FC FUSION PROTEINS WITH CD80 EXTRACELLULAR DOMAIN TO TREAT PD-L1 NEGATIVE TUMORS

Abstract

The present invention relates to a composition comprising fusion molecules with the extracellular domain (ECD) of CD80 for use in the treatment of a PD-L1 negative tumor in a subject, wherein the CD80 ECF fusion molecules comprise a human CD80 ECD and a human IgG Fc domain.

Description

This application demonstrates that fusion proteins comprising an extracellular domain (ECD) of CD80 (B7-1) and a crystallizable fragment (Fe) domain of immunoglobulin are effective for the treatment of tumors, regardless of PD-L1 status. Therefore, such fusion proteins can be advantageously used to treat PD-L1-negative tumors. BACKGROUND PD-1 (programmed cell death protein 1) is an immune checkpoint expressed on activated T lymphocytes. The PD-1 pathway is important in the tumor microenvironment, where tumor-expressed PD-L1 (programmed cell death protein 1 ligand) interacts with PD-1 to suppress T lymphocyte effector functions, thereby overcoming immune surveillance and tumor cell destruction. In addition to tumor cells, PD-L1 can also be expressed by antigen-presenting cells in the tumor microenvironment. PD-1 and PD-L1 antagonists have been approved for cancer treatment. PD-L1 has been reported to bind to CD80 (B7-1) and induce bidirectional inhibitory signaling in the absence of CD28 and CTLA-4 receptors for CD80 (Lí et al., JBC 292:6799-6809 (2017)). Therefore, it has been proposed that CD80 proteins could act therapeutically by antagonizing the PD-L1 / PD-1 inhibitory pathway to trigger potent antitumor immunity (Swanson et al., Cancer Research 78, Abstract 4550 (2018)). Indeed, Fe-extracellular domain (ECD) fusion proteins of CD80 have been shown to generate potent antitumor activity. However, PD-L1 is not expressed in all tumors. As a result, PD-L1 testing is necessary for the treatment of certain indications with PD-1 or PD-L1 inhibitors, meaning that tumors that do not express PD-L1 may not be eligible for treatment. Therefore, treatment methods are needed for PD-L1-negative tumors. COMPENDIUM As demonstrated herein, the antitumor effect generated by fusion proteins comprising the extracellular domain (ECD) of human cluster of differentiation 80 (CD80) and the crystallizable fragment (Fe) domain of human immunoglobulin G1 (IgG1) is mediated by CD28 and CTLA-4, but not by PD-L1. Therefore, these fusion proteins are remarkably capable of treating tumors, regardless of their PD-L1 status. Accordingly, methods for treating PD-L1-negative tumors are provided herein, comprising the delivery of fusion proteins comprising an EDC of CD80 and an Fe domain of human IgG1. In certain respects, a method for treating a PD-L1-negative tumor in a subject comprises administering to the subject a composition comprising fusion molecules with the extracellular domain (ECD) of CD80. In certain respects, the tumor has been determined to be PD-L1-negative prior to administration. In certain respects, the method further comprises determining that the tumor is PD-L1-negative prior to administration. In certain respects, a method for selecting a subject with a tumor for treatment with a composition comprising CD80 ECD fusion molecules comprises determining whether a tumor sample obtained from the subject is PD-L1 negative and selecting the subject for treatment with the composition if the tumor sample is determined to be PD-L1 negative. In certain aspects, a composition comprising CD80 ECD fusion molecules is used in the treatment of a PD-L1-negative cancer tumor in a subject. In certain aspects of the composition used, the subject is selected for treatment by determining that a tumor sample obtained from the subject is PD-L1-negative. In certain aspects, a composition comprising CD80 ECD fusion molecules is used in the treatment of a tumor in a subject, where the tumor has been determined to be PD-L1 negative. In certain respects, an in vitro method for identifying a subject with a tumor that responds to treatment with a composition comprising CD80 ECD fusion molecules comprises determining whether a tumor sample obtained from the subject is PD-L1 negative, wherein the subject is identified as responding to treatment with a CD80 ECD fusion molecule if the tumor sample is determined to be PD-L1 negative. In certain respects, an in vitro use of at least one agent capable of determining that a tumor sample is PD-L1 negative is to identify a subject with a tumor that responds to treatment with a composition comprising a CD80 ECD fusion molecule. In certain aspects of a method, composition, or use provided herein, the tumor has been or is determined to be PD-L1 negative by using an agent capable of detecting the PD-L1 protein. In certain aspects, the agent is an antibody that binds specifically to the PD-L1 protein. In certain aspects, the tumor has been or is determined to be PD-L1 negative by Western blot. In certain aspects, the tumor has been or is determined to be PD-L1 negative by fluorescence-activated cell sorting (FACS). In certain aspects, the tumor has been or is determined to be PD-L1 negative by immunohistochemistry (IHC). In certain aspects, the specimen is a paraffin-embedded specimen. In certain aspects of a method, composition, or use provided herein, the tumor has been or is determined to be PD-L1 negative by using an agent capable of detecting PD-L1 mRNA. In certain aspects, the tumor has been or is determined to be PD-L1 negative by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). In certain aspects, the tumor has been or is determined to be PD-L1 negative by using RNA sequencing. In certain aspects, the tumor has been or is determined to be PD-L1 negative by using a microarray. IVIA / t / ZU¿ I / UOÓZO I In certain aspects of a method, composition, or use provided herein, the tumor is a solid tumor. In certain aspects of a method, composition, or use provided herein, the subject is affected by a cancer selected from the group consisting of colorectal cancer, breast cancer, gastric cancer, non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, and endometrial cancer. In certain aspects of a method, composition, or use provided herein, the subject is affected by a cancer that is recurrent or progressive following therapy consisting of surgery, chemotherapy, radiotherapy, or a combination thereof. In certain aspects of a method, composition, or use provided herein, the CD80 ECD fusion molecules comprise a human CD80 ECD and a human lgG1 Fe domain. In certain aspects of a method, composition, or use provided herein, the composition comprises sialylated CD80 ECD fusion molecules. In certain aspects, the sialylated CD80 ECD fusion molecules comprise at least 15 moles of sialic acid (SA) per mole of fusion protein. In certain aspects, the sialylated CD80 ECD fusion molecules comprise 15–60 moles of SA per mole of fusion protein. In certain aspects, the sialylated CD80 ECD fusion molecules comprise 15–40 moles of SA per mole of fusion protein. In certain aspects, the sialylated CD80 ECD fusion molecules comprise 15–30 moles of SA per mole of fusion protein. In certain aspects, the sialylated CD80 ECD fusion molecules comprise 20–30 moles of SA per mole of fusion protein. In certain aspects of a method, composition, or use provided herein, the CD80 ECD fusion molecules comprise a human CD80 ECD comprising the amino acid sequence of SEQ ID NO:1. In certain aspects of a method, composition, or use provided herein, the CD80 ECD fusion molecules comprise a human lgG1 Fe domain comprising the amino acid sequence of SEQ ID NO:3. In certain aspects of a method, composition, or use provided herein, the human lgG1 Fe domain is linked to the carboxy terminus of the human CD80 ECD. In certain aspects of a method, composition, or use provided herein, the CD80 ECD fusion molecules comprise the amino acid sequence of SEQ ID NO:5. In certain aspects of a method, composition, or use provided herein, the PD-L1 negative tumor has a TPS score of less than 5% or less than 1%. In certain aspects of a method, composition, or use provided herein, the composition alone does not induce a significant release of interferon gamma or TNF alpha from T lymphocytes in vitro. In certain aspects of a method, composition, or use provided herein, the composition alone induces a lesser release of interferon gamma or TNF alpha from T lymphocytes in vitro than TGN1412 alone. In certain aspects of a method, composition, or use provided herein, the composition alone is at least 1000 times less potent in inducing the release of interferon gamma or TNF alpha compared to TGN1412 alone.In certain aspects of a method, composition, or use provided herein, the composition can achieve at least 90% inhibition of tumor growth in at least one syngeneic mouse cancer model for a period of at least one week, 10 days, two weeks, or three weeks following administration of a single dose of the composition at between 0.3 and 0.6 mg / kg. In certain aspects, the syngeneic mouse cancer model is a CT26 tumor model. In certain aspects of a method, composition, or use provided herein, the treatment comprises the administration of approximately 0.07 mg to approximately 70 mg of CD80 ECD fusion molecules. In certain aspects, the treatment comprises the administration of approximately 7.0 mg to approximately 70 mg of CD80 ECD fusion molecules. In certain aspects, the treatment comprises the administration of approximately 70 mg of CD80 ECD fusion molecules. In certain aspects, the treatment comprises the administration of approximately 42 mg of CD80 ECD fusion molecules. In certain aspects, the treatment comprises the administration of approximately 21 mg of CD80 ECD fusion molecules. In certain aspects, the treatment comprises the administration of approximately 7 mg of CD80 ECD fusion molecules.In certain aspects, the treatment comprises the administration of approximately 2.1 mg of CD80 ECD fusion molecules. In certain aspects, the treatment comprises the administration of approximately 0.7 mg of CD80 ECD fusion molecules. In certain aspects, the treatment comprises the administration of approximately 0.21 mg of CD80 ECD fusion molecules. In certain aspects, the treatment comprises the administration of approximately 0.07 mg of CD80 ECD fusion molecules. In certain aspects of a method, composition, or use provided herein, the treatment comprises administration once every three weeks. In certain aspects of a method, composition, or use provided herein, the treatment comprises the intravenous administration of CD80 ECD fusion molecules. In certain aspects of a method, composition, or use provided herein, the subject has not received prior therapy with a PD-1 / PD-L1 antagonist. In certain aspects of a method, composition, or use provided herein, the subject has received prior therapy with at least one anti-angiogenic agent. In certain aspects, the anti-angiogenic agent is sunitinib, sorafenib, pazopanib, axitinib, tivizanib, ramucirumab, or bevacizumab. In certain aspects, the anti-angiogenic agent was administered in an advanced or metastatic setting. In certain aspects of a method, composition, or use that are provided in the In this case, the subject is affected by melanoma that has a BRAF mutation. In certain aspects, the subject has received prior therapy with at least one BRAF inhibitor. In certain aspects, the BRAF inhibitor is vemurafenib or dabrafenib. In certain aspects, the BRAF inhibitor was administered at an advanced or metastatic stage. In certain aspects of a method, composition, or use provided herein, the tumor is recurrent or progressive after a therapy selected from surgery, chemotherapy, radiotherapy, and a combination of these. In certain aspects, a method for treating a PD-L1-negative tumor in a human patient comprises administering to the patient a composition comprising from about 0.07 mg to about 70 mg of CD80 extracellular domain (ECD) fusion molecules comprising the amino acid sequence SEQ ID NO:5. In certain aspects, the tumor has been determined to be PD-L1-negative by IHC prior to administration. In certain aspects, the composition comprises sialylated CD80 ECD fusion molecules, and the sialylated CD80 ECD fusion molecules comprise 15-60 moles of SA per mole of fusion protein. BRIEF DESCRIPTION OF THE FIGURES Figures 1A and 1B: Mouse splenocytes were evaluated for mCD80-Fc docking and receptor occupancy. (A) Subsets of splenic immune cells such as CD11b+ DCs (B220-Thy1.2-CD49b-CD11c+CD11b+), CD11b- DCs (B220-Thy1.2-CD49bCD11c+CD11b-), macrophages (B220-Thy1.2-CD49b-CD11c-CD11b+), NK cells (B220-Thy1.2-CD49b+), and T cells (CD3+CD4+ or CD3+CD8+) were identified by flow cytometry. Representative flow cytometry plots illustrate the sorting strategy. (B) Increasing concentrations of mCD80-Fc were incubated with mouse splenocytes (BALB / c strain in the top row; C57BI / 6 strain in the bottom row), and mCD80-Fc docking was measured by biotin-labeled anti-mlgG followed by Alexa488 streptavidin (“drug bound”) and by detection of CD80 ligands with competing Ab clones (“free”) PD-L1 or CD28 listed as antibody binding capacity (AUC) values.CTLA-4 was not detected in any of the immune cells evaluated (no data shown). LD, AUC detection limit. Graphs illustrate the mean ± SD of n = 3 animals / strain measured in technical duplicates. Statistical tests based on ANOVA were performed to determine drug concentrations versus no drug (0 pg / mL) where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 was considered significant. (See Example 1). Figures 2A and 2B: hCD80-Fc receptor occupancy experiments were performed in lentivirally transduced CHO cells expressing CTLA-4, PD-L1, CD28, or all three CD80 ligands (CHO-CTLA4 / PDL1 / CD28; “CHO-3”). Original, non-transduced CHO cells were used as a negative control. (A) CD80 ligand expression in CHO cell lines was assessed by flow cytometry. Representative stepwise histograms and values ​​of the ML / E / ZuZu / UOJZOI days 4, 7 and 11. mCD80-Fc significantly inhibited tumor growth (p = 0.0004 compared to mlgG2a control; p < 0.0001 compared to the untreated group). (See Example 3). DESCRIPTION OF SPECIFIC MODALITIES 1. Definitions Unless otherwise defined, the scientific and technical terms used in connection with the present invention shall have the meanings commonly accepted by those skilled in the art. Furthermore, unless the context otherwise requires, singular terms shall include plurals and plural terms shall include singular terms. Unless otherwise specifically stated or evident from the context, as used herein, the term "or" is understood to be inclusive. As used in an expression such as "A and / or B" herein, "and / or" is intended to include "A and B," "A or B," and "A and B." Likewise, as used in an expression such as "A, B and / or C," "and / or" is intended to include each of the following combinations: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (only); B (only); and C (only). The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain naturally occurring or non-naturally occurring amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and their fragments are included in the definition. The terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of the present invention, a "polypeptide" refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature) to the natural sequence, provided that the protein retains the desired activity.These modifications can be deliberate, through site-directed mutagenesis, or they can be accidental, such as through mutations in the hosts that produce the proteins or errors due to PCR amplification. A "fusion molecule," as used herein, refers to a molecule composed of two or more different molecules that do not occur together in nature and are joined covalently or non-covalently to form a new molecule. For example, fusion molecules may be composed of a polypeptide and a polymer such as PEG, or of two different polypeptides. A "fusion protein" refers to a fusion molecule composed of two or more polypeptides that are not found as a single molecule in nature. A “CD80 extracellular domain” or “CD80 ECD” refers to a polypeptide of the CD80 extracellular domain, including its natural and modified variants. A CD80 ECD, for example, may comprise, consist essentially of, or consist of the amino acid sequence indicated in SEQ ID NO:1 or 2. A “CD80 ECD fusion molecule” refers to a molecule comprising a CD80 ECD and a fusion partner. The fusion partner may be attached to ML / E / ZuZ / UOoZOt covalently, for example, to the N or C terminus of the CD80 ECD or at an internal location. A 'CD80 ECD fusion protein' is a CD80 ECD fusion molecule comprising a CD80 ECD and another polypeptide not naturally associated with the CD80 ECD, such as an Fe domain. A CD80 ECD fusion protein, for example, may comprise, consist essentially of, or consist of the amino acid sequence indicated in SEQ ID NO: 4 or 5. The term “isolate,” as used herein, refers to a molecule that has been separated from at least some of the components with which it is typically found in nature. For example, a polypeptide is called an “isolate” when it is separated from at least some of the components of the cell in which it was produced. In the case of a cell secreting a polypeptide after expression, the physical separation of the supernatant containing the polypeptide from the cell that produced it is considered to involve “isolating” the polypeptide. Similarly, a polynucleotide is referred to as an “isolate” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) where it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide.Therefore, a DNA polynucleotide contained in a vector within a host cell may be referred to as "isolated", provided that that polynucleotide is not found in that vector in nature. The terms "subject" and "patient" are used interchangeably herein to refer to a human being. In some modalities, methods are also provided for treating other mammals, including, but not limited to, rodents, apes, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sporting animals, and mammalian pets. The term "cancer" is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. A cancer may be a solid tumor, for example, colorectal cancer, breast cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, or endometrial cancer. Terms such as "treated," "treatment," or "treat" refer to therapeutic measures that cure, slow, reduce the symptoms of, and / or halt the progression of a condition or pathological disorder. Therefore, those requiring treatment include individuals already diagnosed with, or suspected of having, the disorder. In certain modalities, a subject is successfully "treated" for cancer according to the methods of the present invention if the patient exhibits one or more of the following: a reduction in the number or total absence of cancer cells; a reduction in tumor size; inhibition or absence of infiltration of cancer cells into peripheral organs, including, for example, spread of cancer into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; and relief of one or more associated symptoms. MA / E / ZUZ1 / uoózo i with the specific cancer; lower morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumorigenic frequency or tumorigenic capacity of a tumor; reduction in the amount or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells from a non-tumorigenic state; increase in progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a shorter time to progression (TTP) or any combination of these. The terms “administer,” “administered,” “administration,” and the like, as used herein, refer to methods that can be used to enable the delivery of a drug, e.g., a CD80 ECD fusion protein or antigen-binding fragment thereof, to the desired site of biological action (e.g., intravenous administration). Delivery techniques that may be employed with the agents and methods described herein are found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington’s, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, PA. The term "therapeutically effective amount" refers to a quantity of a drug, for example, a CD80 ECD fusion protein, that is effective in treating a disease or disorder in a subject. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce tumor size or burden; inhibit, to some extent, the infiltration of cancer cells into peripheral organs; inhibit, to some extent, tumor metastasis; inhibit, to some extent, tumor growth; alleviate, to some extent, one or more of the symptoms associated with cancer; and / or produce a favorable response such as increased progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD), a shorter time to progression (TTP), or any combination thereof. The terms “resistant” or “unresponsive,” when used in the context of treatment with a therapeutic agent, mean that the subject exhibits a diminished response or lack of response to a standard dose of the therapeutic agent, relative to the subject’s response to the standard dose of the therapeutic agent in the past, or relative to the expected response of a similar subject with a similar disorder to the standard dose of the therapeutic agent. Therefore, in some modalities, a subject may be resistant to a therapeutic agent even if the subject has not previously received the therapeutic agent, or the subject may develop resistance to the therapeutic agent after having responded to the agent on one or more previous occasions. A "refractory" cancer is one that progresses even after the patient receives anti-tumor treatment, such as chemotherapy. A "recurrent" cancer is one that has grown back, either in the original location or in a distant location, after a response to initial therapy. The terms "programmed cell death ligand 1 1" and "PD-L1" refer to one of two ML / E / ZuZ / UOJZOI are cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulate T-cell activation and cytokine secretion by binding to PD-1. The term “PD-L1” as used herein includes human PD-L1 (hPD-L1), naturally occurring variants and isoforms of hPD-1, and homologous species of hPD-L1. A mature sequence of hPD-L1 is provided as SEQ ID NO:6. The term "PD-L1-negative tumor" refers to a tumor that does not significantly express PD-L1 on its cell surface. The presence or absence of PD-L1 can be determined, for example, by immunohistochemistry, which can be quantified using a tumor proportion score (TPS). A TPS (%) is equal to [Number of PD-L1-stained tumor cells / Total number of viable tumor cells] x 100. Therefore, a PD-L1-negative tumor may be a tumor with a TPS score of less than 5% or less than 1%. The term "PD-1 / PD-L1 antagonist" refers to a moiety that disrupts the PD-1 / PD-L1 signaling pathway. In some forms, the antagonist inhibits the PD-1 / PD-L1 signaling pathway by binding to PD-1 and / or PD-L1. In some forms, the PD-1 / PD-L1 antagonist also binds to PD-L2. In some forms, a PD-1 / PD-L1 antagonist blocks the binding of PD-1 to PD-L1 and optionally to PD-L2. Non-exhaustive examples of PD-1 / PD-L1 antagonists include PD-1 antagonists, such as antibodies that bind to PD-1 (e.g., nivolumab and pembrolizumab); PD-L1 antagonists, such as antibodies that bind to PD-L1 (e.g., atezolizumab, durvalumab, and avelumab); and fusion proteins, such as AMP-224. and peptides, such as AUR-012. An "anti-angiogenic agent" or "angiogenesis inhibitor" refers to an agent such as a low molecular weight substance, a polynucleotide (including, for example, an inhibitory RNA (RNA or piRNA)), a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or unwanted permeability, either directly or indirectly. An anti-angiogenic agent shall be understood to include agents that bind to and block the angiogenic activity of the angiogenic factor or its receptor.For example, an anti-angiogenic agent is an antibody or other antagonist of an angiogenic agent, such as antibodies against VEGF-A (e.g., bevacizumab (Avastin®)) or the VEGF-A receptor (e.g., the KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec® (imatinib mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, Sutent® / SU11248 (sunitinib malate), AMG706, or those described, for example, in International Patent Application WO 2004 / 113304). Anti-angiogenic agents also include natural inhibitors of angiogenesis, such as angiostatin, endostatin, etc. See, for example, Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3 listing antiangiogenic therapy in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al.(2003) Oncogene 22:6549-6556 (e.g., Table 2 listing anti-angiogenic factors. IVIA / t / ZUZ I / UOÓZO I known); Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1 listing anti-angiogenic agents used in clinical trials), and Jayson (2016) Lancet 338(10043):518-529. The term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective and that does not contain any additional components unacceptably toxic to the subject to whom the formulation would be administered. The formulation may be sterile. A pharmaceutical composition may contain a "pharmaceutical carrier," which refers to a carrier that is non-toxic to the recipients at the doses and concentrations used and is compatible with other ingredients in the formulation. The pharmaceutically acceptable carrier is suitable for the formulation used. For example, if the therapeutic agent is to be administered intravenously, the carrier ideally does not irritate the skin and does not cause a reaction at the injection site. As used herein, the terms "around" and "approximately," when used to modify a numeric value or numeric interval, indicate that deviations of 5% to 10% above and 5% to 10% below the value or interval remain within the intended meaning of the stated value or interval. Any composition or method provided herein may be combined with any other composition or method provided herein. 2. Fe fusion proteins with CD80 extracellular domain Methods for delivering CD80 ECD fusion proteins comprising a CD80 ECD and an Fe domain (a ‘CD80 ECD Fe fusion protein’) are provided herein. Examples of CD80 ECD fusion proteins are provided, for example, in WO 2017 / 079117, which is incorporated herein in full by reference. The CD80 ECD can be, for example, a human CD80 ECD. In certain respects, the human CD80 ECD comprises, essentially consists of, or comprises the amino acid sequence indicated in SEQ ID NO:1. The Fe domain can be the Fe domain of an IgG. The Fe domain can be the Fe domain of a human immunoglobulin. In certain respects, the Fe domain is a human IgG Fe domain. In certain respects, the Fe domain is a human IgG1 Fe domain. In certain respects, the human IgG1 Fe domain comprises, consists essentially of, or consists of the amino acid sequence indicated in SEQ ID NO:4. The CD80 ECD and the Fe domain can be directly linked such that the N-terminus amino acid of the Fe domain immediately follows the C-terminus amino acid of the CD80 ECD. In certain respects, the CD80 ECD and the Fe domain are translated as a single polypeptide from a coding sequence that encodes the CD80 ECD and the Fe domain. In certain respects, the CD80 ECD-Fe fusion protein comprises a human CD80 ECD and a human IgG1 Fe domain. In certain respects, the CD80 ECD-Fe fusion protein comprises, essentially consists of, or is composed of the amino acid sequence indicated in SEQ ID NO:5. IVIA / t / ZUZ I / UOÓZO I Depending on how they are produced, CD80 Fe-ECD fusion proteins can have different levels of specific glycosylation modifications. For example, a CD80 Fe-ECD fusion protein can be sialylated and may have different amounts of sialic acid (SA) residues. In certain aspects, an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprises 10 to 60 SA molecules. In certain aspects, an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprises 15 to 60 SA molecules. In certain aspects, an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprises 10 to 40 SA molecules. In certain respects, a CD80 ECD Fe fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprises 15 to 30 SA molecules.In certain aspects, an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5) comprises 15 to 25 SA molecules. In certain aspects, an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5) comprises 20 to 40 SA molecules. In certain aspects, an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5) comprises 20 to 30 SA molecules. In certain respects, a CD80 ECD Fe fusion protein (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5) comprises 30 to 40 SA molecules.In certain aspects, a CD80 ECD-Fe fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprises 10, 15, 20, 25, 30, 35, or 40 SA molecules. In certain aspects, a CD80 ECD-Fe fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprises at least 15 SA molecules. In certain aspects, a CD80 ECD-Fe fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprises at least 20 SA molecules. In certain respects, a CD80 ECD Fe fusion protein (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5) comprises at least 25 SA molecules.In certain aspects, an Fe fusion protein with CD80 ECD (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5) comprises at least 30 SA molecules. In certain aspects, an Fe fusion protein with CD80 ECD (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID. ML / E / ZuZu / UOÓZO I NO:5) comprises at least 35 SA molecules. In certain respects, a CD80 ECD Fe fusion protein (e.g., comprising a human CD80 ECD and a human IgG 1 Fe domain, or comprising SEQ ID NO:5) comprises at least 40 SA molecules. 3. Pharmaceutical compositions comprising Fe fusion proteins with CD80 extracellular domain Methods are provided herein for administering pharmaceutical compositions comprising Fe-ECD fusion proteins of CD80, for example, that have the desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Such acceptable carriers, excipients, or stabilizers are nontoxic to the recipients at the doses and concentrations employed. (See, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams & Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). Compositions to be used for in vivo administration may be sterile.This is easily achieved by filtration, for example, through sterile filtration membranes. In certain respects, a pharmaceutical composition comprising an Fe fusion protein with CD80 ECD (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) is formulated for intravenous administration. In certain aspects, a pharmaceutical composition comprises 70 mg of an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 42 mg of an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 21 mg of an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5). In certain respects, a pharmaceutical composition comprises 7 mg of an Fe fusion protein with CD80 ECD (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5).In certain aspects, a pharmaceutical composition comprises 2.1 mg of an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 0.7 mg of an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 0.21 mg of an Fe-CD80 ECD fusion protein (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5). In certain aspects, a composition. IVIA / I / UOÓZO I pharmaceutical comprises 0.07 mg of an Fe fusion protein with CD80 ECD (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 0.07 to 70 mg of an Fe fusion protein with a CD80 ECD (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 7 to 70 mg of an Fe fusion protein with a CD80 ECD (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises Fe-CD80 ECD fusion proteins (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprising 10 to 60 moles of SA per mole of Fe-CD80 ECD fusion protein. In certain aspects, a pharmaceutical composition comprises Fe-CD80 ECD fusion proteins (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprising 15 to 60 moles of SA per mole of Fe-CD80 ECD fusion protein. In certain aspects, a pharmaceutical composition comprises Fe fusion proteins with CD80 ECD (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5) comprising 10 to 40 moles of SA per mole of Fe fusion protein with CD80 ECD.In certain aspects, a pharmaceutical composition comprises Fe-CD80 ECD fusion proteins (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprising 15 to 30 moles of SA per mole of Fe-CD80 ECD fusion protein. In certain aspects, a pharmaceutical composition comprises Fe-CD80 ECD fusion proteins (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprising 15 to 25 moles of SA per mole of Fe-CD80 ECD fusion protein. In certain aspects, a pharmaceutical composition comprises Fe fusion proteins with CD80 ECD (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5) comprising 20 to 40 moles of SA per mole of Fe fusion protein with CD80 ECD.In certain aspects, a pharmaceutical composition comprises Fe-CD80 ECD fusion proteins (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprising 20 to 30 moles of SA per mole of Fe-CD80 ECD fusion protein. In certain aspects, a pharmaceutical composition comprises Fe-CD80 ECD fusion proteins (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprising 30 to 40 moles of SA per mole of Fe-CD80 ECD fusion protein. In certain aspects, a pharmaceutical composition comprises Fe-CD80 ECD fusion proteins (e.g., ML / E / ZuZu / UOJZOI comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprising 10, 15, 20, 25, 30, 35, or 40 moles of SA per mole of Fe fusion protein with CD80 ECD. In certain aspects, a pharmaceutical composition comprises Fe fusion proteins with CD80 ECD (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprising at least 15 moles of SA per mole of Fe fusion protein with CD80 ECD. In certain aspects, a pharmaceutical composition comprises Fe fusion proteins with CD80 ECD (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5) comprising at least 20 moles of SA per mole of Fe fusion protein with CD80 ECD.In certain aspects, a pharmaceutical composition comprises Fe-CD80 ECD fusion proteins (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprising at least 25 moles of SA per mole of Fe-CD80 ECD fusion protein. In certain aspects, a pharmaceutical composition comprises Fe-CD80 ECD fusion proteins (e.g., comprising a human CD80 ECD and a human IgG1 Fe domain, or comprising SEQ ID NO:5) comprising at least 30 moles of SA per mole of Fe-CD80 ECD fusion protein. In certain aspects, a pharmaceutical composition comprises Fe fusion proteins with CD80 ECD (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5) comprising at least 35 moles of SA per mole of Fe fusion protein with CD80 ECD.In certain aspects, a pharmaceutical composition comprises Fe fusion proteins with CD80 ECD (e.g., comprising a human CD80 ECD and a human lgG1 Fe domain, or comprising SEQ ID NO:5) comprising at least 40 moles of SA per mole of Fe fusion protein with CD80 ECD. 4. Methods and Uses of Fe-CD80 Extracellular Domain Fusion Proteins Methods for treating a PD-L1-negative tumor (e.g., in a human) are presented herein, comprising administering to a subject in need a CD80 Fe-CD80 extracellular domain fusion protein, or a pharmaceutical composition thereof. The CD80 Fe-CD80 extracellular domain fusion protein may comprise the human CD80 extracellular domain and the Fe domain of human IgG1. In some embodiments, the CD80 Fe-CD80 extracellular domain fusion protein comprises the sequence SEQ ID NO:5. The presence or absence of PD-L1 can be determined using an agent capable of detecting the PD-L1 protein, such as an anti-PD-L1 antibody. Therefore, in some modalities, a tumor can be identified as PD-L1-negative by subjecting a tumor sample to Western blot, fluorescence-activated cell sorting (FACS), or immunohistochemistry (IHC) using such an agent. In some modalities, IHC can be used to quantify the amount of PD-L1 in a tumor sample, for example, by using a tumor proportion score (TPS). A TPS (%) is equal to [Number of PD-L1-stained tumor cells / total number of viable tumor cells] x ML / E / ZuZu / UOJZOI 100. As provided herein, a PD-L1-negative tumor may be a tumor with a TPS score of less than 5%. As provided herein, a PD-L1-negative tumor may be a tumor with a TPS score of less than 1%. The presence or absence of PD-L1 can be determined using an agent capable of detecting PD-L1 mRNA. Therefore, in some modalities, a tumor can be identified as PD-L1 negative by subjecting a tumor sample to quantitative reverse transcriptase (RT) polymerase chain reaction (PCR), RNA sequencing, or microarray analysis. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient approximately 70 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient approximately 42 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient approximately 21 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks.In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient approximately 7 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient approximately 2.1 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient approximately 0.7 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks.In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient approximately 0.21 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. In another aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient approximately 0.07 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient 70 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient 42 mg of a CD80 ECD fusion protein (e.g., comprising In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient 21 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence indicated in SEQ ID NO:5), e.g., once every three weeks. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient 7 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence indicated in SEQ ID NO:5), e.g., once every three weeks. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient 2.1 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence indicated in SEQ ID NO:5), e.g., once every three weeks.In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient 0.7 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient 0.21 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient 0.07 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. In one aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient from about 0.07 mg to about 70 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. In another aspect, a method for treating a PD-L1-negative tumor in a patient comprises administering to the patient from about 7 mg to about 70 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence specified in SEQ ID NO:5), e.g., once every three weeks. According to the methods provided herein, a CD80 ECD fusion protein (e.g., comprising the amino acid sequence indicated in SEQ ID NO:5) can be administered intravenously. According to the methods described herein, a PD-L1-negative tumor may be, for example, a solid tumor, including, for example, an advanced or metastatic solid tumor. In certain cases, a PD-L1-negative tumor is not a primary tumor of the central nervous system. In certain cases, the PD-L1 negative tumor is a renal cell carcinoma. In certain cases, a PD-L1 negative tumor is a melanoma. In certain cases, a PD-L1 negative tumor is a colorectal cancer, breast cancer, Ma / E / ZUZI / uoózo i gastric cancer, non-small cell lung cancer, small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer or endometrial cancer. The patient to be treated according to the methods described herein may have previously received therapy with at least one PD-1 / PD-L1 antagonist selected from a PD-1 antagonist and a PD-L1 antagonist. The PD-1 / PD-L1 antagonist may be, for example, nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab. The PD-1 / PD-L1 antagonist may have been administered in an advanced or metastatic setting. In some cases, the tumor does not respond to this treatment or recurs during or after it. In other cases, the patient to be treated according to the methods described herein has not previously received therapy with a PD-1 / PD-L1 antagonist. The patient to be treated according to the methods provided herein may have previously received therapy with an anti-angiogenic agent. The anti-angiogenic agent may be, for example, sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab. The anti-angiogenic agent may have been administered in an advanced or metastatic setting. The patient to be treated according to the methods provided herein, for example, a patient with melanoma, may have a BRAF mutation. The patient may have received prior therapy with a BRAF inhibitor. The BRAF inhibitor may be, for example, vemurafenib or dabrafenib. The BRAF inhibitor may have been administered in an advanced or metastatic setting. The tumor to be treated according to the methods provided herein may be recurrent or progressive after a therapy selected from surgery, chemotherapy, radiotherapy, and a combination of these. The tumor to be treated according to the methods provided herein may be resistant or unresponsive to a PD-1 / PD-L1 antagonist, such as nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab. The tumor to be treated according to the methods provided herein may be resistant or unresponsive to an anti-angiogenic agent, such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab. The tumor to be treated according to the methods provided herein may be resistant or unresponsive to a BRAF inhibitor, such as vemurafenib or dabrafenib. The tumor to be treated according to the methods provided herein may be refractory to a PD-1 / PD-L1 antagonist, such as nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab. The tumor to be treated according to the methods provided herein may be refractory to an anti-angiogenic agent, such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab. The tumor to be treated according to the methods provided herein may be refractory to a BRAF inhibitor, such as vemurafenib or dabrafenib. The tumor to be treated according to the methods provided herein may recur after treatment with a PD-1 / PD-L1 antagonist, such as nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab. The tumor to be treated according to the methods provided herein may recur after treatment with an anti-angiogenic agent, such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab. The tumor to be treated according to the methods provided herein may recur after treatment with a BRAF inhibitor, such as vemurafenib or dabrafenib. EXAMPLES The examples described below are intended to be purely illustrative of the invention and should not be considered exhaustive in any way. The examples are not intended to represent that the experiments presented below are all or the only experiments performed. Every effort has been made to ensure accuracy with respect to the numbers used (e.g., quantities, temperature, etc.), but some experimental errors and deviations should be expected. Unless otherwise stated, parts are parts by weight, molecular weight is the average molecular weight by weight, temperature is stated in degrees Celsius, and pressure is atmospheric or similar. Example 1: Murine CD80 ECD fusion molecules (mCD80-Fc) do not couple to PD-L1 Adult murine splenocytes of the BALB / cy C57BI / 6 strains were used to determine if a mouse substitute fusion protein comprising the extracellular domain (ECD) of murine CD80 linked to the Fe domain of wild-type mouse lgG2a (mCD80-Fc) couples to CD80 ligands. Mouse splenocytes were prepared from adult BALB / cy C56BI / 6 mice using methods known to those skilled in the art. Splenocytes (2–4 x 10⁶ cells / mL) were pelleted by centrifugation, and the media were discarded. mCD80-Fc was added at various concentrations (0–1000 pg / mL), and the cells were incubated for 40 minutes on ice. Paraformaldehyde (4%) was added to the splenocytes, and they were incubated for 10 minutes at room temperature. The splenocytes were washed and pelleted by centrifugation, followed by the addition of biotin-labeled anti-mouse IgG in FACS buffer and a further 20-minute incubation at room temperature. A mixture of streptavidin-Alexa488 and antibodies targeting CTLA-4, PD-L1, and CD28 was added. Quantum Simply Cellular Bang microspheres were used to develop a standard curve for mCD80-Fc molecules.Sample data were acquired from the LSRII or LSRFortessa databases and analyzed using FlowJo, Excel, and Graphpad Prism. A FACS analysis was performed to determine the coupling of mCD80-Fc to CD11 b-ι- dendritic cells, CD11 b- dendritic cells, macrophages, NK cells, CD4+ T cells, and CD8+ T cells. Figure 1A shows an example of the sorting strategy used in this example. Figure 1B shows that mCD80-Fc binds in a concentration-dependent manner, primarily to CD4+ and CD8+ T lymphocytes in both splenocyte types. However, mCD80-Fc binds to a higher proportion of T lymphocytes from BALB / c-derived splenocytes than from C57BL / 6-derived splenocytes. Additionally, there was a small proportion of mCD80 binding to macrophages in both splenocyte types. Therefore, mCD80-Fc was shown to bind to CD4+ T lymphocytes, CD8+ T lymphocytes, and macrophages, but not to CD11b+ or CD11b- dendritic cells. PD-L1 was detected in the analyzed immune cells, with the highest expression in macrophages. There were no changes in the amount of free PD-L1, even with increasing concentrations of mCD80-Fc, demonstrating that there is no interaction between mCD80-Fc and PD-L1 (Figure 1B). In contrast, CD4+ and CD8+ T lymphocytes were the only immune cells evaluated that showed CD28 expression. With increasing concentrations of mCD80-Fc, there was a significant decrease in the amount of free CD28, demonstrating that mCD80-Fc binds to CD28 (Figure 1B). CTLA-4 was not detected in any of the immune cell types evaluated. These results demonstrate that mCD80-Fc primarily binds to CD4+ T lymphocytes and CD8+ T lymphocytes of BALB / cy C57BI / 6 splenocytes via CD28 coupling, and not PD-L1 coupling. Example 2: Human CD80 ECD fusion molecules (hCD80-Fc) do not couple to PD-L1 Chinese hamster ovary cells (“CHO”) were evaluated to determine hCD80-Fc coupling to human CD80 ligands. CHO cells were modified to express human CTLA4, PD-L1, CD28, or all three CD80 ligands (i.e., CHO-CTLA4 / PD-L1 / CD28; “CHO-3”). The protocol for determining hCD80-Fc coupling to ligands is the same as that used in Example 1. Figure 2A shows CD80 ligand expression in all CHO cell lines by flow cytometry. Representative stepwise histograms are shown, and antibody-binding capacity (ABC) values, which determine the number of Ab-binding sites per cell, are listed in bar graphs on the right. Figure 2B shows that hCD80-Fc bound to CHO-CTLA4, CHO-CD28, and CHO-3 cells in a concentration-dependent manner, with hCD80-Fc binding to CD80 ligands at a minimum of 0.5 pg / mL. Furthermore, hCD80-Fc binding to CD80 ligands resulted in a decrease in free CTLA-4 and CD28 in the respective cell lines (Figure 2B). However, hCD80-Fc did not bind to CHO-PD-L1 or original CHO cells, demonstrating that hCD80-Fc does not couple to PD-L1. These results show that hCD80-Fc binds to CTLA-4 and CD28, but not to PD-L1. Compared to the CHO-3 cell line, hCD80-Fc bound to CTLA-4 and CD28 at levels similar to those in single-expression CHO cell lines. Human PBMCs were evaluated to determine hCD80-Fc coupling on B lymphocytes (CD19+), monocytes (CD14+), NK lymphocytes (CD56+), and T lymphocytes (CD3+CD4+ or CD3+CD8+). Figure ML / E / ZuZu / UOJZOI 3A demonstrates an example of a FACS classification strategy for this study. PD-L1 was detected on T lymphocytes and monocytes, but CD28 was detected primarily on CD4+ and CD8+ T lymphocytes. Figure 3B shows that hCD80-Fc bound in a concentration-dependent manner to CD4+ and CD8+ T lymphocytes, with significant detection of the bound drug at minimum concentrations of 100 pg / mL. hCD80-Fc binding revealed a decrease in free CD28 on CD4+ and CD8+ T lymphocytes, but no changes in PD-L1 levels were detected on T lymphocytes or monocytes. CTLA-4 was not detected on any of the immune cell types assessed. Therefore, these results demonstrate that hCD80-Fc binds primarily to human PBMC CD4+ and CD8+ T lymphocytes via CD28 docking. Human CD4+Teff and CD4+Treg cells expanded in vitro were evaluated to determine the binding of hCD80-Fc to CTLA-4, PD-L1, and CD28. The in vitro-expanded human CD4+Teff and CD4+Treg cells demonstrated expression of CTLA-4, PD-L1, and CD28 (Figure 4A). hCD80-Fc bound to CD4+Teff and Treg cells in a concentration-dependent manner, with hCD80-Fc binding detected at a minimum of 50 pg / mL and saturation at approximately 400 pg / mL (Figure 4B). This binding resulted in decreased free CTLA-4 expression only in CD4+Treg cells, while decreased free CD28 expression was detected in both CD4+Teff and Treg cells (Figure 4B). Free PD-L1 levels did not change even with exposure to increasing concentrations of hCD80-Fc (Figure 4B). These data demonstrate that hCD80-Fc couples to CTLA-4 and CD28 on CD4+Teff and CD4+Treg cells. Example 3: mCD80-Fc inhibits the growth of tumors that do not express PD-L1 in a syngeneic CT26 mouse model Data from Examples 1 and 2 demonstrate that CD80 does not bind to PD-L1. Therefore, to determine whether PD-L1 binding is required for mCD80-Fc to exert its antitumor activity, PD-L1-inactivated CT26 tumor cells were used in an in vivo syngeneic mouse model. Unlike xenograft models, syngeneic mouse models possess a functional immune system and are thus useful for evaluating cancer immunotherapies, which work by harnessing the endogenous immune response. CT26 is a murine colorectal carcinoma derived from BALB / c mice that express high levels of PD-L1. In this study, a genetically modified CT26 tumor (CT26 PD-L1 KO) lacking PD-L1 expression was used. Immunocompetent BALB / c mice were inoculated with PD-L1 KO CT26 tumor cells. Mice were placed into three groups for treatment with the following: (1) mouse lgG2a (control); (2) mCD80-Fc; or (3) no treatment control.Mice were treated with either 0.3 mg / kg of mouse lgG2a (group 1) or 0.3 mg / kg of mCD80-Fc (group 2) on days 4, 7, and 11 (days post-inoculation) by intravenous injection. See Table 1. The mean tumor size at the start of treatment was 90 mm3. The study ended on day 21. Table 1 IVIA / t / ZU¿ I / UOÓZO I Treatment Group Dosage Mice (mg / kg, schedule, route) (n) 1 mouse lgG2a 0.3 mg / kg on D4, D7, D11 200 pL IV 15 2 mCD80-Fc 0.3 mg / kg on D4, D7, D11 200 pL IV 15 3 n / an / a 15 MA / E / ZUZI / uoózo i Figure 5 shows that mean tumor volume increased in the mlgG2a and untreated control groups, but the mCD80-Fc group showed significant inhibition of tumor growth (p = 0.0004 compared to the mouse lgG2a control; p < 0.0001 compared to the untreated group). No adverse effects of either mlgG2a or mCD80-Fc treatment were detected, including changes in body weight (no data shown). These data provide in vivo evidence that mCD80-Fc treatment is surprisingly independent of PD-L1 expression in tumor cells. The scope of the invention is not limited to the specific embodiments described herein. In fact, various modifications of the invention, in addition to those described, will be evident to those skilled in the art from the foregoing description and the accompanying figures. It is intended that these modifications fall within the scope of the appended claims. All references (e.g., publications, patents or patent applications) mentioned herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if it had been specifically and individually stated that each individual reference (e.g., publication, patent or patent application) is incorporated herein in its entirety for all purposes by reference. Other modalities are found in the following claims. SEQUENCE TABLE The table below provides a list of certain sequences referenced herein. SEQ. ID. NO. Description Sequence 1 Human CD80 ECD sequence (without signal sequence) VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMM SGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYE KDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGF PEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTN HSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN 2 Mouse CD80 ECD sequence (without signal sequence) VDEQLSKSVKDKVLLPCRYNSPHEDESEDRIYWQKHDKVVLSV IAGKLKVWPEYKNRTLYDNTTYSLIILGLVLSDRGTYSCVVQKKE RGTYEVKHLALVKLSIKADFSTPNITESGNPSADTKRITCFASGG FPKPRFSWLENGRELPGINTTISQDPESELYTISSQLDFNTTRN signal) HTIKCLIKYGDAHVSEDFTWEKPPEDPPDSKN 3 Fe de lgG1 humana EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEWKFNWYVDGVVHNACTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNCALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSL 4CDKSL de ESPGON 2014 mouse CD80 (part of Fe underlined) VDEQLSKSVKDKVLLPCRYNSPHEDESEDRIYWQKHDKVVLSV IAGKLKVWPEYKNRTLYDNTTYSLIILGLVLSDRGTYSCVVQKKE RGTYEVKHLALVKLSICADFSNITESGNPSATFATTCGTTGC FPKPRFSWLENGRELPGINTTISQDPESELYTISSQLDFNTTRN HTIKCLIKYGDAHVSEDFTWEKPPEDPPDSKNEPRGPTIKPCPP CKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDP DVQISWFVNNVVHTAQSTQTHREDVQLVQ MSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEE MTKKQVTLTCCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDS DGSYFMYSKLRVEKNWVERNSYSCSVVHEGLHNHHTTKSFS RTPGK 5 Fe lgG1 human ECD departed CD0 ( CD8 subgroup) VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMM SGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGF PEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTN HSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNEPKSSDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 6 PD-L1 humano (maduro, sin secuencia señal) FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTH LVILGAILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET Μλ / Ε / ZUZI / uoózo i

Claims

1. A method for treating a PD-L1-negative tumor in a subject, the method comprising administering to the subject a composition comprising fusion molecules with the extracellular domain (ECD) of CD80.

2. The method of claim 1, wherein the tumor has been determined to be negative for PD-L1 prior to administration.

3. The method of claim 1, further comprising determining that the tumor is PD-L1 negative prior to administration.

4. A method for selecting a subject with a tumor for treatment with a composition comprising CD80 ECD fusion molecules, the method comprising determining whether a tumor sample obtained from the subject is PD-L1 negative and selecting the subject for treatment with the composition if the tumor sample is determined to be PD-L1 negative.

5. A composition comprising CD80 ECD fusion molecules for use in the treatment of a PD-L1-negative cancer tumor in a subject.

6. The composition for use of claim 5, wherein the subject is selected for treatment by determining that a tumor sample obtained from the subject is negative for PD-L1.

7. A composition comprising CD80 ECD fusion molecules for use in the treatment of a tumor in a subject, wherein the tumor has been determined to be PD-L1 negative.

8. An in vitro method for identifying a subject with a tumor that responds to treatment with a composition comprising CD80 ECD fusion molecules, the method comprising determining whether a tumor sample obtained from the subject is PD-L1 negative, wherein the subject is identified as responding to treatment with a CD80 ECD fusion molecule if the tumor sample is determined to be PD-L1 negative.

9. An in vitro use of at least one agent capable of determining that a tumor sample is PD-L1 negative, to identify a subject with a tumor that responds to treatment with a composition comprising a CD80 ECD fusion molecule.

10. The method, composition or use of any of claims 2-4 and 6-9, wherein the tumor has been or is determined to be PD-L1 negative by the use of an agent capable of detecting the PD-L1 protein, optionally wherein the agent is an antibody that binds specifically to the PD-L1 protein.

11. The method, composition or use of claim 10, wherein the tumor has been or is determined to be negative for PD-L1 by Western blot.

12. The method, composition or use of claim 10, wherein MLE / E / ZuZu / UOJZOI 25 has been determined or the tumor is determined to be PD-L1 negative by fluorescence-activated cell sorting (FACS).

13. The method, composition or use of claim 10, wherein the tumor has been or is determined to be negative for PD-L1 by immunohistochemistry (IHC), optionally wherein the sample is a paraffin-embedded sample.

14. The method, composition or use of any of claims 2-4 and 6-9, wherein the tumor has been or is determined to be PD-L1 negative by the use of an agent capable of detecting PD-L1 mRNA.

15. The method, composition or use of claim 14, wherein the tumor has been or is determined to be negative for PD-L1 by quantitative reverse transcriptase (RT) polymerase chain reaction (PCR).

16. The method, composition or use of claim 14, wherein the tumor has been or is determined to be negative for PD-L1 by the use of RNA sequencing.

17. The method, composition or use of claim 14, wherein the tumor has been or is determined to be PD-L1 negative by the use of a microarray.

18. The method, composition or use of any of claims 1-17, wherein the tumor is a solid tumor.

19. The method, composition or use of any of claims 1-18, wherein the subject is affected by a cancer selected from the group consisting of colorectal cancer, breast cancer, gastric cancer, non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer and endometrial cancer.

20. The method, composition or use of any of claims 1-19, wherein the subject is affected by a cancer that is recurrent or progressive after therapy consisting of surgery, chemotherapy, radiotherapy, or a combination thereof.

21. The method, composition or use of any of claims 1-20, wherein the CD80 ECD fusion molecules comprise a human CD80 ECD and a human lgG1 Fe domain.

22. The method, composition or use of any of claims 1-21, wherein the composition comprises sialylated CD80 ECD fusion molecules.

23. The method, composition or use of claim 22, wherein the sialylated CD80 ECD fusion molecules comprise at least 15 moles of sialic acid (SA) per mole of fusion protein.

24. The method, composition or use of claim 22, wherein the sialylated CD80 ECD fusion molecules comprise 15-60 moles of SA per mole of fusion protein.

25. The method, composition, or use of claim 22, wherein the sialylated CD80 ECD fusion molecules comprise 15-40 moles of SA per mole of fusion protein. MA / E / ZUZ1 / uoózo i 26. The method, composition or use of claim 22, wherein the sialylated CD80 ECD fusion molecules comprise 15-30 moles of SA per mole of fusion protein.

27. The method, composition or use of claim 22, wherein the sialylated CD80 ECD fusion molecules comprise 20-30 moles of SA per mole of fusion protein.

28. The method, composition or use of any of claims 1-27, wherein the CD80 ECD fusion molecules comprise a human CD80 ECD comprising the amino acid sequence of SEQ ID NO:

1.

29. The method, composition or use of any of claims 1-28, wherein the CD80 ECD fusion molecules comprise a human IgG1 Fe domain comprising the amino acid sequence SEQ ID NO:

3.

30. The method, composition or use of any of claims 1-29, wherein the Fe domain of human lgG1 is linked to the carboxy terminus of the ECD of human CD80.

31. The method, composition or use of any of claims 1-30, wherein the CD80 ECD fusion molecules comprise the amino acid sequence of SEQ ID NO:

5.

32. The method, composition or use of any of claims 1-31, wherein the PD-L1 negative tumor has a TPS score less than 5% or less than 1%.

33. The method, composition or use of any of claims 1-32, wherein the composition alone does not cause a significant release of interferon gamma or TNF alpha from T lymphocytes in vitro.

34. The method, composition or use of any of claims 1-33, wherein the composition alone causes a lower release of interferon gamma or TNF alpha from T lymphocytes in vitro than TGN1412 alone.

35. The method, composition or use of claim 34, wherein the composition alone is at least 1000 times less potent in inducing the release of interferon gamma or TNF alpha compared to TGN1412 alone.

36. The method, composition or use of any of claims 1-35, wherein the composition can achieve an inhibition of tumor growth of at least 90% in at least one syngeneic mouse cancer model for a period of at least one week, 10 days, two weeks or three weeks after administration of a single dose of the composition at between 0.3 and 0.6 mg / kg.

37. The method, composition or use of claim 36, wherein the syngeneic mouse cancer model is a CT26 tumor model.

38. The method, composition or use of any of claims 1-37, wherein the treatment comprises the administration of about 0.07 mg to about 70 mg of the CD80 ECD fusion molecules.

39. The method, composition or use of claim 38, wherein the treatment comprises the administration of about 7.0 mg to about 70 mg of the fusion molecules ML / E / ZuZu / UOJZOI with CD80 ECD.

40. The method, composition or use of claim 39, wherein the treatment comprises the administration of about 70 mg of the CD80 ECD fusion molecules.

41. The method, composition or use of claim 39, wherein the treatment comprises the administration of approximately 42 mg of the CD80 ECD fusion molecules.

42. The method, composition or use of claim 39, wherein the treatment comprises the administration of about 21 mg of the CD80 ECD fusion molecules.

43. The method, composition or use of claim 39, wherein the treatment comprises the administration of about 7 mg of the CD80 ECD fusion molecules.

44. The method, composition or use of claim 38, wherein the treatment comprises the administration of approximately 2.1 mg of the CD80 ECD fusion molecules.

45. The method, composition or use of claim 38, wherein the treatment comprises the administration of about 0.7 mg of the CD80 ECD fusion molecules.

46. ​​The method, composition or use of claim 38, wherein the treatment comprises the administration of about 0.21 mg of the CD80 ECD fusion molecules.

47. The method, composition or use of claim 38, wherein the treatment comprises the administration of about 0.07 mg of the CD80 ECD fusion molecules.

48. The method, composition or use of any of claims 1-47, wherein the treatment comprises administration once every three weeks.

49. The method, composition or use of any of claims 1-48, wherein the treatment comprises the intravenous administration of the CD80 ECD fusion molecules.

50. The method, composition or use of any of claims 1-49, wherein the subject has not received prior therapy with a PD-1 / PD-L1 antagonist.

51. The method, composition or use of any of claims 1-50, wherein the subject has received prior therapy with at least one anti-angiogenic agent.

52. The method, composition or use of any of claim 51, wherein the anti-angiogenic agent is sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab or bevacizumab.

53. The method, composition or use of any of claim 51 or 52, wherein the anti-angiogenic agent was administered in an advanced or metastatic setting.

54. The method, composition or use of any of claims 1-53, wherein the subject is affected by a melanoma having a BRAF mutation.

55. The method, composition or use of claim 54, wherein the subject has received prior therapy with at least one BRAF inhibitor.

56. The method, composition or use of claim 55, wherein the BRAF inhibitor is vemurafenib or dabrafenib.

57. The method, composition or use of any of claim 55 or 56, wherein the BRAF inhibitor was administered in an advanced or metastatic setting.

58. The method, composition or use of any of claims 1-57, wherein the tumor is recurrent or progressive after therapy selected from surgery, chemotherapy, radiotherapy, and a combination thereof.

59. A method for treating a PD-L1-negative tumor in a human patient, the method comprising administering to the patient a composition comprising about 0.07 mg to about 70 mg of fusion molecules with the extracellular domain (ECD) of CD80 comprising the amino acid sequence SEQ ID NO:

5.

60. The method of claim 59, wherein the tumor has been determined to be negative for PD-L1 by IHC prior to administration.

61. The method of claim 59 or 60, wherein the composition comprises sialylated CD80 ECD fusion molecules, and wherein the sialylated CD80 ECD fusion molecules comprise 15-60 moles of SA per mole of fusion protein.