H , and C L aligns with the first constant domain of the heavy chain (C H 1). Specific amino acid residues are thought to form the interface between the variable domains of the light and heavy chains. V Hand V L When paired, a single antigen-binding site is formed. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8. th See Edition, Sties et al. (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6. The light chain from any vertebrate species can be assigned to one of two distinctly different types called kappa and lambda, based on the amino acid sequence of its constant domain. Immunoglobulins have a heavy chain (C H Depending on the amino acid sequence of the constant domain, it can be assigned to a different class or isotype. There are five classes of immunoglobulins, namely IgA, IgD, IgE, IgG, and IgM, each having a heavy chain named α, δ, ε, γ, and μ, respectively. The γ and α classes are C H Based on relatively minor differences in sequence and function, IgG can be further divided into subclasses. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK1.

[0028] "Anti-CD112 antibody" or "anti-CD155 antibody" means an antibody or its antigen-binding fragment that specifically binds to CD112 or CD155, respectively, and inhibits the binding of the respective ligand to the TIGIT receptor. In any treatment, drug, or use of the present invention for a human subject, the anti-CD112 antibody specifically binds to human CD112 and inhibits the binding of human TIGIT to CD112. In any treatment, drug, or use of the present invention for a human subject, the anti-CD155 antibody specifically binds to human CD155 and inhibits the binding of human TIGIT to CD155. The antibody can be a monoclonal antibody, a human antibody, a humanized antibody, or a chimeric antibody, and may contain a human constant region. In some embodiments, the human constant region is selected from the group consisting of the constant regions of IgG1, IgG2, IgG3, and IgG4, and in preferred embodiments, the human constant region is the constant region of IgG1 or IgG4. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab fragments, Fab'-SH fragments, F(ab')2 fragments, scFv fragments, and Fv fragments.

[0029] Antibody "antigen-binding fragments," or "antibody fragments," include a portion of a complete antibody that can still bind to an antigen. Antigen-binding fragments include, for example, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, and Fv fragments, domain antibodies (dAb, e.g., shark and camel antibodies), fragments containing CDRs, single-chain variable fragment antibodies (scFv), single-chain antibody molecules, multispecific antibodies (formed from antibody fragments, maxibodies, nanobodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFv), linear antibodies (see, for example, U.S. Patent No. 5,641,870, Example 2; see Zapata et al. (1995) Protein Eng. 8HO: 1057), and polypeptides containing at least a portion of an immunoglobulin, wherein the portion is sufficient to specifically bind the antigen to the polypeptide. When antibodies are digested with papain, two identical antigen-binding fragments called "Fab fragments" and a residual "Fc" fragment (a name reflecting its ability to easily crystallize) are produced. The Fab fragments consist of one entire L chain plus an H chain (V H ) has a variable region domain and one heavy chain (C H It consists of the first constant domain of 1). Each Fab fragment is monovalent with respect to antigen binding; that is, it has a single antigen-binding site. When the antibody is treated with pepsin, a single large F(ab')2 fragment is produced. This roughly corresponds to two Fab fragments linked by disulfide, each with different antigen-binding activity, and can still crosslink to the antigen. The Fab' fragment contains one or more cysteine ​​from the antibody hinge region. H It differs from the Fab fragment in that it has several additional residues at the carboxyl terminus of one domain. Fab'-SH is the name of Fab', where the cysteine ​​residue in the constant domain has a free thiol group. The F(ab')2 antibody fragment was originally generated as a pair with a Fab' fragment that has a hinged cysteine ​​in between. Other chemical couplings of antibody fragments are also known.

[0030] "Anti-PD-L1 antibody" or "anti-PD-1 antibody" means an antibody or its antigen-binding fragment that prevents PD-L1 expressed on the surface of cancer cells from binding to PD-1. In any treatment, drug, or use of the present invention for a human subject, the anti-PD-L1 antibody specifically binds to human PD-L1 and prevents the binding of human PD-L1 to human PD-1. In any treatment, drug, or use of the present invention for a human subject, the anti-PD-1 antibody specifically binds to human PD-1 and prevents the binding of human PD-L1 to human PD-1. The antibody can be a monoclonal antibody, a human antibody, a humanized antibody, or a chimeric antibody and may contain a human constant region. In some embodiments, the human constant region is selected from the group consisting of the constant regions of IgG1, IgG2, IgG3, and IgG4, and in some embodiments, the human constant region is the constant region of IgG1 or IgG4. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab fragments, Fab'-SH fragments, F(ab')2 fragments, scFv fragments, and Fv fragments.

[0031] "Anti-PD(L)1 antibody" refers to either an anti-PD-L1 antibody or an anti-PD-1 antibody.

[0032] "Anti-TIGIT antibody" means an antibody or its antigen-binding fragment that specifically binds to TIGIT and prevents TIGIT from binding to its ligands, such as CD112 and / or CD155. In some embodiments, the anti-TIGIT antibody prevents TIGIT from binding to either CD112 or CD155. In any treatment, drug, or use of the present invention for treating a human subject, the anti-TIGIT antibody specifically binds to human TIGIT and prevents human TIGIT from binding to human TIGIT ligands, such as CD112 and / or CD155. The antibody can be a monoclonal antibody, a human antibody, a humanized antibody, or a chimeric antibody and may contain a human constant region. In some embodiments, the human constant region is selected from the group consisting of the constant regions of IgG1, IgG2, IgG3, and IgG4, and in preferred embodiments, the human constant region is the constant region of IgG1 or IgG4. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab fragments, Fab'-SH fragments, F(ab')2 fragments, scFv fragments, and Fv fragments.

[0033] "Vintrafusp alfa," also known as M7824, is well understood in this field. Vintrafusp alfa is an anti-PD-L1:TGFβRII fusion protein and is described under CAS registry number 1918149-01-5. Vintrafusp alfa is also described in WO2015 / 118175 and is further described 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, Vintrafusp alfa is a fully human IgG1 monoclonal antibody against human PD-L1 fused to the extracellular domain of human TGF-β receptor II (TGFβRII). Thus, vintrafusp alfa is a bifunctional fusion protein that simultaneously blocks the PD-L1 and TGF-β pathways. In particular, in WO2015 / 118175, Example 1 on page 34 describes vintrafusp alfa as follows (in this context, vintrafusp alfa is also referred to 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 this molecule is identical to the light chain of the anti-PD-L1 antibody (SEQ ID NO: 1). The heavy chain of this molecule (SEQ ID NO: 3) is a fusion protein containing the heavy chain of an anti-PD-L1 antibody (SEQ ID NO: 2) genetically fused to the N-terminus of a soluble TGFβ receptor II (SEQ ID NO: 10) via a flexible (Gly4Ser)4Gly linker (SEQ ID NO: 11). At the fusion junction, the C-terminal lysine residue of the antibody heavy chain is mutated to alanine, reducing cleavage by proteolysis."

[0034] "Biomarkers" generally refer to biomolecules that indicate a disease state, as well as their quantitative and qualitative measurements. "Prognostic biomarkers" correlate with disease outcomes independently of therapy. For example, tumor hypoxia is a negative prognostic marker; the more severe the tumor hypoxia, the greater the likelihood of a poor disease outcome. "Predictive biomarkers" indicate whether a patient is likely to respond positively to a particular therapy. For example, HER2 profiling is commonly used in breast cancer patients to determine whether those patients are likely to respond to Herceptin (trastuzumab, Genentech). "Response biomarkers" provide an indicator of the response to a treatment, thus offering a measure of whether a treatment is working. For example, a decrease in prostate-specific antigen levels generally indicates that anticancer therapy is working well for prostate cancer patients. When a marker is used as a basis for identifying or selecting patients for a treatment described herein, the marker may be measured before and / or during treatment, and the obtained values ​​may be used by a clinician to evaluate the following: (a) the probability or possibility that an individual is suitable for the initial treatment; (b) the probability or possibility that an individual is not suitable for the initial treatment; (c) responsiveness to the treatment; (d) the probability or possibility that an individual is suitable for continuing treatment; (e) the probability or possibility that an individual is not suitable for continuing treatment; (f) dose adjustment; (g) prediction of potential clinical benefit; or (h) toxicity. As will be readily apparent to those skilled in the art, the measurement of a biomarker in a clinical setting is a clear indication that this parameter has been used as a basis for initiating, continuing, adjusting, and / or discontinuing the administration of the treatment described herein.

[0035] "Cancer" means a collection of cells that grow in an abnormal manner. As used herein, the term "cancer" refers to all types of cancer, neoplasms, malignant or benign tumors found in mammals, including leukemia, carcinoma, and sarcoma. Exemplary cancers include breast cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, lung cancer, pancreatic cancer, and glioblastoma. Further examples include brain cancer, lung cancer, non-small cell lung cancer, melanoma, sarcoma, prostate cancer, cervical cancer, gastric cancer, head and neck cancer, uterine cancer, mesothelioma, metastatic bone cancer, medulloblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytopenia, primary macrobuloidemia, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, and endocrine and exocrine pancreatic neoplasms.

[0036] A "CDR" is the amino acid sequence of the complementarity-determining region of an antibody, antibody fragment, or antigen-binding fragment. These are the hypervariable regions of the immunoglobulin heavy and light chains. The variable region of an immunoglobulin contains three heavy chain and three light chain CDRs (or CDR regions).

[0037] "Clinical outcome," "clinical parameter," "clinical response," or "clinical response endpoint" refers to any clinical finding or measurement of a patient's response to 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 adverse events.

[0038] "Combination" as used herein means the presence of one or more additional active modalities in addition to a first active modality (in which case one or more active modalities can be fused). Within the scope of combinations as described herein, consideration is any regimen (such as a combination of PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors) that combine modalities or partners (i.e., active compounds, components, or drugs) contained in one or more compounds and compositions. It is understood that all modalities in a single composition, formulation, or unit dosage form (i.e., a combination of fixed doses) must have the same dosing regimen and delivery route. It is not assumed that multiple modalities must be formulated (e.g., in the same composition, formulation, or unit dosage form) and delivered together. Combined modalities can be manufactured and / or formulated by the same or different manufacturers. Therefore, combination partners can, for example, be completely separate pharmaceutical dosage forms or pharmaceutical compositions, and they can also be marketed independently of each other. In some embodiments, the TGFβ inhibitor is fused with the PD-1 inhibitor so as to be included in a single composition and have the same drug regimen and delivery route.

[0039] "Combination therapy," "in combination with," or "in use with" refers, as used herein, to any form of co-therapy, parallel therapy, concurrent therapy, sequential therapy, or intermittent therapy of at least two different therapeutic modalities (i.e., compounds, components, targeted drugs, or therapeutic agents). Therefore, these terms mean that one therapeutic modality is administered before, during, or after the administration of the other therapeutic modality. The combined modalities may be administered in any order. Therapeutically active modalities are administered together (e.g., simultaneously in the same or different compositions, formulations, or unit dosage forms) or separately (e.g., on the same or different days, in any order according to the appropriate administration protocol for the different compositions, formulations, or unit dosage forms) in the manner and dosing regimen prescribed by the healthcare provider or in accordance with the regulatory authority. Generally, each therapeutic modality is administered in the dose and / or time schedule prescribed for that therapeutic modality. In some cases, four or more modalities may be used in combination therapy. In addition, the combination therapies presented herein can be used in conjunction with other types of treatment. For example, other anti-cancer treatments may be selected from a group consisting of chemotherapy, surgery, radiotherapy (irradiation), and / or hormone therapy, which are among the other treatments related to the current standard of care for the subject.

[0040] "Complete response" or "complete remission" means that all signs of cancer disappear in response to treatment. This does not necessarily mean that the cancer is cured.

[0041] In this specification, “including” is intended to mean that a composition and method includes the elements listed, but does not exclude other elements. “Essentially consisting of” means, when used to define a composition and method, to exclude all elements other than those essential to that composition or method. “Consists of” means to exclude anything more than trace elements of other components for the substantial steps of the composition and method referred to. Embodiments defined by each of these connecting expressions fall within the scope of the invention. Thus, it is intended that a method and composition may include additional steps and components (including), or instead include non-essential steps and compositions (essentially consisting of), or instead mean only the steps or compositions of the method referred to (consists of).

[0042] "Dosage" and "administration" refer to the specific amount of an activating agent or therapeutic agent to be administered. Such an amount is included in the "dosage form." A dosage form means a physically discrete unit suitable as a unit dose for human subjects and other mammals, each unit containing a predetermined amount of the activating agent, calculated to produce the desired effect, tolerability, and therapeutic effect, together with one or more appropriate pharmaceutical excipients (such as carriers).

[0043] "Fc" is a fragment containing the carboxyl-terminal portions of both H chains held together by a disulfide. The effector function of an antibody is determined by the sequence within the Fc region, which is also recognized by the Fc receptor (FcR) found in several types of cells.

[0044] The term "fusion molecule" is well understood in the art, and it will be understood that molecules containing a fused PD-1 inhibitor and a TGFβ inhibitor as referred to in this specification include Ig:TGFβR fusion proteins such as anti-PD-1:TGFβR fusion proteins or anti-PD-L1:TGFβR fusion proteins. An Ig:TGFβR fusion protein is an antibody (in some embodiments, a monoclonal antibody, e.g., in the form of a homodimer) or its antigen-binding fragment fused to a TGF-β receptor. The name anti-PD-L1:TGFβRII fusion protein refers to an anti-PD-L1 antibody or its antigen-binding fragment fused to a TGF-β receptor II or a fragment of its extracellular domain that can bind to TGF-β. The name anti-PD-1:TGFβRII fusion protein refers to an anti-PD-1 antibody or its antigen-binding fragment fused to a TGF-β receptor II or a fragment of its extracellular domain that can bind to TGF-β. The term "anti-PD(L)1:TGFβRII fusion protein" refers to an anti-PD-1 antibody or its antigen-binding fragment, or an anti-PD-L1 antibody or its antigen-binding fragment, fused to a TGF-β receptor II or a fragment of its extracellular domain that is capable of binding to TGF-β.

[0045] "Fv" is the minimal antibody fragment containing one complete site for antigen recognition and antigen binding. This fragment consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly non-covalently bonded. The folding of these two domains gives rise to six hypervariable loops (three of which originate from the H chain and three from the L chain, respectively) that contribute amino acid residues for binding to the antigen, thus giving the antibody antigen-binding specificity. However, even a single variable domain (or half of Fv, containing only three HVRs specific to the antigen) has the ability to recognize and bind to the antigen, albeit with less affinity than the entire binding site.

[0046] A “human antibody” is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or an antibody produced using any technique for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage presentation libraries (see, e.g., Hoogenboom and Winter (1991), JMB 227: 381; Marks et al. (1991) JMB 222: 581). Methods also available for the preparation of human monoclonal antibodies are those 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 an antigen to transgenic animals (e.g., immunized xenomouses (see, for example, U.S. Patents 6,075,181 and 6,150,584 on XENOMOUSE technology)) that have been modified to produce such antigens in response to antigen challenge, but whose endogenous loci have been deactivated. See also, for example, Li et al. (2006) PNAS USA, 103: 3557 on the production of human antibodies by human B-cell hybridoma technology.

[0047] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's HVR are replaced with residues from the HVR of a non-human species (donor antibody) (such as mouse, rat, rabbit, or non-human primate) that have the desired specificity, affinity, and / or capabilities. In some cases, framework ("FR") residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications can further refine the antibody's performance (such as binding affinity). Generally, humanized antibodies contain substantially all of at least one (typically two) variable domains, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin sequence, and all or substantially all of the FR region is the hypervariable loop of the human immunoglobulin sequence, although the FR region may contain one or more individual FR residue substitutions that improve the antibody's performance (binding affinity, isomerization, immunogenicity, etc.). The number of these amino acid substitutions in the FR is typically six or less in the H chain and three or less in the L chain. Humanized antibodies may also contain, in some cases, the immunoglobulin constant region (Fc), typically at least a portion of the Fc of human immunoglobulin. For further details, see, for example, 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. Patents 6,982,321 and 7,087,409.

[0048] "Injection" or "to inject" means introducing a drug-containing solution into the body via a vein for therapeutic purposes. Generally, this is done through an intravenous (IV) bag.

[0049] "Metastatic" cancer refers to cancer that has spread from one part of the body (for example, the lungs) to another part of the body.

[0050] In this specification, "monoclonal antibody" means an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies in that population are identical except for possible native mutations and / or post-translational modifications (e.g., isomerization and amidation) that may be present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic site. In contrast to polyclonal antibody preparations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have advantages in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the nature of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring any special method to produce the antibody. For example, monoclonal antibodies used in accordance with the present invention can be produced 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 nded.;Hammerling et al. (1981) In: Monoclonal Antibodies and T-Cell Hybridomas 563 (Elsevier, NY)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage presentation techniques (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): See 119), and techniques for producing human antibodies or human-like antibodies in the body of animals that have a locus or part or all of a gene of a human immunoglobulin encoding a human immunoglobulin sequence (e.g., WO1998 / 24893; WO1996 / 34096; WO1996 / 33735; WO1991 / 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.See 13: 65-93). Monoclonal antibodies as used herein include, in particular, chimeric antibodies (immunoglobulins) (in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular class or subclass of the antibody, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody originating from a different species or belonging to a different class or subclass of the antibody), as well as fragments of such antibodies (as long as they exhibit the desired biological activity) (see, for example, U.S. Patent No. 4,816,567; Morrison et al. (1984) PNAS USA, 81: 6851).

[0051] "Objective response" refers to a measurable response, which includes complete response (CR) or partial response (PR).

[0052] "Partial response" means that the size of one or more tumors or lesions decreases in response to treatment, or that the spread of cancer within the body decreases.

[0053] The terms “patient” and “subject” are used interchangeably herein and refer to mammals requiring treatment for cancer. Generally, a patient is a person diagnosed with one or more symptoms of cancer, or a person at risk of suffering from such symptoms. In some embodiments, “patient” or “subject” may refer to a non-human mammal (such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat), or an animal used, for example, for screening, characterizing, and evaluating drugs and therapies.

[0054] "PD-1 inhibitor" as used herein means a molecule that inhibits the PD-1 pathway by, for example, inhibiting the interaction between the PD-1 receptor and PD-L1 and / or PD-L2 ligands, such as a PD-1 axis binding partner. Possible effects of such inhibition include the elimination of immunosuppression resulting from signaling on the PD-1 signaling axis. In this context, inhibition does not need to be complete or 100%. Alternatively, inhibition means reducing, decreasing, or inactivating the binding between PD-1 and one or more of its ligands, and / or reducing, decreasing, or inactivating signaling via the PD-1 receptor. In some embodiments, the PD-1 inhibitor binds to PD-L1 or PD-1 and inhibits the interaction between these molecules (e.g., 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 an antibody may be fused to a TGFβ inhibitor, for example, as an anti-PD-L1:TGFβRII fusion protein.

[0055] "PD-L1 expression" as used herein means any detectable level of PD-L1 protein expression on the cell surface or PD-L1 mRNA expression within cells or tissues. PD-L1 protein expression can be detected by IHC assay of tumor tissue sections using a diagnostic PD-L1 antibody, or by flow cytometry. Alternatively, PD-L1 protein expression by tumor cells can be detected by PET imaging using a binder (e.g., antibody fragment, aphid, etc.) that specifically binds to PD-L1. Techniques for detecting and measuring PD-L1 mRNA expression include RT-PCR and real-time quantitative RT-PCR.

[0056] "PD-L1 positive" or "high PD-L1" cancer refers to cancers that contain cells with PD-L1 present on their cell surface, and / or cancers that produce sufficient levels of PD-L1 on the surface of cancer cells for the anti-PD-L1 antibody to have a therapeutic effect by binding to PD-L1. Methods for detecting biomarkers (such as PD-L1) on the surface of cancer or tumors are standard in this art and are considered 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 often expressed as Tumor Proportion Score (TPS) or Combined Positive Score (CPS). TPS describes the percentage of living tumor cells whose membranes are partially or completely stained (e.g., stained for PD-L1). CPS is calculated by dividing the number of PD-L1-stained cells (tumor cells, lymphocytes, macrophages) by the total number of living tumor cells and multiplying by 100. For example, in some embodiments, "high PD-L1" means that 80% or more of tumor cells are PD-L1-positive as determined by the PD-L1 Dako IHC 73-10 assay, or that the Tumor Proportion Score (TPS) as determined by the Dako IHC 22C3 PharmDx assay is 50% or higher. Both the IHC 73-10 assay and the IHC 22C3 assay select similar patient populations at their respective cutoffs. In some embodiments, the level of PD-L1 expression can also be determined using the Ventana PD-L1 (SP263) assay (which largely coincides with the 22C3 PharmDx assay (see Sughayer et al., Appl. Immunohistochem. Mol. Morphol., (2018))). Another assay for determining PD-L1 expression in cancer is the Ventana PD-L1(SP142) assay.In some embodiments, 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 tumor cells express PD-L1.

[0057] "Percent (%) sequence identity" for a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular peptide or polypeptide sequence after sequence alignment and gap introduction, which, if necessary, achieves the maximum percentage of sequence identity and disregards conservative substitutions as part of the sequence identity. Alignment for determining percentage amino acid sequence identity can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, or ALIGN software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0058] "Medically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components, including the formulation, and / or the mammal being treated with it. "Medically acceptable carriers" include any physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders. Examples of medicamentally acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and combinations thereof.

[0059] A "recurrent" cancer is a cancer that has grown again in the original site or a distant site after responding to the initial treatment (such as surgery). A localized "recurrent" cancer is a cancer that has grown again in the same location as the previously treated cancer after treatment.

[0060] "Reduction" (and grammatically equivalent) of one or more symptoms refers to reducing the severity or frequency of a symptom, or eliminating that symptom.

[0061] A "single-stranded Fv" (also abbreviated as "sFv" or "scFv") is an antibody domain V that has been linked together to form a single polypeptide chain. H and V L The antibody fragment contains V. In some embodiments, the sFv polypeptide contains a polypeptide linker that allows the sFv to form a desired structure with respect to antigen binding. H Domain and V L Further inclusions are included between domains. For an overview of sFv, see, for example, Pluckthun (1994), In: The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269.

[0062] "Substantially identical" means (1) a polypeptide in which at least 75%, 85%, 90%, 95%, 99%, or 100% of the amino acid sequence is identical to that of the reference amino acid sequence, or (2) a polypeptide in which 20%, 30%, 20%, 10%, 5%, 1% or less, or 0% of the amino acid positions differ from those of the reference amino acid sequence, where the difference in amino acid positions is due to amino acid substitution, deletion, or addition.

[0063] "Systemic" therapy is a treatment in which drug substances travel through the bloodstream to reach and affect cells throughout the entire body.

[0064] In this specification, "TGFβ inhibitor" means a molecule that inhibits the TGFβ pathway, for example, by inhibiting the interaction between TGFβ and the TGFβ receptor (TGFβR). Possible effects of such inhibition include the elimination of immunosuppression resulting from signaling on the TGFβ signaling axis. In this context, inhibition does not need to be complete or 100%. Alternatively, inhibition means reducing, decreasing, or inactivating the binding between TGF-β and TGFβR, and / or reducing, decreasing, or inactivating signaling via TGFβR. In some embodiments, the TGFβ inhibitor binds to TGFβ or TGFβR to inhibit the interaction between these molecules. In some embodiments, the TGFβ inhibitor comprises TGFβRII or the extracellular domain of a TGFβRII fragment capable of binding to TGF-β. In some embodiments, such a TGFβ inhibitor may be fused to a PD-1 inhibitor, for example, as an anti-PD-L1:TGFβRII fusion protein.

[0065] The terms “TGF-β receptor” (TGFβR), and “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, when referring to such receptors, we include the complete receptor and fragments capable of binding to TGF-β. In some embodiments, this refers to the extracellular domain of the receptor or a fragment of the extracellular domain capable of binding to TGF-β. In some embodiments, the fragment of TGFβRII is selected from the group consisting of SEQ ID NOs: 11, 12, and 13.

[0066] In each case of the present invention, the “therapeutic effective dose” of a PD-1 inhibitor, TGFβ inhibitor, or TIGIT inhibitor means an effective amount, when administered to a patient with cancer, that will produce the intended therapeutic effect (e.g., relief, improvement, reduction, or elimination of one or more signs of cancer in the patient) or any other clinical outcome at the required dose for the required duration. The therapeutic effect does not need to occur with a single dose, but may occur only after a series of doses have been administered. Thus, a therapeutic effective dose can be administered in one or more doses. Such a therapeutic effective dose may vary depending on factors such as the individual’s disease state, age, sex, and weight, and the ability of the PD-1 inhibitor, TGFβ inhibitor, or TIGIT inhibitor to produce the desired response in the individual. The therapeutic effective dose is also the amount in which the therapeutic effective effect outweighs any toxic or adverse effects of the PD-1 inhibitor, TGFβ inhibitor, or TIGIT inhibitor.

[0067] "TIGIT inhibitor" as used herein means a molecule that inhibits the TIGIT pathway by, for example, inhibiting the interaction between the TIGIT receptor and its ligands, e.g., CD155 and / or CD112. Possible effects of such inhibition include the removal of immunosuppression resulting from signaling on the TIGIT signaling axis. In this context, inhibition does not need to be complete or 100%. Alternatively, inhibition means reducing, decreasing, or inactivating the binding between TIGIT and one or more of its ligands, and / or reducing, decreasing, or inactivating signaling via the TIGIT receptor. In some embodiments, the TIGIT inhibitor is an anti-TIGIT antibody that binds to the TIGIT receptor or its ligands CD155 and / or CD112 and inhibits the interaction between these molecules (e.g., an anti-TIGIT antibody, an anti-CD155 antibody, or an anti-CD112 antibody). In some embodiments, the TIGIT inhibitor is an anti-TIGIT antibody, e.g., an anti-TIGIT antibody having a light chain sequence corresponding to SEQ ID NO: 27 and a heavy chain sequence corresponding to SEQ ID NO: 28. In this specification, the anti-TIGIT antibody is also referred to as "H03-12" or "3963H03-12".

[0068] "Treating" or "treating" a disease or patient means a set of steps taken to obtain a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, non-limiting examples of beneficial or desired clinical outcomes include: relief or improvement of one or more symptoms of cancer; reduction of the severity of the disease; delay or slowing of disease progression; improvement, reduction, or stabilization of the disease state; or other beneficial outcomes. Note that references to "treating" or "treating" include not only the prevention of a disease but also the relief of established symptoms. Therefore, "treating" or "treating" a condition, disorder, or disease includes (1) preventing or delaying the onset of the progressing clinical symptoms of the condition, disorder, or disease in a subject who suffers from or is prone to the condition, disorder, or disease but has not yet experienced or shown any clinical or asymptomatic symptoms of the condition, disorder, or disease; (2) suppressing the condition, disorder, or disease, i.e., stopping, reducing, or delaying the progression of the disease or its relapse (in the case of maintenance treatment) or at least one clinical or asymptomatic symptom; or (3) alleviating or reducing the disease, i.e., causing the regression of at least one of the condition, disorder, or disease or its clinical or asymptomatic symptoms.

[0069] In this specification, "unit dosage form" refers to a physically discrete unit of a therapeutic formulation suitable for the target being treated. However, it should be understood that the total daily dose of the compositions of the present invention is determined by the attending physician to the extent of sound medical judgment. The specific effective dose level for any particular subject or organism depends on a variety of factors, including the disorder being treated, its severity; the activity of the specific activator used; the specific composition used; the subject's age, weight, overall health, sex, and diet; the time of administration and the rate of excretion of the specific activator used; the duration of treatment; any drugs and / or additional treatments used in combination with or concurrently with the specific compound used; and similar factors well known in medicine.

[0070] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy chain or light chain of that antibody. The variable domains of the heavy chain and light chain are respectively called "V H " and "V L These domains can be called "antigen-binding sites." These domains are generally the most variable parts of an antibody (compared to other antibodies of the same class) and contain antigen-binding sites.

[0071] In this specification, multiple items, structural elements, components, and / or materials may be presented in general lists for convenience. However, each member of these lists should be interpreted as if each member of the list were identified as an individual, separate, and independent member.

[0072] Concentration, quantity, and other numerical data may be expressed or presented in the form of ranges as specified herein. Since such range forms are used merely for convenience and simplicity, it should be understood that they should be interpreted flexibly to include not only the numerical values ​​explicitly indicated as range boundaries, but also any individual numerical values ​​or subranges included within those ranges, where such numerical values ​​and subranges are explicitly indicated. For example, a numerical range of "approximately 1 to approximately 5" should be interpreted to include not only the explicitly indicated values ​​of approximately 1 to approximately 5, but also the individual values ​​and subranges within the indicated range. Therefore, this numerical range includes individual numerical values ​​(such as 2, 3, and 4) and subranges (such as 1 to 3, 2 to 4, and 3 to 5), as well as the individual values ​​of 1, 2, 3, 4, and 5. This same principle applies to ranges where only one numerical value is indicated as the minimum or maximum value. Furthermore, such interpretations should apply regardless of the width or characteristics of the range described.

[0073] Explanatory Embodiment Combinations of therapies and methods of their use This invention arises in part from the surprising discovery of the beneficial combination of PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors. The treatment schedule and dosage were designed to reveal the potential for synergistic effects. Preclinical data showed that TIGIT inhibitors, when combined with PD-1 inhibitors and TGFβ inhibitors, exhibited synergistic effects.

[0074] Therefore, in one embodiment, the present invention provides a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor for use in a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor to the subject; a method of treating cancer in a subject comprising administering the PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor to the subject; and a use of the PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor for the manufacture of a pharmacopoeia for a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor to the subject. It should be understood that a therapeutically effective dose of the PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor is applied in each therapeutic method. In some embodiments, the PD-1 inhibitor is an anti-PD(L)1 antibody, and the TGFβ inhibitor is a TGFβRII or anti-TGFβ antibody. In some embodiments, the PD-1 inhibitor is fused with the TGFβ inhibitor. For example, the PD-1 inhibitor and the TGFβ inhibitor may be contained in an anti-PD(L)1:TGFβRII fusion protein, such as an anti-PD-L1:TGFβRII fusion protein or an anti-PD-1:TGFβRII fusion protein. In some embodiments, the fusion molecule is an anti-PD-L1:TGFβRII fusion protein, such as an anti-PD-L1:TGFβRII fusion protein in which the light chain sequence and heavy chain sequence correspond to SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In some embodiments, the PD-1 inhibitor is an anti-PD(L)1 antibody, the TGFβ inhibitor is a TGFβRII or anti-TGFβ antibody, and the TIGIT inhibitor is an anti-TIGIT antibody. In some embodiments, the PD-1 inhibitor and the TGFβ inhibitor are fused as an anti-PD-L1:TGFβRII fusion protein, and the TIGIT inhibitor is an anti-TIGIT antibody.

[0075] A PD-1 inhibitor may inhibit the PD-1 pathway, for example, the immunosuppressive signaling pathway of PD-1, by inhibiting the interaction between PD-1 and at least one of its ligands, such as PD-L1 or PD-L2. A 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 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, cintilimab, tislerizumab, tripalimab, semiprimab, and antibodies whose light and heavy chains correspond to SEQ ID NOs. 7 and SEQ ID NOs. 16 or SEQ ID NOs. 15 and SEQ ID NOs. 14, respectively, or antibodies that compete for binding with any of the antibodies in this group. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody is an antibody that can bind to PD-1 or PD-L1 and whose amino acid sequence is substantially identical, for example, has at least 90% sequence identity, to the sequence of one antibody selected from the group consisting of pembrolizumab, nivolumab, avelumab, atezolizumab, durvalumab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, semiprimab, and antibodies whose light and heavy chains correspond to SEQ ID NOs. 7 and SEQ ID NOs. 16 or SEQ ID NOs. 15 and SEQ ID NOs. 14, respectively.

[0076] 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 containing three CDRs having the amino acid sequences of SEQ ID NO: 19 (CDRH1), SEQ ID NO: 20 (CDRH2), and SEQ ID NO: 21 (CDRH3), and a light chain containing three CDRs having the 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 containing three CDRs having the amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3), and a light chain containing three CDRs having the 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 heavy chain variable region of the anti-PD-L1 antibody contain SEQ ID NO: 25 and SEQ ID NO: 26, respectively. In some embodiments, the light and heavy chains of the anti-PD-L1 antibody correspond to SEQ ID NO: 7 and SEQ ID NO: 16, or SEQ ID NO: 15 and SEQ ID NO: 14, respectively.

[0077] In some embodiments, the PD-1 inhibitor is an anti-PD-L1 antibody in which each of the light chain and heavy chain sequences has 80% or more sequence identity with respect to the amino acid sequences of the heavy and light chains of the vintrafusp alpha antibody moiety, for example, 90% or more sequence identity, 95% or more sequence identity, 99% or more sequence identity, or 100% sequence identity, and the PD-1 inhibitor is capable of binding to PD-L1. In some embodiments, the PD-1 inhibitor is an anti-PD-L1 antibody in which each of the light chain and heavy chain sequences has 80% or more sequence identity with respect to the amino acid sequences of the heavy and light chains of the vintrafusp alpha antibody moiety, for example, 90% or more sequence identity, 95% or more sequence identity, 99% or more sequence identity, or 100% sequence identity, and its CDR is completely identical to the CDR of vintrafusp alpha. In some embodiments, the PD-1 inhibitor is an anti-PD-L1 antibody having an amino acid sequence that differs by 50 or fewer amino acid residues, 40 or fewer amino acid residues, 25 or fewer amino acid residues, and 10 or fewer amino acid residues from the heavy chain and light chain of the vintrafusp alpha antibody moiety, and the PD-1 inhibitor is capable of binding to PD-L1. In some embodiments, the PD-1 inhibitor is an anti-PD-L1 antibody having an amino acid sequence that differs by 50 or fewer amino acid residues, 40 or fewer amino acid residues, 25 or fewer amino acid residues, and 10 or fewer amino acid residues from the heavy chain and light chain of the vintrafusp alpha antibody moiety, and its CDR is completely identical to the CDR of vintrafusp alpha.

[0078] In some embodiments, the TGFβ inhibitor is capable of inhibiting the interaction between TGFβ and the TGFβ receptor, and is, for example, a TGFβ receptor or TGFβ ligand- or receptor-blocking antibody, a small molecule that inhibits interaction between TGFβ binding partners, or 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., soluble TGFβ receptor II or III) or a fragment thereof that can bind to TGFβ. In some embodiments, the TGFβ inhibitor is the extracellular domain of human TGFβ receptor II (TGFβRII) or a fragment thereof that can bind to TGFβ. In some embodiments, TGFβRII corresponds to the wild-type human TGF-β2 receptor 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-β2 receptor 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 includes or consists of a sequence corresponding to SEQ ID NO: 11 or a fragment thereof that can bind to TGFβ. For example, the TGFβ inhibitor may correspond to the full-length sequence of SEQ ID NO: 11, or it may be N-terminally deleted. For example, 26 or fewer N-terminal amino acids of SEQ ID NO: 11, e.g., 14 to 21 or 14 to 26 N-terminal amino acids may be deleted. In some embodiments, 14, 19, or 21 N-terminal 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, for example, 90% sequence identical, to one of the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and is capable of binding to TGFβ. In other embodiments, the TGFβ inhibitor is a protein that is substantially identical, for example, 90% sequence identical, to the amino acid sequence of SEQ ID NO: 11, and is capable of binding to TGFβ.In one embodiment, the TGFβ inhibitor is a protein having an amino acid sequence that does not differ from SEQ ID NO: 11 by more than 25 amino acids, and that is capable of binding to TGFβ.

[0079] In some embodiments, the TGFβ inhibitor is a protein that is substantially identical, for example, has at least 90% sequence identity to the TGFβR of vintrafusp alfa, and is capable of binding to TGFβ. In some embodiments, the TGFβ inhibitor is a protein that has an amino acid sequence that differs from the TGFβR of vintrafusp alfa by 50 or fewer, 40 or fewer, 25 or fewer, or 10 or fewer amino acid residues, and is capable of binding to TGFβ. In some embodiments, the TGFβ inhibitor has 100 to 160 amino acid residues or 110 to 140 amino acid residues. In some embodiments, the amino acid sequence of the TGFβ inhibitor is selected from the group consisting of sequences corresponding to positions 1 to 136 of the TGFβR of vintrafusp alfa, sequences corresponding to positions 20 to 136 of the TGFβR of vintrafusp alfa, and sequences corresponding to positions 22 to 136 of the TGFβR of vintrafusp alfa.

[0080] In some embodiments, the TGFβ inhibitor is reldelimumab, XPA681, XPA089, LY2382770, LY3022859, 1D11, 2G7, AP11014, A-80-01, LY364947, LY550410, LY580276, LY566578, SB-505124, SD-093, SD-208, SB-431542, ISTH0036, ISTH0047, garnicertib (LY2157299 monohydrate, a small molecule kinase inhibitor of TGF-βRI), LY3200882 (Pei et al. (2017) CANCER RES 77(13 Suppl):Abstract Small molecule kinase inhibitors disclosed in 955 (TGF-BRI), meterimumab (antibody targeting TGF-β, see Colak et al. (2017) TRENDS CANCER 3(1):56-71), fresolimmab (GC-1008; antibody targeting TGF-β1 and TGF-β2), XOMA 089 (antibody targeting TGF-β1 and TGF-β2, see Mirza et al. (2014) INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE 55:1121), AVlD200 (TGF-β1 and TGF-β3 trap, see Thwaites et al. (2017) BLOOD 130:2532), Trabedersen / AP12009 (TGF-β2 antisense oligonucleotide, see Jaschinski et al. (2011) CURR PHARM The selection is made from the group consisting of TGB-β pathway targeting agents described in BIOTECHNOL. 12(12):2203-13, Belagen-pumatucel-L (a tumor cell vaccine targeting TGF-β2, e.g., Giaccone et al. (2015) EUR J CANCER 51(16):2321-9), Colak et al. (2017), supra (including KI26894, SD208, SM16, IMC-TR1, PF-03446962, TEW-7197, and GW788388).

[0081] In some embodiments, the PD-1 inhibitor and the TGFβ inhibitor are fused, for example, as an anti-PD(L)1:TGFβRII fusion protein. In some embodiments, the fusion molecule is an anti-PD-1:TGFβRII fusion protein or an anti-PD-L1:TGFβRII fusion protein. In some embodiments, the anti-PD(L)1:TGFβRII fusion protein is one of the anti-PD(L)1:TGFβRII fusion proteins described in WO2015 / 118175, WO2018 / 205985, WO2020 / 014285, or WO2020 / 006509. In some embodiments, the N-terminus of the TGFβRII or fragment sequence is fused to the C-terminus of each heavy chain sequence of the antibody or fragment. In some embodiments, the antibody or fragment and the extracellular domain of TGFβRII or fragment are genetically engineered and fused via a linker sequence. In some embodiments, the linker sequence is a short, flexible peptide. In a preferred embodiment, the linker arrangement is (G4S) x G is the case where x is between 3 and 6, for example, between 4 and 5 or 4.

[0082] An exemplary anti-PD-L1:TGFβRII fusion protein is shown in Figure 2. The described heterotetramer consists of two sequences containing two light chain sequences of an anti-PD-L1 antibody and two sequences containing a heavy chain sequence of an anti-PD-L1 antibody, each of which is genetically engineered to be fused via a linker sequence to the N-terminus of the extracellular domain of TGFβRII or a fragment thereof at its C-terminus.

[0083] In one embodiment, the extracellular domain of TGFβRII or a fragment thereof of the anti-PD-L1:TGFβRII fusion protein has an amino acid sequence that differs from SEQ ID NO: 11 by no more than 25 amino acids and is capable of binding to TGFβ. In some embodiments, the anti-PD-L1:TGFβRII fusion protein is one of the anti-PD-L1:TGFβRII fusion proteins described in WO2015 / 118175, WO2018 / 205985, or WO2020 / 006509. For example, the anti-PD-L1:TGFβRII fusion protein may include the light chain sequence and heavy chain sequence of SEQ ID NO: 1 and SEQ ID NO: 3, respectively, of WO2015 / 118175. In another embodiment, the anti-PD-L1:TGFβRII fusion protein is one of the structures described in Table 2 of WO2018 / 205985, for example, structure 9 or 15 of WO2018 / 205985. In another embodiment, an antibody having the heavy chain sequence of SEQ ID NO: 11 and the light chain sequence of SEQ ID NO: 12 of WO2018 / 205985 has a linker sequence (G4S) xIt is fused via G (wherein x is 4-5) to the TGFβRII extracellular domain sequence of Sequence ID No. 14 (wherein x in the linker sequence is 4) or Sequence ID No. 15 (wherein x in the linker sequence is 5) of WO2018 / 205985. In another embodiment, the anti-PD-L1:TGFβRII fusion protein is SHR1701. In yet another embodiment, the anti-PD-L1:TGFβRII fusion protein is one of the fusion molecules disclosed in WO2020 / 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 WO2020 / 006509. In one embodiment, the anti-PD-L1:TGFβRII fusion protein is Bi-PLB-1.2 as disclosed in WO2020 / 006509. In one embodiment, the anti-PD-L1:TGFβRII fusion protein includes SEQ ID NOs. 128 and 95 as disclosed in WO2020 / 006509. In some embodiments, the amino acid sequences of the light chain and heavy chain of the anti-PD-L1:TGFβRII fusion protein correspond to light chain and heavy chain sequences selected from the group consisting of (1) SEQ ID NOs. 7 and 8 as disclosed in this specification, (2) SEQ ID NOs. 15 and 17 as disclosed in this specification, (3) SEQ ID NOs. 15 and 18 as disclosed in this specification, and (4) SEQ ID NOs. 128 and 95 as disclosed in WO2020 / 006509, respectively. In some embodiments, the anti-PD-L1:TGFβRII fusion protein is capable of binding to PD-L and TGFβ, and the amino acid sequences of its light chain and heavy chain are substantially identical, for example, to, light chain and heavy chain sequences selected from the group consisting of (1) SEQ ID NOs: 7 and 8 of this specification, (2) SEQ ID NOs: 15 and 17 of this specification, (3) SEQ ID NOs: 15 and 18 of this specification, and (4) SEQ ID NOs: 128 and 95 disclosed in WO2020 / 006509, respectively, having at least 90% sequence identity.In some embodiments, the amino acid sequences of the light chain and heavy chain of the PD-1 inhibitor of the anti-PD-L1:TGFβRII fusion protein differ from the light chain and heavy chain sequences of the antibody portion of vintrafusp alfa by 50 or fewer, 40 or fewer, 25 or fewer, or 10 or fewer amino acid residues, respectively, and the CDR is completely identical to that of vintrafusp alfa, and / or the PD-1 inhibitor is capable of binding to PD-L1. In some embodiments, the amino acid sequence of the anti-PD-L1:TGFβRII fusion protein is substantially identical to that of vintrafusp alfa, for example, having at least 90% sequence identity, 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 vintrafusp alfa. In some embodiments, the anti-PD-L1:TGFβRII fusion protein is vintrafusp alfa.

[0084] In one particular embodiment, the anti-PD-1:TGFβRII fusion protein is one of the fusion molecules that bind to both PD-1 and TGF-β as described in WO2020 / 014285, for example, in Figure 4 or Example 1 (including Tables 2-9 and 16), and in particular, a fusion protein that binds to both PD-1 and TGF-β, and includes a sequence substantially identical to SEQ ID NO: 15 or SEQ ID NO: 296 of the above specification, for example, a sequence having at least 90% sequence identity, and a sequence substantially identical 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, for example, a sequence having at least 90% sequence identity. In one embodiment, the anti-PD-1:TGFβRII fusion protein includes SEQ ID NO: 15 and SEQ ID NO: 16 of WO2020 / 014285. In one embodiment, the anti-PD-1:TGFβRII fusion protein includes SEQ ID NO: 15 and SEQ ID NO: 143 of WO2020 / 014285. In one embodiment, the anti-PD-1:TGFβIIR fusion protein includes SEQ ID NOs. 15 and 144 of WO2020 / 014285. In one embodiment, the anti-PD-1:TGFβIIR fusion protein includes SEQ ID NOs. 15 and 145 of WO2020 / 014285. In one embodiment, the anti-PD-1:TGFβIIR fusion protein includes SEQ ID NOs. 15 and 294 of WO2020 / 014285. In one embodiment, the anti-PD-1:TGFβIIR fusion protein includes SEQ ID NOs. 15 and 295 of WO2020 / 014285. In one embodiment, the anti-PD-1:TGFβIIR fusion protein includes SEQ ID NOs. 296 and 16 of WO2020 / 014285. In one embodiment, the anti-PD-1:TGFβIIR fusion protein includes SEQ ID NOs. 296 and 143 of WO2020 / 014285. In one embodiment, the anti-PD-1:TGFβIIR fusion protein includes SEQ ID NOs. 296 and 144 of WO2020 / 014285. In one embodiment, the anti-PD-1:TGFβIIR fusion protein includes SEQ ID NOs. 296 and 145 of WO2020 / 014285. In one embodiment, the anti-PD-1:TGFβIIR fusion protein includes SEQ ID NOs. 296 and 294 of WO2020 / 014285.In one embodiment, the anti-PD-1:TGFβIIR fusion protein includes SEQ ID NOs. 296 and 295 of WO2020 / 014285. In a further embodiment, the anti-PD-1:TGFβIIR fusion protein is one of the fusion molecules described in WO2020 / 006509. In one embodiment, the anti-PD-1:TGFβIIR fusion protein is Bi-PB-1, Bi-PB-2, or Bi-PB-1.2 as described in WO2020 / 006509. In one embodiment, the anti-PD-1:TGFβIIR fusion protein is Bi-PB-1.2 as described in WO2020 / 006509. In one embodiment, the anti-PD-1:TGFβIIR fusion protein includes SEQ ID NOs. 108 and 93 as described in WO2020 / 006509.

[0085] In some embodiments, a TIGIT inhibitor can inhibit the TIGIT pathway, e.g., the immunosuppressive signaling pathway of TIGIT, by inhibiting the interaction between the TIGIT receptor and one or more of its ligands, e.g., CD155 and / or CD112. A TIGIT inhibitor may bind to TIGIT or one of its ligands. In one embodiment, a TIGIT inhibitor inhibits the interaction between TIGIT and both CD155 and CD112. In one embodiment, a TIGIT inhibitor binds to TIGIT, CD155, or CD112. In some embodiments, a TIGIT inhibitor binds to TIGIT, CD155, or CD112. In one embodiment, a TIGIT inhibitor binds to TIGIT. In one embodiment, a TIGIT inhibitor is an anti-TIGIT antibody capable of inhibiting the interaction between its ligands CD155 and CD112, or both. In one embodiment, a TIGIT inhibitor is an anti-TIGIT antibody capable of inhibiting the interaction between its ligands CD155 and CD112. In some embodiments, the anti-TIGIT antibody is selected from the group consisting of tiragolumab, MK-7684, and antibodies whose light chain and heavy chain sequences correspond to SEQ ID NOs. 27 and 28, respectively, or antibodies that compete for binding with any of these antibodies. In some embodiments, the anti-TIGIT antibody is substantially identical, for example, to the sequence of tiragolumab, MK-7684, and antibodies selected from the group consisting of antibodies whose light chain and heavy chain sequences correspond to SEQ ID NOs. 27 and 28, respectively, or antibodies that compete for binding with any of these antibodies. In some embodiments, the anti-TIGIT antibody comprises a heavy chain containing three CDRs having the amino acid sequences of SEQ ID NOs. 31 (CDRH1), SEQ ID NOs. 32 (CDRH2), and SEQ ID NOs. 33 (CDRH3), and a light chain containing three CDRs having the amino acid sequences of SEQ ID NOs. 34 (CDRL1), SEQ ID NOs. 35 (CDRL2), and SEQ ID NOs. 36 (CDRL3). In some embodiments, the light chain variable region and heavy chain variable region of the anti-TIGIT antibody include SEQ ID NO: 29 and SEQ ID NO: 30, respectively.

[0086] In one embodiment, the PD-1 inhibitor and TGFβ are fused as an anti-PD(L)1:TGFβIIR fusion protein, and the TIGIT inhibitor is an anti-TIGIT antibody. In another embodiment, the PD-1 inhibitor and TGFβ are fused as an anti-PD-L1:TGFβIIR fusion protein, and the TIGIT inhibitor is an anti-TIGIT antibody. In yet another embodiment, the PD-1 inhibitor and TGFβ are fused as an anti-PD-L1:TGFβIIR fusion protein having a bintrafusp alfa CDR, and the TIGIT inhibitor is an anti-TIGIT antibody having a H03-12 CDR. In yet another embodiment, the PD-1 inhibitor and TGFβ are fused as an anti-PD-L1:TGFβIIR fusion protein having a bintrafusp alfa light chain variable region and a heavy chain variable region, and the TIGIT inhibitor is an anti-TIGIT antibody having a H03-12 light chain variable region and a heavy chain variable region. In one embodiment, the PD-1 inhibitor and TGFβ are fused as an anti-PD-L1:TGFβIIR fusion protein having the amino acid sequence vintrafusp alfa, and the TIGIT inhibitor is an anti-TIGIT antibody having the amino acid sequence H03-12.

[0087] In one embodiment, the therapeutic combination of the present invention is used for the treatment of human subjects. In one embodiment, the PD-1 inhibitor targets human PD-L1. The primary expected benefit of treatment using the therapeutic combination is the gain in the risk / benefit ratio in these human patients. Administration of the combination of the present invention may be more advantageous than individual therapeutic agents in that it may result in one or more improved characteristics compared to individual administrations of a single therapeutic agent alone: ​​i) better anticancer effect than the most active monotherapy agent, ii) synergistic or highly synergistic anticancer activity, iii) dosing protocols that result in enhanced anticancer activity while reducing the side effect profile, iv) reduced toxicity profile, v) increased treatment capacity, and / or vi) increased bioavailability of one or both therapeutic agents.

[0088] In some embodiments, the present invention provides treatments for diseases, disorders, and illnesses characterized by excessive or abnormal cell proliferation. Such diseases include proliferative or hyperproliferative disorders. Examples of proliferative or hyperproliferative disorders include cancer and myeloproliferative disorders.

[0089] In another embodiment, cancer is selected from carcinomas, lymphomas, leukemias, blastomas, and sarcomas. More specifically, such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal cancer, kidney 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, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, biliary tract cancer, and head and neck cancer. The disease or medical disorder in question is preferably selected from any of the following: WO2015118175, WO2018029367, WO2018208720, PCT / US18 / 12604, PCT / US19 / 47734, PCT / US19 / 40129, PCT / US19 / 36725, PCT / US19 / 732271, PCT / US19 / 38600, PCT / EP2019 / 061558.

[0090] In various embodiments, the methods of the present invention are used as primary, secondary, tertiary, or higher-order treatments. The order of treatment refers to the sequence in which a patient receives various pharmaceuticals or other therapies. A primary treatment regimen is the first treatment given, while secondary or tertiary treatments are given after primary or secondary treatments, respectively. Therefore, primary treatment is the first treatment for a disease or illness. In patients with cancer, primary treatment (sometimes called initial treatment or initial therapy) may include surgery, chemotherapy, radiotherapy, or a combination of these therapies. Typically, a patient is given a subsequent chemotherapy regimen (secondary or tertiary treatment) because they did not show a positive clinical outcome to primary or secondary treatment, showed only an asymptomatic response, or relapsed after showing a positive clinical response (sometimes accompanied by a disease that is now resistant to the previous therapy that previously induced a positive response).

[0091] In some embodiments, the therapeutic combinations of the present invention are used as higher-order treatments for cancer, particularly as second-line or higher-order treatments. There is no limit to the number of prior treatments, as long as the subject has previously received at least one round of cancer therapy. A round of prior cancer therapy means a defined schedule / phase for treating the subject using, for example, one or more chemotherapy agents, radiotherapy, or chemoradiotherapy, where such prior treatment has failed, either completed or terminated before the schedule. One possible reason is that the cancer was or became resistant to the prior therapy. Current standards of care (SoCs) for treating cancer patients often involve the administration of toxic and outdated chemotherapy regimens. Such SoCs are associated with a high risk of severe adverse events (such as secondary cancers) that are likely to significantly reduce quality of life. In one embodiment, a combination of a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor may be as effective as, and better tolerated than, an SoC in patients with cancer. Since PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors have different mechanisms of action, it is considered unlikely that administration of the treatment of the present invention will lead to an increase in immune-related adverse events (irAEs).

[0092] In one embodiment, PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors are administered as second-line or higher-order treatment for cancers selected from the group of previously treated recurrent or metastatic NSCLC, unresectable locally advanced NSCLC, previously treated SCLC ED, SCLC unsuitable for systemic treatment, previously treated recurrent or metastatic SCCHN, re-irradiable recurrent SCCHN, and previously treated microsatellite-unstable low (MSI-L) or microsatellite-stable (MSS) metastatic colorectal cancer (mCRC). SCLC and SCCHN, in particular, have been previously treated systemically. MSI-L / MSS mCRC occurs in 85% of all mCRCs.

[0093] In one embodiment, cancer exhibits microsatellite instability (MSI). Microsatellite instability ("MSI") is or includes alterations in the DNA of certain cells (such as tumor cells) in which the number of microsatellites (short repeat sequences of DNA) differs from the number of repeats in the inherited DNA. Microsatellite instability arises from a failure in the repair of replication-related errors due to a defect in the DNA mismatch repair (MMR) system. This failure allows mismatch mutations to persist throughout the genome, but particularly in DNA repeat regions known as microsatellites, leading to an increased mutational load. At least some tumors characterized by high microsatellite instability (MSI-H) have been shown to improve their response 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).

[0094] In some embodiments, cancer has a highly microsatellite-unstable microsatellite-unstable state (e.g., MSI-H state). In some embodiments, cancer has a low microsatellite-unstable microsatellite-unstable state (e.g., MSI-L state). In some embodiments, cancer has a microsatellite-stable microsatellite-unstable state (e.g., MSS state). In some embodiments, the microsatellite-unstable state is evaluated by next-generation sequencing (NGS) assays, immunohistochemistry (IHC) assays, and / or PCR assays. 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.

[0095] In some embodiments, cancer is associated with high tumor mutational burden (TMB). In some embodiments, cancer is associated with high TMB and MSI-H. In some embodiments, cancer is associated with high TMB and MSI-L or MSS. In some embodiments, cancer is endometrial cancer associated with high TMB. In some related embodiments, endometrial cancer is associated with high TMB and MSI-H. In some related embodiments, endometrial cancer is associated with high TMB and MSI-L or MSS.

[0096] In some embodiments, the cancer is a mismatch repair deficiency (dMMR) cancer. Microsatellite instability can also arise from a failure to repair replication-related errors due to defects in the DNA mismatch repair (MMR) system. This failure allows for the persistence of mismatch mutations throughout the genome, particularly in regions of repeating DNA known as microsatellites, leading to an increased mutational load that may improve the response to certain therapeutic agents.

[0097] In some embodiments, the cancer is a highly mutated cancer. In some embodiments, the cancer has a mutation in polymerase epsilon (POLE). In some embodiments, the cancer has a mutation in polymerase delta (POLD).

[0098] In some embodiments, the cancer is endometrial cancer (e.g., MSI-H endometrial cancer or MSS / MSI-L endometrial cancer). In some embodiments, the cancer is an MSI-H cancer with a mutation in POLE or POLD (e.g., an MSI-H non-endometrial cancer with a mutation in POLE or POLD).

[0099] In some embodiments, the cancer is advanced. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is recurrent (e.g., recurrent gynecological cancer (recurrent epithelial ovarian cancer, recurrent fallopian tube cancer, recurrent primary peritoneal cancer, or recurrent endometrial cancer, etc.)). In one embodiment, the cancer is recurrent or advanced.

[0100] In one embodiment, the selection of cancers is made from appendiceal cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer (especially esophageal squamous cell carcinoma), fallopian tube cancer, gastric cancer, glioma (especially diffuse pontine glioma), head and neck cancer (especially head and neck squamous cell carcinoma and / or oropharyngeal cancer), leukemia (especially acute lymphoblastic leukemia, acute myeloid leukemia), lung cancer (especially non-small cell lung cancer), lymphoma (especially Hodgkin lymphoma, non-Hodgkin lymphoma), melanoma, mesothelioma (especially malignant pleural mesothelioma), Merkel cell carcinoma, neuroblastoma, oral cancer, osteosarcoma, ovarian cancer, prostate cancer, kidney cancer, salivary gland tumors, sarcomas (especially Ewing's sarcoma or rhabdomyosarcoma), squamous cell carcinoma, soft tissue sarcoma, thymoma, thyroid cancer, urothelial carcinoma, uterine cancer, vaginal cancer, vulvar cancer, or Wilms' tumor. In a further embodiment, the cancer selection is made 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 carcinoma. In a further embodiment, the cancer selection is made from cervical cancer, endometrial cancer, head and neck cancer (particularly head and neck squamous cell carcinoma and / or oropharyngeal cancer), lung cancer (particularly non-small cell lung cancer), lymphoma (particularly non-Hodgkin lymphoma), melanoma, oral cancer, thyroid cancer, urothelial carcinoma, or uterine cancer. In another embodiment, the cancer selection is made from head and neck cancer (particularly head and neck squamous cell carcinoma and / or oropharyngeal cancer), lung cancer (particularly non-small cell lung cancer), urothelial carcinoma, melanoma, or cervical cancer.

[0101] In one embodiment, the human has a solid tumor. In one embodiment, the solid tumor is an advanced solid tumor. In one embodiment, the cancer selection is made 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 cancer, prostate cancer, colorectal cancer, ovarian cancer, and pancreatic cancer. In one embodiment, the cancer selection is made from the group consisting of colorectal cancer, cervical cancer, bladder cancer, urothelial carcinoma, head and neck cancer, melanoma, mesothelioma, non-small cell lung cancer, prostate cancer, esophageal cancer, and esophageal squamous cell carcinoma. In one embodiment, a human being 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 cancer, mesothelioma (e.g., malignant pleural mesothelioma), and prostate cancer.

[0102] In another embodiment, humans have liquid tumors (such as diffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic lymphoblastic leukemia, follicular lymphoma, acute myeloid leukemia, and chronic myeloid leukemia).

[0103] 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 specific cells called squamous epithelium. Squamous epithelium is found in the outer layers of the skin and mucous membranes (moist tissue that covers 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 head and neck squamous cell carcinoma.

[0104] HNSCC can occur in the mouth (oral cavity), the middle of the throat near the mouth (oropharynx), the space behind the nose (nasal cavity and sinuses), the upper part of the throat near the nasal cavity (nasopharynx), the vocal tract (larynx), or the lower part of the throat near the larynx (hypopharynx). Depending on its location, this cancer may cause abnormal blockage or sores (ulcers) in the mouth or throat, abnormal bleeding or pain in the mouth, unclear sinus congestion, sore throat, earache, pain or difficulty swallowing, hoarseness, difficulty breathing, or enlarged lymph nodes.

[0105] HNSCC can metastasize to other parts of the body (such as lymph nodes, lungs, or liver).

[0106] Smoking and alcohol consumption are the two most important risk factors for the development of HNSCC, and their contributions to risk are synergistic. In addition, human papillomavirus (HPV), particularly HPV-16, is now a well-established independent risk factor. Patients with HNSCC have a relatively poor prognosis. Relapsed / metastatic (R / M) HNSCC is particularly challenging regardless of the HPV status, and there are currently few effective treatment options. HPV-negative HNSCC is associated with a local relapse rate of 19–35% and a distant metastasis rate of 14–22% after standard treatment, compared to 9–18% and 5–12%, respectively, in HPV-positive HNSCC. The median overall survival for patients with R / M disease is 10–13 months in the first-line chemotherapy setting and 6 months in the second-line therapy setting. The current standard treatment is platinum-based dual chemotherapy with or without cetuximab. Second-line standard treatment options include cetuximab, methotrexate, and taxanes. All chemotherapy agents have significant side effects, and only 10-13% of patients respond to treatment. Existing systemic therapies result in transient regression of HNSCC, do not significantly increase survival, and almost all patients succumb to their own malignancies.

[0107] 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 recurrent / refractory (R / R) HNSCC. In one embodiment, the cancer is HPV-negative or HPV-positive HNSCC. In one embodiment, the cancer is locally advanced HNSCC. In one embodiment, the cancer is HNSCC (e.g., (R / M) HNSCC) in PD-L1-positive patients with a CPS of 1% or higher, or a TPS of 50% or higher. CPS or TPS is determined by an FDA or EMA-approved test (e.g., Dako IHC 22C3 PharmDx assay). In one embodiment, the cancer is HNSCC in patients who have experienced PD-1 inhibitors or patients who have not. In one embodiment, the cancer is HNSCC in patients who have experienced PD-1 inhibitors or patients who have not.

[0108] In one embodiment, head and neck cancer is oropharyngeal cancer. In another embodiment, head and neck cancer is oral cancer (i.e., cancer of the mouth).

[0109] In one embodiment, the cancer is lung cancer. In some embodiments, the lung cancer is 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) (e.g., squamous cell NSCLC). In some embodiments, the lung cancer is ALK translocation lung cancer (e.g., ALK translocation NSCLC). In some embodiments, the cancer is NSCLC with an identified ALK translocation. In some embodiments, the lung cancer is EGFR mutation lung cancer (e.g., EGFR mutation NSCLC). In some embodiments, the cancer is NSCLC with an identified EGFR mutation. In one embodiment, the cancer is NSCLC in a PD-L1-positive patient with a TPS of ≥1% or a TPS of 50%. TPS is determined by an FDA or EMA-approved test (e.g., Dako IHC 22C3 PharmDx assay or VENTANA PD-L1(SP263) assay).

[0110] In one embodiment, the cancer is melanoma. In some embodiments, the melanoma is advanced melanoma. In some embodiments, the melanoma is metastatic melanoma. In some embodiments, the melanoma is MSI-H melanoma. In some embodiments, the melanoma is MSS melanoma. In some embodiments, the melanoma is POLE mutation melanoma. In some embodiments, the melanoma is POLD mutation melanoma. In some embodiments, the melanoma is high TMB melanoma.

[0111] In one embodiment, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is advanced colorectal cancer. In some embodiments, the colorectal cancer is metastatic colorectal cancer. In some embodiments, the colorectal cancer is MSI-H colorectal cancer. In some embodiments, the colorectal cancer is MSS colorectal cancer. In some embodiments, the colorectal cancer is POLE mutation colorectal cancer. In some embodiments, the colorectal cancer is POLD mutation colorectal cancer. In some embodiments, the colorectal cancer is high TMB colorectal cancer.

[0112] In some embodiments, cancer is gynecological cancer (i.e., 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, non-limiting examples of cancer of the female reproductive system include ovarian cancer, fallopian tube cancer, peritoneal cancer, and breast cancer.

[0113] In some embodiments, the cancer is ovarian cancer (e.g., serous ovarian cancer or clear cell ovarian cancer). In some embodiments, the cancer is fallopian tube cancer (e.g., serous fallopian tube cancer or clear cell fallopian tube cancer). In some embodiments, the cancer is primary cancer (e.g., serous primary peritoneal cancer or clear cell primary peritoneal cancer).

[0114] In some embodiments, ovarian cancer is epithelial cancer. Epithelial cancer accounts for 85-90% of ovarian cancers. Historically, it has been considered to begin on the surface of the ovary, but new evidence suggests that at least some ovarian cancers begin in specialized cells within a portion of the fallopian tube. The fallopian tubes are small tubes that connect a woman's ovaries to the uterus and are part of the female reproductive system. A normal female reproductive system has two fallopian tubes, one located on each side of the uterus. Cancer cells that begin in the fallopian tube may initially travel to the surface of the ovary. The term “ovarian cancer” is often used to describe epithelial cancers that begin within the ovary, epithelial cancers that begin within the fallopian tube, and epithelial cancers that begin in the lining of the abdominal cavity called the peritoneum. In some embodiments, the cancer is or includes germ cell tumors. Germ cell tumors are a type of ovarian cancer that develops within the egg-producing cells of the ovary. In some embodiments, the cancer is or includes stromal tumors. Stromal tumors develop within the connective tissue cells that hold the ovary together and are sometimes the tissue that produces the female hormone called estrogen. In some embodiments, the cancer is or includes granulosa cell tumors. Granulosa cell tumors can secrete estrogen, resulting in abnormal vaginal bleeding at the time of diagnosis. In some embodiments, gynecological cancers are associated with homologous recombination repair (HRD) and / or BRCA1 / 2 mutations. In some embodiments, gynecological cancers are platinum-sensitive. In some embodiments, gynecological cancers responded to platinum-based therapy. In some embodiments, gynecological cancers have developed resistance to platinum-based therapy. In some embodiments, gynecological cancers showed a temporary partial or complete response to platinum-based therapy (e.g., partial or complete response to the last or second-to-last platinum-based therapy). In some embodiments, gynecological cancers are currently resistant to platinum-based therapy.

[0115] In some embodiments, the cancer is breast cancer. Typically, breast cancer arises within cells known as lobules of the mammary gland or within the milk ducts. Less frequently, breast cancer may begin in the stromal tissue. The stromal tissue includes the fatty connective tissue and fibrous connective tissue of the breast. Over time, breast cancer cells can invade neighboring tissues (such as the lymph nodes in the armpit or the lungs) in a process known as metastasis. The stage of breast cancer, the size of the tumor, and its growth rate are all factors that determine the type of treatment offered. Treatment options include surgery to remove the tumor, drug therapy including chemotherapy and hormone therapy, radiation therapy, and immunotherapy. Prognosis and survival rates vary widely, with the 5-year relative survival rate ranging from 98% to 23% depending on the type of breast cancer that develops. Breast cancer is the second most common cancer worldwide, with approximately 1.7 million new cases in 2012, and is the fifth leading cause of cancer death, with approximately 521,000 deaths. Approximately 15% of these cases are triple-negative, meaning they do not express estrogen receptor, progesterone receptor (PR), or HER2. In some embodiments, triple-negative breast cancer (TNBC) is characterized by breast cancer cells that are negative for estrogen receptor expression (less than 1% of cells), negative for progesterone receptor expression (less than 1% of cells), and negative for HER2. In one embodiment, this cancer is a TNBC in a PD-L1-positive patient in which 1% or more tumor-infiltrating immune cells (ICs) express PD-L1. ICs are determined by FDA or EMA-approved tests (such as the Ventana PD-L1 (SP142) assay).

[0116] 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 breast cancer, or TNBC. In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is advanced breast cancer. In some embodiments, the cancer is stage II, stage III, or stage IV breast cancer. In some embodiments, the cancer is stage IV breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer.

[0117] In one embodiment, the cancer is endometrial cancer. Endometrial cancer is the most common cancer of the female reproductive tract, accounting for 10 to 20 cases per 100,000 women per year. The number of new endometrial cancers (EC) worldwide is estimated at approximately 325,000 per year. Furthermore, EC is most common in postmenopausal women. Approximately 53% of endometrial cancers occur in developed countries. In 2015, approximately 55,000 cases of EC were diagnosed in the United States, but there are currently no approved therapies targeting EC for use. There is a need for drugs and programs to improve survival in advanced and recurrent EC in 1L and 2L settings. In the United States, approximately 10,170 people were projected to die from EC in 2016. The most common histological form is endometrioid adenocarcinoma, accounting for approximately 75-80% of diagnosed cases. Other histological morphologies include serous uterine body (less than 10%), clear cell (4%), mucinous (1%), squamous (less than 1%), and mixed (approximately 10%).

[0118] From a pathological standpoint, endometrioid carcinoma (EC) is divided into two distinct types: so-called Type I and Type II. Type I tumors are low-grade, estrogen-associated endometrioid carcinomas (EEC), while Type II tumors are non-endometrioid carcinomas (NEEC) (primarily serous and clear cell carcinomas). The World Health Organization has updated its pathological classification of EC, recognizing nine different subtypes, but EEC and serous carcinoma (SC) account for the majority of cases. EEC is an estrogen-associated cancer that occurs in postmenopausal women and is preceded by precancerous lesions (endometrial hyperplasia / endometrioid intraepithelial neoplasia). Microscopically, low-grade EEC (EEC 1-2) contains tubular glands somewhat resembling proliferative endometrium, exhibiting structural complexity due to these glands and a cribriform pattern. High-grade EEC shows a firm growth pattern. In contrast, SC occurs in postmenopausal women who are not estrogen-excess. At the microscopic level, SCs present as thick, fibrous or edematous papillary projections with marked stratification of tumor cells, cell budding, and undifferentiated cells with large eosinophilic cytoplasm. The majority of EECs are low-grade tumors (grades 1 and 2) and are associated with a good prognosis when confined to the uterus. Grade 3 EECs (EEC3) are invasive tumors with an increased frequency of lymph node metastasis. SCs are highly invasive, not associated with estrogen stimulation, and occur mainly in older women. EEC3 and SCs are considered high-grade tumors. SCs and EEC3s were compared using data from the 1998-2001 surveillance, epidemiology and End Results (SEER) program. They account for 10% and 15% of ECs, respectively, but represent 39% and 27% of cancer deaths, respectively. Endometrial cancer can also be classified into four molecular subgroups. Specifically, (1) hypermutation / POLE mutation; (2) high-frequency mutation MSI+ (e.g., MSI-H or MSI-L); (3) low copy number / microsatellite stable (MSS); and (4) high copy number / serous. Approximately 28% of cases are high MSI. (Murali, Lancet Oncol. (2014)). In some embodiments, the patient has a 2L mismatch repair deficiency subset of endometrial cancer. In some embodiments, the endometrial cancer is metastatic endometrial cancer.In some embodiments, the patient has MSS endometrial cancer. In some embodiments, the patient has MSI-H endometrial cancer.

[0119] In one embodiment, the cancer is cervical cancer. In some embodiments, the cervical cancer is advanced cervical cancer. In some embodiments, the cervical cancer is metastatic cervical cancer. In some embodiments, the cervical cancer is MSI-H cervical cancer. In some embodiments, the cervical cancer is MSS cervical cancer. In some embodiments, the cervical cancer is POLE mutation cervical cancer. In some embodiments, the cervical cancer is POLD mutation cervical cancer. In some embodiments, the cervical cancer is high TMB cervical cancer. In one embodiment, the cancer is cervical cancer in PD-L1-positive patients with a CPS of 1% or higher. CPS is determined by an FDA or EMA-approved test (such as the Dako IHC 22C3 PharmDx assay).

[0120] In one embodiment, the cancer is uterine cancer. In some embodiments, the uterine cancer is advanced uterine cancer. In some embodiments, the uterine cancer is metastatic uterine cancer. In some embodiments, the uterine cancer is MSI-H uterine cancer. In some embodiments, the uterine cancer is MSS uterine cancer. In some embodiments, the uterine cancer is POLE mutation uterine cancer. In some embodiments, the uterine cancer is POLD mutation uterine cancer. In some embodiments, the uterine cancer is high TMB uterine cancer.

[0121] In one embodiment, the cancer is urothelial carcinoma. In some embodiments, the urothelial carcinoma is advanced urothelial carcinoma. In some embodiments, the urothelial carcinoma is metastatic urothelial carcinoma. In some embodiments, the urothelial carcinoma is MSI-H urothelial carcinoma. In some embodiments, the urothelial carcinoma is MSS urothelial carcinoma. In some embodiments, the urothelial carcinoma is POLE-mutated urothelial carcinoma. In some embodiments, the urothelial carcinoma is POLD-mutated urothelial carcinoma. In some embodiments, the urothelial carcinoma is high-TMB urothelial carcinoma. In one embodiment, the cancer is urothelial carcinoma in a PD-L1-positive patient with a CPS of 10% or more. CPS is 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 a PD-L1-positive patient with 5% or more tumor-infiltrating immune cells (ICs) expressing PD-L1. IC is determined by a test approved by the FDA or EMA (such as the Ventana PD-L1(SP) assay).

[0122] In one embodiment, the cancer is thyroid cancer. In some embodiments, the thyroid cancer is advanced thyroid cancer. In some embodiments, the thyroid cancer is metastatic thyroid cancer. In some embodiments, the thyroid cancer is MSI-H thyroid cancer. In some embodiments, the thyroid cancer is MSS thyroid cancer. In some embodiments, the thyroid cancer is POLE mutation thyroid cancer. In some embodiments, the thyroid cancer is POLD mutation thyroid cancer. In some embodiments, the thyroid cancer is high TMB thyroid cancer.

[0123] As tumors, hematopoietic (or blood, blood system, or blood-related) cancers (for example, cancers originating from blood cells or immune cells, which can be called "liquid tumors") are possible. Specific examples of clinical conditions based on hematological malignancies include leukemia (such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and acute lymphocytic leukemia); plasma cell malignancies (such as multiple myeloma, monoclonal immunoglobulinemia of unknown significance (MGUS), and Waldenström macroglobulinemia); and lymphoma (such as non-Hodgkin lymphoma and Hodgkin lymphoma).

[0124] In one embodiment, the cancer is gastric cancer (GC) or gastroesophageal junction cancer (GEJ). In one embodiment, the cancer is GC or GEJ in PD-L1-positive patients with a CPS of 10% or higher. CPS is determined by an FDA or EMA-approved test (such as the Dako IHC 22C3 PharmDx assay).

[0125] In one embodiment, the cancer is esophageal squamous cell carcinoma (ESCC). In one embodiment, the cancer is ESCC in PD-L1-positive patients with a CPS of 10% or higher. CPS is determined by an FDA or EMA-approved test (such as the Dako IHC 22C3 PharmDx assay).

[0126] Cancer can be any cancer diagnosed as a hematological malignancy, characterized by an abnormal number of blast cells or unwanted cell proliferation, and including both lymphoid and myeloid malignancies. Non-exclusive examples of myeloid malignancies include acute myeloid (or myeloid, myeloid, or myeloblastic) leukemia (undifferentiated or differentiated), acute promyeloid (or promyelocytic, promyeloid, or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia, and megakaryoblastic (or megakaryoblastic) leukemia. These leukemias can be collectively referred to as acute myeloid (or myeloid, or myeloid) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPDs), non-limiting examples of which include chronic myeloid (or myeloid or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polycythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndromes or MDS), which include refractory anemia (RA), refractory anemia with blast plaque (RAEB), and refractory anemia with blast plaque in the transitional phase (RAEBT); and myelofibrosis (MFS), with or without idiopathic myeloid metaplasia.

[0127] In one embodiment, the cancer is non-Hodgkin lymphoma. Hematopoietic carcinomas also include lymphoid malignancies, which may invade the lymph nodes, spleen, bone marrow, peripheral blood, and / or extranodal sites. Lymphoid carcinomas include B-cell malignancies, and a non-limiting example of this is B-cell non-Hodgkin lymphoma (B-NHL). B-NHL can be smoldering (or low-grade), intermediate-grade (or invasive), or high-grade (highly invasive). Low-grade B-cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL) (including nodular MZL, extranodal MZL, splenic MZL, and splenic MZL with hairy lymphocytes); lymphoplasmacytic lymphoma (LPL); and mucosa-associated lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-NHL includes mantle cell lymphoma (MCL) with or without leukemia, diffuse large B-cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B-NHL includes Burkitt lymphoma (BL), Burkitt-like lymphoma, small non-incisional nuclear cell lymphoma (SNCCL), and lymphoblastic lymphoma. Other B-NHL includes immunoblastic lymphoma (or immunocytoma), primary exudative lymphoma, HIV-related (or AIDS-related) lymphoma, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cell malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenström macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoblastic (or lymphocytic or lymphoblastic) leukemia, and Castleman disease. NHL can also include T-cell non-Hodgkin lymphoma (T-NHL), but are not limited to, non-specific T-cell non-Hodgkin lymphoma (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphadenopathy (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma / delta lymphoma, cutaneous T-cell lymphoma, mycosis fungoides, and Sézary syndrome.

[0128] Hematopoietic cancers include Hodgkin lymphoma (or disease), which includes classical Hodgkin lymphoma, nodular sclerosing Hodgkin lymphoma, mixed Hodgkin lymphoma, lymphocyte-predominant (LP) Hodgkin lymphoma, nodular LP Hodgkin lymphoma, and lymphopenic Hodgkin lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers such as multiple myeloma (MM), which include smoldering MM, monoclonal gammaglobulinemia of unknown significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenström 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 containing hematopoietic cells and referred to herein as “hematopoietic tissue” include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues (such as the spleen, lymph nodes, mucosal lymphoid tissues (e.g., intestinal lymphoid tissue), tonsil-associated, Peierls' patch-associated, and appendiceal lymphoid tissues, and other mucosal lymphoid tissues (e.g., bronchial lining)).

[0129] In one embodiment, the treatment is a first-line or second-line treatment for HNSCC. In one embodiment, the treatment is a first-line or second-line treatment for recurrent / metastatic HNSCC. In one embodiment, the treatment is a first-line treatment for recurrent / metastatic (1L R / M) HNSCC. In one embodiment, the treatment is a first-line treatment for PD-L1-positive 1L R / M HNSCC. In one embodiment, the treatment is a second-line treatment for recurrent / metastatic (2L R / M) HNSCC.

[0130] In one embodiment, the treatment is primary, secondary, tertiary, quaternary, or quintuple treatment for HNSCC that has not experienced PD-1 / PD-L1. In another embodiment, the treatment is primary, secondary, tertiary, quaternary, or quintuple treatment for HNSCC that has experienced PD-1 / PD-L1.

[0131] In some embodiments, the cancer treatment is primary cancer treatment. In one embodiment, the cancer treatment is secondary cancer treatment. In some embodiments, the treatment is tertiary cancer treatment. In some embodiments, the treatment is quaternary cancer treatment. In some embodiments, the treatment is quintuple cancer treatment. In some embodiments, the treatment prior to the aforementioned secondary, tertiary, quaternary, or quintuple cancer treatment includes one or more of the following: radiotherapy, chemotherapy, surgery, or chemoradiotherapy.

[0132] In one embodiment, previous treatments include diterpenoids (such as paclitaxel, nab-paclitaxel, or docetaxel); vinca alkaloids (such as vinblastine, vincristine, or vinorelbine); platinum-coordinate 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); actinomycin (such as dactinomycin); anthrocyclines (such as daunorubicin or doxorubicin); bleomycin; and epipodophyllotoxin. (e.g., etoposide or teniposide); antimetabolite antineoplastic agents (e.g., fluorouracil, methotrexate, cytarabine, mecaptopurine, thioguanine, or gemcitabine); methotrexate; camptothecin (e.g., irinotecan or topotecan); rituximab; ofatumumab; trastuzumab; cetuximab; bexarotene; sorafenib; erbB inhibitors (e.g., lapatinib, erlotinib, or gefitinib); pertuzumab; ipilimumab; nivolumab; FOLFOX; capecitabine; FOLFIRI; bevacizumab; atezolizumab; sericrelumab; obinotuzumab, or any combination thereof. In one embodiment, the treatment prior to the aforementioned second, third, fourth, or fifth treatment of cancer includes ipilimumab and nivolumab. In one embodiment, the treatment prior to the aforementioned second, third, fourth, or fifth treatment of cancer includes FOLFOX, capecitabine, FOLFIRI / bevacizumab, and atezolizumab / sericrelumab. In one embodiment, the treatment prior to the aforementioned second, third, fourth, or fifth treatment of cancer includes carboplatin / nab-paclitaxel. In one embodiment, the treatment prior to the aforementioned second, third, fourth, or fifth treatment of cancer includes nivolumab and electrochemotherapy. In one embodiment, the treatment prior to the aforementioned second, third, fourth, or fifth treatment of cancer includes radiotherapy, cisplatin, and carboplatin / paclitaxel.

[0133] In one embodiment, the treatment is a first- or second-line treatment for head and neck cancer (particularly head and neck squamous cell carcinoma and / or oropharyngeal cancer). In one embodiment, the treatment is a first- or second-line treatment for recurrent / metastatic HNSCC. In one embodiment, the treatment is a first-line treatment for recurrent / metastatic (1L R / M) HNSCC. In one embodiment, the treatment is a first-line treatment for PD-L1-positive 1L R / M HNSCC. In one embodiment, the treatment is a second-line treatment for recurrent / metastatic (2L R / M) HNSCC.

[0134] In one embodiment, the treatment is primary, secondary, tertiary, quaternary, or quintuple treatment for HNSCC that has not experienced PD-1 / PD-L1. In another embodiment, the treatment is primary, secondary, tertiary, quaternary, or quintuple treatment for HNSCC that has experienced PD-1 / PD-L1.

[0135] In some embodiments, the treatment results in one or more of the following compared to pre-treatment levels (e.g., baseline levels): an increase in tumor-infiltrating lymphocytes (including cytotoxic T cells, helper T cells, and NK cells), an increase in T cells, an increase in granzyme B+ cells, a decrease in proliferating tumor cells, and an increase in activated T cells. Activated T cells can be observed by greater expression of OX40 and human leukocyte antigen DR. In some embodiments, the treatment results in upregulation of PD-1 and / or PD-L1 compared to pre-treatment levels (e.g., baseline levels).

[0136] In one embodiment, the method of the present invention further comprises administering at least one neoplastic agent or cancer adjuvant to the human. The method of the present invention may also be used in conjunction with other cancer treatments.

[0137] Typically, any anticancer agent or adjuvant cancer agent with activity against the tumor (such as a susceptible tumor under treatment) can be administered concurrently in the cancer treatment according to the present invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology, 10th edition (December 5, 2014), edited by VT Devita, TS Lawrence, and SA Rosenberg, published by Lippincott Williams & Wilkins Publishers.

[0138] In one embodiment, the human has previously been treated with one or more different cancer treatment modalities. In some embodiments, at least some 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 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-order cancer therapies can be identified as a 2L cancer patient (e.g., a 2L NSCLC patient). In some embodiments, the patient has received two or more-order cancer therapies (e.g., a 2L+ cancer patient, such as a 2L+ endometrial cancer patient). In some embodiments, the patient has not previously been treated with antibody therapy (e.g., anti-PD-1 therapy). In some embodiments, the patient has previously received at least one-order cancer therapy (e.g., the patient has previously received at least one or at least two-order cancer therapies). In some embodiments, the patient has previously received at least one-order treatment for metastatic cancer (e.g., the patient has previously received one or at least two-order treatment for metastatic cancer). In some embodiments, the subject is resistant to treatment with PD-1 inhibitors. In some embodiments, the subject is refractory to treatment with PD-1 inhibitors. In some embodiments, the method described herein makes the subject sensitive to treatment with PD-1 inhibitors.

[0139] In some embodiments, the cancer to be treated is PD-L1 positive. For example, in some embodiments, the cancer to be treated exhibits PD-L1+ expression (e.g., high PD-L1 expression). Methods for detecting biomarkers (such as PD-L1) on the surface of cancer or tumors are common in the art, and such common methods are intended herein. Non-limiting examples include immunohistochemistry, immunofluorescence, and fluorescence-activated cell sorting (FACS). In some embodiments, subjects or patients with PD-L1 hypercancer are treated by intravenous administration of anti-PD-L1:TGFβRII fusion protein at a dose of approximately 1200 mg once every two weeks. In some embodiments, subjects or patients with PD-L1 hypercancer are treated by intravenous administration of anti-PD-L1:TGFβRII fusion protein at a dose of approximately 1800 mg once every three weeks. In some embodiments, subjects or patients with PD-L1 hypercancer are treated by intravenous administration of anti-PD-L1:TGFβRII fusion protein at a dose of approximately 2100 mg once every three weeks. In some embodiments, subjects or patients with PD-L1 hypercancer are treated with intravenous administration of anti-PD-L1:TGFβRII fusion protein at a dose of approximately 2400 mg once every three weeks. In some embodiments, subjects or patients with PD-L1 hypercancer are treated with intravenous administration of anti-PD-L1:TGFβRII fusion protein at a dose of approximately 15 mg / kg once every three weeks.

[0140] In one embodiment, the drug regimen is a dosage of approximately 0.01 to 3000 mg (for example, approximately 0.01 mg; approximately 0.08 mg; approximately 0.1 mg; approximately 0.24 mg; approximately 0.8 mg; approximately 1 mg; approximately 2.4 mg; approximately 8 mg; approximately 10 mg; approximately 20 mg; approximately 24 mg; approximately 30 mg; approximately 40 mg; approximately 48 mg; approximately 50 mg; approximately 60 mg; approximately 70 mg; approximately 80 mg; approximately 90 mg; approximately 100 mg; approximately 160 mg; approximately 200 mg; approximately 240 mg; approximately 300 mg; approximately 400 mg; approximately 500 mg; approximately 600 mg; approximately 700 mg; approximately 800 mg This includes administration of an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, in doses of mg; approximately 900 mg; approximately 1000 mg; approximately 1100 mg; approximately 1200 mg; approximately 1300 mg; approximately 1400 mg; approximately 1500 mg; approximately 1600 mg; approximately 1700 mg; approximately 1800 mg; approximately 1900 mg; approximately 2000 mg; approximately 2100 mg; approximately 2200 mg; approximately 2300 mg; approximately 2400 mg; approximately 2500 mg; approximately 2600 mg; approximately 2700 mg; approximately 2800 mg; approximately 2900 mg; or approximately 3000 mg. In some embodiments, the dose of the anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is about 0.001 to 100 mg / kg. In some embodiments, the dose is about 0.001 mg / kg. In some embodiments, the dose is about 0.003 mg / kg. In some embodiments, the dose is about 0.01 mg / kg. In some embodiments, the dose is about 0.03 mg / kg. In some embodiments, the dose is about 0.1 mg / kg. In some embodiments, the dose is about 0.3 mg / kg. In some embodiments, the dose is about 1 mg / kg. In some embodiments, the dose is about 2 mg / kg.In some embodiments, the dose is approximately 3 mg / kg. In some embodiments, the dose is approximately 10 mg / kg. In some embodiments, the dose is approximately 15 mg / kg. In some embodiments, the dose is approximately 30 mg / kg. In some embodiments, the dose is approximately 500 mg. In some embodiments, the dose is approximately 1200 mg. In some embodiments, the dose is approximately 2400 mg.

[0141] All fixed doses disclosed herein are considered equivalent to body weight doses based on a reference body weight of 80 kg. Therefore, in the case of a fixed dose of 2400 mg, a body weight dose of 30 mg / kg is also disclosed.

[0142] In some embodiments, the light chain and heavy chain sequences of the anti-PD-L1:TGFβRII fusion protein correspond to SEQ ID NOs. 15 and 17 or SEQ ID NOs. 15 and 18, respectively, and the dose of the anti-PD-L1:TGFβRII fusion protein is 30 mg / kg.

[0143] In one embodiment, an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered once every 2 to 6 weeks (e.g., 2, 3, or 4 weeks, particularly 3 weeks). In one embodiment, an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered once every 2 weeks. In one embodiment, an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered once every 3 weeks. In one embodiment, an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered once every 6 weeks. In one embodiment, an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered once every 3 weeks for 2 to 6 administration cycles (e.g., the first 3, 4, or 5 administration cycles, particularly the first 4 administration cycles).

[0144] In some embodiments, the light and heavy chain sequences of the anti-PD-L1:TGFβRII fusion protein correspond to SEQ ID NOs. 15 and 17 or SEQ ID NOs. 15 and 18, respectively, and the anti-PD-L1:TGFβRII fusion protein is administered once every three weeks.

[0145] In one embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered to the subject once every two weeks. In another embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered to the subject once every three weeks.

[0146] In some embodiments, the light and heavy chain sequences of the anti-PD-L1:TGFβRII fusion protein correspond to SEQ ID NOs. 15 and 17 or SEQ ID NOs. 15 and 18, respectively, and the anti-PD-L1:TGFβRII fusion protein is administered at a dose of 30 mg / kg once every three weeks.

[0147] In one embodiment, the drug regimen is a dosage of approximately 0.01 to 3000 mg (for example, approximately 0.01 mg; approximately 0.08 mg; approximately 0.1 mg; approximately 0.24 mg; approximately 0.8 mg; approximately 1 mg; approximately 2.4 mg; approximately 8 mg; approximately 10 mg; approximately 20 mg; approximately 24 mg; approximately 30 mg; approximately 40 mg; approximately 48 mg; approximately 50 mg; approximately 60 mg; approximately 70 mg; approximately 80 mg; approximately 90 mg; approximately 100 mg; approximately 160 mg; approximately 200 mg; approximately 240 mg; approximately 300 mg; approximately 400 mg; approximately 500 mg; approximately 600 mg; approximately 700 mg; approximately 800 mg This includes administration of an anti-TIGIT antibody, such as H03-12, in doses of approximately 1,000 mg, In some embodiments, the dose of the anti-TIGIT antibody, for example H03-12, is approximately 0.001 to 100 mg / kg. In some embodiments, the dose is approximately 0.001 mg / kg. In some embodiments, the dose is approximately 0.003 mg / kg. In some embodiments, the dose is approximately 0.01 mg / kg. In some embodiments, the dose is approximately 0.03 mg / kg. In some embodiments, the dose is approximately 0.1 mg / kg. In some embodiments, the dose is approximately 0.125 mg / kg. In some embodiments, the dose is approximately 0.375 mg / kg. In some embodiments, the dose is approximately 1.25 mg / kg. In some embodiments, the dose is approximately 3.75 mg / kg. In some embodiments, the dose is approximately 11.25 mg / kg.In some embodiments, the dose is approximately 20 mg / kg. In some embodiments, the dose is selected from the group consisting of approximately 10 mg, approximately 30 mg, approximately 100 mg, approximately 300 mg, approximately 900 mg, and approximately 1600 mg. In some embodiments, the dose is approximately 10 mg. In some embodiments, the dose is approximately 30 mg. In some embodiments, the dose is approximately 100 mg. In some embodiments, the dose is approximately 300 mg. In some embodiments, the dose is approximately 900 mg. In some embodiments, the dose is approximately 1600 mg.

[0148] In one embodiment, an anti-TIGIT antibody, e.g., H03-12, is administered once every 2 to 6 weeks (e.g., every 2, 3, or 4 weeks, especially every 3 weeks). In one embodiment, an anti-TIGIT antibody, e.g., H03-12, is administered once every 2 weeks. In one embodiment, an anti-TIGIT antibody, e.g., H03-12, is administered once every 3 weeks. In one embodiment, an anti-TIGIT antibody, e.g., H03-12, is administered once every 4 weeks. In one embodiment, an anti-TIGIT antibody, e.g., H03-12, is administered once every 6 weeks. In one embodiment, an anti-TIGIT antibody, e.g., H03-12, is administered once every 3 weeks for 2 to 6 administration cycles (e.g., the first 3, 4, or 5 administration cycles, especially the first 4 administration cycles).

[0149] In one embodiment, approximately 300 mg of anti-TIGIT antibody, for example H03-12, is administered to the subject once every two weeks. In another embodiment, approximately 900 mg of anti-TIGIT antibody, for example H03-12, is administered to the subject once every two weeks. In another embodiment, approximately 1600 mg of anti-TIGIT antibody, for example H03-12, is administered to the subject once every two weeks. In another embodiment, approximately 300 mg of anti-TIGIT antibody, for example H03-12, is administered to the subject once every three weeks. In another embodiment, approximately 900 mg of anti-TIGIT antibody, for example H03-12, is administered to the subject once every three weeks. In yet another embodiment, approximately 1600 mg of anti-TIGIT antibody, for example H03-12, is administered to the subject once every three weeks. In yet another embodiment, approximately 300 mg of anti-TIGIT antibody, for example H03-12, is administered to the subject once every four weeks. In one embodiment, approximately 900 mg of anti-TIGIT antibody, for example H03-12, is administered to the subject once every four weeks. In another embodiment, approximately 1600 mg of anti-TIGIT antibody, for example H03-12, is administered to the subject once every four weeks.

[0150] In one embodiment, the anti-PD-L1:TGFβRII fusion protein is administered in doses of approximately 0.01 to 3000 mg, and the anti-TIGIT antibody in doses of approximately 0.01 to 3000 mg. In another embodiment, the anti-PD-L1:TGFβRII fusion protein is administered in doses of approximately 600 to 3000 mg, and the anti-TIGIT antibody in doses of approximately 5 to 2000 mg. In yet another embodiment, the anti-PD-L1:TGFβRII fusion protein is administered in doses of approximately 1200 mg, and the anti-TIGIT antibody in doses of approximately 300 mg. In yet another embodiment, the anti-PD-L1:TGFβRII fusion protein is administered in doses of approximately 1200 mg, and the anti-TIGIT antibody in doses of approximately 900 mg. In yet another embodiment, the anti-PD-L1:TGFβRII fusion protein is administered in doses of approximately 1200 mg, and the anti-TIGIT antibody in doses of approximately 1600 mg. In one embodiment, the anti-PD-L1:TGFβRII fusion protein is administered at a dose of approximately 2400 mg, and the anti-TIGIT antibody at a dose of approximately 300 mg. In another embodiment, the anti-PD-L1:TGFβRII fusion protein is administered at a dose of approximately 2400 mg, and the anti-TIGIT antibody at a dose of approximately 900 mg. In yet another embodiment, the anti-PD-L1:TGFβRII fusion protein is administered at a dose of approximately 2400 mg, and the anti-TIGIT antibody at a dose of approximately 1600 mg.

[0151] In one embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered in doses of 0.01 to 3000 mg, and the anti-TIGIT antibody H03-12 is administered in doses of 0.01 to 3000 mg. In another embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered in doses of 600 to 3000 mg, and the anti-TIGIT antibody H03-12 is administered in doses of 5 to 2000 mg. In yet another embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered in doses of 1200 mg, and the anti-TIGIT antibody H03-12 is administered in doses of 300 mg. In yet another embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered in doses of 1200 mg, and the anti-TIGIT antibody H03-12 is administered in doses of 900 mg. In one embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered at a dose of 1200 mg, and the anti-TIGIT antibody H03-12 is administered at a dose of 1600 mg. In one embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered at a dose of 2400 mg, and the anti-TIGIT antibody H03-12 is administered at a dose of 300 mg. In one embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered at a dose of 2400 mg, and the anti-TIGIT antibody H03-12 is administered at a dose of 900 mg. In one embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered at a dose of 2400 mg, and the anti-TIGIT antibody H03-12 is administered at a dose of 1600 mg.

[0152] In one embodiment, the anti-PD-L1:TGFβRII fusion protein is administered once every 2 to 6 weeks (e.g., 2, 3, or 4 weeks, especially 3 weeks), and the anti-TIGIT antibody is administered once every 2 to 6 weeks (e.g., 2, 3, or 4 weeks, especially 3 weeks). In another embodiment, the anti-PD-L1:TGFβRII fusion protein is administered once every 2 weeks, and the anti-TIGIT antibody is administered once every 2 weeks. In yet another embodiment, the anti-PD-L1:TGFβRII fusion protein is administered once every 3 weeks, and the anti-TIGIT antibody is administered once every 3 weeks. In yet another embodiment, the anti-PD-L1:TGFβRII fusion protein is administered once every 6 weeks, and the anti-TIGIT antibody is administered once every 6 weeks.

[0153] In one embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every 2 to 6 weeks (e.g., 2, 3, or 4 weeks, especially 3 weeks), and the anti-TIGIT antibody H03-12 is administered once every 2 to 6 weeks (e.g., 2, 3, or 4 weeks, especially 3 weeks). In one embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every 2 weeks, and the anti-TIGIT antibody H03-12 is administered once every 2 weeks. In one embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every 3 weeks, and the anti-TIGIT antibody H03-12 is administered once every 3 weeks. In one embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every 6 weeks, and the anti-TIGIT antibody H03-12 is administered once every 6 weeks.

[0154] In one embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein is administered every two weeks, and approximately 300 mg of anti-TIGIT antibody is administered every two weeks. In another embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein is administered every two weeks, and approximately 900 mg of anti-TIGIT antibody is administered every two weeks. In yet another embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein is administered every two weeks, and approximately 1600 mg of anti-TIGIT antibody is administered every two weeks. In yet another embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein is administered every two weeks, and approximately 300 mg of anti-TIGIT antibody is administered every three weeks. In one embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein is administered every two weeks, and approximately 900 mg of anti-TIGIT antibody is administered every three weeks. In another embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein is administered every two weeks, and approximately 1600 mg of anti-TIGIT antibody is administered every three weeks. In yet another embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein is administered every three weeks, and approximately 300 mg of anti-TIGIT antibody is administered every two weeks. In yet another embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein is administered every three weeks, and approximately 900 mg of anti-TIGIT antibody is administered every two weeks. In one embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein is administered once every three weeks, and approximately 1600 mg of anti-TIGIT antibody is administered once every two weeks. In another embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein is administered once every three weeks, and approximately 300 mg of anti-TIGIT antibody is administered once every three weeks. In yet another embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein is administered once every three weeks, and approximately 900 mg of anti-TIGIT antibody is administered once every three weeks. In yet another embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein is administered once every three weeks, and approximately 1600 mg of anti-TIGIT antibody is administered once every three weeks.

[0155] In one embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every two weeks, and approximately 300 mg of anti-TIGIT antibody H03-12 is administered once every two weeks. In another embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every two weeks, and approximately 900 mg of anti-TIGIT antibody H03-12 is administered once every two weeks. In yet another embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every two weeks, and approximately 1600 mg of anti-TIGIT antibody H03-12 is administered once every two weeks. In one embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every two weeks, and approximately 300 mg of anti-TIGIT antibody H03-12 is administered once every three weeks. In another embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every two weeks, and approximately 900 mg of anti-TIGIT antibody H03-12 is administered once every three weeks. In yet another embodiment, approximately 1200 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every two weeks, and approximately 1600 mg of anti-TIGIT antibody H03-12 is administered once every three weeks. In one embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every three weeks, and approximately 300 mg of anti-TIGIT antibody H03-12 is administered once every two weeks. In another embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every three weeks, and approximately 900 mg of anti-TIGIT antibody H03-12 is administered once every two weeks. In yet another embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every three weeks, and approximately 1600 mg of anti-TIGIT antibody H03-12 is administered once every two weeks.In one embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every three weeks, and approximately 300 mg of anti-TIGIT antibody H03-12 is administered once every three weeks. In another embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every three weeks, and approximately 900 mg of anti-TIGIT antibody H03-12 is administered once every three weeks. In yet another embodiment, approximately 2400 mg of anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa is administered once every three weeks, and approximately 1600 mg of anti-TIGIT antibody H03-12 is administered once every three weeks.

[0156] In addition to the combined treatments of the present invention, additional concomitant therapies deemed necessary for the patient's well-being may be given at the discretion of the treating physician. In some embodiments, the present invention provides a method for treating, stabilizing, or reducing the severity or progression of one or more diseases or disorders described herein, the method comprising administering to a patient in need a PD inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor in combination with additional therapies, such as chemotherapy, radiotherapy, or chemoradiotherapy.

[0157] In one embodiment, diterpenoids such as paclitaxel, nab-paclitaxel, or docetaxel; vinca alkaloids such as vinblastine, vincristine, or vinorelbine; platinum-coordinated 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; anthrocyclines such as daunorubicin or doxorubicin; bleomycin; epipodophyllotoxins such as etoposide or teniposide; fluorouracil, pemetrexed, methotrexed Antimetabolite antitumor agents such as Xate, cytarabine, mecaptopurine, thioguanine, or gemcitabine; methotrexate; camptothecines 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; sericrelumab; obinotuzumab; or any combination thereof, administered in conjunction with or sequentially with PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors.

[0158] In one embodiment, chemotherapy is further administered in conjunction with or sequentially with a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor. In one embodiment, chemotherapy is further administered in conjunction with or sequentially with a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT 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, the chemotherapy is platinum bi-chemotherapy comprising cisplatin or carboplatin and one of pemetrexed, paclitaxel, gemcitabine, or fluorouracil. In one embodiment, patients who have not received PD-1 inhibitor treatment are further administered chemotherapy in combination with or sequentially with a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor.

[0159] In one embodiment, a PD-1 inhibitor, a TGFβ inhibitor, a TIGIT inhibitor, and chemotherapy are administered once every three weeks for, for example, six cycles, and then the PD-1 inhibitor, TGFβ inhibitor, and TIGIT inhibitor are administered once every three weeks for, for example, 35 cycles.

[0160] In one embodiment, a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor are administered jointly or sequentially to a PD-L1-positive patient.

[0161] In one embodiment, radiotherapy is further administered in conjunction with or sequentially with a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor. In some embodiments, radiotherapy is selected from the group consisting of whole-body radiotherapy, external beam radiotherapy, image-guided radiotherapy, tomotherapy, stereotactic radiosurgery, stereotactic radiotherapy, and proton therapy. In some embodiments, radiotherapy includes external beam radiotherapy, internal radiotherapy (proximity radiotherapy), or whole-body radiotherapy. See, for example: Amini et al., Radiat Oncol. “Stereotactic body radiation therapy (SBRT) for lung cancer patients previously treated with conventional radiation therapy: a review” 9:210 (2014); Baker et al., Radiat Oncol. “A critical review of recent developments in radiation therapy 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).

[0162] In some embodiments, radiotherapy includes external beam radiotherapy, which includes intensity-modulated radiotherapy (IMRT), image-guided radiotherapy (IGRT), tomotherapy, stereotactic radiosurgery, stereotactic radiotherapy, proton therapy, or other charged particle beams.

[0163] In some embodiments, radiotherapy includes stereotactic radiotherapy.

[0164] PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors are administered in any amount and via any route of administration that is effective in treating or alleviating the severity of the disorders described above. The exact amount required will vary from patient to patient depending on their race, age, and overall condition, the severity of the infection, the specific drug, and the method of administration.

[0165] In some embodiments, PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors are administered simultaneously, separately, or sequentially and in any order. PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors are administered to the patient in any order (i.e., simultaneously or sequentially), and these 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, PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors are administered simultaneously or sequentially in any order, in amounts that are therapeutically effective in combination (e.g., synergistically effective amounts), for example, in doses corresponding to the amounts described herein, daily or intermittently. Each of the compounds combined with PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors may be administered separately or jointly at different times during the course of treatment. Typically, in such combination therapies, the individual compounds are formulated into separate pharmaceutical compositions or pharmaceuticals. When these compounds are formulated separately, the individual compounds can be administered simultaneously or sequentially, possibly via different routes for each compound. Depending on the circumstances, the treatment regimens for PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors may have different but overlapping delivery regimens, for example, one administered daily or twice daily, and the other administered as a single dose or weekly. In some embodiments, PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors are administered simultaneously in the same composition containing these PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors. In some embodiments, PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors are administered simultaneously in separate compositions, for example, by administering each of the PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors simultaneously in separate unit dosage forms. In some embodiments, the PD-1 inhibitor and TGFβ inhibitor are fused and administered in a separate unit dosage form from the TIGIT inhibitor, and the PD-1 inhibitor and TGFβ inhibitor are administered simultaneously with the TIGIT inhibitor or sequentially in any order. It should be understood that PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors may be administered on the same day or on different days in any order, according to the appropriate dosing protocol.Therefore, the present invention should be understood to encompass all regimens of such simultaneous or sequential treatments, and the term “administer” should be interpreted accordingly.

[0166] In some embodiments, the anti-PD-L1:TGFβRII fusion protein and the anti-TIGIT antibody are administered simultaneously, separately, or sequentially in any order. The anti-PD-L1:TGFβRII fusion protein and the anti-TIGIT antibody are administered to the patient in any order (i.e., simultaneously or sequentially), and these 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 anti-PD-L1:TGFβRII fusion protein and the anti-TIGIT antibody are administered simultaneously or sequentially in any order, in a therapeutically effective amount (e.g., a synergistically effective amount), for example, in doses corresponding to the amounts described herein, daily or intermittently. The individual compounds combined with the anti-PD-L1:TGFβRII fusion protein and the anti-TIGIT antibody may be administered separately or jointly at different times during the course of treatment. Typically, in such combination therapies, the individual compounds are formulated into separate pharmaceutical compositions or pharmaceuticals. When these compounds are formulated separately, the individual compounds can be administered simultaneously or sequentially, and possibly via different routes for each compound. The therapeutic regimens for anti-PD-L1:TGFβRII fusion proteins and anti-TIGIT antibodies may have different but overlapping delivery regimens, for example, one administered daily or twice daily, and the other as a single dose or weekly. Anti-PD-L1:TGFβRII fusion proteins may be administered before, substantially simultaneously with, together with, or after anti-TIGIT antibodies. In some embodiments, anti-PD-L1:TGFβRII fusion proteins are administered simultaneously in the same composition containing these anti-PD-L1:TGFβRII fusion proteins and anti-TIGIT antibodies. In some embodiments, anti-PD-L1:TGFβRII fusion proteins and anti-TIGIT antibodies are administered simultaneously in separate compositions, for example, by administering anti-PD-L1:TGFβRII fusion proteins and anti-TIGIT antibodies in separate unit dosage forms. It should be understood that the anti-PD-L1:TGFβRII fusion protein and the anti-TIGIT antibody may be administered on the same day or on different days in any order, according to the appropriate dosing protocol.

[0167] In some embodiments, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa and the anti-TIGIT antibody H03-12 are administered simultaneously, separately, or sequentially and in any order. The anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa and the anti-TIGIT antibody H03-12 are administered to the patient in any order (i.e., simultaneously or sequentially), and these 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 anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa and the anti-TIGIT antibody H03-12 are administered simultaneously or sequentially in any order, in a dose that is therapeutically effective in combination (e.g., a synergistically effective dose), for example, in doses corresponding to the amounts described herein, daily or intermittently. The individual partners of the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa and the anti-TIGIT antibody H03-12 may be administered separately or jointly at different times during the course of treatment. Typically, in such combination therapies, the individual compounds are formulated into separate pharmaceutical compositions or pharmaceuticals. When these compounds are formulated separately, the individual compounds may be administered simultaneously or sequentially, and possibly via different routes for each compound. The treatment regimens for the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa and the anti-TIGIT antibody H03-12 may have different but overlapping delivery regimens, for example, one administered daily or twice daily, and the other as a single dose or weekly. The anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa may be administered before, substantially simultaneously with, together with, or after the anti-TIGIT antibody H03-12. In one embodiment, anti-PD-L1:TGFβRII fusion proteins having the amino acid sequence of vintrafusp alpha are administered simultaneously by the same composition containing these anti-PD-L1:TGFβRII fusion proteins having the amino acid sequence of vintrafusp alpha and the anti-TIGIT antibody H03-12.In one embodiment, the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa and the anti-TIGIT antibody H03-12 are administered simultaneously in separate compositions, for example, by administering the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa and the anti-TIGIT antibody H03-12 simultaneously in separate unit dosage forms. For example, the anti-PD-L1:TGFβRII fusion protein and the anti-TIGIT antibody are administered simultaneously in separate compositions, for example, by administering the anti-PD-L1:TGFβRII fusion protein and the anti-TIGIT antibody H03-12 simultaneously in separate unit dosage forms. It will be understood that the anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa and the anti-TIGIT antibody H03-12 are administered in any order on the same day or on different days, according to an appropriate drug administration protocol.

[0168] In some embodiments, one or more of the PD-1 inhibitor, TGFβ inhibitor, and TIGIT inhibitor are administered to a patient requiring treatment at a first dose at a first interval during a first period, and at a second dose at a second interval during a second period. These first and second periods may be the induction and duration of treatment. Between the first and second periods, there may be a drug-free period during which one or more of the PD-1 inhibitor, TGFβ inhibitor, and TIGIT inhibitor in the combination are not administered to the patient. In some embodiments, there is a drug-free period between the first and second periods. In some embodiments, the drug-free period is between 1 and 30 days. In some embodiments, the drug-free 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 drug-free 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.

[0169] In some embodiments, the first and second doses are the same. In some embodiments, the first and second doses are different.

[0170] In some embodiments, the first and second doses of the anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, are approximately 1200 mg. In some embodiments, the first and second doses of the anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, are approximately 2400 mg. In some embodiments, the first dose of the anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is approximately 1200 mg, and the second dose is approximately 2400 mg. In some embodiments, the first dose of the anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is approximately 2400 mg, and the second dose is approximately 1200 mg.

[0171] In some embodiments, the first and second doses of the anti-TIGIT antibody, for example, H03-12, are approximately 300 mg. In some embodiments, the first and second doses of the anti-TIGIT antibody, for example, H03-12, are approximately 900 mg. In some embodiments, the first and second doses of the anti-TIGIT antibody, for example, H03-12, are approximately 1600 mg. In some embodiments, the first dose of the anti-TIGIT antibody, for example, H03-12, is approximately 900 mg, and the second dose is approximately 1600 mg. In some embodiments, the first dose of the anti-TIGIT antibody, for example, H03-12, is approximately 1600 mg, and the second dose is approximately 900 mg. In some embodiments, the first dose of the anti-TIGIT antibody, for example, H03-12, is approximately 900 mg, and the second dose is approximately 300 mg. In some embodiments, the first dose of the anti-TIGIT antibody, for example, H03-12, is about 300 mg and the second dose is about 900 mg. In some embodiments, the first dose of the anti-TIGIT antibody, for example, H03-12, is about 300 mg and the second dose is about 1600 mg. In some embodiments, the first dose of the anti-TIGIT antibody, for example, H03-12, is about 1600 mg and the second dose is about 300 mg.

[0172] In some embodiments, the first and second intervals are the same. In some embodiments, the first and second intervals are once every two weeks. In some embodiments, the first and second intervals are once every three weeks. In some embodiments, the first and second intervals are once every six weeks. In some embodiments, the first and second intervals are different. In some embodiments, the first interval is once every two weeks and the second interval is once every three weeks. In some embodiments, the first interval is once every three weeks and the second interval is once every six weeks.

[0173] In some embodiments, an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered at a first dose of 1200 mg once every two weeks for a first period of 2 to 6 administration cycles (e.g., the first 3, 4, or 5 administration cycles, in particular the first 4 administration cycles), and then at a second dose of 2400 mg once every three weeks until treatment is discontinued (depending on disease progression, side effects, or physician's discretion). In some embodiments, an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered at a first dose of 1200 mg once every two weeks for the first 3 administration cycles, and then at a second dose of 2400 mg once every three weeks until treatment is discontinued (depending on disease progression, side effects, or physician's discretion). In some embodiments, an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered at a first dose of 1200 mg once every two weeks for the first four administration cycles, and then at a second dose of 2400 mg once every three weeks until treatment is discontinued (depending on disease progression, side effects, or physician's discretion). In some embodiments, an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered at a first dose of 1200 mg once every two weeks for the first five administration cycles, and then at a second dose of 2400 mg once every three weeks until treatment is discontinued (depending on disease progression, side effects, or physician's discretion).

[0174] It is understood that after an initial treatment with one or two compounds from among TIGIT inhibitors, PD-1 inhibitors, and TGFβ inhibitors, treatment with all three compounds may follow. Between administering a TIGIT inhibitor, PD-1 inhibitor, TGFβ inhibitor, or a fused PD-1 inhibitor and TGFβ inhibitor to a patient as monotherapy and administering a PD-1 inhibitor, TGFβ inhibitor, and TIGIT inhibitor as the combination therapy described herein, a period of no treatment or no administration may be implemented, for example, for a certain number of cycles. For example, after the initial administration of monotherapy, the patient may not receive treatment for one or two cycles of 3, 6, or 12 weeks, and then receive the combination therapy described herein. Thus, in one embodiment, the patient may initially receive a TIGIT inhibitor as monotherapy, then not receive treatment for one or two cycles of 3, 6, or 12 weeks, and then receive a TIGIT inhibitor and a PD-1 inhibitor and TGFβ inhibitor as the combination therapy described herein. In one embodiment, the patient is initially administered a PD-1 inhibitor and / or a TGFβ inhibitor as monotherapy, followed by one or two cycles of 3, 6, or 12 weeks without treatment, after which the PD-1 inhibitor, TGFβ inhibitor, and TIGIT inhibitor may be administered in combination therapy as described herein.

[0175] In some embodiments, the patient may first be administered an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, as a monotherapy regimen at a dose of about 1200 mg, and then the anti-PD-L1:TGFβRII fusion protein may be administered as a combination therapy regimen with an anti-TIGIT antibody, for example, H03-12, at a dose of about 300 mg. In some embodiments, the patient may first be administered an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, as a monotherapy regimen at a dose of about 1200 mg, and then the anti-PD-L1:TGFβRII fusion protein may be administered as a combination therapy regimen with an anti-TIGIT antibody, for example, H03-12, at a dose of about 1200 mg. In some embodiments, the patient may initially receive an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, as a monotherapy regimen at a dose of about 1200 mg, followed by a combination therapy regimen of the anti-PD-L1:TGFβRII fusion protein at a dose of about 1200 mg with an anti-TIGIT antibody, for example, H03-12, at a dose of about 900 mg. In some embodiments, the patient may initially receive an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, as a monotherapy regimen at a dose of about 2400 mg, followed by a combination therapy regimen of the anti-PD-L1:TGFβRII fusion protein at a dose of about 2400 mg with an anti-TIGIT antibody, for example, H03-12, at a dose of about 300 mg. In some embodiments, the patient may first be administered an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, as a monotherapy regimen at a dose of approximately 2400 mg, and then the anti-PD-L1:TGFβRII fusion protein may be administered at a dose of approximately 2400 mg in combination with an anti-TIGIT antibody, for example, H03-12, at a dose of approximately 600 mg.In some embodiments, the patient may first be administered an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, as a monotherapy regimen at a dose of approximately 2400 mg, and then the anti-PD-L1:TGFβRII fusion protein may be administered at a dose of approximately 2400 mg in combination with an anti-TIGIT antibody, for example, H03-12, at a dose of approximately 900 mg.

[0176] In some embodiments, the patient may initially receive an anti-TIGIT antibody, for example, H03-12, at a dose of about 300 mg as a monotherapy regimen, followed by a combination therapy regimen in which the anti-TIGIT antibody is administered at a dose of about 300 mg in combination with an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, at a dose of about 1200 mg. In some embodiments, the patient may initially receive an anti-TIGIT antibody, for example, H03-12, at a dose of about 900 mg as a monotherapy regimen, followed by a combination therapy regimen in which the anti-TIGIT antibody is administered at a dose of about 900 mg in combination with an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, at a dose of about 1200 mg. In some embodiments, the patient may initially receive an anti-TIGIT antibody, for example, H03-12, at a dose of about 1600 mg as a monotherapy regimen, followed by a combination therapy regimen in which the anti-TIGIT antibody is administered at a dose of about 1600 mg in combination with an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa. In some embodiments, the patient may initially receive an anti-TIGIT antibody, for example, H03-12, at a dose of about 300 mg as a monotherapy regimen, followed by a combination therapy regimen in which the anti-TIGIT antibody is administered at a dose of about 300 mg in combination with an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa. In some embodiments, the patient may first be administered an anti-TIGIT antibody, for example H03-12, at a dose of about 900 mg as a monotherapy regimen, and then the anti-TIGIT antibody may be administered at a dose of about 900 mg together with an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, at a dose of about 2400 mg as a combination therapy regimen.In some embodiments, the patient may first be administered an anti-TIGIT antibody, for example H03-12, at a dose of about 1600 mg as a monotherapy regimen, and then the anti-TIGIT antibody may be administered at a dose of about 1600 mg together with an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, at a dose of about 2400 mg as a combination therapy regimen.

[0177] In some embodiments, the combination regimen includes (a) the step of the subject taking a PD-1 inhibitor and a TGFβ inhibitor before taking a first dose of the TIGIT inhibitor under the direction or supervision of a healthcare professional; and (b) the step of the subject taking a TIGIT inhibitor under the direction or supervision of a healthcare professional. In some embodiments, the combination regimen includes (a) the step of the subject taking a TIGIT inhibitor before taking a first dose of the PD-1 inhibitor and a TGFβ inhibitor under the direction or supervision of a healthcare professional; and (b) the step of the subject taking a PD-1 inhibitor and a TGFβ inhibitor under the direction or supervision of a healthcare professional. In some embodiments, the combination regimen includes (a) the step of the subject taking a PD-1 inhibitor before taking a first dose of the TGFβ inhibitor and a TIGIT inhibitor under the direction or supervision of a healthcare professional; and (b) the step of the subject taking a TGFβ inhibitor and a TIGIT inhibitor under the direction or supervision of a healthcare professional. In some embodiments, the combination regimen includes (a) the step of the subject taking a TGFβ inhibitor and a TIGIT inhibitor before taking a first dose of the PD-1 inhibitor under the direction or supervision of a healthcare professional; and (b) the step of the subject taking a PD-1 inhibitor under the direction or supervision of a healthcare professional. In some embodiments, the combination regimen includes (a) the step of the subject taking a TGFβ inhibitor before taking a first dose of the PD-1 inhibitor and a TIGIT inhibitor under the direction or supervision of a healthcare professional; and (b) the step of the subject taking a PD-1 inhibitor and a TIGIT inhibitor under the direction or supervision of a healthcare professional. In some embodiments, the combination regimen includes (a) the step of the subject taking a PD-1 inhibitor and a TIGIT inhibitor before taking a first dose of the TGFβ inhibitor under the direction or supervision of a healthcare professional; and (b) the step of the subject taking a TGFβ inhibitor under the direction or supervision of a healthcare professional.

[0178] In some embodiments, the combined regimen includes (a) the step of the subject ingesting an anti-PD(L)1 antibody and TGFβRII or anti-TGFβ antibody before the subject ingests a first dose of anti-TIGIT antibody under the direction or supervision of a healthcare professional; and (b) the step of the subject ingesting an anti-TIGIT antibody under the direction or supervision of a healthcare professional. In some embodiments, the combined regimen includes (a) the step of the subject ingesting an anti-PD(L)1 antibody before the subject ingests a first dose of TGFβRII or anti-TGFβ antibody and anti-TIGIT antibody under the direction or supervision of a healthcare professional; and (b) the step of the subject ingesting TGFβRII or anti-TGFβ antibody and anti-TIGIT antibody under the direction or supervision of a healthcare professional. In some embodiments, the combined regimen includes (a) the step of the subject ingesting TGFβRII or anti-TGFβ antibody before the subject ingests a first dose of anti-PD(L)1 antibody and anti-TIGIT antibody under the direction or supervision of a healthcare professional; and (b) the step of the subject ingesting anti-PD(L)1 antibody and anti-TIGIT antibody under the direction or supervision of a healthcare professional.

[0179] In some embodiments, the combined regimen includes (a) the step of the subject ingesting an anti-PD-L1:TGFβRII fusion protein before the subject ingests a first dose of anti-TIGIT antibody under the direction or supervision of a healthcare professional; and (b) the step of the subject ingesting an anti-TIGIT antibody under the direction or supervision of a healthcare professional. In some embodiments, the combined regimen includes (a) the step of the subject ingesting an anti-PD-L1:TGFβRII fusion protein before the subject ingests a first dose of anti-PD-L1:TGFβRII fusion protein under the direction or supervision of a healthcare professional; and (b) the step of the subject ingesting an anti-PD-L1:TGFβRII fusion protein under the direction or supervision of a healthcare professional. In some embodiments, the combined regimen includes (a) the step of the subject ingesting an anti-PD-L1:TGFβRII fusion protein before the subject ingests a first dose of anti-TIGIT antibody under the direction or supervision of a healthcare professional; and (b) the step of the subject ingesting an anti-TIGIT antibody under the direction or supervision of a healthcare professional. In some embodiments, the combined regimen includes (a) the step of the subject ingesting an anti-TIGIT antibody before ingesting a first dose of anti-PD-L1:TGFβRII fusion protein under the direction or supervision of a healthcare professional; and (b) the step of the subject ingesting an anti-PD-L1:TGFβRII fusion protein under the direction or supervision of a healthcare professional.

[0180] In some embodiments, the combined regimen includes (a) the step of the subject ingesting an anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alpha before the subject ingests a first dose of anti-TIGIT antibody H03-12 under the direction or supervision of a healthcare professional; and (b) the step of the subject ingesting an anti-TIGIT antibody H03-12 under the direction or supervision of a healthcare professional. In some embodiments, the combined regimen includes (a) the step of the subject ingesting an anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alpha before the subject ingests a first dose of anti-TIGIT antibody H03-12 under the direction or supervision of a healthcare professional; and (b) the step of the subject ingesting an anti-TIGIT antibody H03-12 under the direction or supervision of a healthcare professional.

[0181] Furthermore, combinations comprising a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor are also provided. Combinations comprising an anti-PD(L)1 antibody, TGFβRII or an anti-TGFβ antibody, and an anti-TIGIT antibody are also provided. Combinations comprising a TIGIT inhibitor and a fused PD-1 inhibitor and TGFβ inhibitor are also provided. Furthermore, combinations comprising an anti-PD-L1:TGFβRII fusion protein and an anti-TIGIT antibody are also provided. In some embodiments, any of the above combinations are intended for use as pharmaceuticals or for use in the treatment of cancer.

[0182] In the various embodiments described above, the PD-1 inhibitor and the TGFβ inhibitor may be fused, for example, as an anti-PD(L)1:TGFβRII fusion protein or an anti-PD-L1:TGFβRII fusion protein.

[0183] Pharmaceutical preparations and kits The PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors described herein may be in the form of pharmaceutical formulations or kits.

[0184] 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 a TGFβ inhibitor. In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising an anti-PD-L1:TGFβRII fusion protein. In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising an anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa. In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising a TIGIT inhibitor. In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising an anti-TIGIT antibody. 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 TIGIT inhibitor. In some embodiments, the present invention provides a pharmaceutical composition comprising a PD-1 inhibitor and a TIGIT inhibitor. In some embodiments, the present invention provides a pharmaceutical composition comprising a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor. In some embodiments, the present invention provides a pharmaceutical composition comprising a TIGIT inhibitor and a fused PD-1 inhibitor and a TGFβ inhibitor. In some embodiments, the present invention provides a pharmaceutical composition comprising an anti-PD-L1:TGFβRII fusion protein and an anti-TIGIT antibody. In some embodiments, the present invention provides a pharmaceutical composition comprising an anti-PD-L1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa and an anti-TIGIT antibody H03-12. A pharmaceutically acceptable composition may further comprise at least a pharmaceutically acceptable excipient or auxiliary agent, such as a pharmaceutically acceptable carrier.

[0185] In some embodiments, a composition containing a fused PD-1 inhibitor and a TGFβ inhibitor, for example, an anti-PD-L1:TGFβRII fusion protein, is separate from a composition containing a TIGIT inhibitor. In some embodiments, the PD-1 inhibitor and the TGFβ inhibitor are fused, for example, as an anti-PD-L1:TGFβRII fusion protein and are present in the same composition as the TIGIT inhibitor.

[0186] Examples of such pharmaceutically acceptable compositions are further described below in this specification.

[0187] The compositions of the present invention can be in various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as solutions (e.g., injectable and injectable solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The compositions of the present invention are administered orally, parenterally, by inhalation spray, topically, rectally, nasally, orally, vaginally, or via an implanted reservoir. In this specification, the term “parenteral” includes techniques of injection or infusion into the subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intrafocal, and intracranial regions. In some embodiments, the compositions are administered orally, intraperitoneally, subcutaneously, or intravenously. In one embodiment, the composition is administered by intravenous infusion or injection. In another embodiment, the composition is administered by intramuscular or subcutaneous injection. In one embodiment, the anti-PD-L1:TGFβRII fusion protein is administered by intravenous infusion or injection. In another embodiment, the anti-PD-L1:TGFβRII fusion protein is administered by intramuscular or subcutaneous injection. In one embodiment, the anti-TIGIT antibody is administered by intravenous infusion or injection. In yet another embodiment, the anti-TIGIT antibody is administered by intramuscular or subcutaneous injection.

[0188] In some embodiments, the anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered intravenously (e.g., as an intravenous infusion) or subcutaneously. In some embodiments, the anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered by intravenous infusion. In some embodiments, the anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered intravenously in doses of about 1200 mg or about 2400 mg. In some embodiments, the anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered intravenously once every two weeks in doses of about 1200 mg. In some embodiments, the anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered intravenously once every three weeks in doses of about 2400 mg. In some embodiments, an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, is administered intravenously once every three weeks at a dose of approximately 15 mg / kg.

[0189] In some embodiments, the anti-TIGIT antibody, e.g., H03-12, is administered intravenously (e.g., as an intravenous infusion) or subcutaneously. In some embodiments, the anti-TIGIT antibody, e.g., H03-12, is administered by intravenous infusion. In some embodiments, the anti-TIGIT antibody, e.g., H03-12, is administered intravenously in doses of about 300 mg, about 900 mg, or about 1600 mg. In some embodiments, the anti-TIGIT antibody, e.g., H03-12, is administered intravenously once every two weeks in a dose of about 300 mg. In some embodiments, the anti-TIGIT antibody, e.g., H03-12, is administered intravenously once every two weeks in a dose of about 900 mg. In some embodiments, the anti-TIGIT antibody, e.g., H03-12, is administered intravenously once every two weeks in a dose of about 1600 mg. In some embodiments, an anti-TIGIT antibody, e.g., H03-12, is administered intravenously at a dose of approximately 300 mg once every three weeks. In some embodiments, an anti-TIGIT antibody, e.g., H03-12, is administered intravenously at a dose of approximately 900 mg once every three weeks. In some embodiments, an anti-TIGIT antibody, e.g., H03-12, is administered intravenously at a dose of approximately 1600 mg once every three weeks.

[0190] Non-limiting examples of pharmaceutically acceptable carriers, auxiliaries, or vehicles used in the compositions of the present invention include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering 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, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0191] Non-limiting examples of liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. Liquid dosage forms may also contain inert diluents (e.g., water or other solvents), solubilizers, and emulsifiers commonly used in the art, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof. Oral compositions may also contain auxiliary agents (such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances) in addition to inert diluents.

[0192] Injectable preparations, such as aqueous or oily sterile injection suspensions, can be formulated according to known techniques using appropriate dispersants or wetting agents and suspending agents. Sterile injection preparations may also include sterile solutions, suspensions, or emulsions for injection in non-toxic, parenterally acceptable diluents or solvents (e.g., solutions in 1,3-butanediol). Acceptable vehicles and solvents that may be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile non-volatile oils are commonly used as solvents or suspension media. For this purpose, any non-irritating non-volatile oil (including synthetic glycerides or diglycerides) can be used. Furthermore, fatty acids (such as oleic acid) are used in injection preparations.

[0193] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a bactericide in the form of a sterilized solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0194] To prolong the effects of the compounds of the present invention, it is often desirable to delay absorption from subcutaneous or intramuscular injection. This can be achieved by using suspensions of crystalline or amorphous materials with poor water solubility. The absorption rate then depends on the dissociation rate, which may depend on the crystal size and morphology. Alternatively, delayed absorption of parenterally administered PD-1 inhibitors, TGFβ inhibitors, and / or TIGIT inhibitors can be achieved by dissolving or suspending the compounds in an oil vehicle. Injectable depot formulations are prepared by forming a microencapsulated matrix of PD-1 inhibitors, TGFβ inhibitors, and / or TIGIT inhibitors in a biodegradable polymer (such as polylactide-polyglycolide). The release rate of the compound can be controlled depending on the ratio of the compound to the polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydride). Depot injection formulations can also be prepared by encapsulating the compounds in liposomes or microemulsions that conform to body tissues.

[0195] The composition for rectal or vaginal administration may be a suppository. The suppository can be prepared by mixing the compound of the present invention with a suitable non-irritating excipient or carrier (such as cocoa butter, polyethylene glycol, or suppository wax (which is solid at ambient temperature but liquid at body temperature, and therefore melts in the rectum or vagina to release the active compound)).

[0196] Dosage forms for oral administration include capsules, tablets, pills, powders, and granules, as well as 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 bulking agents (such as starch, lactose, sucrose, glucose, mannitol, and silicic acid), b) binders (such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic), c) humectants (such as glycerol), and d) disintegrants (such as agar and calcium carbonate). These include (e) potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, etc., (f) dissolution retarders (such as paraffin), (g) absorption accelerators (such as quaternary ammonium compounds), (g) wetting agents (such as 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 glycol, sodium lauryl sulfate, etc., and mixtures thereof). In the case of capsules, tablets, and pills, the dosage form may also include buffers.

[0197] Similar types of solid compositions can also be used as fillers in gelatin soft and hard capsules using excipients such as lactose or lactose, as well as high molecular weight polyethylene glycol. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells (such as enteric coatings or other coatings well known in the pharmaceutical formulation field). These may optionally contain opacifiers and can be formulated to release only the active ingredient, or, in some cases, the active ingredient preferentially to a specific part of the intestinal tract, with a delay. Examples of usable embedding compositions include polymers and waxes.

[0198] PD-1 inhibitors, TGFβ inhibitors, and / or TIGIT inhibitors can also be microencapsulated with one or more excipients, as noted above. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells (such as enteric coatings, controlled-release coatings, and other coatings well known in the field of pharmaceutical formulation). In such solid dosage forms, PD-1 inhibitors, TGFβ inhibitors, and / or TIGIT inhibitors can be mixed with at least one inert diluent (such as sucrose, lactose, or starch). Such dosage forms may also include additional substances other than inert diluents (e.g., tablet lubricants and other tablet adjuvants (such as magnesium stearate and microcrystalline cellulose)), as is customary. In the case of capsules, tablets, and pills, the dosage form may also include buffers. These may optionally contain opacifiers and may be composed to release only the active ingredient, or preferentially to a certain part of the intestinal tract, and possibly with a delay. Examples of usable embedding compositions include polymeric substances and waxes.

[0199] Dosage forms for topical or transdermal administration of PD-1 inhibitors, TGFβ inhibitors, and / or TIGIT inhibitors include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed, if necessary, with a pharmaceutically acceptable carrier and any necessary preservatives or buffers under sterile conditions. Typical carriers for topical administration of these compounds are mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying waxes, and water. Alternatively, the pharmaceutically acceptable compositions provided can be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Non-limiting examples of suitable carriers include mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Ophthalmic formulations, ear drops, and eye drops are also considered to be within the scope of the present invention. In addition, the present invention considers the use of transdermal patches, which have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms are prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flow of the compound through the skin. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0200] The pharmaceutically acceptable compositions of the present invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to well-known techniques in the field of pharmaceutical formulation, and are prepared as solutions in physiological saline using benzyl alcohol or other suitable preservatives, absorption enhancers to increase bioavailability, fluorocarbons, and / or other common solubilizers or dispersants.

[0201] In further embodiments, the present invention relates to a PD-1 inhibitor and a kit comprising a package insert containing instructions for using the PD-1 inhibitor in combination with a TIGIT inhibitor and a TGFβ inhibitor to treat or delay the progression of cancer in a subject. A kit is also provided comprising a TIGIT inhibitor and a package insert containing instructions for using the TIGIT inhibitor in combination with a PD-1 inhibitor and a TGFβ inhibitor to treat or delay the progression of cancer in a subject. A kit is also provided comprising a TGFβ inhibitor and a package insert containing instructions for using the TGFβ inhibitor in combination with a PD-1 inhibitor and a TIGIT inhibitor to treat or delay the progression of cancer in a subject. Furthermore, a kit is also provided comprising an anti-PD-L1 antibody and a package insert containing instructions for using the anti-PD-L1 antibody in combination with an anti-TIGIT antibody and a TGFβRII or anti-TGFβ antibody to treat or delay the progression of cancer in a subject. Furthermore, kits are provided that include an anti-TIGIT antibody, and a package insert containing instructions for using the anti-TIGIT antibody in combination with an anti-PD-L1 antibody and a TGFβRII or anti-TGFβ antibody to treat or delay the progression of cancer in the target population. Furthermore, kits are provided that include a TGFβRII or anti-TGFβ antibody, and a package insert containing instructions for using the TGFβRII or anti-TGFβ antibody in combination with an anti-PD-L1 antibody and an anti-TIGIT antibody to treat or delay the progression of cancer in the target population. Furthermore, kits are provided that include a PD-1 inhibitor and a TGFβ inhibitor, and a package insert containing instructions for using the PD-1 inhibitor and TGFβ inhibitor in combination with a TIGIT inhibitor to treat or delay the progression of cancer in the target population. Furthermore, a kit is also provided that includes an anti-PD-L1 antibody and TGFβRII or anti-TGFβ antibody, as well as a package insert containing instructions for using the anti-PD-L1 antibody and TGFβRII or anti-TGFβ antibody in combination with an anti-TIGIT antibody to treat or delay the progression of cancer in the target population.Furthermore, kits are provided that include an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, and a package insert containing instructions for using the anti-PD-L1:TGFβRII fusion protein in combination with an anti-TIGIT antibody, for example, H03-12, to treat or delay the progression of cancer in a subject. Furthermore, kits are provided that include a PD-1 inhibitor and a TIGIT inhibitor, and a package insert containing instructions for using the PD-1 inhibitor and the TIGIT inhibitor in combination with a TGFβ inhibitor to treat or delay the progression of cancer in a subject. Furthermore, kits are provided that include a TGFβ inhibitor and a TIGIT inhibitor, and a package insert containing instructions for using the TGFβ inhibitor and the TIGIT inhibitor in combination with a PD-1 inhibitor to treat or delay the progression of cancer in a subject. Furthermore, kits are provided that include an anti-PD-L1 antibody and an anti-TIGIT antibody, as well as a package insert containing instructions for using the anti-PD-L1 antibody and the anti-TIGIT antibody in combination with a TGFβRII or anti-TGFβ antibody to treat or delay the progression of cancer in the target population. Furthermore, kits are provided that include an anti-PD-L1 antibody and an anti-TIGIT antibody, as well as a package insert containing instructions for using the anti-PD-L1 antibody in combination with a TGFβRII or anti-TGFβ antibody and anti-TIGIT antibody to treat or delay the progression of cancer in the target population. Furthermore, kits are provided that include a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor, as well as a package insert containing instructions for using the PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor to treat or delay the progression of cancer in the target population. Furthermore, kits are also provided that include an anti-PD-L1 antibody, TGFβRII or anti-TGFβ antibody and anti-TIGIT antibody, as well as a package insert containing instructions for using the anti-PD-L1 antibody, TGFβRII or anti-TGFβ antibody and anti-TIGIT antibody to treat or delay the progression of cancer in the target population.A kit is also provided which includes an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, an anti-TIGIT antibody, for example, H03-12, and a package insert which includes instructions for using the anti-PD-L1:TGFβRII fusion protein and the anti-TIGIT antibody to treat or delay the progression of cancer in a subject. The kit may include a first container, a second container, a third container, and a package insert, wherein the first container contains at least one dose of a PD-1 inhibitor, the second container contains at least one dose of a TIGIT inhibitor, and the third container contains at least one dose of a TGFβ inhibitor, and the package insert may include instructions for using these three compounds to treat cancer in a subject. In some embodiments, the kit may comprise a first container, a second container, and a package insert, wherein the first container comprises at least one dose of an anti-PD-L1:TGFβRII fusion protein, e.g., a fusion protein having the amino acid sequence of vintrafusp alfa, and the second container comprises at least one dose of an anti-TIGIT antibody, e.g., H03-12, and the package insert may comprise instructions for using these two compounds to treat cancer in a subject. The first, second, and third containers may comprise the same or different shapes (e.g., vials, syringes, and bottles) and / or materials (e.g., plastic or glass). The kit may further comprise other materials that may be useful for administering the pharmacopoeia, e.g., diluents, filters, IV bags and tubes, needles and syringes. The instructions may state that the pharmacopoeia is intended for use in the treatment of a subject having cancer that has been tested to be positive for PD-L1 by immunohistochemical (IHC) assay, FACS, or LC / MS / MS.

[0202] Further diagnosis, prediction, prognosis, and / or treatment methods This disclosure further provides diagnostic, predictive, prognostic, and / or therapeutic methods using PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors. Such methods are at least in part based on elucidating the characteristics of the expression levels of markers 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 using the therapeutic combination of the present invention.

[0203] Any suitable sample can be used for this method. Non-limiting examples include one or more serum samples, plasma samples, whole blood, pancreatic juice samples, tissue samples, tumor lysates, or tumor samples, which can be isolated from needle biopsies, core biopsies, and needle aspirates. For example, tissue, plasma, or serum samples are collected from patients before and possibly during treatment with the therapeutic combination of the present invention. The expression levels obtained during treatment are compared to values ​​obtained before the patient's treatment began. The information obtained may predict the prognosis in the sense that it can indicate whether the patient responded favorably or unfavorably to cancer therapy.

[0204] It should be understood that the information obtained using the diagnostic assays described herein can be used alone or in combination with other information (such as, but not limited to, the expression levels of other genes, clinical chemical parameters, histopathological parameters, or the age, sex, and weight of the subject). When the information obtained using the diagnostic assays described herein is used alone, it is useful for determining or identifying the clinical outcome of treatment, selecting patients to treat, or treating patients. Conversely, when the information obtained using the diagnostic assays described herein is used in combination with other information, it is useful for assisting in determining or identifying the clinical outcome of treatment, assisting in selecting patients to treat, or assisting in treating patients. In one particular embodiment, expression levels can be used in a diagnostic panel, each of which contributes to the final diagnosis, prognosis, or treatment selected for the patient.

[0205] Using any appropriate method, the levels of PD-L1 protein, DNA, RNA, or other suitable readouts can be measured. Examples thereof are described herein and / or are well known to those skilled in the art.

[0206] In some embodiments, measuring PD-L1 levels involves determining PD-L1 expression. In some embodiments, PD-L1 levels are determined by the concentration of PD-L1 protein in a patient sample, for example, using a ligand (such as an antibody or specific binding partner) specific to PD-L1. Binding events can be detected, for example, by competitive or non-competitive methods. Methods include the use of a labeled ligand, or a moiety specific to PD-L1 (e.g., an antibody), or a labeled competitive moiety that competes with the marker protein for its binding event (including labeled PD-L1 criteria or TGFβ criteria). If the marker-specific ligand can form a complex with PD-L1, the complex formation may indicate PD-L1 expression in the sample. In various embodiments, levels of biomarker proteins are determined by methods including quantitative Western blotting, multiplex immunoassays, ELISA, immunohistochemistry, histochemistry, or FACS analysis of tumor lysates, immunofluorescence staining, bead-based suspension immunoassays, Luminex technology, or proximity ligation assays. In one embodiment, PD-L1 expression is determined by immunohistochemistry using one or more primary anti-PD-L1 antibodies or primary anti-TGFβ antibodies.

[0207] In another embodiment, the level of biomarker RNA is determined by a method including a microarray chip, RT-PCR, qRT-PCR, multiplex qPCR, or in-situ hybridization. In one embodiment of the present invention, the DNA array or RNA array includes a polynucleotide arrangement presented by a PD-L1 gene immobilized on a solid surface, or a polynucleotide arrangement that hybridizes to that gene. For example, limited to elucidating PD-L1 mRNA, the mRNA of a sample can be isolated after sufficient sample preparation steps (e.g., tissue homogenization) if necessary, and hybridized with a marker-specific probe on a microarray platform, with or without amplification, or hybridized with primers for a PCR-based detection method (e.g., PCR extension labeling using a probe specific to a portion of the marker mRNA).

[0208] 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 uses a simple binary endpoint of positive or negative for PD-L1 expression, with a positive result defined as the percentage of tumor cells showing histological evidence of staining of the cell surface membrane.

[0209] The expression level of PD-L1 mRNA can be compared to the mRNA expression levels of one or more reference genes (such as ubiquitin C) frequently used in quantitative RT-PCR. In some embodiments, the level of PD-L1 expression (protein and / or mRNA) by malignant cells and / or infiltrating immune cells within a tumor is determined to be "overexpressed" or "elevated" based on a comparison with the level of PD-L1 expression (protein and / or mRNA) by a suitable control. For example, the control PD-L1 protein or mRNA expression level can be the level quantified in sections from non-malignant cells of the same type or from matched normal tissue.

[0210] In one embodiment, the effectiveness of the therapeutic combination of the present invention is predicted by the expression of PD-L1 in tumor samples. Immunohistochemistry using an anti-PD-L1 primary antibody was performed on serial sections of formalin-fixed paraffin-embedded samples from patients treated with the anti-PD-L1 antibody.

[0211] This disclosure also provides a kit for determining whether the combination of the present invention is suitable for the treatment of cancer patients, comprising means for determining the 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 embodiment, the kit further comprises a PD-L1 antibody for immunotherapy. In one embodiment of the present invention, a determined high level of PD-L1 indicates that when the patient is treated with the therapeutic combination of the present invention, PFS or OS will increase. In one embodiment of the kit, the means for determining the PD-L1 protein level are, respectively, antibodies that specifically bind to PD-L1.

[0212] In yet another aspect, the present invention provides a method for promoting a PD-1 inhibitor in combination with a TGFβ inhibitor and a TIGIT inhibitor, comprising promoting to a target audience the use of the combination to treat subjects having cancer, optionally selected based on PD-L1 expression in a sample obtained from a subject. In yet another aspect, the present invention provides a method for promoting a TIGIT inhibitor in combination with a PD-1 inhibitor and a TGFβ inhibitor, where the PD-1 inhibitor and the TGFβ inhibitor are fused, comprising promoting to a target audience the use of the combination to treat subjects having cancer, optionally selected based on PD-L1 expression in a sample obtained from a subject. In yet another aspect, the present invention provides a method for promoting a TGFβ inhibitor in combination with a PD-1 inhibitor and a TIGIT inhibitor, comprising promoting to a target audience the use of the combination to treat subjects having cancer, optionally selected based on PD-L1 expression in a sample obtained from a subject. In yet another aspect, the present invention provides a method for promoting an anti-PD-L1:TGFβRII fusion protein, for example, a fusion protein having the amino acid sequence of vintrafusp alfa, in combination with an anti-TIGIT antibody, for example, H03-12, comprising promoting to a target audience the use of the combination to treat subjects having cancer, optionally selected based on PD-L1 expression in a sample obtained from a subject. In yet another aspect, the present invention provides a method for promoting a combination of a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor, comprising promoting to a target audience the use of the combination to treat subjects having cancer, optionally selected based on PD-L1 expression in a sample obtained from a subject. Promotion can be carried out by any available means. In some embodiments, promotion is by means of a package insert accompanying a market formulation of the therapeutic combination of the present invention.Furthermore, the promotion may be provided by a package insert accompanying a marketed formulation of a PD-1 inhibitor, a TGFβ inhibitor, or a TIGIT inhibitor or other pharmaceutical (where the treatment is a therapeutic combination of the present invention and further pharmaceuticals). In some embodiments, the promotion is provided by a package insert providing instructions for treatment with the therapeutic combination of the present invention, in some embodiments with another pharmaceutical, after measuring the PD-L1 expression level. In some embodiments, the patient is treated with the therapeutic combination of the present invention, with or without another pharmaceutical, following the promotion. In some embodiments, the package insert indicates that if the patient's cancer sample is characterized by high PD-L1 biomarker levels, the therapeutic combination of the present invention is used to treat the patient. In some embodiments, the package insert indicates that if the patient's cancer sample is characterized by low PD-L1 biomarker levels, the therapeutic combination of the present invention is not used to treat the patient. In some embodiments, high PD-L1 biomarker levels mean that, if the patient is treated with the therapeutic combination of the present invention, the measured PD-L1 levels correlate with the likelihood of increased PFS and / or OS, and vice versa. In some embodiments, PFS and / or OS are reduced compared to patients not treated with the therapeutic combination of the present invention. In some embodiments, the enhancement is provided by a package insert that provides instructions for treatment with an anti-PD-L1:TGFβRII fusion protein in combination with a TIGIT inhibitor, after first measuring PD-L1 expression levels. In some embodiments, the enhancement is followed by treatment of the patient with an anti-PD-L1:TGFβRII fusion protein in combination with a TIGIT inhibitor, with or without another pharmacopoeia.

[0213] All references cited herein are incorporated by reference within the disclosure of the present invention.

[0214] The present invention can be carried out or tested using methods and materials similar to or equivalent to those described herein, but suitable examples are provided below. Within the scope of the examples, standard reagents and buffers that are free of contaminating activity are used (where possible). The examples are not limited to the explicitly demonstrated combinations of features, and the illustrative features should be interpreted as being able to be combined again without limitation as long as the technical problem of the invention is solved. Similarly, features of any claim can be combined with features of one or more other claims. The present invention as described in the abstract and specification is illustrative and is not limited by the following examples. [Examples]

[0215] Example 1: Immune cell activation by combination therapy using anti-TIGIT antibody and vintrafusp alfa. The ability of the anti-TIGIT antibody H03-12 and vintrafusp alfa combination to activate immune cells was evaluated in an allogeneic two-way MLR assay by measuring IFN-γ in the supernatant of co-cultured PBMCs derived from two different human donors two days after treatment. H03-12 dose-dependently enhanced IFN-γ production compared to isotype controls, and EC 50 The concentration was shown to be 158.9 ± 185.0 ng / mL (1.065 ± 1.240 nM) (see Figure 3A). The effect of H03-12 on IFN-γ production was further enhanced by the addition of vintrafusp alfa (see Figure 3B). These results suggest that H03-12 promotes immune cell activation, and that the combination with vintrafusp alfa further enhances this activation.

[0216] The ability of the H03-12 and vintrafusp alfa combination to enhance T cell activation was further investigated in a one-way MLR assay. H03-12 dose-dependently enhanced IFN-γ production in these cells compared to isotype controls, with an EC50 of 136.9 ± 114.6 ng / mL (0.917 ± 0.768 nM) (see Figure 3C). The H03-12 and vintrafusp alfa combination further enhanced T cell activation (see Figure 3D).

[0217] Example 2: Flow cytometry analysis of samples treated with vintrafusp alfa Cell subsets from CT26-KSA tumor-bearing mice were analyzed by flow cytometry after vintrafusp alfa treatment. Vintrafusp alfa monotherapy (24.6 mg / kg) showed a significant antitumor effect in CT26-KSA tumor-bearing mice compared to isotype controls (P=0.0029, day 22) (see Figure 4A). Flow cytometry analysis of spleens and tumors from separate mice treated with vintrafusp alfa showed that the proportion of TIGIT-expressing spleen CD4+ T cells (P<0.0001), CD8+ T cells (P=0.0001), and Tregs (P<0.0001) increased with vintrafusp alfa monotherapy compared to isotype controls. Vintrafusp alfa also tended to increase the proportion of TIGIT+ tumor-infiltrating CD4+ T cells, CD8+ T cells, NK cells, and Tregs (see Figure 4B). This data suggests that increased TIGIT expression on immune subsets induced by vintrafusp alfa therapy may induce resistance to vintrafusp alfa therapy.

[0218] Example 3: Antitumor effect of combination therapy with anti-TIGIT antibody and vintrafusp alfa in a CT26-KSA tumor model in BALB / c mice The antitumor effects of the anti-mouse TIGIT antibody 18G10 and / or vintrafusp alfa, which contain the light chain sequence of SEQ ID NO: 39 and the heavy chain sequence of SEQ ID NO: 40, were investigated in a CT26-KSA tumor model using BALB / c mice. Vintrafusp alfa (24.6 mg / kg) monotherapy resulted in moderate tumor growth inhibition (49.5%) compared to isotype controls (P<0.0001, day 21). 18G10 monotherapy resulted in greater tumor growth inhibition (TGI=85.3%) compared to isotype controls (P<0.0001, day 21). However, the combination of 18G10 and vintrafusp alfa (TGI=110.1%) further enhanced the antitumor effect compared to 18G10 monotherapy (P<0.0001, day 33) or vintrafusp alfa monotherapy (P<0.0001, day 21) (see Figure 5A). In fact, complete tumor regression was observed in 70% of mice treated with the 18G10 and vintrafusp alfa combination therapy on day 33 (7 / 10 mice, see Figure 5C).

[0219] Combination therapy with 18G10 and vintrafusp alfa significantly extended median survival compared to 18G10 monotherapy (50 days, P<0.0001) or vintrafusp alfa monotherapy (35 days, P=0.0002) (uncertain) (see Figure 5B).

[0220] Example 4: Antitumor effect of combination therapy with anti-TIGIT antibody and vintrafusp alfa in an MC38 tumor model in C57BL / 6 mice The antitumor effects of anti-TIGIT antibodies and / or vintrafusp alfa were investigated in an MC38 tumor model using C57BL / 6 mice. Vintrafusp alfa monotherapy (24.6 mg / kg) resulted in tumor growth inhibition (TGI = 40.2%) compared to the isotype control (P < 0.0001, day 24). 18G10 monotherapy resulted in milder tumor growth inhibition (TGI = 18.2%) compared to the isotype control (P = 0.0219, day 24). However, the combination of 18G10 and vintrafusp alfa further enhanced the antitumor effect compared to 18G10 monotherapy (P < 0.0001, day 24) and vintrafusp alfa monotherapy (TGI = 58.8%) (P = 0.0169, day 24) (see Figures 6A and 6C).

[0221] The median survival time was also slightly longer with the combination therapy of 18G10 and vintrafusp alfa (34 days) compared with 18G10 monotherapy (29.5 days) or isotype control (26 days) (see Figure 6B).

[0222] Example 5: Antitumor effect of combination therapy with anti-TIGIT antibody and vintrafusp alfa in an MC38 tumor model using B-huTIGIT knock-in mice The antitumor effect of the combination of the anti-human TIGIT antibody H03-12-muIgG2c and vintrafusp alfa was evaluated in MC38 tumor-carrying B-huTIGIT knock-in mice. Anti-PD-L1 antibodies with the light chain sequence of SEQ ID NO: 7 and SEQ ID NO: 16, respectively, were used as further controls.

[0223] Because H03-12 lacks cross-reactivity with the mouse TIGIT protein, the mouse extracellular domain of TIGIT was replaced with the human extracellular domain of TIGIT in mice with a C57BL / 6 genetic background. Specifically, the coding region of exon 2 of mouse TIGIT, from amino acids 22 to 131, was replaced with a human coding sequence using CRISPR / Cas9 technology. Furthermore, to achieve effector function while avoiding potential immunogenicity in mice, an H03-12 mouse chimeric antibody (H03-12-muIgG2c, in which the human IgG1 fragment crystallizable (Fc) region of H03-12 is replaced with mouse IgG2c Fc) was developed. The light and heavy chain sequences of H03-12-muIgG2c are reflected in SEQ ID NOs. 37 and 38, respectively.

[0224] As reflected in Figure 7 and Table 1, the trap control (a variant of vintrafusp alfa that can no longer bind to PD-L1, with its light and heavy chain sequences reflected in SEQ ID NOs. 47 and 48, respectively) showed no antitumor effect compared to anti-HEL isotype controls and inactive anti-PD-L1 isotype controls. H03-12-muIgG2c monotherapy, anti-PD-L1 monotherapy, and vintrafusp alfa monotherapy all inhibited tumor growth (TGI = 36.65%, 61.87%, and 66.3% at day 32 compared to isotype controls, with P<0.0001 for all three monotherapys), and extended median survival (47, 47, and 51 days, respectively, P=0.01, P=0.0008, and P=0.0004) compared to isotype controls (39 days).

[0225] Tumor growth inhibition was enhanced in the combination of H03-12-muIgG2c and a trap control (TGI = 64.26%) compared to H03-12-muIgG2c monotherapy (P = 0.0055, day 32) and trap control monotherapy (P < 0.0001, day 32). The combination of H03-12-muIgG2c and anti-PD-L1 further enhanced the antitumor effect (TGI=80.07%) compared to H03-12-muIgG2c alone (P=0.0001, day 32) and tended to increase the antitumor effect compared to anti-PD-L1 monotherapy (P<0.2215, day 32). The antitumor effect of the combination of H03-12-muIgG2c and vintrafusp alfa (TGI=88.2%) was enhanced compared to H03-12-muIgG2c monotherapy (P<0.0001, day 32) and tended to increase the antitumor effect compared to vintrafusp alfa monotherapy (P=0.0658, day 32).

[0226] Treatment with H03-12-muIgG2c combined with a trap control, anti-PD-L1, or vintrafusp alfa also extended median survival (51, 54.5, and 74 days, respectively). On day 39, the mean tumor volume for treatment with H03-12-muIgG2c combined with a trap control, anti-PD-L1, or vintrafusp alfa was 437.58 mm².3 , 285.35 mm 3 , and 193.19 mm 3 That was the case.

[0227] [Table 1]

[0228] Combined, the combination therapy using H03-12-muIgG2c and vintrafusp alfa enhanced antitumor activity and extended survival compared to each monotherapy. The efficacy of the H03-12-muIgG2c and vintrafusp alfa combination was superior to that of the H03-12-muIgG2c and anti-PD-L1 combination.

[0229] Example 6: TIGIT / CD226 expression changes in the tumor microenvironment (TME) after anti-TIGIT antibody therapy and vintrafusp alfa therapy. To understand the mechanism of action (MOA) of the antitumor effect of H03-12-muIgG2c + vintrafusp alfa therapy, changes in TIGIT / CD226 expression in the tumor microenvironment (TME) after H03-12-muIgG2c treatment and vintrafusp alfa treatment were investigated using flow cytometry analysis. Anti-PD-L1 antibodies with the light chain sequence of SEQ ID NO: 7 and SEQ ID NO: 16, and the heavy chain sequence of SEQ ID NO: 16, respectively, were used as controls.

[0230] In subsets of CD4+ T cells, CD8+ T cells, and Treg cells, H03-12-muIgG2c dramatically reduced TIGIT expression compared to isotype controls, while trap controls and anti-PD-L1 tended to increase TIGIT expression, and vintrafusp alfa treatment significantly increased TIGIT expression. Adding H03-12-muIgG2c treatment to anti-PD-L1 or vintrafusp alfa treatment can reduce the risk of Treg activation initiated by anti-PD-L1 or vintrafusp alfa monotherapy (see Figure 8A).

[0231] Monotherapy with H03-12-muIgG2c, a trap control, anti-PD-L1, and vintrafusp alfa tended to increase CD226 expression in subsets of CD4+ T cells, CD8+ T cells, and Treg cells. Compared to the combinations of H03-12-muIgG2c + trap control and H03-12-muIgG2c + anti-PD-L1, treatment with H03-12-muIgG2c + vintrafusp alfa tended to increase CD226 expression (see Figure 8B).

[0232] The dual combinations of H03-12-muIgG2c + trap control, H03-12-muIgG2c + anti-PD-L1, and H03-12-muIgG2c + vintrafusp alfa all promoted the expression of both TIGIT and CD226 in subsets of CD4+ T cells, CD8+ T cells, and Treg cells compared to isotype controls. The ratio of CD226 expression to TIGIT expression in the three dual combination groups in the immune subsets was the same as the ratio observed after H03-12-muIgG2c monotherapy, but greater than that of monotherapy with trap control, anti-PD-L1, and vintrafusp alfa (see Figure 8C).

[0233] These data suggest that adding H03-12-muIgG2c to vintrafusp alfa can reverse the increase in TIGIT expression caused by vintrafusp alfa monotherapy and further increase the ratio of CD226 expression to TIGIT expression in immune cells. Although TIGIT and CD226 compete for the same receptors CD155 and CD112, they exhibit opposite immunomodulatory effects. Specifically, TIGIT expression can suppress the proliferation and cytotoxicity of immune cells, while CD226 can promote immune activation and killing effects. The increased ratio of CD226 expression to TIGIT expression indicates a polarization of immunomodulation from the immunosuppressive TIGIT pathway to the immunoactivating CD226 pathway.

[0234] Example 7: Tumor Invasion Immunoprofen in MC38 Tumors of B-huTIGIT Knock-in Mice After Anti-TIGIT Antibody Therapy and Vintrafusp Alpha Therapy Immunophenotypic signatures in TME after H03-12-muIgG2c treatment and vintrafusp alfa treatment were examined by flow cytometry. Anti-PD-L1 antibodies with light chain and heavy chain sequences of SEQ ID NO: 7 and SEQ ID NO: 16, respectively, were used as controls.

[0235] Compared to isotype controls, monotherapy with trap controls, anti-PD-L1, and H03-12-muIgG2c slightly increased CD8+ T cell infiltration, while vintrafusp alfa monotherapy significantly increased CD8+ T cell infiltration. The H03-12-muIgG2c + trap control combination therapy promoted CD8+ T cell infiltration compared to any of the monotherapy arms, while the H03-12-muIgG2c + anti-PD-L1 combination did not further increase CD8+ T cell infiltration. The amount of infiltrating CD8+ T cells in the H03-12-muIgG2c + vintrafusp alfa combination group was similar to that in the vintrafusp alfa monotherapy group, but greater than that in the H03-12-muIgG2c + anti-PD-L1 combination. All monotherapy and combination therapy groups also promoted Treg infiltration to some degree. The combination therapy of H03-12-muIgG2c + vintrafusp alfa significantly increased the ratio of CD8+ T cells to Tregs compared to the combination of H03-12-muIgG2c + trap control, the combination of H03-12-muIgG2c + anti-PD-L1, and either monotherapy (see Figure 9A).

[0236] Compared to isotype controls, monotherapy with trap controls, anti-PD-L1, and H03-12-muIgG2c tended to increase cytotoxicity, while treatment with vintrafusp alfa significantly increased cytotoxicity of CD4+ T cells, CD8+ T cells, and NK cells. The combination of H03-12-muIgG2c and trap controls enhanced cytotoxicity in immune subsets compared to any of the monotherapys. The combination of H03-12-muIgG2c and trap controls did not further promote cytotoxicity of immune cells. The cytotoxicity of the H03-12-muIgG2c + vintrafusp alfa combination was the same as that of vintrafusp alfa monotherapy, but stronger than that of the H03-12-muIgG2c + anti-PD-L1 combination (Figure 8B).

[0237] An increased ratio of CD8+ T cells to Treg cells, along with increased cytotoxicity of T cells and NK cells, indicated that TMEs were converted from immunosuppressive to a more immunotolerant phenotype after combination therapy with H03-12-muIgG2c + vintrafusp alfa.

[0238] In summary, within the range of the H03-12-muIgG2c and vintrafusp alfa combination, each monotherapy agent may contribute to the enhancement of antitumor immunity in a complementary manner. The complementary mechanisms of H03-12 and vintrafusp alfa work together to produce organized antitumor activity.

[0239] Example 8: Research on Second Chance A re-challenge study was conducted in MC38 tumor-carrying B-huTIGIT knock-in mice that showed complete tumor regression for at least 3 months after combination therapy with H03-12-muIgG2c and vintrafusp alfa. Mice that were "cured" after combination therapy with H03-12-muIgG2c and vintrafusp alfa (n=4) were challenged with MC38 tumor cells on the opposite side of the initial injection site. None of these mice developed tumors for at least 36 days (0 / 4, 0%), whereas all naive B-huTIGIT knock-in mice injected with MC38 cells (n=10) developed tumors (10 / 10, 100%) (see Figure 10). These results suggest that combination therapy with H03-12-muIgG2c and vintrafusp alfa provides tumor antigen-specific protective immunity in B-huTIGIT knock-in mice.

[0240] Example 9: Repeated dose escalation study of TIGIT inhibitors and anti-PD-L1:TGFβRII fusion protein in participants with metastatic tumors or locally advanced unresectable solid tumors. The purpose of this study is to investigate the safety, tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of the combination administration of the anti-TIGIT antibody H03-12 and the anti-PD-L1:TGFβRII fusion protein vintrafusp alfa.

[0241] Study participants receive intravenous infusions of H03-12 at increasing doses on day 1 of each cycle (each cycle consisting of 14 days) every two weeks until they reach their maximum tolerated dose (MTD) or an exacerbation is observed (parts 1A and 1B of the study). In addition, in part 1B of the study, patients receive intravenous infusions of vintrafusp alfa at day 1 of each cycle every two weeks until an exacerbation is observed.

[0242] The selection criteria for research participants are as follows: - Participants have histologically or cytologically proven locally advanced or advanced solid malignancies that are refractory or progressing under standard treatment and for which there are no other treatment options known to provide clinical benefit. - Participants with an Eastern Cooperative Oncology Group Performance Status (ECOGPS) of 0-1 at the time of screening. - Participants with an average life expectancy of at least 12 weeks - Participants with measurable diseases according to the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) - Sufficient blood, liver, and kidney function as specified in the protocol. - Other protocols specified in the selection criteria may apply. The exclusion criteria for study participants are as follows: - Participants whose grade of persistent toxicity related to previous treatment is greater than 1 according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v 5.0, but whose grade of alopecia, sensory neuropathy is 2 or less, or whose other non-immune-related grades are 2 or less and do not constitute a safety risk. - Participants with a history of previous organ transplants, including allogeneic stem cell transplants. - Participants with a prior toxicity grade of 3 or higher on the NCICTCAE v 5.0 scale related to immune checkpoint inhibitors (except in cases where the grade was 1 or lower before inclusion in the study). - Participants who currently have a significant cardiac conduction abnormality (including a prolonged corrected QT interval (QTcF, corrected using the Fridericia correction formula) of more than 450 milliseconds (ms), or impaired cardiovascular function, ventricular tachycardia, or hypokalemia), or a history of paroxysmal atrial fibrillation, serious arrhythmias, or a family history of sudden death or long QT syndrome, i.e., vascular, cardiovascular, or cerebrovascular disorders, cerebrovascular attack / stroke (within 6 months prior to registration), myocardial infarction (within 6 months prior to registration), unstable angina, congestive heart failure (New York Heart Association Classification Class II or higher), deep vein thrombosis (within 3 months prior to registration), or pulmonary embolism / embolism (within 3 months prior to registration). - Other protocols specified in the exclusion criteria may apply.

[0243] The primary outcome measures (in both Part 1A and Part 1B of the study) include the following: - Occurrence of dose-restricted toxicity (DLT) during the DLT observation period (28 days) - Occurrence of adverse events (TEAEs) and treatment-related adverse events (TRAEs) during investigational treatments, according to the National Cancer Institute Common Terminology Criteria of Adverse Events (NCI-CTCAE) version 5. - Number of participants who experienced TEAE, based on severity and death. - Changes from baseline in clinical laboratory measurements - Changes from baseline in electrocardiogram (ECG) - Changes from baseline in vital signs - Changes from baseline in ECOGPS

[0244] The following are included as secondary outcome measures (in both Part 1A and Part 1B of the study): - Area below the H03-12 serum concentration-time curve from time zero to the last sampling time (AUC 0-t). - Area below the serum concentration-time curve for H03-12 from time zero to infinity (AUC 0-inf) - Area below the H03-12 serum concentration-time curve from time zero to tau (τ) (AUCτ) for one dosing interval. - Maximum observed serum concentration (Cmax) in H03-12 - Serum concentration observed immediately before the next dose of H03-12 (Ctrough) - Time until H03-12 reaches its maximum serum concentration (Tmax) - Apparent terminal phase half-life (t1 / 2) of H03-12 - Excretion rate constant of H03-12 - Immunogenicity of H03-12 as measured by anti-drug antibody (ADA) assay - Change from baseline in the QT interval - Best overall result as assessed by the researcher according to the Response Criteria in Solid Tumors Version 1.1 (RECIST 1.1) - Duration of response as assessed by the researcher according to RECIST 1.1 - Time to response to tumor, as assessed by the investigator according to RECIST 1.1. - Disease control as assessed by the investigator according to RECIST 1.1 - Progression-free survival as assessed by the researcher according to RECIST 1.1 - Overall survival.

[0245] Furthermore, Part 1B of the study includes the following secondary outcome measures: - Maximum observed serum concentration (Cmax) of vintrafusp alfa - Serum concentration (Ctrough) observed immediately before the next dose of vintrafusp alfa

[0246] Further embodiments of this disclosure are as follows: 1. PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors for use in a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor, TGFβ inhibitor, and TIGIT inhibitor to the subject.

[0247] 2. PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors for use in a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor, TGFβ inhibitor, and TIGIT inhibitor to the subject; and, PD-1 inhibitors are anti-PD(L)1 antibodies, TGFβ inhibitors are TGFβRII or anti-TGFβ antibodies, and TIGIT inhibitors are anti-TIGIT antibodies; these are PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors.

[0248] 3. PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors for use in a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor, TGFβ inhibitor, and TIGIT inhibitor to the subject; and, PD-1 inhibitors and TGFβ inhibitors are fused as anti-PD(L)1:TGFβRII fusion proteins, and TIGIT inhibitors are anti-TIGIT antibodies.

[0249] 4. A PD-1 inhibitor for use in a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor to the subject in combination with a TGFβ inhibitor and a TIGIT inhibitor.

[0250] 5. A TGFβ inhibitor for use in a method of treating cancer in a subject, the method comprising administering the TGFβ inhibitor to the subject in combination with a PD-1 inhibitor and a TIGIT inhibitor.

[0251] 6. A TIGIT inhibitor for use in a method of treating cancer in a subject, the method comprising administering the TIGIT inhibitor to the subject in combination with a PD-1 inhibitor and a TGFβ inhibitor.

[0252] 7. A PD-1 inhibitor and a TGFβ inhibitor for use in a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor and the TGFβ inhibitor to the subject in combination with a TIGIT inhibitor; and the PD-1 inhibitor and the TGFβ inhibitor being fused.

[0253] 8. A method of treating cancer in a subject, the method comprising administering a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor to the subject.

[0254] 9. A method of treating cancer in a subject, the method comprising administering a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor to the subject; wherein the PD-1 inhibitor is an anti-PD(L)1 antibody, the TGFβ inhibitor is a TGFβRII or an anti-TGFβ antibody, and the TIGIT inhibitor is an anti-TIGIT antibody.

[0255] 10. A method of treating cancer in a subject, the method comprising administering a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor to the subject; wherein the PD-1 inhibitor and the TGFβ inhibitor are fused as an anti-PD(L)1:TGFβRII fusion protein, and the TIGIT inhibitor is an anti-TIGIT antibody.

[0256] 11. Use of a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor in the manufacture of a medicament for a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor, the TGFβ inhibitor, and the TIGIT inhibitor to the subject.

[0257] 12. Use of PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors for the manufacture of a pharmacopoeia for a method of treating cancer in a subject, the method comprising administering a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor to a subject; wherein the PD-1 inhibitor is an anti-PD(L)1 antibody, the TGFβ inhibitor is TGFβRII or an anti-TGFβ antibody, and the TIGIT inhibitor is an anti-TIGIT antibody.

[0258] 13. Use of PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors for the manufacture of a pharmacopoeia for a method of treating cancer in a subject, the method comprising administering PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors to a subject; wherein the PD-1 inhibitor and TGFβ inhibitor are fused as anti-PD(L)1:TGFβRII fusion proteins, and the TIGIT inhibitor is an anti-TIGIT antibody.

[0259] 14. Use of a PD-1 inhibitor for the manufacture of a pharmaceutical product for a method of treating cancer in a subject, wherein the method comprises administering the PD-1 inhibitor to the subject in combination with a TGFβ inhibitor and a TIGIT inhibitor.

[0260] 15. Use of a TGFβ inhibitor for the manufacture of a medicament for a method of treating cancer in a subject, wherein the method comprises administering the TGFβ inhibitor to the subject in combination with a PD-1 inhibitor and a TIGIT inhibitor.

[0261] 16. Use of a TIGIT inhibitor for the manufacture of a medicament for a method of treating cancer in a subject, wherein the method comprises administering the TIGIT inhibitor to the subject in combination with a PD-1 inhibitor and a TGFβ inhibitor.

[0262] 17. Use of PD-1 inhibitors and TGFβ inhibitors for the manufacture of a pharmacopoeia for a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor and TGFβ inhibitor in combination with a TIGIT inhibitor to the subject; wherein the PD-1 inhibitor and TGFβ inhibitor are fused.

[0263] 18. PD-1 inhibitors are compounds, therapeutic methods, or uses described in any one of items 1 to 17 that can inhibit the interaction between PD-1 and PD-L1.

[0264] 19. PD-1 inhibitors are anti-PD(L)1 antibodies, compounds for use, methods of treatment, or uses as described in item 18.

[0265] 20. PD-1 inhibitors are anti-PD-L1 antibodies, compounds for use, methods of treatment, or uses as described in item 19.

[0266] 21. The anti-PD-L1 antibody comprises a heavy chain sequence having the sequence of SEQ ID NO: 1, CDRH2 having the sequence of SEQ ID NO: 2, and CDRH3 having the sequence of SEQ ID NO: 3, and a light chain sequence having the sequence of SEQ ID NO: 4, CDRL1 having the sequence of SEQ ID NO: 5, and CDRL3 having the sequence of SEQ ID NO: 6, as described in item 20 for use, method of treatment, or use.

[0267] 22. A TGFβ inhibitor is a compound, therapeutic method, or use for use as described in any one of items 1 to 21, which can inhibit the interaction between TGFβ and the TGFβ receptor.

[0268] 23. A TGFβ inhibitor is a TGFβ receptor or fragment thereof capable of binding to TGFβ, a compound for use, method of treatment, or use as described in any one of items 1 to 22.

[0269] 24. The TGFβ receptor is a TGFβ receptor II or a fragment thereof that can bind to TGFβ, as described in item 23 for the use, method of treatment, or use.

[0270] 25. The TGFβ receptor is the extracellular domain of TGFβ receptor II or a fragment thereof that is capable of binding to TGFβ, as described in item 24 for the use of the compound, therapeutic method, or use.

[0271] 26. The TGFβ inhibitor has at least 80%, 90%, 95%, or 100% sequence identity to any one of the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and is capable of binding to TGFβ. It is a compound, treatment method, or use for the use described in any one of Items 1 to 25.

[0272] 27. 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 to TGFβ. It is a compound, treatment method, or use for the use described in any one of Items 1 to 26.

[0273] 28. The TGFβ inhibitor contains any one of the sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. It is a compound, treatment method, or use for the use described in any one of Items 1 to 25.

[0274] 29. The TGFβ inhibitor contains the sequence of SEQ ID NO: 11. It is a compound, treatment method, or use for the use described in Item 28.

[0275] 30. The PD-1 inhibitor and the TGFβ inhibitor are fused. It is a compound, treatment method, or use for the use described in any one of Items 1 to 29.

[0276] 31. The PD-! inhibitor and the TGFβ inhibitor are fused to a molecule containing (a) an antibody or a fragment thereof that is capable of binding to PD-L1 and inhibiting the interaction between PD-1 and PD-L1, and (b) the extracellular domain of TGFβRII or a fragment thereof that is capable of binding to TGFβ and inhibiting the interaction between TGFβ and the TGFβ receptor. It is a compound, treatment method, or use for the use described in any one of Items 1 to 30.

[0277] 32. The fusion molecule is one of each fusion molecule disclosed in WO2015 / 118175 or WO2018 / 205985. It is a compound, treatment method, or use for the use described in Item 31.

[0278] 33. The extracellular domain of TGFβRII or a fragment thereof is fused to each of the heavy chain sequences of the antibody or fragment thereof, as described in item 31 for use, a compound for use, a therapeutic method, or use.

[0279] 34. The fusion between the extracellular domain of TGFβRII or a fragment thereof and the heavy chain sequence of the antibody or fragment thereof occurs via a linker sequence, as described in item 33 for the use of the compound, therapeutic method, or use.

[0280] 35. The amino acid sequence of the light chain sequence and the sequence comprising the heavy chain sequence and the extracellular domain of TGFβRII or a fragment thereof correspond to sequences selected from the group consisting of (1) SEQ ID NOs: 7 and SEQ ID NOs: 8, (2) SEQ ID NOs: 15 and SEQ ID NOs: 17, and (3) SEQ ID NOs: 15 and SEQ ID NOs: 18, respectively, for the compounds, therapeutic methods, or uses described in item 34.

[0281] 36. A compound, therapeutic method, or use for use as described in any one of items ~35, wherein a PD-1 inhibitor and a TGFβ inhibitor are fused, and the fusion protein has at least 80%, 90%, 95%, or 100% sequence identity with respect to the amino acid sequence of vintrafusp alfa.

[0282] 37. A PD-1 inhibitor and a TGFβ inhibitor are fused, and this fusion protein is vintrafusp alfa, a compound for use, therapeutic method, or use as described in any one of items ~35.

[0283] 38. A TIGIT inhibitor is an anti-TIGIT antibody, a compound for use, a method of treatment, or use as described in any one of items 1 to 37.

[0284] 39. The anti-TIGIT antibody has at least 80%, 90%, 95%, or 100% sequence identity with tiragolumab, MK-7684, and one amino acid sequence of the antibody whose light chain sequence and heavy chain sequence correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively, for use as described in item [item], a compound for use, a therapeutic method, or use.

[0285] 40. The light chain variable region and heavy chain region of the anti-TIGIT antibody correspond to SEQ ID NO: 29 and SEQ ID NO: 30, respectively, or the anti-TIGIT antibody comprises a heavy chain sequence including CDRH1 having the sequence of SEQ ID NO: 31, CDRH2 having the sequence of SEQ ID NO: 32, and CDRH3 having the sequence of SEQ ID NO: 33, and a light chain sequence including CDRL1 having the sequence of SEQ ID NO: 34, CDRL2 having the sequence of SEQ ID NO: 35, and CDRL3 having the sequence of SEQ ID NO: 36, as described in item 40. Compounds for use, therapeutic methods, or uses as described in item 40. The light chain variable region and heavy chain region of the anti-TIGIT antibody correspond to SEQ ID NO: 29 and SEQ ID NO: 30, respectively, or the anti-TIGIT antibody comprises a heavy chain sequence including CDRH1 having the sequence of SEQ ID NO: 31, CDRH2 having the sequence of SEQ ID NO: 32, and CDRH3 having the sequence of SEQ ID NO: 33, and a light chain sequence including CDRL1 having the sequence of SEQ ID NO: 34, CDRL2 having the sequence of SEQ ID NO: 35, and CDRL3 having the sequence of SEQ ID NO: 36.

[0286] 41. A TIGIT inhibitor for use in a method of treating cancer in a subject, the method comprising administering the TIGIT inhibitor to the subject in combination with a PD-1 inhibitor and a TGFβ inhibitor; and, The PD-1 inhibitor and the TGFβ inhibitor are fused, and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of vintrafusp alfa; and, TIGIT inhibitors are anti-TIGIT antibodies whose light chain sequence and heavy chain sequence correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0287] 42. PD-1 inhibitors and TGFβ inhibitors for use in a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor and TGFβ inhibitor in combination with a TIGIT inhibitor to the subject; and, The PD-1 inhibitor and the TGFβ inhibitor are fused, and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of vintrafusp alfa; and, TIGIT inhibitors are PD-1 inhibitors and TGFβ inhibitors that are anti-TIGIT antibodies whose light chain sequence and heavy chain sequence correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0288] 43. A method for treating cancer in a subject, the method comprising administering a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor to the subject; and, The PD-1 inhibitor and the TGFβ inhibitor are fused, and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of vintrafusp alfa; and, The TIGIT inhibitor is an anti-TIGIT antibody whose light chain sequence and heavy chain sequence correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0289] 44. Use of PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors for the manufacture of a pharmacopoeia for a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor, TGFβ inhibitor, and TIGIT inhibitor to the subject; The PD-1 inhibitor and the TGFβ inhibitor are fused, and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of vintrafusp alfa; and, TIGIT inhibitors are anti-TIGIT antibodies whose light chain sequence and heavy chain sequence correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0290] 45. Use of a TIGIT inhibitor for the manufacture of a medicament for a method of treating cancer in a subject, the method comprising administering the TIGIT inhibitor to the subject in combination with a PD-1 inhibitor and a TGFβ inhibitor; The PD-1 inhibitor and the TGFβ inhibitor are fused, and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of vintrafusp alfa; and, TIGIT inhibitors are anti-TIGIT antibodies whose light chain sequence and heavy chain sequence correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0291] 46. ​​Use of PD-1 inhibitors and TGFβ inhibitors for the manufacture of a pharmacopoeia for a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor and TGFβ inhibitor to the subject in combination with a TIGIT inhibitor; The PD-1 inhibitor and the TGFβ inhibitor are fused, and the amino acid sequence of the fusion molecule corresponds to the amino acid sequence of vintrafusp alfa; and, TIGIT inhibitors are anti-TIGIT antibodies whose light chain sequence and heavy chain sequence correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0292] 47. Cancer is a compound, treatment method, or use described in any one of items 1 to 46, selected from the group consisting of carcinoma, lymphoma, leukemia, blastoma, and sarcoma.

[0293] 48. Cancer is selected from the group consisting of squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal (tubal) cancer, kidney 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, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, biliary tract cancer, and head and neck cancer, and is a compound for use, treatment method, or use as described in any one of items 1 to 47.

[0294] 49. PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors are compounds, therapeutic methods, or uses described in any one of items 1 to 48, administered in the first-line treatment of cancer.

[0295] 50. The subject is a compound, treatment method, or use for use described in any one of items 1 to 48, who has previously received at least one cancer treatment.

[0296] 51. Cancer that is resistant to or has become resistant to previous cancer therapies, the compound, treatment method, or use described in item 50.

[0297] 52. PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors are compounds, therapeutic methods, or uses described in any one of items 1 to 48, administered in the second-line or higher-level treatment of cancer.

[0298] 53. The cancer is selected from the group consisting of previously treated recurrent metastatic NSCLC, unresectable locally advanced NSCLC, previously treated SCLC ED, SCLC unsuitable for systemic treatment, previously treated recurrent or metastatic SCCHN, re-irradiable recurrent SCCHN, and previously treated microsatellite-unstable-L (MSI-L) or microsatellite-stable-stable (MSS) metastatic colorectal cancer (mCRC), and the compounds, treatment methods, or uses for use as described in item 52.

[0299] 54. A compound, therapeutic method, or use for use described in any one of items 1 to 53, in which a PD-L1 inhibitor and a TGFβ inhibitor are fused and administered by intravenous infusion.

[0300] 55. A compound, therapeutic method, or use for use described in any one of items 1 to 54, in which a PD-L1 inhibitor and a TGFβ inhibitor are fused and administered in a dose of approximately 1200 mg or approximately 2400 mg.

[0301] 56. A compound, treatment method, or use described in any one of items 1 to 54, in which a PD-L1 inhibitor and a TGFβ inhibitor are fused and administered in doses of approximately 1200 mg once every two weeks or approximately 2400 mg once every three weeks.

[0302] 57. TIGIT inhibitors are administered by intravenous infusion, as described in any one of items 1 to 56, for use, treatment, or use.

[0303] 58. TIGIT inhibitors are administered in doses of approximately 300 mg, approximately 900 mg, or approximately 1600 mg, as described in any one of items 1 to 57, for use, treatment, or use.

[0304] 59. TIGIT inhibitors are administered in doses of approximately 300 mg once every two weeks, approximately 900 mg once every two weeks, approximately 1600 mg once every two weeks, approximately 300 mg once every three weeks, approximately 900 mg once every three weeks, or approximately 1600 mg once every three weeks, as described in any one of items 1 to 58.

[0305] 60. Compounds, therapeutic methods, or uses described in any one of items 1 to 59, including an induction period, and in some cases, a duration of action continuing after the induction period.

[0306] 61. The compounds, therapeutic methods, or uses described in item 60, wherein the compound agents are administered concurrently during either the induction or sustained period, and possibly non-concurrently during the other period, or these compounds are administered non-concurrently during the induction and sustained periods, or two or more of the compounds are administered concurrently during the induction and sustained periods, with the others administered non-concurrently.

[0307] 62. Co-administration of compounds, therapeutic methods, or uses for use described in item 61, occurring sequentially, in any order, or substantially simultaneously.

[0308] 63. A compound, therapeutic method, or use described in any one of items 60-62, in which a PD-1 inhibitor and a TGFβ inhibitor are fused, and the duration of use includes administration of the fused PD-1 inhibitor and TGFβ inhibitor alone, or in combination with a TIGIT inhibitor.

[0309] 64. The induction period includes the co-administration of PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors, and any of the compounds, therapeutic methods, or uses described in any one of items 60-63.

[0310] 65. A compound, therapeutic method, or use described in any one of items 1 to 64, selected based on PD-L1 expression in a sample obtained from a subject.

[0311] 66. A pharmaceutical composition comprising a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor, and at least a pharmaceutically acceptable excipient or adjuvant.

[0312] 67. A pharmaceutical composition comprising a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor and at least a pharmaceutically acceptable excipient or adjuvant; A pharmaceutical composition in which the PD-1 inhibitor is an anti-PD(L)1 antibody, the TGFβ inhibitor is TGFβRII or an anti-TGFβ antibody, and the TIGIT inhibitor is an anti-TIGIT antibody.

[0313] 68. A pharmaceutical composition comprising a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor and at least a pharmaceutically acceptable excipient or adjuvant; A pharmaceutical composition in which a PD-1 inhibitor and a TGFβ inhibitor are fused as an anti-PD(L)1:TGFβRII fusion protein, and the TIGIT inhibitor is an anti-TIGIT antibody.

[0314] 69. A pharmaceutical composition comprising a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor and at least a pharmaceutically acceptable excipient or adjuvant; A pharmaceutical composition comprising a PD-1 inhibitor and a TGFβ inhibitor fused as an anti-PD(L)1:TGFβRII fusion protein having the amino acid sequence of vintrafusp alfa, and a TIGIT inhibitor being an anti-TIGIT antibody whose light chain sequence and heavy chain sequence correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0315] 70. A pharmaceutical composition according to any one of items 66 to 69, for use in therapy, for example, in the treatment of cancer.

[0316] 71. PD-1 inhibitors; and, Package insert including instructions for using the PD-1 inhibitor in combination with a TIGIT inhibitor and a TGFβ inhibitor to treat or delay the progression of cancer in the target population; A kit that includes this.

[0317] 72. TIGIT inhibitors; and, Package insert including instructions for using the TIGIT inhibitor in combination with a PD-1 inhibitor and a TGFβ inhibitor to treat or delay the progression of cancer in the target population; A kit that includes this.

[0318] 73. TGFβ inhibitors; and, Package insert including instructions for using the TGFβ inhibitor in combination with a PD-1 inhibitor and a TIGIT inhibitor to treat or delay the progression of cancer in the target population; A kit that includes this.

[0319] 74. PD-1 inhibitors; and, Package insert including instructions for using the PD-1 inhibitor in combination with a TIGIT inhibitor and a TGFβ inhibitor to treat or delay the progression of cancer in the target population; A kit that includes, The kit contains a PD-1 inhibitor (an anti-PD(L)1 antibody), a TGFβ inhibitor (TGFβRII or an anti-TGFβ antibody), and a TIGIT inhibitor (an anti-TIGIT antibody).

[0320] 75. TIGIT inhibitors; and, Package insert including instructions for using the TIGIT inhibitor in combination with a PD-1 inhibitor and a TGFβ inhibitor to treat or delay the progression of cancer in the target population; A kit that includes, The kit contains a PD-1 inhibitor (an anti-PD(L)1 antibody), a TGFβ inhibitor (TGFβRII or an anti-TGFβ antibody), and a TIGIT inhibitor (an anti-TIGIT antibody).

[0321] 76. TGFβ inhibitors; and, Package insert including instructions for using the TGFβ inhibitor in combination with a PD-1 inhibitor and a TIGIT inhibitor to treat or delay the progression of cancer in the target population; A kit that includes, The kit contains a PD-1 inhibitor (an anti-PD(L)1 antibody), a TGFβ inhibitor (TGFβRII or an anti-TGFβ antibody), and a TIGIT inhibitor (an anti-TIGIT antibody).

[0322] 77. A kit comprising a PD-1 inhibitor, a TGFβ inhibitor, and a package insert containing instructions for using the PD-1 inhibitor and the TGFβ inhibitor in combination with a TIGIT inhibitor to treat or delay the progression of cancer in a subject, This kit contains a PD-1 inhibitor and a TGFβ inhibitor fused as an anti-PD(L)1:TGFβRII fusion protein, and a TIGIT inhibitor as an anti-TIGIT antibody.

[0323] 78. A kit as described in any one of items 71-77, wherein the package insert states that the drug is intended for use in the treatment of subjects with cancer that is tested to be positive for PD-L1 expression.

[0324] 79. A method for promoting a PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor, comprising promoting to a target audience the use of the combination to treat subjects having cancer, for example, cancer selected based on PD-L1 expression in a sample obtained from a subject.

[0325] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] Table 2-5 Table 2-6 Table 2-7 Table 2-8

Claims

1. PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors for use in methods of treating cancer in the subject, The method includes administering PD-1 inhibitors, TGFβ inhibitors, and TIGIT inhibitors to the target population. PD-1 inhibitors are anti-PD-L1 antibodies or fragments thereof that can bind to PD-L1; TGFβ inhibitors are TGFβRII or fragments thereof that can bind to TGFβ, or anti-TGFβ antibodies or fragments thereof that can bind to TGFβ; and TIGIT inhibitors are anti-TIGIT antibodies or fragments thereof that can bind to TIGIT.

2. The anti-PD-L1 antibody or fragment thereof comprises a heavy chain sequence including CDRH1 having the sequence of SEQ ID NO: 1, CDRH2 having the sequence of SEQ ID NO: 2, and CDRH3 having the sequence of SEQ ID NO: 3, and a light chain sequence including CDRL1 having the sequence of SEQ ID NO: 4, CDRL2 having the sequence of SEQ ID NO: 5, and CDRL3 having the sequence of SEQ ID NO: 6; or The anti-PD-L1 antibody or fragment thereof comprises a heavy chain sequence including CDRH1 having the sequence of SEQ ID NO: 19, CDRH2 having the sequence of SEQ ID NO: 20, and CDRH3 having the sequence of SEQ ID NO: 21, and a light chain sequence including CDRL1 having the sequence of SEQ ID NO: 22, CDRL2 having the sequence of SEQ ID NO: 23, and CDRL3 having the sequence of SEQ ID NO:

24. The compound for use as described in claim 1.

3. The compound for use according to claim 1 or 2, wherein the TGFβ inhibitor is the extracellular domain of TGFβRII or a fragment thereof that can bind to TGF-β.

4. The compound for use according to any one of claims 1 to 3, wherein the PD-1 inhibitor and the TGFβ inhibitor are fused as an anti-PD-L1:TGFβRII fusion protein.

5. The compound for use according to claim 4, wherein the light chain sequence and heavy chain sequence of the anti-PD-L1:TGFβRII fusion protein have at least 90% sequence identity with light chain sequences and heavy chain sequences selected from the group consisting of (1) SEQ ID NOs: 7 and SEQ ID NOs: 8, (2) SEQ ID NOs: 15 and SEQ ID NOs: 17, and (3) SEQ ID NOs: 15 and SEQ ID NOs:

18.

6. The compound for use according to claim 4, wherein the amino acid sequence of the anti-PD-L1:TGFβRII fusion protein corresponds to the amino acid sequence of vintrafusp alfa.

7. The compound for use according to any one of claims 4 to 6, wherein the anti-PD-L1:TGFβRII fusion protein is administered in a dose of 1200 mg once every two weeks or 2400 mg once every three weeks.

8. The compound for use according to any one of claims 1 to 7, wherein the TIGIT inhibitor is an anti-TIGIT antibody whose heavy chain comprises the amino acid sequences of SEQ ID NO: 31 (CDRH1), SEQ ID NO: 32 (CDRH2), and SEQ ID NO: 33 (CDRH3), and whose light chain comprises the amino acid sequences of SEQ ID NO: 34 (CDRL1), SEQ ID NO: 35 (CDRL2), and SEQ ID NO: 36 (CDRL3).

9. The compound for use according to claim 8, wherein the TIGIT inhibitor is an anti-TIGIT antibody whose light chain sequence and heavy chain sequence have at least 90% sequence identity with the light chain sequence and heavy chain sequence of SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

10. The compound for use according to any one of claims 1 to 9, wherein the TIGIT inhibitor is administered in doses of approximately 300 mg once every two weeks, approximately 900 mg once every two weeks, approximately 1600 mg once every two weeks, approximately 300 mg once every three weeks, approximately 900 mg once every three weeks, or approximately 1600 mg once every three weeks.

11. A PD-1 inhibitor, a TGFβ inhibitor, and a TIGIT inhibitor for use in a method of treating cancer in a subject, the method comprising administering the PD-1 inhibitor, the TGFβ inhibitor, and the TIGIT inhibitor to the subject, PD-1 inhibitors and TGFβ inhibitors are fused to molecules having the amino acid sequence of vintrafusp alfa, and TIGIT inhibitors are anti-TIGIT antibodies whose light chain sequence and heavy chain sequence correspond to SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

12. The compound for use according to any one of claims 1 to 11, 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 lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal (tubal) cancer, kidney 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, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, biliary tract cancer, and head and neck cancer.

13. Anti-PD-L1:TGFβRII fusion protein; and, Package insert including instructions for using the anti-PD-L1:TGFβRII fusion protein in combination with an anti-TIGIT antibody or fragment thereof that can bind to TIGIT, in order to treat or delay the progression of cancer in the subject; A kit that includes this.

14. Anti-TIGIT antibodies or fragments thereof that can bind to TIGIT; and, A package insert containing instructions for using an anti-TIGIT antibody or fragment thereof, capable of binding to TIGIT, in combination with an anti-PD-L1:TGFβRII fusion protein to treat or delay the progression of cancer in a target; A kit that includes this.

Images